Amel Guenifi

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

SummaryInsulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 μU/ng islet DNA in GK rats and 28.8±2.5 μU/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 μ · ng islet DNA−1· h−1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of 3H2O from [5-3 H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA−1 · h−1) than in control islets (1.3±0.1 pmol · ng islet DNA−1 · h−1; p<0.05). In contrast, glucose oxidation, estimated as the production of 14CO2 from [U-14C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with 3H2O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.

Glucagon-like peptide I enhances the insulinotropic effect of glibenclamide in NIDDM patients and in the perfused rat pancreas.

OBJECTIVE To investigate the acute effects of glibenclamide and glucagon-like peptide I (GLP-I) and their combination in perfused isolated rat pancreas and in patients with secondary failure to sulfonylureas. RESEARCH DESIGN AND METHODS Rat islets were perfused with 10 nmol/l GLP-I in combination with 2 μumol/l glibenclamide. In human experiments, GLP-I (0.75 pmol. kg−1 · min−1) was given as a continuous infusion during 240 min, while glibenclamide (3.5 mg) was administered orally. Eight patients participated in the study (age 57.6 ± 2.7 years, BMI 28.7 ± 1.5 kg/m2, mean ± SE). In all subjects, blood glucose was first normalized by insulin infusion administered by an artificial pancreas (Biostator). RESULTS GLP-I increased the insulinotropic effect of glibenclamide fourfold in the perfused rat pancreas. In human experiments, treatment with GLP-I alone and in combination with glibenclamide significantly decreased basal glucose levels (5.1 ± 0.4 and 4.5 ± 0.1 vs. 6.0 ± 0.3 mmol/l, P < 0.01), while with only glibenclamide, glucose concentrations remained unchanged. GLP-I markedly decreased total integrated glucose response to the meal (353 ± 60 vs. 724 ± 91 mmol μ l−1 · min−1, area under the curve [AUC] [–30–180 min], P < 0.02), whereas glibenclamide had no effect (598 ± 101 mmol · l−1 · min−1, AUC [–30–180 min], NS). The combined treatment further enhanced the glucose lowering effect of GLP-I (138 ± 24 mmol · l−1 · min, AUC [–30–180 min], P < 0.001). GLP-I, glibenclamide, and combined treat-stimulated meal-induced insulin release as reflected by insulinogenic indexes (control 1.44 ± 0.4; GLP-I 6.3 ± 1.6, P < 0.01; glibenclamide 6.8 ± 2.1, P < 0.01; combination 20.7 ± 5.0, P < 0.001). GLP-I inhibited basal but not postprandial glucagon responses. Using paracetamol as a marker for gastric emptying rate of the test meal, treatment with GLP-I decreased gastric emptying at 180 min by ∼50% compared with the control subjects (P < 0.01). CONCLUSIONS In acute experiments on overweight patients with NIDDM, GLP-I exerted a marked antidiabetogenic action on the basal and postprandial state. The peptide stimulated insulin, suppressed basal glucagon release, and prolonged gastric emptying. The glucose-lowering effect of GLP-I was further enhanced by glibenclamide. This action may be at least partially accounted for by a synergistic effect of these two compounds on insulin release. Glibenclamide per se enhanced postprandial but not basal insulin release and exerted a less pronounced antidiabetogenic effect compared with GLP-I.

Impact of Diabetic Inheritance on Glucose Tolerance and Insulin Secretion in Spontaneously Diabetic GK-Wistar Rats

The impact of genetic factors and maternal diabetes on glucose tolerance and pancreatic β-cell function was studied in first generation (F1) offspring generated in crosses between the spontaneously diabetic Goto-Kakizaki (GK)-Wistar rat and normoglycemic control Wistar rats (W). The (GK × W) F1 hybrids were offspring of either male GK (mGK) and female Wistar (fW) (mGK × fW) or male Wistar (mW) and female GK (fGK) (mW × fGK) rats. Already at 8 days of age, blood glucose levels were elevated in GK (7.6 ± 0.5 vs. 4.8 ± 0.3 mM in W; P < 0.001) and in F1 rats (6.0 ± 0.3 in mGK × fW and 6.6 ± 0.4 mM in mW × fGK; both P < 0.01 vs. W). In 2-month-old male rats, glucose (2 g/kg, intraperitoneally) markedly increased blood glucose levels after 60 min in GK rats (18.1 ± 0.6 vs. 5.5 ± 0.3 mM in W; P < 0.001) and moderately increased levels in F1 rats (9.9 ± 0.9 in mGK × fW and 11.6 ± 1.0 mM in mW × fGK, both P < 0.01 vs. W). Similar patterns were obtained in female rats. Repeated backcrossing of F1 with W rats successively improved glucose tolerance. In perfused pancreases of male rats, the 20-min insulin response to 16.7 mM glucose was −7.44 ± 5.18 pmol in GK rats, 71.57 ± 12.25 pmol in W rats, 9.00 ± 0.89 pmol in mGK × fW rats, and 18.20 ± 3.97 pmol in mW × fGK rats. In female W rats, the glucose-induced insulin response was significantly lower than in males (P < 0.05). However, as in males, insulin responses to glucose were impaired in both GK and F1 female rats. Arginine-induced insulin release was similar in all groups. In mGK × fW, glucose-stimulated somatostatin release was 50% of that in W rats, whereas arginine-stimulated responses of glucagon and somatostatin were not different from W rats. Pancreatic contents of insulin and glucagon were similar in mGK × fW and W rats, whereas somatostatin content was lower in mGK × fW rats (P < 0.05). In conclusion, the diabetic state in GK and F1 rats was evident early in life. Hybrid rats were intermediate between W and GK rats with regard to glucose tolerance and glucose-stimulated insulin response in vitro, but had normal pancreatic insulin content. Results of repeated backcrossing of F1 rats with W rats showed that genes in >1 locus contribute to the diabetic state. Furthermore, the absence of significant differences between impact of maternal and paternal origin of the GK genes for glucose intolerance suggests that hyperglycemia in utero does not influence the severity of diabetes in the F1 offspring.

Impaired Coupling of Glucose Signal to the Exocytotic Machinery in Diabetic GK Rats

The GK rat is a spontaneous model of NIDDM. The insulin response to 16.7 mmol/l glucose was markedly impaired in both isolated perfused pancreas and isolated islets from GK rats compared with control Wistar rats. Depolarization with 30 mmol/l KC1 in the presence of 3.3 mmol/l glucose and 250 μmol/l diazoxide induced similar insulin responses in perfused pancreases of GK and control rats. In contrast, the glucose-stimulated insulin release was also severely impaired in GK pancreases in the depolarized state. Forskolin (1 μmol/l) markedly enhanced insulin release at 3.3 mmol/l glucose in GK but not control pancreases (54 ± 15 vs. 3 ± 1 pmol/l0 min, P < 0.001). Dibutyryl cAMP (1 mmol/l) exerted effects similar to forskolin on insulin release in the perfused pancreas. In depolarized pancreases of GK but not control rats, forskolin also induced a marked insulin response at 3.3 mmol/l glucose (163 ± 48 vs. 16 ± 1 pmol/20 min,< P < 0.03). Similarly, in studies on isolated islets from GK rats cultured in 5.5 or 16.7 mmol/l glucose for 48 h, forskolin (5 μmol/l) restored insulin release in response to 16.7 mmol/l glucose but had no effect on islet glucose utilization at 3.3 or 16.7 mmol/l glucose. Forskolin markedly stimulated insulin release at 3.3 mmol/l glucose in GK but not control rat islets cultured for 48 h in 5.5 mmol/l glucose, whereas 20 mmol/l arginine had an almost identical effect in both islet varieties. However, in islets cultured in 16.7 mmol/l glucose, forskolin stimulated insulin release similarly both in control and GK islets at 3.3 mmol/l glucose. In conclusion, this study suggests that the insulinotropic effects of glucose are coupled to a direct regulation of the exocytotic machinery in the pancreatic 3-cell. This pathway is markedly impaired in GK rats, contributing to defective insulin response to glucose. In this model, cAMP generation restores the insulin response to 16.7 mmol/l glucose and exerts a marked insulin release even at 3.3 mmol/l glucose.

Preserved β-Cell Density in the Endocrine Pancreas of Young, Spontaneously Diabetic Goto-Kakizaki (GK) Rats

The Goto-Kakizaki (GK) rat represents a spontaneous animal model of non-insulin-dependent diabetes mellitus (NIDDM) characterized by impaired glucose-stimulated insulin release from the pancreatic β cells. To study whether an alteration in their islet β-cell numbers occurs in parallel with the impairment of insulin secretion in this model, the relative volume density of β cells was determined by means of conventional point sampling in immunostained 4-μm-thick sections of the pancreata from 8-week-old GK rats. The pancreata of nondiabetic Wistar rats were used as control parenchyma. In the GK pancreata the majority of islets was found to have a normal structure; only a few of the islets demonstrated an irregular shape (starfish-shaped islets) with fibrosis. The relative volume of the total endocrine parenchyma was found to be 2.0 ± 0.6% (mean ± SEM) of the whole pancreatic parenchyma in GK rats. In the control rats the corresponding value was 2.3 ± 0.5%. The islet β-cell density was also similar in GK and control rat islets, amounting to 15.2 ± 8.5 and 66.9 ± 6.6%, respectively. Thus, the total relative volume of β cells was 1.5 ± 0.5% in GK rats and 1.6 ± 0.4% in controls. In conclusion, the density of β cells is preserved in the pancreata of the young, diabetic GK rats, suggesting the absence of a causal relationship between the relative pancreatic β-cell volume and the impaired glucose-induced insulin secretion in this NIDDM animal model.

Adenylyl cyclase isoform expression in non-diabetic and diabetic Goto-Kakizaki (GK) rat pancreas. Evidence for distinct overexpression of type-8 adenylyl cyclase in diabetic GK rat islets

Abstract Glucose-induced insulin release is markedly decreased in the spontaneously diabetic Goto-Kakizaki (GK) rat pancreas. This defect was recently shown to be reversed by forskolin which markedly enhances cAMP generation in GK islets. These effects of forskolin were associated with overexpression of type-3 adenylyl cyclase (AC) mRNA due to the presence of two functional point mutations in the promoter region of AC3 gene in GK rat. Nine AC isoforms have been described, but their expression pattern in relation to the main pancreatic islet cell types, as well as their involvement in the diabetic state, is still unknown. Using antibodies raised against AC1–8, we have studied by double immunofluorescence the localisation of these AC isoforms in different endocrine cell types in both normal and diabetic GK rat pancreas. Our results demonstrated a clear immunoreaction (IR) to AC1–4 and 6 in normal and GK islet β-cells, while a smaller number of ACs were expressed in α- and δ-cells. No AC-IR was observed in pancreatic polypeptide cells. Moreover, we have found an increased IR of the Ca2+-stimulated AC1, AC3 and AC8 in diabetic β- and α-cells, compared with the corresponding IR in control pancreas. Most noticeable was the eliciting of a markedly enhanced AC8-IR in GK rat β- and α-cells, in contrast to a barely discernible AC8-IR in corresponding normal cells. In conclusion, AC expression exhibits a complex pattern in the endocrine pancreas, with specific differences between the normal and diabetic state.

Carbachol restores insulin release in diabetic GK rat islets by mechanisms largely involving hydrolysis of diacylglycerol and direct interaction with the exocytotic machinery.

In several models of insulin resistance, cholinergically induced insulin secretion is augmented. We studied here whether this also is present in the spontaneously diabetic GK (Goto–Kakizaki) rat pancreas. Using carbachol (50 &mgr;mol/L), enhanced insulin release was elicited in perfused pancreas under normal or depolarized conditions in GK compared with control rats at 3.3 mmol/L glucose (p < 0.03). Carbachol fully normalized insulin secretion in GK rats at 16.7 mmol/L glucose through an effect abolished by atropine. Similarly, direct stimulation of protein kinase C (PKC) with the DAG-permeable compound 1-oleoyl-2-acetyl-sn-glycerol (OAG, 300 &mgr;mol/L) induced more pronounced insulin release in GK islets than in control islets. The diacylglycerol (DAG) lipase inhibitor RHC-80267 (35 &mgr;mol/L) significantly reduced carbachol effects in control and GK islets, but had no effect on OAG-induced insulin release. The enhanced insulinotropic effects of carbachol in GK islets was not accompanied by increased cyclic adenosine monophosphate (cAMP) or arachidonic acid (AA) formation in GK when compared with control islets. In conclusion, cholinergic stimulation induced enhanced insulin release in diabetic GK islets. This is largely mediated through mechanisms involving hydrolysis of DAG to AA and interaction with exocytotic steps of insulin release.

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production.

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.

Differential effects of glucagon-like peptide-1 (7-36)amide versus cholecystokinin on arginine-induced islet hormone release in vivo and in vitro.

We compared the effects of two incretin hormones, glucagon-like peptide-1 (7-36)amide (GLP-1) and cholecystokinin (CCK), on islet hormone secretion. GLP-1 strongly potentiated glucose-stimulated insulin secretion in the perfused rat pancreas and in vivo in mice (p < 0.001). In contrast, GLP-1 did not enhance arginine-induced insulin release under these experimental conditions. In the perfused rat pancreas, GLP-1 also potentiated glucose-stimulated somatostatin secretion but, again, had no effect on arginine-induced somatostatin release. However, GLP-1 promptly inhibited the arginine-induced glucagon release (p < 0.02). In contrast, CCK enhanced insulin release in response to arginine both in the perfused rat pancreas and in vivo in mice (p < 0.001). In conclusion, GLP-1, in contrast to CCK, failed to enhance arginine-induced insulin release both in vitro and in vivo. This suggests that a signal generated by nutrient metabolism is required for the potentiation of insulin secretion by GLP-1. Furthermore, GLP-1 directly inhibited arginine-induced glucagon release as no concurrent increase in insulin or somatostatin release was noted.

Glucose enhances adenylyl cyclase responses in normal but not diabetic GK rat islets

In the GK rat model of type 2 diabetes, adenylyl cyclase (AC) expression and stimulation are increased. Whether the prevalent glucose level has any effects on AC responses is, however, unclear. We have studied concurrent insulin release and cyclic adenosine monophosphate (cAMP) generation in response to 5 &mgr;M forskolin in islets cultured for 48 hours in 5.5 or 11 m M glucose. Insulin release was impaired in GK rat islets, irrespective of culture condition, in response to 3.3 and 16.7 m M glucose and was fully restored by forskolin through exaggerated insulin responses. Stimulation of normal islets with 5 &mgr;M forskolin elicited different islet cAMP responses, which were dependent on the dose of glucose in the culture medium. Thus in normal islets cultured in 11 m M glucose, forskolin increased cAMP levels fivefold to sixfold at 3.3 and 16.7 m M glucose, whereas forskolin increased cAMP levels only twofold in islets cultured at 5.5 m M glucose. In GK islets, forskolin induced a consistently exaggerated approximately eightfold increase in cAMP generation irrespective of glucose concentration in the culture medium. In conclusion, culturing normal islets at hyperglycemic glucose levels (11 m M) primed and markedly enhanced cAMP generation in response to forskolin.