{ } THE PURINA PET INSTITUTE SYMPOSIUM

Advancing Life Through Diet Restriction

LESS
WHEN

IS
MORE
{ } THE PURINA PET INSTITUTE SYMPOSIUM

Advancing Life Through Diet Restriction

September 20–21, 2002

Hyatt Regency Union Station
St. Louis, Missouri, USA
 Published by The Gloyd Group, Inc.
Wilmington, Delaware, USA

©2002 by Nestléu.
All rights reserved.
Printed in the United States of America.
{ } TA B L E O F C O N T E N T S
■ Dietary Restriction and Aging: An Historical Overview
/ Richard Weindruch, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
■ Effects of Diet Restriction on Life Span and Age-Related Changes in Dogs:
Experimental Design / Richard D. Kealy, PhD, and Dennis F. Lawler, DVM . . . . . . . . . . 11
General Sessions

■ Diet Restriction and Longevity: Chronic Diseases and Causes of Mortality

/ Dennis F. Lawler, DVM, Richard H. Evans, DVM, MS, Richard D. Kealy, PhD,
Jay M. Harrison, MS, MA, and Brian T. Larson, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
■ Influences of Limit-Feeding and Aging on Antioxidant Status of Pair-Fed
Labrador Retrievers / Howard D. Stowe, DVM, PhD, Diplomate ACVN,
Richard D. Kealy, PhD, and Dennis F. Lawler, DVM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
■ A Longitudinal Study of Immunosenescence: Does Diet Restriction
Ameliorate the Aging Process in Dogs? / Elizabeth H. Greeley, PhD,
Joan M. Ballam, MS, Jay M. Harrison, MS, MA, Richard D. Kealy, PhD,
Dennis F. Lawler, DVM, and Mariangela Segre, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ Diet Restriction and Osteoarthritis in Animals / George Lust, PhD . . . . . . . . . . . . . 16

■ Effects of Restricted Feeding on Onset, Incidence, and Severity of
Osteoarthritis

Hip Dysplasia and Osteoarthritis in Dogs: Diagnostic, Therapeutic,

and Genetic Ramifications / Gail K. Smith, VMD, PhD, Diplomate ACVS,
Darryl N. Biery, DVM, Richard D. Kealy, PhD, and Dennis F. Lawler, DVM . . . . . . . . . . . . . 21
■ Clinical Significance of Osteoarthritis and Hip Dysplasia Findings in
the Restricted Feeding Trial / Gail K. Smith, VMD, PhD, Diplomate ACVS,
Darryl N. Biery, DVM, Richard D. Kealy, PhD, and Dennis F. Lawler, DVM . . . . . . . . . . . . . 27

■ Diet Restriction, Carbohydrate Metabolism, and the Retardation of

Senescence / Edward J. Masoro, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Carbohydrate
Metabolism

■ Diet Restriction and Aging: Canine Carbohydrate and Lipid Metabolism

/ Brian T. Larson, PhD, Dennis F. Lawler, DVM, Jay M. Harrison, MS, MA, and
Richard D. Kealy, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
■ Life is Shorter if You Eat Dessert First: Clinical Implications of the
Purina 448 Study / Deborah S. Greco, DVM, PhD, Diplomate ACVIM . . . . . . . . . . . . . . . 35

■ The Aging Cardiovascular System: Alterations Induced by Dietary

Restriction / Jeremiah T. Herlihy, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Cardiology

■ Electrocardiography in a Study of Diet Restriction and Aging in

Labrador Retrievers / Walter E. Weirich, DVM, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
■ Systemic Arterial Blood Pressure: The Silent Killer that Never
Should Be! / Robert L. Hamlin, DVM , PhD, Diplomate ACVIM . . . . . . . . . . . . . . . . . . . . 54

■ Estimated Body Composition Values of Control-Fed Versus Restricted-Fed

Labrador Retrievers in a Life Span Study / Richard D. Kealy, PhD,
Composition

Dennis F. Lawler, DVM, Brian T. Larson, PhD, and Edward C. Hume, BS . . . . . . . . . . . . . . . 55

Body

■ Body Composition and Dietary Restriction in Rhesus Macaques

/ Joseph W. Kemnitz, PhD, and Ricki J. Colman, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
■ Controlling Body Composition: Who, When, and How
/ D. P. Laflamme, DVM, PhD, Diplomate ACVN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

continued
{ } TA B L E O F C O N T E N T S continued

■ Brief Calorie Restriction Leads to Enhanced Insulin Signaling in

Skeletal Muscle / Gregory D. Cartee, PhD, Carrie E. McCurdy, BS,
Robert T. Davidson, PhD, and Edward B. Arias, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
■ Calorie Restriction Provides Insights into Insulin Signaling in
Skeletal Muscle / Heidi K. Ortmeyer, PhD, Noni L. Bodkin, PhD,
and Barbara C. Hansen, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
■ Effects of Calorie Restriction in Long-Lived, Growth Hormone
(GH)-Resistant GHR/GHBP-Knockout (KO) Mice / Michael Bonkowski, MS,
Khalid Al-Regaiey, DVM, and Andrzej Bartke, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
■ Age-Related Changes in Canine CD8 Memory Cells: A Longitudinal
Aging/Dietary Restriction Study / Elizabeth H. Greeley, PhD,
Posters

Joan M. Ballam, MS, Jay M. Harrison, MS, MA, Richard D. Kealy, PhD,
Dennis F. Lawler, DVM, and Mariangela Segre, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
■ Inhibition of Th-2 Cytokines and Prolongation of Life Span of Lupus
Disease Prone Mice by a Combination of Dietary n-3 Fatty Acids and
Food Restriction / Gabriel Fernandes, PhD, Christopher Jolly, PhD,
Richard Lawrence, PhD, and Dongxu Sun, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
■ Energy Requirements of Old Cats / Gerardo Pérez-Camargo, MRCVS, PhD,
and Robert Rudnick, MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
■ A Gene Expression Signature for Delayed Aging in Mice
/ Richard A. Miller, MD, PhD, Yayi Chang, BS, Andrzej T. Galecki, MD, PhD,
Khalid Al-Regaiey, DVM, John J. Kopchick, PhD, and Andrzej Bartke, PhD . . . . . . . . . . . . . 71
■ Comparison of Indirect Calorimetry Methods in Dogs:
Open Flow Cage Versus Mask / Sharon A. Center, DVM, Diplomate ACVIM,
Karen L. Warner, AS, LVT, and D.P. Carey, DVM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

ARTICLES REPRINTED FROM THE JOURNAL OF THE AMERICAN VETERINARY MEDICAL

ASSOCIATION AND THE AMERICAN JOURNAL OF VETERINARY RESEARCH

■ Effects of Limited Food Consumption on the Incidence of Hip Dysplasia

in Growing Dogs / Richard D. Kealy, PhD, Sten E. Olsson, DVM, MD, PhD,
K.L. Monti, PhD, Dennis F. Lawler, DVM, Darryl N. Biery, DVM, R.W. Helms, PhD,
George Lust, PhD, Gail K. Smith, VMD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
■ Five-Year Longitudinal Study on Limited Food Consumption and
Appendix I

Development of Osteoarthritis in Coxofemoral Joints of Dogs /

Richard D. Kealy, PhD, Dennis F. Lawler, DVM, Joan M. Ballam, MS,
George Lust, PhD, Gail K. Smith, VMD, PhD, Darryl N. Biery, DVM,
Sten E. Olson, DVM, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
■ Within- and Between-Examiner Repeatability of Distraction Indices
of the Hip Joints in Dogs / Gail K. Smith, VMD, PhD, Elizabeth LaFond, DVM,
Thomas P. Gregor, BS, Dennis F. Lawler, DVM, and Robert C. Nie . . . . . . . . . . . . . . . . . . . . 91
■ Influence of Restricted Food Intake on Estrous Cycles and
Pseudopregnancies in Dogs / Dennis F. Lawler, DVM,
Shirley D. Johnston , DVM, PhD, D. Glenn Keltner, PhD, Joan M. Ballam, MS,
Richard D. Kealy, PhD, Thomas Bunte, PhD, George Lust, PhD, Sandra L. Mantz,
and Robert C. Nie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

continued
{ } TA B L E O F C O N T E N T S continued

ARTICLES REPRINTED FROM THE JOURNAL OF THE AMERICAN VETERINARY MEDICAL

Appendix I (continued) ASSOCIATION AND THE AMERICAN JOURNAL OF VETERINARY RESEARCH

■ Evaluation of the Effect of Limited Food Consumption on Radiographic

Evidence of Osteoarthritis in Dogs / Richard D. Kealy, PhD,
Dennis F. Lawler, DVM, Joan M. Ballam, MS, George Lust, PhD,
Darryl N. Biery, DVM, Gail K. Smith, VMD, PhD, and Sandra L. Mantz . . . . . . . . . . . . . . . . 105
■ Effects of Diet Restriction on Life Span and Age-Related Changes
in Dogs / Richard D. Kealy, PhD, Dennis F. Lawler, DVM, Joan M. Ballam, MS,
Sandra L. Mantz, Darryl N. Biery, DVM, Elizabeth H. Greeley, PhD,
George Lust, PhD, Mariangela Segre, DSc, Gail K. Smith, VMD, PhD,
and Howard D. Stowe, DVM, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

NESTLÉ PURINA BODY CONDITION SYSTEMS

Appendix II

■ Canine Body Condition System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

■ Feline Body Condition System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
■ DIETARY RESTRICTION AND AGING: AN HISTORICAL OVERVIEW
Richard Weindruch, PhD
Department of Medicine, University of Wisconsin, and
Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center,
Madison, Wisconsin

THE EARLY YEARS OF DIETARY RESTRICTION RESEARCH Dr. Richard Weindruch is

a Professor of Medicine at the
It has been known for more than 60 years that dietary restriction (DR), if properly
University of Wisconsin and an
executed to avoid malnutrition, will greatly extend the maximum life span of laboratory investigator with the Geriatric
rodents and decrease the incidence of cancer and other late-life diseases. This field Research, Education and
began with observations published in 1935 from McCay et al1 on rats whose growth Clinical Center at the VA
Hospital in Madison. He earned
was stunted by DR, which as they aged appeared much younger than
his BS and MS in Biology at the
age-matched, ad libitum-fed animals and lived far longer, too. Next, studies were University of Illinois (Urbana)
published by Tannenbaum2 in the 1940s and 1950s on reduced spontaneous and and his PhD (1978) in
induced cancer incidence in calorie-restricted mice. In the 1960s and 1970s, some Experimental Pathology at
UCLA under the direction of Dr.
of the most important work was published by Ross, who described longevity and
Roy Walford. For 27 years, he
spontaneous cancer patterns in rats on various DR regimens.3 Thus, the initial phase has studied caloric restriction,
of DR research was focused on its effects on survival and cancer, but this began to which is known to slow the
broaden in the early 1970s with studies on the effects of DR on immune system aging aging process in experimental
animals. He has received several
in mice initiated by Walford et al4 and Good et al.5
awards for his research includ-
During most of that period, these impressive actions of DR were regarded more ing the Gerontological Society
as an interesting curiosity than as a unique model system for investigating the biology of America's Kleemeir Award
(1998), the Glenn Foundation
of decelerated aging. Interest in DR-induced phenomena increased markedly in the Award (2000) and the National
mid-1970s, and even more so thereafter, due in part to increased focus on the inves- Institute on Aging's Nathan
tigation of biological aging in the United States and other developed countries as well Shock Award (2000).
as the widening appreciation of the striking efficacy of DR to retard a broad spectrum
of age-associated biological and pathological changes. Notable here are the efforts of
Masoro’s group, which began in the late 1970s to characterize multiple responses of
male Fischer 344 rats to DR.6

THE 1980S

This was a decade of rapidly increasing interest in and research funding for DR. The
National Institute on Aging (NIA) established a colony of some 30,000 rats and mice,
which were either fed normally or subjected to DR, in order to support study by NIA-
funded grantee laboratories to establish the validity and usefulness of potential bio-
markers of aging. The 1980s also brought substantial progress on three other fronts:
1. characterizing the effects of DR on age-sensitive physiological and patho-
logical processes in laboratory rodents;
2. studies in rodents on the mechanisms underlying aging retardation by DR; and
3. initiating long-term trials of DR in monkeys at the NIA7 and at the University
of Wisconsin–Madison.8

7 General Sessions
{ } >>> WEINDRUCH

In 1988, this author and his mentor, Dr. Roy Walford, published a book which reviewed all published data
on DR.9 As discussed therein, it is noteworthy that life-span extension by DR has also been observed in fish,
spiders, Daphnia (water-fleas), and other non-rodent species which suggests a broad relationship between
caloric intake and the aging process. Four other books on DR were published between 1989 and 1995.10–13
Since 1980, the DR field has expanded rapidly. To illustrate, the number of publications found on
Medline having in the title or abstract any of these terms — “diet restriction,” “dietary restriction,” “food
restriction,” “caloric restriction,” “calorie restriction,” or “energy restriction” — was 35 for the period
1966–1969, 297 for 1970–1979, 1162 for 1980–1989, 2158 for 1990–1999, and 655 for 2000 until
mid-May 2002.

THE 1990S AND BEYOND

Over the last 12 years, there has been continuing emphasis on investigating underlying mechanisms of
aging retardation in laboratory rodents14 and, quite recently, in simpler animal models such as yeast,15
nematodes,16 and fruit flies.17 In rodents, there is accumulating evidence to suggest that age-associated
increases in oxidative stress and damage of mitochondrial origin may represent a primary aging process in
postmitotic tissues, which is attenuated by DR.18 Our recent experiments in mouse skeletal muscle19 and
brain20 using microarrays to conduct gene expression profiling support this notion and reveal two types of
effects of DR on gene expression:
1. prevention of age-associated changes, and
2. shifting the expression level of genes that do not change with aging (transcriptional reprogramming).
A new area of inquiry is the attempt to develop drug or nutrient interventions that may mimic the
actions of DR.21 A challenge in that regard is that the current “gold standard” of life extension for screening
interventions to retard the aging process has some major shortcomings: the assay requires ~4 years to
complete and provides imprecise data on the rate of aging in individual organ systems (e.g., did the
candidate mimetic retard aging in the heart?). Importantly, advances in genomics provide exciting new
opportunities and have created the new field of nutrigenomics, which can be applied to this problem. Our
data strongly suggest that gene expression profiles (which consist of hundreds of markers of aging at the
transcriptional level) can be used to determine the biological age of a tissue19,20 and thereby allow one to
determine whether interventions can retard aging on an organ-specific basis. Further, one can quantify the
ability of a candidate mimetic to induce the transcriptional reprogramming that is associated with DR.
Will DR retard the aging process in primates? The recent discoveries by Nestlé Purina scientists that DR
in dogs increases average life span and health span represent an important extension to a larger mammalian
species.22 The observations from the studies in rhesus monkeys subjected to DR23, 24 and limited human
epidemiologic data9, 14, 25 support the notion of human translatability. Compared with age-matched, normally
fed controls, rhesus monkeys on DR for over a decade show increased insulin sensitivity, favorable changes
in plasma lipid profiles, and reduced free radical damage in skeletal muscle. Definitive data on longevity and
disease patterns in these populations will require another 15 to 20 years of study. The NIA will soon launch
DR studies in nonobese people to explore the extent to which people can adhere to a targeted 20% to 30%
lowering of caloric intake. However, whether or not DR retards human aging, the prolongation of the health
span and life span of rodents by DR has major implications for many disciplines, and raises important
questions about the desirability of ad libitum feeding.

8
DIETARY RESTRICTION AND AGING: AN HISTORICAL OVERVIEW

ACKNOWLEDGMENT

The author thanks Jennifer Christensen and Roger Klopp for their contributions to his research program.
This author’s research is supported by the National Institutes of Health (P01 AG-11915, R01 AG-18922). This
is Publication No. 02-05 from the Veterans Administration Geriatric Research Education and Clinical Center,
Madison, Wisconsin.

REFERENCES
1. McCay CM, Crowell MF, Maynard LA. The effect of retarded growth upon the length of the life span
and upon the ultimate body size. J Nutr. 1935;10:63–79.
2. Tannenbaum A. Effects of varying caloric intake upon tumor incidence and tumor growth. Ann N Y
Acad Sci. 1947;49:5–17.
3. Ross MH. Nutritional regulation of longevity. In: Behnke JA, Finch CE, Moment GB (eds): The Biology
of Aging. New York: Plenum Press. 1978:173–189.
4. Walford RL, Liu RK, Mathies M, et al. Influence of caloric restriction on immune function: Relevance
for an immunologic theory of aging. In: Chavez A, Bourges H, Basta S (eds): Proc IXth Intl Congr Nutr
(Book of Abstracts). Basel: S. Karger. 1975:374–381.
5. Good RA, Fernandes G, Yunis EJ, et al. Nutritional deficiency, immunologic function, and disease. Am
J Pathol. 1976;84:599–614.
6. Masoro EJ, Shimokawa I, Yu BP. Retardation of the aging processes in rats by food restriction. Ann NY
Acad Sci 1991;621:337–352.
7. Ingram DK, Cutler RG, Weindruch R, et al. Dietary restriction and aging: The initiation of a primate
study. J Gerontol Biol Sci. 1990;45:B148–B163.
8. Kemnitz JW, Weindruch R, Roecker EB, et al. Dietary restriction of adult male rhesus monkeys: Design,
methodology, and preliminary findings from the first year of study. J Gerontol Biol Sci.
1993;48:B17–B26.
9. Weindruch R, Walford RL. The Retardation of Aging and Disease by Dietary Restriction. Springfield,
IL: CC Thomas. 1988.
10. Snyder DL (ed). Dietary Restriction and Aging. New York: Alan R. Liss. 1989.
11. Fishbein L (ed). Biological Effects of Dietary Restriction. New York: Springer–Verlag. 1991.
12. Yu BP (ed). Modulation of Aging Processes by Dietary Restriction. Boca Raton, FL: CRC Press. 1994.
13. Hart RW, Neuman DA, Robinson RT (eds). Dietary Restriction: Implications for the Design and
Interpretation of Toxicity and Carcinogenicity Studies. Washington, DC: ILSI Press. 1995.
14. Weindruch R, Sohal RS. Caloric intake and aging. N Engl J Med. 1997;337:986–994.
15. Lin SJ, Defossez PA, Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie
restriction in Saccharomyces cerevisiae. Science. 2000;289:2126–2128.
16. Lakowski B, Hekimi S. The genetics of caloric restriction in Caenorhabditis elegans. Proc Natl Acad Sci
USA. 1998;95:13091–13096.
17. Pletcher SD, Macdonald SJ, Marguerie R, et al. Genome-wide transcript profiles in aging and
calorically restricted Drosophila melanogaster. Curr Biol. 2002;12:712–723.
18. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273:59–63.
19. Lee CK, Klopp RG, Weindruch R, Prolla TA. Gene expression profile of aging and its retardation by
caloric restriction. Science. 1999;285:1390–1393.
20. Lee CK, Weindruch R, Prolla TA. Gene expression profile of the aging brain in mice. Nature Genet.
2000;25:294–297.
21. Weindruch R, Keenan KP, Carney JM, et al. Caloric restriction mimetics: Metabolic interventions.
J Gerontol Biol Sci. 2001;56A(Special Issue I):20–33.
22. Kealy RD, Lawler DE, Ballam JM, et al: Effects of diet restriction on life span and age-related changes
in dogs. J Am Vet Med Assoc. 2002;220:1315–20.

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 >>> WEINDRUCH

23. Ramsey JJ, Colman RJ, Binkley NC, et al. Dietary restriction and aging in rhesus monkeys: The
University of Wisconsin study. Exp Gerontol. 2000;35:1131–1149.
24. Roth GS, Ingram DK, Black A, Lane MA. Effects of reduced energy intake on the biology of aging: The
primate model. Eur J Clin Nutr. 2000;54(Suppl 3):S15–S20.
25. Kagawa Y. Impact of westernization on the nutrition of Japanese: Changes in physique, cancer,
longevity, and centenarians. Prev Med. 1978;7:205–217.

10
■ EFFECTS OF DIET RESTRICTION ON LIFE SPAN AND
AGE-RELATED CHANGES IN DOGS: EXPERIMENTAL DESIGN
Richard D. Kealy, PhD, and Dennis F. Lawler, DVM
Pet Nutrition Research Department, Nestlé Purina PetCare Company, St. Louis, Missouri

ABSTRACT: Dr. Richard D. Kealy is a

Purina Research Fellow at
This study was conducted with 48 Labrador retrievers from 7 litters, in a paired
Nestlé Purina PetCare Company,
feeding design. The dogs were paired at age 6 weeks by gender and body weight within where he has been an animal
litter, and assigned at random to dietary treatment. At age 8 weeks, one pair-mate nutritionist for 35 years. His BS
began ad libitum (AL) feeding, while the other (restricted-fed) pair-mate received a degree was in Agriculture
Education at the University of
quantity equal to 75% of the amount of food that its counterpart consumed the
Nebraska, MS degree in Poultry
previous day. When the dogs were age 3.25 years, two adjustments were incorporated Nutrition at the University of
into the feeding protocol. All dogs were switched from the growth formula (27% protein) Nebraska, and he later com-
to an adult formula (21% protein), and the amount of food was reduced and fed at a pleted a PhD degree in Animal
Nutrition at the University of
constant amount to prevent insidious development of obesity in the dogs that were
Arkansas. Dr. Kealy has served
fed AL. This was done by estimating the ideal body weight for each dog that was fed as a member of the National
AL, based on skeletal size in reference to other dogs of the same breed. This group of Research Council panel on
dogs then was fed 62.1 kcal of metabolizable energy (ME) per kg of estimated ideal feline nutrition. He currently
holds three patents, two that
body weight (maintenance requirement for large breed dogs), while each of their
relate to nutritional influences
respective restricted pair-mates received 25% less. After 3.25 years, the group that on canine hip dysplasia. He has
was fed AL was referred to as the control-fed group. The dogs were weighed weekly authored or coauthored over
as pups, periodically as adolescents, and then weekly as adults. They were evaluated 800 studies at Purina on nutri-
tional needs of dogs and cats.
for body condition annually from age 6 years, using a 9-point scale ranging from 1
His expertise is in the area of
(emaciated) to 9 (severely obese), to assess degree of leanness/fatness. Amounts of orthopedic nutrition in dogs
lean, fat, and bone mass were estimated annually from age 6 years, using a Lunar and he has lectured interna-
Model DPX alpha dual-energy x-ray absorptiometer (DEXA). Analyses of serum were tionally and domestically at
veterinary conferences and
done annually for glucose, cholesterol, and triglycerides, using a Ciba-Corning Blood
veterinary schools primarily on
Chemistry Analyzer, Model 550 Express. Serum triiodothyronine (T3) analyses were this subject.
done annually from age 4 years by radioimmunoassay. Samples were obtained by
jugular venipuncture after overnight fast. Intravenous glucose tolerance tests (IVGTT)
were done annually from age 9 years, by injection of 2.0 mL/kg body weight of 50%
glucose solution, with samples obtained by jugular venipuncture at 0, 5, 30, 45, 60 and
120 minutes. Plasma insulin was estimated by radioimmunoassay of the same samples.
The dogs were monitored daily throughout life for signs of illness or abnormality.
When necessary, appropriate therapeutic measures consistent with established colony
protocols were taken under supervision of the attending veterinarian. Health
management and euthanasia protocols were pre-established for the entire facility.
Similar conditions among dogs were managed as uniformly as possible. Dietary treat-
ments were not adjusted because of illness, and choices of therapeutic measures were
not influenced by dietary treatment. Most mortality (46 of 48) occurred by euthanasia,
according to the dictates of humane treatment. Humane euthanasia was carried out
only after extensive diagnostic evaluation, careful monitoring and assessment of

11 General Sessions
 >>> K E A LY, L AW L E R

response to treatment, serial evaluations of clinical condition, and consideration of prognosis, according to
the practices established for the entire colony. Response variables were examined with a mixed-effects
analysis of variance model for a repeated measures design. Feeding regimen, age, and their interaction were
considered to be the fixed effects of interest. Random effects accounted for variation among litters, pairs
within litters, and their interaction with age. The repeated measures aspect of the design was addressed by
assigning blocks to individual dogs. The Wilcoxon signed rank test for paired data was used to evaluate
differences in median life span. The paired Prentice-Wilcoxon test was used to evaluate differences in
median time to treatment for osteoarthritis and other chronic conditions.

12
■ DIET RESTRICTION AND LONGEVITY: CHRONIC DISEASES AND
CAUSES OF MORTALITY
Dennis F. Lawler, DVM,1 Richard H. Evans, DVM, MS,3
Richard D. Kealy, PhD,1 Jay M. Harrison, MS, MA,2 and Brian T. Larson, PhD1
1
Pet Nutrition Research Department, 2Statistical Services Department, Nestlé Purina PetCare
Company, St. Louis, Missouri; and 3Veterinary Pathology Services, Aliso Viejo, California

ABSTRACT: Dr. Dennis Lawler received

his DVM degree from University
Labrador retriever dogs (48) were studied lifetime to assess effects of diet restriction
of Illinois in 1974, and joined
on longevity and physiological parameters. Restricted-fed (RF) dogs received 75% of Ralston Purina Company in
the same diet consumed by control-fed (CF) pair-mates. Median life span of RF dogs 1979. In addition to his extensive
(13.0 years) was greater (P < 0.01) than CF dogs (11.2 years). Chronic diseases includ- experience with primary patient
care, his research interests
ed osteoarthritis (43 by radiographic diagnosis); malignant neoplasia (21 tumors, 17
include nutrition, population
dogs); benign mammary gland neoplasia (35 tumors, 12 dogs); benign non-mamma- medicine, reproduction, pediatrics,
ry neoplasia (7 tumors, 6 dogs); recurring skin disease (19); liver disease (11); cystic and aging. He has published
endometrial hyperplasia, pyometra, or recurring severe pseudopregnancy (11); over 60 scientific articles,
abstracts, and book chapters in
hypothyroidism (4); and seizures (4). Primary causes of mortality concentrated in mus-
these areas, and has been
culoskeletal (26) and digestive (12) systems; neoplastic disease of these (4) and other Section Editor three times for
body systems (7) accounted for 11 mortalities. Among 26 total mortalities from mus- Consultations in Feline Internal
culoskeletal causes, 12 were RF dogs (mean 13.1 years at death) and 14 were CF dogs Medicine. Dr. Lawler served
as primary clinician for
(mean 10.6 years at death). Hazard of death from musculoskeletal diseases differed
Nestlé Purina's 14-year Diet
between RF and CF dogs (P=0.002). Twelve dogs died from digestive system diseases, Restriction and Longevity Study.
including 7 RF (mean 10.9 years at death) and 5 CF (mean 10.7 years at death,
P > 0.05). Eleven dogs died from neoplastic diseases, including 5 RF (mean 11.6 years
at death) and 6 CF (mean 9.7 years at death, P > 0.05). Neoplastic diseases were not
independent of musculoskeletal and digestive systems mortalities, but a numerical
trend was found for neoplastic causes of mortality. Restricted feeding influenced
median life span, but mortality causes generally were similar by body systems.

13 General Sessions
 ■ INFLUENCES OF LIMIT-FEEDING AND AGING ON ANTIOXIDANT
STATUS OF PAIR-FED LABRADOR RETRIEVERS
Howard D. Stowe, DVM, PhD, Diplomate ACVN,1 Richard D. Kealy, PhD,2
and Dennis F. Lawler, DVM2
1
Professor Emeritus, Michigan State University, East Lansing, Michigan; and
2
Nestlé Purina PetCare. Company, St. Louis, Missouri

Dr. Howard D. Stowe is ABSTRACT:

Professor Emeritus of the
Twenty-four littermate pairs of 8-week-old Labrador retrievers were assigned to
Department of Large Animal
Clinical Sciences, College of an experiment in 1987 to evaluate biological markers of aging in dogs and determine
Veterinary Medicine, at Michigan the effects of limit-feeding (75% of control-fed pair) on the quality and span of their
State University (MSU). Dr. lives. Between 1992 and the end of the study, the antioxidant status of these dogs was
Stowe earned his BS degree in
monitored by annual clinical assays of serum, plasma, or whole blood for retinol,
Animal Husbandry at the
University of Massachusetts, retinyl palmitate, vitamin E, selenium, copper, ceruloplasmin, ascorbic acid, uric acid,
and MS degree in Animal total peroxyl-radical trapping activity, glutathione peroxidase, superoxide dismutase,
Nutrition, DVM, and PhD in and catalase. For the period covered by this report (ages 5 through 10 years), limit-
Nutritional Pathology in the
feeding reduced serum retinol (P < 0.05), vitamin E (P < 0.05), ceruloplasmin (P < 0.01),
College of Veterinary Medicine
at MSU. Dr. Stowe’s primary and copper (P < 0.01). Aging was associated with decreases (P < 0.01) in serum retinyl
research interests involved the palmitate, serum vitamin E, serum selenium, and whole blood catalase, and with
use of purified-type diets to increases in serum retinol (P < 0.01), serum copper (P < 0.01), plasma uric acid
help define nutrient require-
(P < 0.05), and whole blood glutathione peroxidase (P < 0.01). Female dogs were found
ments of animals, particularly
selenium and vitamins A and E to have lower (P < 0.05) serum retinyl palmitate, ceruloplasmin, and copper than male
in horses and swine. His equine dogs. Litter effects (P < 0.01), presumably reflecting genetic influences, were observed
research was a major impetus for serum vitamin E, serum copper, plasma uric acid, and whole blood glutathione
in establishing the current
peroxidase. The limit-feeding effects on serum retinol, vitamin E, and copper can
Equine Nutrition and Physiology
Society and his comparative reflect the reduced daily per-dog intake of each of these nutrients, compared with
nutrition interests contributed control-fed dogs. This indicates, in the case of the serum retinol and copper, that these
to the establishment of the two variables in Labrador retrievers are not as homeostatically regulated by hepatic
American College of Veterinary
storage as is reported for other species. The decreases in retinyl palmitate, vitamin E,
Nutrition of which he is a
charter diplomate. Dr. Stowe’s and selenium with aging may signal factors especially important in canine geriatric
interest in practical and accu- diets. The lower serum retinol, retinyl palmitate, and copper values in females versus
rate assays for the clinical males might be expected to negatively affect pregnancy and/or lactation. These
assessment of the antioxidant
antioxidant profiles provide some new and contemporary clinical reference data
status of animals was a primary
factor in establishing the which can enhance our understanding of canine nutrition, have practical application
Clinical Nutrition Section of the in formulating canine diets, and help reveal how limit-feeding has a positive effect on
MSU Animal Health Diagnostic the life span of dogs.*
Laboratory. He is a widely cited
author in the animal nutrition
area and has served on several
National Academy of Science
publication committees.

*See Kealy RD, Lawler DE, Ballam JM, et al. Effects of diet restriction on life span and age-related
changes in dogs. JAVMA. 2002;220:1315-1320, which is reprinted with permission in Appendix I in
these proceedings.

General Sessions 14
■ A LONGITUDINAL STUDY OF IMMUNOSENESCENCE: DOES DIET
RESTRICTION AMELIORATE THE AGING PROCESS IN DOGS?
Elizabeth H. Greeley, PhD,1 Joan M. Ballam, MS,2 Jay M. Harrison, MS, MA,2
Richard D. Kealy, PhD,2 Dennis F. Lawler, DVM,2 and Mariangela Segre, DSc1
1
Department of Veterinary Pathobiology, University of Illinois, Urbana, Ilinois; and
2
Nestlé Ralston Purina Co., St. Louis, Missouri

ABSTRACT: Dr. Elizabeth Greeley

received her undergraduate
The effects of dietary restriction (DR) on the canine immune system have been
degree from the University of
investigated in a longitudinal study. A group of Labrador retrievers (30 females, 18 Illinois Medical Center in
males), divided into age- and gender-matched pairs, were maintained on a diet Chicago and her PhD in
restriction protocol from age 8 weeks until death. The restricted-fed (RF) dog received Microbiology and Immunology
from Duke University; she
75% of the total food consumed by its control-fed (CF) pair-mate. The immune status
completed a postdoctoral
of these dogs was evaluated at established time intervals using a battery of immune fellowship there in 1977. She
parameters including lymphoproliferative responses to the mitogens concanavalin A joined the Department of
(Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM); natural killer (NK) Veterinary Pathobiology at the
University of Illinois in 1978
cell activity; white blood cell (WBC) counts and lymphocyte subset distributions; and
as a Research and Teaching
neutrophil phagocytic activity. Data were evaluated as a function of age, diet, and Associate. Since 1992, she has
gender. Lymphoproliferative responses to all three mitogens declined with age. Diet- held the position of Research
restricted dogs exhibited a slower rate of decline, an effect that was more pronounced Scientist and Clinical Assistant
Professor. In 1996, she received
in females. Age-related alterations of lymphocyte subset distribution in peripheral
the Academic Professional
blood included decreases in CD4-cell and B-cell percentages, with RF dogs demonstrating Excellence Award from the
lower B-cell percentages than CF dogs. Age-related increases in T-cell percentages University of Illinois College of
were observed overall, but the rate of increase in RF females was not significant. Age- Veterinary Medicine. Her major
research interests include
related increases in CD8 T-cell percentages were also observed. The absolute numbers
immunoregulation, immuno-
of all monitored lymphocyte subsets declined with age; RF dogs had lower numbers toxicity, and immunosenes-
of B cells than their CF pair-mates. Diet restriction slowed the rate of decline for CD4, cence. She has authored 16
CD8, and total T-cell numbers. Natural killer cell activity declined with age, but was articles appearing in refereed
journals, made 8 presentations
not affected by diet; gender differences were observed with males having higher levels
at national meetings, served
of activity. The phagocytic capacity of neutrophils was not significantly affected by on several graduate student
age, diet, or gender. Diet restriction was indeed beneficial in retarding several age- committees, and advises gradu-
related immune changes including the declines in lymphoproliferative responses and ate students on their research
projects on a regular basis. She
numbers of CD4, CD8, and total T cells, and the increase in T-cell percentages.
teaches immunology in two
Correlation of immune findings with decreased risk of death revealed that an immune graduate-level courses as well
profile of high lymphoproliferative responses, a high percentage and number of CD8 as in the first-year professional
cells, and high numbers of T cells favored increased survival. Three of these predictors curriculum.
of increased survival were positively affected by diet restriction. This study seems to
suggest that the female immune system derives greater benefit from dietary restriction
than the male immune system.

15 General Sessions

Dr. George Lust received
his PhD from Cornell University.
He began his career at the
James A. Baker Institute for
Animal Health at Cornell, and
has held the position of
Professor of Physiological
Chemistry there since 1983. Dr.
Lust is head of the John M. Olin
Laboratory for the Study of
Canine Bone and Joint Diseases
at the Baker Institute, and for
the past 30 years has actively
pursued the study of the cause,
pathogenesis, diagnosis, and
treatment of canine hip dyspla-
sia and osteoarthritis. He is
interested in the early diagnosis
of canine hip dysplasia, and in
pursuit of this goal, he and his
colleagues developed a new
radiographic method of diagno-
sis called the dorsolateral sub-
luxation test. Dr. Lust and col-
leagues are also involved in
seeking out a genetic marker
for canine hip dysplasia, a
research project actively sup-
ported by Purina since 1991. A
large, informative pedigree of
disease-free and dysplastic
dogs has been developed to aid
in this search. In addition to
teaching, and being a major
advisor to numerous graduate
students and postdoctoral
associates, he is the author or
co-author of 148 publications,
with additional publications
currently in press or preparation.
Osteoarthritis 16
■ DIET RESTRICTION AND OSTEOARTHRITIS IN ANIMALS
George Lust, PhD
James A. Baker Institute for Animal Health, College of Veterinary Medicine,
Cornell University, Ithaca, New York
WHAT IS OSTEOARTHRITIS?
Osteoarthritis (OA) is a disease of diarthrodial joints characterized by pain, stiff-
ness, and limitation of motion. At present the terms osteoarthritis, osteoarthrosis, and
degenerative joint disease are used interchangeably. When the underlying predispos-
ing factor is unknown OA is referred to as primary or idiopathic, but in the vast major-
ity of cases a pathogenic cause can be identified and then it is called secondary. It can
occur as a result of trauma, in association with malformations in the region of the
joint, or as part of the aging process. Osteoarthritis most often is diagnosed in the
older animals; however, it can appear at any age. The disease can affect a single joint
or several joints. The large weight-bearing joints of the hip, stifle, shoulder, elbow, or
ankle are frequently affected, and the joints of the spine (vertebrae) and extremities
are often abnormal. Although multiple joints may be diseased, pain and disability are
usually greatest in one joint. Osteoarthritis is one of the most prevalent causes of
disability in humans,1 and it occurs in primates. It is a serious veterinary medical problem
in animals2 and is commonly diagnosed in dogs, horses, and pigs, and has been well
described in mice, guinea pigs, and chickens (Table 1).
Joint disease that results from trauma clearly is not inherited, but other cases
have a strong genetic association. Osteoarthritis is often observed in littermates and
in the progeny of affected parents, and environmental effects such as time, physical
activity, and nutrition influence the expression of disease. Injuries or malformations
that cause joints to become unstable or alter their weight-bearing function promote
the development of OA. Hip dysplasia is a frequent predisposing condition in humans
and in dogs. Other causes of OA include misalignment of the kneecap and unfused
bones in joints. Separation of articular cartilage from the subchondral bone, a condi-
tion called osteochondrosis, results in OA in shoulder, stifle, elbow, and hock joints and
is observed in rapidly growing animals.3
An early sign of OA is an animal’s reluctance to ambulate normally. Less weight is
put onto affected limbs and animals may limp or appear stiff. Rising from a lying position
is difficult and an animal may whine or snap when an affected joint is manipulated.
Intense activity can aggravate the pain in joints and thereby reveal signs of disease in
animals that otherwise appear normal. The pain often is made worse by cold or a
change in the weather.
DIAGNOSIS AND SIGNS OF DISEASE
Observation can raise suspicions of OA, but diagnosis is done by radiographic exami-
nation of the affected joint.1 Evidence on an x-ray photograph includes joint capsule
DIET RESTRICTION AND OSTEOARTHRITIS IN ANIMALS

TABLE 1. Animal Species Affected with Osteoarthritis

SPECIES JOINTS INVOLVED

Human Hip, knee, shoulder, vertebrae, elbow, ankle, fingers
Dog Hip, stifle, shoulder, elbow, vertebrae
Pig Hip, stifle, shoulder, elbow
Horse Stifle, fetlock, carpal, shoulder
Primate Hip, knee, shoulder, elbow
Goat/Sheep Stifle
Mouse Stifle
Guinea Pig Stifle
Chicken Hip, hock, stifle

thickening, mineral deposits in tissues surrounding the joint, osteophytes at the edge of the joint,
subchondral bone sclerosis, and joint space narrowing signaling loss of cartilage. Synovial fluid can also be
examined to provide more information. Disease-free joints contain only a small amount of clear viscous
fluid. An increased amount or discoloration of the fluid, or an increase in its content of discarded synovial
cells suggests disease. Osteoarthritic joints characteristically do not have elevated numbers of white blood cells.
The progression of OA is similar in most animals. The articular cartilage that covers the ends of bones
is affected early. Cartilage functions to distribute loads over a wide area and also provides low-friction sur-
faces for movement of joints and any impairment in this tissue leads to disease. The normally smooth and
elastic cartilage becomes soft, then rough, and often is worn away exposing underlying bone.4 Capsule and
ligaments become stretched and enlarged and even muscles in the region of a joint are weakened. Opposing
bones articulate abnormally causing pressure, deformation, and spurs called osteophytes and inflammation
and further injury. Movement becomes impaired and painful.

SOURCES OF PAIN

The principal symptom of OA is pain which arises after joint movement and is relieved by rest.1,2 Pain arises
from noncartilaginous intra-articular tissues and periarticular structures and not from the cartilage itself;
it is aneural. Pain emanates from the periosteum, the osteophytes, trabecular microfractures, subchondral
bone exposed by eburnation, ligamentous and capsular distention, and synovitis.

OVERWEIGHT AND DIET RESTRICTION

The role of obesity in the pathogenesis of OA is complex and is of great interest for scientists, clinicians,
clients, and the affected animals. Obesity in animals with OA may be a consequence of reduced activity
because of joint pain, but studies have suggested that it is likely that obesity has a direct role in the initia-
tion and progression of OA. For example, weight change was directly associated with OA risk, and weight
loss was correlated to a reduced risk for OA.1 In addition to the biomechanical effects of extra weight as the
cause of OA, a metabolic role for biochemical constituents has been proposed and a number of hormones
and other biochemicals have been implicated in OA development (e.g., lipids, insulin-like growth factor [IGF-1],
insulin, as well as sex hormones). Definitive progress in this area has been slow, but many studies are ongo-
ing and further research will help clarify the pertinent interrelationships between altered body metabolism
and the pathogenesis of OA.
A survey of the nutritional literature identifies numerous studies on diet and weight reduction in
humans and also in experimental animal models such as mice. However, there is a paucity of reports of

17 Osteoarthritis
{ } >>> LUST

TABLE 2. Diet Restriction and Osteoarthritis in Three Species

SPECIES WEIGHT JOINTS IMPROVED

REDUCTION
(%)
Growing Dogs5 23% OA in hip and other joints: dramatic radiograph
improvement in frequency and severity
Adult obese dogs6 14% OA in hip; improved mobility (clinical)
Guinea pigs7 28% Knee OA improved 40% (pathology)
Chickens8 10% Hock OA and proteoglycan content improved

controlled studies on diet restriction and OA development in animals. A few good studies do exist and
results of four of these are summarized in Table 2.
Three species, dogs, guinea pigs and chickens, were fed reduced quantities of their respective control
diets and weight loss was recorded and signs of OA were analyzed. A diet reduction of 25% in growing dogs
(up to 5 years) genetically at risk for canine hip dysplasia substantially lowered the frequency and severity
of hip joint OA as diagnosed radiographically.5 In another study, overweight adult dogs were put on a reducing
diet that resulted in an average weight loss of 14% (in 10–19 weeks). Symptoms of hip joint OA in these
dogs were reduced clinically as assessed by increased mobility and range of motion in the hip joint.6
Apparently weight loss in the adult dogs compared with controls reduced the stress on their weight bear-
ing joint, easing pain and reducing symptoms of the disease. A third study utilized guinea pigs, and a 28%
reduction in weight as compared with controls resulted in an impressive average 40% reduction of pathology
in the OA of stifles in guinea pigs as assessed by histologic examination of stifle joint tissue.7 The fourth
report concerned broiler fowl. Even chickens fed a smaller quantity of food that resulted in a 10% weight
reduction had the evidence for OA of hock joints reduced.8 Concomitantly the content of proteoglycan in
the articular cartilage of the hock joint in these chickens was observed to be at disease-free levels, where-
as control-fed OA chicken cartilage had diminished proteoglycan content.9 Loss of proteoglycans from the
articular cartilage of OA joints is a characteristic feature commonly observed in all species that have been
examined.2,4 Thus, the results reviewed in Table 2 clearly substantiate that diet restriction in three species
of animals with OA leads to improved joint function as assessed clinically, radiographically, and histologi-
cally. The data suggested that in the case of the young growing dogs development of OA of the hip even
has been prevented as disclosed by a lower frequency of OA in the hip joints of the diet-restricted dogs.5
The progression of OA in hip joints of two groups dogs that were genetically predisposed for canine hip
dysplasia is presented in Table 3. The cardinal feature of hip dysplasia is displacement of the femoral head
away from the acetabulum as observed on a pelvic radiograph. With few exceptions OA is the invariant

TABLE 3. Occurrence of Hip Osteoarthritis in Dogs with Agea

PERCENT AFFECTED DOGS AT:

2 YEARS 2 YEARS 5 YEARS 8 YEARS
HIP DYSPLASIA10 OA10 OA5 OA11
Control-fed dogs 67% 33% 53% 71%
Restricted-fed dogs 29% 4% 13% 15%
Percentage change b
57% 88% 75% 78%
due to diet reduction
a
25% Diet Restriction Study (Nestlé Purina Co.) included 24 dogs in each group. Osteoarthritis was diagnosed radiographically
as described in References 5, 10, and 11.
b
Percent change = difference between control-fed and restricted-fed divided by control-fed, times 100.

18
DIET RESTRICTION AND OSTEOARTHRITIS IN ANIMALS

TABLE 4. Lifetime Diet Restriction, Weight, 50% Mortality, and Osteoarthritis in Dogsa
% CHANGE
AGE AT DATA CONTROL- RESTRICTED- DUE TO DIET
VARIABLE COLLECTION FED FED REDUCTIONb
Body weight12 2–12 years 33.2 kg 25.8 kg –22%
Restricted-fed dogs 0–13 years 11.2 years 12.9 years +15
mortality12
50% of dogs clinically — 9.8 years 12.6 years +29
treated for OA12
>2 joints with OA11 8 years 86% 24% –72%
a
25% Diet Restriction Study (Nestlé Purina Co.) included 24 dogs in each group. Osteoarthritis was diagnosed radiographically
as described in Reference 11.
b
Percent change due to diet reduction = difference between control-fed and restricted-fed divided by control-fed, times 100.

consequence of the trait called canine hip dysplasia.2,4 Results were that 67% of the control-fed dogs had
evidence for hip dysplasia at 2 years of age. Strikingly different, only 29% of the dogs in the restricted-fed
group had hip dysplasia.10 Thus the results in Table 3 suggest that the overall chondro-osseous conforma-
tion of hip joints in the restricted-fed group was better, or more normal, i.e., less disconformity, than for the
control group. Also at 2 years of age, 33% of control-fed dogs actually had hip OA (e.g., osteophytes and
other bone changes in the femoral head and acetabulum), whereas only 4% of the restricted-fed dogs had
definitive radiographic signs of OA.10 At 5 and 8 years of age 52% and 71% of control-fed dogs, respective-
ly, had hip OA, but only 13% and 15% of restricted-fed dogs, respectively, had OA.5,11 These results suggest
that the restricted diet effects ameliorate the underlying joint conformation (displacement, i.e., the dys-
plastic trait) by 57%, but an additional improvement resulted for signs of OA because the radiographic evi-
dence for OA specifically was reduced even more. The underlying chondro-osseous joint instability is the
trait called hip dysplasia, but other factors including time and physical activity interacting with the geno-
type of susceptible dogs lead from joint instability to overt OA in dogs with hip dysplasia.
Some of the data from the lifetime diet restriction study in dogs that also have relevance to the status
of OA in these dogs are presented in Table 4.12 A 25% restriction in food consumption resulted in a mean
22% reduction in weight over the lifetime for the restricted dogs when compared with the control group
of dogs. As reported previously, the 50% survival age was extended by 1.8 years in the restricted dogs and
the corresponding 10% survival age was extended about 1 year. The severity of OA was evaluated when the
dogs were 8 years old in hip, shoulder, elbow, and stifle joints. Eighty-six percent of the dogs in the control-
fed group at 8 years of age had two or more joints with radiographic evidence of OA.11 In marked contrast,
the restricted-fed group had only 24% of the dogs with OA in two or more joints. This represented a reduc-
tion of 72% in the joint pathology that was attributable to diet reduction. Of significance was that the radi-
ographic improvements in the restricted-fed group in fact were consistent with the clinical behavior of the
dogs. The lessened joint pathology in each dog was translated into a meaningful clinical observation in that
the age at which 50% of the dogs required treatment for OA was delayed to 12.6 years when compared
with control-fed dogs that needed OA therapy at a mean age of 9.8 years.12 Taken together these results
provide compelling evidence that diet restriction by 25% resulted in biological and clinically meaningful
improvements in joint function in these dogs.

SUMMARY AND CONCLUSIONS

Obesity is a common form of malnutrition particularly among elderly humans,13 but animals of all ages
are similarly affected, including dogs, pigs, and some rodents. Excessive weight gain is associated with

19 Osteoarthritis
{ } >>> LUST

decreased exercise and a decline in caloric needs for weight maintenance. Overweight animals are at
increased risk for cardiac disease, diabetes, and for OA. Weight loss mitigates these conditions, but it has
some potentially adverse effects as well, such as excessive loss of lean body mass, possible electrolyte imbal-
ance, and liver abnormalities including elevated bile acids in blood.14 The adverse effects of dieting usually
are considered not severe enough to contraindicate a reducing diet for weight loss.
The data reviewed here lend strong support to the concept that reduced food consumption resulting in
a reduction in body weight with its concomitant altered body metabolism is clearly beneficial to lessen the
severity of OA and even prevent its occurrence if fed to young, growing animals. This effect was substantiated
in dogs, guinea pigs, and chickens. The lessening of the signs of OA, either clinically or pathologically, was
achieved by reducing diets of both long and short duration and in growing as well as in adult animals.
Restricted food consumption resulted in a striking amelioration of OA.

REFERENCES
1. Moskowitz RW, Holderbaum D. Clinical and laboratory findings in osteoarthritis. In: Koopman WJ (ed):
Arthritis and Allied Conditions. Philadelphia: Lippincott Williams and Wilkins. 2001:2216–2245.
2. Burton-Wurster N, Todhunter RJ, Lust G. Animal models of osteoarthritis. In: Woessner JF, Howell DS
(eds): Joint Cartilage Degradation: Basic and Clinical Aspects. New York: Marcel Dekker.
1993:347–384.
3. Olsson SE. Osteochondrosis in domestic animals. Introduction. Acta Radiol. 1987;358 (Suppl):9–14.
4. Burton-Wurster N, Farese JP, Todhunter RJ, Lust G. Site-specific variation in femoral head cartilage
composition in dogs at high and low risk for development of osteoarthritis: Insights into cartilage
degeneration. Osteoarthr Cart.1999;7:486–497.
5. Kealy RD, Lawler DF, Ballam JM, et al. Five-year longitudinal study on limited food consumption and
development of osteoarthritis in coxofemoral joints of dogs. J Am Vet Med Assoc. 1997;210:222–225.
6. Impellizeri JA, Tetrick MA, Muir P. Effect of weight reduction on clinical signs of lameness in dogs with
hip osteoarthritis. J Am Vet Med Assoc. 2000;216:1089–1091.
7. Bendele AM, Hulman JF. Effects of body weight restriction on the development and progression of
spontaneous osteoarthritis in guinea pigs. Arthritis Rheum. 1991;34:1180–1184.
8. Venkatesan N, Thorp BH, Hulmes DJ. Articular cartilage proteoglycan metabolism in avian degenerative
joint disease: Effects of strain selection and body weight. Connect Tissue Res. 1999;40:199–208.
9. Anderson-MacKenzie JM, Hulmes DJ, Thorp BH. Effects of body mass and genotype on avian degenerative
joint disease pathology and articular cartilage proteoglycan distribution. Clin Exp Rheumatol.
1998;16:403–408.
10. Kealy RD, Olsson SE, Monti KL, et al. Effects of limited food consumption on the incidence of hip
dysplasia in growing dogs. J Am Vet Med Assoc. 1992;201:857–863.
11. Kealy RD, Lawler DF, Ballam JM, et al. Evaluation of the effect of limited food consumption on
radiographic evidence of osteoarthritis in dogs. J Am Vet Med Assoc. 2000;217:1678–1680.
12. Kealy RD, Lawler DF, Ballam JM, et al. Effects of diet restriction on life span and age-related changes
in dogs. J Am Vet Med Assoc. 2002;220:1315–1320.
13. Seim HC, Holtmeier KB. Treatment of obesity in the elderly. Am Family Physician. 1993;47:1183–1189.
14. Pi-Sunyer FX. Short-term medical benefits and adverse effects of weight loss. Ann Intern Med.
1993;119:722–726.

20
■ EFFECTS OF RESTRICTED FEEDING ON ONSET, INCIDENCE, AND
SEVERITY OF HIP DYSPLASIA AND OSTEOARTHRITIS IN DOGS:
DIAGNOSTIC, THERAPEUTIC, AND GENETIC RAMIFICATIONS
Gail K. Smith, VMD, PhD, Diplomate ACVS,1 Darryl N. Biery, DVM,1
Richard D. Kealy, PhD,2 and Dennis F. Lawler, DVM2
1
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania; 2Department of Research & Development, Nestlé Purina PetCare
Company, St. Louis, Missouri

Osteoarthritis (OA) is a common, often-debilitating disease of both animals and Dr. Gail Smith received his
humans. In dogs a highly prevalent form of OA, known as canine hip dysplasia (CHD), VMD from the University
of Pennsylvania, completed
is capable of causing pain and dysfunction in affected animals. The prevalence of CHD
orthopaedic surgical residency
is as high as 75% in some breeds of dogs. Canine hip dysplasia is a developmental training, and was awarded
disease recognized to have complex inheritance, a so-called polygenic disease. It is a PhD in engineering. He is cur-
understood that the incidence and severity of such genetic diseases can be influenced rently Professor of Orthopaedic
Surgery and Chairman of the
considerably by environmental factors,1–4 meaning any “nongenetic” factor, such as
Department of Clinical Studies
diet, lifestyle, housing, or trauma. Dogs that develop CHD are born with apparently at the University of Pennsylvania
normal hip joints, but after they have reached a few weeks of age the earliest signs, School of Veterinary Medicine.
such as hip joint laxity, can be demonstrated by necropsy studies.1,5,6 Dr. Smith has published more
than 150 research articles, book
It is well accepted that hip laxity plays a prominent role in the pathogenesis of chapters, and proceedings. Dr.
CHD.7 Initial phenotypic expression of CHD is recognized radiographically as femoral Smith’s research interests have
head subluxation, often followed by progressive degenerative joint disease (DJD). focused on orthopaedic prob-
lems of the spine, the knee and
Femoral head subluxation, subjectively scored on the hip-extended radiograph, and the hip. A clinically compelling
the distraction index, directly measured on the distraction radiograph, are two types product of his research is a
of passive hip laxity. They are static phenomena. Osteoarthritis, however, is thought to stress-radiographic diagnostic
be caused by functional hip laxity, the dynamic phenomenon associated with the method capable of predicting
the susceptibility to hip dysplasia
femoral head slipping partially out of the acetabulum during weight bearing.8 It has in dogs as young as 16 weeks of
been shown that joints with excessive passive hip laxity are susceptible to traumatic age. Dr. Smith founded PennHIP®,
subluxation of the femoral head.9 Tight joints are not. Hip joint laxity and femoral head a cooperative scientific initiative
subluxation, if functional, produce pathologic consequences that include abnormal representing multiple collabo-
rative centers in North America
loading during weight bearing causing cartilage damage and bony remodeling. The and Canada, trained to test the
end result of this self-perpetuating cycle of abnormal loading and remodeling is new hip dysplasia diagnostic
osteoarthritis, characterized by synovitis, increased joint fluid volume, joint cartilage technology. Research continues
erosion, elongation and edema of the round ligament, thickening of the joint capsule, to support the role of the
PennHIP phenotype to effectively
and osteophyte formation.6,7,10 select breeding stock to reduce
Degenerative changes develop in joints of many dogs as they age; however, more the frequency of this most-
prevalent osteoarthritic disease.
severely affected dogs will show radiographic signs of OA well before the geriatric
period, often by 1 year of age or younger. Joint laxity associated with CHD is the most
important risk factor for OA in canine coxofemoral joints.9 Trauma or metabolic
dysfunctions are also thought to affect disease expression.9,10 One theory of pathogenesis
for OA includes the idea that excessive body weight leads to mechanical stress on
joints, facilitating the transformation of passive hip laxity to functional hip laxity,
thereby initiating OA. Excessive body weight has been documented as a risk factor for
OA development in humans, guinea pigs, mice, and dogs.9,11–16

21 Osteoarthritis
 { }
Dr. Darryl N. Biery is
Professor of Radiology at the
University of Pennsylvania’s
School of Veterinary Medicine
and a Diplomate of the
American College of Veterinary
Radiology. He received his DVM
degree at The Ohio State
University, intern training at
The Animal Medical Center in
New York City, and his radiology
specialty training in the
Graduate School of Medicine at
the University of Pennsylvania.
Dr. Biery has published more
than 125 papers, abstracts, and
textbook chapters, plus is the
co-author of the book
Radiographic Interpretation for
the Small Animal Clinician. Dr.
Biery is a past President of the
American College of Veterinary
Radiology, an officer in the
International Veterinary Radiology
Association, and the recipient
of awards from Radiological
Society of North American,
American Animal Hospital
Association, Investigative Radi-
ology, The Animal Medical
Center in New York, and the
European Association of
Veterinary Diagnostic Imaging.
He has been a speaker at
numerous local, national, and
international scientific meet-
ings and continuing education
programs. Dr. Biery has been
especially interested in musculo-
skeletal diseases and actively
involved in canine hip dysplasia
and osteoarthritis research.
22
>>> S M I T H , B I E R Y, K E A LY, L AW L E R
The studies summarized here were conducted to evaluate the effect of food
restriction on development of hip joint laxity during growth and subsequent occur-
rence of OA in coxofemoral joints during adulthood. In addition, the effect of food
restriction on development of OA in joints other than hips was evaluated.
EFFECT OF LIMITED FOOD CONSUMPTION ON HIP DYSPLASIA IN
GROWING DOGS
In this study,17 48 8-week-old Labrador retriever puppies from seven litters were allotted
by pairing to two groups of 24 dogs each. This pairing created two groups of genetically
similar dogs. One group was fed ad libitum (full fed) and each member of the other
group (restricted) was given 25% less of the same food given to the full-fed pair mate.
Radiography of the hip joints was done when the dogs were ages 30, 42, 54, 78, and
104 weeks. Subluxation was measured using the Norberg angle on radiographs made
with the dog in the standard, hip-extended position. The same radiographs were
evaluated for evidence of CHD by independent, blinded radiologists, one using the
Swedish scoring system and the other, the Orthopedic Foundation for Animals (OFA)
scoring system.
Independent of the age at which radiography was performed, hip joint quality
was better in the diet-restricted dogs, as a group. Radiographs taken when dogs were
age 104 weeks revealed less subluxation and less OA in restricted-fed dogs, irrespec-
tive of whether the Norberg angle, Swedish scoring system, or OFA scoring system was
utilized. Using the Swedish method, CHD at 2 years of age was diagnosed in 5 of 24
restricted dogs and 18 of 24 full-fed dogs. Using the OFA method, CHD was diagnosed
in 7 of 24 restricted dogs and 16 of 24 full-fed dogs. These findings support a clinical
recommendation to avoid excessive food intake in growing dogs, particularly in breeds
prone to CHD.
EFFECT OF LIMITED FOOD CONSUMPTION OVER 5 YEARS ON
COXOFEMORAL JOINT OSTEOARTHRITIS
The same Labrador retrievers from the growth study were continued on test for an
additional 3 years and additional radiographic evaluations were recorded yearly.18 At 3.25
years of age two adjustments were made in the feeding protocol. All dogs were switched
from a 27% protein puppy growth formula to a 21% protein adult formula, and the
amount of food was reduced to prevent insidious development of obesity in full-fed dogs.
We estimated the ideal body weight for each full-fed dog on the basis of skeletal size in
reference to other dogs of the same breed. These dogs then were fed 62.1 kcal metaboliz-
able energy/kg of estimated body weight. Restricted dogs continued to receive 75%
of the amount fed to the corresponding full-fed pair-mate, and thus the intake rela-
tionship between the two groups remained the same as prior to thediet adjustment.
Radiographic evaluations of coxofemoral joints for frequency and severity of OA
were done at yearly intervals up to age 5 years. Three investigators evaluated each
radiograph without knowledge of group assignments, using a scoring system based on
radiographic features of OA.19-23 The median value obtained by the three investigators
was assigned as the score for each dog.
EFFECTS OF RESTRICTED FEEDING ON ONSET, INCIDENCE AND
S E V E R I T Y O F H I P D I S P L A S I A A N D O S T E O A RT H R I T I S I N D O G S

Frequency and severity of OA were greater in full-fed dogs. By age 52 weeks, differences between the
groups for frequency and severity of OA were statistically significant. The prevalence of DJD in hip joints
increased through age 5 years. By age 5 years, 12 of 23 full-fed dogs and 3 of 23 restricted dogs had
radiographic signs of OA. Body weight correlated significantly with OA scores at age 5 years.
Dogs varied considerably with respect to individual maintenance energy requirements, and so it is not
feasible to specify a universal energy intake to achieve the benefits observed in the restricted dogs. It is
recommended, however, that growing puppies and adult dogs be fed throughout their lives to maintain lean
body conformation to minimize the development of DJD with advancing age.

EFFECT OF LIMITED FOOD CONSUMPTION ON MULTIPLE JOINT OSTEOARTHRITIS

In this study, 24 restricted dogs continued to receive 75% of the amount fed to the corresponding full-fed
pair-mate through age 8 years. Hip, shoulder, elbow, and stifle joints were evaluated radiographically at this
time. Radiographs were evaluated by one radiologist without knowledge of dietary treatment.
Osteoarthritis affecting multiple joints was significantly more common in full-fed dogs. Ten of 22 (45%)
full-fed dogs had OA in two different joints, and seven (29%) had OA in three joints, whereas only one of
21 (5%) restricted dogs had DJD in two joints, and one (5%) had OA in three joints. Five restricted dogs had
no evidence of OA in any evaluated joint, while only two full-fed dogs lacked evidence of OA in the
evaluated joints.
Theoretically development of OA at a primary site such as the hip could produce aberrant compensatory
biomechnical forces acting on other joints; the altered weight bearing and ambulation leading to multiple
joint OA. In our study, hip joints were not always affected first; some dogs had radiographic evidence of
shoulder or elbow lesions without hip joint involvement. Thus, alternatively it may be hypothesized that OA
has a systemic cause, with variable expression in different joints. This might explain in part the development
of OA in joints that are not subject to large forces associated with weight-bearing, such as lumbar inter-
vertebral joints in dogs and joints in the skeleton of sharks.25 Alternatively, a humoral substance from an
affected joint may affect tissues in other joints. This concept is supported by the recent finding that extract
of human arthritic bone tissue induced abnormalities in disease-free articular cartilage.26 Additional support
for the concept of variability in tissue susceptibility is derived from a recent report that shoulder joints in
young adult dogs at high risk for CHD have histopathologic articular cartilage abnormalities similar to those
found in the corresponding hip joints.27 Yet another hypothesis might be that OA among different joints
represents phenomena that are not interrelated or interdependent.

ABSTRACT (UNPUBLISHED DATA): EFFECT OF RESTRICTED FEEDING AND AGE ON HIP

OSTEOARTHRITIS AND HIP SCORE: A LIFE-LONG STUDY IN LABRADOR RETRIEVERS

It is generally agreed that there has been slow progress in reducing the incidence of CHD by selective
breeding of normal dogs.28 The conventional diagnosis of CHD has been based on subjective radiographic
findings of subluxation of the coxofemoral joint, or secondary OA as seen on evaluation of the hip-extended,
ventrodorsal radiographic view of the pelvis. In the USA this analysis is performed by the OFA after dogs are
2 years of age. In much of Europe a similar analysis is made after 1 year of age. In the published reports
cited above it was shown that the condition of excessive hip laxity (Norberg angle measured from the hip-
extended radiograph) can be marginally reduced by caloric intake and this tightening of the joint prevents
or delays the expression of OA in some dogs predisposed to the condition.17,18,24 It has been generally assumed
that the subjective scoring of hip phenotype at 1 or 2 years of age accurately reflects the true phenotype
of the dog. No lifelong studies have been conducted to document the accuracy of the one- or two-year

23 Osteoarthritis
{ } >>> S M I T H , B I E R Y, K E A LY, L AW L E R

evaluation to predict the end-of-life hip phenotype. It was the purpose of the present investigation to test
the influence of food restriction on hip phenotype and to compare end-of-life hip phenotypes with OFA
scores, PennHIP scores, and OA scores at 2 years of age.
Materials and Methods. Forty-eight 8-week-old Labrador retriever puppies from seven litters were allotted
by pairing to two groups of 24 dogs each. One group was fed ad libitum (full-fed) and each member of the
other group (limit-fed) was given 25% less of the same food given to the full-fed pair-mate. Radiography
(ventrodorsal, hip-extended) of the hip joints was done when the dogs were ages 30 and 54 weeks and yearly
thereafter for life. Subluxation was measured using the Norberg angle on radiographs made with the dog
in the standard, hip-extended position. The same radiographs were evaluated for evidence of CHD and OA
by a board-certified radiologist using the scoring system of the OFA. At 2 years of age the hips were scored
using the PennHIP method.
Results. Limit-feeding had a profound positive effect on the hip phenotype of Labrador retrievers. Limit-
fed dogs had significantly lower incidence and severity of CHD and OA compared with full-fed pair-mates.
This health benefit continued for the life of the dogs. In the pooled sample of 48 dogs, the prevalence of
hip OA increased linearly throughout the study, from 15% at 2 years of age to 67% at end-of-life. For the
full-fed dogs, end-of-life OA prevalence was 83% and for the limit-fed dogs, 50%. At 2 years of age, OFA-
type scoring found 19 of the 48 dogs in the study to be “dysplastic” while 29 dogs were scored as “normal.”
The 19 dysplastic dogs remained dysplastic for life, with OFA scores increasing in severity for many of the
dogs. However, of the 29 dogs scored “normal,” 16 (55%) were scored dysplastic by end-of-life, representing a
55% false-negative rate of diagnosis. PennHIP results showed that all the dogs included in this investigation
were susceptible to OA (distraction index [DI] >0.36, range 0.36–0.92). Kaplan-Meier curves of disease-free
interval showed that for dogs with a low DI the onset of OA was much later in life than dogs with a large
DI. For dogs with DI ≤0.4 the median disease-free interval was 12 years of age compared with dogs with
DI >0.6, whose median disease-free interval was only 3 years of age.
Discussion. Results of this lifelong study showed that by keeping dogs lean the onset of OA was delayed
and its severity and prevalence was reduced significantly. In addition, OA prevalence in other joints of the
lean dogs was decreased. Such findings are both statistically and clinically significant. The linear increase in
OA incidence over the life of these Labrador retrievers refutes popular dogma that holds that hip OA occurs
either early in life, in the case of dysplastic dogs, or much later in the geriatric years in the case of “old age
OA.” The principal risk factor, if not the cause, for the development of hip OA has been shown to be joint
laxity irrespective of age. In the hip-extended view this laxity is underestimated and often masked
completely, leading to false-negative diagnoses. This results in some dogs appearing phenotypically normal
although they are genotypically abnormal. Evidence for this impression derives from the continued high
frequency of CHD in many breeds of dogs despite systematic mating of normal parents. The life-long study
reported here provides additional evidence. Hip phenotypes in this sample were much worse at the end of
life than at 2 years of age. The “normal” designation of hips at 2 years of age was wrong more than it was
right (55% false-negative rate). The dogs in this study derived from lines of dogs with hip dysplasia and
therefore there was a high probability to express OA. Dogs that were permitted to become overweight
(mean body condition scores of 6) expressed OA much more, 83%, than those kept lean (OA 50%) (mean
body condition score of 4). The PennHIP distraction index indicated that all dogs in this study (at 2 years of age)
were susceptible to OA and therefore genotypically abnormal. This predicted susceptibility was borne out by
the observed pattern of OA incidence later in life. In this regard the distraction index was not influenced by
the effects of the diet. This finding represents an extremely desirable characteristic of a genetic test.

24
EFFECTS OF RESTRICTED FEEDING ON ONSET, INCIDENCE AND
S E V E R I T Y O F H I P D I S P L A S I A A N D O S T E O A RT H R I T I S I N D O G S

REFERENCES
1. Henricson B, Norberg I, Olsson SE. On the etiology and pathogenesis of hip dysplasia: A comparative
review. J Small Anim Pract. 1966;7:673–688.
2. Hutt FB. Genetic selection to reduce the incidence of hip dysplasia in dogs. J Am Vet Med Assoc.
1967;151:1041–1048.
3. Jessen CR, Spurrell FA. Heritability of canine hip dysplasia. In: Proceedings of the Canine Hip Dysplasia
Symposium and Workshop. St. Louis, MO.1972:53–61.
4. Hedhammar A, Olsson SE, Andersson SA, et al. Canine hip dysplasia: A study of heritability in 401 litters
of German Shepherd dogs. J Am Vet Vet Assoc. 1979;174:1012–1016.
5. Mansson J, Norbert I. Hoftledsdysplasi hos hund (Hip dysplasia in the dog). J Swed Vet Assoc.
1961;12:1–8.
6. Riser WH. The dysplastic hip joint: Its radiographic and histologic development. J Am Vet Radiol Soc.
1973;14:35–50.
7. Olsson SE, Lasstrom H. Etiology and pathogenesis of canine hip dysplasia: Introduction to a new
concept. In: Proceedings of the Canine Hip Dysplasia Symposium and Workshop. St. Louis, MO.
1972:1–52
8. Smith GK, Gregor TP, Rhodes WH, Biery DN. Coxofemoral joint laxity from distraction radiography and
its contemporaneous and prospective correlation with laxity, subjective score and evidence of degen-
erative joint disease from conventional hip-extended radiography. Am J Vet Res. 1993;54:1021–1042.
9. Smith GK, Popovitch CA, Gregor TP, et al. Evaluation of risk factors for degenerative joint disease
associated with hip dysplasia in dogs. J Am Vet Med Assoc. 1995;206:642–647.
10. Lust G, Rendano VT, Summers BA. Canine hip dysplasia: Concepts and diagnosis. J Am Vet Med Assoc.
1985;187:638–640.
11. Davis MA, Ettinger WH, Neuhaus JM, et al. Sex differences in osteoarthritis of the knee.
Am J Epidemiol. 1988;127:1019–1030.
12. van Saase JLCM, Vandenbroucke JP, van Romunde LKJ, et al. Osteoarthritis and obesity in the general
population: A relationship calling for an explanation. J Rheumatol. 1988;15:1152–1158.
13. Anderson JJ, Felson DT. Factors associated with osteoarthritis of the knee in the first national health
and nutrition examination survey (Hanes I). Am J Epidemiol. 1988;128:179–189.
14. Bendele AM, Hulman JF. Effects of body weight restriction on the development and progression of
spontaneous osteoarthritis in guinea pigs. Arthritis Rheum. 1991;34:1180–1184.
15. Wilhelmi R, Maier R. Beobachtungen uber den einflub von druckbelastung und bewegung auf die
gelenke am modell arthrosedisponieter Mause. Akt Rheumatol. 1987;12:161–167.
16. Popovitch CA, Smith GK, Gregor TP, et al. Comparison of susceptibility for hip dysplasia between
Rotweillers and German Shepherd dogs. J Am Vet Med Assoc. 1995;206:648–650.
17. Kealy RD, Olsson SE, Monti KL, et al. Effects of limited food consumption on the incidence of hip
dysplasia in growing dogs. J Am Vet Med Assoc. 1992;201:857–863.
18. Kealy RD, Lawler DF, Ballam JM, et al. Five-year longitudinal study on limited food consumption and
development of osteoarthritis in coxofemoral joints of dogs. J Am Vet Med Assoc. 1997;210:222–225.
19. Whittington K, Banks WC, Carlson WD, et al. Report of panel on canine hip dysplasia. J Am Vet Med
Assoc. 1961;139:791–806.
20. Owens JM, Biery DN. Radiographic Interpretation for the Small Animal Clinician. St. Louis, MO:
Ralston Purina Co. 1982:40–43.
21. Lust G, Geary JC, Sheffy BE. Development of hip dysplasia in dogs. Am J Vet Res. 1973;34:87–91.
22. Riser WH. The dog as a model for the study of hip dysplasia. Vet Pathol. 1975;12:234–334.
23. Smith GK, Gregor TP, Rhodes WH, et al. Coxofemoral joint laxity from distraction radiography and its
contemporaneous and prospective correlation with laxity, subjective score, and evidence of degenerative
joint disease from conventional hip-extended radiography in dogs. Am J Vet Res. 1993;54:1021–1042.
24. Kealy RD, Lawler DF, Ballam JM, et al. Evaluation of the effect of limited food consumption on
radiographic evidence of osteoarthritis in dogs. J Am Vet Med Assoc. 2000;217:1678–1680.

25 Osteoarthritis
 >>> S M I T H , B I E R Y, K E A LY, L AW L E R

25. Sokoloff L. The pathology of osteoarthritis and the role of aging. In: Nuki G (ed): The Aetiopathogeneis
of Osteoarthritis. Tunbridge Wells, UK: Pitman Medical Publishing Co. 1980:1–15.
26. Wastacott CI, WEbb GR, Warnock MG, et al. Alteration of cartilage metabolism by cells from
osteoarthric bone. Arthritis Rheum. 1997;40:1282–1291.
27. Farquhar T, Bertram J, Todhunter RJ, et al. Variations in composition of cartilage from the shoulder
joints of young adult dogs at risk for developing canine hip dysplasia. J Am Vet Med Assoc.
1997;210:1483–1485.
28. Corley EA. Role of the Orthopedic Foundation for Animals in the control of canine hip dysplasia.
Vet Clin North Am: Small Anim Pract. 1992;201:857–863.

26
■ CLINICAL SIGNIFICANCE OF OSTEOARTHRITIS AND HIP
DYSPLASIA FINDINGS IN THE RESTRICTED FEEDING TRIAL
Gail K. Smith, VMD, PhD, Diplomate ACVS,1 Darryl N. Biery, DVM,1
Richard D. Kealy, PhD,2 and Dennis F. Lawler, DVM2
1
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania; 2 Department of Research & Development, Nestlé Purina PetCare
Company, St. Louis, Missouri

The studies abstracted in the preceding article in these proceedings have Dr. Gail Smith received his
profound clinical significance for the practicing veterinarian, orthopaedic surgeon, VMD from the University
of Pennsylvania, completed
dog breeder, and pet owner: Dogs suspected to be susceptible to canine hip dysplasia
orthopaedic surgical residency
(CHD) should be kept lean for life and dogs selected for breeding should have hip training, and was awarded
evaluation at regular intervals throughout life. Although the studies were performed a PhD in engineering. He is cur-
in one breed of dog, the Labrador retriever, it is reasonable to consider the results rently Professor of Orthopaedic
Surgery and Chairman of the
clinically applicable to similar breeds, such as other retriever breeds and even
Department of Clinical Studies
Rottweilers and German Shepherd dog breeds. This opinion finds support from at the University of Pennsylvania
studies that have shown body weight to be a risk factor for hip osteoarthritis (OA).1,2 School of Veterinary Medicine.
Owners of dogs of OA-susceptible breeds should strive to keep body condition at score Dr. Smith has published more
than 150 research articles, book
5 or below (on a scale of 1 to 9 where 9 represents extreme obesity). For tight-hipped
chapters, and proceedings. Dr.
breeds of dogs known to not be susceptible to hip OA, such as performance grey- Smith’s research interests have
hounds and Borzois, it is unnecessary to adhere to these dietary recommendations. focused on orthopaedic prob-
However, as will be shown in other parts of this symposium, keeping body condition lems of the spine, the knee and
the hip. A clinically compelling
scores at 5 or below has other health benefits unrelated to OA.
product of his research is a
The foregoing studies demonstrated that controlling body weight delayed, lessened stress-radiographic diagnostic
the severity, or prevented the development of OA in some dogs. Consistent with these method capable of predicting
the susceptibility to hip dysplasia
radiographic findings was a parallel benefit in clinical signs within this pool of dogs. in dogs as young as 16 weeks of
Dogs that were kept lean required less pain medication (nonsteroidal anti-inflammatory age. Dr. Smith founded PennHIP®,
drugs) starting later in life than dogs in the full-fed group. The mean therapy-free a cooperative scientific initiative
interval for the lean dogs with OA was 10.3 years, and for the restricted-fed dogs, 13.3 representing multiple collabo-
rative centers in North America
years (P < 0.01).3 Further support for this experimental finding was published recently and Canada, trained to test the
in studies showing that reducing body weight and body score in obese dogs with clinical new hip dysplasia diagnostic
signs of hip dysplasia resulted in a substantial improvement in clinical lameness4 and technology. Research continues
in gait5 (as demonstrated by force plate testing). These findings add to the growing to support the role of the
PennHIP phenotype to effectively
pool of data in humans showing similar beneficial effects of weight reduction in select breeding stock to reduce
alleviating the discomfort of OA. the frequency of this most-
prevalent osteoarthritic disease.
Hip dysplasia has long been understood to be a disease of complex inheritance.
Environmental factors can influence the expression of diseases of complex inheritance
and in this investigation, food consumption and body weight were found to be potent
factors. Keeping dogs lean certainly did not change the genes of dogs predisposed to
hip dysplasia. Rather, leanness was shown to delay or prevent the expression of
radiographic signs of CHD in dogs prone to CHD, likely by antagonizing the conversion of
passive hip laxity to functional hip laxity that ultimately leads to OA. Some would argue
that such environmental manipulation is contraindicated because it “masks” the disease
phenotype, thereby confounding diagnosis and related breeding recommendations.

27 Osteoarthritis
 >>> S M I T H , B I E R Y, K E A LY, L AW L E R

Others, however, would counter that for the comfort of the dog, one should invoke all known
environmental measures to delay the onset or lessen the severity of CHD. It is conceivable if the dogs in this
study were stressed even more (environmentally) or if they had lived longer, that all of them might have
expressed OA. Clearly a diagnostic test for hip dysplasia that is not confounded by environmental factors is
needed. The measure of such a test is embodied in the concept of “heritability” and the higher the estimate
of heritability the better. Comparative studies estimating the heritability of hip phenotype have shown the
distraction index (DI) to have higher heritability than subjective hip scores. It is encouraging that in this
study the PennHIP distraction index was not confounded by the diet and that, consistent with observed
lifelong hip phenotypes, the distraction index put all of the dogs into the OA-susceptible category. These
findings support the view that the estiomate of heritability for the distraction index is likely higher than the
subjective hip score.
Longitudinal studies, such as this one, are essential to understanding the true biological behavior of a
disease as complex as CHD. To the authors’ knowledge no similar studies have been published. Of particular
interest and importance is the observation that OA prevalence and OFA score increased linearly long after
2 years of age, the accepted convention for phenotypic expression. This understanding raises questions
about the diagnostic accuracy of the standard radiographic method of hip scoring, which is usually per-
formed shortly after 2 years of age. For scoring methods performed at 1 year of age, the diagnostic error
would likely be even larger. Hitherto unappreciated, these studies draw critical attention to the magnitude
of change in hip score with aging. Of dogs graded normal by OFA-type scoring at 2 years of age, 55%
became dysplastic by the end of life. Conventional subjective hip scoring at 2 years of age, therefore, under-
estimates the frequency of hip dysplasia in dogs and this observation provides partial explanation for the
recognized slow progress in reducing the frequency and severity of CHD by selective breeding. There
currently is no requirement for dogs that are OFA certified at 2 years of age to undergo repeat evaluations
to validate the 2-year score. Minimally, this new data warrants a strong clinical recommendation for hip
films well beyond 2 years of age, and in the case of breeding dogs, hips should be evaluated at regular
intervals for life.

REFERENCES
1. Smith GK, Popovitch CA, Gregor TP, et al. Evaluation of risk factors for degenerative joint disease asso-
ciated with hip dysplasia in dogs. J Am Vet Med Assoc. 1995;206:642–647.
2. Popovitch CA, Smith GK, Gregor TP, et al. Comparison of susceptibility for hip dysplasia between
Rotweillers and German Shepherd dogs. J Am Vet Med Assoc. 1995;206:648–650.
3. Kealy RD, Lawler DF, Ballam JM, et al. Effects of diet restriction on life span and age-related changes
in dogs. J Am Vet Med Assoc. 2002;220:1315–1320
4. Impellizeri JA, Tetrick MA, Muir P. Effect of weight reduction on clinical signs of lameness in dogs with
hip osteoarthritis. J Am Vet Med Assoc. 2000;216:1089–1091.
5. Burkholder WJ, Taylor L, Hulse DA. Weight loss to optimal body condition increases ground reactive
force in dogs with osteoarthritis. Proceedings, 2000 Purina Nutrition Forum. 2001;23:74.

28
■ DIET RESTRICTION, CARBOHYDRATE METABOLISM, AND THE
RETARDATION OF SENESCENCE
Edward J. Masoro, PhD
Professor Emeritus, Department of Physiology,
University of Texas Health Science Center, San Antonio, Texas

Studies with laboratory rodents have convincingly demonstrated that the reduction Dr. Edward J. Masoro
of caloric intake is the nutritional factor primarily responsible for the life-extending received both his PhD in
Physiology and AB from the
and anti-aging actions of diet restriction1 and this has resulted in gerontologists more
University of California at
frequently using the term caloric restriction (CR) rather than diet restriction when Berkeley. He chaired the
referring to this anti-aging phenomenon. These findings also logically led most Department of Physiology at
gerontologists to the conclusion that a reduction in metabolic rate was the basis of the University of Texas Health
Science Center at San Antonio
the life-extending effect of CR. This concept has required revision, however, in light of
(UTHSCSA) and then became
the findings of Duffy et al2 with mice and McCarter and Palmer3 with rats showing Founding Director of the Aging
that CR can increase life span without decreasing metabolic rate per unit of lean body Research and Education Center
mass or per unit of “metabolic mass.” at UTHSCSA. Currently a
Professor Emeritus, he serves in
CALORIC RESTRICTION AND CARBOHYDRATE METABOLISM IN RODENTS the Research Development Core
of the National Institute on
One line of thinking led to the hypothesis that CR has its life-prolonging effect by Aging-funded Nathan Shock
altering the characteristics of carbohydrate fuel use. The fact that hyperglycemia and Center for Excellence in the
hyperinsulinemia damage mammalian organisms and that this damage has some Biology of Aging at UTHSCSA.
He has served as President of
resemblance to what occurs in senescence are the triggers that led to this hypothesis.
the Gerontological Society of
Early Studies America, Chair of the Board of
Scientific Counselors of the
Even before this hypothesis was proposed, the effect of CR on carbohydrate metabolism National Institute on Aging,
was the subject of considerable study. Long-term CR was found to decrease the editor of the Journal of
fasting plasma levels of glucose and insulin in mice and rats.4–6 In addition, Reaven and Gerontology: Biological Sciences,
and President of the
Reaven7 made a single determination of plasma insulin between 1400 and 1600 hours
Association of Chairmen of
in 3- and 12-month-old rats and found that animals on a reduced caloric intake have Departments of Physiology. His
lower levels of plasma insulin. CR also was found to decrease the postprandial level of research in biological gerontology
plasma insulin in rats.8 has focused on the anti-aging
action of caloric restriction in
Early studies also focused on glucose utilization. Reaven et al5 studied the effect rodent models, for which he
of insulin on glucose utilization in anesthetized 12- to 13-month-old rats fasted for received the Allied-Signal
4 hours using a method involving continuous infusion of epinephrine, propranolol, Achievement Award in Aging,
the Robert W. Kleemeier Award,
glucose, and insulin. They found that rats on a low caloric intake were less insulin
the Irving Wright Award of
resistant than those on a higher caloric intake. To the contrary, Kalant et al9 used the Distinction, and the Glenn
same technique to measure the effect of insulin on glucose utilization in anesthetized Foundation Award.
4-, 12-, 18-, and 24-month-old rats that had been fasted overnight and found that
long-term CR decreased the ability of insulin to stimulate glucose utilization.
Ivy et al10 measured glucose utilization by rat hindlimb preparations; they found that
preparations from rats on long-term CR had an increased ability to utilize glucose.
These early studies are difficult to interpret in regard to the anti-aging action of
CR because they do not provide information on carbohydrate metabolism and insulin

29 Carbohydrate Metabolism
{ } >>> MASORO

action under conditions similar to those in which animals in longevity studies spend their lifetime. Rather
these measurements were made on rats and mice that were either fasted or anesthetized or surgically
traumatized or subjected to a combination of these stressors.
Lifetime Longitudinal Study of Carbohydrate Metabolism in Rats

A longitudinal study of male F344 rats was carried out in my laboratory with the aim of determining plas-
ma glucose and insulin levels and glucose utilization over the lifetime under usual daily living conditions.11
The study included a detailed analysis of the circadian pattern of plasma glucose concentration and the
measurement of plasma insulin concentration at the daily maximum and minimum glucose concentrations.
Throughout most of the day, plasma glucose concentration in the rats on CR was significantly lower than
that of the rats fed ad libitum. Only during the 2 to 3 hours following receipt of their daily allotment of
food did the plasma level of the CR rats approach that of those fed ad libitum. This circadian pattern of
plasma glucose concentration continued throughout the life span of the ad libitum-fed rats. (0f course,
because the rats on CR lived much longer than those fed ad libitum, there was a considerable period of the
life of the CR rats during which there were no ad libitum-fed rats of the same age alive for comparison.)
There were some age-associated quantitative differences; the mean 24-hour difference in plasma glucose
concentration ranged from 13 to 21 mg/dL with greatest difference occurring in rats in 9- to 13-month age
range and the smallest difference in rats in the 21- to 25-month age range. The average difference in the
24-hour mean plasma glucose concentration between the two groups over the lifetime of the ad libitum-
fed rats was 12%. Plasma insulin levels were measured during the daily periods of the maximum and min-
imum plasma glucose levels; at all ages in common, the plasma insulin concentration was markedly lower
at both times of day in the CR rats compared with those fed ad libitum. The difference, which varied with
the age of the rat and the time of day, ranged from 45% to 80% lower concentrations in the CR rats.
McCarter and Palmer3 had measured the oxygen consumption of male rats throughout the life span in
their home cages under what was their usual living conditions and found that over most of the life span,
the rate of oxygen consumption per kilogram of “metabolic mass” was the same for ad libitum-fed and CR
rats. They also found that over the lifetime of these rats that the respiratory quotient (RQ) averaged 0.89
for both groups of rats, which is the expected value if the fuel mixture eaten is the same as the fuel mixture
utilized. This finding was not surprising because, for most of their lives, the rats are in a near steady state
in regard to body mass and body composition. Based on this information and on the plasma glucose insulin
levels in our longitudinal study, it is evident that the rate of glucose fuel use per kilogram of “metabolic
mass” is the same for rats on the CR regimen and those fed ad libitum even though our study showed that
CR rats maintain lower plasma glucose levels and markedly lower plasma insulin levels. We decided that this
information was so important that it should be confirmed with a group of rats in which the oxygen con-
sumption, RQ, and plasma glucose and insulin levels were measured simultaneously. Male F344 rats aged
8 to 10 months fed ad libitum or on a CR regimen from 6 weeks of age were used. The two dietary groups
had a similar daily RQ and rate of oxygen consumption per kilogram “metabolic mass.” During the 2-hour
peak period of plasma glucose concentration, CR rats had an oxygen consumption of 0.94 L/kg “metabolic
mass,” a RQ of 0.93, plasma glucose of 155 mg/dL, and plasma insulin of 4.4 ng/mL, while the ad libitum-
fed rats had an oxygen consumption of 0.92 L/kg “metabolic mass,” a RQ of 0.90, plasma glucose of 159
mg/dL, and plasma insulin of 8.1 ng/mL. Based on the sum total of these findings, our conclusion is that CR
either increases glucose effectiveness or insulin sensitivity, or both.

30
D I E T R E S T R I C T I O N , C A R B O H Y D R AT E M E TA B O L I S M ,
A N D T H E R E TA R D AT I O N O F S E N E S C E N C E

In Vitro Studies on Glucose Effectiveness and Insulin Sensitivity

McCarter et al12 used the rat epitroclearis muscle to determine the effect of CR on glucose effectiveness.
They found that muscle preparations from rats on long-term CR exhibited increased glucose effectiveness.
Using the same rat skeletal muscle preparation, Cartee et al13 found that CR enhances insulin stimulation of
glucose transport. CR also enhances insulin’s ability to promote glucose metabolism by isolated adipocytes.14
In Vivo Studies on Glucose Metabolism by Specific Tissue and Organ Sites

It is important that the effect of CR and insulin action on carbohydrate metabolism be determined in
specific tissues and organs as they function in the intact organism. Escriva et al15 did the pioneering work in
this regard. They used the 2-deoxyglucose method and studied glucose uptake by various skeletal muscle
and adipose tissue sites in anesthetized 70-day-old Wistar rats under basal and hyperinsulinemic conditions.
They found that CR increased insulin-mediated glucose uptake at all these sites.
Wetter et al16 also used the 2-deoxyglucose method but made a major advancement by carrying out
their study on 11- to 12-month-old (F344 × BN)F1 rats under as usual living conditions as possible. They
found that although CR maintained low levels of plasma insulin, it did not affect glucose uptake by
cerebellum, lung, kidney, soleus muscle, and diaphragm and it enhanced glucose uptake by brown adipose
tissue, white adipose tissue, and the epitroclearis, plantaris, and gastrocnemius muscles.
Role of Altered Carbohydrate Metabolism in Anti-Aging Action of Caloric Restriction

Initial attention focused on the potential role of the lifetime lowering of plasma glucose levels as a factor
in the life-prolonging action of CR. The reasons for this focus were two. First, hyperglycemia results in
functional and morphologic deteriorations similar to those of senescence.17 Second, Cerami18 proposed that
nonenzymatic glycation and subsequent reactions involving proteins and nucleic acids are causally involved
in senescence and that increasing levels of plasma glucose concentration promote their formation. CR has
been found to decrease the extent of glycation of proteins and the age-associated increase in advanced
glycation end-products in rodents.19,20 It still remains to be established, however, that glycation and
subsequent reactions play a major role in aging.
Recently, the focus has shifted to the potential role of the marked reduction in plasma insulin levels in
CR rodents as the factor most responsible for the anti-aging action of CR. One reason for this focus is the
evidence that long-term hyperinsulinemia causes premature senescence-like damage.21 The main reason,
however, is the recent research on genetic manipulations that extend the life span of the nematode
Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Loss of function mutations in genes
coding for components of the insulin-signaling pathway of this nematode species22 and this fruit fly
species23 extend the life span of these invertebrate animal models. It has been suggested that there is an
evolutionary link between the effects of these loss of function mutations in these invertebrate animal models
and the life extending effect of CR in rodent models.24 My initial reaction was negative to the hypothesis
that these findings on nematodes and fruit flies relate to the life-extending action of CR in rats and mice
because CR does not decrease but rather enhances the insulin signaling system in the skeletal muscle and
adipose tissue of rodents. The recently reported findings of Wolkow et al,25 however, made me reconsider
this hypothesis. These investigators restored insulin signaling in specific tissues of a nematode strain that has a
mutation resulting in a loss of function in insulin signaling and found that restoring insulin signaling in the
nervous system of these animals eliminated the increased longevity but that restoring it in muscle and intestine
did not. Indeed, if CR does not enhance or attenuate insulin signaling in the central nervous system of rodents,
the sustained low levels of circulating plasma insulin may have the same effect on insulin signaling in the

31 Carbohydrate Metabolism
{ } >>> MASORO

central nervous system of rodents as that of the loss of function mutations in C. elegans. Although the
proponents of the hypothesis that insulin signaling is a universal component of aging in all animal species seem
overly exuberant given the current state of knowledge, the concept is certainly worthy of further study.

CALORIC RESTRICTION AND CARBOHYDRATE METABOLISM IN RHESUS MONKEYS

Three studies are ongoing on the effect of diet restriction on aging of rhesus monkeys; these studies
are being conducted at the National Institute on Aging, the University of Maryland, and the University of
Wisconsin. As yet, none has provided definitive evidence that CR increases the length of life of these
monkeys but they have shown that many of the phenotypic effects of CR in rodents also occur in rhesus
monkeys.26 Long-term CR has been found to decrease fasting plasma glucose levels in two of these
studies27,28 but not in one of the studies.29 CR decreased fasting plasma insulin levels and increased insulin
sensitivity in all three studies.27–29 In all three studies, however, insulin sensitivity was determined in fasted
monkeys under light anesthesia. Thus, as of now, there is no information on the effects of CR on carbohydrate
metabolism and insulin action in rhesus monkeys under usual living conditions.

REFERENCES
1. Masoro EJ. Dietary restriction: An experimental approach to the study of the biology of aging. In:
Masoro EJ, Austad SN (eds): Handbook of the Biology of Aging, 5th ed. San Diego: Academic Press.
2001;396–420.
2. Duffy PH, Feuers RJ, Leakey JEA, et al. Chronic caloric restriction in old female mice: Changes in
circadian rhythms of physiological and behavioral variables. In: Fishbein L (ed): Biological Effects of
Dietary Restriction. Berlin: Springer-Verlag. 1991;245–263.
3. McCarter RJM, Palmer J. Energy metabolism and aging: A lifelong study of Fischer 344 rats.
Am J Physiol. 1992;263:E448–E452.
4. Masoro EJ, Compton C, Yu BP, et al. Temporal and compositional dietary restrictions modulate
age-related changes in serum lipids. J Nutr. 1983;113:880–885.
5. Reaven E, Wright C, Mondon CE, et al. Effect of age and diet on insulin secretion and insulin action
in the rat. Diabetes. 1983;32:175–180.
6. Koizumi A, Wada Y, Tsukada M, et al. Low blood glucose levels and small islets of Langerhans in the
pancreas of calorie-restricted mice. Age. 1989;12:93–96.
7. Reaven GM, Reaven EP. Prevention of age-related hypertriglyceridemia by caloric restriction and
exercise training in the rat. Metabolism.1981;30:982–986.
8. Belage M, Grizard J, Manin M. Effect of calorie restriction on skeletal muscle and liver insulin binding
in growing rats. Horm Metab Res. 1990;22:207–214.
9. Kalant N, Winslow JR, Reynolds RW. Effect of diet restriction on glucose metabolism. Mech Ageing
Dev. 1988;46:89–104.
10. Ivy JL, Young JC, Craig BW, et al. Aging, exercise, and food restriction: Effect on skeletal muscle
glucose uptake. Mech Ageing Dev. 1991;61:123–133.
11. Masoro EJ, McCarter RJM, Katz MS, et al. Dietary restriction alters characteristics of glucose fuel use.
J Gerontol: Biol Sci. 1992;47:B202–B208.
12. McCarter RJ, Masoro E J, Mejia WJ. Effects of dietary restriction on glucose utilization of skeletal
muscle of Fischer 344 rats. Gerontologist. 1994;34:356.
13. Cartee GD, Kietzke EW, Briggs-Tung C. Adaptation of muscle glucose transport with caloric restriction
in adult, middle-aged and old rats. Am J Physiol. 1994;266:R1443–R1447.
14. Craig BW, Garthwaite SM, Holloszy JO. Adipocyte insulin resistance. Effects of aging, obesity, exercise,
and food restriction. J Appl Physiol. 1987;62:95–100.
15. Escriva F, Rodriquez C, Cacho J, et al. Glucose utilization and insulin action in adult rats submitted to
prolonged food restriction. Am J Physiol. 1992;263:E1–E7.

32
D I E T R E S T R I C T I O N , C A R B O H Y D R AT E M E TA B O L I S M ,
A N D T H E R E TA R D AT I O N O F S E N E S C E N C E

16. Wetter TG, Gazdag AC, Dean DJ, et al. Effect of caloric restriction on in vivo glucose metabolism
by individual tissues in rats. Am J Physiol. 1999;276:E-728–E-738.
17. Rossetti L, Giaccari A, DeFronzo RA. Glucose toxicity. Diabetes Care. 1990;13:610–630.
18. Cerami A. Hypothesis: Glucose as a mediator of aging. J Am Geriatr Soc. 1985;33:610–630.
19. Cefalu WR, Bell-Farrow AD, Wang ZQ, et al. Caloric restriction decreases age-dependent
accumulation of the glycoxidation products, Nε-(carboxymethyl)lysine and pentosidine in rat
skin collagen. J Gerontol: Biol Sci. 1995;50A:B337–B341.
20. Sell DR, Monnier VM. Age-related association of tail tendon break time with tissue pentosidine
in DBA/2 vs. C57BL/6 mice. The effect of dietary restriction. J Gerontol: Biol Sci.
1997;52A:B277–B284.
21. Parr T. Insulin exposure controls rate of mammalian aging. Mech Ageing Dev. 1997;88:75–82.
22. Vanfleteren JR, Braeckman BD. Mechanisms of life span determination in Caenorhabditis
elegans. Neurobiol Aging. 1999;20:487–502
23. Clancy DJ, Gems D, Harshman LG, et al. Extension of life-span by loss of CHICO, a Drosophila
insulin receptor substrate protein. Science. 2001;292:104–106.
24. Kimura KD, Tissenbaum HA, Lu Y, et al. daf-2, an insulin receptor-like gene that regulates
longevity and diapause in Caenorhabditis elegans. Science. 1997;277:942–946.
25. Wolkow CA, Kimura KD, Lee MS, et al. Regulation of C. elegans life-span by insulin-like signaling
in the nervous system. Science. 2000;290:147–150.
26. Roth GS, Ingram DK, Lane MA. Caloric restriction in primates: Will it work and how will we know?
J Am Geriatr Soc. 1999;47:896–903.
27. Kemnitz JW, Roecker EB, Weindruch R, et al. Dietary restriction increases insulin sensitivity and
lowers blood sugar in rhesus monkeys. Am J Physiol. 1994;266:E540–E547.
28. Lane MA, Ball SS, Ingram DK, et al. Diet restriction in rhesus monkeys lowers fasting and glucose-
stimulated glucoregulatory end points. Am J Physiol. 1995;E-941–E-948.
29. Bodkin NL, Ortmeyer HA, Hansen BC. Long-term dietary restriction in older-aged rhesus
monkeys: Effects on insulin resistance. J Gerontol: Biol Sci. 1995;50A:B142–B147.

33 Carbohydrate Metabolism
 ■ DIET RESTRICTION AND AGING: CANINE CARBOHYDRATE AND
LIPID METABOLISM
Brian T. Larson, PhD,1 Dennis F. Lawler, DVM,1 Jay M. Harrison, MS, MA,2
and Richard D. Kealy, PhD1
1
Pet Nutrition Research Department, 2Statistical Services Department,
Nestlé Purina PetCare Company, St. Louis, Missouri

Dr. Brian Larson was ABSTRACT:

employed by Ralston Purina
Labrador retrievers (48) were used to assess diet restriction effects on carbohydrate
Company in 2000 as a Nutrition
Scientist in Research and and lipid metabolism. Restricted-fed (RF) dogs were fed 75% of the same diet
Development. Dr. Larson com- consumed by control-fed (CF) pair-mates. An intravenous glucose tolerance test
pleted his undergraduate (IVGTT) was performed annually for years 9 to 12. Dietary treatment, age, and inter-
education and MS degree in
actions were fixed effects. Statistical procedures used when appropriate included
nutrition from the Department
of Animal Sciences at the mixed-model, repeated-measures ANOVA; least-squares means; Tukey’s multiple
University of Minnesota, where comparison; paired t-test; Spearman rank correlation; and Cox proportional hazards
he served on the Animal regression. The following IVGTT indicators improved (P < 0.001) in RF dogs compared
Science faculty as an Extension
with CF dogs: glucose and insulin means, peaks, ∆’s, return-to-basal times, areas-
Livestock Specialist with state-
wide programming and teaching under-curve; glucose decline rate; and insulin sensitivity overall average and for each
responsibilities in nutrition. Dr. year 9, 10, and 11. The following IVGTT indicators were not significantly different
Larson completed a PhD in the (P > 0.05) for RF versus CF dogs: insulin decline rate; year 12 insulin sensitivity; and
Animal Science Department at
insulinogenic index. Peak and ∆ glucose were positively correlated and insulin
the University of Missouri,
where he focused his efforts on sensitivity negatively correlated with body condition score (BCS), fat percentage and
dopamine receptors’ adverse mass, and body weight. Serum cholesterol and triglyceride were not remarkably
cellular effects initiated by correlated with glucose/insulin indices nor associated with survival. Median survival
consumption of ergot alkaloids
time tended to be greater with lower basal glucose and insulin (P = 0.065, P = 0.096).
and received his primary
research training with the Time-to-first osteoarthritis treatment or death was greater in dogs with lower basal
Pharmacology Department of glucose and higher insulin sensitivity (P = 0.021, P = 0.023); however, diet restriction
the School of Medicine. Dr. explained most of the relationship variation. Higher insulinogenic indices were
Larson subsequently joined the
associated with greater median survival (P = 0.053) and those with higher insulin
Animal Sciences faculty at the
University of Kentucky to continue sensitivity had less (P = 0.018) hazard of dying or receiving chronic disease treatment.
his work on biogenic amine These insulin indices added more information than diet restriction alone (P = 0.057,
receptor toxicology and with P = 0.055). Lifelong diet-restricted glucose disposal efficiency and insulin response
byproduct sources of proteins
were associated with increased quality and quantity of life.
and carbohydrates, teach
undergraduate nutrition and
chair the successful effort to
conceptualize, design, build,
equip, and staff the new animal
research center. Dr. Larson’s
companion animal nutrition
research at Nestlé Purina
PetCare Company has concen-
trated on carbohydrate and
protein metabolism interface
with cellular and endocrine
mechanisms.

Carbohydrate Metabolism 34
■ LIFE IS SHORTER, IF YOU EAT DESSERT FIRST:
CLINICAL IMPLICATIONS OF THE PURINA 448 STUDY
Deborah S. Greco, DVM, PhD, Diplomate ACVIM
Professor, Small Animal Internal Medicine, College of Veterinary Medicine and Biomedical
Sciences, Colorado State University, Fort Collins, Colorado

“Life is short, eat dessert first” — Anonymous Dr. Deborah S. Greco is a

Professor of Small Animal
We live to eat and eat to live. When food is plentiful, we store “fuel” as fat. Glucose Internal Medicine at Colorado
is absorbed from carbohydrate sources in food and insulin is secreted to allow storage State University. She received
of glucose as fat and glycogen. During periods of starvation, however, our metabolic her DVM from the University of
California and completed an
machinery changes from “storage phase” to “starvation phase.” Insulin secretion is
internship in small animal med-
diminished and fat deposits are used as an energy source to conserve glucose for icine and surgery at Louisiana
insulin-independent organs such as the brain. Do we have the mechanisms for State University. She became a
preventing obesity and its many detrimental effects? Does body composition— lean Diplomate of the American
College of Veterinary Internal
versus fat mass— make a difference? Is the onset of disease heralded by the onset of
Medicine after completing an
perturbations in carbohydrate metabolism? What are the lessons learned from the internal medicine residency at
Purina 448 study? This article will address the clinical consequences of the Purina 448 Texas A&M. After completion of
study for today’s veterinary practitioner. her medicine residency at Texas
A&M she received a PhD in
OBESITY veterinary physiology and
pharmacology. Dr. Greco is a
Humans are not the only species beset by the problem of obesity. It is estimated that past president of the Society for
approximately 35% to 40% of cats, 25% to 35% of dogs, and more than 50% of Comparative Endocrinology and
humans are obese.1–3 In humans, and probably in cats, obesity is a precursor to served as the ACVIM Forum
program chairperson from
diabetes mellitus (type 2). Obese cats are four times more likely to develop diabetes,
1994–1997. Current research
five times more likely to develop lameness, and three times more likely to have interests are in the fields of
non-allergic skin conditions compared with cats of optimal body condition.4 To say canine hyperadrenocorticism,
that obesity in the pet population is a problem is a gross understatement. Until only canine hypothyroidism, and
treatment of feline diabetes
recently, however, veterinarians had no real “smoking gun” to convince owners to
mellitus. Dr. Greco just com-
watch their pet’s weight. pleted a 6-month sabbatical at
The Animal Medical Center
THE SET POINT focusing on treatment of feline
Studies have shown that weight loss and neuroendocrine mechanisms revolve around diabetes mellitus with a high
protein, low carbohydrate diet.
a body set point. Perturbations from the “normal” set point, which may be genetically
determined, result in weight gain and redistribution of body mass (lean versus fat).
This set point can be altered by changes in diet caloric content and composition
(protein versus carbohydrate). Studies in humans have shown that subjects consuming
a high-glycemic index diet for 9 days have higher levels of serum leptin, a greater
decrease in resting energy expenditure (REE), and more negative nitrogen balance
than those consuming a low-glycemic index diet.3
Obese diabetic cats fed a restricted carbohydrate diet lost body fat (based on dual
energy X-ray absorbtiometry), gained lean body mass, became normoglycemic, and
lost insulin dependence.5 Overweight dogs fed 20% calories from protein lost twice as
much lean body mass as overweight dogs fed 30% or 39% calories from protein.6

35 Carbohydrate Metabolism
{ } >>> GRECO

Obese cats fed 35% metabolizable energy as protein showed weight loss consisting of 20% as lean body
mass (LBM) and 79% as fat; however, the cats fed 45% energy as protein lost only 11% as LBM and 88% as
fat.7 Clearly, shifting to a diet which is lower in carbohydrates, lower in glycemic index, and higher in protein
has a beneficial effect on metabolism leading to maintenance of lean body mass and loss of body fat.
Finally, as shown in the Purina 448 study, dogs fed ad libitum had a higher percentage of body fat, pertur-
bations in carbohydrate metabolism, and significantly shorter life span than dogs fed a restricted-calorie diet.8

CLINICAL IMPLICATIONS

One of the problems associated with obesity management is the lack of an objective, practical method for
accurately assessing body composition (LBM and percent body fat). The most useful research tool for assess-
ment of body composition is dual energy x-ray absorptiometry. Most veterinarians, however, do not have
access to such tools. Therefore, body condition score systems have been developed for both cats and dogs
and correlated with laboratory methods such as DEXA.9,10 The nine-point system developed by Laflamme
provides a simple, repeatable method of assessing body composition in small animals.9,10 (See Appendix II
at end of these proceedings for the canine and feline body condition score charts.)
One of the easiest and most practical methods is to take a digital or Polaroid photograph (head on and
profile view) of the dog or cat on a yearly basis. Photographic images can be imported into the animal’s
medical record thus providing serial images of body condition over the years. Body weight and body
condition scores should be recorded on a yearly, if not quarterly, basis. Dogs and cats with heavy haircoats
should be palpated carefully to assess changes in body condition which may not be visually apparent.
Changes in insulin, glucose, and thyroid metabolism were evident in the Purina 448 study.8 Controlled-
feeding dogs had higher fasted serum insulin concentrations and greater area under the curve (AUC) for
glucose after an intravenous glucose tolerance test (IVGTT) than restricted-feeding dogs.8 From a practical
standpoint, IVGTTs are not easily performed in a practice setting for routine annual examinations. However,
a serial record of annual fasting blood glucose values for an individual animal may be very illuminating.
Particularly in cats, because the risk of type 2 diabetes is so great, annual fasting and postprandial blood
glucose values might be of assistance in identifying early type 2 diabetic cats. Dietary intervention at this
point could prevent the development of insulin dependence.
Another parameter that might be beneficial, particularly in cats, is serum fructosamine. Fructosamine
is albumin and other serum proteins which have become glycosylated by a nonenzymatic process. Increased
serum fructosamine values may presage the onset of overt diabetes mellitus in cats and dogs. Again, serial
fructosamine values on an annual basis may provide a more accurate reflection of normal versus abnormal
for a particular patient.
Changes in serum triiodothyronine (T3) concentrations were also noted in the Purina 448 study; in fact,
higher serum triiodothyronine (TT3) were evident in the controlled-feeding group.8 Higher serum TT3 has
been shown to be a consequence of over-feeding because of an increase in deiodination of thyroxine (T4)
to T3.11 In contrast, carbohydrate restriction and weight reduction cause a decrease in serum T3 concentrations.
This brings up the question of whether serial measurements of serum T3 and T4 might be beneficial in
monitoring and prevention of obesity in pet animals.
The problem with this strategy, particularly in dogs, is the tendency to over-diagnose thyroid disorders.
Thyroid metabolism is complex and the finding of low serum T4 in an otherwise healthy animal may not be
of much importance. In dogs, if thyroid hormone is low, endogenous thyroid-stimulating hormone (eTSH)
concentrations should be measured. The findings of a high serum eTSH combined with a low TT4 will ensure

36
L I F E I S S H O RT E R I F Y O U E AT D E S S E RT F I R S T

against the misdiagnosis of hypothyroidism. Serial measurement of T4 and T3, while not detrimental, probably
do not provide any additional information for the management and prevention of obesity. Obviously,
hypothyroid dogs should have these parameters measured on an annual basis.

STRATEGIES TO COPE WITH OBESITY

In a recent article examining factors involved in feline obesity, the top four main factors contributing to
body condition were neuter status, age, frequency of treat feeding, and ad libitum feeding.1 Because
neutering has health benefits and prevents pet overpopulation, it seems unlikely that current recommendations
for neutering will change. Age cannot be eliminated as a factor, although increased vigilance in middle age
seems appropriate as this age group is most at risk for obesity.
Treat feeding is a source of calories and a known contributor to obesity in small animals. If animals are
fed a diet that satisfies them, or treats that satisfy their hunger, overeating will become less of an issue. In
cats and dogs, owners should provide a food with adequate protein for maintenance of lean body mass and
in the case of cats, enough fat to provide satiety. In dogs, higher protein levels will maintain body mass,
even during calorie restriction for weight loss. In cats, the author has had success in reducing body fat in
both diabetic and nondiabetic cats using a high protein, low carbohydrate canned formulations such Purina
DM® Diet (Nestlé Purina PetCare Company, St. Louis, MO).5
Just as in humans, portion size should be regulated. A true measuring cup of dry food is a small amount.
Clients should not use a jumbo sized cup or giant scoop to measure dry food amounts. Clients need to realize
how much food they are feeding— a “Big Gulp” cup contains about four actual cups of food. Portions may
be more accurately assessed using canned formulations. The expense of canned foods also helps limit over-
feeding and waste of excess food. In the author’s experience, cats are more satisfied with foods that are
higher in fat and protein and lower in carbohydrate content. This “satiety” factor seems to help limit
over-eating in this species.
The most obvious change that veterinarians can promote is a change in feeding regimens and the
easiest change to implement is to eliminate free-choice or ad libitum feeding. Ad libitum feeding (which is
not available in nature, by the way) has been shown to increase the incidence of osteoarthritis in young
growing dogs and increase obesity in cats.1,12 Veterinarians can now cite the Purina 448 study which showed
that ad libitum feeding has detrimental effects—namely, a decrease in life span!8

CONCLUSIONS

If you told your clients there was a way to prolong their dog or cat’s life by almost 20% would they listen
to you? The answer is a resounding “yes.” It is up to us, as veterinary professionals, to bring the enlightening
information of the Purina 448 study to the pet-owning public.

REFERENCES
1. Russell K, Sabin R, Holt S, et al. J Small Anim Pract. 2000;41:12–17.
2. Edny ATB, Smith PM. Study of obesity—The clinical nutritionist’s experience. Int J Obesity.
1994;18:S29–S35.
3. Agus MSD, Swain JF, Larson CL, et al. Dietary composition and physiologic adaptations to energy
restriction. Am J Clin Nutr. 2000;71:901–907.
4. Scarlett JM, Donoghue S, Saidla J, Wills J. Overweight cats: Prevalence and risk factors. Int J Obesity.
1994;212:1725–1731.

37 Carbohydrate Metabolism
 >>> GRECO

5. Mazzaferro E, Greco DS Turner AS. High protein diet and acarbose for the treatment of diabetes
mellitus in cats. J Vet Intern Med. May 2000;14:345.
6. Hannah SS, Laflamme DP. Increased dietary protein spares lean body mass during weight loss in dogs.
J Vet Intern Med. 1998;12:224.
7. Hannah SS, Laflamme DP. Increased dietary protein spares lean body mass during weight loss in cats.
Proc Br Small Anim Vet Assoc. Birmingham, England. 1998.
8. Kealy RD, Lawler DF, Ballam JM, et al. Effects of diet restriction on life span and age-related changes
in dogs. J Am Vet Med Assoc. 2002;220:1315–1320.
9. Laflamme DP. Development and validation of a body condition score system for dogs. Canine Pract.
1997;22:10–15.
10. Laflamme DP. Development and validation of a body condition score system for cats. Feline Pract.
1997;22:13–18.
11. Immam K, Sowers JR. Obesity and hypertension: A review. Henry Ford Hosp Med J. 1988;36:82–87.
12. Kealy RD, Olsson S-E, Monti KL, et al. Effects of limited food consumption on the incidence of hip
dysplasia in growing dogs. J Am Vet Med Assoc 1992;201:857–863.

38
■ THE AGING CARDIOVASCULAR SYSTEM:
ALTERATIONS INDUCED BY DIETARY RESTRICTION
Jeremiah T. Herlihy, PhD
Department of Physiology, University of Texas Health Science Center at San Antonio,
San Antonio, Texas

Physiologic deterioration and a rise in pathology of organs and organ systems Dr. Jeremiah Herlihy
characterize aging. Dietary restriction (DR) retards the age-related physiologic decline received his PhD in Physiology
from the University of Virginia.
and inhibits the appearance of pathology associated with aging. The task of this report
His predoctoral work involved
is to review the effects of DR on the aging cardiovascular system in all species other the mechanics of vascular
than the dog. At first sight, the scope of the review seems exceptionally broad. In reality, smooth muscle, followed by a
however, virtually all the available data are confined to the rat or mouse. These species postdoctoral fellowship in the
Department of Physiology at
have relatively short life spans, can be used in cross-sectional as well as longitudinal
the University of Virginia,
studies, and allow for invasive measurements. Studies utilizing these species are less focusing on the characterization
expensive to complete than those involving larger species such as humans or nonhuman of the contractile proteins of
primates. Moreover, food intake is more easily controlled in the rodent, thereby vascular smooth muscle and
the regulation of coronary
minimizing a large source of variability in the experimental conditions. Finally, because
blood flow. In 1975 he joined
of the size differential, the rat is preferred over the mouse when functional measure- the faculty in the Department
ments are made on the cardiovascular system. Therefore, with the exception of the of Physiology at the University
studies performed by Purina on dogs, most of the available data on the effects of DR of Texas Health Science Center
at San Antonio, where he is
on the aging cardiovascular system are confined to rats.
currently Associate Professor of
Physiology. His specific research
BODY WEIGHT/HEART WEIGHT
focus investigates the alter-
Heart weight increases with age in both ad libitum-fed (AL) and calorie-restricted (DR) ations of vascular smooth
rats.1,2 At all ages, the heart weights of the DR rats are less than those of the AL muscle sensitivity in response
to stretch and also aging and
animals.1,2 In contrast, the heart weight to body weight ratio of AL rats decreases from
calorie restriction. Current
6 to 12 months of age, while the ratio for DR hearts increases with age such that the interests also include ischemia-
ratio for the DR hearts is always higher than that of the AL rats (Table 1). This difference reperfusion insult and the
may indicate that the heart is spared the loss in mass with DR at the expense of other protective effects of sodium
nitroprusside in the isolated,
organs. The functional ramifications of the elevated heart weight/body weight ratio
perfused rat heart. For many
are not known. It would be interesting to determine whether the apparent cardiac years he served on the editorial
hypertrophy seen with DR parallels that seen in trained athletes where an elevated board of the American Journal
stroke volume preserves cardiac output at a lower heart rate. In fact, DR is associated of Physiology, Heart and
Circulation, and is currently
with lower heart rates in rats (see below). Not all studies report an increase in the
on the editorial board of
TABLE 1. Body Weight and Heart Weight Experimental Biology and
Medicine.
AGE BODY WEIGHT HEART WEIGHT HEART WEIGHT/BODY WEIGHT
(MONTHS) (g) (mg) (mg/g)
AL DR AL DR AL DR
6 342±8 224±2 895±17 659±9 2.62±0.03 2.94±0.06
12 485±20 273±2 1148±61 813±15 2.36±0.04 2.98±0.06
18 552±14 305±3 1314±24 938±31 2.39±0.06 3.07±0.10
24 530±11 321±3 1291±17 995±21 2.45±0.06 3.10±0.06
AL = fed ad libitum; DR = diet restricted.

39 Cardiology
{ } >>> HERLIHY

0.6
AL

(ml/min/g tissue/mmHg)
* DR

CONDUCTANCE
0.4

0.2

0.0
0 6 12 18 24 30
AGE (months)

FIGURE 1. Effects of age and DR on the coronary conductance of isolated hearts from Fischer
344 rats. Ad libitum–fed controls (AL) are denoted by open circles; dietary-restricted (DR) rats
are denoted by closed circles. Circles and bars represent the means ± SEM for 5 hearts in each
group. * Represents a significant (P < 0.05) difference from the AL group of the same age. Drawn
from data obtained from Reference 17.

heart weight/body weight ratio. Dietary restriction for shorter durations (5 to 6 weeks) does not elevate the
heart weight/body weight ratios for young adult, female, Sprague-Dawley rats, although the body weights
and left ventricular weights decreased upon DR.3–6

CARDIOMYOPATHY

The age-related increase in heart mass is often associated with the appearance of cardiomyopathy involving
loss of cardiocytes, hypertrophy of surviving cardiocytes, and infiltration of fibrotic tissue.7–11 Early studies
by Berg and Simms7,8 indicated that DR drastically reduced the age-related cardiomyopathy in Sprague-
Dawley rats. Similar protective effects of DR were observed in the Fischer 344 male rat subjected to 40%
DR,11 even when the DR was initiated during young adulthood.12 DR was also effective in ameliorating the
appearance of cardiomyopathy in the SHR and WKY rat models.13 This study is extremely interesting in that
DR exerted its anti-aging actions (increased life span, decreased incidence and severity of pathologic
lesions) without altering the rise in blood pressure seen in the control SHR rats! Thus, DR prevented
cardiac and renal degeneration that was associated with high blood pressure without affecting the blood
pressure itself.13 The mechanism by which DR bestows protection against end-organ damage in the face of
genetically determined pathology is not known. Given the clinical importance of hypertension in developed
countries, it would seem important to explore and expand the findings obtained in Lloyd’s study.13

CORONARY CIRCULATION

Concurrent alterations beset the coronary circulation where aging leads to increases in minimal coronary
vascular resistance and decreases in coronary flow reserve.9,10,14,15 These age-related functional changes in the
coronary circulation are thought to contribute to the structural and functional alterations observed in the
aging heart.14 In fact, Vandewoude and Buyssens15 suggested that vascular deficiency, ultimately leading to
ischemia and cell loss, might contribute to the physiologic deterioration seen in the aging heart. They also
noted15 that extreme malnutrition led to regression of age-related morphologic changes in the vasculature,
thereby offering some protection to the aging cardiocyte by enhancing metabolic exchange. A fortuitous
observation made in our laboratory was that isolated, perfused hearts from 14-month-old Fischer 344 male
rats subjected to 40% DR exhibited higher coronary conductance than control hearts.16 This observation was
extended throughout the life span as shown in Figure 1.17 Because the degree of coronary tone present in
the various age and diet groups during perfusion was not assessed, it is not certain whether the higher

40
T H E A G I N G C A R D I O VA S C U L A R S Y S T E M

0.5 8
ab
0.4
b
MDA PRODUCTION

6
(nmol/mg Pr/min)

(pmol/mg Pr/min)
DCF FORMATION
0.3 * 4
a

0.2

2
0.1

0.0 0
AL DR 6AL 6DR 24AL 24DR

FIGURE 2. Malondialdehyde (MDA) content FIGURE 3. Effects of age (6 and 24 months)

of cardiac mitochondria from 19-month-old
AL and DR hearts. Heights of the columns and
vertical bars represent the means ± SEM for
5 hearts in each group. * Represents a
significant (P < 0.05) difference from AL.
Redrawn from Reference 24.
and DR on the mitochondrial membrane
production of reactive oxygen species
(ROS) induced by tert-butyl hydroperoxide
addition. 2’,7’-dichlorofluorescein-diacetate
(DCF) is used as an index of ROS production.
Heights of columns and bars represent means
± SEM for 5 samples in each group. a and b
represent significant differences between the
means of the groups of the same diet (age
effect) and same age (diet effect), respectively.
Redrawn from Reference 25.

conductance seen with DR was due to morphologic changes in the vasculature or differences arising from
active changes in coronary tone. Nevertheless, the enhanced coronary conductance seen in DR hearts may
contribute to the lower occurrence of age-related cardiomyopathies, as suggested above,15 and this topic
represents a fruitful area for future research.

OXIDATIVE DAMAGE

The oxidative stress theory of aging proposes that the progressive deterioration and time- associated alter-
ations arising during aging are the cumulative result of incessant free radical generation occurring in the
course of normal cellular metabolism.18,19 Dietary restriction possesses a remarkable ability to reduce oxida-
tive damage20–23 and this protective attribute has been proposed as a possible mechanism by which DR exerts
its anti-aging action. We have examined the effects of DR on age-related changes in oxidative damage to
cardiac mitochondrial membranes and antioxidant defenses in cardiac muscle.24,25 In the earlier study,24 DR
decreased the malondialdehyde production rate of cardiac mitochondrial membranes indicating that the
degree of lipid peroxidation in these membranes was decreased (Figure 2). The later study25 confirmed the
protective effect of DR. Here, 2’,7’-dichlorofluorescein-diacetate (DCF) formation, an index of reactive oxygen
species production and an indicator of membrane lipid peroxidation, increased from 6 to 24 months of age
in the AL group (Figure 3). Even at 6 months of age, the DCF formation of the DR group was lower than
the AL group and, in fact, remained lower than the 6-month-old group even up to 24 months of age
(Figure 3). Measurement of the fatty acid composition indicated that membranes from control hearts
exhibited a decrease in the peroxidizability index at 24 months of age, whereas in the DR group no decline
in this index was observed over the same time period (Figure 4). In agreement with these results,
membrane fluidity decreased with age in the AL group, while DR restriction prevented the decline (Figure 5).

ANTIOXIDANT PROTECTION

Part of these protective effects of DR may arise from dietary-induced changes in the antioxidant status of
the myocardium.24 DR increased the activity of the following cardiac cytosolic enzymes (Figure 6): Cu/Zn

41 Cardiology
{ } >>> HERLIHY

180 4.0

PEROXIDIZABILITY INDEX

MEMBRANE FLUIDITY
3.5
150

(1/P)
120
3.0
*
2.5

90 2.0
6AL 6DR 24AL 24DR 6AL 6DR 24AL 24DR

FIGURE 4. Effects of age (6 and 24 months)
and DR on the peroxizability index of cardiac
mitochondrial membranes. Heights of the
columns and bars represent means ± SEM for
5 samples in each group. * represents a
significant difference between the
24-month-old AL group and all other groups.
Data from Reference 25.
FIGURE 5. Effects of age and DR on the
membrane fluidity of cardiac mitochondria.
Fluidity is expressed on the ordinate as the
inverse of membrane fluorescence polariza-
tion detected with 1-(4-trimethylammonium
phenyl)-6-phenyl-1,3,5 hexatriene. Heights
of the columns and bars represent means ±
SEM for 5 samples in each group. * represents
a significant difference between the
24-month-old AL group and all other groups.
Data from Reference 25.

superoxide dismutase (SOD), glutathione S-transferase (GST), and selenium-dependent glutathione peroxidase
(GSH). DR exerted no effect on catalase (CAT) in this study (Figure 6).

MYOSIN ISOZYMES

The aging myocardium exhibits a profound shift in the myosin isoenzyme profile from the fast isoform (V1)
to the slow isoform (V3).26 Work from our laboratory27 has confirmed this age-associated shift in the profile.
The V1 content decreased from a value of about 60% at 6 months of age to approximately 40% at 24
months of age (Figure 7A). Surprisingly, the effect of life-long DR was actually an enhancement, rather
than an inhibition, of the age-related decline in V1 isozyme content. At every age the V1 content of the DR
hearts was lower than that of the AL controls. Similar effects were observed with the slower V3 isozyme
(Figure 7B) where V3 increased with age in the AL group and DR increased the levels at all ages. The effect
of diet depended only upon calorie input and was unaffected by changes in carbohydrate ingestion.27
Interestingly, older AL rats (16 months) retained the ability to respond to DR by decreasing V1 and increasing
V3 isozyme contents.

40
*
30
ENZYME ACTIVITY

20
*
10
*
0
AL DR AL DR AL DR AL DR
CAT SOD GST GSH

FIGURE 6. Cardiac cytosolic antioxidant enzyme activity from 19-month-old AL and DR hearts.
CAT = catalase; SOD = superoxide dismutase; GST = glutathione S-transferase; GSH = selenium-
dependent glutathione peroxidase. Consult the original reference for actual enzyme activity
values. Heights of the columns and vertical bars represent the means ± SEM for 5 hearts in each
group. * represents a significant (P < 0.05) difference from AL. Redrawn from Reference 24.

42
T H E A G I N G C A R D I O VA S C U L A R S Y S T E M

80 AL Late DR 80 AL
V1 MYOSIN ISOZYME CONTENT

V1 MYOSIN ISOZYME CONTENT

DR Low Carbohydrate DR
60 60 Late DR
Low
(% Total)

(% Total)
Carbohydrate
40 40

20 20

0 0
0 6 12 18 24 0 6 12 18 24
Age (months) Age (months)

FIGURE 7. A, Effects of age and DR on the cardiac V1 myosin isozyme content expressed as a
percentage of total myosin content. Circles and vertical bars represent the means ± SEM for 10
hearts. Squares and vertical bars represent the means ± SEM for 5 hearts. At 16 months, AL rats
were subjected to DR (red squares). At 16 months, DR rats were subjected to low carbohydrate
feeding (blue squares). B, Effects of age and DR on the cardiac V3 myosin isozyme content
expressed as a percentage of total myosin content. Redrawn from Reference 27.

Dietary restriction applied for shorter periods elicits the same results. Dillmann et a128 and Haddad et al4
showed that a decrease in the Ca++-activated myosin ATPase accompanied the diet-induced decrease in
the V1 isozyme content. Baldwin’s group3–6 confirmed and extended these results to include decreases in
α-myosin heavy chain protein content and its mRNA.

CARDIAC MECHANICS

Transition from the fast V1 to the slower V3 isozyme profile is accompanied by a number of functional
alterations, including prolongation of both the rising29–31 as well as the falling30,31 phase of the cardiac twitch.
Aging itself is characterized by similar changes in the cardiac contraction cycle.31 The effects of long-term
DR (9 to 12 months) on the cardiac mechanics were investigated in the isolated perfused hearts of Fischer
344 male rats.32 Contraction times (time to peak pressure) and relaxation times (time to 1/2 relaxation) were
measured in control and DR hearts under conditions of high inotropic states induced by high pefusate calcium
and isoproterenol and under two intraventricular volumes. The results are shown in Table 2. Regardless of
the inotropic agent used (calcium or isoproterenol) or the intraventricular volume, DR hearts required a
longer time to reach maximum pressure. They also took a longer time to reach the 1/2 relaxation point. Just
as DR appeared to enhance the age-related change in the myosin isozyme profile (see earlier), so too with
contraction and relaxation times; DR accelerated the age-related increases in cardiac twitch contraction
and relaxation times.
Both developed pressure as well as diastolic pressure are affected by DR.32 At low calcium ion concentrations
DR hearts developed greater pressure than AL hearts. This difference disappeared at higher perfusate calcium
concentrations (Figure 8A). DR hearts also exhibited greater developed pressure than AL hearts when

TABLE 2. Effects of Dietary Restriction (DR) on Time to Peak Pressure (TPP) and
1/2 Relaxation Timea

Isoproterenol Calcium
Volume AL DR AL DR
Low 58.3±0.9 61.0±0.8b 62.9±0.5 65.1±0.3b
High 66.6±0.9 72.1±0.5b 73.9±0.3 75.4±0.3b
AL = fed ad libitum; DR = diet restricted.
a
Time in msec. bDifferent from its respective AL group in same volume and with the same inotropic agent.

43 Cardiology
{ } >>> HERLIHY

160 160
AL AL
DR DR *
*

PRESSURE (mmHg)

PRESSURE (mmHg)
120 120

* * * *
80 80

40 40

0 0
.85 1.0 1.5 3.0 0 1 3 10
[Ca] mM [Isoproterenol] nM

FIGURE 8. A, Effect of DR on the maximum developed pressure over a range of calcium ion con-
centrations in the perfusate. Heights of the columns and bars represent means ± SEM for 10 and
7 hearts from the AL and DR groups, respectively. * represents a significant (P < 0.05) difference
from the AL group. B, Effect of DR on the maximum developed pressure over a range of
isoproterenol concentrations in the perfusate. Heights of the columns and bars represent means
± SEM for 10 and 7 hearts from the AL and DR groups, respectively. * represents a
significant (P < 0.05) difference from the AL group. Redrawn from Reference 32.

stimulated with isoproterenol (1 to 10 nM) (Figure 8B). A decrease in diastolic pressure was observed during
inotropic stimulation as a result of elevated calcium in the pefusate (Figure 9A) or addition of isoproterenol
(Figure 9B) and DR enhanced the decline. These results indicate that the sensitivity of the heart to inotropic
agents, which is known to decrease with age, is enhanced with DR.

CARDIAC ENERGETICS

The mechanism by which DR exerts its anti-aging action is not known. One hypothesis is that it increases
the efficiency of energy utilization in one or more organs.33 Enhanced efficiency would be beneficial from
two points of view. First, it may lead to a lower metabolic rate for that organ or tissue and therefore lower
the levels of injurious metabolic end-products. Second, the energy saved with greater efficiency may be
utilized for greater repair and maintenance. We have examined the effects of DR on cardiac energetics to
determine whether the DR heart utilizes energy more efficiently than the AL control hearts.16,34 The energy
utilization by the heart can be divided into three basic components of approximately equal magnitude:
energy usage for mechanical activity, for activation (contractility), and for basal metabolism.

20 AL 20
AL
15
DR 15 DR
* *
PRESSURE (mmHg)

PRESSURE (mmHg)

10

5
10

5
*
0 0

-5 .85 1.0 1.5 3.0 -5 0 1 3 10

[Ca] mM [Isoproterenol] nM
-10 -10

FIGURE 9. A, Effect of DR on the end-diastolic pressure over a range of calcium ion

concentrations in the perfusate. Heights of the columns and bars represent means ± SEM for 10
and 7 hearts from the AL and DR groups, respectively. * represents a significant (P < 0.05)
difference from the AL group. B, Effect of DR on the end-diastolic pressure over a range of
isoproterenol concentrations in the perfusate. Heights of the columns and bars represent means
± SEM for 10 and 7 hearts from the AL and DR groups, respectively. * represents a
significant (P < 0.05) difference from the AL group. Redrawn from Reference 32.

44
T H E A G I N G C A R D I O VA S C U L A R S Y S T E M

6 6
V̇O2 OF TENSION DEVELOPMENT

V̇O2 OF TENSION DEVELOPMENT

AL DR AL DR
(nl 02/mmHg/g/beat)

(nl 02/mmHg/g/beat)
4 4

2 2

0 0
.85 1.0 1.5 3.0 .85 1.0 1.5 3.0
[Ca] mM [Isoproterenol] nM

FIGURE 10. A, Effect of DR on the oxygen cost of tension development over a range of
contractile states induced by different calcium ion concentrations. B, Effect of DR on the
oxygen cost of tension development over a range of contractile states induced by different
isoproterenol concentrations. In A and B, symbols are the same as those shown in Figure 9A.
Redrawn from Reference 16.

The oxygen cost of tension development represents the energy consumed for mechanical activity. The
greater the tension developed by the muscle, the greater the oxygen consumption. Over a wide range of
developed pressures, the oxygen consumption was unaffected by DR. Whether pressure was altered by
changes in external calcium (Figure 10A) or β-adrenergic stimulation (Figure 10B) no differences in
oxygen consumption could be detected between AL and DR hearts. Thus, DR did not affect the efficiency of
energy utilization for mechanical activity.
The oxygen cost of contractility represents the energy utilized for electrical and excitation-coupling
processes, the latter arising mainly from energy required for calcium pumping into the sarcoplasmic
reticulum. DR did not alter the oxygen cost of contractility when calcium was used as the inotropic agent
to change contractility (Figure 11). Interestingly, the DR heart exhibited a lower efficiency than AL hearts
when isoproterenol was used to change contractility. As shown in Figure 11, the oxygen consumption due
to isoproterenol-induced changes in contractility was greater for the DR hearts than that of the AL hearts.
Finally, the basal metabolic rate represents the oxygen consumption under conditions of zero tension
development and zero contractility. This rate can be measured directly when the external calcium is
completely removed from the perfusate. Figure 12 demonstrates that DR had no effect on the basal
metabolic rate of the heart when compared with that of the AL controls.
In summary, these studies provide no evidence that long-term DR enhances the energy efficiency of
the heart.

CARDIAC ADRENERGIC FUNCTION

The decline in β-adrenergic receptor responsiveness is a hallmark of the aging process. It has been
documented in most organs studied and the age-related decrease in cardiac responsiveness has been
observed consistently.35 Both short-term and long-term DR enhance the cardiac response to β-adrenergic
stimulation.2,32,36 The effects of DR on the β-adrenergic receptors and post-receptor activity have not
been explored.

CARDIAC SYMPATHETIC NERVES

The aging heart, like most aging organs, exhibits profound changes in its sympathetic innervation,37-39
including a loss in the norepinephrine content. An early study by McLean et al40 showed that the cardiac

45 Cardiology
{ } >>> HERLIHY

100 AL 0.08
*

V̇O2 TENSION DEVELOPMENT

DR

BASAL METABOLIC RATE

[nl 02/min/g (mmHg/sec)]
80
0.06

(ml O2/min/g)
60
0.04
40

0.02
20

0 0.00
CALCIUM ISOPROTERENOL AL DR

FIGURE 11. Effect of DR on the oxygen cost
of contractility induced by changing
perfusate calcium or isoproterenol concentra-
tions. Symbols are the same as those shown
in Figure 9A. Redrawn from Reference 16.
FIGURE 12. Effect of DR on the basal
metabolic rate of the heart. Symbols are the
same as those shown in Figure 9A. Redrawn
from Reference 16.

sympathetic axons degenerated with age and suggested that part of the decline arose from the loss of
sympathetic nerves to the heart. We also have noted a decline in cardiac norepinephrine content with age
and this decline is prevented by long-term DR (Figure 13). The mechanism by which DR prevents the
age-related decline in norepinephrine content is not known. It may be related, however, to preservation of
sympathetic axons or the lower sympathetic nervous system activity that occurs with DR (see below) may
also contribute to the elevated content. Roberts’ group41,42 has examined the effects of age and DR on the
handling of norepinephrine by cardiac synaptosomes. Norepinephrine release from as well as its uptake into
the synaptosomes decline as the heart ages and DR was shown to inhibit the aging effects. These results
agree with our findings,43 where DR of 4.5 months’ duration increased the norepinephrine uptake by
cardiac synaptosomes (Figure 14).

AUTONOMIC NERVOUS SYSTEM

It is widely believed that the activity of the sympathetic nervous system increases with age (for reviews see
References 37–39 and 44) and this belief has led to the idea that “old age may represent a hyperadrener-
gic state.”44 Short-term DR or fasting decreases sympathetic nervous system activity.45 The effects of long-
term DR on this activity have not been studied.
Very few data are available on the effects of DR on the parasympathetic innervation of the aging heart.
Results from our laboratory (see below) suggest that DR rats possess a higher resting parasympathetic tone
than AL controls.

HEART RATE

Resting heart rate is not markedly affected by age in humans46 or in rodents.38 Of the 34 studies reviewed
earlier38 over 70% reported that heart rate did not change substantially with age and the remaining 30%
observed a decrease. DR has been shown to consistently lower resting heart rate. The dietary-induced
decline in heart rate was noted in the earliest studies (McCay as quoted by Comfort47). In our own laboratory,48,49
DR rats exhibited significantly lower heart rates than AL rats at 7, 14, and 21 months of age (Figure 15).
Heart rate is regulated by both the sympathetic and parasympathetic components of the autonomic
nervous system. Removal of both components results in the intrinsic rate of the heart. Most studies report
that intrinsic heart rate either decreases or at least shows a tendency to decrease with age. Blockade of the

46
T H E A G I N G C A R D I O VA S C U L A R S Y S T E M

9 1.2
AL DR

* * * 0.9 *

(pmol/mg Pr/min)
[NE] (nmol/g)

NE UPTAKE
0.6

3
0.3

0 0.0
0 6 12 18 24 AL DR
Age (months)

FIGURE 13. Effects of age and DR on the nor-
epinephrine content of the heart. Circles and
bars represent the mean ± SEM for 10 hearts
in each group. * represents a significant
(P < 0.05) difference from the AL group at the
same age. Redrawn from Reference 54.
FIGURE 14. Effect of DR (4.5 months) on the
norepinephrine uptake of cardiac synapto-
somes. Heights of the columns and bars
represent the means ± SEM for 5 hearts in
each group. Redrawn from Reference 43.

parasympathetic nervous system with atropine results in a greater increase in heart rate in the DR group,
suggesting that the DR groups possess a higher parasympathetic tone than the AL group (Figure 16). Total
blockade of the autonomic nervous system with both atropine (parasympathetic) and propranolol
(sympathetic) decreased heart rate to a lower level in the AL group than the DR group, indicating that DR
may inhibit the age-related decline in intrinsic heart rate.

BLOOD PRESSURE

In humans, aging is associated with increasing blood pressure. In modern industrial societies, elevated
systolic, diastolic, and mean arterial pressures are observed50 and are associated with an increasing preva-
lence of hypertension. Comparative studies have shown, however, that minimal changes in diastolic and
mean pressures occur with age in rural subjects in contrast to their urban counterparts.26 This suggests that
the age-related changes occurring in diastolic and mean pressures are more related to environmental
factors, such as diet and stress, rather than to aging per se. The rise in systolic pressure is associated with
an age-related decline in arterial compliance. This stiffening of the arteries appears to be a primary aging
process and the consequent impedance mismatch contributes to the cardiomyopathies observed in the
aging heart.26

450
AL
DR
* * *
HEART RATE

400
(beats/min)

350

300
0 7 14 21
AGE (months)

FIGURE 15. Effects of age and DR on the resting heart rate of Fischer 344 rats. Red and bue
circles represent means for the AL and DR rats, respectively. Bars represent the SEM for 6 to 8
rats in each group. * indicates a significant (P < 0.05) difference between the means. Redrawn
from References 48 and 49.

47 Cardiology
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450 AL
DR
400

HEART RATE
(beats/min)
350

300

250
BASAL ATR ATR + PROP

FIGURE 16. Effect of DR (12.5 months) on the intrinsic heart rate of Fischer 344 rats. Heights
of the columns and bars represent means ± SEM for 3 rats in each group. * represents a
significant (P < 0.05) difference between the means of the two groups. ATR = atropine, PROP
= propranolol. Redrawn from Reference 37.

Dietary restriction leading to weight loss is considered a major nonpharmacologic therapy for controlling
high blood pressure, especially in overweight subjects. The decline in pressure with dietary restriction
appears to be independent of salt intake.51 In most studies weight loss accompanies DR and it is commonly
believed that DR exerts its hypotensive action through its effect on body weight rather than the effect of
energy restriction per se.
In laboratory animals, the data on the changes in blood pressure with age are inconclusive.38 In this
review, most studies examined reported no changes in pressure with age. Where changes did occur, the
method of blood pressure measurement appeared to qualitatively affect the outcome. For example, with the
tail cuff method, pressure increases were generally reported, whereas pressure decreases with age were
observed when measured by direct cannulation. In the Fischer 344 rat, Yu et al1 reported an increase in tail
cuff pressure between 9 and 19 months of age. In contrast, from the same rat colony we have observed48,49
a moderate, but progressive, decrease in mean arterial pressure during aging (Figure 17).
The data on the effects of DR on blood pressure in the rat are also inconclusive. Most studies report
that DR elicits either no change or a decrease in blood pressure. When DR exerted a hypotensive action, the
study usually utilized hypertensive rats and this observation is consistent with the observation from clinical
studies that the higher the initial pressure the greater the hypotensive action of DR. Very little change in
blood pressure is observed in normotensive rats subjected to DR.38 The longitudinal study of Yu et al1
observed no effect of DR on the tail cuff pressures measured throughout life. Our own data48,49 generally
confirmed these results except at 22 months where a slight decrease in mean pressure was observed in the
DR group (Figure 17).

BAROREFLEX

The arterial baroreflex is the major mechanism responsible for buffering acute changes in blood
pressure. In humans52 and laboratory rodents53 the responsiveness of the reflex declines with age. Very little
work has been done on the effects of DR on the age-related changes in baroreflex sensitivity. We have
examined the sensitivity of the cardiac component of the reflex48,49 by measuring the increase in heart rate
that results from hypotensive episodes induced by nitroprusside administration (Figure 18). A decrease in
sensitivity occurred with age in AL control rats. At every age, the sensitivity of the DR group was higher than

48
T H E A G I N G C A R D I O VA S C U L A R S Y S T E M

150
AL
MEAN ARTERIAL PRESSURE

DR
125

*
(mmHg)

100

75
0 7 14 21
AGE (months)

FIGURE 17. Effects of age and DR on the mean arterial pressure of Fischer 344 rats. Red and
blue circles represent means for the AL and DR animals, respectively. Bars
represent the SEM for 6 to 8 rats in each group. * indicates a significant (P < 0.05) difference
between the means. Redrawn from References 48 and 49.

that of the AL group. Interestingly, the sensitivity of the DR group declined with age, albeit at a higher
level (Figure 18).

SUMMARY

It is clear that DR alters many age-related changes that occur in the cardiovascular system. In some cases,
DR exerts an anti-aging action in that it reverses or at least inhibits age-related changes. In other cases, DR
seems to enhance the aging process in that it itself produces alterations that mimic the age-related
changes. Although these paradoxical effects make it difficult to erect a unified picture of cardiovascular
aging and the effect of DR, they do offer hints on future avenues of research that may prove fruitful in
unraveling the effect of age on the cardiovascular system.

ACKNOWLEDGMENTS

Dr. Herlihy’s work was supported by NIA Grant AG01188 and NIA Grant T32AG00205. The secretarial
assistance of Ms. Nancy Markham in the preparation of this manuscript is greatly appreciated.

5
AL
* *
BAROREFLEX SENSITIVITY

4 DR
(Beats/min/-mmHg)

3
*
2

0
0 7 14 21
AGE (months)

FIGURE 18. Effects of age and DR on the baroreflex sensitivity of Fischer 344 rats. Sensitivity is
defined as the increase in heart rate per decrease in mm Hg induced by nitroprusside adminis-
tration. Red and blue circles represent means for the AL and DR animals, respectively. Bars
represent the SEM for 6 to 8 rats in each group. * indicates a significant (P < 0.05) difference
between the means.Redrawn from References 48 and 49.

49 Cardiology
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Mech Ageing Dev. 1998;103:1–12
3. Swoap SJ, Haddad F, Bodell P, et al. Effect of chronic energy deprivation on cardiac thyroid
hormone receptor and myosin isoform expression. Am J Physiol. 1994;266:E254–E260.
4. Haddad F, Bodell PW, McCue SA, et al. Food restriction-induced transformations in cardiac
functional and biochemical properties in rats. J Appl Physiol. 1993;74:606–612.
5. Swoap SJ, Haddad F, Bodell P, et al. Control of beta-myosin heavy chain expression in systemic hyper-
tension and caloric restriction in the rat heart. Am J Physiol. 1995;269:C1025–C1033.
6. Swoap SJ, Boddell P, Baldwin KM. Interaction of hypertension and caloric restriction on cardiac mass
and isomyosin expression. Am J Physiol. 1995;268:R33–R39.
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J Am Coll Cardiol. 1986;8:1441–1448.
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12. Maeda H, Gleiser CA, Masoro EJ, et al. Nutritional influences on aging of Fischer 344 rats: II. Pathology.
J Gerontol. 1985;40:671–688.
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16. Klebanov S. Effects of Food Restriction on Cardiac Biochemistry, Mechanics and Energetics. PhD
Dissertation, San Antonio, University of Texas Health Science Center at San Antonio, 1996.
17. Kelley GR. Effects of Age and Diet on the β-Adrenergic Responsiveness of the Isolated Rat Heart.
MS Thesis, San Antonio, University of Texas Health Science Center at San Antonio, 1997.
18. Harman D. The aging process. Proc Natl Acad Sci USA. 1981;78:7124–7128.
19. Yu BP. Cellular defenses against damage from reactive oxygen species. Physiological Reviews.
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20. Enesco HE, Kruk P. Dietary restriction reduces fluorescent age pigment accumulation in mice. Exp
Gerontol. 1981;16:350–357.
21. Chipalkatti S, De AK, Aiyar SS. Effect of diet restriction on some biochemical parameters related to
aging in mice. J Nutr. 1983;113:940–944.
22. Koizumi A, Weindruch R, Walford RL. Influences of dietary restriction and age on liver enzyme
activities and lipid peroxidation in mice. J Nutr. 1987;117:361–367.
23. Yu BP. Aging and oxidative stress: Modulation by dietary restriction. Free Rad Biol Med.
1996;21:651–668.
24. Kim JD, Yu BP, McCarter RJ, et al. Exercise and diet modulate cardiac lipid peroxidation and antioxidant
defenses. Free Rad Biol Med. 1996;20:83–88.
25. Lee J, Yu BP, Herlihy JT. Modulation of cardiac mitochondrial membrane fluidity by age and calorie
restriction. Free Rad Biol Med. 26:260–265, 1999.

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26. Lakatta EG: Heart and Circulation, in Schneider EL, Rowe JW (eds): Handbook of the Biology of Aging,
ed 3. San Diego: Academic Press. 1990;181–216.
27. Klebanov S, Herlihy JT. Effect of life-long food restriction on cardiac myosin composition.
J Gerontol. 1997;52A:B184–B189.
28. Dillmann WH, Berry S, Alexander NM. A physiological dose of triiodothyronine normalizes
cardiac myosin adenosine triphosphatase activity and changes myosin isoenzyme distribution in
semistarved rats. Endocrinology. 1983;112:2081–2087.
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molecular characteristics. Am J Physiol. 1993;265:H183–H190.
30. Capasso JM, Malhotra A, Scheuer J, et al. Myocardial biochemical, contractile, and electrical
performance after imposition of hypertension in young and old rats. Circ Res. 1986;58:445–460.
31. Effron MB, Bhatnagar GM, Spurgeon HA, et al. Changes in myosin isoenzymes, ATPase activity, and
contraction duration in rat cardiac muscle with aging can be modulated by thyroxine. Circ Res. 1987;
60:238–245.
32. Klebanov S, Herlihy JT, Freeman GL. Effect of long-term food restriction on cardiac mechanics.
Am J Physiol. 1997;273:H2333–H2342.
33. Weindruch R, Walford R. The Retardation of Aging and Disease by Dietary Restriction. Springfield, IL:
Charles C Thomas. 1988.
34. Klebanov S, Freeman GL, Herlihy JT. Assessing effects of long-term food restriction on myocardial
energetics in isolated heart preparation. Mech Ageing Dev. In press.
35. Lakatta EG. Diminished beta-adrenergic modulation of cardiovascular function in advanced age.
Cardiology Clinics. 1986;4:185–200.
36. Herlihy JT. Dietary manipulation of cardiac and aortic smooth muscle reactivity to isoproterenol. Am
J Physiol. 1984;246:H369–H373.
37. Herlihy JT, Thomas JN. The aging of the cardiovascular system: modulation by dietary restriction. Age
& Nutrition. 1992;3:185–191.
38. Herlihy JT, Kim SW. Modulation of the aging cardiovascular system by dietary restriction. In: Yu BP
(ed): Modulation of Aging Processes by Dietary Restriction. Boca Raton, FL: CRC Press. 1994;57–88.
39. Young JB. Effects of aging on the sympathoadrenal system. In: Masoro EJ, Austad SN (eds): Handbook
of the Biology of Aging, ed 5. San Diego: Academic Press. 2001;269–296.
40. McLean MR, Goldberg PB, Roberts J. An ultrastructural study of the effects of age on sympathetic
innervation and atrial tissue in the rat. J Mol Cell Cardiol. 1983;15:75–92.
41. Snyder DL, Gayheart-Walsten PA, Rhie S, et al. Effect of age, gender, rat strain, and dietary restriction,
on norepinephrine release from cardiac synaptosomes. J Gerontol. 1998;53:B33–B41.
42. Snyder DL, Aloyo VJ, Wang W, et al. Influence of age and dietary restriction on norepinephrine uptake
into cardiac synaptosomes. J Cardiovasc Pharmacol. 1998;32:896–901.
43. Kim SW, Yu BP, Sanderford M, et al. Dietary restriction modulates the norepinephrine content and
uptake of the heart and cardiac synaptosomes. Proc Soc Exp Biol Med. 1994;207:43–47.
44. Rowe JW, Troen BR. Sympathetic nervous system and aging in man. Endocrine Rev. 1980; 1:167–179.
45. Landsberg L, Krieger DR. Obesity, metabolism, and the sympathetic nervous system. Am J Hypertens.
1989;2:125S–132S.
46. Fleg JL. Alterations in cardiovascular function with advancing age. Am J Cardiol. 1986; 57:33C–44C.
47. Comfort A. The Biology of Senescence, ed 3. Amsterdam: Elsevier/North Holland. 1979;186.
48. Thomas J, Bertrand H, Stacy C, et al. Long-term caloric restriction improves baroreflex sensitivity in
aging Fischer 344 rats. J Gerontol. 1993;48:B151–B155.
49. Herlihy JT, Stacy C, Bertrand HA. Long-term calorie restriction enhances baroreflex responsiveness in
Fischer 344 rats. Am J Physiol. 1992;263:H1021–H1025.
50. Kannel WB, Brand N, Skinner JJ Jr, et al. The relation of adiposity to blood pressure and
development of hypertension. Ann Intern Med. 1985;67:40–48.

51 Cardiology
 >>> HERLIHY

51. Reisin E. Weight reduction in the management of hypertension: epidemiologic and mechanistic
evidence. Can J Physiol Pharmacol. 1986;64:810–818.
52. Gribben B, Pickering TG, Sleight P, et al. Effect of age and high blood pressure on baroreflex
sensitivity in man. Circ Res. 1971;29:420–424.
53. Rothbaum DA, Shaw DJ, Angell CS, et al. Age differences in baroreceptor responses of rats.
J Gerontol. 1974;29:480–488.
54. Herlihy JT, Bertrand HA. Effects of age and caloric restriction on tissue catecholamine restriction. The
Gerontologist. 1991;31:287A.

52
■ ELECTROCARDIOGRAPHY IN A STUDY OF DIET RESTRICTION
AND AGING IN LABRADOR RETRIEVERS
Walter E. Weirich, DVM, PhD
Professor Emeritus, Surgery and Cardiology
School of Veterinary Medicine, West Lafayette, Indiana

ABSTRACT: Walter E. Weirich, DVM,

PhD, is Professor Emeritus of
The electrocardiogram (ECG) is the “gold standard” for the rhythm of the heart. Cardiology and Surgery at the
The length of the intervals between the waveforms and the shape, frequency, and School of Veterinary Medicine
duration of the waveforms reveal a great deal about the ability of the heart to deliver at Purdue University. Dr.
blood to the tissues of the body. While the ECG is the “accepted” measure of the Weirich is the past president of
the Academy of Veterinary
heart’s electrical activity, it can also provide information on the size and shape of the Cardiology, American Association
various heart chambers. Clinicians rely on the ECG to allow them to sort out arrhythmias of Veterinary Clinicians, the
that may be produced by the patient’s disease or may be side effects of the drugs they National Society of Phi Zeta,
have prescribed to treat the disease. Serial ECG tracings recorded over the course of a and the Veterinary Medical
Data Base. He was a co-founder
patient’s cardiac disease reveal changes that are helpful to the clinician in making of Vetronics, Inc. which developed
medication adjustments as well as providing prognostic information to the client. the ECG Analyzer, a computerized
ECG used routinely in this manner has provided a wealth of information about what system for recording and inter-
ECG changes are likely to occur in dogs as they age with an abnormal and/or preting the electrocardiograms
of dogs and cats. In addition to
compromised cardiovascular system. The Purina 448 study is the first time a group of cardiology and electrocardio-
normal dogs were followed routinely with ECG tracings as they aged through all graphy Dr. Weirich’s interests
stages of their lives. ECG tracings were done at the beginning of this study and at least include cardiopulmonary surgery
once each year until these animals died. The tracings of each of these animals were in domestic species and soft
tissue surgery of other
evaluated year to year with the changes noted over time for each animal. The tracings body systems.
were also compared within their sex and feeding trial groups. This 14-year study
provides ECG aging information in a controlled situation that heretofore has never
been reported. The ECG changes seen in these dogs, although often not outside what
is considered normal ranges for dogs, provide valuable insight on how the electrical
system within the heart changes over the life span of a Labrador retriever.

53 Cardiology
 ■ SYSTEMIC ARTERIAL BLOOD PRESSURE: THE SILENT KILLER
THAT NEVER SHOULD BE!
Robert L. Hamlin, DVM, PhD, Diplomate ACVIM
(Cardiology/Internal Medicine)
Stanton Youngberg Professor of Veterinary Physiology/Pharmacology, and
Professor of Biomedical Engineering, Ohio State University, Columbus, Ohio

Dr. Robert Hamlin received ABSTRACT:

his DVM and PhD degrees from
The prevalence, pathophysiology, and diagnosis of systemic arterial hypertension
The Ohio State University
(OSU). Dr. Hamlin is a Diplomate in dogs remain problematic. We assume that a blood pressure exceeding 165 mmHg
of American College of in systole or 135 mmHg in diastole is above normal. Most animals with hypertension
Veterinary Internal Medicine have abnormal renal function, but it is not known whether it is the cause or the
(Cardiology/Internal Medicine).
consequence of hypertension. Hyperadrenocorticism is a common cause of hypertension.
Dr. Hamlin is a Stanton
Youngberg Professor of Veterinary Hypertension results in renal (glomerular disease with proteinuria), ocular (retinal
Physiology/Pharmacology and hemorrhage, edema and detachment), and possible cardiac consequences (ventricular
Professor of Biomedical hypertrophy). Dogs fed a diet restricted in calories tend to have lower blood pressure,
Engineering at OSU. His clinical
lower pressure pulse, slower heart rates, and much lower rate-pressure products—a
interests are cardiopulmonary
medicine and physical exami- prime determinant of myocardial oxygen consumption. Caloric restriction also tends
nation, while his research interests to decrease pulse pressure, indicating a decrease in arterial impedance. Considering
include pathophysioloogy of extrapolations from experience with human hypertension and presuming that all dogs
heart failure and arrhythmias,
would respond as Labrador retrievers, caloric reduction in dogs and reduction in
comparative electrocardiography,
and drug-induced ventricular arterial pressure and arterial pulse pressure should translate into decreased morbidity
arrhythmias. Dr. Hamlin’s teach- and mortality.
ing responsibilities include
cardiovascular physiology, pul-
monary pathophysiology, elec-
trocardiography, noninvasive
diagnosis of cardiopulmonary
diseases, and diagnosis and
management of heart failure.
He has received the Mahanna
Award of the American Heart
Association, Career Development
Award of NIH, Robert Kirk
Award from ACVIM, National
and International Waltham
Awards, and Distinguished
Teaching Award at OSU. He has
written more than 250 scientific
publications and 12 textbook
chapters.

Cardiology 54
■ ESTIMATED BODY COMPOSITION VALUES OF CONTROL-FED VERSUS
RESTRICTED-FED LABRADOR RETRIEVERS IN A LIFE SPAN STUDY
Richard D. Kealy, PhD, Dennis F. Lawler, DVM, Brian T. Larson, PhD,
and Edward C. Hume, BS
Pet Nutrition Research Department, Nestlé Purina PetCare Company, St. Louis, Missouri

ABSTRACT: Dr. Richard D. Kealy is a

Purina Research Fellow at
A study was designed to evaluate the effect of diet restriction on body lean, fat,
Nestlé Purina PetCare Company,
and bone mass in Labrador retrievers throughout their life span. Eight-week-old pups where he has been an animal
were allotted to two treatment groups in a paired-feeding design with 24 pairs. One nutritionist for 35 years. His BS
pair-mate was control-fed and the other pair-mate received 25% less food on a daily degree was in Agriculture
Education at the University of
basis. Body lean, fat, and bone mass were collected by dual-energy x-ray absorptiometry
Nebraska, MS degree in Poultry
(DEXA) starting at 6 years of age. Pair-mates received the same nutritionally complete Nutrition at the University of
diet through their entire life span. Control-fed (CF) dogs received an average of 1,745 Nebraska, and he later com-
kcal metabolizable energy (ME) per day, whereas the restricted-fed (RF) dogs received pleted a PhD degree in Animal
Nutrition at the University of
an average of 1,352 kcal ME per day (P < 0.01) through 12 years. Caloric intake data
Arkansas. Dr. Kealy has served
after 12 years was erratic due to increased incidence of illness, especially in the CF as a member of the National
group. Mean body weight of RF dogs was, on average, 26% lower than the CF group Research Council panel on
(P < 0.01). Mean body condition score (range: 1 to 9, emaciated to grossly obese) feline nutrition. He currently
holds three patents, two that
taken during the 6- to 12-year period was 6.7 in the CF group as opposed to 4.6 in
relate to nutritional influences
the RF group. Mean lean body mass remained constant from 6 through 9 years of age on canine hip dysplasia. He has
for CF dogs and from 6 through 11 years of age for RF dogs. From 6 through 9 years authored or coauthored over
of age, mean lean body mass was significantly (P < 0.01) greater for CF dogs than for 800 studies at Purina on nutri-
tional needs of dogs and cats.
the RF group. A progressive decrease in lean body mass was detected among CF dogs
His expertise is in the area of
after 9 years of age, but a similar decrease was not detected among RF dogs until after orthopedic nutrition in the
11 years of age. Mean percentage lean body mass decreased significantly (P < 0.05) in canine and he has lectured
both groups from 6 through 12 years of age; however, the RF dogs always had a internationally and domestically
at veterinary conferences and
significantly (P < 0.01) greater mean percentage lean body mass. Mean absolute and
veterinary schools primarily on
percentage body fat mass increased significantly (P < 0.05) in both groups from this subject.
6 through 12 years of age. Body fat mass, expressed as an absolute (i.e., grams of fat
tissue) or as a percentage of body mass, always was significantly (P < 0.01) higher
among CF dogs than among RF dogs. Mean percentage body fat mass for the entire
period from 6 through 12 years of age was significantly (P < 0.01) higher for the CF
dogs (29.9%) than for the RF dogs (16.8%). Results for bone mass were similar to
results for lean body mass. Dogs in the CF group had significantly (P < 0.05) higher
bone mass than did RF dogs from 6 through 9 years of age. After 9 years of age, bone
mass among CF dogs decreased significantly (P < 0.05), whereas bone mass among RF
dogs remained constant. The decrease in lean body mass late in life (after 9 years
of age among CF dogs and after 11 years of age in RF dogs) might have been a
consequence of deteriorating physiologic function associated with aging or disease,
with delayed expression among RF dogs. The range in estimated body fat for the RF
group ranged from 10 to 22% and suggested that body fat less than 22% is a
reasonable goal for attaining maximum quality and quantity of life in dogs.

55 Body Composition

Dr. Joe Kemnitz received
his PhD from the University of
Wisconsin-Madison and joined
the staff of the Wisconsin
Regional Primate Research
Center (WRPRC) immediately
thereafter. His early work with
rhesus monkeys focused on
hypothalamic mechanisms for
the regulation of energy
balance and the influences of
gonadal hormones on food
intake and body composition.
He has also examined these
relationships in free-ranging
baboons and in zoo-housed
orangutans. Since 1989, he has
been characterizing the changes
that occur in middle age and
older adulthood of rhesus mon-
keys, and assessing the effects
of dietary restriction on aging
in this species. He was elected
Fellow in the Gerontological
Society of America in 1993,
and is an active member in
the American Society for
Nutritional Sciences, the
American Physiological Society,
the North American Association
for the Study of Obesity, the
Endocrine Society, and the
American Diabetes Association.
He is now Director of the
WRPRC and Professor in the
Department of Physiology and
the Interdepartmental Graduate
Program in Nutritional Sciences
at the University of Wisconsin-
Madison.
Body Composition 56
■ BODY COMPOSITION AND DIETARY RESTRICTION IN
RHESUS MACAQUES
Joseph W. Kemnitz, PhD, and Ricki J. Colman, PhD
Wisconsin Primate Research Center at the University of Wisconsin, Madison, Wisconsin
BODY COMPOSITION
Overview
Body composition is defined as the relative contributions of different components to
the whole body mass. Such components include, but are not limited to, fat, muscle, organ,
water, and bone. The definition of the components of body composition relies on the use
of body composition models. The classic two-compartment model of body composition
divides body weight into fat mass and fat-free mass. The direct measurement of fat
mass is a significant challenge for most body composition techniques. Given a
measurement of fat-free mass, however, fat mass can be estimated based on the two-
compartment model as the difference between body weight and fat-free mass. Three
methods traditionally exist for two-compartment body composition analysis:
> measurement of body density by underwater weighing,
> measurement of body cell mass by whole-body potassium counting, and
> measurement of total body water by isotope dilution.
As more measurement techniques were developed the basic two-compartment
model evolved into multicompartment models of body composition. Currently, a
comprehensive five-level, multicompartment system is used to systematically characterize
all of the major body-composition components. According to this system the more
than 30 major body composition components are organized into five levels: atomic,
molecular, cellular, tissue-system, and whole body. Each component is distinct, clearly
identified, and occupies only one level within the hierarchy.1,2
Importance
Body composition is an important variable of interest in many circumstances. By itself,
body composition is a measure of energy economy reflecting the difference between
energy intake and expenditure. A change in an individual component may be defined
as pathology (e.g., the loss of skeletal muscle mass suggesting sarcopenia, or the loss
of bone mineral indicating osteoporosis). In addition, body composition is often used
as a covariate for normalizing variables of interest for statistical analysis (e.g., the use
of lean body mass as a covariate in the analysis of metabolic rate).
Of major importance, body composition is independently an excellent marker of
health risk and disease. For example, increased body fat, especially increased visceral
adiposity, is related to cardiovascular risks such as hypertension and hyperlipidemia,
and to glucose regulation problems such as hyperglycemia, hyperinsulinemia, insulin
resistance, and diabetes. Therefore, the anatomic distribution of body fat is an
additional important factor for predicting morbidity and mortality.
BODY COMPOSITION AND DIETARY RESTRICTION IN RHESUS MACAQUES

Body composition is known to change as a function of age. Progressive loss of skeletal muscle mass is
a well-established characteristic of aging. This loss of muscle mass is associated with decreased strength
and increased physical frailty. With age, the ratio of fat tissue mass to lean tissue mass tends to increase
even with constant weight. Men undergo a progressive loss of lean body mass (primarily skeletal muscle)
and a steady gain of body fat. Early in the aging process there is often a net gain of body weight and a
considerable increase in whole body energy store because of the greater energy content of adipose tissue
compared with muscle. Similar changes in body composition occur for women with advancing age, but the
rate of change is slower than for men, and it begins later in life. These changes lead to an overall increase
in percent body fat with advancing age and a decrease in lean tissue and bone mass with age.
Measurement

All in vivo measurements of body composition are by nature indirect and are therefore approximations. The
only way to assess body composition directly is by dissection and chemical analysis. Although the results
can be very accurate, this approach can be done only once and as a terminal procedure.
The most basic, traditional in vivo assessments of body composition are somatometric indices (e.g., body
weight, height, skinfold thicknesses, body circumferences) and derived estimates (e.g., body mass index for
estimating body fat). These measurements are easy to acquire, cost effective, and require little specialized
equipment or training. Although such indices have been very useful, they can misrepresent the true body
composition (e.g., very high body mass index in body builders reflects increased muscle, not fat mass) and
can be difficult to translate to animal models.
Underwater weighing for the measurement of body volume was developed in the 1940s based on the
two-compartment model of body composition. Although still used as the standard in many laboratories, this
technique has several limitations. The major technical difficulty with this method is that the subject must
be completely submerged under water and air in the lungs must be quantified. A second major problem with
this technique is the assumption that the density of fat-free mass is constant, while in fact the density of
fat-free mass is known to vary with many factors including age, gender, and ethnicity.
Unlike the tissue density, the water content of fat-free mass is roughly constant (73.2%) in healthy
individuals. This provides the basis for the assessment of fat-free mass from total body water measurements.
This method involves the administration of a known dose of a stable isotope of water, a mixing period, and
then assessment of the tracer concentration in bodily fluids. The major limitation of this method is in the
cost of the isotope and spectroscopic analysis.
Bioelectrical impedance analysis is an alternative method of body water analysis based on the electrical
properties of tissues. This technique is based on the premise that when electrical current is passed through
the body, the voltage drop between the two electrodes is proportional to the body’s fluid volume in that
region. This method is inexpensive and easy to perform; however, its biological interpretation may be limited as
it is based on many, sometimes inaccurate, assumptions.
Similar to bioelectrical impedance analysis, total body electrical conductivity is based on the electrical
conductivity properties of tissues. Specifically, the subject is placed inside a large electromagnetic coil and
the amount of energy absorbed is recorded. The amount of energy absorbed is related to the quantity of
conducting material present in the tissue. This rarely used method suffers from the same limitations as
bioelectrical impedance analysis in addition to being costly.
More recently three imaging techniques have been applied to body composition analysis: computed
tomography (CT), magnetic resonance imaging (MRI), and dual-energy x-ray absorptiometry (DEXA). These

57 Body Composition
{ } >>> KEMNITZ

methods allow for relatively rapid, precise, and accurate multicompartment analysis of body composition.
Their limitations are cost of the equipment and the need for highly trained technical staff. In addition, CT
involves relatively high radiation exposure and entails risk for children, women of childbearing years, or use
in longitudinal studies. Despite its limitations, DEXA has become a well-established tool for the assessment
of soft tissue composition and bone mineral in humans and animals.

THE WISCONSIN DIETARY RESTRICTION STUDY

Dietary restriction (DR), or reduced caloric intake without malnutrition, is the only intervention that has
repeatedly and strikingly increased the maximum life span and retarded the rate of aging in laboratory
rodents. The ability of DR to increase life span has also been shown in fish, spiders, water fleas, and other
lower animals.3 Study into DR dates to the early 1900s. The first studies reported reduced tumor growth
under conditions of severe DR. These were followed by numerous studies documenting DR’s effectiveness
in reducing cancer and increasing life span in mice and rats. An ongoing phase of research, begun in 1970,
shows that rodents on DR stay younger longer. Another phase of DR research concerns the mechanisms by
which DR retards disease and aging in rodents. Finally, the prospective study of DR in primates has recent-
ly begun, with one such study underway at the Wisconsin Primate Research Center at the University of
Wisconsin-Madison (UW study)4,5 and another at the National Institute on Aging (NIA study).6 The UW Study
has three major aims:
> to advance the development of the rhesus monkey as a model for aging research,
> to determine the influence of DR on the rate of aging in this primate species, and finally
> to identify and validate biomarkers of aging in the rhesus monkey.
Approximately 165 rhesus monkeys, both males and females, are being used in the two primate DR
studies. To date, results from these two studies are remarkably similar even given differences in experimental
design. Two major differences between these two studies are in the age of the animals at the onset of DR
and the way in which the reduced diet was calculated. The NIA study began with animals in three age
groups: juvenile, adult and old. The juveniles were all clearly still growing and it is likely that the adult males
(3 to 5 years old at onset of DR) were still growing as well. The UW study began with all fully adult animals
(8 to 14 years of age at onset of DR). Because the NIA animals were still growing, total ad libitum dietary
intake was based upon National Research Council Guidelines, while for the UW study, individual baseline
intakes were determined for each animal. Both studies proceeded to impose a 30% restriction of ad libitum
intake on the subjects.
Specifically, the UW study began in 1989 with a group of 30 adult male rhesus macaques (Macaca
mulatta; Group 1). In 1994, a group of 30 adult females (Group 2) and an additional group of 16 adult males
(Group 3) were added. Within each group, half of the animals were randomly assigned to the control group
(allowed ad libitum access to food for 6 to 8 hours per day) and the remainder assigned to the DR group
(70% of individual ad libitum food intake).
Animals are fed a pelleted, semi-purified diet (Teklad, Madison, WI) which contains 15% lactalbumin,
10% corn oil, and approximately 65% carbohydrate in the form of sugars and corn starch. The macronutri-
ent content of the control and restricted diets is similar, but the restricted diet is supplemented with an
additional 30% of the vitamin and mineral content to assure that the groups, on average, consume a
similar amount of micronutrients. Food intake is measured daily for each animal.
In addition to daily food intake measurements, animals are weighed weekly, assessed for body compo-
sition and overall health semi-annually, and studied in a six-week battery of specialized tests yearly. Body

58
BODY COMPOSITION AND DIETARY RESTRICTION IN RHESUS MACAQUES

composition is measured by somatometric assessment (body lengths, trunk and limb circumferences,
skinfold thicknesses) and DEXA (DPX-L, GE/Lunar, Madison, WI). Overall health checks include, among other
measurements, serum chemistries and complete blood counts, blood pressure, physical examination, and
dental examination. During the six-week annual assessment period, additional measurements include an
electrocardiogram and echocardiographic examination of cardiac function, radiographic examination for
osteoarthritis, energy expenditure by indirect calorimetry methods, physical activity, as well as glucose
tolerance and insulin sensitivity testing. In addition, serum, plasma, and urine samples are banked twice
yearly for assessment of among other analytes, reproductive hormones, lipid profiles, and biochemical
markers of bone metabolism and skeletal relevance.
As predicted from rodent studies, monkeys on DR show rapid alterations in body composition. Body
weight is significantly lower in restricted compared with control monkeys in all three groups. The DEXA data
suggest (and are confirmed by somatometrics) that the difference in body weight between control and
restricted animals is primarily attributable to differences in body fat.7, 8 Restricted animals have significantly less
body fat compared with controls at all time points following restriction and a difference is seen between
groups in longitudinal comparisons of rate of change in body fat over time. In all animals, abdominal fat mass
accounted for ~50% of the difference in total body fat mass between groups. This difference in abdominal
fat mass is clear in several somatometric variables as well.7,8 In addition, in Group 1, there has been an
emerging difference in fat distribution between control and restricted animals in which the restricted group
has a lower percent abdominal fat compared with controls.7 In light of the health problems associated with
abdominal fat, this reduction can be seen as a positive health benefit of DR. In the near future we plan to
begin acquiring CT images of the abdomen and mid-thigh for more direct measurement of visceral fat mass
and fat content of skeletal muscle, respectively. In Group 1 animals show an additional treatment difference
in lean body mass due primarily to an increase in the control group.7,9 In contrast to Group 1, in Groups 2
and 3 lean body mass differences to this point have been very slight. In all groups, DEXA-measured bone
mass is lower in DR compared with control animals. We believe this lower bone mass is expected as a direct
consequence of lower body weight, i.e., less mechanical load.

SUMMARY

Body composition is of interest for many reasons including its important direct relationship with overall
health. Many techniques have been established to assess body composition based upon both the original
basic two-compartment model as well as the more complex multicompartment models. Our study of DR in
rhesus macaques has shown that an approximately 30% DR can be safely maintained long-term in both
male and female rhesus macaques, and that restricted animals have favorable changes in body composition
and improved health compared with controls.

REFERENCES
1. Wang Z, Wang ZM, Heymsfield SB. History of the study of human body composition: A brief review.
Am J Human Biol. 1999;11:157–165.
2. Pietrobelli A, Heymsfield SB, Wang ZM, et al. Multi-compartment body composition models: Recent
advances and future directions. Eur J Clin Nutr. 2001; 55: 69–75.
3. Weindruch R, Walford RL. The Retardation of Aging and Disease by Dietary Restriction. Springfield,
IL: Charles C Thomas. 1988.
4. Kemnitz JW, Weindruch R, Roecker EB, et al. Dietary restriction of adult male rhesus monkeys: Design,
methodology, and preliminary findings from the first year of study. Journal of Gerontology: Biological
Sciences. 1993; 48: B17–B26.

59 Body Composition
 >>> KEMNITZ

5. Ramsey JJ, Colman RJ, Binkley NC, et al. Dietary restriction and aging in rhesus monkeys: The
University of Wisconsin study. Experimental Gerontology. 2000; 35: 1131–1149.
6. Roth GS, Ingram DK, Cutler RG, et al. Biological basis of lifespan modulation by nutrition: The NIA pri-
mate study, in Dall JLC (Ed). Adaptations in Aging, vol. 24, The 1988 Sandoz Lectures in Gerontology.
London/San Diego: Academic Press. 1995.
7. Colman RJ, Ramsey JJ, Roecker EB, et al. Body fat distribution with long-term dietary restriction in
adult male rhesus macaques. Journal of Gerontology: Biological Sciences. 1999; 54A:B283–290.
8. Colman RJ, Roecker EB, Ramsey JJ, et al. The effect of dietary restriction on body composition in adult
male and female rhesus macaques. Aging: Clinical and Experimental Research. 1998;10:83–92.
9. Gresl TA, Colman RJ, Roecker EB, et al. Dietary restriction and glucose regulation in aging rhesus mon-
keys: A follow-up report at 8.5 years. Am J Physiol Endocrinol Metabol. 2001;281:E757–765.

60
■ CONTROLLING BODY CONDITION: WHY, WHEN AND HOW?
D. P. Laflamme, DVM, PhD, Diplomate ACVN
Nestlé Purina PetCare Company, St. Louis, Missouri

Food restriction is the only nutritional intervention that has been shown effective Dr. Dottie Laflamme is a
in delaying morbidity and mortality. This has been documented in numerous species, Research Fellow at Nestlé
Purina PetCare Company. She
now including dogs.1–5 One might question whether it is food restriction that is the
earned an MS degree in rumi-
dietary intervention, or if overfeeding is the dietary intervention. After all, obesity is nant nutrition, a DVM degree,
a disease of domestication. Abundant availability of food and reduced activity are and a PhD specializing in
hallmarks of modern life and are risk factors for obesity. Rodents kept in cages and canine nutrition, all from the
University of Georgia. She was
provided with food ad libitum, which is typical of “control” groups in food-restriction
selected in 1988 to receive the
studies, hardly reflect rats or mice in a “natural” environment. Nevertheless, it is not ALPO Postdoctoral Fellowship in
unlike life for humans and pets in developed countries. While the semantics and Clinical Nutrition. Dr. Laflamme
implications of this point could be argued several ways, the present discussion begins is a Diplomate and Past-
President of the American
with the acceptance that lean body condition promotes longevity whereas excess
College of Veterinary Nutrition.
body fat is associated with enhanced morbidity and reduced longevity. She is the author of more than
100 papers, abstracts, and text-
WHEN SHOULD CALORIE RESTRICTION BE PRACTICED? book chapters; and has been a
According to the recent canine findings, even a mild degree of excess body fat can be speaker at a number of veteri-
nary, research, and continuing
a risk factor for disease and early mortality.2 The quantitative benefit of calorie restriction,
education programs worldwide.
e.g., enhanced life span, is greatest when started early in life,3 but calorie restriction Her recent research has focused
initiated in adulthood provides significant benefits as well.3,6,7 Similar controlled studies on therapeutic nutrition and
initiated in adulthood are not available for dogs, but the evidence that does exist obesity management.
suggests a similar pattern. Dogs that were obese at one year of age had a greater risk
for developing mammary cancer later in life.8,9 Puppies overfed during growth have a
greater likelihood of developing hip dysplasia or other orthopedic diseases.10,11
Restricting calories, along with limited exercise, to achieve weight loss in overweight,
lame dogs resulted in enhanced mobility.12 Thus, while benefits can be derived from
controlling body condition throughout life, it is believed that achieving and
maintaining lean body condition can be of benefit at any time.

WHEN SHOULD CALORIE RESTRICTION BE AVOIDED?

The studies that have demonstrated a health benefit to calorie restriction mostly
involved select strains of rodents. The sole canine study involved a single breed of dog,
the Labrador retriever, which is an obese-prone breed. While most rodent studies have
shown beneficial results from calorie restriction, at least one study showed a
difference based on genetic strain. Mean and maximum life spans were extended by
food restriction in male B6CBAF1 hybrid mice and in genetically obese (ob/ob) B6
mice, but the exact opposite effect was observed when male B6(C57BL/6J) mice were
food restricted.1 Whether or not such strain or breed differences may be observed in
other species remains to be seen.

61 Body Composition
{ } >>> LAFLAMME

While postweaning calorie restriction generally provides health benefits, preweaning and in utero
nutrient restriction appears to have the opposite effect. There is now considerable evidence that nutritional
restriction during reproduction can induce health risks in the offspring that may not be evident until
months to years later.5 Human infants and rodents that are calorie or protein restricted in utero or as
neonates experience shortened life spans13 or have increased risk for developing diabetes mellitus or
cardiovascular disease later in life.14,15 Puppies whose dams were protein restricted during gestation showed
delayed evidence of compromised immune function during adolescence.16 In separate research with mice it
was documented that calorie restriction enhanced, but protein restriction compromised, survival.17
From these studies, it can be seen that calorie restriction should not extend to restriction of other
nutrients, and that calorie restriction should not be practiced during reproduction or before weaning.
Whether this is appropriate for all breeds of dogs remains to be seen, yet obesity appears to be a major
health risk in all breeds and species evaluated to date. Thus, restriction of calories to minimize obesity seems
a reasonable goal.

HOW SHOULD CALORIE RESTRICTION BE APPLIED TO PET DOGS?

One of the issues with applying calorie restriction outside the research laboratory is the question, “restric-
tion from what?”.
The maintenance energy requirements (MER) of individual dogs vary greatly. Even among adult dogs of
similar breed, age, gender, activity level, and body condition, MER can vary by 50% or more. Differences
among breeds can further increase this variation. In addition, total energy needs of healthy dogs can be
increased by activity, reproduction, growth, and cold environments or decreased by neutering and age.
Further complicating the issue is the physiologic response to varying energy intake: basal metabolic rate,
heat increment, and intestinal nutrient uptake associated with food consumption will increase or decrease
in most species in response to excess or restricted energy intake.18–20
Thus, for practical purposes, one cannot simply apply “calorie restriction” for individual dogs. Rather,
one must feed individual dogs to whatever calorie intake is needed to maintain an ideal body condition. This
may be accomplished by feeding initially using a calculated estimate of average MER (e.g., metabolizable
energy/kg body weight = 110 · kg 0.75), and then adjusting based on the dog’s response to that intake.

WHAT CAN VETERINARIANS DO TO PROMOTE LEAN BODY CONDITION IN PETS?

Dogs with an ideal body condition score (BCS) between 4 and 5 (using the Purina 9-point BCS system) lived
15% longer than dogs with a BCS between 6 and 7.2 Currently, at least one in four dogs and cats seen by
practitioners are overweight or obese.21 Yet many pet owners do not realize their pets are overweight or at
risk for health problems. In a recent survey involving 200 dogs and their owners, it was demonstrated that
owners do not recognize their own dog as overweight.22 In that survey, the mean BCS determined by trained
pet experts was 6.3 while the mean BCS determined by the dog owners was 5.3. About 27% of the owners
underestimated BCS by 2 units: 2 units on this BCS system correlates to 20% to 30% excess body weight.23
In other research, it was shown that the primary way that dog owners recognized their pet as overweight
was based on their veterinarian’s assessment.24
Thus, veterinarians should begin or continue to evaluate BCS on all patients, and to discuss with clients
the importance of maintaining an ideal BCS. Veterinarians may want to provide illustrated BCS charts for
their clients, or to post them within their clinic. A videotape teaching pet owners how to monitor and
control BCS can be a valuable client education tool that practitioners may wish to provide.

62
C O N T R O L L I N G B O D Y C O N D I T I O N : W H Y, W H E N A N D H O W ?

Puppy owners should be taught how to assess BCS in their puppies, and advised to adjust food
allowances to maintain a lean body condition while promoting a slow, healthy rate of weight gain.
Neutering in both sexes is associated with a reduction in energy requirements. All pet owners should be
advised to alter the feeding management of their pet following spay or castration.
To assure that all essential nutrients are provided despite energy control, it is important that the caloric
density of the diet fed be appropriate to the energy needs of the individual pet. Those dogs with very low
energy requirements should be fed products with an enhanced nutrient:calorie ratio, such as properly
formulated “lite” or weight management diets. Dogs with high energy needs may benefit from performance
or high calorie foods. Consideration must be given to calories provided from sources other than complete
and balanced pet foods, to reduce the risk of nutrient dilution as well as calorie excess.

WHAT FURTHER RESEARCH IS NEEDED?

One of the questions that remains to be answered involves the relative roles of lean body mass (LBM) compared
with fat mass. Does increasing LBM provide incremental health benefits? In the canine food restriction
study, LBM decreased later in the food-restricted group coincident with delayed morbidity; and the decrease
in LBM appeared to precede an increase in morbidity in both groups of dogs. It is not currently known
whether the decrease in LBM is a marker of subclinical disease, a contributor to disease, or an unrelated
event. Assuming a causal relationship, can disease be delayed further by nutritional enhancements to LBM, such
as increased dietary protein and metabolically active amino acids? These areas require further investigation.
Do the canine, rodent, and primate findings apply to cats as well? Calorie restriction studies have not
been completed in cats. However, studies have shown similar associations between excess body condition
and increased morbidity in cats. Scarlett showed that obesity in middle-aged cats was associated with early
mortality.26 Overweight cats also demonstrate compromised insulin sensitivity and have a fourfold increased
risk for developing diabetes mellitus.25–27 Pending further research findings, it seems reasonable to feed
cats to maintain a lean, healthy body condition. For cats of any age, a BCS of 5 (of 9) appears to be an
appropriate target.

REFERENCES
1. Harrison DE, Archer JR. Genetic differences in effects of food restriction on aging in mice. J Nutr.
1987; 117:376–382.
2. Kealy RD, Lawler DF, Ballam JM, et al. Effects of diet restriction on life span and age-related changes
in dogs. J Am Vet Med Assoc. 2002;220:1315–1320.
3. Maeda H, Gleiser CA, Masoro EJ, et al. Nutritional influences on aging of Fischer 344 rats. II. Pathology.
J Gerontol. 1985;40:671–688.
4. Masoro EJ, Shimokawa I, Yu BP. Retardation of the aging process in rats by food restriction. Ann NY
Acad Sci. 1991;621:337–352.
5. Sayer A, Cooper C. Getting started right: The influence of early growth and nutrition on aging.
Compend Contin Educ Pract Vet. 2002;24(Suppl):4–7
6. Goto S, Takahashi R, Araki S, Nakamoto H. Dietary restriction initiated in late adulthood can reverse
age-related alterations of protein and protein metabolism. Ann NY Acad Sci. 2002;959:50–56.
7. Lane MA, Tilmont EM, DeAngelis H, et al. Short-term calorie restriction improves disease-related
markers in older male rhesus monkeys (Macaca mulatta). Mech Aging Dev. 2000;112:185–196.
8. Perez-Alenza MD, Pena L, Del Castillo N, Nieto AI. Factors influencing the incidence and prognosis of
canine mammary tumors. J Small Anim Pract. 2000;41:287–291.
9. Sonnenschein E, Glickman L, McKee L, Goodschmidt M. Nutritional risk factors for spontaneous breast
cancer in pet dogs: A case-controlled study. Am J Epidemiol. 1987;126:736

63 Body Composition
 >>> LAFLAMME

10. Kealy RD, Olsson SE, Monti KL, et al. Effects of limited food consumption on the incidence of hip
dysplasia in growing dogs. J Am Vet Med Assoc. 1992;201:857–863.
11. Meyer H, Zentek J. Energy requirements of growing Great Danes. J Nutr. 1991;121:S35–36.
12. Burkholder WJ, Taylor L, Hulse DA. Weight loss to optimal body condition increases ground reactive
force in dogs with osteoarthritis. Compend Contin Educ Pract Vet. 2001;23(Suppl):74.
13. Sayer A, Dunn R, Langley-Evans S, Cooper C. Prenatal exposure to a maternal low protein diet shortens
life span in rats. Gerontology. 2001;47:9–14
14. Ozanne SE, Hales CN. The long-term consequences of intra-uterine protein malnutrition for glucose
metabolism. Proc Nutr Soc. 1999;58:615–619.
15. Stein CE, Fall CH, Kumaran K, et al. Fetal growth and coronary heart disease in south India. Lancet.
1996;348:1269–1273.
16. Newberne PM. The influence of nutrition response to infectious disease. Adv Vet Sci Comp Med.
1973;17:265–289.
17. Peck MD, Babcock GF, Alexander JW. The role of protein and calorie restriction in outcome from
Salmonella infection in mice. J Parenter Enter Nutr. 1992;16:561–565.
18. Ferraris RP, Cao Q, Prabhakaram S. Chronic but not acute energy restriction increases intestinal
nutrient transport in mice. J Nutr. 2001;131:779–786.
19. Pouteau EB, Mariot SM, Martin LJ, et al. Rate of carbon dioxide production and energy expenditure
in fed and food-deprived adult dogs determined by indirect calorimetry and isotopic methods. Am J
Vet Res. 2002;63:111–118.
20. Weyer C, Walford RL, Harper IT, et al. Energy metabolism after 2 years of energy restriction: The
Biosphere 2 experiment. Am J Clin Nutr. 2000;72:946–953.
21. Armstrong PJ, Lund EM. Changes in body composition and energy balance with aging. Vet Clin Nutr.
1996;3:83–87.
22. Singh R, Laflamme DP, Sidebottom-Nielsen M. Owner perceptions of canine body condition score.
J Vet Intern Med. 2002;16:362
23. Laflamme DP. Development and validation of a body condition score system for dogs: A clinical tool.
Canine Pract. 1997;22:10–15.
24. Jackson M, Ballam JM, Laflamme DP. Client perceptions and canine weight loss. Compend Contin
Educ Pract Vet. 2001;23(Suppl):90.
25. Scarlett JM, Donoghue S. Associations between body condition and disease in cats. J Am Vet Med
Assoc. 1998;212:1725–1731.
26. Appleton DJ, Rand JS, Sunvold GD. Insulin sensitivity decreases with obesity, and lean cats with low
insulin sensitivity are at greatest risk of glucose intolerance with weight gain. J Feline Med Surg.
2001;3:211–228.
27. Fettman MJ, Stanton CA, Banks LL, et al. Effects of weight gain and loss on metabolic rate, glucose
tolerance, and serum lipids in domestic cats. Res Vet Sci. 1998;64:11–16.

64
■ BRIEF CALORIE RESTRICTION LEADS TO ENHANCED INSULIN
SIGNALING IN SKELETAL MUSCLE
Gregory D. Cartee, PhD, Carrie E. McCurdy, BS, Robert T. Davidson, PhD,
and Edward B. Arias, PhD
Biodynamics Laboratory, Department of Kinesiology, and Department of Nutritional Sciences,
University of Wisconsin-Madison, Madison, Wisconsin

Improved insulin sensitivity is a hallmark of a moderate calorie restriction

(CR; 20% to 40% below ad libitum, AL) in many species, including mice, rats,
nonhuman primates, and humans. In rat skeletal muscle, we have found that the
effect of CR on glucose transport is specific to the insulin-stimulated pathway
(i.e., neither basal glucose transport nor activation by in vitro hypoxia, an insulin-
independent stimulus, is increased by CR). The increased insulin-stimulated glucose
transport is attributable to an enhanced recruitment of the GLUT4 glucose transporter
to the cell surface, without any increase in total GLUT4 abundance. In this context, we
suspect that CR enhances insulin action secondary to an amplification of the insulin
signaling pathway. Activation of phosphatidylinositol 3-kinase (PI3K) is essential for
insulin-stimulated glucose transport under normal conditions, but we have found that
brief CR (20 days) results in no significant change in insulin-stimulation of insulin
receptor substrate1 (IRS-1)-, IRS-2-, or phosphotyrosine-PI3K compared with AL
controls. However, by incubating muscles with PI3K inhibitor (wortmannin) we were
able to completely eliminate the CR-induced increase in insulin-stimulated glucose
transport. These findings led us to assess a key post-PI3K step in insulin signaling: Akt
(also called protein kinase B). We found that CR did not alter abundance of the
predominant isoforms expressed by skeletal muscle (Akt1 and Akt2). However, insulin
stimulation of Akt phosphorylation was significantly higher in muscles from CR
versus AL animals. In conclusion, these findings indicate that brief CR can enhance
insulin action in skeletal muscle by a wortmannin-inhibitable and presumably
PI3K-dependent mechanism which likely involves enhanced phosphorylation of Akt.

65 Posters
 ■ CALORIE RESTRICTION PROVIDES INSIGHTS INTO INSULIN
SIGNALING IN SKELETAL MUSCLE
Heidi K. Ortmeyer, PhD, Noni L. Bodkin, PhD, and Barbara C. Hansen, PhD
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland

As rhesus monkeys age, a large number of them become obese and develop
type 2 diabetes. We have shown that there is a step-wise decrease in insulin sensitivity
(as assessed by whole-body insulin-mediated glucose disposal rates) as monkeys
progress in time from young/normal to older/obese/hyperinsulinemic to
old/obese/type 2 diabetic. In these monkeys, insulin sensitivity is significantly
positively correlated to in vivo insulin action on skeletal muscle enzyme activity
including (1) insulin receptor substrate-1 (IRS-1)–dependent phosphatidylinositol
3-kinase (PI3K) activity, (2) atypical (z/l/t) protein kinase C (PKC) activity, and (3) glycogen
synthase fractional activity. Prevention of obesity by caloric restriction prevents the
development of insulin resistance; calorie-restricted monkeys, although similar in age
to the old type 2 diabetic monkeys, have normal insulin action to increase whole-body
glucose disposal, normal IRS-1–dependent PI3K activity, and normal atypical PKC
activity during a euglucemic hyperinsulinemic clamp. These findings suggest that
decreased glucose disposal rate, and concomitantly decreased ability of in vivo insulin
to activate IRS-1–dependent-PI3K and atypical PKC activity, are secondary to obesity.
Interestingly, calorie-restricted monkeys have significantly higher basal skeletal
muscle glycogen synthase activity compared with any of the ad libitum-fed groups.
These findings suggest that protein kinase B (PKB) is not involved in the regulation of
glycogen synthase in rhesus monkeys. In addition, calorie-restricted monkeys do not
have normal insulin activation of skeletal muscle glycogen synthase during a
euglycemic hyperinsulinemic clamp. Thus a defect in insulin activation of glycogen
synthase is not likely to be secondary to obesity. We conclude that examining the
skeletal muscle of calorie-restricted primates will provide invaluable information on
insulin signaling pathways and the mechanisms by which calorie restriction improves
insulin sensitivity.

Posters 66
■ EFFECTS OF CALORIC RESTRICTION IN LONG-LIVED, GROWTH
HORMONE (GH)–-RESISTANT GHR/GHBP-KNOCKOUT (KO) MICE
Michael Bonkowski, MS, Khalid Al-Regaiey, DVM, and Andrzej Bartke, PhD
Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois

Caloric restriction (CR) in laboratory mice significantly increases life span and
prevents age-associated disease. GHR/GHBP-KO, “Laron” mice live significantly longer
than normal (N) animals, but the mechanism that is responsible for delayed aging is
unknown. We are studying the effects of CR on longevity of GHR-KO mice. Thirty
percent CR was initiated at the age of approximately 2 months and caused the
expected reduction in body weight (BW). In both N and KO animals food consumption
expressed in terms of metabolic BW showed that CR significantly increased the
efficiency of food utilization. At nine months of age plasma glucose levels were
reduced by CR in both N and KO mice with a significant interaction between
genotype and treatment (P < 0.0003). Insulin levels were also significantly reduced by
CR in both N and KO animals with a significant interaction between genotype and
treatment (P < 0.0001). As in previous studies, plasma insulin levels in KO mice were
markedly reduced in comparison with levels measured in N animals (0.128 ± 0.013 vs.
0.225 ± 0.028 ng/mL). Insulin levels in N-CR and KO ad libitum (AL) groups were
indistinguishable (0.122 ± 0.019 vs. 0.128 ± 0.013 ng/mL). Plasma corticosterone
levels were significantly elevated by CR in N mice, but not affected in KO animals. The
results allow for comparison of the effects of CR in GHR-KO animals which are
GH-resistant, insulin-like growth factor-1 (IGF-1)-deficient, hypoinsulinemic, and
long-lived with the effects of the same treatment in normal mice from the same line.
In both N and KO animals, CR improved feed efficiency and reduced plasma glucose
and insulin levels. However, CR failed to alter plasma corticosterone levels in KO mice.
It remains to be determined whether CR will affect longevity of GHR-KO animals.

Supported by the Illinois CFAR and NIH (AG19899).

67 Poster
 ■ AGE-RELATED CHANGES IN CANINE CD8 MEMORY CELLS: A
LONGITUDINAL AGING/DIETARY RESTRICTION STUDY
Elizabeth H. Greeley, PhD,1 Joan M. Ballam, MS,2 Jay M. Harrison, MS, MA,2
Richard D. Kealy, PhD,2 Dennis F. Lawler, DVM,2 and Mariangela Segre, DSc1
1
Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois;
2
Nestlé Purina PetCare Company, St Louis, Missouri

Percentages of CD8 memory cells were examined as a function of age in a group

of 46 Labrador retrievers. At birth dogs were divided into age- and sex-matched pairs
and, from age 8 weeks, restricted dogs received 75% of the total calories consumed
by their maintenance-fed pair-mates. Commencing at 4 years of age, immunologic
parameters were examined annually and periodic lymphocyte samples were cryo-
preserved. Lymphocyte subset analysis (B, T, CD4, CD8) from 4 to 13 years revealed
significant age-related decreases in percentages of CD4 cells and B cells, with restricted
dogs demonstrating lower B-cell percentages. Age-related increases in CD8 and T-cell
percentages were observed overall, but the rate of increase in T-cell percentages in
restricted females was not significant. While it was not initially feasible to monitor
canine memory cells, subsequent availability of a suitable CD44 reagent allowed us to
examine immune memory as a function of diet and age using the cryopreserved cells.
For each dog pair, samples from three “freeze” dates, spanning an age range of 4 to
13 years, were evaluated on the same test date. The percentages of CD8 memory cells
(as defined by CD44 bright staining) increased markedly with age; diet restriction was
found to be minimally beneficial in retarding this increase.

This work was supported by Nestlé Purina PetCare Company, St. Louis, Missouri.

Posters 68
■ INHIBITION OF Th-2 CYTOKINES AND PROLONGATION OF LIFE
SPAN OF LUPUS DISEASE PRONE MICE BY A COMBINATION OF
DIETARY n-3 FATTY ACIDS AND FOOD RESTRICTION
Gabriel Fernandes, PhD, Christopher Jolly, PhD, Richard Lawrence, PhD,
and Dongxu Sun, MD, PhD
University of Texas Health Science Center at San Antonio, San Antonio, Texas

Moderate food, energy, and/or calorie restriction delays age-related immune

dysfunction and prolongs life span in multiple animal models. The amount and type
of dietary fatty acids can also profoundly affect life span. Marine-derived fish oils
contain (n-3) fatty acids which have potent anti-inflammatory properties. We therefore
examined the influence of food restriction (FR) (40% overall reduction in intake of all
of the dietary components) combined with substitution of fish oil (FO) for corn oil (CO)
(5%). Autoimmune-prone female (NZB x NZW)F(1) (B/W) mice, which develop fatal
autoimmune renal disease, were used. The food-restricted/fish oil diet maximally
extended median life span to 645 days (vs. 494 days for the food-restricted corn oil
diet). Similarly, fish oil prolonged life span in the ad libitum (AL)-fed mice to 345 days
(vs. 242 for the ad libitum/corn oil diet). Increased life span was partially associated
with decreased body weight, decreased renal proinflammatory cytokine (interferon-g,
interleukins-10 and –12, and tumor necrosis factor-a) mRNA levels, and lower nuclear
factor kappa B (NF-kB). Reductions in NF-kB were preceded by enhanced superoxide
dismutase, catalase, and glutathione peroxidase activities. We report here that CO/FR
and FO/FR and to a lesser extent FO/AL offset disease-associated losses in Th-1
cytokine production, CD69 expression, and NF-kB activation in splenic T lymphocytes
activated ex vivo. Similarly, CO/FR and FO/FR prevented the disease-dependent rise in
Th-2 cytokine production ex vivo and CD69 expression in vivo. In summary, the
T-lymphocyte phenotype in the old CO/FR and FO/FR groups was identical to that in
the young disease-free mice. Taken together, the data suggest that both CO/FR and
FO/FR increase life span, in part, by maintaining a youthful immune phenotype in
autoimmune prone mice.

Supported by NIH Grants R01 AG14541 (to G.F.) and F32 AG05826 (to C.J.).

69 Posters
 ■ ENERGY REQUIREMENTS OF OLD CATS
Gerardo Pérez-Camargo, MRCVS, PhD, and Robert Rudnick, MS
Nestlé Purina Product Technology Center, Nestec Ltd., St. Joseph, Missouri

There is a general belief that, as cats age, energy requirements decrease,

which is attributed to a decrease in activity. Old age–associated body weight (BW)
losses often contradict this belief, however, suggesting that it might be more prudent
to assess old cats individually for the best feeding practice. Data records were analyzed
to investigate energy requirements of colony cats. Daily energy requirements of cats
(n = 138) from 1 to 15 years of age were calculated from the calorie intake required
to maintain BW (± 3%) over a 4-week period. A significant (P < 0.001) decline in
energy requirements with age was observed and suggests a 20% lower energy
requirements for cats over 7 years of age. A plot (n = 444) of BW by age (1 to 20 years)
shows a significant (P > 0.001) BW decline with age. The decline primarily appears to
affect cats after the age of 12, with an increased incidence of underweight cats
(< 2 kg) in that age group. Cats showed a slow and progressive loss of BW starting at
least two years prior to their natural death, irrespective of cause of death. Average BW
losses from one to two years prior to death were over 6% and losses over 10% were
seen during the year prior to death when the cause of death was cancer, chronic renal
failure, or hyperthyroidism. These data suggest that assessment of energy require-
ments over a 4-week weight maintenance period may not be a suitable method of
assessing energy requirements in old cats because BW declines slowly over longer
periods of time. Cats appear to undergo two old age life stages: senior, from 7 years
to approximately 12 years, and geriatric, over 12 years. The energy requirements for
these two life stages may be different. Further work is needed to investigate the
causes of general loss of BW in geriatric cats, which contributes to the decline of their
quality of life.

Posters 70
■ A GENE EXPRESSION SIGNATURE FOR DELAYED AGING IN MICE
Richard A. Miller, MD, PhD, Yayi Chang, BS, Andrzej T. Galecki, MD, PhD,
Khalid Al-Regaiey, DVM, John J. Kopchick, PhD, and Andrzej Bartke, PhD
Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois

To gain insight into the pathways by which caloric restriction (CR) slows aging,
gene expression levels were assessed for each of 2352 genes in liver of 9-month-old
CR and control mice. 352 genes were found to be significantly increased or decreased
by CR. The distribution of affected genes among functional classes was similar to the
distribution of genes within the test set. Surprisingly, a disruption or knockout of the
gene for the growth hormone receptor (GHR-KO), which also produces life extension,
had a much smaller effect on gene expression, with no more than 10 genes meeting
the selection criterion. There was, however, an interaction between GHR-KO mutation
and the CR diet: the effects of CR on gene expression were significantly lower in
GHR-KO mice than in control mice. Of the 352 genes altered significantly by CR, 29
had shown a significant and parallel alteration in expression in a previous study of
liver gene expression that compared mice of the long-lived Snell dwarf stock (dw/dw)
to controls. These 29 genes, altered both by CR and in dwarf mice, provide a list of
biochemical features common to both models of delayed aging, and thus merit
confirmation and more detailed study.

71 Posters
 ■ COMPARISON OF INDIRECT CALORIMETRY METHODS IN DOGS:
OPEN FLOW CAGE VERSUS MASK
Sharon A. Center, DVM, Diplomate ACVIM,1 Karen L. Warner, AS, LVT,1
and D.P. Carey, DVM2
1
College of Veterinary Medicine, Cornell University, Ithaca, New York; and
2
The Iams Company, Lewisburg, Ohio

A number of studies using a bonnet/mask technique have been published where

estimation of resting energy expenditure (REE) is done in dogs. Since gas measurement
can be profoundly influenced by inconsistent gas flow and mixing, we hypothesized
that a mask method of indirect calorimetry could produce data significantly different
from a flow controlled/equilibrated open flow cage system.
Methods and Materials. Twenty-five healthy dogs of various breeds, body size, and
condition (>20 kg [n = 12], <20 kg [n = 13]) had REE measured on a single day by the
same operators; cage studies preceded mask studies. Dogs were fasted for 12 hours prior
to calorimetry; body weights were recorded on a calibrated scale to the nearest 0.1 kg.
Daily variation (n = 9) was investigated using a cooperative 20-kg dog, body condition
2.5/5, maintained on a consistent measured energy intake at a stable body weight.
Masks were custom made and fitted to each dog with shapes suited to their muzzle
physique. An attempt was made to provide consistent mask-face contact during
measurements. Airtight cages (except for entry and exit portals) were made either
from modified air flight cages or were specially constructed of Plexiglas. Cage “dead
space” was reduced with styrofoam inserts. Room temperature was controlled to 18˚C.
Initial gas flow was provided at a minimum of 750 mL/min/kg0.67 and was modified to
achieve CO2 change between 0.4 and 0.8%. Animal movement/cooperation during
recordings were recorded; only recordings during quiet intervals were used.
Cage gas flow was determined with calibrated mass flow controllersa and
confirmed with nitrogen dilution. Mask flow was maintained at the same rate as cage
flow (mass flow meter) immediately after cage measurements. Water absorbentb was
positioned in line before mass flow meters, CO2 absorbentc before the O2 analyzer, in
series following the CO2 analyzer. CO2 concentrations were determined with an
infrared-based analyzerd and O2 concentrations with a disposable fuel cell.e Gas
analyzers were calibrated with pure nitrogen and a span gas (20.0% O2, 5.0% CO2) with
guaranteed analysis before each session. After habituation to chamber confinement
or mask (15 to 30 minutes for most), expiratory gases were continuously sampled and
recorded every 6 seconds, for 30 minutes. A customized software program automated
chamber and baseline samplings throughout recording sessions.f Total energy
expenditure was calculated using VO2 and VCO2 and the equation of DeWeir:

TEE (kcal/day) ≈ {[3.94 × VO2 (mL/min)] + [1.10 × VCO2 (mL/min)]} × 1.44

Respiratory quotient (RQ) = VCO2 /VO2

Posters 72
C O M PA R I S O N O F I N D I R E C T C A L O R I M E T RY M E T H O D S
IN DOGS: OPEN FLOW CAGE VERSUS MASK

Statistical Analysis. Since most data were non-gaussian (box and whisker plots), significant differences
in REE, REE/kg, REE/kg0.67, and RQ were evaluated with the Wilcoxon signed rank test (two-tailed);
P values < 0.05 were considered significant. Interassay differences between methods were evaluated similarly.
Bland-Altman plots were used to demonstrate data variation.
Results. Descriptive statistics of REE, REE/kg, and REE/kg0.67 showed lowest values for cage measurements
in 15 dogs. Significantly lower REE, REE/kg, and REE/kg0.67 occurred in dogs <20 kg. Bland-Altman plots
displayed discordant values at high REE. The interassay study yielded significant differences in REE, REE/kg,
REE/kg0.67, and RQ values; mask values were higher.
Conclusion. This study shows that mask collections can generate data significantly different from a
calibrated open flow cage system in dogs ≤20 kg. In addition, interassay variability with the mask method
may importantly confuse data interpretation when REE and RQ measurements are made on different days.

a
Sierra Series 840 mass flow controllers; Sierra Instruments, Inc., Monterey, California.
b
Drierite, anhydrous CaSO4; WA Hammond Drierite Company, Xenia, Ohio.
c
Ascarite II; Thomas Scientific, Swedesboro, New Jersey.
d
Sable Systems CA-1 carbon dioxide analyzer; Sable Systems, Henderson, Nevada.
e
Sable Systems PC-1 oxygen analyzer; Sable Systems, Henderson, Nevada.
f
Sable Systems software; Sable Systems, Henderson, Nevada.

BIBLIOGRAPHY
DeWeir V: New methods for calculating metabolic rate with special reference to protein metabolism.
J Physiol. 1949:109.
Ferrannini E. The theoretical basis of indirect calorimetry: A review. Metabolism. 1988;37:287–301.
Fedak MA, Rome L, Seeherman HJ. One-step N2 dilution technique for calibrating open-circuit VO2
measuring systems. J Appl Physiol. 1981;51:772–776.
Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical
measurement. The Lancet. 1986;1:307–310.

73 Posters
 Ribs, lumbar vertebrae, pelvic bones and all bony prominences
evident from a distance. No discernible body fat. Obvious loss
of muscle mass.
]
UNDERFED

Ribs, lumbar vertebrae and pelvic bones easily visible.

1
No palpable fat. Some evidence of other bony prominence.
Minimal loss of muscle mass.

Ribs easily palpated and may be visible with no palpable fat.

Tops of lumbar vertebrae visible. Pelvic bones becoming
prominent. Obvious waist and abdominal tuck.
]
Ribs easily palpable, with minimal fat covering. Waist easily
3
noted, viewed from above. Abdominal tuck evident.
IDEAL

Ribs palpable without excess fat covering. Waist observed

behind ribs when viewed from above. Abdomen tucked up
when viewed from side.
]
Ribs palpable with slight excess fat covering. Waist is
discernible viewed from above but is not prominent.
5
Abdominal tuck apparent.

Ribs palpable with difficulty; heavy fat cover. Noticeable fat

]
OVERFED

deposits over lumbar area and base of tail. Waist absent or

barely visible. Abdominal tuck may be present.

Ribs not palpable under very heavy fat cover, or palpable

only with significant pressure. Heavy fat deposits over lumbar
7
area and base of tail. Waist absent. No abdominal tuck.
Obvious abdominal distention may be present.

Massive fat deposits over thorax, spine and base of tail.

Waist and abdominal tuck absent. Fat deposits on neck
and limbs. Obvious abdominal distention.
]
The BODY CONDITION SYSTEM was developed at the Nestlé Purina Pet Care Center and has been
validated as documented in the following publications:
9
Mawby D, Bartges JW, Moyers T, et. al. Comparison of body fat estimates by dual-energy x-ray
absorptiometry and deuterium oxide dilution in client owned dogs. Compendium 2001; 23 (9A): 70

Laflamme DP. Development and Validation of a Body Condition Score System for Dogs. Canine Practice
July/August 1997; 22:10-15

Kealy, et. al. Effects of Diet Restriction on Life Span and Age-Related Changes in Dogs. JAVMA 2002;

Call 1-800-222-VETS (8387), weekdays, 8:00 a.m. to 4:30 p.m. CT

 Ribs visible on shorthaired cats; no palpable fat;
1 severe abdominal tuck; lumbar vertebrae and wing of
ilia easily palpated.
]
1
UNDERFED

2 Shared characteristics of BCS 1 and 3.

Ribs easily palpable with minimal fat covering; lumbar

3 vertebrae obvious; obvious waist behind ribs; minimal

abdominal fat.
]
3
4 Shared characteristics of BCS 3 and 5.
IDEAL

5 Well-proportioned; observe waist behind ribs; ribs palpable

with slight fat covering; abdominal fat pad minimal. ]
5
6 Shared characteristics of BCS 5 and 7.

Ribs not easily palpated with moderate fat covering;

7 ]
OVERFED

waist poorly discernible; obvious rounding of abdomen;

moderate abdominal fat pad.

7
8 Shared characteristics of BCS 7 and 9.

Ribs not palpable under heavy fat cover; heavy fat

9 deposits over lumbar area, face and limbs; distention of
abdomen with no waist; extensive abdominal fat deposits. ]
The BODY CONDITION SYSTEM was developed at the Nestlé Purina Pet Care Center and has been
validated as documented in the following publications:
9
Laflamme DP. Development and Validation of a Body Condition Score System for Cats: A Clinical Tool.
Feline Practice 1997; 25:13-17

Laflamme DP, Hume E, Harrison J. Evaluation of Zoometric Measures as an Assessment of Body

Composition of Dogs and Cats. Compendium 2001; 23(Suppl 9A):88

Call 1-800-222-VETS (8387), weekdays, 8:00 a.m. to 4:30 p.m. CT

VET 5789