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 micromol/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 micromol/L) induced more pronounced insulin release in GK islets than in control islets. The diacylglycerol (DAG) lipase inhibitor RHC-80267 (35 micromol/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.

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 microM forskolin in islets cultured for 48 hours in 5.5 or 11 mM glucose. Insulin release was impaired in GK rat islets, irrespective of culture condition, in response to 3.3 and 16.7 mM glucose and was fully restored by forskolin through exaggerated insulin responses. Stimulation of normal islets with 5 microM 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 mM glucose, forskolin increased cAMP levels fivefold to sixfold at 3.3 and 16.7 mM glucose, whereas forskolin increased cAMP levels only twofold in islets cultured at 5.5 mM 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 mM) primed and markedly enhanced cAMP generation in response to forskolin.

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.

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 beta-cells, while a smaller number of ACs were expressed in alpha- and delta-cells. No AC-IR was observed in pancreatic polypeptide cells. Moreover, we have found an increased IR of the Ca2+-stimulated ACl, AC3 and AC8 in diabetic beta- and alpha-cells, compared with the corresponding IR in control pancreas. Most noticeable was the eliciting of a markedly enhanced AC8-IR in GK rat beta- and alpha-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.

Preserved initiatory and potentiatory effect of alpha-ketoisocaproate on insulin release in islets of glucose intolerant rats.

Insulin responses to glucose and non-glucose secretagogues were studied in short-term cultured pancreatic islets and perfused pancreata of the glucose intolerant F1 hybrid rats of spontaneously diabetic Goto-Kakizaki and control Wistar rats. After culture at 5.5 mmol/l glucose, hybrid islet responses to 11.1, 16.7 and 27.0 mmol/l glucose were between 60 and 40% of control islet responses. A combination of 1 mmol/l isobutylmethylxanthine and 16.7 mmol/l glucose induced a pronounced insulin release, which was of similar magnitude in hybrid and control rat islets. This response was not further augmented by addition of glibenclamide and arginine. The slope of potentiation of arginine (10 mmol/l)-stimulated insulin secretion by glucose (5.5-16.7 mmol/l) was greatly impaired in hybrid islets. In contrast to glucose, alpha-ketoisocaproate (KIC), which is metabolized in Krebs cycle, dose-dependently stimulated insulin secretion to similar levels in hybrid and control islets, cultured at 5.5 mmol/l glucose. Also in hybrid islets depolarized by potassium chloride (30 mmol/l) and with adenosine triphosphate-sensitive K+-channels kept open by diazoxide, insulin responses to glucose were greatly impaired but intact to KIC. Furthermore, KIC potentiated normally the insulin response to arginine in hybrid islets. In the isolated perfused pancreas, KIC induced similar insulin responses in hybrid rats and control rats. The potentiating effect by 5.5 mmol/l glucose on the KIC-stimulated insulin responses was, however, greatly reduced in isolated islets and absent in the perfused pancreata of hybrid rats. Taken together, these findings suggest an intact capacity for insulin release, although the initiating and potentiating effect by glucose on insulin release are defective in the Goto-Kakizaki-hybrid rats. An abnormal beta-cell glucose metabolism proximal to the Krebs cycle is likely to account for the impairment of insulin release.

Mutations in the promoter of adenylyl cyclase (AC)-III gene, overexpression of AC-III mRNA, and enhanced cAMP generation in islets from the spontaneously diabetic GK rat model of type 2 diabetes.

Glucose-induced insulin release is decreased in the spontaneously diabetic GK rat, a nonobese rodent model of type 2 diabetes. Forskolin restores the impaired insulin release in both the isolated perfused pancreas and isolated islets from these rats (Abdel-Halim et al., Diabetes 45:934-940, 1996). We demonstrate here that the insulinotropic effect of forskolin in the GK rat is due to increased generation of cAMP and that it is associated with overexpression of adenylyl cyclase (AC)-III mRNA and gene mutations. The AC-III mRNA overexpression was demonstrated by in situ hybridization using oligonucleotide probes binding to different regions of the rat AC-III mRNA. It was associated with the presence of two point mutations identified at positions -28 bp (A --> G) and -358 bp (A --> C) of the promoter region of the AC-III gene and was demonstrable in both GK rat islets and peripheral blood cells. Transfection of COS cells with a luciferase reporter gene system revealed up to 25-fold increased promoter activity of GK AC-III promoter when compared with normal rat promoter (P < 0.0001). In conclusion, forskolin restores the impaired insulin release in islets of the GK rat through enhanced cAMP generation. This is linked to overexpression of AC-III mRNA in GK islets due to two functional point mutations in the promoter region of the AC-III gene.

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 mumol/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.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 approximately 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.

Impaired coupling of glucose signal to the exocytotic machinery in diabetic GK rats: a defect ameliorated by cAMP.

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 KCl in the presence of 3.3 mmol/l glucose and 250 micromol/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 micromol/l) markedly enhanced insulin release at 3.3 mmol/l glucose in GK but not control pancreases (54 +/- 15 vs. 3 +/- 1 pmol/10 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 pmol/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 beta-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.

Increased amounts of a high molecular weight insulin-like growth factor II (IGF-II) peptide and IGF-II messenger ribonucleic acid in pancreatic islets of diabetic Goto-Kakizaki rats.

Insulin-like growth factor II (IGF-II), a member of the insulin family, regulates cell growth and differentiation. The IGF-II gene is localized close to the insulin gene in man and rat. IGF-II peptide binds weakly to the insulin receptor and exerts insulin-like effects on the blood glucose level. We studied IGF-II in endocrine pancreas in an animal model of noninsulin-dependent diabetes mellitus, the Goto-Kakizaki (GK) rat. At the age of 2 months, these rats have structural islet changes, with fibrosis and irregular configuration, so-called starfish-shaped islets. Immunohistochemical investigation revealed IGF-II immunoreactivity in the beta-cells in both GK and control rats. Pancreatic extraction, followed by size separation using gel chromatography, disclosed a high mol wt form of IGF-II in all animals, and RIA measurements revealed a considerably larger amount of the IGF-II peptide in the 2-and 6-month-old GK rats than in the 1-month GK and control rats. In situ hybridization of 3-month-old GK rats showed increased IGF-II messenger RNA expression in the starfish-shaped islets of GK rats than in the islets with normal structure in both diabetic and control animals. The reason for the increased amount of IGF-II is unclear. As the animals are diabetic before the islet changes occur, it might be a compensatory effect in response to hyperglycemia, but could also be a cause of the islet fibrosis.

Preserved beta-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 beta cells. To study whether an alteration in their islet beta-cell numbers occurs in parallel with the impairment of insulin secretion in this model, the relative volume density of beta cells was determined by means of conventional point sampling in immunostained 4-microns-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 beta-cell density was also similar in GK and control rat islets, amounting to 75.2 +/- 8.5 and 66.9 +/- 6.6%, respectively. Thus, the total relative volume of beta cells was 1.5 +/- 0.5% in GK rats and 1.6 +/- 0.4% in controls. In conclusion, the density of beta cells is preserved in the pancreata of the young, diabetic GK rats, suggesting the absence of a causal relationship between the relative pancreatic beta-cell volume and the impaired glucose-induced insulin secretion in this NIDDM animal model.

Development of decreased insulin-induced glucose transport in skeletal muscle of glucose-intolerant hybrids of diabetic GK rats.

1. The effect of glucose intolerance on insulin-stimulated glucose transport in isolated skeletal muscles was investigated in male F1 hybrids of spontaneously diabetic GK (Goto-Kakizaki) and control Wistar rats at 1 and 2 months of age. 2. Hybrid rats are characterized by markedly impaired glucose-induced insulin secretion. The area under the blood glucose curve was significantly higher following an intraperitoneal glucose injection (2 g/kg) in hybrid rats in both age groups than in the control rats (P < 0.001). In 2-month-old hybrid rats the incremental area under the insulin curve during the intraperitoneal glucose tolerance test was not different from that of control rats. Serum cholesterol, triacylglycerol or plasma free fatty acid levels did not differ between the groups. Fasting and post-prandial plasma glucose concentrations were elevated in 2-month-old hybrid rats compared with control rats (54%, P < 0.05, and 27%, P < 0.05, respectively), but were not different in 1-month-old rats. Plasma insulin did not differ between the hybrid and control rats in the fasting or post-prandial state at either age studied. 3. The insulin dose-response curves for 3-O-methylglucose transport did not differ between 1-month-old hybrid and control rats for either the soleus or epitrochlearis muscle. The insulin dose-response curve for the epitrochlearis, but not for the soleus, muscle from 2-month-old hybrid rats was shifted to the right compared with the curve from the control animals (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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 beta-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 x W) F1 hybrids were offspring of either male GK (mGK) and female Wistar (fW) (mGK x fW) or male Wistar (mW) and female GK (fGK) (mW x 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 x fW and 6.6 +/- 0.4 mM in mW x 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 x fW and 11.6 +/- 1.0 mM in mW x 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 x fW rats, and 18.20 +/- 3.97 pmol in mW x fGK rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Both somatostatin and insulin responses to glucose are impaired in the perfused pancreas of the spontaneously noninsulin-dependent diabetic GK (Goto-Kakizaki) rats.

We have studied the responses of insulin and somatostatin to glucose and arginine in the perfused pancreas of GK rats, which spontaneously develop mild noninsulin-dependent diabetes without concomitant obesity. Our GK rats have been obtained after at least 42 generations of inbreeding of Wistar rats with initial selection for increased blood glucose levels during glucose tolerance tests. During perfusion with a high (16.7 mmol l-1) glucose concentration, normal responses of insulin and somatostatin were found in pancreata from non-diabetic control rats. In GK pancreata, however, these responses were virtually abolished. When the glucose concentration of the perfusion medium was switched from 16.7 to 3.3 mmol l-1 glucose, a transient increase- 'off-response' -in both insulin and somatostatin secretion was noted in GK but not in control pancreata. During the subsequent stimulation with arginine (20 mmol l-1), insulin and somatostatin responses were similar in pancreata from non-diabetic and GK rats. The pancreatic content of insulin did not differ between non-diabetic and GK rats, whereas the content of somatostatin was increased by 56% (P < 0.025) in GK glands. In conclusion, abnormalities assigned to pancreatic hormone secretion in the GK rat comprise not only markedly impaired insulin but also somatostatin response to glucose. Since there was no decrease in pancreatic content of these hormones in GK rats, the cause of glucose insensitivity of the hormone-producing cells is likely to reside in a defective stimulus-secretion coupling rather than decreased availability of the hormones.

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

Insulin 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 microU/ng islet DNA in GK rats and 28.8 +/- 2.5 microU/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 microU.ng islet DNA-1.h-1 in GK rat islets and 0.04 +/- 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 [5-3H]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.(ABSTRACT TRUNCATED AT 250 WORDS)