Swine Models of Type 2 Diabetes Mellitus: Insulin Resistance, Glucose Tolerance, and Cardiovascular Complications

Dwight A. Bellinger, Elizabeth P. Merricks, and Timothy C. Nichols

Abstract
In humans, diabetes mellitus (DM) is considered a heterogeneous metabolic disorder. Swine have been used as a model for many human conditions including type 1 (insulindeficient) and type 2 (insulin-resistant) DM research because of their phenotypic similarities to humans including: cardiovascular anatomy and function, metabolism, lipoprotein profile, size, tendency to obesity, and omnivorous habits. There is phenotypic overlap between the two types of DM and pig models show characteristics and complications of both. Streptozotocin and alloxan have been used to create insulin deficient diabetes in pigs. One of the most unique and useful phenotypes is that these insulin-deficient pigs develop more severe coronary atherosclerosis than nondiabetic controls. It is not fully understood why patients with either type 1 or type 2 DM have increased severity and diffuseness of atherosclerosis compared with nondiabetic patients. The current human epidemic of type 2 DM and its attendant cardiovascular complications underscore the unmet need for creating a useful, readily available animal model of type 2 insulin resistant DM that also develops coronary artery atherosclerosis. The phenotypic susceptibility to coronary atherosclerosis makes pigs an attractive species to identify causative mechanisms. Suggested criteria for validation of an animal model of humanoid type 2 DM are described. The goal would be to develop a useful animal model for mechanistic studies as well as to develop and test novel therapeutics both for type 2 DM as well as its cardiovascular complications. Key Words: animal model; atherosclerosis; cardiovascular disease; glucose intolerance; insulin resistance; minipig; obesity; swine; type 2 diabetes mellitus

Introduction
iabetes mellitus (DM1) comprises a heterogeneous group of disorders with the common characteristic of elevated blood glucose (Harris 2004). The range of normal, prediabetic and diabetic glucose values for humans is shown in Figure 1 (Diagnosis and Classification of Diabetes Mellitus 2005). These values are derived from large population studies and are consensus driven. These values are also relevant to pigs and provide a framework for establishing the diagnosis of DM in experimental studies. A discussion of some of the similarities and differences in glucose metabolism and glucose tolerance between pigs and humans is given in a review of type 1 DM in the minipig (Larsen and Rolin 2004). Type 1 DM is due to pancreatic -cell destruction leading to insulin deficiency and is generally characterized by notable symptoms (weight loss, polyuria, and polydipsia), abrupt onset at a young age but usually after puberty, immune-mediated loss of -cells by anti-islet cell antibodies, and a need for exogenous insulin therapy. Type 2 DM is characterized by insulin resistance (IR1) and hyperglycemia and differs from type 1 DM in that patients are generally overweight and asymptomatic in the early stages. Type 2 DM usually has a slow onset and, until recently, most often occurred in adults (Kaufman 2005). IR is defined as a decreased biological response to normal concentrations of serum insulin that over time leads to compensatory hypersinsulinemia (Kohen-Avramoglu et al. 2003). Type 2 DM is often preceded by hyperinsulinemia and impaired glucose tolerance with 50% of patients with hyperinsulinemia progressing to develop diabetes (Kaufman 2005). Although fully manifested diabetes mellitus confers a greater risk for cardiovascular disease and other complications than isolated IR without hyperglycemia, the presence of IR is a major independent risk factor with relative risk

Dwight A. Bellinger, D.V.M., Ph.D., is a Professor in the Department of Pathology and Laboratory Medicine and Clinical Veterinarian in the Division of Laboratory Animal Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina (UNC-CHSM). Elizabeth P. Merricks, B.S., is a Graduate Student in the Department of Pathology and Laboratory Medicine, UNC-CH-SM. Timothy C. Nichols, M.D., is a Professor in the Departments of Medicine and Pathology and Laboratory Medicine and Director of the Francis Owen Blood Research Laboratory, UNC-CH-SM.

Abbreviations used in this article: DM, diabetes mellitus; FH, familial hypercholesterolemia; GLP glucagon-like peptide; GLUT4, glucose transporter 4; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; IGF, insulin growth factor; INSR, insulin receptor; IR, insulin resistance; IRS, insulin receptor substrate; LDL, low-density lipoprotein; LO, lipoxygenase; PLD, phospholipase-dependent; PPAR, peroxisomeproliferative-activated receptor; SMC, smooth muscle cell; SZT, streptozotocin; TG, triglyceride; TNF, tumor necrosis factor; VLDL, very lowdensity lipoprotein.

Volume 47, Number 3

2006

243

Figure 1 Glucose tolerance categories. The diagnosis of diabetes mellitus is based on fasting and 2-hr plasma glucose values on oral glucose tolerance test (OGTT). *The diagnosis of diabetes can be based on unequivocal symptoms and a random glucose measurement of 200 mg/dL. Adapted from the American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care January 28, 2005, Suppl 1:S37-S42.

ratios between 2.2 and 2.7 when multifactorial linear regression analysis is utilized to assess risk (Howard 1999; Kohen-Avramoglu et al. 2003; Taskinen 1995). Thus, both IR and hyperglycemia appear to mediate the pathophysiology of type 2 DM independently. This review focuses on different pig strains and models of diabetes mellitus with an emphasis on their relationship to insulin resistance and type 2 DM (Table 1). We include some pig models in which diabetes was induced with alloxan or streptozotocin (SZT1). Although these pigs may be considered to model type 1 DM (acute onset and low insulin levels), the features often overlap with many characteristics of type 2 DM (insulin resistance, dyslipidemia, and low insulin levels in the late stages). We also focus on the cardiovascular complications because other complications such as retinopathy, neuropathy, and nephropathy have not been extensively reported in these pig models.

verse consequences of these complications. Induction and maintenance of diabetes require adequate support. With adequate monitoring, good husbandry practices, and appropriate veterinary care, it is possible to minimize adverse effects and to produce new information that will benefit both experimental animals and humans.

Background
There is a growing epidemic of type 2 DM in children and adults and the associated diabetic complications, especially cardiovascular disease (Kohen-Avramoglu et al. 2003; Zimmet et al. 2001). Strategies to ameliorate IR have been hampered by a poor understanding of the genotypes predisposed to this phenotype and, specifically, the pathophysiological mechanisms that when activated in IR patients accelerate atherosclerosis and lead to other complications. Research has focused on the loss of peripheral insulin sensitivity in muscle that is associated with an abnormal increase in hepatic glucose output leading to changes in the rate of peripheral glucose disposal (Cline et al. 1999; Lofgren et al. 2000). Chronic hyperglycemia has deleterious effects on both insulin secretion and insulin activity (glucotoxicosis). Thus, hyperglycemia may be partially responsible for the defective insulin mediated glucose disposal in both type 1 and type 2 DM. In addition, it has been proposed that IR leads to -cell dysfunction, including decreased and delayed insulin secretion. Multiple murine models have been useful in studying the consequences of genetic manipulations that induce IR (Michael et al. 2000). The most informative among these have been structural changes in the insulin receptor (INSR1) and the downstream signaling molecules insulin receptor substrate (IRS1)-1 and IRS-2 (Kido et al. 2000; Michael et
ILAR Journal

Legal and Ethical Issues

Animal welfare laws vary considerably around the world. The literature includes numerous articles in which the use of animals in research is discussed (e.g., Akins et al. 2005; Tuffery 1995). It should be noted that there are those who believe that animals should not be used for experimental purposes. Nevertheless, it is possible to minimize distress and have humane endpoints with well-designed studies. Research involving animals should be a balance between knowledge gained and potential harm to animals. The utilization of pigs in diabetes research, whether induced experimentally or developed through breeding for selected traits, must take into account the potential adverse effects. To study the complications associated with diabetes, mechanisms should be in place to prevent or alleviate ad244

Volume 47, Number 3

Table 1 Characteristics of pig strains used as models of type 2 diabetes mellitusa Characteristics of diabetic state hyperglycemia, hyperinsulinemia Lipid profile
FFAHigh K mean 863 mEq/L Low K mean 1094 mEq/dL TGHigh K mean 145 mg/dL Low K 127-133 mg/dL Control approx 50 kg Become obese when fed ad lib Cholesterol (Dixon et al. 2002) Control 79 mg/dL HF 358 mg/dL Diabetic HF 419 mg/dL LDL cholesterol Control 35 mg/dL HF 197 mg/dL Diabetic HF 255 mg/dL HDL Control 32 mg/dL HF 127 mg/dL Diabetic HF 102 mg/dL HDL/LDL Control 1.03 HF 1.53 Diabetic HF 2.54 TG in overfed pigs Body wt Control 51 kg Overfed 107 kg Porcine Obesity index Control 0.47 Overfed 0.97 Body wt (Mokelke et al. 2005a) Control 60 kg Diabetic HF 69 kg

2006

Strain Diet
NA High K group K >3.5 Low K group K <1.5 Low K-hyperglycemic during lactation. glucose tolerance with high-fat diet Fasting glucose (Dixon et al. 2002) Control 49 mg/dL HF 43 mg/dL Diabetic HF 316 mg/dL

S/A

Body weight/ body composition Genetic alterations and complications

Use
Produced by selective breeding Not currently available

Yucatan (Panepinto et al. 1982; Phillips et al. 1982)

NA

Yucatan (Boullion et al. 2003; Korte et al. 2005; Mokelke et al. 2005a; Mokelke et al. 2003; Mokelke et al. 2002; Phillips et al. 1980) HF

Modified pancreatic receptor or postreceptor response Thickened capillary membranes in muscle sections Coronary artery and myocardial dysfunction Diabetic high-fat-fed pigs had increased atherosclerotic plaques in aorta Retinal capillary basement membrane thickening

Model of diabetic dyslipidemia and can be used to study mechanisms, interventions, and atherosclerosis. Model for early retinal microvascular change (intervention and mechanism)

Yucatan (Sebert et al. 2005a; Sebert et al. 2005b Overfed (1.5 cals)

Fasting glucose in overfed pigs (4.5 vs. 3.7 mmol/L) Glucose infusion rate (insulin sensitivity) Control 14.5 mg/kg/min Overfed 6.4 mg/kg/min

Modification of gene expression in skeletal muscles

Model for childhood obesity and insulin sensitivity

245

246

Table 1 (continued) Characteristics of pig strains used as models of type 2 diabetes mellitusa Characteristics of diabetic state hyperglycemia, hyperinsulinemia Lipid profile Genetic alterations and complications
Vascular dysfunction, mild carotid Atherosclerosis 12-mo-old body weight not given

Strain Diet
HF HC Fasting glucose Control 3 mmol/L HF 4 mmol/L Diabetic HF 17 mmol/L Fructosamine Control 175 mmol/L Diabetic HF 300 mmol/L

S/A

Body weight/ body composition Use

Sinclair (Dixon et al. 1999; Roberts et al. 2001)

Model for pharmacotherapy for prevention of vascular disease associated with diabetes

Gottingen (Johansen et al. 2001; Larsen and Rolin 2004; Larsen et al. 2002) HF HE

NA

TG Control 0.34 mmol/L HF 0.38 mmol/L Diabetic HF 0.99 mmol/L Cholesterol Control 1.9 mmol/L HF 7.76 mmol/L Diabetic HF 13 mmol/L LDL Control 0.98 mmol/L HF 3.54 mmol/L Diabetic HF 7.96 mmol/L Cholesterol & Control 1.82 mmol/L HF-HE 2.03 mmol/L TG Control 0.13 mmol/L HF-HE 0.24 mmol/L (TG with age) Body wt Control 21 kg HF-HE 26.8 kg (visibly obese) Control 25 kg HF-HE 33 kg % total body fat Control 6.1% HF-HE 13.2% Body wt Range 14-32 kg (multiple protocols) None given

Obese model that shares metabolic impairments with humans and may serve as a model of insulin resistance and metabolic syndrome

Go ttingen (Kjems et al. 2001; Larsen et al. 2003) NA NA

Fasting glucose Control 3.6 mM HF-HE 4.3 mM Insulin Control 23 pM HF-HE 80 pM Insulin response to IV glucose (AUC) Control 771 pM min HF-HE 6741 pM min Fasting glucose (S 125 + NIA 67 mg/kg) Control (pre) 3.6 mM Diabetic (post) 5.0 mM Fasting insulin Control (pre) 54.3 pM Diabetic (post) 25.5 pM Impaired glucose tolerance Impaired pulsatile insulin secretion

Reduced -cell mass Hyperglucagonemia

Model suitable for studying pathophysiology and agents for treatment of diabetes. Varying degrees of glucose intolerance and diabetes dependent on the dose of NIA + S

ILAR Journal

Volume 47, Number 3

Table 1 (continued) Characteristics of pig strains used as models of type 2 diabetes mellitusa

2006

Strain Diet
HF HS Fasting glucose (Yin et al. 2004) Control 101 mg/dL HFHS 165 mg/dL Fasting insulin Control 11 mU/mL HFHS 20 mU/mL HFHS insulin sensitivity Body wt Control 25 kg HFHS 40 kg HFHS in abdominal fat

S/A

Characteristics of diabetic state hyperglycemia, hyperinsulinemia Lipid profile Body weight/ body composition Genetic alterations and complications
Hepatic steatosis, mild aortic atherosclerosis HFHS plasma TNF

Use
Model of diabetic dyslipidemia for the study of treatments and pathophysiology.

Guizhou (Xi et al. 2004; Yin et al. 2004)

NA

Ossabaw (Dyson et al. 2006; Hausman et al. 1983; Meserole and Etherton 1984; Mokelke et al. 2005b) HF HF Fasting glucose Controls and HF 3.3-6.1 mmol/L (not different) Diabetic and Diabetic HF 3-4 times that of controls Cholesterol Control and Diabetic 4 mmol/L HF and Diabetic HF 18 mmol/L TG Control and Diabetic and HF 25 mmol/L Diabetic HF 70 mmol/L

NA

glucose clearance Insulin-resistant Hyperinsulinemia

Cholesterol Control 73 mg/dL HFHS 211 mg/dL HDL cholesterol Control 37 mg/dL HFHS 41 mg/dL TG Control 67 mg/dL HFHS 203 mg/dL FFA Control 118 mol/L HFHS 406 mol/L Compared with Yorkshire Triglyceride Cholesterol VLDL-C Obese 88 kg Two carcass adiposity Backfat 5.97 cm

Model for metabolic syndrome

Yorkshire (Askari et al. 2002; Gerrity et al. 2001; Natarajan et al. 2002; Suzuki et al. 2001)

10- to 12-mo-old Yorkshire pigs weigh 175 kg Backfat 2.64 cm

Obese Ossabaw become hypertensive neointima hyperplasia Glucose intolerance exacerbated with HF diet Dyslipidemia Severe Coronary and aortic atherosclerosis

Study of accelerated atherosclerosis in diabetes

247

a NA, not applicable; K, value, rate of glucose clearance; S, streptozotocin; A, Alloxan; HF, high fat; HS, high sucrose; HE, high energy; HC, high cholesterol; FFA, free fatty acids; IR, insulin resistance; LDL, low density lipoprotein; HDL, high density lipoprotein; TG, triglyceride; AUC, area under curve; NIA, nicotinamide.

Model of familial hypercholesterolemia Coronary and aortic atherosclerosis Potential for chemically induced diabetes and/or insulin resistance

al. 2000). These studies have shown that mutations altering insulin-stimulated glucose transport in muscle lead to secondary changes in fat cells that are similar to those that occur in type 2 DM. Likewise, loss of insulin sensitivity in muscle or fat also results in stimulation of hepatic glucose output. Recently, novel studies with peroxisomeproliferative-activated receptor (PPAR1) , , and genes and agonists have led to the discovery of new regulatory interactions between metabolism, inflammation, and atherosclerosis (Lee et al. 2003; Levi et al. 2003; Li et al. 2000; Tordjman et al. 2001). These genetically manipulated murine models have been very useful in characterizing the effects of altering the function/signaling of the INSR and PPAR pathways, but they do not recapitulate all of the essential elements of the predisposing phenotype (e.g., IR, obesity, hypertriglyceridemia, and hypercholesterolemia) that lead to impaired glucose tolerance and the development of type 2 DM. Notably, rodent models mimic some of the changes that occur in human IR, but the development of hyperlipidemia and atherosclerosis requires an additional genetic manipulation such as deletion of apolipoprotein-E or low-density lipoprotein (LDL1) receptor (Hasty et al. 2001; Zhang et al. 1992). The atherosclerosis that develops in these mice has minimal proliferative changes and occurs almost exclusively in proximal ascending aorta. Although adding or subtracting various genetic traits is useful in looking at genetic interactions, this requirement for multiple genetic manipulations may complicate the use of rodents for testing the role of subtle changes that IR might exert on diabetes complications. There is an unmet need for a useful animal model to determine the mechanisms that mediate the development of complications in IR type 2 DM patients (Kohen-Avramoglu et al. 2003; Zimmet et al. 2001). The rationale for choosing pigs is based on several issues. Humans and pigs have similar pharmacokinetics after subcutaneous drug administration, gastrointestinal structure and function, pancreas morphology, and overall metabolic status (Larsen and Rolin 2004). Moreover, pigs have been informative in studies of cardiovascular, renal, and ophthalmic complications associated with SZT-induced type 1 DM (Askari et al. 2002; Gerrity et al. 2001; Hainsworth et al. 2002; Marshall et al. 1980; Natarajan et al. 2002; Suzuki et al. 2001; Zhang et al. 2003). Pigs fed a high-fat high-cholesterol diet develop coronary, aortic, iliac, and carotid atherosclerotic lesions, anatomical locations extremely relevant to the human condition. Most importantly, these lesions recapitulate the histopathology seen in humans: proliferative lesions consisting of smooth muscle cells, macrophages, lymphocytes, foam cells, calcification, fibrous caps, necrotic and apoptotic cells, plaque hemorrhage, and expanded extracellular matrices (Brodala et al. 2005; Gerrity et al. 1979; Nichols et al. 1992; Prescott et al. 1991, 1995). Results of testing medicines (e.g., statins) and devices (e.g., stents) in pigs have been regarded as having a high positive predictive value for subsequent translation to humans (Hasler-Rapacz et al. 1996; Johnson et al. 1999).
ILAR Journal

Genetic alterations and complications Table 1 (continued) Characteristics of pig strains used as models of type 2 diabetes mellitusa Body weight/ body composition Characteristics of diabetic state hyperglycemia, hyperinsulinemia Diet S/A Strain Lipid profile
248

Use

Familial NA Hypercholesterolemic (FH) (Prescott et al. 1995; Prescott et al. 1991)

NA

Not developed

FH Cholesterol 316 mg/dL HDL-C 22 mg/dL LDL-C 275 mg/dL LDL/HDL 8.1 Triglycerides 56 mg/dL Normal Cholesterol 105 mg/dL HDL-C 33 mg/dL LDL-C 67 mg/dL LDL/HDL 2.2 Triglycerides 29 mg/dL

Mixed commercial breed background

Missense mutation (C253 T253) substituting Arg94 Cys94 in the LDL receptor and is inherited in an autosomal fashion.

However, human diabetics have not enjoyed the same benefit of drug-eluting stents as nondiabetic patients due to accelerated disease progression (Finn et al. 2005; Flaherty and Davidson 2005; Iakovou et al. 2005). The mechanism for this accelerated disease progression is unknown but is likely due to the systemic metabolic perturbations that occur in DM. Such mechanisms may also be operative in the development of diffuse atherosclerosis in peripheral (e.g., iliac, femoral, and carotid) arteries. Thus, pigs have great potential as a relevant animal model of insulin-resistant type 2 DM to identify mechanisms that lead to the development of diabetic complications and to develop and test novel therapeutic approaches.

Yucatan Minipigs
The Yucatan pig is native to the Yucatan Peninsula of Mexico. Its early development and characterization for laboratory use took place at Colorado State University (Panepinto 1996). Selective breeding has been reported to be a successful strategy for producing pigs with impaired glucose tolerance. During the 1970s, researchers at Colorado State University used this strategy to develop two lines of Yucatan minipigs with altered glucose tolerance, low K with impaired tolerance and high K with enhanced tolerance (Phillips and Panepinto 1986; Phillips et al. 1982). The impaired glucose tolerance was due to a decrease in peripheral insulin concentration resulting from decreased insulin secretion in response to a glucose challenge. The low serum insulin levels in this line did not appear to be due to impaired synthesis and storage of insulin but were consistent with a modified pancreatic receptor or postreceptor response as suggested by the finding that these pigs had normal insulin release in response to isoproterenol challenge. These pigs also had normal numbers of islets and -cells (Phillips and Panepinto 1986). Females from this strain rapidly became obese, and some developed insulin resistance and diabetes and exhibited both hyperinsulinemia and hyperglycemia during gestation and lactation. When these pigs were fed a high-fat diet and sucrose as the carbohydrate source, they had a marked increase in peripheral insulin concentration and prolonged elevation of plasma glucose (Phillips et al. 1981). In this study, impaired glucose tolerance or dietary changes did not affect serum lipids except for an elevation in fasting free fatty acid concentration. Although selective breeding was shown to be feasible, several generations were required. Glucose intolerance was not observed in the F7 generation of the low-K line (Hand et al. 1987), and these pigs are not currently available. Thus, chemical induction is an attractive alternative and has been the primary means for creating diabetic pigs. Recently a Yucatan minipig model of diabetic dyslipidemia at the University of Missouri at Coumbia has been described in which alloxan (175 mg/kg intravenously) was given to induce diabetes. Feed and fluids were used to maintain positive energy balance and prevent wasting (Boullion
Volume 47, Number 3 2006

et al. 2003). Plasma glucose was maintained at 300 to 400 mg/dL with supplemental insulin, dyslipidemia with plasma cholesterol levels at 280 to 405 mg/dL, and triglyceride levels at 55 to 106 mg/dL. Pigs maintained normal weight (lean) or gained two times normal weight (obese). The advantage of this model is that it is not a wasting disease as can occur in other hyperglycemic models. This diabetic swine model (alloxan and fat diet in the Yucatan strain) has been used to examine the effect of Atorvastatin on diabetes and atherogenesis by researchers at the University of Missouri (Dixon et al. 2002; Otis et al. 2003). Compared with dyslipidemic nondiabetic pigs, the diabetic swine had larger LDL that resembled intermediate-density lipoprotein (IDL1), increased coronary atherosclerosis as determined by intravascular ultrasound, and increased collagen deposition in internal mammary artery. Atorvastatin decreased total and very low-density lipoprotein (VLDL1) triglycerides and protected from or reduced the extent of atherosclerosis but had minimal effects on dyslipidemic profile or LDL/HDL ratio in the doses used. The authors suggest that Atorvastatin may have other antiatherogenic effects in diabetes perhaps by altering inflammation (Youssef et al. 2002). This diabetic model is one of the few pig studies in which microvascular disease has been examined and reported. After 20 wk, microvascular retinal changes were evaluated (Hainsworth et al. 2002). The diabetic pigs had thicker retinal capillary basement membranes than nondiabetic pigs. The dyslipidemia induced by feeding a highfat diet did not appear to contribute to the basement membrane thickening. This model can be useful in identifying mechanisms in diabetic retinopathy and evaluating early interventions. Researchers at the University of Missouri have extended the use of this model to show enhanced contractibility in coronary arteries of diabetic pigs and that this enhanced contractibility is prevented by exercise (Mokelke et al. 2005a). Diabetes was induced with alloxan (125 mg/kg intravenously), and animals were maintained at fasting blood glucose levels between 300 and 400 mg/dL and total cholesterol >300 mg/dL (high-fat, high-cholesterol, and sucrose diet) as before. Pigs that were trained to exercise on a treadmill exhibited a resting bradycardia and a lower heart rate in response to work load consistent with improved fitness. Coronary blood flow was lower in diabetic dyslipidemic pigs than in controls (nondiabetic, high-fat diet) and exercise on the treadmill prevented the decrease. Both sedentary and exercised diabetic dyslipidemic pigs had a lower response to bradykinin (endothelial-dependent relaxation) than controls (nondiabetic, high-fat diet). In prostaglandin F2-constricted vessels, dilation was depressed in sedentary diabetic dyslipidemic animals compared with the nondiabetic control and exercised diabetic dyslipidemic pigs. Adenosine (endothelial-independent) dilation was depressed in diabetic dyslipidemic compared with control and diabetic dyslipidemic exercised pigs. In vivo microvascular
249

tone was greater in diabetic dyslipidemic animals, and this was prevented by exercise. Recently, a model for childhood obesity and insulin resistance without chemical induction was created by overfeeding (1.5 times the recommended amount of calories) a Western style diet that was high in saturated fat and carbohydrates with a high glycemic index to 4-mo-old Yucatan minipigs until adulthood (Sebert et al. 2005a). The overfed pigs became obese and had a porcine obesity index (based on abdominal and neck measurements) and respective weight that were approximately twice those of controls fed normally. The overfed pigs also developed lower insulin sensitivity as measured by the euglycemic hyperinsulinemic clamp method than did control-fed pigs. Gene expression of glucose transporter 4 (GLUT 41), INSR, and PPAR was lower in muscle of adult overfed pigs compared with normal fed pigs consistent with increased insulin resistance. In adipose tissue, GLUT4 transcription was increased, indicating possible enhanced glucose uptake or lipogenesis. In addition, leptin expression was increased. Plasma insulin growth factor 1 (IGF-11) was increased in the younger overfed pigs compared with normally fed pigs. Overfed pigs had higher LDL-cholesterol:HDL-cholesterol ratios than pigs fed normally.

Go ttingen Minipigs
The Gttingen minipig was developed in the 1960s at the Institute of Animal Breeding and Genetics of the University of Gttingen, Germany, by crossbreeding the Vietnamese, Hormel, and German improved Landrace swine. Researchers at Novo Nordisk with others have developed and used the Gttingen minipig to study physiological aspects of diabetes and pharmacological therapies for diabetes. A summary of the Gttingen minipig as a model of diabetes was provided in a recent ILAR Journal issue (Larsen and Rolin 2004). The reference values for glucose and lipid homeostasis have been well characterized for the Gttingen minipig. Plasma glucose, leptin, fructosamine, insulin, c-peptide, triglyceride, free fatty acids, and cholesterol were measured as pigs aged and became heavier, revealing that plasma glucose, fructosamine, and triglycerides increase with age (Larsen et al. 2001). When fed a high-fat high-energy diet, the Gttingen minipig may serve as a model for the metabolic syndrome (Johansen et al. 2001). Female Gttingen minipigs fed a high-fat high-energy diet to induce obesity had increased body weight and fat content. Although preprandial plasma glucose and insulin concentrations were not altered, insulin response to intravenous glucose was increased (Johansen et al. 2001). Male Gttingen minipigs fed a high-fat high-energy diet also became obese (increased weight and body fat) and had increased fasting blood glucose and insulin levels compared with normally fed controls (Larsen et al. 2001). Although oral glucose tolerance was not changed, insulin response to intravenous glucose was increased while glucose clearance was unchanged. The Gttingen minipig with reduced -cell mass following nicotinamide (67 mg/kg intravenously to protect the -cells partially) and SZT (125 mg/kg intravenously) or alloxan (80 mg/kg intravenously) was used to examine changes in pulsatile insulin secretion. In this strain, a decrease in -cell mass leads to impaired pulsatile insulin secretion, reduced postprandial hepatic insulin clearance, and relative hyperglucagonemia (Kjems et al. 2001). Other studies with this strain have also demonstrated reduced insulin pulsatility. However, the authors indicate that neither -cell mass nor the slight hyperglycemia seen in this model completely accounted for the disturbed insulin pulsatility observed in human type 2 DM (Larsen et al. 2003). In addition to physiological studies, this model has been used to examine therapies for hyperglycemia. A glucagonlike peptide 1 (GLP11) derivative NN2211 was evaluated for treatment in hyperglycemic minipigs (Ribel et al. 2002). GLP1 is an effective but unstable antidiabetic agent, and NN2211 is a long-acting derivative of GLP1. After treatment with NN2211, more glucose was required in the hyperglycemic clamp procedure to maintain plasma glucose 1.5 to 2.0 mmol above baseline. Insulin variations were higher, and glucagon levels were suppressed. In oral glucose tolerance testing, NN2211 reduced glucose excursion with no change in insulin. This type of study demonstrates
ILAR Journal

Sinclair Minipigs
The Sinclair miniature pig was originally developed at the Hormel Institute of the University of Minnesota in the 1950s by breeding four feral strains. A Yorkshire domestic pig was later introduced into the strain. Significant characterization and development took place later at the Sinclair Comparative Medicine Research Farm at the University of Missouri (Panepinto 1996). Researchers at the University of Missouri have used alloxan (175 mg/kg intravenously)induced diabetic Sinclair minipigs to examine the relationship of diabetes to vascular dysfunction. The induced diabetic state was characterized by a postprandial hyperglycemia (6 times that of controls) and reduced insulin response to an intravenous glucose load, which generally did not require insulin support. Vascular function in diabetic Sinclair minipigs fed an atherogenic diet was compared with that of nondiabetic pigs fed an atherogenic diet and nondiabetic pigs fed a control diet (Dixon et al. 1999). The diabetic pigs fed the high-fat diet had lipoprotein profiles similar to human diabetics, with a shift of the cholesterol into the LDL fraction. In diabetic pigs fed the high-fat diet, 75% of coronary artery segments showed contractile oscillations in response to prostaglandin F2 compared with 25% for the high fat-fed nondiabetic pigs and 10% for nondiabetic pigs. Endothelial-dependent relaxation in response to bradykinin was nearly abolished in brachial arteries of the diabetic pigs fed the high-fat diet while remaining unchanged in the high-fat controls. Carotid artery atherosclerosis was increased in the high fat-fed diabetic pigs compared with the other groups.
250

the potential value of hyperglycemic swine in the development of new therapies for glucose control.

Yorkshire and Yorkshire-crossed Strains

The Yorkshire is an old breed developed in the Yorkshire area of northern England, and it was first imported into the United States in 1893. This commercial breed is noted for its large frame, long body, and bacon-type conformation (Anderson and Kiser 1967). This swine model has been well characterized over the past 20 yr for its use in atherosclerosis research. A Yorkshire swine model of SZT-induced diabetes (50 mg/kg/day intravenously for 3 days) maintained without supplemental insulin was established at the Medical College of Georgia with others by reducing -cell numbers to approximately 20% of normal (Askari et al. 2002; Gerrity et al. 2001; Natarajan et al. 2002; Suzuki et al. 2001). The hyperglycemia (fasting glucose levels 3-4 times that of controls) that resulted did not affect plasma cholesterol levels. These swine, like others with chemically induced diabetes, were reported to have impaired glucose tolerance to oral glucose tolerance testing. Compared with controls, coronary atherosclerosis was exacerbated, with lesions containing calcification and a higher rate of smooth muscle proliferation. Occlusive lesions were also reported in the small epicardial branches of the coronary arteries in diabetic pigs, with induction of dyslipidemia by feeding a high-fat diet. Monocyte/macrophage function that is altered by hyperlipidemia was further altered by diabetes. These diabetic dyslipidemic pigs had elevations in macrophage lipid metabolism. Monocyte acyl-CoA-cholesterol acyltransferase activity was increased with enhanced 12 lipoxogenase and H2O2 production. Although this model is established by the acute destruction of -cells that is characteristic of type 1 DM, the lack of insulin dependence, increased triglycerides, and glucose intolerance resemble late-stage type 2 DM to some extent. Because increased cardiovascular complications are seen in both type 1 and type 2 human diabetics, classification may be less important than understanding the mechanisms that mediate these increased complication rates. One of the most important aspects of using pigs to study vascular disease in diabetics is the ability to define the precise biochemical changes and mechanisms that initiate and perpetuate lesion progression, which are unique to the insulin-resistant and/or hyperglycemic state. There appears to be a lack of direct effect of glucose on smooth muscle cell (SMC1) proliferation in vitro. Studies were undertaken to determine the mechanism for the increase in smooth muscle accumulation and proliferation seen in the diabetic pigs (Askari et al. 2002). Diabetes increased plasma triglycerides that contained palmitic, linoleic, stearic, oleic, and arachidonic fatty acids. IGF-1 is abundant in atherosclerotic lesions of diabetic pigs, whereas arteries from nondiabetic controls contain little. Oleate and linoleate markedly enhance the growth-promoting effects of IGF-1 through phospholipaseVolume 47, Number 3 2006

dependent (PLD1) pathways in SMCs isolated from these diabetic dyslipidemic animals (Askari et al. 2002). The authors proposed that the diabetic accelerated atherosclerosis was caused in part by increased IGF-1-induced PLD activity and generation of diacylglycerol fueled by triglycerides containing oleate and linoleate. A valuable aspect of the swine model is that it can be used to extend the in vitro data to the in vivo situation, and to identify molecular mechanisms. The lipoxygenase (LO1) pathway is associated with oxidant stress and the pathogenesis of atherosclerosis. LO, a protein product of arachidonate metabolism, has been postulated to play a role in the accelerated atherosclerosis seen in diabetes. Immunostaining of abdominal and coronary arteries from diabetic pigs fed a high-fat diet demonstrated increased 12-LO protein compared with dyslipidemic nondiabetic pigs (Natarajan et al. 2002). Leukocyte-type 12-LO protein and 12-LO mRNA expression were also significantly greater in the arteries of the diabetic pigs. Increased oxidant stress as measured by the production of hydrogen peroxide after phorbol-myristate acetate stimulation was also observed in monocytes from peripheral blood in the dyslipidemic diabetic pigs compared with dyslipidemic-only and diabetic-only pigs, indicating a synergistic effect. This work then identified a mechanism by which oxidant stress may mediate diabetic complications. One study (Otieno et al. 2005) related to commercial pork production and involving a Yorkshire and Berkshire intercross was designed to determine whether polymorphisms of genes implicated in human hyperglycemia and insulin resistance were associated with porcine muscle and fat measurements. Although carried out for the potential enhancement of lean pork production, results may prove useful in identifying additional characteristics of swine for modeling type 2 DM. Investigators examined the genes for human proteins MAPK8, RETN, HSD11B1, and AKT2, which are implicated in type 2 DM. MAPK8 is implicated in type 2 DM by mediating tumor necrosis factor (TNF1)-induced insulin resistance. Disruption of MAPK8 in mice increases insulin sensitivity. RETN inhibits glucose uptake. HSD11B1 converts inactive glucose to active form, and inhibition of HSD11B1 decreases glucose concentrations in mice. AKT2 binds GLUT4 and mediates glucose binding in muscle and fat cells. In this study, polymorphisms of the MAPK8, RETN, and HSD11B1 genes were identified that were associated with fat deposition traits in pigs. Selective breeding for some of these polymorphisms may well produce better swine models of type 2 DM.

Chinese Guizhou Minipig

The Chinese Guizhou minipig is one of numerous strains of miniature pigs native to China, and it has proven suitable for biomedical research (Panepinto 1996). This strain has also been used to study the interaction of type 2 DM and atherosclerosis. Chinese Guizhou minipigs fed a high-fat highsucrose diet for 6 mo produced a mild elevation in plasma
251

total cholesterol and triglycerides without chemical destruction of islets (Xi et al. 2004). The animals were heavier and fatter. Fasting blood glucose was elevated compared with control diet animals, and plasma fasting insulin levels, although elevated initially, were not significantly different from controls. The pigs fed a high-fat high-sucrose diet were less sensitive to insulin than the control pigs. The authors speculated that this reduced sensitivity may represent induction of insulin resistance followed by progressive decline in pancreatic -cell function. These high-fat highsucrose-fed pigs were glucose intolerant and had impaired acute insulin secretion in oral glucose tolerance testing. They had higher plasma total cholesterol, triglycerides, and free fatty acids. They also had increased serum TNF, TNF, and free fatty acids, which are modulators of glucose metabolism. Obese humans and animals have increased TNF mRNA expression in adipose tissue. Histological analyses demonstrated adipose hypertrophy, high triglyceride staining, hepatic steatosis, and a decrease in pancreatic -cell mass. Atherosclerosis fatty streak lesions were present in the aorta of the pigs fed a high-fat high-sucrose diet. This Chinese Guizhou minipig fed a high-fat highsucrose diet has also been useful in new drug testing. A synthetic lipoprotein lipase activator (N0-1886) tested in this model inhibited adipose enlargement, suppressed plasma TNF, free fatty acids, and glucose, and increased glucose clearance and insulin response to intravenous loading. It also reduced total cholesterol and increased HDL cholesterol (Yin et al. 2004). This model should be advantageous in studying the effects of diabetes on atherosclerosis.

Ossabaw Pigs
Ossabaw pigs hold promise to overcome the lack of genetically determined swine models of type 2 DM (Buhlinger et al. 1978; Cote et al. 1982; Wangsness et al. 1980). Ossabaw pigs are believed to be the descendants of pigs brought from Spain that escaped and were isolated on the Ossabaw Islands off the coast of Georgia (Brisbin et al. 1977). Thus, this unique strain of pigs has lived in relative genetic isolation for centuries surviving on abundant food in the fall and relative starvation conditions in the winter. Such a scenario may have selected for a thrifty genotype, which has been offered as an explanation for the high prevalence of type 2 DM in certain populations (Lee et al. 2005; Prentice et al. 2005). The Ossabaw pigs have also been reported to demonstrate insulin resistance. It has been shown that insulin binding to liver microsomes during growth decreased in Yorkshire but not in Ossabaw pigs (Meserole and Etherton 1984). The binding affinity was lower for liver microenzymes in Ossabaw pigs. These findings have suggested that the liver in Ossabaw pigs is moderately insensitive to insulin. Plasma lipoproteins of Ossabaw pigs have been characterized in 10- to 12-mo-old animals. Ossabaw pigs have
252

increased triglycerides, cholesterol, and VLDL. The VLDL in Ossabaw pigs are two fold larger than Yorkshire. No differences were seen in LDL. The VLDL correlated with backfat thickness, and adiposity correlated with highdensity lipoprotein (HDL1) (Etherton and Kris-Etherton 1980). In contrast to contemporary commercial pigs, which are selected for leanness, Ossabaw pigs are considered obese, thus exhibiting an important risk factor for the development of type 2 DM. Notably, the backfat measures 2.64 cm in Yorkshire whereas it measures 5.97 cm in age-matched Ossabaw pigs (Hausman and Martin 1981). Dietary restrictions to 65% that of ad libitum dietary intake for 20 wk resulted in a seven fold decrease in adipose tissue, a six fold decrease in number of adipocytes, and a 50% decrease in muscle mass for Yorkshire pigs (Etherton and Kris-Etherton 1980; Etherton et al. 1982). However, in Ossabaw pigs this dietary restriction resulted in only a 2.6 % decrease in adipose mass, no change in number of adipocytes, and no loss of muscle mass. It has been reported that in Ossabaw pigs lipolytic enzyme adaptation occurs in adipose tissue but not liver, and that the enzymatic response to fasting and refeeding is more dynamic in lean pigs than Ossabaw pigs (Buhlinger et al. 1978). Ossabaw pigs are, however, not a large size. Yorkshire lean commercial pigs weigh 175 kg at 10 to 12 mo, whereas Ossabaw pigs weigh 88 kg at the same age. Yorkshire pigs are fast growing, whereas Ossabaw pigs are slow growing and have lower plasma growth hormone (Kasser et al. 1981; Wangsness et al. 1977). Recently Ossabaw pigs have been shown to be a novel model of metabolic syndrome (obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension) (Dyson et al. 2006). When fed a high-fat high-cholesterol diet for 20 to 40 wk, Ossabaw pigs were more obese, had higher peak glucose and insulin following glucose tolerance testing, had increased serum triglycerides, and developed higher blood pressure than Yucatan pigs fed the same diet (Dyson et al. 2005; Mokelke et al. 2005b). In addition, hyperlipidemic Ossabaw pigs that appear to model the metabolic syndrome had greater neointimal hyperplasia in stented segments of coronary arteries compared with Yucatan pigs (Zafar et al. 2005). These findings make the Ossabaw pig an exciting model for continued studies to investigate the accelerated cardiovascular complications associated with diabetes without chemical induction.

Familial Hypercholesterolemic Pigs

Pigs with familial hypercholesterolemia (FH1) created by Drs. Jan and Judith Rapacz in Madison, Wisconsin, spontaneously exhibit hypercholesterolemia and develop severe coronary and abdominal aortic atherosclerosis while being fed low fat pig chow (Prescott et al. 1991, 1995). The pig FH causative mutation represents a missense mutation (C253T253), in which Arg94Cys94 is substituted in the LDL receptor and is inherited in an autosomal fashion
ILAR Journal

(Grunwald et al. 1999; Hasler-Rapacz et al. 1998). Like FH humans, FH pigs have hypercholesterolemia mostly from increased LDL cholesterol; however, they also have depressed HDL cholesterol and moderate triglyceridemia (Grunwald et al. 1999; Hasler-Rapacz et al. 1994, 1998). This spontaneous dyslipidemia is also characteristic of that found in IR humans and thus is desirable scientifically (Garvey et al. 2003; Goff et al. 2005). Because these pigs exhibit this hyperlipidemia on low-fat pig chow, they are significantly less expensive to maintain than normocholesterolemic pigs fed a high-fat diet. Currently, efforts are under way at the University of North Carolina at Chapel Hill to develop a pig model of type 2 DM in FH pigs.

seven-step process to characterize an animal model of human type 2 DM is listed in Table 2. The criteria that each step addresses are discussed briefly below.

Step 1: Document Fasting Hypersinsulinemia, Insulin Sensitivity, and Serum Glucose Levels
Linking insulin resistance to a given diabetic complication requires documenting its severity during the study period. Fasting and postprandial glucose and insulin levels, insulin clamping studies, and frequently sampled insulin glucose tolerance testing have been used (Garvey et al. 2003; Goff et al. 2005). These assays must be reproducible and sensitive to variables that alter insulin sensitivity. For example, large weight gains or losses would be predicted to be accompanied by inverse changes in insulin sensitivity. To measure long-term glucose control, hemoglobin A1C is not applicable in pigs because pig red blood cells are relatively impermeable to glucose (Higgins et al. 1982). Instead, fructosamine is used as a measure of glucose concentrations over the preceding 1 to 3 wk.

Low-Birth-Weight Pigs and Type 2 Diabetes Mellitus

In humans, low birth weight is associated with an increased risk of glucose intolerance and type 2 DM and cardiovascular disease in adult life (Poore and Fowden 2004). The thrifty genotype hypothesis proposes that suboptimal nutrition in utero causes adaptations to occur in the fetus to slow growth but these adaptations are detrimental once full nutrition is achieved. Notably, low-birth-weight piglets have abnormal cardiovascular function and poor glucose metabolism (Poore et al. 2002). Insulin sensitivity measured as a decrease in plasma glucose after intravenous injection of insulin was reduced in pigs as adults that were smaller in the neonatal period. The study suggests that postnatal growth in pigs, rather than low birth weight per se, was an important determinant of insulin sensitivity as an adult.

Step 2: Determine Lipoprotein Concentration and Particle Size

Detailed lipoprotein analyses are essential to understanding the relevance of a given animal model to the dyslipidemia present in the majority of human type 2 DM. The human IR phenotype is characterized by accumulation of small dense LDL particles as opposed to large LDL particles (Carr et al. 1999; Gardner et al. 1996; Kohen-Avramoglu et al. 2003) and by suppression of large HDL particles (Brown et al. 1990; Deeb et al. 2003; Garvey et al. 2003; Goff et al. 2005; Howard et al. 2003). Thus, not only are the magnitude of lipoprotein changes important, but the distribution and size of particles are also important in macrovascular disease.

Criteria for Validating Models of Type 2 Insulin-resistant Diabetes Mellitus

Complications due to diabetes mellitus generally take years to develop in humans. Cardiovascular complications remain the most common cause of death among diabetics, but considerable morbidity and mortality are also associated with an increased risk of complications in renal, ophthalmological, gastrointestinal, and central and peripheral nerve tissues. Modeling these complications in animals also requires allowing sufficient time. The National Institutes of Healthsponsored website for the Animal Models of Diabetic Complications Consortium (AMDCC) has many detailed protocols listed for characterizing renal, neurological, retinal, and cardiovascular complications in rodent models of diabetes that can be extrapolated to pigs: https:// www.amdcc.org/index.aspx. Carefully designed studies in validated models are needed to determine whether future mechanistic and intervention studies could be done in a shorter time frame and thus with less expense. Such studies must account for the pathophysiological changes that occur in type 2 DM to link these changes to the complications. A
Volume 47, Number 3 2006

Step 3: Obtain Total Body Fat Measurements

An estimate of total body fat will determine whether there is a correlation between total body fat and IR and whether changes in total body fat over time correlate with changes in Table 2 Steps for validating models of type 2 insulin-resistant diabetes mellitus
1. Document fasting hyperinsulinemia, insulin sensitivity, and serum glucose levels. 2. Determine lipoprotein concentration and particle size. 3. Obtain total body fat measurements. 4. Characterize serum markers. 5. Measure arterial blood pressure. 6. Document end-organ damage due to type 2 insulin-resistant diabetes mellitus. 7. Document reduction in end-organ damage due to type 2 insulin-resistant diabetes mellitus with intervention

253

IR to determine the influence of total body fat on the degree of IR and diabetes complications. As in humans, the distribution of body fat in swine may be important in determining the risk associated with obesity, so the distribution should be analyzed (Dyson et al. 2006). Noninvasive measures of body fat allow for serial measurements throughout the course of a study. Recent studies in Yucatan swine have shown that simple measures can be predictive (Witczak et al. 2005).

Step 4: Characterize Serum Markers

Regulatory factors secreted from adipose tissue (adipokines) are the subject of increased research to determine their role in obesity and type 2 DM. C-reactive protein, interleukin-6, TNF, leptin, plasminogen activator inhibitor-1, adiponectin, resistin, and fibrinogen are frequently elevated in human type 2 DM (Harris et al. 1999; Hunt et al. 2002). In the currently available animal models of type 2 DM, it is not yet known whether (1) changes in the levels of these markers can distinguish between insulin sensitive and IR animals, (2) there is a progressive change in inflammatory markers over time, (3) these changes correlate with the severity of complications, (4) the changes in these markers correlate with changes in fat mass and changes in insulin resistance, and importantly (5) which measurements correlate best with the changes that have been measured in affected humans.

is the most common cause of death and disability among human type 2 diabetics. Thus, measuring atherosclerosis in a given animal model must account for severity and diffuseness as well as the rate of progression. Endothelial function studies and assessment of vascular reactivity as described in studies above are also important to consider. Screening the urine for protein and performing definitive histological analysis are required to determine whether IR is associated with the development of renal disease. The histopathological characterization would include light and electron microscopic detection of renal histopathology and immunohistochemical detection of immunoglobulin and complement deposition. Characterization of changes in cardiac muscle, retinal arteries, and central and peripheral nerves depends on study design and target organs. Current animal models of diabetic microangiopathy have limitations. Although the pig has proven useful for studying macrovascular disease, there are very few references in the literature to microvascular disease in diabetic pigs. It is necessary to collect tissues, especially from long-term studies, to evaluate the microvasculature. Finally, documenting changes in expression levels of insulin-sensitive genes in liver (e.g., FOXO-1 and PEPCK) as well as fat and skeletal muscle will be very important for the design of subsequent mechanistic studies that will lower insulin resistance.

Step 5: Measure Arterial Blood Pressure

A large percentage of human type 2 diabetics are hypertensive, therefore control of blood pressure is essential to retard or prevent complications such as renal failure, retinopathy, or accelerated atherosclerosis (Bell et al. 2005; Buch et al. 2005; Cueto-Manzano et al. 2005; McFarlane et al. 2005; Teitelbaum et al. 2005). Validated noninvasive methods for measuring blood pressure are available in several species (Krege et al. 1995; Mesangeau et al. 2000; Springett et al. 2001). The range of pig arterial blood pressure values is very close to that of humans and varies to some extent with age, gender, and strain (Chow et al. 1999; Goodrich et al. 2001). Of note, blood pressure appears to be dysregulated in pigs with SZT-induced, insulin-deficient DM (Mesangeau et al. 2000). It should be noted that most studies in humans that establish levels considered hypertensive are considerably larger than is feasible in most experimental pig herds. Thus, comparison of diabetic versus controls (i.e., concurrent nondiabetic pigs or changes with induction of DM) will help establish the impact of changes in blood pressure on the specific complication(s) being studied.

Step 7. Document Reduction in End-organ Damage Due to Type 2 Insulin-resistant Diabetes Mellitus with Intervention
To document that an animal exhibiting these criteria is a valid model of human type 2 DM and its complications requires mechanistic studies in appropriate numbers of treated and control animals showing that treatment of the DM is accompanied by prevention or reduction of the severity of a given complication.

Summary and Future Directions

Pigs have many attributes that make them suitable for creating an animal model of type 2 DM to determine the mechanisms that mediate cardiovascular complications. Important examples of attributes include the following: Pigs develop atherosclerosis in anatomical locations that are relevant to the human condition (Brodala et al. 2005; Hasler-Rapacz et al. 1995). These animals represent an ideal model for identifying causative mechanisms and testing new treatment strategies in these vascular beds, especially in the presence of type 2 DM. Pigs develop atherosclerotic lesions that recapitulate the histopathology seen in humans (Brodala et al. 2005; Gerrity et al. 1979; Nichols et al. 1992; Prescott et al. 1991, 1995). The strains of pigs reviewed in this article docuILAR Journal

Step 6: Document End-organ Damage Due to Type 2 Insulin-resistant Diabetes Mellitus

Development of severe and diffuse coronary, aortic, and peripheral (e.g., iliac, femoral, and carotid) atherosclerosis
254

ment that pigs recapitulate many of the metabolic abnormalities present in type 2 DM. The current limitations pigs impose are mostly due to size and expense. The Ossabaw pigs hold promise for providing animals of a more suitable size and an inherited predilection to insulin resistance. FH pigs exhibit a spontaneous dyslipidemia characteristic of that found in IR humans, obviating the need for and expense of additional dietary supplementation. It should be emphasized that defining the precise biochemical changes that occur in type 2 DM and correlating these changes with specific complications will be essential for identifying potential mechanisms that initiate and perpetuate complications that are unique to the insulin-resistant and/or hyperglycemic state. The pig model appears to be ideal for using these mechanistic data to develop and test potential new treatment strategies. The ultimate validation of an animal model of diabetic complications will require reducing the severity of a given complication with successful treatment of the DM, including metabolic control with decreased insulin resistance or hyperglycemia, implantation of devices (e.g., stents), or other novel therapeutics.

Acknowledgments
We acknowledge support in part from the National Institutes of Health grant RO1 HL069364 in the preparation of this manuscript, as well as the contributions of John Buse in the preparation of Figure 1.

References
Akins CK, Panicker S, Cunningham CL. 2005. Laboratory Animals in Research and Teaching: Ethics, Care and Methods. Washington DC: American Psychological Association. Anderson AL, Kiser JJ. 1967. Introductory Animal Science. New York: The MacMillan Co. p 481-511. Askari B, Carroll MA, Capparelli M, Kramer F, Gerrity RG, Bornfeldt KE. 2002. Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells. J Biol Chem 277: 36338-36344. Bell RA, Camacho F, Duren-Winfield VT, Bonds DE, Anderson RT, Konen JC, Goff DC Jr. 2005. Improving diabetes care among low-income North Carolinians: Project IDEAL. N C Med J 66:96-102. Boullion RD, Mokelke EA, Wamhoff BR, Otis CR, Wenzel J, Dixon JL, Sturek M. 2003. Porcine model of diabetic dyslipidemia: Insulin and feed algorithms for mimicking diabetes mellitus in humans. Comp Med 53:42-52. Brisbin IL, Geiger RA, Graves HB, Pinder JE III, Sweeney JM, Sweeney JR. 1977. Morphological characterization of two populations of feral swine. Acta Theriol 22:75-85. Brodala N, Merricks EP, Bellinger DA, Damrongsri D, Offenbacher S, Beck J, Madianos P, Sotres D, Chang YL, Koch G, Nichols TC. 2005. Porphyromonas gingivalis bacteremia induces coronary and aortic atherosclerosis in normocholesterolemic and hypercholesterolemic pigs. Arterioscler Thromb Vasc Biol 25:1456-1451. Brown G, Albers JJ, Fisher LD, Schaefer SM, Lin JT, Kaplan C, Zhao XQ, Bisson BD, Fitzpatrick VF, Dodge HT. 1990. Regression of coronary

artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. N Engl J Med 323:1289-1298. Buch HN, Barton DM, Varughese GI, Bradbury S, Scarpello JH, Walker AB. 2005. An assessment of the coverage of a district-wide diabetic retinopathy screening service. Diabetic Med 22: 840-841. Buhlinger CA, Wangsness PJ, Martin RJ, Ziegler JH. 1978. Body composition, in vitro lipid metabolism and skeletal muscle characteristics in fast-growing, lean and in slow-growing, obese pigs at equal age and weight. Growth 42:225-236. Carr MC, Hokanson JE, Deeb SS, Purnell JQ, Mitchell ES, Brunzell JD. 1999. A hepatic lipase gene promoter polymorphism attenuates the increase in hepatic lipase activity with increasing intra-abdominal fat in women. Arterioscler Thromb Vasc Biol 19:2701-2707. Chow PK, Ng TH, Heng D, Mack PO. 1999. A simple method of blood pressure measurement in the pig using a neonatal cuff. Ann Acad Med Singapore 28:15-19. Cline GW, Petersen KF, Krssak M, Shen J, Hundal RS, Trajanoski Z, Inzucchi S, Dresner A, Rothman DL, Shulman GI. 1999. Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes. N Engl J Med 341:240-246. Cote PJ, Wangsness PJ, Varela-Alvarez H, Griel LC, Jr., Kavanaugh JF. 1982. Glucose turnover in fast-growing, lean and in slow-growing, obese swine. J Anim Sci 54:89-94. Cueto-Manzano AM, Cortes-Sanabria L, Martinez-Ramirez HR, RojasCampos E, Barragan G, Alfaro G, Flores J, Anaya M, Canales-Munoz JL. 2005. Detection of early nephropathy in Mexican patients with type 2 diabetes mellitus. Kidney Int Suppl S40-S45. Deeb SS, Zambon A, Carr MC, Ayyobi AF, Brunzell JD. 2003. Hepatic lipase and dyslipidemia: Interactions among genetic variants, obesity, gender, and diet. J Lipid Res 44:1279-1286. Diagnosis and Classification of Diabetes Mellitus. 2005. Diabetes Care 28:S37-S42. Dixon JL, Shen S, Vuchetich JP, Wysocka E, Sun GY, Sturek M. 2002. Increased atherosclerosis in diabetic dyslipidemic swine: Protection by atorvastatin involves decreased VLDL triglycerides but minimal effects on the lipoprotein profile. J Lipid Res 43:1618-1629. Dixon JL, Stoops JD, Parker JL, Laughlin MH, Weisman GA, Sturek M. 1999. Dyslipidemia and vascular dysfunction in diabetic pigs fed an atherogenic diet. Arterioscler Thromb Vasc Biol 19:2981-2992. Dyson MC, Alloosh M, Boullion RD, Mokelke EA, Sturek M. 2005. Glucose intolerance and insulin resistance in Ossabaw compared to Yucatan swine. Swine in Biomedical Research Conference (http:// www.conference.uiuc.edu/Swine/swine_Abstract_Booklet.pdf). Dyston MC, Alloosh M, Vuchetich JP, Mokelke EA, Sturek M. 2006. Components of metabolic syndrome and coronary artery disease in female Ossabaw swine fed excess atherogenic diet. Comp Med 56:3545. Etherton TD, Kris-Etherton PM. 1980. Characterization of plasma lipoproteins in swine with different propensities for obesity. Lipids 15:823829. Etherton TD, Wangsness PJ, Hammers VM, Ziegler JH. 1982. Effect of dietary restriction on carcass composition and adipocyte cellularity of swine with different propensities for obesity. J Nutr 112:2314-2323. Finn AV, Palacios IF, Kastrati A, Gold HK. 2005. Drug-eluting stents for diabetes mellitus: A rush to judgment? J Am Coll Cardiol 45:479-483. Flaherty JD, Davidson CJ. 2005. Diabetes and coronary revascularization. Jama 293:1501-1508. Gardner CD, Fortmann SP, Krauss RM. 1996. Association of small lowdensity lipoprotein particles with the incidence of coronary artery disease in men and women. Jama 276:875-881. Garvey WT, Kwon S, Zheng D, Shaughnessy S, Wallace P, Hutto A, Pugh K, Jenkins AJ, Klein RL, Liao Y. 2003. Effects of insulin resistance and type 2 diabetes on lipoprotein subclass particle size and concentration determined by nuclear magnetic resonance. Diabetes 52:453462. Gerrity RG, Naito HK, Richardson M, Schwartz CJ. 1979. Dietary induced atherogenesis in swine: Morphology of the intima in prelesion stages. Am J Pathol 95:775-792.

Volume 47, Number 3

2006

255

Gerrity RG, Natarajan R, Nadler JL, Kimsey T. 2001. Diabetes-induced accelerated atherosclerosis in swine. Diabetes 50:1654-1665. Goff DC Jr, DAgostino RB Jr, Haffner SM, Otvos JD. 2005. Insulin resistance and adiposity influence lipoprotein size and subclass concentrations. Results from the Insulin Resistance Atherosclerosis Study. Metabolism 54:264-270. Goodrich JA, Lackland DT, Del Signore MJ, Swindle MM. 2001. Noninvasive measurement of blood pressures in the Yucatan micropig (Sus scrofa domestica), with and without midazolam-induced sedation. Comp Med 51:13-15. Grunwald KA, Schueler K, Uelmen PJ, Lipton BA, Kaiser M, Buhman K, Attie AD. 1999. Identification of a novel Arg--Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia in pigs. J Lipid Res 40:475-485. Hainsworth DP, Katz ML, Sanders DA, Sanders DN, Wright EJ, Sturek M. 2002. Retinal capillary basement membrane thickening in a porcine model of diabetes mellitus. Comp Med 52:523-529. Hand MS, Surwit RS, Rodin J, Van Order P, Feinglos MN. 1987. Failure of genetically selected miniature swine to model NIDDM. Diabetes 36:284-287. Harris MI. 2004. Definition and classification of diabetes mellitus and the criteria for diagnosis. In: Diabetes Mellitus: A Fundamental and Clinical Text. Philadelphia: Lippincott Williams & Wilkins. p 457-467. Harris TB, Ferrucci L, Tracy RP, Corti MC, Wacholder S, Ettinger WH, Jr., Heimovitz H, Cohen HJ, Wallace R. 1999. Associations of elevated interleukin-6 and C-reactive protein levels with mortality in the elderly. Am J Med 106:506-512. Hasler-Rapacz J, Ellegren H, Fridolfsson AK, Kirkpatrick B, Kirk S, Andersson L, Rapacz J. 1998. Identification of a mutation in the low density lipoprotein receptor gene associated with recessive familial hypercholesterolemia in swine. Am J Med Genet 76:379-386. Hasler-Rapacz J, Kempen HJ, Princen HM, Kudchodkar BJ, Lacko A, Rapacz J. 1996. Effects of simvastatin on plasma lipids and apolipoproteins in familial hypercholesterolemic swine. Arterioscler Thromb Vasc Biol 16:137-143. Hasler-Rapacz J, Prescott MF, Von Linden-Reed J, Rapacz JM, Hu Z, Rapacz J. 1995. Elevated concentrations of plasma lipids and apolipoproteins B, C-III, and E are associated with the progression of coronary artery disease in familial hypercholesterolemic swine. Arterioscler Thromb Vasc Biol 15:583-592. Hasler-Rapacz JO, Nichols TC, Griggs TR, Bellinger DA, Rapacz J. 1994. Familial and diet-induced hypercholesterolemia in swine: Lipid, ApoB, and ApoA-I concentrations and distributions in plasma and lipoprotein subfractions. Arterioscler Thromb 14:923-930. Hasty AH, Shimano H, Osuga J, Namatame I, Takahashi A, Yahagi N, Perrey S, Iizuka Y, Tamura Y, Amemiya-Kudo M, Yoshikawa T, Okazaki H, Ohashi K, Harada K, Matsuzaka T, Sone H, Gotoda T, Nagai R, Ishibashi S, Yamada N. 2001. Severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor. J Biol Chem 276:37402-37408. Hausman GJ, Campion DR, Thomas GB. 1983. Adipose tissue cellularity and histochemistry in fetal swine as affected by genetic selection for high or low backfat. J Lipid Res 24:223-228. Hausman GJ, Martin RJ. 1981. Subcutaneous adipose tissue development in Yorkshire (lean) and Ossabaw (obese) pigs. J Anim Sci 52:14421449. Higgins PJ, Garlick RL, Bunn HF. 1982. Glycosylated hemoglobin in human and animal red cells: Role of glucose permeability. Diabetes 31:743-748. Howard BV. 1999. Insulin resistance and lipid metabolism. Am J Cardiol 84:28J-32J. Howard BV, Criqui MH, Curb JD, Rodabough R, Safford MM, Santoro N, Wilson AC, Wylie-Rosett J. 2003. Risk factor clustering in the insulin resistance syndrome and its relationship to cardiovascular disease in postmenopausal white, black, hispanic, and Asian/Pacific Islander women. Metabolism 52:362-371. Hunt KJ, Pankow JS, Offenbacher S, Kritchevsky SB, Duncan BB, Shahar E, Sharrett AR, Heiss G. 2002. B-mode ultrasound-detected carotid

artery lesions with and without acoustic shadowing and their association with markers of inflammation and endothelial activation: The atherosclerosis risk in communities study. Atherosclerosis 162:145-155. Iakovou I, Schmidt T, Bonizzoni E, Ge L, Sangiorgi GM, Stankovic G, Airoldi F, Chieffo A, Montorfano M, Carlino M, Michev I, Corvaja N, Briguori C, Gerckens U, Grube E, Colombo A. 2005. Incidence, predictors, and outcome of thrombosis after successful implantation of drug-eluting stents. JAMA 293:2126-2130. Johansen T, Hansen HS, Richelsen B, Malmlof R. 2001. The obese Gttingen minipig as a model of the metabolic syndrome: Dietary effects on obesity, insulin sensitivity, and growth hormone profile. Comp Med 51:150-155. Johnson GJ, Griggs TR, Badimon L. 1999. The utility of animal models in the preclinical study of interventions to prevent human coronary artery restenosis: Analysis and recommendations. On behalf of the Subcommittee on Animal, Cellular and Molecular Models of Thrombosis and Haemostasis of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 81:835-843. Kasser TR, Martin RJ, Gahagan JH, Wangsness PJ. 1981. Fasting plasma hormones and metabolites in feral and domestic newborn pigs. J Anim Sci 53:420-426. Kaufman FR. 2005. Type 2 diabetes in children and youth. Endocrinol Metab Clin N Am 34:659-676, ix-x. Kido Y, Burks DJ, Withers D, Bruning JC, Kahn CR, White MF, Accili D. 2000. Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2. J Clin Invest 105:199-205. Kjems LL, Kirby BM, Welsh EM, Veldhuis JD, Straume M, McIntyre SS, Yang D, Lefebvre P, Butler PC. 2001. Decrease in beta-cell mass leads to impaired pulsatile insulin secretion, reduced postprandial hepatic insulin clearance, and relative hyperglucagonemia in the minipig. Diabetes 50:2001-2012. Kohen-Avramoglu R, Theriault A, Adeli K. 2003. Emergence of the metabolic syndrome in childhood: An epidemiological overview and mechanistic link to dyslipidemia. Clin Biochem 36:413-420. Korte FS, Mokelke EA, Sturek M, McDonald KS. 2005. Exercise improves impaired ventricular function and alterations of cardiac myofibrillar proteins in diabetic dyslipidemic pigs. J Appl Physiol 98:461-47. Krege JH, Hodgin JB, Hagaman JR, Smithies O. 1995. A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25:1111-1115. Larsen MO, Gotfredsen CF, Wilken M, Carr RD, Porksen N, Rolin B. 2003. Loss of beta-cell mass leads to a reduction of pulse mass with normal periodicity, regularity and entrainment of pulsatile insulin secretion in Gttingen minipigs. Diabetologia 46:195-202. Larsen MO, Rolin B. 2004. Use of the Gttingen minipig as a model of diabetes, with special focus on type 1 diabetes research. ILAR J 45: 303-313. Larsen MO, Rolin B, Wilken M, Carr RD, Svendsen O. 2002. High-fat high-energy feeding impairs fasting glucose and increases fasting insulin levels in the Gttingen minipig: Results from a pilot study. Ann N Y Acad Sci 967:414-423. Larsen MO, Rolin B, Wilken M, Carr RD, Svendsen O, Bollen P. 2001. Parameters of glucose and lipid metabolism in the male Gttingen minipig: Influence of age, body weight, and breeding family. Comp Med 51:436-442. Lee CH, Chawla A, Urbiztondo N, Liao D, Boisvert WA, Evans RM, Curtiss LK. 2003. Transcriptional repression of atherogenic inflammation: Modulation by PPARdelta. Science 302:453-457. Lee HK, Park KS, Cho YM, Lee YY, Pak YK. 2005. Mitochondria-based model for fetal origin of adult disease and insulin resistance. Ann N Y Acad Sci 1042:1-18. Levi Z, Shaish A, Yacov N, Levkovitz H, Trestman S, Gerber Y, Cohen H, Dvir A, Rhachmani R, Ravid M, Harats D. 2003. Rosiglitazone (PPARgamma-agonist) attenuates atherogenesis with no effect on hyperglycaemia in a combined diabetes-atherosclerosis mouse model. Diabetes Obes Metab 5:45-50. Li AC, Brown KK, Silvestre MJ, Willson TM, Palinski W, Glass CK.

256

ILAR Journal

2000. Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice. J Clin Invest 106:523-531. Lofgren P, van Harmelen V, Reynisdottir S, Naslund E, Ryden M, Rossner S, Arner P. 2000. Secretion of tumor necrosis factor-alpha shows a strong relationship to insulin-stimulated glucose transport in human adipose tissue. Diabetes 49:688-692. Marshall M, Oberhofer H, Staubesand J. 1980. Early micro- and macroangiopathy in the streptozotocin diabetic minipig. Res Exp Med (Berl) 177:145-158. McFarlane SI, Castro J, Kaur J, Shin JJ, Kelling D, Jr., Farag A, Simon N, El-Atat F, Sacerdote A, Basta E, Flack J, Bakris G, Sowers JR. 2005. Control of blood pressure and other cardiovascular risk factors at different practice settings: Outcomes of care provided to diabetic women compared to men. J Clin Hypertens (Greenwich) 7:73-80. Mesangeau D, Laude D, Elghozi JL. 2000. Early detection of cardiovascular autonomic neuropathy in diabetic pigs using blood pressure and heart rate variability. Cardiovasc Res 45:889-899. Meserole VK, Etherton TD. 1984. Insulin binding to liver microsomes from lean and obese swine during growth to market weight. J Anim Sci 59:650-657. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR. 2000. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell 6:87-97. Mokelke EA, Dietz NJ, Eckman DM, Nelson MT, Sturek M. 2005a. Diabetic dyslipidemia and exercise affect coronary tone and differential regulation of conduit and microvessel K+ current. Am J Physiol Heart Circ Physiol 288:H1233-H1241. Mokelke EA, Dyson MC, Zafar MN, Alloosh M, Boullions RD, Kaser S, Sturek M. 2005b. Ossabaw minature swine as a novel model of the metabolic syndrome and subsequent cardiovascular disease. Swine in Biomedical Research Conference (http://www.conference.uiuc.edu/ Swine/swine_Abstract_Booklet.pdf). Mokelke EA, Hu Q, Song M, Toro L, Reddy HK, Sturek M. 2003. Altered functional coupling of coronary K+ channels in diabetic dyslipidemic pigs is prevented by exercise. J Appl Physiol 95:1179-1193. Mokelke EA, Wang M, Sturek M. 2002. Exercise training enhances coronary smooth muscle cell sodium-calcium exchange activity in diabetic dyslipidemic Yucatan swine. Ann N Y Acad Sci 976:335-337. Natarajan R, Gerrity RG, Gu JL, Lanting L, Thomas L, Nadler JL. 2002. Role of 12-lipoxygenase and oxidant stress in hyperglycaemia-induced acceleration of atherosclerosis in a diabetic pig model. Diabetologia 45:125-133. Nichols TC, Bellinger DA, Davis KE, Koch GG, Reddick RL, Read MS, Rapacz J, Hasler-Rapacz J, Brinkhous KM, Griggs TR. 1992. Porcine von Willebrand disease and atherosclerosis: Influence of polymorphism in apolipoprotein B100 genotype. Am J Pathol 140:403-415. Otieno CJ, Bastiaansen J, Ramos AM, Rothschild MF. 2005. Mapping and association studies of diabetes related genes in the pig. Anim Genet 36:36-42. Otis CR, Wamhoff BR, Sturek M. 2003. Hyperglycemia-induced insulin resistance in diabetic dyslipidemic Yucatan swine. Comp Med 53:5364. Panepinto LM. 1996. Minature swine breeds used worldwide in research. In: Tumbleson SL, ed. Advances in Swine in Biomedical Research. New York: Plenum Press. p 681-691. Panepinto LM, Phillips RW, Westmoreland NW, Cleek JL. 1982. Influence of genetics and diet on the development of diabetes in Yucatan miniature swine. J Nutr 112:2307-2313. Phillips RW, Panepinto LM. 1986. Swine as a model for human diabetes. In: Swine in Biomedical Research. Vol 1. New York: Plenum Press. p 549-560. Phillips RW, Westmoreland N, Panepinto LM, Case GL. 1981. Dietary effects on metabolism of Yucatan miniature swine selected for low and high glucose utilization. J Nutr 112:104-111. Phillips RW, Panepinto LM, Spangler R, Westmoreland N. 1982. Yucatan

miniature swine as a model for the study of human diabetes mellitus. Diabetes 31:30-36. Phillips RW, Panepinto LM, Will DH, Case GL. 1980. The effects of alloxan diabetes on Yucatan miniature swine and their progeny. Metabolism 29:40-45. Poore KR, Forhead AJ, Gardner DS, Giussani DA, Fowden AL. 2002. The effects of birth weight on basal cardiovascular function in pigs at 3 months of age. J Physiol 539:969-978. Poore KR, Fowden AL. 2004. Insulin sensitivity in juvenile and adult large white pigs of low and high birthweight. Diabetologia 47:340-348. Prentice AM, Rayco-Solon P, Moore SE. 2005. Insights from the developing world: Thrifty genotypes and thrifty phenotypes. Proc Nutr Soc 64:153-161. Prescott MF, Hasler-Rapacz J, von Linden-Reed J, Rapacz J. 1995. Familial hypercholesterolemia associated with coronary atherosclerosis in swine bearing different alleles for apolipoprotein B. Ann N Y Acad Sci 748:283-292. Prescott MF, McBride CH, Hasler-Rapacz J, Von Linden J, Rapacz J. 1991. Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for apolipoprotein B. Am J Pathol 139:139-147. Ribel U, Larsen MO, Rolin B, Carr RD, Wilken M, Sturis J, Westergaard L, Deacon CF, Knudsen LB. 2002. NN2211: A long-acting glucagonlike peptide-1 derivative with anti-diabetic effects in glucose-intolerant pigs. Eur J Pharmacol 451:217-225. Roberts TM, Sturek M, Dixon JL, Hardin CD. 2001. Alterations in the oxidative metabolic profile in vascular smooth muscle from hyperlipidemic and diabetic swine. Mol Cell Biochem 217:99-106. Sebert SP, Lecannu G, Kozlowski F, Siliart B, Bard JM, Krempf M, Champ MM. 2005a. Childhood obesity and insulin resistance in a Yucatan mini-piglet model: Putative roles of IGF-1 and muscle PPARs in adipose tissue activity and development. Int J Obes Relat Metab Disord 29:324-333. Sebert SP, Lecannu G, Sene S, Hucteau S, Chetiveaux M, Ouguerram K, Champ MM. 2005b. Obesity induced during sexual maturation is linked to LDL-triacylglycerols in Yucatan miniature swine. Br J Nutr 94:282-289. Springett R, Sakata Y, Delpy DT. 2001. Precise measurement of cerebral blood flow in newborn piglets from the bolus passage of indocyanine green. Phys Med Biol 46:2209-2225. Suzuki LA, Poot M, Gerrity RG, Bornfeldt KE. 2001. Diabetes accelerates smooth muscle accumulation in lesions of atherosclerosis: Lack of direct growth-promoting effects of high glucose levels. Diabetes 50: 851-860. Taskinen MR. 1995. Insulin resistance and lipoprotein metabolism. Curr Opin Lipidol 6:153-160. Teitelbaum BA, Roberts DK, Ecklund Winters J, Castells DD, Clark Alexander C. 2005. Blood pressure control in an African American sample with diabetes mellitus in an urban eye clinic. Optometry 76: 653-656. Tordjman K, Bernal-Mizrachi C, Zemany L, Weng S, Feng C, Zhang F, Leone TC, Coleman T, Kelly DP, Semenkovich CF. 2001. PPARalpha deficiency reduces insulin resistance and atherosclerosis in apoE-null mice. J Clin Invest 107:1025-1034. Tuffery AA. 1995. Laboratory Animals: An Introduction for Experimenters. New York: John Wiley & Sons Inc. Wangsness PJ, Martin RJ, Gahagan JH. 1977. Insulin and growth hormone in lean and obese pigs. Am J Physiol 233:E104-E108. Wangsness PJ, Martin RJ, Gatchel BB. 1980. Insulin induced growth hormone response in fast-growing, lean and in slow-growing, obese pigs. Growth 44:318-326. Witczak CA, Mokelke EA, Boullion R, Wenzel J, Keisler DH, Sturek M. 2005. Noninvasive measures of body fat percentage in male Yucatan swine. Comp Med 55:445-451. Xi S, Yin W, Wang Z, Kusunoki M, Lian X, Koike T, Fan J, Zhang Q. 2004. A minipig model of high-fat/high-sucrose diet-induced diabetes and atherosclerosis. Int J Exp Pathol 85:223-231. Yin W, Liao D, Kusunoki M, Xi S, Tsutsumi K, Wang Z, Lian X, Koike

Volume 47, Number 3

2006

257

T, Fan J, Yang Y, Tang C. 2004. NO-1886 decreases ectopic lipid deposition and protects pancreatic beta cells in diet-induced diabetic swine. J Endocrinol 180:399-408. Youssef S, Stuve O, Patarroyo JC, Ruiz PJ, Radosevich JL, Hur EM, Bravo M, Mitchell DJ, Sobel RA, Steinman L, Zamvil SS. 2002. The HMGCoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 420: 78-84. Zafar MN, Kaser S, Mokelke EA, Alloosh M, Dyson MC, Sturek M. 2005. Ossabaw swine having the metabolic syndrome exhibit greater neointimal hyperplasia after coronary stent placement than lean Yucatan

swine. Swine in Biomedical Research Conference (http://www .conference.uiuc.edu/Swine/swine_Abstract_Booklet.pdf). Zhang L, Zalewski A, Liu Y, Mazurek T, Cowan S, Martin JL, Hofmann SM, Vlassara H, Shi Y. 2003. Diabetes-induced oxidative stress and low-grade inflammation in porcine coronary arteries. Circulation 108: 472-478. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. 1992. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258:468-471. Zimmet P, Alberti KG, Shaw J. 2001. Global and societal implications of the diabetes epidemic. Nature 414:782-787.

258

ILAR Journal