05-10-0480.
qxp 4/11/2006 1:25 PM Page 1383
Physical characteristics, blood hormone
concentrations, and plasma lipid concentrations
in obese horses with insulin resistance
Nicholas Frank, DVM, PhD; Sarah B. Elliott, BSc; Laura E. Brandt, BSc; Duane H. Keisler, PhD
Objective—To compare obese horses with insulin
resistance (IR) with nonobese horses and determine
whether blood resting glucose, insulin, leptin, and
lipid concentrations differed between groups and
were correlated with combined glucose-insulin test
(CGIT) results.
Animals—7 obese adult horses with IR (OB-IR group)
and 5 nonobese mares.
Procedures—Physical measurements were taken, and
blood samples were collected after horses had accli-
mated to the hospital for 3 days. Response to insulin
was assessed by use of the CGIT, and maintenance of
plasma glucose concentrations greater than the prein-
jection value for ≥ 45 minutes was used to define IR.
Area under the curve values for glucose (AUCg) and
insulin (AUCi) concentrations were calculated.
Results—Morgan, Paso Fino, Quarter Horse, and
Tennessee Walking Horse breeds were represented in
the OB-IR group. Mean neck circumference and BCS
differed significantly between groups and were posi-
tively correlated with AUC values. Resting insulin and
leptin concentrations were 6 and 14 times as high,
respectively, in the OB-IR group, compared with the
nonobese group, and were significantly correlated
with AUCg and AUCi. Plasma nonesterified fatty acid,
very low-density lipoprotein, and high-density lipopro-
tein-cholesterol (HDL-C) concentrations were signifi-
cantly higher (86%, 104%, and 29%, respectively) in
OB-IR horses, and HDL-C concentrations were posi-
tively correlated with AUC values.
Conclusions and Clinical Relevance—Measurements
of neck circumference and resting insulin and leptin
concentrations can be used to screen obese horses for
IR. Dyslipidemia is associated with IR in obese horses.
(J Am Vet Med Assoc 2006;228:1383–1390)
O besity is a major health concern in humans
because of its association with IR, type 2 diabetes
mellitus, and coronary heart disease.1 Insulin resis-
tance has also been associated with obesity in horses,2
and this condition may be of particular concern
because of the putative link between laminitis and
glucose metabolism.3 This link is supported by anec-
dotal observations that obese horses are more likely
From the Department of Large Animal Clinical Sciences, College of
Veterinary Medicine, University of Tennessee, Knoxville, TN
37996 (Frank, Elliott, Brandt); and the Division of Animal
Sciences, University of Missouri, Columbia, MO 65211 (Keisler).
Supported by a grant from the University of Tennessee Centers of
Excellence in Livestock Diseases and Animal Health.
The authors thank Dr. Mickey Coleman for assistance with this study.
Address correspondence to Dr. Frank.
JAVMA, Vol 228, No. 9, May 1, 2006
ABBREVIATIONS
IR Insulin resistance
EQUINE
PPID Pituitary pars intermedia dysfunction
CGIT Combined glucose-insulin test
BCS Body condition score
VLDL Very-low-density lipoprotein
PL Phospholipid
HDL High-density lipoprotein
DEX-TRH Dexamethasone-thyrotropin releasing
hormone
AUCg Area under the curve for glucose
AUCi Area under the curve for insulin
NEFA Nonesterified fatty acid
to develop laminitis and that dietary changes such as
grazing on lush pasture can trigger episodes of
laminitis. Other support is provided by in vitro evi-
dence that hoof tissues have a high requirement for
glucose.3 Pass et al3 reported that hoof explants
undergo epidermal-dermal separation readily when
glucose concentrations are low in the medium. It has
also been reported that ponies with laminitis are less
responsive to insulin than healthy ponies4 and that
ponies with both obesity and laminitis are more
insulin resistant than those that are just obese.5
Laminitis has also been associated with PPID, and IR
often accompanies this condition.6,7
Obesity, hyperinsulinemia, and laminitis some-
times develop concurrently in horses without
detectable PPID, and this group of disorders has been
referred to as syndrome X or equine metabolic syn-
drome.6,8,9 The latter term is used most commonly but
may not be appropriate because metabolic syndrome is
defined by the presence of IR or type 2 diabetes melli-
tus as well as at least 2 of 4 additional risk factors,
including obesity or visceral adiposity, dyslipidemia,
microalbuminemia, or arterial hypertension.10 To the
authors’ knowledge, microalbuminemia and hyperten-
sion have not been reported in obese horses, but blood
lipid concentrations have been measured in ponies and
donkeys with IR, and many of them were obese.11,12
Plasma triglyceride and total cholesterol concentra-
tions do not differ between clinically normal and
hyperinsulinemic ponies,11 but a positive correlation
(r = 0.55; P = 0.002; n = 31) exists between plasma
insulin and triglyceride concentrations in donkeys
with naturally occurring hyperlipidemia.12
When methods of assessing IR in horses and
ponies were recently reviewed, available tests were
divided into those that provide nonspecific indications
of IR and those that give specific quantitative measure-
ments of insulin sensitivity.13 The CGIT used in the
Scientific Reports: Original Study 1383
05-10-0480.qxp 4/11/2006 1:25 PM Page 1384
present study was not discussed, but this test should be
included in the first category because it does not pro-
vide values for insulin sensitivity, glucose effectiveness,
or endogenous insulin secretion.13,14 However, delayed
return of blood glucose concentrations to the preinjec-
tion concentrations can be attributed to IR when the
CGIT is used because glucose concentrations should
rapidly decrease in response to exogenous insulin
administration.14 The CGIT was also used in the pre-
sent study because it is easily interpreted, has low cost,
and is readily applicable to clinical practice.14
In this study, obese horses were selected on the
basis of BCS and IR was defined by CGIT results. The
EQUINE
purpose of the study reported here was to compare
obese horses with IR with nonobese horses to deter-
mine whether blood resting glucose, insulin, leptin,
and lipid concentrations differed between groups and
were correlated with CGIT results. We also wanted to
identify variables that predicted CGIT responses when
only a single measurement is available and determine
whether obesity, IR, and dyslipidemia occur concur-
rently in horses.
Materials and Methods
Horses—Client-owned horses were admitted to the
University of Tennessee Large Animal Hospital and remained
there for a 7-day evaluation period. Horses were evaluated
between July 2003 and July 2004. Seven obese horses (5
mares, 2 geldings) with CGIT results indicative of IR (OB-IR
group) were compared with 5 nonobese mares with a BCS of
4/9 to 6/9 and normal insulin sensitivity (NO group). Median
age for the OB-IR group was 11 years (range, 10 to 16 years),
and Morgan Horse (n = 3), Paso Fino (1), Quarter Horse (2),
and Tennessee Walking Horse (1) breeds were represented.
Median age of the NO group was 10 years (range, 7 to 16
years), and Quarter Horse (n = 3), Appaloosa (1), and
Mustang (1) breeds were represented. Obesity was defined as
a BCS ≥ 7 on a 1 to 9 scale.15 Insulin resistance was arbitrari-
ly defined by plasma glucose concentrations that remained
greater than the preinjection concentration for ≥ 45 minutes
during the CGIT (ie, attenuation of the negative phase).14
The study protocol was approved by the University of
Tennessee Institutional Animal Care and Use Committee,
and signed owner consent was obtained for each horse.
Experimental design—Horses were admitted on day 0,
and weight, height at the withers, girth, and neck circumfer-
ence were measured. Physical examinations, CBC, and serum
biochemical analyses were performed. An adipose tissue
biopsy specimen was also collected on day 0, and flunixin
megluminea was administered orally for the next 3 days. Each
horse was then housed in a 3.7 X 3.7-m stall within the teach-
ing hospital, provided with mixed timothy-orchard grass hay
and water for ad libitum intake, and acclimated to the envi-
ronment for 3 days.
Blood samples were collected for resting hormone and
blood lipid measurements via jugular venipuncture between
8 AM and 9 AM on the fourth day. An IV catheter was placed,
and a CGIT was performed the next day. Horses were
screened for PPID by use of the combined DEX-TRH sup-
pression test16 on subsequent days, with the exception of 3
horses from the OB-IR group that were excluded at the own-
ers’ request. Resting plasma ACTH concentrations were mea-
sured in these horses instead. The OB-IR horses were dis-
charged 1 week after admission.
Neck circumference measurements—Horses were
restrained so that the head and neck were maintained in a
1384 Scientific Reports: Original Study
normal upright position, and the distance along a straight
line from the poll to the cranial aspect of the withers was
measured with a measuring tape (Figure 1). Neck circumfer-
ence was measured perpendicular to this line at 0.25, 0.50,
and 0.75 of the distance from the withers to the poll with a
measuring tape (Figure 1). Mean neck circumference values
were calculated.
CGIT—Horses were provided with ad libitum grass hay
and water before and during the procedure. A 14-gauge
polypropylene catheter was inserted into the left jugular vein,
and an injection cap and infusion setb (length, 30 cm; inter-
nal diameter, 0.14 cm) were attached. The CGIT was per-
formed as described.14 Briefly, glucose (150 mg/kg) was
infused as a 50% dextrose solutionc through the infusion line
and catheter, followed by heparinized saline (0.9% NaCl)
solution and regular insulind (0.1 U/kg). Blood samples were
collected via the catheter at 1, 5, 15, 25, 35, 45, 60, 75, 90,
105, 120, 135, and 150 minutes after insulin injection. At
each time point, 3 mL of blood was withdrawn from the infu-
sion line and discarded. A 6-mL blood sample was collected,
followed by infusion of 5 mL of heparinized saline solution.
Blood was transferred into tubes containing sodium fluoride
and potassium oxalate that were immediately cooled on ice
and refrigerated or transferred into glass tubes containing no
anticoagulant that were left at 21oC for 1 hour to allow clot
formation. Serum or plasma was subsequently harvested
after low-speed centrifugation (1,000 X g) at 4oC for 20 min-
utes and stored at –20oC until further analysis.
Isolation and quantification of lipoproteins—Plasma
lipoprotein fractions were isolated by use of the sequential
ultracentrifugation method of Havel et al.17 Briefly, blood was
centrifuged (1,000 X g) at 4oC for 20 minutes to separate plas-
ma from chilled blood samples. Six-milliliter plasma samples
were placed in a fixed-angle rotore for ultracentrifugation at
112,000 X g for 18 hours at 10oC. A 1-mL fraction of plasma
(density < 1.006 g/mL) was isolated from each tube. This frac-
tion was referred to as VLDL. Triglyceride, PL, and total cho-
lesterol components of VLDL were measured with enzymatic
colorimetric reagentsf in an automated discrete analyzer.g
Lipoprotein lipase, phospholipase D, and cholesterol oxidase,
respectively, were the principal reagents of the triglyceride,
Figure 1—Illustration of a procedure used to measure mean
neck circumference in horses. a = 0.25 of the distance from poll
to withers b = 0.50 of the distance from poll to withers. c = 0.75
of the distance from poll to withers. Reprinted with permission.
JAVMA, Vol 228, No. 9, May 1, 2006
05-10-0480.qxp 4/11/2006 1:25 PM Page 1385
PL, and total cholesterol assays. Protein content of VLDL was
analyzed by use of bovine serum albumin standards and a
spectrophotometerh in accordance with a modified method of
the Lowry18 procedure.19 Plasma VLDL concentrations were
calculated by summing concentrations of lipid (triglyceride,
total cholesterol, PL) and protein components.
Low-density lipoprotein was isolated from the remain-
ing plasma by ultracentrifugation under the same conditions
after addition of potassium bromide to raise the plasma den-
sity to 1.063 g/mL. A 1.063 g/mL solution of potassium bro-
mide prepared in EDTA saline solution was also added to
each sample to increase the volume to 6 mL. After ultracen-
trifugation, a 1-mL fraction of plasma (density < 1.063 g/mL)
was isolated from each tube and dialyzed overnight in 0.5M
ammonium bicarbonate solution to remove dissolved salts.
Postdialysis volumes were recorded for each sample, and
duplicate samples were processed; this fraction was referred
to as LDL. Compositional analysis of dialyzed LDL samples
was performed as described. Plasma LDL concentrations
were adjusted to account for addition of potassium bromide
solution and volume expansion during dialysis. High-densi-
ty lipoprotein was isolated and quantified by use of the same
methods as described for LDL, with the exception that the
plasma density was increased to 1.21 g/mL. Equine VLDL,
LDL, and HDL have established density limits of < 1.006,
1.019 to 1.063, and 1.063 to 1.21 g/mL, respectively.20
Measurement of plasma glucose and lipid concentra-
tions—Glucose concentrations were measured by use of a
colorimetric assayi on an automated discrete analyzer.g
Concentrations of plasma triglyceride and total cholesterol
were measured by use of enzymatic colorimetric reagentsf
and the automated discrete analyzer.g Plasma NEFA concen-
trations were measured by use of an in vitro enzymatic col-
orimetric test kitf on a microtiter plate reader.j For all lipid,
protein, and glucose measurements, duplicate assays were
performed and intra-assay coefficients of variation < 5% were
required for acceptance of results.
Screening for PPID—A DEX-TRH test was performed in
4 of 7 horses in the OB-IR group and all of the horses in the
NO group according to the method of Eiler at al.16 Briefly, a
baseline blood sample was collected, and 40 µg of DEXk/kg
was injected IV. After collection of another blood sample at 3
hours, 1.2 mg of TRHl was injected IV and blood samples
were collected at 3.5, 4, and 24 hours after DEX injection.
Measurement of blood hormone concentrations—
Serum or EDTA plasma was obtained via low-speed centrifu-
gation (1,000 X g) at 4oC for 20 minutes. Insulin concentra-
tions were measured in serum by use of a radioimmunoassay
Table 1—Measured variables (median [range]) in 5 nonobese horses and 7 obese
horses with IR (OB-IR).
Variable
Weight (kg)
BCS (scale, 1–9)
Height (cm)
Girth (cm)
Neck circumference (cm) 87.7 (85.3–95.7)
Plasma leptin (ng/mL)
Resting plasma glucose (mg/dL) 66.9 (49.8–74.1)
Resting serum insulin (µU/mL)
Resting glucose-to-insulin ratio
AUCg (X 103 mg·dL–1·min–1)
AUCi (X 103 µU·mL–1·min–1) 12.8 (10.6–13.2)
*Includes only 6 observations.
JAVMA, Vol 228, No. 9, May 1, 2006
kitm validated for equine insulin.21 Serum samples were ana-
lyzed for leptin with double-antibody radioimmunoassay
procedures validated for equine leptin.22 Plasma cortisol con-
centrations were measured by use of a radioimmunoassay kitn
validated for equine cortisol.21
Measurement of plasma ACTH concentrations—Assay
of equine ACTH was performed on EDTA plasma by use of a
commercially available immunoradiometric assay for
ACTH.23,o Endogenous plasma ACTH concentrations were
measured in blood collected from 3 OB-IR horses that did not
undergo combined DEX-TRH testing. For all hormone
assays, duplicate measurements were performed and an
intra-assay coefficient of variance < 10% was required for
acceptance of results.
EQUINE
Statistical analysis—The AUCg and AUCi concentra-
tions were calculated from total concentrations by use of the
trapezoidal method and computer software.o Groups were
compared by use of the Mann-Whitney U test after distribu-
tions were examined and Shapiro-Wilk tests were performed,
and median, range, mean, and SD values were calculated.
Correlations among variables were examined by calculating
Spearman correlation coefficients. Statistical tests were per-
formed with computer software.p Significance was defined at
a value of P < 0.05.
Results
No abnormalities were detected via physical exam-
ination, and results of CBC and serum biochemical
tests were within reference ranges for all horses. Four
of 7 horses in the OB-IR group had a history of chron-
ic laminitis, and abnormal growth rings were seen on
hooves, but these horses were not lame at the time of
examination. Groups differed significantly with respect
to BCS, girth, and neck circumference, but did not dif-
fer in weight or height (Table 1).
In the OB-IR group, plasma glucose concentra-
tions were greater than baseline concentrations for ≥
45 minutes in 1 horse, 75 minutes in 2 horses, 90 min-
utes in 1 horse, 120 minutes in 1 horse, 135 minutes in
1 horse, and > 150 minutes in 1 horse (Figure 2). In
contrast, plasma glucose concentrations returned to a
value less than the baseline value within 25 minutes in
5 horses in the NO group.
Compared with the NO group, median AUCg and
AUCi values for CGIT curves were 68% and 81% high-
er, respectively, in the OB-IR group (Table 1). Resting
Nonobese OB-IR
(n = 5) (7) P
473 (444–476) 518 (426–610) 0.223
6 (4–6) 7 (7–9) 0.004
145 (142–158) 151 (143–155)* 0.927
179 (172–183) 189 (182–205)* 0.011
105.8 (94.3–110.3)* 0.010
0.8 (0.5–11.0) 11.0 (1.0–30.5) 0.028
83.9 (76.0–97.5) 0.005
9.1 (6.4–21.1) 50.5 (17.0–93.4) 0.007
6.4 (3.2–11.6) 1.7 (0.9–5.3) 0.007
9.2 (7.7–10.1) 15.5 (13.1–22.6) 0.005
23.2 (18.1–86.6) 0.005
Scientific Reports: Original Study 1385
05-10-0480.qxp 4/12/2006 1:30 PM Page 1386

serum insulin and plasma leptin concentrations were 6
times (P = 0.007) and 14 times (P = 0.028) greater,
respectively, in the OB-IR group, but ranges over-
lapped. These single measurements of insulin (r = 0.76;
P = 0.004) and leptin (r = 0.75; P = 0.005) were posi-
tively correlated with BCS. Compared with NO horses,
plasma NEFA, VLDL, and HDL-cholesterol concentra-
tions were 86%, 104%, and 29% greater, respectively, in
OB-IR horses (Table 2).
EQUINE
Variables significantly correlated with both AUCg
and AUCi included BCS, neck circumference, leptin
concentration, glucose concentration, insulin concen-
tration, glucose-to-insulin ratio, and HDL-cholesterol
(Table 3). In this study, AUCg and AUCi were most
closely correlated with BCS (r = 0.84; P < 0.001) and
neck circumference (r = 0.88; P < 0.001), respectively.
Plasma cortisol concentrations were < 10 ng/mL at
24 hours after DEX administration in all of the horses
evaluated by use of the DEX-TRH test, with the excep-
tion of 1 OB-IR horse that had a concentration of 10.7
ng/mL. For baseline and 30-minute post-TRH injection
plasma cortisol concentrations, 1 horse from each

group had responses (74% and 150%) that exceeded

the 66% cutoff established for unaffected horses.16
However, both horses had typical suppression at 24
hours in response to administration of DEX. Median
plasma ACTH concentrations for the 3 OB-IR horses
tested ranged from 2.5 to 3.7 pmol/L, and these values
were less than the 10 pmol/L upper limit of the refer-
ence range provided by the laboratory.q

Figure 2—Mean ± SD serum insulin concentrations (A) and plas-
ma glucose concentrations (B) for 5 nonobese horses (squares)
and 7 obese horses with IR (diamonds) during CGITs. Time 0 =
Time of insulin and glucose injection.
Table 2—Plasma lipid and lipoprotein concentrations (median [range]) in 5 nonobese
horses and 7 obese horses with IR.
Discussion
A small population of obese horses with IR was
assembled for this study, and these horses had signifi-
cantly greater resting glucose, insulin, and leptin con-
centrations than healthy nonobese horses. High VLDL,
VLDL-triglyceride, and HDL-cholesterol concentra-
tions were also detected in OB-IR horses, suggesting
that obesity, IR, and dyslipidemia occur concurrently
in certain horses.
Mares predominated in the OB-IR group, but anec-
dotal reports and results of other studies2,5 suggest that
geldings are equally affected by obesity. Although vari-
ous breeds were represented in the OB-IR group,
clients consistently referred to their horses as “easy
keepers” because they appeared to require fewer calo-

Nonobese OB-IR
Variable (n = 5) (7) P
NEFA (µmol/L) 197.1 (151.5–268.3) 366.5 (187.8–634.0) 0.028
Total cholesterol (mg/dL) 75.3 (73.0–88.1) 78.6 (60.4–86.0) 0.570
Triglyceride (mg/dL) 18.9 (11.0–22.6) 34.6 (13.8–134.8) 0.123
VLDL (mg/dL) 24.4 (19.8–34.6) 49.7 (29.0–162.0) 0.012
VLDL-triglyceride (mg/dL) 15.6 (11.2–23.2) 30.6 (17.4–97.1) 0.019
LDL (mg/dL) 30.3 (13.1–46.8) 30.9 (16.6–54.6) 0.465
LDL-cholesterol (mg/dL) 11.8 (5.2–18.3) 13.5 (6.8–22.4) 0.223
HDL (mg/dL) 139.7 (106.4–169.4) 110.8 (96.4–160.9) 0.168
HDL-cholesterol (mg/dL) 27.5 (24.8–34.2) 35.4 (32.2–52.3) 0.029

Table 3—Variables significantly correlated with plasma AUCg and serum AUCi values
for CGITs in horses.

AUCg AUCi
No. of
Variable horses r P r P
BCS 12 0.84 < 0.001 0.84 < 0.001
Neck circumference 11 0.71 0.015 0.88 < 0.001
Resting leptin concentration 12 0.76 0.004 0.69 0.013
Resting glucose concentration 12 0.83 < 0.001 0.72 0.008
Resting insulin concentration 12 0.66 0.020 0.83 < 0.001
Resting glucose-to-insulin ratio 12 –0.62 0.033 –0.87 < 0.001
HDL-cholesterol concentration 12 0.60 0.039 0.69 0.013

1386 Scientific Reports: Original Study JAVMA, Vol 228, No. 9, May 1, 2006
05-10-0480.qxp 4/11/2006 1:25 PM Page 1387
ries than most horses to maintain body weight. It has
been suggested that some horses are genetically predis-
posed to obesity because of adaptations to survival on
poorer quality forages.8,24 According to this theory, con-
sumption of concentrated feeds or grazing on rich pas-
tures might therefore promote obesity in susceptible
horses. Genetic and environmental factors are likely to
be important in the development of obesity in horses,
and it is interesting that all of the OB-IR horses in this
study were 10 years of age or greater, which suggests
that time is required for environmental factors to alter
glucose metabolism.
Because it is likely that diet contributes substantial-
ly to the development of obesity, the diet consumed by
horses prior to admission may have been a confounding
factor in this study. No attempt was made to control the
diet of horses prior to their arrival, so horses had access
to pasture and were consuming hay from various
sources and sometimes a small quantity of a concentrate
feed as a treat. Hoffman et al2 detected the effects of diet
on response to insulin by determining that horses are
less responsive to insulin after consuming a diet com-
posed primarily of starch and sugar instead of fat and
fiber. The adipose biopsy procedure performed on day 0
may have also been a source of variability, but this was
discounted because all horses underwent the same pro-
cedure and CGIT results for nonobese horses were con-
sistent with those from another study.14 Effects of flunix-
in meglumine on response to insulin have not been
investigated, but administration of phenylbutazone for 5
days did not affect glucose tolerance or insulin secretion
in geldings.25
Obesity was defined by BCS in this study with the
scale developed by Henneke et al.15 Alternative mea-
sures of obesity include cadaver dissection,26 rump-fat
thickness,15,26 body mass index,27 and estimation of total
body water by use of bioelectric impedance analysis or
indicator dilution.28 Of these procedures, BCS and
rump-fat thickness are the easiest to perform. A BCS is
assigned after visual appraisal and palpation of fat at 6
sites on the body, including the neck, withers, back,
tailhead, ribs, and area caudal to the shoulder.15 Rump-
fat thickness is measured via B-mode ultrasonography
at a site approximately 5 cm lateral from the midline at
the center of the pelvic bone, and percentage fat is cal-
culated with an established formula.29
Mean neck circumference was examined to assess
regional adiposity because many OB-IR horses have
enlargement of adipose tissues at this location, which
is sometimes referred to as a cresty neck. In humans,
regional adiposity develops within the abdomen and an
enlarged waist circumference has been identified as a
risk factor for metabolic syndrome.1 Of the physical
measurements collected in this study, mean neck cir-
cumference was most closely correlated with AUCi,
which suggested that enlargement of the neck is a risk
factor for IR in horses.
The CGIT was selected for use in this study
because of its simplicity and low cost.14 The frequent-
sample IV glucose tolerance test and the euglycemic-
hyperinsulinemic clamp test may have been better
choices because both provide specific quantitative
measures of insulin sensitivity,13 but these tests are
JAVMA, Vol 228, No. 9, May 1, 2006
technically more challenging and therefore less applic-
able to clinical practice. These tests also require more
frequent sampling and are therefore more expensive. In
this study, the time required for blood glucose concen-
trations to enter the negative phase was used to define
IR. This definition is easily applied, and only blood
glucose measurements are required. Use of the baseline
measurement as a reference point also allows handheld
glucometers to be used because variability between
glucometers does not affect results. This procedure is
also applicable to ambulatory practice because the
CGIT can be abbreviated to 60 minutes.
Higher resting serum insulin concentrations and
EQUINE
lower resting glucose-to-insulin ratios were detected in
horses from the OB-IR group, compared with nonobese
horses, suggesting that these measures may be useful
screening tests for IR. Hyperinsulinemia is a feature of
IR in humans and occurs when insulin secretion from
the pancreas increases to compensate for reduced
response to insulin.30 In the present study, resting
serum insulin concentrations and glucose-to-insulin
ratios were closely correlated with AUCg and AUCi,
suggesting that the same compensatory responses
occur in horses. However, it should be noted that a
resting serum insulin concentration of 17.0 µU/mL was
detected in 1 OB-IR horse, and this value was within
the range detected for the nonobese group. This find-
ing therefore underscores the importance of perform-
ing challenge tests such as the CGIT when evaluating
horses with suspected IR.
Resting glucose concentrations were positively
correlated with AUCg values, indicating that blood glu-
cose concentrations increased as IR progressed.
However, it is doubtful that resting blood glucose con-
centrations will serve as a useful measure of response
to insulin in clinical practice because of the confound-
ing effects of stress. Administration of epinephrine31 or
dexamethasone11,32 increases blood glucose concentra-
tions in horses, and stressful procedures such an endo-
scopic examination can markedly alter CGIT results.14
In this study, horses were acclimated to the hospi-
tal environment for 3 days before the first blood sam-
ples were collected and mixed timothy-orchard grass
hay was fed ad libitum before and during the CGIT to
reduce stress. Horses were not tested until 3 days after
their arrival because transportation increases blood
cortisol concentrations and induces hyperglycemia and
hyperinsulinemia in donkeys.33 A 3-day acclimation
period was selected for the present study on the basis
of previous experiences with the CGIT14 and because
blood glucose and insulin concentrations returned to
baseline concentrations within 22 hours in donkeys
that were transported.33 Grass hay was fed to horses
because this feed was assumed to have a low glycemic
index.34 The glycemic index of the mixed timothy-
orchard grass hay fed to horses in this study was not
determined, but Bermuda hay has a mean glycemic
index of 23%, compared with oats (100%).34 Hay fed to
horses in the present study was not analyzed, but the
mean nonstructural carbohydrate content of hay pur-
chased from the same farm was 12.8% of dry matter
when evaluated as part of another study35 performed
during the same time period (January to October). The
Scientific Reports: Original Study 1387
05-10-0480.qxp 4/11/2006 1:25 PM Page 1388
decision to permit hay consumption during testing was
also influenced by the observation that obese horses
become particularly agitated when deprived of feed.
An association between obesity and IR has been
reported in horses2,36,37 and ponies.5,11 When Hoffman et
al2 studied 3 obese Thoroughbred geldings by use of
the frequently-sampled IV glucose tolerance test, mean
insulin sensitivity was < 20% of the value in nonobese
geldings. Obesity and lack of exercise are primary risk
factors for IR in humans, and the risk of developing
type 2 diabetes mellitus increases with the severity of
obesity.38 Blood NEFA concentrations increase with
obesity and lack of physical activity in humans because
EQUINE
adipose tissues reach their maximum capacity for fat
storage and the inhibitory effects of insulin on hor-
mone-responsive lipase are reduced.39 As a result, the
influx of fatty acids into tissues (including those of
skeletal muscle) increases, which causes diacylglyc-
erols to accumulate in cells.39 This phenomenon is
sometimes referred to as lipotoxicity because intracel-
lular lipids disrupt insulin receptor signaling in
myocytes or β-cell function in the pancreas.39,40 In the
study reported here, plasma NEFA concentrations were
greater in OB-IR horses than in NO horses and were
significantly correlated with AUCi values.
Plasma concentrations of VLDL and VLDL-triglyc-
eride were significantly greater in OB-IR horses than in
NO horses, and the median VLDL concentration (49.7
mg/dL) was also greater than mean concentrations of
28.5 and 19.1 mg/dL detected in adult nonobese
mares.41,42 Increased uptake of fatty acids by the liver
should increase the availability of triglyceride for
VLDL synthesis and stimulate production of this
lipoprotein.1 Oversecretion of triglyceride-rich VLDL
particles is associated with abdominal obesity in
humans and attributed to an increase in hepatic fatty
acid uptake.1 Increased VLDL production and synthe-
sis of triglyceride-rich VLDLs have also been associat-
ed with feed deprivation and hyperlipemia in horses,
which are conditions that develop in response to
increased mobilization of NEFA from adipose
stores.41,43
Hypertriglyceridemia (≥ 150 mg/dL) and low
HDL-cholesterol concentrations are 2 criteria used to
define metabolic syndrome in humans.10 These vari-
ables are associated because triglyceride carried by
VLDLs and chylomicrons is exchanged for cholesterol
when these triglyceride-rich lipoproteins interact with
HDL in the blood.44 Consequently, as VLDL concentra-
tions increase, interactions with HDL increase, and
HDL-cholesterol concentrations decrease in humans.1
However, VLDL and HDL-cholesterol concentrations
were greater in OB-IR horses than in NO horses, which
differs from responses detected in humans.1 This find-
ing may be explained by the fact that transfer of cho-
lesterol from HDL to VLDL is catalyzed by cholesterol
ester transfer protein in humans,1 whereas there is a
near absence of cholesterol ester transfer protein activ-
ity in equine blood.45 Higher plasma HDL-cholesterol
concentrations have been associated with increased
lipoprotein lipase activity in horses fed high-fat diets,46
which suggests that the higher HDL-cholesterol con-
centrations detected in OB-IR horses may be attribut-
1388 Scientific Reports: Original Study
able to an increase in lipoprotein lipase activity. A pos-
itive correlation between plasma HDL and triglyceride
concentrations has also been detected in Shetland
ponies.44
Results of the present study provide evidence of an
association between hyperleptinemia and IR in obese
horses. Resting plasma leptin concentrations ranged
from 1.0 to 30.5 ng/mL in OB-IR horses and were pos-
itively correlated with AUCg and AUCi values. Leptin is
a protein hormone synthesized by adipose tissues that
is secreted in greater quantities when the body is in a
positive energy balance.47 Buff et al22 determined that
serum leptin concentrations were positively correlated
(r = 0.64; P < 0.001) with BCS in a herd of 71 Quarter
Horses, which indicates that blood leptin concentra-
tions reflect body-fat mass in horses. Plasma leptin
concentrations and BCS were also positively correlated
in the present study. Hyperleptinemia has also been
associated with glucose metabolism disturbances in
horses.32 Cartmill et al32 identified obese horses with
higher serum leptin concentrations and found that glu-
cose tolerance test results were abnormal in these hors-
es. When obese (BCS ≥ 7.5) horses were divided into
low-leptin (< 5 ng/mL) and high-leptin (7 to 20
ng/mL) groups, significantly greater insulin responses
to IV glucose infusion were detected in the high-leptin
group.32
Results of the present study indicate that resting
serum insulin and leptin concentrations are useful
screening tests for IR in horses, but other factors
including time of day and season must be considered
when interpreting these values. Gordon and
McKeever48 determined that glucose and insulin
responses to feeding are higher in the morning, and
Fitzgerald and McManus49 determined that serum lep-
tin concentrations were highest in mature mares dur-
ing the late summer and early fall and lowest during
the winter months in Kentucky. When the same group
measured serum insulin concentrations in obese mares
across a 12-month period, mean insulin concentrations
were also higher during the summer and early fall.36
Because most of the horses included in the present
study were evaluated during the summer, concentra-
tions of these hormones may have been higher than at
other times of the year.
Horses included in this study were screened for
PPID because this endocrinopathy has been associated
with IR in horses.7 Three of 9 horses had abnormal
responses when combined DEX-TRH tests were per-
formed. However, no clinical signs of PPID were
observed, with the exception of laminitis, which has
been associated with this endocrinopathy in horses,6
and the 2 components of the test did not agree in any
of the horses tested. These may have been false-posi-
tive responses, and the time of year that tests were per-
formed may have influenced results. This issue has not
been examined extensively, but Donaldson et al50
recently determined that false-positive results are more
likely to occur when DEX suppression tests are per-
formed in the month of September. Alternatively, hors-
es with positive test results in the present study may
have had early or mild PPID that affected only 1 com-
ponent of the test.
JAVMA, Vol 228, No. 9, May 1, 2006
05-10-0480.qxp 4/11/2006 1:25 PM Page 1389
In the present study, examination of a small popu-
lation of OB-IR horses revealed that mean neck cir-
cumference should be measured in addition to BCS to
identify horses at risk for IR and that single measure-
ments of blood insulin and leptin concentrations are
useful indicators of IR in obese horses. Horses concur-
rently affected by obesity, IR, and dyslipidemia were
identified.
a. VedaGesic, Phoenix Scientific Inc, St Joseph, Mo.
b. Butterfly, Abbott Laboratories, North Chicago, Ill.
c. Dextrose 50% injection, Abbott Laboratories, North Chicago, Ill.
d. Humulin R, Eli Lilly & Co, Indianapolis, Ind.
e. Beckman Instruments Inc, Fullerton, Calif.
f. Wako Chemicals USA, Richmond, Va.
g. Cobas Mira, Roche Diagnostic Systems Inc, Somerville, NJ.
h. UV-160, Shimadzu, Kyoto, Japan.
i. Glucose, Roche Diagnostic Systems Inc, Somerville, NJ.
j. ELx800 Microplate Reader, Bio-Tek, Winooski, Vt.
k. Dexamethasone solution, Pro Labs Ltd, St Joseph, Mo.
l. P2161 thyrotropin-releasing hormone acetate, Sigma Chemical
Co, St Louis, Mo.
m. Coat-A-Count Insulin, Diagnostic Products Corp, Los Angeles,
Calif.
n. Coat-A-Count Cortisol, Diagnostic Products Corp, Los Angeles,
Calif.
o. ACTH adrenocorticotropic hormone, Nichols Institute
Diagnostics, San Clemente, Calif.
p. SAS, version 9.1, SAS Institute Inc, Cary, NC.
q. Diagnostic Center for Population and Animal Health, College
of Veterinary Medicine, Michigan State University, East
Lansing, Mich.
References
1. Carr MC, Brunzell JD. Abdominal obesity and dyslipidemia
in the metabolic syndrome: importance of type 2 diabetes and famil-
ial combined hyperlipidemia in coronary artery disease risk. J Clin
Endocrinol Metab 2004;89:2601–2607.
2. Hoffman RM, Boston RC, Stefanovski D, et al. Obesity and
diet affect glucose dynamics and insulin sensitivity in Thoroughbred
geldings. J Anim Sci 2003;81:2333–2342.
3. Pass MA, Pollitt S, Pollitt CC. Decreased glucose metabo-
lism causes separation of hoof lamellae in vitro: a trigger for lamini-
tis? Equine Vet J Suppl 1998:133–138.
4. Coffman JR, Colles CM. Insulin tolerance in laminitic
ponies. Can J Comp Med 1983;47:347–351.
5. Jeffcott LB, Field JR, McLean JG, et al. Glucose tolerance and
insulin sensitivity in ponies and Standardbred horses. Equine Vet J
1986;18:97–101.
6. Donaldson MT, Jorgensen AJ, Beech J. Evaluation of sus-
pected pituitary pars intermedia dysfunction in horses with lamini-
tis. J Am Vet Med Assoc 2004;224:1123–1127.
7. Garcia MC, Beech J. Equine intravenous glucose tolerance
test: glucose and insulin responses of healthy horses fed grain or hay
and of horses with pituitary adenoma. Am J Vet Res 1986;47:570–572.
8. Johnson PJ. The equine metabolic syndrome peripheral
Cushing’s syndrome. Vet Clin North Am Equine Pract 2002;18:271–293.
9. Kronfeld DS. Equine syndrome X, the metabolic syndrome,
and equine grain-associated disorders: nomenclature and dietetics.
J Equine Vet Sci 2003;23:567–569.
10. Marchesini G, Forlani G, Cerrelli F, et al. WHO and ATPIII
proposals for the definition of the metabolic syndrome in patients
with Type 2 diabetes. Diabet Med 2004;21:383–387.
11. Freestone JF, Wolfsheimer KJ, Ford RB, et al. Triglyceride,
insulin, and cortisol responses of ponies to fasting and dexametha-
sone administration. J Vet Intern Med 1991;5:15–22.
12. Forhead AJ, French J, Ikin P, et al. Relationship between
plasma insulin and triglyceride concentrations in hypertriglyceri-
daemic donkeys. Res Vet Sci 1994;56:389–392.
13. Kronfeld DS, Treiber KH, Geor RJ. Comparison of nonspecif-
ic indications and quantitative methods for the assessment of insulin
resistance in horses and ponies. J Am Vet Med Assoc 2005;226:712–719.
JAVMA, Vol 228, No. 9, May 1, 2006
14. Eiler H, Frank N, Andrews FM, et al. Physiologic assess-
ment of blood glucose homeostasis via combined intravenous glu-
cose and insulin testing in horses. Am J Vet Res 2005;66:1598–1604.
15. Henneke DR, Potter GD, Kreider JL, et al. Relationship
between condition score, physical measurements and body fat per-
centage in mares. Equine Vet J 1983;15:371–372.
16. Eiler H, Oliver JW, Andrews FM, et al. Results of a combined
dexamethasone suppression/thyrotropin-releasing hormone stimula-
tion test in healthy horses and horses suspected to have a pars inter-
media pituitary adenoma. J Am Vet Med Assoc 1997;211:79–81.
17. Havel RJ, Eder HA, Bragdon JH. The distribution and chem-
ical composition of ultracentrifugally separated lipoproteins in
human serum. J Clin Invest 1955;34:1345–1353.
18. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measure-
ment with the Folin phenol reagent. J Biol Chem 1951;193:265–275.
19. Markwell MA, Haas SM, Bieber LL, et al. A modification of
EQUINE
the Lowry procedure to simplify protein determination in membrane
and lipoprotein samples. Anal Biochem 1978;87:206–210.
20. Watson TD, Burns L, Love S, et al. The isolation, character-
isation and quantification of the equine plasma lipoproteins. Equine
Vet J 1991;23:353–359.
21. Freestone JF, Wolfsheimer KJ, Kamerling SG, et al. Exercise
induced hormonal and metabolic changes in Thoroughbred horses:
effects of conditioning and acepromazine. Equine Vet J 1991;23:219–223.
22. Buff PR, Dodds AC, Morrison CD, et al. Leptin in horses:
tissue localization and relationship between peripheral concentra-
tions of leptin and body condition. J Anim Sci 2002;80:2942–2948.
23. McFarlane D, Beech J, Cribb A. Alpha-melanocyte stimulating
hormone release in response to thyrotropin releasing hormone in
healthy horses, horses with pituitary pars intermedia dysfunction and
equine pars intermedia explants. Domest Anim Endocrinol 2005;in press.
24. Robie SM, Janson CH, Smith SC, et al. Equine serum lipids:
serum lipids and glucose in Morgan and Thoroughbred horses and
Shetland ponies. Am J Vet Res 1975;36:1705–1708.
25. Zicker SC, Brumbaugh GW. Effects of phenylbutazone on
glucose tolerance and on secretion of insulin in healthy geldings. Am
J Vet Res 1989;50:743–746.
26. Kearns CF, McKeever KH, Abe T. Overview of horse body
composition and muscle architecture: implications for performance.
Vet J 2002;164:224–234.
27. Donaldson MT, McFarlane D, Jorgensen AJ, et al.
Correlation between plasma alpha-melanocyte-stimulating hormone
concentration and body mass index in healthy horses. Am J Vet Res
2004;65:1469–1473.
28. Forro M, Cieslar S, Ecker GL, et al. Total body water and
ECFV measured using bioelectrical impedance analysis and indica-
tor dilution in horses. J Appl Physiol 2000;89:663–671.
29. Kearns CF, McKeever KH, Kumagai K, et al. Fat-free mass is
related to one-mile race performance in elite standardbred horses. Vet
J 2002;163:260–266.
30. Reaven GM. Compensatory hyperinsulinemia and the
development of an atherogenic lipoprotein profile: the price paid to
maintain glucose homeostasis in insulin-resistant individuals.
Endocrinol Metab Clin North Am 2005;34:49–62.
31. Geor RJ, Hinchcliff KW, McCutcheon LJ, et al. Epinephrine
inhibits exogenous glucose utilization in exercising horses. J Appl
Physiol 2000;88:1777–1790.
32. Cartmill JA, Thompson DL Jr, Storer WA, et al. Endocrine
responses in mares and geldings with high body condition scores
grouped by high vs. low resting leptin concentrations. J Anim Sci
2003;81:2311–2321.
33. Forhead AJ, Smart D, Smith RF, et al. Transport-induced stress
responses in fed and fasted donkeys. Res Vet Sci 1995;58:144–151.
34. Kronfeld D, Rodiek A, Stull C. Glycemic indices, glycemic
loads, and glycemic dietetics. J Equine Vet Sci 2004;24:399–404.
35. Frank N, Andrews FM, Elliott SB, et al. Effects of rice bran
oil on plasma lipid concentrations, lipoprotein composition, and glu-
cose dynamics in mares. J Anim Sci 2005;83:2509–2518.
36. Fitzgerald BP, Reedy SE, Sessions DR, et al. Potential signals
mediating the maintenance of reproductive activity during the non-
breeding season of the mare. Reprod Suppl 2002;59:115–129.
37. Powell DM, Reedy SE, Sessions DR, et al. Effect of short-
term exercise training on insulin sensitivity in obese and lean mares.
Equine Vet J Suppl 2002;34:81–84.
Scientific Reports: Original Study 1389
05-10-0480.qxp 4/11/2006 1:25 PM Page 1390

38. Hawley JA. Exercise as a therapeutic intervention for the
prevention and treatment of insulin resistance. Diabetes Metab Res
Rev 2004;20:383–393.
39. Boden G, Laakso M. Lipids and glucose in type 2 diabetes:
what is the cause and effect? Diabetes Care 2004;27:2253–2259.
40. Poitout V, Robertson RP. Minireview: Secondary beta-cell
failure in type 2 diabetes—a convergence of glucotoxicity and lipo-
toxicity. Endocrinology 2002;143:339–342.
41. Frank N, Sojka JE, Latour MA. Effects of hypothyroidism
and withholding of feed on plasma lipid concentrations, concentra-
tion and composition of very-low-density lipoprotein, and plasma
lipase activity in horses. Am J Vet Res 2003;64:823–828.
42. Frank N, Sommardahl CS, Eiler H, et al. Effects of oral
administration of levothyroxine sodium on concentrations of plasma
lipids, concentration and composition of very-low-density lipopro-
EQUINE
tein metabolism in the horse (Equus caballus). Comp Biochem Physiol B
1993;104:45–53.
45. Watson TD, Packard CJ, Shepherd J. Plasma lipid transport
in the horse (Equus caballus). Comp Biochem Physiol B 1993;106:
27–34.
46. Geelen SN, Jansen WL, van Oldruitenborgh-Oosterbaan
MM, et al. Fat feeding increases equine heparin-released lipoprotein
lipase activity. J Vet Intern Med 2001;15:478–481.
47. Buff PR, Morrison CD, Ganjam VK, et al. Effects of short-
term feed deprivation and melatonin implants on circadian patterns
of leptin in the horse. J Anim Sci 2005;83:1023–1032.
48. Gordon ME, McKeever KH. Diurnal variation of ghrelin,
leptin, and adiponectin in Standardbred mares. J Anim Sci 2005;
83:2365–2371.
49. Fitzgerald BP, McManus CJ. Photoperiodic versus metabolic

teins, and glucose dynamics in healthy adult mares. Am J Vet Res
2005;66:1032–1038.
43. Watson TD, Burns L, Love S, et al. Plasma lipids, lipopro-
teins and post-heparin lipases in ponies with hyperlipaemia. Equine
Vet J 1992;24:341–346.
44. Watson TD, Burns L, Freeman DJ, et al. High density lipopro-
signals as determinants of seasonal anestrus in the mare. Biol Reprod
2000;63:335–340.
50. Donaldson MT, McDonnell SM, Schanbacher BJ, et al.
Variation in plasma adrenocorticotropic hormone concentration and
dexamethasone suppression test results with season, age, and sex in
healthy ponies and horses. J Vet Intern Med 2005;19:217–222.

Selected abstract for JAVMA readers from the

American Journal of Veterinary Research
Correlation of magnetic resonance images with anatomic features of the equine tarsus
Rafael Latorre et al

Objective—To correlate anatomic features of the equine tarsus identified in plastinated sections with
images obtained via magnetic resonance imaging (MRI). May 2006
Animals—4 horses.
Procedures—MRI (1.5-Tesla magnet) of the tarsus was performed on the pelvic limbs of 4 clinically normal
horses following euthanasia. After imaging, tarsocrural joint spaces and vasculature were injected with colored
See the midmonth
latex. Sagittal and transverse sections of the tarsi were plastinated to facilitate interpretation of MR images. issues of JAVMA
Results—Relevant anatomic structures were identified and labeled on the plastinated tissue slices
for the expanded table
and corresponding MRI images. Results indicated high correlations between MRI findings and those of of contents
plastinated sections. for the AJVR
Conclusions and Clinical Relevance—The data obtained provided certain reference standards for or log on to
normal anatomic structure sizes and positions in the equine tarsus. This information may aid future avmajournals.avma.org
physiologic or clinical studies of this joint. (Am J Vet Res 2006;67:756–761) for access
to all the abstracts.

1390 Scientific Reports: Original Study JAVMA, Vol 228, No. 9, May 1, 2006