Christian L. Brand

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

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 β‐cell mass in rat models of β‐cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in 60% pancreatectomized rats. When liraglutide was dosed s.c. at 150 μg 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). Histological analyses revealed that β‐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 β‐cell mass was significantly increased compared to overtly diabetic vehicle‐treated animals, while β‐cell proliferation was unaffected. 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 β‐cell mass was observed in these virtually normoglycemic animals. In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of β‐cell deficiencies, and the in vivo effect of liraglutide on β‐cell mass may in part depend on the metabolic state of the animals.

Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats.

SummaryThe role of glucagon in diabetic hyperglycaemia has been a matter of controversy because of difficulties in the production of selective glucagon deficiency. We developed a high-capacity (40 nmol/ ml), high-affinity (0.6·1011 l/mol) monoclonal glucagon antibody (Glu-mAb) and gave i.v. injections (4 ml/kg) to rats in order to study the effect of selective glucagon deficiency on blood glucose. Controls received a mAb against trinitrophenyl. Glu-mAb completely abolished the hyperglycaemic effect of 2.86 nmol/kg glucagon in normal rats (p<0.05, n=6). In moderately hyperglycaemic rats injected with streptozotocin as neonates (N-STZ), Glu-mAb abolished a postprandial increase in blood glucose (from 11.2±0.7 mmol/l to 17.3±1.8 mmol/l in controls vs 10.5±0.9 mmol/l to 9.3±1.0 mmol/l; cross-over: n=6, p<0.05). No significant effect of Glu-mAb treatment was observed in more hyperglycaemic N-STZ rats (cross-over, n=4) and in severely hyperglycaemic rats injected with STZ as adults (n=6), but after insulin treatment of the latter, at doses partially restoring blood glucose levels (12.7±4.3 mmol/l), Glu-mAb administration almost normalized blood glucose (maximal difference: 6.0±3.8 mmol/l; cross-over: n=5, p<0.05). In conclusion, our results provide strong additional evidence for the hypothesis that glucagon is involved in the pathogenesis of diabetes. The hormone plays an important role in the development of STZ-diabetic hyperglycaemia, but glucagon neutralization only leads to normoglycaemia in the presence of insulin.

Capsaicin-sensitive sensory fibers in the islets of Langerhans contribute to defective insulin secretion in Zucker diabetic rat, an animal model for some aspects of human type 2 diabetes

The system that regulates insulin secretion from β‐cells in the islet of Langerhans has a capsaicin‐sensitive inhibitory component. As calcitonin gene‐related peptide (CGRP)‐expressing primary sensory fibers innervate the islets, and a major proportion of the CGRP‐containing primary sensory neurons is sensitive to capsaicin, the islet‐innervating sensory fibers may represent the capsaicin‐sensitive inhibitory component. Here, we examined the expression of the capsaicin receptor, vanilloid type 1 transient receptor potential receptor (TRPV1) in CGRP‐expressing fibers in the pancreatic islets, and the effect of selective elimination of capsaicin‐sensitive primary afferents on the decline of glucose homeostasis and insulin secretion in Zucker diabetic fatty (ZDF) rats, which are used to study various aspects of human type 2 diabetes mellitus. We found that CGRP‐expressing fibers in the pancreatic islets also express TRPV1. Furthermore, we also found that systemic capsaicin application before the development of hyperglycemia prevents the increase of fasting, non‐fasting, and mean 24‐h plasma glucose levels, and the deterioration of glucose tolerance assessed on the fifth week following the injection. These effects were accompanied by enhanced insulin secretion and a virtually complete loss of CGRP‐ and TRPV1‐coexpressing islet‐innervating fibers. These data indicate that CGRP‐containing fibers in the islets are capsaicin sensitive, and that elimination of these fibers contributes to the prevention of the deterioration of glucose homeostasis through increased insulin secretion in ZDF rats. Based on these data we propose that the activity of islet‐innervating capsaicin‐sensitive fibers may have a role in the development of reduced insulin secretion in human type 2 diabetes mellitus.

Glucagon receptor knockout mice display increased insulin sensitivity and impaired beta-cell function

In previous studies, glucagon receptor knockout mice (Gcgr−/−) display reduced blood glucose and increased glucose tolerance, with hyperglucagonemia and increased levels of glucagon-like peptide (GLP)-1. However, the role of glucagon receptor signaling for the regulation of islet function and insulin sensitivity is unknown. We therefore explored β-cell function and insulin sensitivity in Gcgr−/− and wild-type mice. The steady-state glucose infusion rate during hyperinsulinemic-euglycemic clamp was elevated in Gcgr−/− mice, indicating enhanced insulin sensitivity. Furthermore, the acute insulin response (AIR) to intravenous glucose was higher in Gcgr−/− mice. The augmented AIR to glucose was blunted by the GLP-1 receptor antagonist, exendin-3. In contrast, AIR to intravenous administration of other secretagogues was either not affected (carbachol) or significantly reduced (arginine, cholecystokinin octapeptide) in Gcgr−/− mice. In islets isolated from Gcgr−/− mice, the insulin responses to glucose and several insulin secretagogues were all significantly blunted compared with wild-type mice. Furthermore, glucose oxidation was reduced in islets from Gcgr−/− mice. In conclusion, the present study shows that glucagon signaling is required for normal β-cell function and that insulin action is improved when disrupting the signal. In vivo, augmented GLP-1 levels compensate for the impaired β-cell function in Gcgr−/− mice.

A novel high-affinity peptide antagonist to the insulin receptor

In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.

Evidence for a Major Role for Glucagon in Regulation of Plasma Glucose in Conscious, Nondiabetic, and Alloxan-Induced Diabetic Rabbits

Effects of glucagon immunoneutralization on plasma glucose, insulin, and glucagon were studied 2–4 h after intravenous injection of a high-affinity, monoclonal glucagon antibody into normal as well as moderately and severely alloxan (ALX)-induced diabetic rabbits (n = 5–7). A monoclonal trinitrophenyl antibody was used in control studies. Endogenous glucagon was completely neutralized as evidenced by undetectable levels of free glucagon and high plasma glucagon-binding capacities. In postabsorbtive normal rabbits, glucagon neutralization decreased plasma glucose by 2.2 ± 0.3 mmol/l, and the resulting plasma levels of insulin and glucagon (indirectly measured) were 8 ± 3 and 640 ± 129% of baseline, respectively. However, when euglycemia was maintained by means of glucose infusion (steady-state plasma glucose and glucose infusion rate: 6.6 ± 0.1 mmol/l and 3.0 ± 0.4 mg · kg−1 · min−1), both plasma insulin and glucagon remained unaltered. Thus, the glucose infusion rate accurately reflects glucagons contribution to postabsorbtive glucose production. In both moderately and severely diabetic rabbits, immunoneutralization of glucagon decreased plasma glucose by ∼8 mmol/l, leading to euglycemia (7.3 ± 1.1 mmol/l) and reduced hyperinsulinemia (41 ± 9% of baseline) in the former and to partial restoration of euglycemia (12.7 ± 1.8 mmol/l) and unchanged insulin levels in the latter group of diabetic rabbits (P < 0.05 vs. controls in all studies). No significant changes were observed in control studies. In conclusion, glucagon is an important regulator of postabsorbtive glucose production in normal rabbits and plays an important role in the maintenance of hyperglycemia in ALX-induced diabetic rabbits.

The dual PPARα/γ agonist, ragaglitazar, improves insulin sensitivity and metabolic profile equally with pioglitazone in diabetic and dietary obese ZDF rats

1 In 6‐ and 10‐week‐old obesity‐prone (fa/fa) Zucker diabetic fatty (ZDF) rats, effects of prevention and intervention therapies, respectively, were compared between PPARα/γ agonist, ragaglitazar (RAGA) and separate PPARγ and α agonists, pioglitazone (PIO) and bezafibrate (BF). 2 In a separate study, lean (+/+) ZDF rats fed highly palatable chow to induce dietary obesity and insulin resistance were treated similarly. To test insulin‐secretory capacity, all animals underwent a hyperglycaemic clamp. 3 Insulin sensitivity was improved equally by RAGA and PIO in fa/fa rats subjected to both prevention and intervention treatments (e.g., prevention HOMA‐IR: −71 and −72%, respectively), as was hyperglycaemia (both −68%). BF had no effect on either parameter in any study. Plasma lipids were markedly reduced (by 48–77%) by RAGA in all studies, equivalent to PIO, but to a greater extent than BF. 4 RAGA improved β‐cell function (HOMA‐β) more than three‐fold with prevention and intervention therapies, whereas PIO showed improvement only in intervention therapy. Consistent with improved insulin sensitivity, glucose infusion rate during the clamp was 60% higher in RAGA‐treated animals subjected to prevention therapy, but there was little additional insulin‐secretory response, suggesting that insulin secretion was already maximal. 5 Thus, RAGA and PIO equally improve metabolic profile in ZDF rats, particularly when administered early in the course of diabetes. They also improve β‐cell function, although this is better demonstrated through indices incorporating fasting insulin and glucose concentrations than through the hyperglycaemic clamp technique in this model.

Immunoneutralization of Endogenous Glucagon Reduces Hepatic Glucose Output and Improves Long-Term Glycemic Control in Diabetic ob/ob Mice

In type 2 diabetes, glucagon levels are elevated in relation to the prevailing insulin and glucose levels. The relative hyperglucagonemia is linked to increased hepatic glucose output (HGO) and hyperglycemia. Antagonizing the effects of glucagon is therefore considered an attractive target for treatment of type 2 diabetes. In the current study, effects of eliminating glucagon signaling with a glucagon monoclonal antibody (mAb) were investigated in the diabetic ob/ob mouse. Acute effects of inhibiting glucagon action were studied by an oral glucose tolerance test (OGTT) and by measurement of HGO. In addition, the effects of subchronic (5 and 14 days) glucagon mAb treatment on plasma glucose, insulin, triglycerides, and HbA1c (A1C) levels were investigated. Glucagon mAb treatment reduced the area under the curve for glucose after an OGTT, reduced HGO, and increased the rate of hepatic glycogen synthesis. Glucagon mAb treatment for 5 days lowered plasma glucose and triglyceride levels, whereas 14 days of glucagon mAb treatment reduced A1C. In conclusion, acute and subchronic neutralization of endogenous glucagon improves glycemic control, thus supporting the contention that glucagon antagonism may represent a beneficial treatment of diabetes.

Effects of high-fat feeding and fasting on ghrelin expression in the mouse stomach.

Ghrelin is a peptide identified as an endogenous ligand for the growth hormone secretagogue receptor. Studies have shown that ghrelin stimulates growth hormone, promotes food intake and decreases energy expenditure. Furthermore, feeding status seems to influence plasma ghrelin levels, as these are increased during fasting, whereas feeding and oral glucose intake reduce plasma ghrelin. This study examined whether standardized obesity and fasting affect cellular expression of ghrelin. Specimens from the gastrointestinal tract of fed or 18-h fasted, low-fat or high-fat fed (10 weeks on diet) C57BL/6J mice were studied by immunocytochemistry (ICC) for ghrelin and in situ hybridization (ISH) for ghrelin mRNA. Ghrelin was expressed in especially the corpus but also the antrum of the stomach of all groups studied. Cells positive for ghrelin and ghrelin mRNA in the stomach were reduced in high-fat fed mice. In contrast, ghrelin expression was not affected by fasting. The reduction in ghrelin expression in the high-fat fed mice was associated with a reduction in plasma levels of ghrelin, whereas after fasting, when expression rate was not altered, there was an increase in plasma ghrelin. In conclusion, ghrelin is highly expressed in the corpus and antrum of the stomach of C57BL/6J mice. This expression is reduced in obesity, whereas fasting has no effect.

A Novel Glucagon Receptor Antagonist, NNC 25-0926, Blunts Hepatic Glucose Production in the Conscious Dog

Elevated glucagon is associated with fasting hyperglycemia in type 2 diabetes. We assessed the effects of the glucagon receptor antagonist (2R)-N-[4-({4-(1-cyclohexen-1-yl)[(3,5-dichloroanilino)carbonyl]anilino}methyl)benzoyl]-2-hydroxy-b-alanine (NNC 25-0926) on hepatic glucose production (HPG) in vivo, using arteriovenous difference and tracer techniques in conscious dogs. The experiments consisted of equilibration (–140 to –40 min), control (40–0 min), and experimental [0–180 min, divided into P1 (0–60 min) and P2 (60–180 min)] periods. In P1, NNC 25-0926 was given intragastrically at 0 (veh), 10, 20, 40, or 100 mg/kg, and euglycemia was maintained. In P2, somatostatin, basal intraportal insulin, and 5-fold basal intraportal glucagon (2.5 ng/kg/min) were infused. Arterial plasma insulin levels remained basal throughout the study in all groups. Arterial plasma glucagon levels remained basal during the control period and P1 and then increased to ∼70 pg/ml in P2 in all groups. Arterial plasma glucose levels were basal in the control period and P1 in all groups. In P2, the arterial glucose level increased to 245 ± 22 and 172 ± 15 mg/dl in the veh and 10 mg/kg groups, respectively, whereas in the 20, 40, and 100 mg/kg groups, there was no rise in glucose. Net hepatic glucose output was ∼2 mg/kg/min in all groups during the control period. In P2, it increased by 9.4 ± 2 mg/kg/min in the veh group. In the 10, 20, 40, and 100 mg/kg groups, the rise was only 4.1 ± 0.9, 1.6 ± 0.6, 2.4 ± 0.7, and 1.5 ± 0.3 mg/kg/min, respectively, due to inhibition of glycogenolysis. In conclusion, NNC 25-0926 effectively blocked the ability of glucagon to increase HGP in the dog.