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.     

Alterations in adenylate cyclase activity due to streptozotocin-induced diabetic cardiomyopathy

The effect of chronic experimental diabetes on the adenylate cyclase system (AC) in the rat heart was investigated. Rats were made diabetic by an intravenous injection of streptozotocin (65 mg/kg), hearts were removed 8 weeks later and washed cell particles were isolated. AC activity was measured in the absence and presence of different concentrations of forskolin, NaF, GTP analogue [Gpp(NH)p] or epinephrine. A significant depression in the epinephrine stimulated AC activity was observed in diabetic hearts. Basal AC activity and stimulation of AC with forskolin, NaF and Gpp(NH)p were not significantly different between control and diabetic preparations. These results indicate no apparent alterations in the regulatory or catalytic properties of AC in hearts from chronic diabetic rats. The observed depression in epinephrine stimulated AC activity may account for the depressed inotropic action of catecholamines in the diabetic cardiomyopathy.     

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Background

Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra).

Methodology/Principal Findings

Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia.

Conclusions/Significance

GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the β-cell microenvironment and contribute to progressive type 2 diabetic β-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent β-cell dysfunction in type 2 diabetes.     

Hypoglycemic effect of polysaccharides produced by submerged mycelial culture of Laetiporus sulphureus on streptozotocininduced diabetic rats

The hypoglycemic effect of the crude extracellular polysaccharides (EPS) produced from submerged mycelial culture of an edible mushroom Laetiporus sulphureus var. miniatus in streptozotocin (STZ)-induced diabetic rat was investigated. Hypoglycemic effect of EPS was evaluated in STZ-induced diabetic rats, and its possible mechanism was suggested by the results of western blot analysis and immunohistochemical staining. The results revealed that orally administrated EPS, when given 48 h after STZ treatment exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in EPS-fed rats to 43.5% of STZ-treated rats. The plasma levels of total cholesterol and triglyceride were significantly increased upon STZ treatment and they were markedly reduced by oral administration of EPS to near-normal levels. The results of immunohistochemical staining of the pancreatic tissues showed that EPS treatment considerably increased the insulin antigenesity of diabetic islet β-cells, suggesting the possibility of β-cell proliferation or regeneration by EPS therapy. Moreover, immunoblotting study revealed that protein levels of iNOS was increased and SOD2, catalase, GPx were significantly increased after EPS treatments, suggesting alleviated oxidative stress mediated by STZ. Orally administrated EPS exhibited considerable hypoglycemic effect in STZ-induced diabetic rats and that these EPS may be useful for the management of diabetes mellitus.     

Age-induced changes in pancreatic islet blood flow: evidence for an impaired regulation in diabetic GK rats

The present study aimed to compare longitudinal variations in islet blood perfusion in rats with different degrees of impairment of glucose metabolism. For this purpose, mildly diabetic Goto-Kakizaki (GK) rats, glucose intolerant F(1) hybrids of GK and Wistar (W) rats (H), and control W rats were examined at 5 wk, 12 wk, or 1 yr of age, using the microsphere technique for blood flow measurements. W rats showed progressively increasing islet blood flow (IBF) throughout the experiment. Both GK and H rats demonstrated increasing IBF between 5 and 12 wk. However, H rats showed no further increment in IBF at 1 yr, whereas GK rats displayed a pronounced decrease in IBF between 12 wk and 1 yr of age. The augmented IBF seen in older W rats may constitute an adaptation to the increasing demand for insulin secretion in aging rats. The inability to adapt to the increased demand for insulin secretion by upregulation of islet blood flow could contribute to the progressive deterioration of glucose metabolism seen in the aging GK rat.     

Hexose Metabolism in Pancreatic Islets: Glycogen Synthase and Glycogen Phosphorylase Activities

The activity of glycogen synthase and glycogen phosphorylase was measured in rat pancreatic islet homogenates. For this purpose, the sensitivity of current radioisotopic procedures for the assay of these enzymes in liver extracts was increased by about two orders of magnitude. Even so, the measurement of glycogen synthase and phosphorylase in islet homogenates was hampered by a potent amylase-like activity, resulting in the hydrolysis of preformed or newly formed ¹⁴C-labeled glycogen. Acarbose suppressed the latter phenomenon which was found attributable to both minute contamination of isolated islets by acinar cells and genuine α-amylase activity in purified islet β-cells. As measured by the more sensitive method in the presence of acarbose, the a/(a + b) ratio for glycogen synthase activity in islet homogenates was increased in islets preincubated in the presence as distinct from absence of D-glucose and decreased after preincubation with forskolin. These changes represented a mirror image of those evoked by D-glucose and forskolin in the a/(a + b) ratio for glycogen phosphorylase activity. It is concluded that glycogen synthesis and breakdown are regulated in the endocrine pancreas in a manner qualitatively comparable to that prevailing in hepatocytes, the possible participation of an amylase-like activity to glycogen metabolism in intact islet β-cells requiring further investigation.     

Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1

Background

A 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 Findings

We 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 N^G-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.

Conclusion

The 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.     

Does IGF-I stimulate pancreatic islet cell growth?

Both IGF-I and its receptor (IGF-IR) are specifically expressed in various cell types of the endocrine pancreas. IGF-I has long been considered a growth factor for islet cells as it induces DNA synthesis in a glucose-dependent manner, prevents Fas-mediated autoimmune β-cell destruction and delays onset of diabetes in non-obese diabetic (NOD) mice. Islet-specific IGF-I overexpression promotes islet cell regeneration in diabetic mice. However, in the last few years, results from most gene-targeted mice have challenged this view. For instance, combined inactivation of insulin receptor and IGF-IR or IGF-I and IGF-II genes in early embryos results in no defect on islet cell development; islet β-cell-specific inactivation of IGF-IR gene causes no change in β-cell mass; liver- and pancreatic-specific IGF-I gene deficiency (LID and PID mice) suggests that IGF-I exerts an inhibitory effect on islet cell growth albeit indirectly through controlling growth hormone release or expression of Reg family genes. These results need to be evaluated with potential gene redundancy, model limitations, indirect effects and ligand-receptor cross-activations within the insulin/IGF family. Although IGF-I causes islet β-cell proliferation and neogenesis directly, what occur in normal physiology, pathophysiology or during development of an organism might be different. Locally produced and systemic IGF-I does not seem to play a positive role in islet cell growth. Rather, it is probably a negative regulator through controlling growth hormone and insulin release, hyperglycemia, or Reg gene expression. These results complicate the perspective of an IGF-I therapy for β-cell loss.     

The distribution of PKC isoforms in enteric neurons, muscle and interstitial cells of the human intestine

In many organs, different protein kinase C (PKC) isoforms are expressed in specific cell types, suggesting that the different PKCs have cell-specific roles, and also that drugs acting on a particular PKC may have effects on the whole organ that are distinguishable from drugs that target other isoforms. Previous studies of the guinea-pig and mouse intestine indicate that there are cell-specific expressions of PKC isoforms in neurons, muscle and the interstitial cells of Cajal. In the present study we have investigated the expression of different PKCs in human intestine. Immunohistochemical studies showed that the forms that are prominent in human enteric neurons are PKCs γ and ε and in muscle the dominant form is PKCδ. Neurons were weakly stained for PKCβI. These observations parallel findings in guinea-pig and mouse, except that in human PKCγ-IR was not present in the same types of neurons that express it in the guinea-pig. Enteric glial cells were strongly immunoreactive for PKCα, which is also the major isoform in enteric glial cells of guinea-pig. In human and guinea-pig, glial cells also express PKCβI. Spindle-shaped cells in the mucosa were immunoreactive for PKCα and PKCγ and in the muscle layers similar cells had PKCγ-IR and PKCθ-IR. The spindle-shaped cells were similar in morphology to interstitial cells of Cajal. Western analysis and RT-PCR confirmed the presence of the PKC isoform proteins and mRNA in the tissue. We conclude that there is cell-type specific expression of different PKCs in enteric neurons and intestinal muscle in human tissue, and that there are strong similarities in patterns of expression between laboratory animals and human, but some clear differences are also observed.     

Defective protein histidine phosphorylation in islets from the Goto-Kakizaki diabetic rat

We recently described novel regulatory roles for protein histidine phosphorylation of key islet proteins (e.g., nucleoside diphosphate kinase and succinyl thiokinase) in insulin secretion from the islet beta-cell (Kowluru A. Diabetologia 44: 89-94, 2001; Kowluru A, Tannous M, and Chen HQ. Arch Biochem Biophys 398: 160-169, 2002). In this context, we also characterized a novel, ATP- and GTP-sensitive protein histidine kinase in isolated beta-cells that catalyzed the histidine phosphorylation of islet (endogenous) proteins as well as exogenously added histone 4, and we implicated this kinase in the activation of islet endogenous G proteins (Kowluru A. Biochem Pharmacol 63: 2091-2100, 2002). In the present study, we describe abnormalities in ATP- or GTP-mediated histidine phosphorylation of nucleoside diphosphate kinase in islets derived from the Goto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes. Furthermore, we provide evidence for a marked reduction in the activities of ATP- or GTP-sensitive histidine kinases in GK rat islets. On the basis of these observations, we propose that alterations in protein histidine phosphorylation could contribute toward insulin-secretory abnormalities demonstrable in the diabetic islet.     

Cytokine-induced selective increase of high-molecular-weight bFGF isoforms and their subcellular kinetics in cultured rat hippocampal astrocytes

Cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and epidermal growth factor (EGF) are probable factors responsible for up-regulation of basic fibroblast growth factor (bFGF) expression in reactive astrocytes following brain damage, however the effect of these cytokines on the expression of each bFGF-isoform has not been elucidated. Western biot analysis revealed the expression of 18, 22 and 24-kD bFGF isoforms in cultured rat hippocampal astrocytes, and the expression of high molecular weight (HMW)-isoforms (22 and 24-kD isoforms) but not of 18-kD isoform was selectively increased by cytokines. Immunofluorescent analysis demonstrated that bFGF content in the cytoplasm of astrocytes is initially increased by cytokines followed by nuclear targeting and localization in agreement with the previous evidence that HMW-isoforms possess a nuclear targeting signal. The present results suggest the important role of HMW-bFGF isoforms in the response of nervous tissue to injury.     

The mouse adducin gene family: alternative splicing and chromosomal localization

Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10. Received: 1 June 1999 / Accepted: 25 August 1999     

Endocrine pancreatic cells of postatal “diabetes” (DB) mice in cell culture

Summary Ultrastructural characteristics as well as secretory and biosynthetic behavior of monolayer pancreatic cell cultures established from 4-day-old C57BL/KsJ misty diabetic (m db/ m db) mice have been studied in comparison to normal littermate controls. Hypersecretion of glucagon by α-cells from BL/Ks misty diabetic mice after 2 days in vitro was found to precede any hyperfunction of the insulin-secreting β-cells. The increased level of glucagon-release in BL/Ks cell cultures from diabetic mice was accompanied by a greatly enhanced level of incorporation of [³H] tryptophan into glucagon-like molecules whose specific radioactivity was up to 15-fold higher than that observed in cultures from genetic controls. The finding of an α-cell dysfunction in cultures established from preweaning diabetic BL/Ks mice suggests that glucagon could play an early role in shaping the events that culminate in the expression of frank diabetes in this inbred strain. This study was supported in part by NIH Grants AM 17631 and AM 14461, and a grant from the Juvenile Diabetes Foundation. The Jackson Laboratory is fully accredited by the American Association for Accrediation of Laboratory Animal Care.     

Down-regulated expression of exocytotic proteins in pancreatic islets of diabetic GK rats

Exocytosis is regulated by exocytotic proteins, which are present in insulin-secreting beta-cells and play regulatory roles in insulin secretion. Non-insulin dependent diabetes mellitus (type 2 diabetes) is a disease characterized by impaired insulin secretion and insulin resistance. Exocytotic protein immunoreactivities were studied in pancreatic islets of type 2 diabetic Goto-Kakizaki (GK) rats using immunofluorescence histochemistry. The immunoreactivities for vesicle-associated membrane protein-2 (VAMP-2), synaptotagmin III, cysteine string protein (CSP), mammalian homologue of the unc-18 gene (Munc-18), alpha-soluble N-ethylmaleimide-sensitive attachment protein (alpha-SNAP), N-ethylmaleimide-sensitive factor (NSF) and synaptosomal-associated protein of 25 kDa (SNAP-25) exhibited weaker immunofluorescence intensity in islets of GK rats as compared to control Wistar rats. Insulin immunoreactivity was also decreased in GK rat beta-cells, whereas no detectable alterations in the expression of actin immunoreactivity could be detected. The data suggest that reduced expression of exocytotic proteins and decreased insulin content may contribute to the diabetic syndrome in the GK rat.     

Alpha-, Delta- and PP-cells: Are They the Architectural Cornerstones of Islet Structure and Co-ordination?

Islet non-β-cells, the α- δ- and pancreatic polypeptide cells (PP-cells), are important components of islet architecture and intercellular communication. In α-cells, glucagon is found in electron-dense granules; granule exocytosis is calcium-dependent via P/Q-type Ca²⁺-channels, which may be clustered at designated cell membrane sites. Somatostatin-containing δ-cells are neuron-like, creating a network for intra-islet communication. Somatostatin 1-28 and 1-14 have a short bioactive half-life, suggesting inhibitory action via paracrine signaling. PP-cells are the most infrequent islet cell type. The embryologically separate ventral pancreas anlage contains PP-rich islets that are morphologically diffuse and α-cell deficient. Tissue samples taken from the head region are unlikely to be representative of the whole pancreas. PP has anorexic effects on gastro-intestinal function and alters insulin and glucagon secretion. Islet architecture is disrupted in rodent diabetic models, diabetic primates and human Type 1 and Type 2 diabetes, with an increased α-cell population and relocation of non-β-cells to central areas of the islet. In diabetes, the transdifferentiation of non-β-cells, with changes in hormone content, suggests plasticity of islet cells but cellular function may be compromised. Understanding how diabetes-related disordered islet structure influences intra-islet cellular communication could clarify how non-β-cells contribute to the control of islet function.     

Age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats

Previous studies have demonstrated that experimental type 1 diabetes induced by streptozotocin causes alterations in the biochemical and functional properties of several receptor systems in the rat bladder. However, the exact mechanism involved in the pathophysiology of voiding dysfunction in type 2 diabetic patients is unknown. Because the GK rat is a widely accepted genetically determined rodent model for human type 2 diabetes, we investigated diabetes-induced changes in the bladder smooth muscle of the GK rats at several time points. Male GK rats and age-matched Wistar rats, as controls, were maintained for 4, 8, 16, and 32 weeks. Contractile responses to KCl, carbachol, ATP, and electrical field stimulation (EFS) were measured by using the isolated muscle bath techniques. Acetylcholine (ACh) release induced by EFS from bladder muscle strips was measured by using high-performance liquid chromatography coupled with a microdialysis procedure. Maximum contractile responses to carbachol and ATP, the release of ACh, and tissue sorbitol levels were similar in bladders from GK and control rats until 8 weeks of age. At 16 weeks of age, however, the contractile responses to carbachol and ATP, and tissue sorbitol levels were increased, and the EFS-induced ACh release was decreased in GK rats compared with controls. Although the maximum contractile responses to EFS were unchanged until 16 weeks of age, they were decreased in 32-week-old GK rats, compared with controls. Our data indicate the presence of age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats.