Erica Nishimura

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.

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.

Fibroblast Growth Factor 21 (FGF21) and Glucagon-Like Peptide 1 Contribute to Diabetes Resistance in Glucagon Receptor–Deficient Mice

Mice genetically deficient in the glucagon receptor (Gcgr−/−) show improved glucose tolerance, insulin sensitivity, and α-cell hyperplasia. In addition, Gcgr−/− mice do not develop diabetes after chemical destruction of β-cells. Since fibroblast growth factor 21 (FGF21) has insulin-independent glucose-lowering properties, we investigated whether FGF21 was contributing to diabetes resistance in insulin-deficient Gcgr−/− mice. Plasma FGF21 was 25-fold higher in Gcgr−/− mice than in wild-type mice. FGF21 was found to be expressed in pancreatic β- and α-cells, with high expression in the hyperplastic α-cells of Gcgr−/− mice. FGF21 expression was also significantly increased in liver and adipose tissue of Gcgr−/− mice. To investigate the potential antidiabetic actions of FGF21 in insulin-deficient Gcgr−/− mice, an FGF21-neutralizing antibody was administered prior to oral glucose tolerance tests (OGTTs). FGF21 neutralization caused a decline in glucose tolerance in insulin-deficient Gcgr−/− mice during the OGTT. Despite this decline, insulin-deficient Gcgr−/− mice did not develop hyperglycemia. Glucagon-like peptide 1 (GLP-1) also has insulin-independent glucose-lowering properties, and an elevated circulating level of GLP-1 is a known characteristic of Gcgr−/− mice. Neutralization of FGF21, while concurrently blocking the GLP-1 receptor with the antagonist Exendin 9-39 (Ex9-39), resulted in significant hyperglycemia in insulin-deficient Gcgr−/− mice, while blocking with Ex9-39 alone did not. In conclusion, FGF21 acts additively with GLP-1 to prevent insulinopenic diabetes in mice lacking glucagon action.

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.

Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding†

Conjointly, the solvent-exposed residues of the central alpha-helix of the B chain form a well-defined ridge, which is flanked and partly overlapped by the two described insulin receptor binding surfaces on either side of the insulin molecule. To evaluate the importance of this interface in insulin receptor binding, we developed a new powerful method that allows us to introduce all the naturally occurring amino acids into a given position and subsequently determine the receptor binding affinities of the resulting insulin analogues. The total amino acid scanning mutagenesis was performed at positions B9, B10, B12, B13, B16, and B17, and the vast majority of the insulin analogue precursors were expressed and secreted in amounts close to that of the wild-type (human insulin) precursor. The analogue binding data revealed that positions B12 and B16 were the two positions most affected by the amino acid substitutions. Interestingly, the receptor binding affinities of the B13 analogues were also markedly affected by the amino acid substitutions, suggesting that GluB13 indeed is a part of insulins binding surface. The B10 library screen generated analogues covering a wide range of (20-340%) of relative binding affinities, and the results indicated that a structural stabilization of the central alpha-helix and thereby a more rigid presentation of the binding epitope at the insulin receptor is important for receptor recognition. In conclusion, systematic amino acid scanning mutagenesis allowed us to confirm the importance of the B chain alpha-helix as a central recognition element serving as a linker of a continual binding surface.

Novel glucagon receptor antagonists with improved selectivity over the glucose-dependent insulinotropic polypeptide receptor.

Optimization of a new series of small molecule human glucagon receptor (hGluR) antagonists is described. In the process of optimizing glucagon receptor antagonists, we counter-screened against the closely related human gastric inhibitory polypeptide receptor (hGIPR), and through structure activity analysis, we obtained compounds with low nanomolar affinities toward the hGluR, which were selective against the hGIPR and the human glucagon-like peptide-1 receptor (hGLP-1R). In the best cases, we obtained a >50 fold selectivity for the hGluR over the hGIPR and a >1000 fold selectivity over the hGLP-1R. A potent and selective glucagon receptor antagonist was demonstrated to inhibit glucagon-induced glycogenolysis in primary rat hepatocytes as well as to lower glucagon-induced hyperglycemia in Sprague-Dawley rats. Furthermore, the compound was shown to lower blood glucose in the ob/ob mouse after oral dosing.

Free Fatty Acid-Induced PP2A Hyperactivity Selectively Impairs Hepatic Insulin Action on Glucose Metabolism

In type 2 Diabetes (T2D) free fatty acids (FFAs) in plasma are increased and hepatic insulin resistance is “selective”, in the sense that the insulin-mediated decrease of glucose production is blunted while insulins effect on stimulating lipogenesis is maintained. We investigated the molecular mechanisms underlying this pathogenic paradox. Primary rat hepatocytes were exposed to palmitate for twenty hours. To establish the physiological relevance of the in vitro findings, we also studied insulin-resistant Zucker Diabetic Fatty (ZDF) rats. While insulin-receptor phosphorylation was unaffected, activation of Akt and inactivation of the downstream targets Glycogen synthase kinase 3α (Gsk3α and Forkhead box O1 (FoxO1) was inhibited in palmitate-exposed cells. Accordingly, dose-response curves for insulin-mediated suppression of the FoxO1-induced gluconeogenic genes and for de novo glucose production were right shifted, and insulin-stimulated glucose oxidation and glycogen synthesis were impaired. In contrast, similar to findings in human T2D, the ability of insulin to induce triglyceride (TG) accumulation and transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was unaffected. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs (aPKCs). The activity of the Akt-inactivating Protein Phosphatase 2A (PP2A) was increased in the insulin-resistant cells. Furthermore, inhibition of PP2A by specific inhibitors increased insulin-stimulated activation of Akt and phosphorylation of FoxO1 and Gsk3α. Finally, PP2A mRNA levels were increased in liver, muscle and adipose tissue, while PP2A activity was increased in liver and muscle tissue in insulin-resistant ZDF rats. In conclusion, our findings indicate that FFAs may cause a selective impairment of insulin action upon hepatic glucose metabolism by increasing PP2A activity.

Human glucagon receptor antagonists with thiazole cores. A novel series with superior pharmacokinetic properties.

The aim of the work presented here was to design and synthesize potent human glucagon receptor antagonists with improved pharmacokinetic (PK) properties for development of pharmaceuticals for the treatment of type 2 diabetes. We describe the preparation of compounds with cyclic cores (5-aminothiazoles), their binding affinities for the human glucagon and GIP receptors, as well as affinities for rat, mouse, pig, dog, and monkey glucagon receptors. Generally, the compounds had slightly less glucagon receptor affinity compared to compounds of the previous series, but this was compensated for by much improved PK profiles in both rats and dogs with high oral bioavailabilities and sustained high plasma exposures. The compounds generally showed species selectivity for glucagon receptor binding with poor affinities for the rat, mouse, rabbit, and pig receptors. However, dog and monkey glucagon receptor affinities seem to reflect the human situation. One compound of this series, 18, was tested intravenously in an anesthetized glucagon-challenged monkey model of hyperglucagonaemia and hyperglycaemia and was shown dose-dependently to decrease glycaemia. Further, high plasma exposures and a long plasma half-life (5.2 h) were obtained.

Insulin detemir is a fully efficacious, low affinity agonist at the insulin receptor

Aim: To compare the properties of insulin detemir with human insulin or insulin aspart in various in vitro and in vivo experiments, thereby highlighting the importance of performing dose–response studies when investigating insulin analogues, in this study specifically insulin detemir.

Insulin Delivery Into the Peripheral Circulation: A Key Contributor to Hypoglycemia in Type 1 Diabetes.

Hypoglycemia limits optimal glycemic control in type 1 diabetes mellitus (T1DM), making novel strategies to mitigate it desirable. We hypothesized that portal (Po) vein insulin delivery would lessen hypoglycemia. In the conscious dog, insulin was infused into the hepatic Po vein or a peripheral (Pe) vein at a rate four times of basal. In protocol 1, a full counterregulatory response was allowed, whereas in protocol 2, glucagon was fixed at basal, mimicking the diminished α-cell response to hypoglycemia seen in T1DM. In protocol 1, glucose fell faster with Pe insulin than with Po insulin, reaching 56 ± 3 vs. 70 ± 6 mg/dL (P = 0.04) at 60 min. The change in area under the curve (ΔAUC) for glucagon was similar between Pe and Po, but the peak occurred earlier in Pe. The ΔAUC for epinephrine was greater with Pe than with Po (67 ± 17 vs. 36 ± 14 ng/mL/180 min). In protocol 2, glucose also fell more rapidly than in protocol 1 and fell faster in Pe than in Po, reaching 41 ± 3 vs. 67 ± 2 mg/dL (P < 0.01) by 60 min. Without a rise in glucagon, the epinephrine responses were much larger (ΔAUC of 204 ± 22 for Pe vs. 96 ± 29 ng/mL/180 min for Po). In summary, Pe insulin delivery exacerbates hypoglycemia, particularly in the presence of a diminished glucagon response. Po vein insulin delivery, or strategies that mimic it (i.e., liver-preferential insulin analogs), should therefore lessen hypoglycemia.