GK Islets Research Articles

Kynurenine-3-monooxygenase expression is activated in the pancreatic endocrine cells by diabetes and its blockade improves glucose-stimulated insulin secretion

Type 2 diabetes is associated with an inflammatory phenotype in the pancreatic islets. We previously demonstrated that proinflammatory cytokines potently activate the tryptophan/kynurenine pathway (TKP) in INS-1 cells and in normal rat islets. Here we examined: (1) the TKP enzymes expression in the diabetic GK islets; (2) the TKP enzymes expression profiles in the GK islets before and after the onset of diabetes; (3) The glucose-stimulated insulin secretion (GSIS) in vitro in GK islets after KMO knockdown using specific morpholino-oligonucleotides against KMO or KMO blockade using the specific inhibitor Ro618048; (4) The glucose tolerance and GSIS after acute in vivo exposure to Ro618048 in GK rats. We report a remarkable induction of the kmo gene in GK islets and in human islets exposed to proinflammatory conditions. It occurred prominently in beta cells. The increased expression and activity of KMO reflected an acquired adaptation. Both KMO knockdown and specific inhibitor Ro618048 enhanced GSIS in vitro in GK islets. Moreover, acute administration of Ro618048 in vivo improved glucose tolerance, GSIS and basal blood glucose levels in GK rats. These results demonstrate that targeting islet TKP is able to correct defective GSIS. KMO inhibition could represent a potential therapeutic strategy for type 2 diabetes.

IL-1 antagonism reduces hyperglycemia and tissue inflammation in the type 2 diabetic GK rat

Recent studies suggest an inflammatory process, characterized by local cytokine/chemokine production and immune cell infiltration, regulates islet dysfunction and insulin resistance in type 2 diabetes. However, the factor initiating this inflammatory response is not known. Here, we characterized tissue inflammation in the type 2 diabetic GK rat with a focus on the pancreatic islet and investigated a role for IL-1. GK rat islets, previously characterized by increased macrophage infiltration, displayed increased expression of several inflammatory markers including IL-1beta. In the periphery, increased expression of IL-1beta was observed primarily in the liver. Specific blockade of IL-1 activity by the IL-1 receptor antagonist (IL-1Ra) reduced the release of inflammatory cytokines/chemokines from GK islets in vitro and from mouse islets exposed to metabolic stress. Islets from mice deficient in IL-1beta or MyD88 challenged with glucose and palmitate in vitro also produced significantly less IL-6 and chemokines. In vivo, treatment of GK rats with IL-1Ra decreased hyperglycemia, reduced the proinsulin/insulin ratio, and improved insulin sensitivity. In addition, islet-derived proinflammatory cytokines/chemokines (IL-1beta, IL-6, TNFalpha, KC, MCP-1, and MIP-1alpha) and islet CD68(+), MHC II(+), and CD53(+) immune cell infiltration were reduced by IL-1Ra treatment. Treated GK rats also exhibited fewer markers of inflammation in the liver. We conclude that elevated islet IL-1beta activity in the GK rat promotes cytokine and chemokine expression, leading to the recruitment of innate immune cells. Rather than being directly cytotoxic, IL-1beta may drive tissue inflammation that impacts on both beta cell functional mass and insulin sensitivity in type 2 diabetes.

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 β-cells over time.

Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

BackgroundA distinctive feature of type 2 diabetes is inability of insulin-secreting β-cells to properly respond to elevated glucose eventually leading to β-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of β-cell dysfunction.Principal FindingsWe show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon.ConclusionThe results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms.

Phenotyping of individual pancreatic islets locates genetic defects in stimulus secretion coupling to Niddm1i within the major diabetes locus in GK rats.

The major diabetes quantitative trait locus (Niddm1), which segregates in crosses between GK rats affected with spontaneous type 2-like diabetes and normoglycemic F344 rats, encodes at least two different diabetes susceptibility genes. Congenic strains for the two subloci (Niddm1f and Niddm1i) have been generated by transfer of GK alleles onto the genome of F344 rats. Whereas the Niddm1f phenotype implicated insulin resistance, the Niddm1i phenotype displayed diabetes related to insulin deficiency. Individual islets from 16-week-old congenic rats were characterized for insulin release and oxygen tension (pO(2)). In the presence of 3 mmol/l glucose, insulin release from Niddm1f and Niddm1i islets was approximately 5 pmol. g(-1). s(-1) and pO(2) was 120 mmHg. Similar recordings were obtained from GK and F344 islets. When glucose was raised to 11 mmol/l, insulin release increased significantly in Niddm1f and F344 islets but was essentially unchanged in islets from GK and Niddm1i. The high glucose concentration lowered pO(2) to the same extent in islets from all strains. Addition of 1 mmol/l tolbutamide to the perifusion medium further increased pulsatile insulin release threefold in all islets. The pulse frequency was approximately 0.4 min(-1). alpha-Ketoisocaproate (11 mmol/l) alone increased pulsatile insulin release eightfold in islets from Niddm1f, Niddm1i, and control F344 rats but had no effect on insulin release from GK islets. These secretory patterns in response to alpha-ketoisocaproate were paralleled by reduction of pO(2) in Niddm1f, Niddm1i, and control F344 islets and no change of pO(2) in GK islets. The results demonstrate that Niddm1i carries alleles of gene(s) that reduce glucose-induced insulin release and that are amenable to molecular identification by genetic fine mapping.

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.

Gαolf Identification by RT-PCR in Purified Normal Pancreatic B Cells and in Islets from Rat Models of Non-insulin-Dependent Diabetes

Recent reports using immunohistochemistry have shown that Gαolf which shares 88% homology with Gαs was expressed in pancreatic islets. To test the specificity of the expression of this G protein isotype in rat islet cells, B and non-B cells were separated by flow cytometry. The expression of Gαolf and adenylyl cyclases (AC) of types II, III, V, and VI was evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). Since alterations in the expression of AC III were recently reported in the GK rat (a model of non-insulin-dependent diabetes mellitus, NIDDM), we also have analyzed the mRNA expression of Gαolf and AC isoforms in pancreatic islets from GK rats and from adult rats neonatally treated by streptozotocin (nSTZ rats), another model of NIDDM. Southern blots of amplicons generated with specific primers of Gαolf revealed the presence of a 540-bp band only in B cells. AC of types II, III, V, and VI were expressed both in B and non-B cells. However, AC III mRNA was clearly more abundant in non-B than in B cells. Moreover, in B cells the expression of AC VI was higher than that of AC V, whereas equal expressions of AC V and AC VI were found in non-B cells. In GK rat islets, the mRNA expressions of Gαolf, AC II, and AC III were clearly increased and no change in AC V and AC VI was found. In nSTZ rat islets, Gαolf expression was barely detectable, but AC II and AC III mRNA levels were higher than those observed in controls. In conclusion, Gαolf mRNA appeared specifically expressed in islet B cells and was increased in GK islets. The steady-state mRNA levels of AC II and AC III were clearly increased in the islets of the two rat models of NIDDM. Thus, alterations in the expression of G protein isotypes and AC isoforms could contribute to the diabetic phenotype.

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.

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 &amp;lt; 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,&amp;lt; P &amp;lt; 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.

Deficiency of pyruvate dehydrogenase activity in pancreatic islets of diabetic GK rats.

We investigated the role of islet pyruvate dehydrogenase (PDH) enzyme activity and fatty acid oxidation in the impaired insulin secretion in spontaneously diabetic GK rats. Blood glucose levels were elevated in 2- to 3-month-old GK rats (8.7 +/- 0.5 vs. 6.5 +/- 0.3 mM in control Wistar rats; P < 0.01), whereas serum insulin levels were comparable to those in control rats. Insulin and DNA contents were similar in freshly isolated islets from GK and control rats, whereas insulin responses to 27 mM glucose from GK islets were reduced by 52%. The effect of acetate or pyruvate on insulin responses evoked by succinate monomethylester (SAM) were compared to indirectly assess deficient generation of acetyl-coenzyme A from pyruvate. Acetate potentiated SAM-induced insulin secretion similarly in GK and control islets, whereas 10 mM pyruvate (which supplies acetyl-coenzyme A through PDH enzyme activity) failed to normally potentiate insulin secretion in GK islets (92% of SAM-induced response in GK vs. 154% in control islets). The PDH activity (active form) was decreased in GK islets by 35% (P < 0.001). The proportion of active form PDH to total PDH activity was reduced in GK islets (56% vs. 71% in control islets; P < 0.01). The activity of PDH kinase (which inactivates PDH by phosphorylation) was increased in GK islets, the rate of ATP-dependent inactivation of PDH was -0.29 +/- 0.02 vs. -0.19 +/- 0.02/min in control islets (P < 0.05). Culturing GK islets for 48 h at 5.5 mM glucose failed to correct the impaired insulin response to glucose and the decreased PDH activity. Serum FFA levels and islet triglyceride contents did not differ between GK and control rats. Etomoxir (1.0 and 10 microM), a carnitine palmitoyl transferase I inhibitor, failed to enhance glucose-induced insulin release in GK islets. The following conclusions were reached: 1) a kinase-mediated decrease in PDH activity in islets of GK rats may in part account for the decreased ratio of oxidized to utilized glucose and impaired insulin release in these islets; and 2) impaired insulin release in the GK rats is not linked to an inhibitory influence of islet fatty acid oxidation.

Spontaneously-developed Diabetic GK Rat—Pathogenesis and Diabetic Complications

Spontaneously diabetic GK rats were produced from rats of Wistar strain by repeating the selective breeding using glucose tolerance as a selection indicator. Of 211 normal rats 18 were selected by oral glucose tolerance test (GTT) and by the breeding 162 first generation rats were obtained. This procedure was repeated in each generation. The mean glucose tolerance curve became more diabetic with advance of generation and all of 9th generation rats had a diabetic character. The diabetic rats are not obese and there is no sex difference as regards occurrence of severity of the diabetic state. Significantly lower insulin secretion in GTT was observed in GK rats as compared with Wistar rats. Pancreas perfusion studies revealed that GK rats lacked the first response and then showed delayed and low insulin secretion. In addition, there was significantly reduced insulin secretion in isolated GK islets as compared to Wistar rats. Histological studies showed severe deformity, atrophy and fibrosis of GK islets. These histological abnormalities progressed with age. Immunohistochemistry to insulin and glucagon displayed reduced number of B cells and malorientation of A cells within GK islets. Glycolytic pathway enzyme activities in GK livers showed significantly higher than those of Wistar rats. Insulin insensitivity in GK livers may probably suggest the existence of insulin resistance. GK rats have the perilphera neuropathy and the thickening of glomerular basement membranes. GK rat is a promising model of non-insulin dependent diabetes mellitus and a good model of diabetic complications, as well.