The human GLP-1 analogs liraglutide and semaglutide: absence of histopathological effects on the pancreas in nonhuman primates.

Increased pancreas mass and glucagon-positive adenomas have been suggested to be a risk associated with sitagliptin or exenatide therapy in humans. Novo Nordisk has conducted extensive toxicology studies, including data on pancreas weight and histology, in Cynomolgus monkeys dosed with two different human glucagon-like peptide-1 (GLP-1) receptor agonists. In a 52-week study with liraglutide, a dose-related increase in absolute pancreas weight was observed in female monkeys only. Such dose-related increase was not found in studies of 4, 13, or 87 weeks' duration. No treatment-related histopathological abnormalities were observed in any of the studies. Quantitative histology of the pancreas from the 52-week study showed an increase in the exocrine cell mass in liraglutide-dosed animals, with normal composition of endocrine and exocrine cellular compartments. Proliferation rate of the exocrine tissue was low and comparable between groups. Endocrine cell mass and proliferation rates were unaltered by liraglutide treatment. Semaglutide showed no increase in pancreas weight and no treatment-related histopathological findings in the pancreas after 13 or 52 weeks' dosing. Overall, results in 138 nonhuman primates showed no histopathological changes in the pancreas associated with liraglutide or semaglutide, two structurally different GLP-1 receptor agonists.

Improved insulin sensitivity and islet function after PPARdelta activation in diabetic db/db mice.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Several reports have shown that PPARdelta is involved in lipid metabolism, increasing fat oxidation and depleting lipid accumulation. Whether PPARdelta is involved in the regulation of glucose metabolism is not completely understood. In this study, we examined effects of long-term PPARdelta activation on glycemic control, islet function and insulin sensitivity in diabetic db/db mice. Male db/db mice were administered orally once daily with a selective and partial PPARdelta agonist (NNC 61-5920, 30 mg/kg) for eight weeks; control mice received vehicle. Fasting and non-fasting plasma glucose were reduced, reflected in reduced hemoglobinA(1c) (3.6+/-1.6% vs. 5.4+/-1.8 in db/db controls, P<0.05) and furthermore, the AUC(glucose) after oral glucose (3g/kg) was reduced by 67% (P<0.05) after long-term PPARdelta activation. Following intravenous glucose (1g/kg), glucose tolerance was improved after PPARdelta activation (K(G) 1.3+/-0.6 vs. -0.05+/-0.7 %/min, P=0.048). Insulin sensitivity, measured as the glucose clearance after intravenous injection of glucose (1g/kg) and insulin (0.75 or 1.0 U/kg), during inhibition of endogenous insulin secretion by diazoxide (25mg/kg), was improved (K(G) 2.9+/-0.6 vs. 1.3+/-0.3 %/min in controls, P<0.05) despite lower insulin levels. Furthermore, islets isolated from PPARdelta agonist treated mice demonstrated improved glucose responsiveness as well as improved cellular topography. In conclusion, PPARdelta agonism alleviates insulin resistance and improves islet function and topography, resulting in improved glycemia in diabetic db/db mice. This suggests that activation of PPARdelta improves glucose metabolism and may therefore potentially be target for treatment of type 2 diabetes.

Liraglutide, but not vildagliptin, restores normoglycaemia and insulin content in the animal model of type 2 diabetes, Psammomys obesus.

In order to investigate the effect and mechanism of liraglutide and vildagliptin in diabetic Psammomys obesus, we examined proliferation and apoptosis of beta-cells, beta-cell mass (BCM), and pancreatic insulin content after zero, six and fourteen days of treatment compared to control groups. One group of animals was kept on low-energy diet and seven groups were given high-energy diet (HED) that induced diabetes over a four week period. Non-fasting morning blood glucose, body weight, HbA(1C) and pancreatic insulin content were measured and beta cell mass (BCM), proliferation and apoptosis frequencies were determined using stereological point counting. Liraglutide significantly reduced blood glucose and even normalized it in all animals treated for six days and in 11 out of 17 animals treated for fourteen days. HED increased BCM and treatment with liraglutide did not change this. However, compared to the vehicle-treated animals pancreatic insulin content was normalized in animals treated for six and fourteen days with liraglutide. In contrast, vildagliptin, in doses causing full inhibition of plasma DPP-IV activity, neither reduced blood glucose nor altered HED-induced increases in BCM or pancreatic insulin content. These results suggest that liraglutide restores normoglycaemia and improves glycaemic control in P. obesus by increasing their insulin content and improving the function of the beta-cells. In contrast, vildagliptin does not improve glycaemic control in P. obesus nor affect beta-cell insulin content.

Liraglutide, a long-acting glucagon-like peptide-1 analog, reduces body weight and food intake in obese candy-fed rats, whereas a dipeptidyl peptidase-IV inhibitor, vildagliptin, does not.

Metabolic effects of the glucagon-like peptide-1 analog liraglutide and the dipeptidyl peptidase-IV inhibitor vildagliptin were compared in rats made obese by supplementary candy feeding. Female Sprague-Dawley rats were randomized to 12-week diets of chow or chow plus candy. The latter were randomized for 12 further weeks to continue their diet while receiving 0.2 mg/kg liraglutide twice daily subcutaneously, 10 mg/kg vildagliptin twice daily orally, or vehicle or to revert to chow-only diet. Energy expenditure was measured, and oral glucose tolerance tests (OGTTs) were performed. Body composition was determined by dual-energy X-ray absorptiometry scanning, and pancreatic beta-cell mass was determined by histology. Candy feeding increased weight, fat mass, and feeding-associated energy expenditure. Liraglutide or reversal to chow diet fully reversed weight and fat gains. Liraglutide was associated with decreased calorie intake and shifted food preference (increased chow/decreased candy consumption). Despite weight loss, liraglutide-treated rats did not decrease energy expenditure compared with candy-fed controls. Vildagliptin affected neither weight, food intake, nor energy expenditure. OGTTs, histology, and blood analyses indirectly suggested that both drugs increased insulin sensitivity. Liraglutide and vildagliptin inhibited obesity-associated increases in beta-cell mass. This was associated with weight and fat mass normalization with liraglutide, but not vildagliptin, where the ratio of beta-cell to body mass was low.

Measurements of insulin responses as predictive markers of pancreatic beta-cell mass in normal and beta-cell-reduced lean and obese Göttingen minipigs in vivo.

At present, the best available estimators of beta-cell mass in humans are those based on measurement of insulin levels or appearance rates in the circulation. In several animal models, these estimators have been validated against beta-cell mass in lean animals. However, as many diabetic humans are obese, a correlation between in vivo tests and beta-cell mass must be evaluated over a range of body weights to include different levels of insulin sensitivity. For this purpose, obese (n = 10) and lean (n = 25) Göttingen minipigs were studied. Beta-cell mass had been reduced (n = 16 lean, n = 5 obese) with a combination of nicotinamide (67 mg/kg) and streptozotocin (125 mg/kg), acute insulin response (AIR) to intravenous glucose and/or arginine was tested, pulsatile insulin secretion was evaluated by deconvolution (n = 30), and beta-cell mass was determined histologically. AIR to 0.3 (r(2) = 0.4502, P < 0.0001) or 0.6 g/kg glucose (r(2) = 0.6806, P < 0.0001), 67 mg/kg arginine (r(2) = 0.5730, P < 0.001), and maximum insulin concentration (r(2) = 0.7726, P < 0.0001) were all correlated to beta-cell mass when evaluated across study groups, and regression lines were not different between lean and obese groups except for AIR to 0.3 g/kg glucose. Baseline pulse mass was not significantly correlated to beta-cell mass across the study groups (r(2) = 0.1036, NS), whereas entrained pulse mass did show a correlation across groups (r(2) = 0.4049, P < 0.001). This study supports the use of in vivo tests of insulin responses to evaluate beta-cell mass over a range of body weights in the minipig. Extensive stimulation of insulin secretion by a combination of glucose and arginine seems to give the best correlation to beta-cell mass.

Long-term AICAR administration and exercise prevents diabetes in ZDF rats.

Lifestyle interventions including exercise programs are cornerstones in the prevention of obesity-related diabetes. The AMP-activated protein kinase (AMPK) has been proposed to be responsible for many of the beneficial effects of exercise on glucose and lipid metabolism. The effects of long-term exercise training or 5-aminoimidazole-4-carboxamide-1-beta-d-riboruranoside (AICAR) treatment, both known AMPK activators, on the development of diabetes in male Zucker diabetic fatty (ZDF) rats were examined. Five-week-old, pre-diabetic ZDF rats underwent daily treadmill running or AICAR treatment over an 8-week period and were compared with an untreated group. In contrast to the untreated, both the exercised and AICAR-treated rats did not develop hyperglycemia during the intervention period. Whole-body insulin sensitivity, as assessed by a hyperinsulinemic-euglycemic clamp at the end of the intervention period, was markedly increased in the exercised and AICAR-treated animals compared with the untreated ZDF rats (P < 0.01). In addition, pancreatic beta-cell morphology was almost normal in the exercised and AICAR-treated animals, indicating that chronic AMPK activation in vivo might preserve beta-cell function. Our results suggest that activation of AMPK may represent a therapeutic approach to improve insulin action and prevent a decrease in beta-cell function associated with type 2 diabetes.

Improved beta-cell survival and reduced insulitis in a type 1 diabetic rat model after treatment with a beta-cell-selective K(ATP) channel opener.

Treatment with ATP-sensitive K(+) channel openers (KCOs) leads to inhibition of insulin secretion and metabolic "rest" in beta-cells. It is hypothesized that in type 1 diabetes this may reduce beta-cell death resulting from metabolic stress as well as reduce the immunogenicity of the beta-cells during autoimmune beta-cell destruction. We have investigated whether the beta-cell-selective KCO compound, NN414, can be used to improve beta-cell survival in DR-BB rats rendered diabetic by modulation of their immune system. The rats were treated three times daily on days 1-19 with NN414, diazoxide, or vehicle. On day 21, an intravenous glucose tolerance test was conducted to assess beta-cell function. Postmortem histological analysis of rats' pancreata assessed the degree of insulitis and beta-cell volume. Among NN414-treated rats, 46% (16 of 35) were found to have a beta-cell mass similar to that of nondiabetic controls and significant glucose-stimulated C-peptide values, whereas only 11% (4 of 36) of vehicle-treated rats possessed a normal beta-cell mass and function (P < 0.002, by chi(2) test). Furthermore, responsive NN414-treated rats were almost free of insulitis. Thus, this study demonstrated that treatment with KCO compounds can indeed lead to preservation of beta-cell function and reduction of insulitis in a rat diabetes model.

Valine pyrrolidide preserves intact glucose-dependent insulinotropic peptide and improves abnormal glucose tolerance in minipigs with reduced beta-cell mass.

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important in blood glucose regulation. However, both incretin hormones are rapidly degraded by the enzyme dipeptidyl peptidase IV (DPPIV). The concept of DPPIV inhibition as a treatment for type 2 diabetes was evaluated in a new large animal model of insulin-deficient diabetes and reduced beta-cell mass, the nicotinamide (NIA) (67 mg/kg) and streptozotocin (STZ) (125 mg/kg)-treated minipig, using the DPPIV inhibitor, valine pyrrolidide (VP) (50 mg/kg). VP did not significantly affect levels of intact GLP-1 but increased levels of intact GIP (from 4543 +/- 1880 to 9208 +/- 3267 pM x min; P <.01), thus improving glucose tolerance (area under the curve [AUC] for glucose reduced from 1904 +/- 480 to 1582 +/- 353 mM x min; P =.05). VP did not increase insulin levels during the oral glucose tolerance test (OGTT) but increased the insulinogenic index in normal animals (from 83 +/- 42 to 192 +/- 108; P <.05), but not after NIA + STZ, possibly because of less residual insulin secretory capacity in these animals. GIP seems to contribute to the antihyperglycemic effect of VP in this model; however, additional mechanisms for the effect of DPPIV inhibition cannot be excluded. The authors conclude that DPPIV inhibitors may be useful to treat type 2 diabetes, even when this is due to reduced beta-cell mass.

GLP-1 derivative liraglutide in rats with beta-cell deficiencies: influence of metabolic state on beta-cell mass dynamics.

(1) Liraglutide is a long-acting GLP-1 derivative, designed for once daily administration in type II diabetic patients. To investigate the effects of liraglutide on glycemic control and beta-cell mass in rat models of beta-cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in 60% pancreatectomized rats. (2) When liraglutide was dosed s.c. at 150 microg kg-1 b.i.d. for 6 weeks in ZDF rats 6-8 weeks of age at study start, diabetes development was markedly attenuated. Blood glucose was approximately 12 mm lower compared to vehicle (P<0.0002), and plasma insulin was 2-3-fold higher during a normal 24-h feeding period (P<0.001). Judged by pair feeding, approximately 53% of the antihyperglycemic effect observed on 24-h glucose profiles was mediated by a reduction in food intake, which persisted throughout the study and averaged 16% (P<0.02). (3) Histological analyses revealed that beta-cell mass and proliferation were significantly lower in prediabetic animals still normoglycemic after 2 weeks treatment compared to vehicle-treated animals that had begun to develop diabetes. When the treatment period was 6 weeks, the liraglutide-treated animals were no longer completely normoglycemic and the beta-cell mass was significantly increased compared to overtly diabetic vehicle-treated animals, while beta-cell proliferation was unaffected. (4) In the experiments with 60% pancreatectomized rats, 8 days treatment with liraglutide resulted in a significantly lower glucose excursion in response to oral glucose compared to vehicle treatment. Again, part of the antihyperglycemic effect was due to reduced food intake. No effect of liraglutide on beta-cell mass was observed in these virtually normoglycemic animals. (5) In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of beta-cell deficiencies, and the in vivo effect of liraglutide on beta-cell mass may in part depend on the metabolic state of the animals.

Dual PPARalpha /gamma activation provides enhanced improvement of insulin sensitivity and glycemic control in ZDF rats.

Improvement of insulin sensitivity and lipid and glucose metabolism by coactivation of both nuclear peroxisome proliferator-activated receptor (PPAR)gamma and PPARalpha potentially provides beneficial effects over existing PPARgamma and alpha preferential drugs, respectively, in treatment of type 2 diabetes. We examined the effects of the dual PPARalpha/gamma agonist ragaglitazar on hyperglycemia and whole body insulin sensitivity in early and late diabetes stages in Zucker diabetic fatty (ZDF) rats and compared them with treatment with the PPARgamma preferential agonist rosiglitazone. Despite normalization of hyperglycemia and Hb A(1c) and reduction of plasma triglycerides by both compounds in both prevention and early intervention studies, ragaglitazar treatment resulted in overall reduced circulating insulin and improved insulin sensitivity to a greater extent than after treatment with rosiglitazone. In late-intervention therapy, ragaglitazar reduced Hb A(1c) by 2.3% compared with 1.1% by rosiglitazone. Improvement of insulin sensitivity caused by the dual PPARalpha/gamma agonist ragaglitazar seemed to have beneficial impact over that of the PPARgamma-preferential activator rosiglitazone on glycemic control in frankly diabetic ZDF rats.

Measurements of insulin secretory capacity and glucose tolerance to predict pancreatic beta-cell mass in vivo in the nicotinamide/streptozotocin Göttingen minipig, a model of moderate insulin deficiency and diabetes.

Knowledge about beta-cell mass and/or function could be of importance for the early diagnosis and treatment of diabetes. However, measurement of beta-cell function as an estimate of beta-cell mass is currently the only method possible in humans. The present study was performed to investigate different functional tests as predictors of beta-cell mass in the Göttingen minipig. beta-cell mass was reduced in the Göttingen minipig with a combination of nicotinamide (100 [n = 6], 67 [n = 25], 20 [n = 2], or 0 mg/kg [n = 4]) and streptozotocin (125 mg/kg). Six normal pigs were included. An oral glucose tolerance test (OGTT) (n = 43) and insulin secretion test (n = 30) were performed and pancreata obtained for stereological determination of beta-cell mass. During OGTT, fasting glucose (r(2) = 0.1744, P < 0.01), area under the curve for glucose (r(2) = 0.2706, P < 0.001), maximum insulin secretion (r(2) = 0.2160, P < 0.01), and maximum C-peptide secretion (r(2) = 0.1992, P < 0.01) correlated with beta-cell mass. During the insulin secretion test, acute insulin response to 0.3 g/kg (r(2) = 0.6155, P < 0.0001) and 0.6 g/kg glucose (r(2) = 0.7321, P < 0.0001) and arginine (67 mg/kg) (r(2) = 0.7732, P < 0.0001) and maximum insulin secretion (r(2) = 0.8192, P < 0.0001) correlated with beta-cell mass. This study supports the use of functional tests to evaluate beta-cell mass in vivo and has established a validated basis for developing a mathematical method for estimation of beta-cell mass in vivo in the Göttingen minipig.

The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases beta-cell mass in diabetic mice.

NN2211 is a long-acting, metabolically stable glucagon-like peptide-1 (GLP-1) derivative designed for once daily administration in humans. NN2211 dose dependently reduced the glycemic levels in ob/ob mice, with antihyperglycemic activity still evident 24 h postdose. Apart from an initial reduction in food intake, there were no significant differences between NN2211 and vehicle treatment, and body weight was not affected. Histological examination revealed that beta-cell proliferation and mass were not increased significantly in ob/ob mice with NN2211, although there was a strong tendency for increased proliferation. In db/db mice, exendin-4 and NN2211 decreased blood glucose compared with vehicle, but NN2211 had a longer duration of action. Food intake was lowered only on day 1 with both compounds, and body weight was unaffected. beta-Cell proliferation rate and mass were significantly increased with NN2211, but with exendin-4, only the beta-cell proliferation rate was significantly increased. In conclusion, NN2211 reduced blood glucose after acute and chronic treatment in ob/ob and db/db mice and was associated with increased beta-cell mass and proliferation in db/db mice. NN2211 is currently in phase 2 clinical development.

Nateglinide, but not repaglinide, stimulates growth hormone release in rat pituitary cells by inhibition of K channels and stimulation of cyclic AMP-dependent exocytosis.

OBJECTIVE: GH causes insulin resistance, impairs glycemic control and increases the risk of vascular diabetic complications. Sulphonylureas stimulate GH secretion and this study was undertaken to investigate the possible stimulatory effect of repaglinide and nateglinide, two novel oral glucose regulators, on critical steps of the stimulus-secretion coupling in single rat somatotrophs. METHODS: Patch-clamp techniques were used to record whole-cell ATP-sensitive K(+) (K(ATP)) and delayed outward K(+) currents, membrane potential and Ca(2+)-dependent exocytosis. GH release was measured from perifused rat somatotrophs. RESULTS: Both nateglinide and repaglinide dose-dependently suppressed K(ATP) channel activity with half-maximal inhibition being observed at 413 nM and 13 nM respectively. Both compounds induced action potential firing in the somatotrophs irrespective of whether GH-releasing hormone was present or not. The stimulation of electrical activity by nateglinide, but not repaglinide, was associated with an increased mean duration of the action potentials. The latter effect correlated with a reduction of the delayed outward K(+) current, which accounts for action potential repolarization. The latter effect had a K(d) of 19 microM but was limited to 38% inhibition. When applied at concentrations similar to those required to block K(ATP) channels, nateglinide in addition potentiated Ca(2+)-evoked exocytosis 3.3-fold (K(d)=3 microM) and stimulated GH release 4.5-fold. The latter effect was not shared by repaglinide. The stimulation of exocytosis by nateglinide was mimicked by cAMP and antagonized by the protein kinase A inhibitor Rp-cAMPS. CONCLUSION: Nateglinide stimulates GH release by inhibition of plasma membrane K(+) channels, elevation of cytoplasmic cAMP levels and stimulation of Ca(2+)-dependent exocytosis. By contrast, the effect of repaglinide was confined to inhibition of the K(ATP) channels.

Mild streptozotocin diabetes in the Göttingen minipig. A novel model of moderate insulin deficiency and diabetes.

Nonrodent models of diabetes are needed for practical and physiological reasons. Induction of mild insulin-deficient diabetes was investigated in male Göttingen minipigs by use of streptozotocin (STZ) alone (75, 100, and 125 mg/kg) or 125 mg/kg combined with pretreatment with nicotinamide (NIA; 0, 20, 67, 100, 150, and 230 mg/kg). Use of NIA resulted in a less steep slope of the regression line between fasting plasma glucose and changing doses compared with STZ [-7.0 +/- 1.4 vs. 29.7 +/- 7.0 mM. mg(-1). kg(-1), P < 0.0001]. Intermediate NIA doses induced moderate changes of glucose tolerance [glucose area under the curve increased from 940 +/- 175 to 1,598 +/- 462 mM. min, P < 0.001 (100 mg/kg) and from 890 +/- 109 to 1,669 +/- 691 mM. min, P = 0.003 (67 mg/kg)] with reduced insulin secretion [1,248 +/- 602 pM. min after 16 days and 1,566 +/- 190 pM. min after 60 days vs. 3,251 +/- 804 pM. min in normal animals (P < 0.001)] and beta-cell mass [5.5 +/- 1.4 mg/kg after 27 days and 7.9 +/- 4.1 mg/kg after 60 days vs. 17.7 +/- 4.7 mg/kg in normal animals (P = 0.009)]. The combination of NIA and STZ provided a model characterized by fasting and especially postprandial hyperglycemia and reduced, but maintained, insulin secretion and beta-cell mass. This model holds promise as an important tool for studying the pathophysiology of diabetes and development of new pharmacological agents for treatment of the disease.