REVIEW URRENT C OPINION
The nonhuman primate as a model for type 2 diabetes Lynley D. Pound a, Paul Kievit a, and Kevin L. Grove a,b
Purpose of the review Although rodent models provide insight into the mechanisms underlying type 2 diabetes mellitus (T2DM), they are limited in their translatability to humans. The nonhuman primate (NHP) shares important metabolic similarities with the human, making it an ideal model for the investigation of type 2 diabetes and use in preclinical trials. This review highlights the key contributions in the field over the last year using the NHP model. Recent findings The NHP has not only provided novel insight into the normal and pathological processes that occur within the islet, but has also allowed for the preclinical testing of novel pharmaceutical targets for obesity and T2DM. Particularly, administration of fibroblast growth factor-21 in the NHP resulted in weight loss and improvements in metabolic health, supporting rodent studies and recent clinical trials. In addition, the NHP was used to demonstrate that a novel melanocortin-4 receptor agonist did not cause adverse cardiovascular effects. Finally, this model has been used to provide evidence that glucagon-like peptide-1based therapies do not induce pancreatitis in the healthy NHP. Summary The insight gained from studies using the NHP model has allowed for a better understanding of the processes driving T2DM and has promoted the development of well tolerated and effective treatments. Keywords fibroblast growth factor-21, glucagon-like peptide-1, melanocortin-4 receptor, nonhuman primate, type 2 diabetes
INTRODUCTION In recent decades, the prevalence of type 2 diabetes mellitus (T2DM) has risen dramatically, paralleling an alarming rise in obesity. Approximately 11% of the US adult population currently has T2DM, while another 35% is afflicted with impaired glucose tolerance and prediabetes [1]. Furthermore, obesity and insulin resistance are often accompanied by dyslipidemia, hypertension and central adiposity, collectively termed the metabolic syndrome. T2DM risk is influenced by environmental factors and lifestyle choices, and increases with age, decreased physical activity, adiposity and consumption of a Western-style diet (WSD) high in cholesterol, saturated fat and fructose. Rodent models have provided insight into the mechanisms underlying T2DM and therapeutic targets but have been limited in their translatability to humans. The rodent differs significantly from the human in the pathophysiology of T2DM, glucose metabolism, islet morphology and function [2,3]
and blood–brain barrier permeability [4]. Typically, rodents are not naturally susceptible to spontaneous T2DM or hypertension development and most strains are resistant to diet-mediated T2DM [5]. Rather, genetic models have often been used; however, these may not accurately recapitulate the heterogenic nature of the disease in humans. Thus, the nonhuman primate (NHP) has emerged as an alternative to human studies with a number of advantages over both rodent models and human trials. Metabolic disease in the NHP bears important similarities to that in the human, including obesity, a
Division of Diabetes, Obesity, & Metabolism and bDivision of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, Oregon, USA Correspondence to Kevin L. Grove, 505 NW 185th Ave., Beaverton, OR 97006, USA. Tel: +1 503 690 5380; fax: +1 503 466 3820; e-mail: [email protected] Curr Opin Endocrinol Diabetes Obes 2014, 21:89–94 DOI:10.1097/MED.0000000000000043
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Diabetes and the endocrine pancreas I
High fructose-fed nonhuman primate
KEY POINTS The NHP bears important metabolic similarities to the human, particularly in the pathophysiology of type 2 diabetes, and thus has emerged as a preclinical model in the metabolic field. Over the last year, the NHP model has been particularly advantageous for the preclinical testing of novel pharmaceutical agents, particularly FGF21, GLP-1-based agonists and MC4R agonists. Administration of FGF21 analogs, and particularly longer-acting analogs, in the NHP supports both studies in rodents and recent human clinical trials demonstrating weight loss and metabolic health improvements, though longer-term studies are needed to confirm efficacy. A novel MC4R agonist was developed that promoted weight loss, but unlike previous drugs, did not cause adverse cardiovascular effects in the obese NHP, an effect that may be reflective of differences in blood–brain barrier penetration. GLP-1-based therapies do not appear to induce pancreatitis in the healthy NHP model; however, because pancreatitis is more prevalent in obese and diabetic populations, further studies need to be performed in these models.
central adiposity, insulin resistance, dyslipidemia and hypertension [6 ,7]. NHPs spontaneously develop T2DM, specially with age and when exposed to a hypercaloric diet. Furthermore, the use of NHPs allows for controlled longitudinal studies on the natural progression of obesity and T2DM and, because of the larger body size, frequent biological measurements. Unlike human trials, however, NHP studies benefit from a well controlled environment during the experimental period and allow extensive postmortem sampling of key metabolic organs, thus facilitating the correlation of molecular mechanisms with physiological readouts. Taken together, this makes the NHP a valuable experimental model, particularly in the investigation and preclinical testing of novel T2DM therapeutic agents, with a high potential for translatability to humans. &
COMMON NONHUMAN PRIMATE MODELS OF TYPE 2 DIABETES MELLITUS A number of models are in use for the study of T2DM in the NHP and thus, care should be taken when interpreting the data and extrapolating to the human. Highlighted in this review are some of the common models currently being used in this field. 90
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Fructose consumption by humans has increased markedly coincident with the rise of obesity and insulin resistance. Recently, Bremer et al. [8] characterized a high fructose-fed (HFr) rhesus macaque (Macaca mulatta) model that was maintained on a healthy chow diet (11% kcal from fat) supplemented with a fructose-sweetened beverage (30% kcal) for 1 year. All animals on their study developed some degree of metabolic syndrome within 6–12 months on the diet, particularly central adiposity, dyslipidemia and insulin resistance, whereas a subset developed frank T2DM thus mirroring the human response to high fructose. In contrast, Kavanagh et al. [9 ] characterized a HFr NHP model and determined that when caloric intake was controlled between groups, fructose consumption did not result in obesity or other aspects of the metabolic syndrome, but was associated with biomarkers of liver damage. Thus, it is unclear whether the adverse metabolic effects described by Bremer et al. were because of the fructose per se or were simply a result of increased caloric consumption. In addition, unlike a typical Westernized diet in humans, this model does not include high dietary fat, particularly saturated fat, or cholesterol. &
High fat, Western-style diet-fed nonhuman primate Our group has previously published a number of studies utilizing the WSD NHP model, particularly for investigation of pharmaceutical interventions for obesity, metabolic syndrome and T2DM [6 ,10 ,11]. Like humans consuming a WSD, NHPs develop a range of metabolic complications with a subset of NHPs remaining relatively lean and metabolically healthy, and others developing obesity, adiposity, insulin resistance and dyslipidemia. Furthermore, like the HFr model, a subset will develop T2DM. Because the WSD is highly palatable, it is a particularly useful model for testing drugs that aim to reduce food intake. An important consideration of this model, however, is that a diet high in saturated fat and cholesterol suppresses endogenous cholesterol synthesis [12], making it of limited use when targeting this pathway. &
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Aged spontaneous type 2 diabetes mellitus nonhuman primate Like the human, the NHP has an increased propensity to develop obesity and T2DM with increasing age [13 ]. This group consumes a healthy diet and thus the likelihood of developing T2DM is presumably driven by an accumulation of single nucleotide &
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polymorphisms affecting disease risk, an important consideration in the use of this model. Consumption of a healthy control diet may not be reflective of the calorie dense foods and lack of physical activity that often accompanies the development of T2DM in the human. However, this model does provide insight into genetic and aged onset mechanisms driving T2DM development, though the length of time, and thus cost required to develop this model, may be a technical limitation.
USE OF THE NONHUMAN PRIMATE IN ISLET BIOLOGY Recent studies using NHPs have provided insight into the dynamic interactions of islet function in healthy, insulin resistant and T2DM conditions [14 ,15 ]. As the impairment in glucose tolerance worsens, there is a compensatory increase in insulin secretion, placing additional strain on the pancreatic islet. Thus, a better understanding of the pathological processes that occur at the islet is critical to the treatment of T2DM. As there are significant differences between rodents and primates in islet structure and function, the use of NHPs for islet studies is particularly advantageous because of its similarities to the human. Rodent islets consist of a b-cell core surrounded by a mantle of a-cells. In contrast, the human and NHP islet consists of interspersed b-cells and a-cells with a higher proportion of a-cells. Thus, islet cell–cell contact differs between species and may affect intraislet communication. Furthermore, compared with the rodent, human islets have distinct glucoseregulated gene expression and glucose-stimulated insulin secretion (GSIS) [16,17]. Two recent studies have sought to clarify the normal and pathogenic processes occurring in the islet in a NHP model. Guardado-Mendoza et al. [14 ] demonstrated that both obese and insulin-resistant states are associated with increased a-cell volume, which occurs prior to changes in b-cell volume, and correlates with obesity duration and severity. The authors propose that increased a-cell mass will lead to increased glutamate secretion and may stress the b-cell, which is particularly vulnerable to cytotoxicity induced by glutamate treatment. In addition, Wang et al. [15 ] utilized a graded glucose infusion to characterize b-cell function in vivo in healthy and spontaneously dysmetabolic and diabetic NHP islets. Using deconvolution of serial C-peptide concentrations, the authors identify true insulin secretion prior to clearance at the liver. Like humans, dysmetabolic cynomolgus macaques (Macaca fascicularis) displayed increased fasting hyperinsulinemia, whereas diabetic macaques &&
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displayed impaired GSIS. Dysmetabolic animals did not display a significant increase in GSIS, but rather showed a reduction in clearance at the liver. These studies underscore the importance of the use of NHPs particularly in longitudinal studies that will provide insight into the natural progression of the pathophysiology of T2DM. Understanding the mechanisms leading to b-cell failure and the identification of interventions that improve b-cell function in NHP models are key to the development of novel clinical therapeutics in humans.
PRECLINICAL TESTING IN THE NONHUMAN PRIMATE Recently, the NHP has been used to assess safety and efficacy of a number of pharmaceutical agents in the obesity and T2DM field. Here, we discuss three key targets of current preclinical testing in the NHP.
Fibroblast growth factor-21 analogs Fibroblast growth factor-21 (FGF-21) has emerged as a promising therapeutic candidate not only for the treatment of hyperglycemia, but also for the symptoms of metabolic syndrome that often accompany T2DM. Particularly, FGF-21 treatment improves insulin sensitivity, obesity, b-cell function, glycemia and lipidemia in diabetic rodents [18 ,19–21]. In this respect, FGF-21 and its analogs may be superior to available therapies that aim to alleviate hyperglycemia, but fail to address the broader spectrum of associated metabolic complications. Importantly, these metabolic benefits have not been accompanied by the adverse side-effects typically observed with metabolic pharmaceuticals, including body weight gain, increased adiposity, hypoglycemia, mitogenecity or nausea [18 ]. Recently, a number of studies have investigated the effect of variants of FGF-21 in the NHP model [18 ,22–24 ]. Treatment with human recombinant FGF-21 (hrFGF21) had been previously shown to improve fasting plasma glucose, triglycerides, insulin and glucagon levels in the aged diabetic NHP model [25]; however, native FGF-21 has a short halflife thus requiring frequent dosing and driving a need for longer-acting analogs of this molecule [18 ,22,24 ,26]. Three such molecules have been identified: Fc-FGF21(RG), a FGF21 analog [18 ]; mimAb1, a monoclonal antibody that activates the bKlotho/FGF receptor-1c (FGFR-1c) complex [22]; and C3201-HSA, a bispecific protein that simultaneously activates both bKlotho and FGFR-1c [24 ]. FGF21 binds the transmembrane protein, bKlotho, and can then bind a number of FGFRs, but particularly FGFR1c, 2c and 3c. bKlotho, unlike
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the FGFR family, is highly specific to the liver, adipose and pancreas, thereby reducing the likelihood of general activation of FGF signaling and thus making it an appealing drug target [22]. All three molecules induced approximately 10–15% weight loss and generally improved metabolic health in the spontaneously obese, nondiabetic macaque [18 ,22,24 ]. Similarly, Adams et al. [23 ] generated an additional FGF-21 variant, LY2405319, which in the aged diabetic rhesus macaque significantly reduced body weight and plasma glucose, triglycerides, cholesterol and insulin. In contrast, in the spontaneously obese model, Fc-FGF21(RG), hrFGF21 and C3201-HSA failed to improve fasting blood glucose levels [18 ,24 ]; however, these animals are generally metabolically healthy and already possess glucose values within a normal range. Though the improvements reported in obese but metabolically healthy NHPs are certainly noteworthy, obesity and T2DM in humans are typically accompanied by the consumption of an obesogenic, Westernized diet. Thus, studies of FGF-21 variants in the context of a WSD are warranted. Despite the clear metabolic benefits of FGF-21 treatment, there are noteworthy caveats that have emerged from the studies in the NHP. In the diabetic cohort, food intake was drastically reduced by approximately 75% [23 ] that would at least partially explain the 12.5–23.6% weight loss in these animals and is likely indicative of nausea. In contrast, a recent clinical study demonstrated that the same molecule promotes minimal weight loss in humans (1.5% loss) [27]. Low dose mimAb1 treatment, however, caused approximately 10% body weight reduction with only a modest, transient effect on food intake, whereas food intake was not reported following Fc-FGF21(RG) or C3201-HSA treatment [18 ,24 ]. Whether the loss of body weight is due to loss of fat or lean mass is not discussed, but a dramatic reduction in lean mass would likely have unintended adverse effects. It is unclear to what degree the metabolic improvements observed in the NHP were a direct effect of the compound or were due to the substantial reduction in food intake and subsequent weight loss. However, some metabolic benefits certainly precede the reduction in food intake. Specifically, the diabetic NHPs displayed a rapid increase in adiponectin levels, a known insulin sensitizer [23 ]. Similarly, LY2405319 treatment in humans improves adiponectin levels as well as fasting insulin and dyslipidemia [27]. In addition, FGF-21 levels have been previously reported to be elevated in humans [28] and NHPs [10 ] in the context of insulin resistance and may be indicative of FGF-21 resistance [10 ,29]. Though this &&
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clearly can be overcome by pharmacological doses, the longer-term efficacy of pharmaceutical FGF-21 treatment still needs to be investigated particularly as many of the currently available therapies are known to suffer from a decline in efficacy over time [30].
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Melanocortin-4 receptor agonists The melanocortin-4 receptor (MC4R) plays a key role in energy homeostasis, an effect mediated by a-melanocortin-stimulating hormone (a-MSH), which, upon binding to MC4R, stimulates energy expenditure and inhibits food intake [31–35]. Inactivating mutations in the human MC4R gene result in a drastic increase in appetite, leading to increased body weight and adiposity [31]. Pharmacological agonists have been shown to mimic these effects; however, recent reports suggest that MC4R agonist treatment may also be associated with harmful effects to the cardiovascular system. Specifically, Greenfield et al. [36] indicated that short-term treatment with a MC4R agonist, LY2112688, resulted in a dose-dependent increase in blood pressure. Furthermore, MC4R-deficient patients are paradoxically protected from increased blood pressure typically associated with severe obesity [36]. Our group tested a novel MC4R agonist, RM493, to determine its efficacy and influence on the cardiovascular system in NHPs consuming a WSD. RM-493 treatment resulted in greater than 10% weight loss. Though this was accompanied by a transient reduction in food intake during the early phase of treatment (weeks 1–2), weight loss was sustained throughout the 8-week treatment period. Thus, weight loss can likely be attributed to the increased basal metabolic rate. In contrast to the effect of LY2112688 treatment, RM-493 treatment did not increase heart rate or blood pressure [6 ]. These data may be explained by differences in brain penetration with LY2112688 having greater permeability than RM-493, and thus broader activation of the sympathetic system. It is important to note that species differences, particularly between rodents and primates, have previously been demonstrated in the brain penetrability of pharmaceutical molecules [4], thus underscoring the value of NHPs in preclinical testing of drugs targeted to the brain. &
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Glucagon-like peptide-1 based therapies Glucagon-like peptide-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 inhibitors (DPP4i) have been used to treat T2DM based on their ability to activate incretin receptor signaling, thereby enhancing GSIS and lowering glycemia with a low Volume 21 Number 2 April 2014
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risk of hypoglycemia [37]. More recently, however, NHPs have been used to further investigate potential adverse side-effects of GLP-1-based therapies. Specifically, a number of recent studies have reported an increased incidence of pancreatitis in rodents and human patients being treated with exenatide [38–41], liraglutide (both GLP-1R agonists) [37] and sitagliptin (DPP4i) [41], though the overall occurrence is low. Consistent with these findings, a recent study in humans indicated that patients taking either sitagliptin or exenatide displayed expansion of both the endocrine and exocrine pancreas, increased pancreatic mass and dysplastic pancreatic intraepithelial neoplastic lesions [42]. However, the low overall incidence of pancreatitis has made causality conclusions in human follow-up studies challenging. Furthermore, treatment and follow-up periods have typically been short and compliance has not been adequately assessed [43]. Thus, Nyborg et al. [44] used an NHP model to investigate the role of GLP-1-based therapies in the occurrence of acute pancreatitis. In a healthy cohort of cynomolgus macaques, the authors reported no evidence of pancreatitis or of preneoplastic proliferative lesions after 87 weeks of treatment with liraglutide at doses 60-fold higher than maximal clinical doses in humans. Although this was in a relatively small cohort of animals (n ¼ 5/group), the lack of evidence is a compelling argument against an increased incidence of pancreatitis following GLP1R agonist treatment in a healthy cohort, though it is still unclear whether the same is true in an obese and/or T2DM population. Indeed, studies in humans have indicated that obesity and T2DM are associated with an increased risk of pancreatitis and pancreatic cancer [45], though it is unknown whether the use of GLP-1-based therapies confers an additional risk to these patients. Thus, investigation of GLP-1-based therapies in a higher risk population of NHPs is warranted.
CONCLUSION The use of NHPs in biomedical research faces some challenges, not least of which are the expense and relatively few laboratories that are equipped to accommodate such studies. However, NHPs have been instrumental in the characterization of normal and T2DM pathological processes. Additionally, they provide a valuable resource for the assessment of safety and efficacy of therapeutic interventions prior to implementation in clinical trials. The insight gained from NHP studies advances us toward the well tolerated and effective future treatment of T2DM.
Acknowledgements The authors would like to thank Victoria Roberts for discussions. The author’s work in this area has been supported by NIH funding: R24 DK090964, RC4 DK090956 (both to KLG) and P51 OD011092 (partial salary support for KLG). Conflicts of interest L.D.P. and P.K. declare no conflicts of interest. K.L.G. is a paid consultant to Ember Pharmaceuticals, Eli Lilly, and Rhythm Pharmaceuticals. He is a member of the Scientific Advisory Board for Novo Nordisk, and currently receives research funding from Novo Nordisk, Janssen Research and Development, Ipsen, Acceleron and Rhythm.
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Diabetes and the endocrine pancreas I 14. Guardado-Mendoza R, Jimenez-Ceja L, Majluf-Cruz A, et al. Impact of obesity severity and duration on pancreatic beta- and alpha-cell dynamics in normoglycemic nonhuman primates. Int J Obesity 2013; 37:1071–1078. Islet morphology was investigated in the obese normoglycemic baboon. The authors determined that obesity and insulin resistance are associated with increased a-cell volume with little effect on the b-cell, suggesting that changes in a-cell mass precede the loss of b-cells. 15. Wang X, Hansen BC, Shi D, et al. Quantification of beta-cell insulin secretory && function using a graded glucose infusion with C-peptide deconvolution in dysmetabolic, and diabetic cynomolgus monkeys. Diabetol Metab Syndr 2013; 5:40. Using C-peptide deconvolution, this study demonstrated that dysmetabolic cynomolgus macaques display impaired hepatic insulin clearance in addition to insulin hypersecretion. 16. Dai C, Brissova M, Hang Y, et al. Islet-enriched gene expression and glucoseinduced insulin secretion in human and mouse islets. Diabetologia 2012; 55:707–718. 17. Mueller KR, Balamurugan AN, Cline GW, et al. Differences in glucosestimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin. Xenotransplantation 2013; 20:75–81. 18. Veniant MM, Komorowski R, Chen P, et al. Long-acting FGF21 has enhanced && efficacy in diet-induced obese mice and in obese rhesus monkeys. Endocrinology 2012; 153:4192–4203. This study tested a newly developed long-acting FGF21 analog in obese rhesus macaques and demonstrated weight loss, improved metabolic health and greater efficacy than human recombinant FGF21. 19. Kharitonenkov A, Beals JM, Micanovic R, et al. Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319. PLoS One 2013; 8:e58575. 20. Iglesias P, Selgas R, Romero S, Diez JJ. Biological role, clinical significance, and therapeutic possibilities of the recently discovered metabolic hormone fibroblastic growth factor 21. Eur J Endocrinol 2012; 167:301–309. 21. Zhao Y, Dunbar JD, Kharitonenkov A. FGF21 as a therapeutic reagent. Adv Exp Med Biol 2012; 728:214–228. 22. Foltz IN, Hu S, King C, et al. Treating diabetes and obesity with an FGF21mimetic antibody activating the betaKlotho/FGFR1c receptor complex. Sci Transl Med 2012; 4:162ra153. 23. Adams AC, Halstead CA, Hansen BC, et al. LY2405319, an Engineered && FGF21 Variant, Improves the Metabolic Status of Diabetic Monkeys. PLoS One 2013; 8:e65763. This study tested the effect of a novel FGF21 variant, LY2405319 in the aged spontaneously diabetic rhesus macaque. Administration of LY2405319 led to dramatic weight loss and improvements in metabolic health. 24. Smith R, Duguay A, Bakker A, et al. FGF21 can be mimicked in vitro and in vivo && by a novel anti-FGFR1c/beta-Klotho bispecific protein. PLoS One 2013; 8:e61432. This study developed a novel bispecific artifical protein that simultaneously binds both bKlotho and FGFR1c to induce weight loss and improve metabolic health in the obese cynomolgus macaque. 25. Kharitonenkov A, Wroblewski VJ, Koester A, et al. The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21. Endocrinology 2007; 148:774–781. 26. Hecht R, Li YS, Sun J, et al. Rationale-based engineering of a potent longacting FGF21 analog for the treatment of type 2 diabetes. PLoS One 2012; 7:e49345. &&
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The nonhuman primate as a model for type 2 diabetes Lynley D. Pound a, Paul Kievit a, and Kevin L. Grove a,b
Purpose of the review Although rodent models provide insight into the mechanisms underlying type 2 diabetes mellitus (T2DM), they are limited in their translatability to humans. The nonhuman primate (NHP) shares important metabolic similarities with the human, making it an ideal model for the investigation of type 2 diabetes and use in preclinical trials. This review highlights the key contributions in the field over the last year using the NHP model. Recent findings The NHP has not only provided novel insight into the normal and pathological processes that occur within the islet, but has also allowed for the preclinical testing of novel pharmaceutical targets for obesity and T2DM. Particularly, administration of fibroblast growth factor-21 in the NHP resulted in weight loss and improvements in metabolic health, supporting rodent studies and recent clinical trials. In addition, the NHP was used to demonstrate that a novel melanocortin-4 receptor agonist did not cause adverse cardiovascular effects. Finally, this model has been used to provide evidence that glucagon-like peptide-1based therapies do not induce pancreatitis in the healthy NHP. Summary The insight gained from studies using the NHP model has allowed for a better understanding of the processes driving T2DM and has promoted the development of well tolerated and effective treatments. Keywords fibroblast growth factor-21, glucagon-like peptide-1, melanocortin-4 receptor, nonhuman primate, type 2 diabetes
INTRODUCTION In recent decades, the prevalence of type 2 diabetes mellitus (T2DM) has risen dramatically, paralleling an alarming rise in obesity. Approximately 11% of the US adult population currently has T2DM, while another 35% is afflicted with impaired glucose tolerance and prediabetes [1]. Furthermore, obesity and insulin resistance are often accompanied by dyslipidemia, hypertension and central adiposity, collectively termed the metabolic syndrome. T2DM risk is influenced by environmental factors and lifestyle choices, and increases with age, decreased physical activity, adiposity and consumption of a Western-style diet (WSD) high in cholesterol, saturated fat and fructose. Rodent models have provided insight into the mechanisms underlying T2DM and therapeutic targets but have been limited in their translatability to humans. The rodent differs significantly from the human in the pathophysiology of T2DM, glucose metabolism, islet morphology and function [2,3]
and blood–brain barrier permeability [4]. Typically, rodents are not naturally susceptible to spontaneous T2DM or hypertension development and most strains are resistant to diet-mediated T2DM [5]. Rather, genetic models have often been used; however, these may not accurately recapitulate the heterogenic nature of the disease in humans. Thus, the nonhuman primate (NHP) has emerged as an alternative to human studies with a number of advantages over both rodent models and human trials. Metabolic disease in the NHP bears important similarities to that in the human, including obesity, a
Division of Diabetes, Obesity, & Metabolism and bDivision of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, Oregon, USA Correspondence to Kevin L. Grove, 505 NW 185th Ave., Beaverton, OR 97006, USA. Tel: +1 503 690 5380; fax: +1 503 466 3820; e-mail: [email protected] Curr Opin Endocrinol Diabetes Obes 2014, 21:89–94 DOI:10.1097/MED.0000000000000043
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High fructose-fed nonhuman primate
KEY POINTS The NHP bears important metabolic similarities to the human, particularly in the pathophysiology of type 2 diabetes, and thus has emerged as a preclinical model in the metabolic field. Over the last year, the NHP model has been particularly advantageous for the preclinical testing of novel pharmaceutical agents, particularly FGF21, GLP-1-based agonists and MC4R agonists. Administration of FGF21 analogs, and particularly longer-acting analogs, in the NHP supports both studies in rodents and recent human clinical trials demonstrating weight loss and metabolic health improvements, though longer-term studies are needed to confirm efficacy. A novel MC4R agonist was developed that promoted weight loss, but unlike previous drugs, did not cause adverse cardiovascular effects in the obese NHP, an effect that may be reflective of differences in blood–brain barrier penetration. GLP-1-based therapies do not appear to induce pancreatitis in the healthy NHP model; however, because pancreatitis is more prevalent in obese and diabetic populations, further studies need to be performed in these models.
central adiposity, insulin resistance, dyslipidemia and hypertension [6 ,7]. NHPs spontaneously develop T2DM, specially with age and when exposed to a hypercaloric diet. Furthermore, the use of NHPs allows for controlled longitudinal studies on the natural progression of obesity and T2DM and, because of the larger body size, frequent biological measurements. Unlike human trials, however, NHP studies benefit from a well controlled environment during the experimental period and allow extensive postmortem sampling of key metabolic organs, thus facilitating the correlation of molecular mechanisms with physiological readouts. Taken together, this makes the NHP a valuable experimental model, particularly in the investigation and preclinical testing of novel T2DM therapeutic agents, with a high potential for translatability to humans. &
COMMON NONHUMAN PRIMATE MODELS OF TYPE 2 DIABETES MELLITUS A number of models are in use for the study of T2DM in the NHP and thus, care should be taken when interpreting the data and extrapolating to the human. Highlighted in this review are some of the common models currently being used in this field. 90
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Fructose consumption by humans has increased markedly coincident with the rise of obesity and insulin resistance. Recently, Bremer et al. [8] characterized a high fructose-fed (HFr) rhesus macaque (Macaca mulatta) model that was maintained on a healthy chow diet (11% kcal from fat) supplemented with a fructose-sweetened beverage (30% kcal) for 1 year. All animals on their study developed some degree of metabolic syndrome within 6–12 months on the diet, particularly central adiposity, dyslipidemia and insulin resistance, whereas a subset developed frank T2DM thus mirroring the human response to high fructose. In contrast, Kavanagh et al. [9 ] characterized a HFr NHP model and determined that when caloric intake was controlled between groups, fructose consumption did not result in obesity or other aspects of the metabolic syndrome, but was associated with biomarkers of liver damage. Thus, it is unclear whether the adverse metabolic effects described by Bremer et al. were because of the fructose per se or were simply a result of increased caloric consumption. In addition, unlike a typical Westernized diet in humans, this model does not include high dietary fat, particularly saturated fat, or cholesterol. &
High fat, Western-style diet-fed nonhuman primate Our group has previously published a number of studies utilizing the WSD NHP model, particularly for investigation of pharmaceutical interventions for obesity, metabolic syndrome and T2DM [6 ,10 ,11]. Like humans consuming a WSD, NHPs develop a range of metabolic complications with a subset of NHPs remaining relatively lean and metabolically healthy, and others developing obesity, adiposity, insulin resistance and dyslipidemia. Furthermore, like the HFr model, a subset will develop T2DM. Because the WSD is highly palatable, it is a particularly useful model for testing drugs that aim to reduce food intake. An important consideration of this model, however, is that a diet high in saturated fat and cholesterol suppresses endogenous cholesterol synthesis [12], making it of limited use when targeting this pathway. &
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Aged spontaneous type 2 diabetes mellitus nonhuman primate Like the human, the NHP has an increased propensity to develop obesity and T2DM with increasing age [13 ]. This group consumes a healthy diet and thus the likelihood of developing T2DM is presumably driven by an accumulation of single nucleotide &
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polymorphisms affecting disease risk, an important consideration in the use of this model. Consumption of a healthy control diet may not be reflective of the calorie dense foods and lack of physical activity that often accompanies the development of T2DM in the human. However, this model does provide insight into genetic and aged onset mechanisms driving T2DM development, though the length of time, and thus cost required to develop this model, may be a technical limitation.
USE OF THE NONHUMAN PRIMATE IN ISLET BIOLOGY Recent studies using NHPs have provided insight into the dynamic interactions of islet function in healthy, insulin resistant and T2DM conditions [14 ,15 ]. As the impairment in glucose tolerance worsens, there is a compensatory increase in insulin secretion, placing additional strain on the pancreatic islet. Thus, a better understanding of the pathological processes that occur at the islet is critical to the treatment of T2DM. As there are significant differences between rodents and primates in islet structure and function, the use of NHPs for islet studies is particularly advantageous because of its similarities to the human. Rodent islets consist of a b-cell core surrounded by a mantle of a-cells. In contrast, the human and NHP islet consists of interspersed b-cells and a-cells with a higher proportion of a-cells. Thus, islet cell–cell contact differs between species and may affect intraislet communication. Furthermore, compared with the rodent, human islets have distinct glucoseregulated gene expression and glucose-stimulated insulin secretion (GSIS) [16,17]. Two recent studies have sought to clarify the normal and pathogenic processes occurring in the islet in a NHP model. Guardado-Mendoza et al. [14 ] demonstrated that both obese and insulin-resistant states are associated with increased a-cell volume, which occurs prior to changes in b-cell volume, and correlates with obesity duration and severity. The authors propose that increased a-cell mass will lead to increased glutamate secretion and may stress the b-cell, which is particularly vulnerable to cytotoxicity induced by glutamate treatment. In addition, Wang et al. [15 ] utilized a graded glucose infusion to characterize b-cell function in vivo in healthy and spontaneously dysmetabolic and diabetic NHP islets. Using deconvolution of serial C-peptide concentrations, the authors identify true insulin secretion prior to clearance at the liver. Like humans, dysmetabolic cynomolgus macaques (Macaca fascicularis) displayed increased fasting hyperinsulinemia, whereas diabetic macaques &&
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displayed impaired GSIS. Dysmetabolic animals did not display a significant increase in GSIS, but rather showed a reduction in clearance at the liver. These studies underscore the importance of the use of NHPs particularly in longitudinal studies that will provide insight into the natural progression of the pathophysiology of T2DM. Understanding the mechanisms leading to b-cell failure and the identification of interventions that improve b-cell function in NHP models are key to the development of novel clinical therapeutics in humans.
PRECLINICAL TESTING IN THE NONHUMAN PRIMATE Recently, the NHP has been used to assess safety and efficacy of a number of pharmaceutical agents in the obesity and T2DM field. Here, we discuss three key targets of current preclinical testing in the NHP.
Fibroblast growth factor-21 analogs Fibroblast growth factor-21 (FGF-21) has emerged as a promising therapeutic candidate not only for the treatment of hyperglycemia, but also for the symptoms of metabolic syndrome that often accompany T2DM. Particularly, FGF-21 treatment improves insulin sensitivity, obesity, b-cell function, glycemia and lipidemia in diabetic rodents [18 ,19–21]. In this respect, FGF-21 and its analogs may be superior to available therapies that aim to alleviate hyperglycemia, but fail to address the broader spectrum of associated metabolic complications. Importantly, these metabolic benefits have not been accompanied by the adverse side-effects typically observed with metabolic pharmaceuticals, including body weight gain, increased adiposity, hypoglycemia, mitogenecity or nausea [18 ]. Recently, a number of studies have investigated the effect of variants of FGF-21 in the NHP model [18 ,22–24 ]. Treatment with human recombinant FGF-21 (hrFGF21) had been previously shown to improve fasting plasma glucose, triglycerides, insulin and glucagon levels in the aged diabetic NHP model [25]; however, native FGF-21 has a short halflife thus requiring frequent dosing and driving a need for longer-acting analogs of this molecule [18 ,22,24 ,26]. Three such molecules have been identified: Fc-FGF21(RG), a FGF21 analog [18 ]; mimAb1, a monoclonal antibody that activates the bKlotho/FGF receptor-1c (FGFR-1c) complex [22]; and C3201-HSA, a bispecific protein that simultaneously activates both bKlotho and FGFR-1c [24 ]. FGF21 binds the transmembrane protein, bKlotho, and can then bind a number of FGFRs, but particularly FGFR1c, 2c and 3c. bKlotho, unlike
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the FGFR family, is highly specific to the liver, adipose and pancreas, thereby reducing the likelihood of general activation of FGF signaling and thus making it an appealing drug target [22]. All three molecules induced approximately 10–15% weight loss and generally improved metabolic health in the spontaneously obese, nondiabetic macaque [18 ,22,24 ]. Similarly, Adams et al. [23 ] generated an additional FGF-21 variant, LY2405319, which in the aged diabetic rhesus macaque significantly reduced body weight and plasma glucose, triglycerides, cholesterol and insulin. In contrast, in the spontaneously obese model, Fc-FGF21(RG), hrFGF21 and C3201-HSA failed to improve fasting blood glucose levels [18 ,24 ]; however, these animals are generally metabolically healthy and already possess glucose values within a normal range. Though the improvements reported in obese but metabolically healthy NHPs are certainly noteworthy, obesity and T2DM in humans are typically accompanied by the consumption of an obesogenic, Westernized diet. Thus, studies of FGF-21 variants in the context of a WSD are warranted. Despite the clear metabolic benefits of FGF-21 treatment, there are noteworthy caveats that have emerged from the studies in the NHP. In the diabetic cohort, food intake was drastically reduced by approximately 75% [23 ] that would at least partially explain the 12.5–23.6% weight loss in these animals and is likely indicative of nausea. In contrast, a recent clinical study demonstrated that the same molecule promotes minimal weight loss in humans (1.5% loss) [27]. Low dose mimAb1 treatment, however, caused approximately 10% body weight reduction with only a modest, transient effect on food intake, whereas food intake was not reported following Fc-FGF21(RG) or C3201-HSA treatment [18 ,24 ]. Whether the loss of body weight is due to loss of fat or lean mass is not discussed, but a dramatic reduction in lean mass would likely have unintended adverse effects. It is unclear to what degree the metabolic improvements observed in the NHP were a direct effect of the compound or were due to the substantial reduction in food intake and subsequent weight loss. However, some metabolic benefits certainly precede the reduction in food intake. Specifically, the diabetic NHPs displayed a rapid increase in adiponectin levels, a known insulin sensitizer [23 ]. Similarly, LY2405319 treatment in humans improves adiponectin levels as well as fasting insulin and dyslipidemia [27]. In addition, FGF-21 levels have been previously reported to be elevated in humans [28] and NHPs [10 ] in the context of insulin resistance and may be indicative of FGF-21 resistance [10 ,29]. Though this &&
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clearly can be overcome by pharmacological doses, the longer-term efficacy of pharmaceutical FGF-21 treatment still needs to be investigated particularly as many of the currently available therapies are known to suffer from a decline in efficacy over time [30].
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Melanocortin-4 receptor agonists The melanocortin-4 receptor (MC4R) plays a key role in energy homeostasis, an effect mediated by a-melanocortin-stimulating hormone (a-MSH), which, upon binding to MC4R, stimulates energy expenditure and inhibits food intake [31–35]. Inactivating mutations in the human MC4R gene result in a drastic increase in appetite, leading to increased body weight and adiposity [31]. Pharmacological agonists have been shown to mimic these effects; however, recent reports suggest that MC4R agonist treatment may also be associated with harmful effects to the cardiovascular system. Specifically, Greenfield et al. [36] indicated that short-term treatment with a MC4R agonist, LY2112688, resulted in a dose-dependent increase in blood pressure. Furthermore, MC4R-deficient patients are paradoxically protected from increased blood pressure typically associated with severe obesity [36]. Our group tested a novel MC4R agonist, RM493, to determine its efficacy and influence on the cardiovascular system in NHPs consuming a WSD. RM-493 treatment resulted in greater than 10% weight loss. Though this was accompanied by a transient reduction in food intake during the early phase of treatment (weeks 1–2), weight loss was sustained throughout the 8-week treatment period. Thus, weight loss can likely be attributed to the increased basal metabolic rate. In contrast to the effect of LY2112688 treatment, RM-493 treatment did not increase heart rate or blood pressure [6 ]. These data may be explained by differences in brain penetration with LY2112688 having greater permeability than RM-493, and thus broader activation of the sympathetic system. It is important to note that species differences, particularly between rodents and primates, have previously been demonstrated in the brain penetrability of pharmaceutical molecules [4], thus underscoring the value of NHPs in preclinical testing of drugs targeted to the brain. &
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Glucagon-like peptide-1 based therapies Glucagon-like peptide-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 inhibitors (DPP4i) have been used to treat T2DM based on their ability to activate incretin receptor signaling, thereby enhancing GSIS and lowering glycemia with a low Volume 21 Number 2 April 2014
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risk of hypoglycemia [37]. More recently, however, NHPs have been used to further investigate potential adverse side-effects of GLP-1-based therapies. Specifically, a number of recent studies have reported an increased incidence of pancreatitis in rodents and human patients being treated with exenatide [38–41], liraglutide (both GLP-1R agonists) [37] and sitagliptin (DPP4i) [41], though the overall occurrence is low. Consistent with these findings, a recent study in humans indicated that patients taking either sitagliptin or exenatide displayed expansion of both the endocrine and exocrine pancreas, increased pancreatic mass and dysplastic pancreatic intraepithelial neoplastic lesions [42]. However, the low overall incidence of pancreatitis has made causality conclusions in human follow-up studies challenging. Furthermore, treatment and follow-up periods have typically been short and compliance has not been adequately assessed [43]. Thus, Nyborg et al. [44] used an NHP model to investigate the role of GLP-1-based therapies in the occurrence of acute pancreatitis. In a healthy cohort of cynomolgus macaques, the authors reported no evidence of pancreatitis or of preneoplastic proliferative lesions after 87 weeks of treatment with liraglutide at doses 60-fold higher than maximal clinical doses in humans. Although this was in a relatively small cohort of animals (n ¼ 5/group), the lack of evidence is a compelling argument against an increased incidence of pancreatitis following GLP1R agonist treatment in a healthy cohort, though it is still unclear whether the same is true in an obese and/or T2DM population. Indeed, studies in humans have indicated that obesity and T2DM are associated with an increased risk of pancreatitis and pancreatic cancer [45], though it is unknown whether the use of GLP-1-based therapies confers an additional risk to these patients. Thus, investigation of GLP-1-based therapies in a higher risk population of NHPs is warranted.
CONCLUSION The use of NHPs in biomedical research faces some challenges, not least of which are the expense and relatively few laboratories that are equipped to accommodate such studies. However, NHPs have been instrumental in the characterization of normal and T2DM pathological processes. Additionally, they provide a valuable resource for the assessment of safety and efficacy of therapeutic interventions prior to implementation in clinical trials. The insight gained from NHP studies advances us toward the well tolerated and effective future treatment of T2DM.
Acknowledgements The authors would like to thank Victoria Roberts for discussions. The author’s work in this area has been supported by NIH funding: R24 DK090964, RC4 DK090956 (both to KLG) and P51 OD011092 (partial salary support for KLG). Conflicts of interest L.D.P. and P.K. declare no conflicts of interest. K.L.G. is a paid consultant to Ember Pharmaceuticals, Eli Lilly, and Rhythm Pharmaceuticals. He is a member of the Scientific Advisory Board for Novo Nordisk, and currently receives research funding from Novo Nordisk, Janssen Research and Development, Ipsen, Acceleron and Rhythm.
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