PACAP is an islet neuropeptide which contributes to glucose-stimulated insulin secretion

Pituitary adenylate cyclase activating peptide (PACAP) is a ubiquitously distributed neuropeptide which also is localized to pancreatic islets and stimulates insulin secretion. We examined whether endogenous PACAP within the islets might contribute to glucose-stimulated insulin secretion by immunoneutralizing endogenous PACAP. Immunocytochemistry showed that PACAP immunoreactivity is expressed in nerve terminals within freshly isolated rat islets, but not in islets that had been cultured for 48 h. In contrast, islet endocrine cells did not display PACAP immunoreactivity. Addition of either of two specific PACAP antisera markedly inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from freshly isolated rat islets, whereas a control rabbit serum did not affect glucose-stimulated insulin secretion. In contrast, the PACAP antisera had no effect on glucose-stimulated insulin secretion in cultured islets. Based on these results we therefore suggest that PACAP is an islet neuropeptide which is required for the normal insulinotropic action of glucose.     

Expression of islet inducible nitric oxide synthase and inhibition of glucose-stimulated insulin release after long-term lipid infusion in the rat is counteracted by PACAP27

Chronic exposure of pancreatic islets to elevated plasma lipids (lipotoxicity) can lead to beta-cell dysfunction, with overtime becoming irreversible. We examined, by confocal microscopy and biochemistry, whether the expression of islet inducible nitric oxide synthase (iNOS) and the concomitant inhibition of glucose-stimulated insulin release seen after lipid infusion in rats was modulated by the islet neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)27. Lipid infusion for 8 days induced a strong expression of islet iNOS, which was mainly confined to beta-cells and was still evident after incubating islets at 8.3 mmol/l glucose. This was accompanied by a high iNOS-derived NO generation, a decreased insulin release, and increased cyclic GMP accumulation. No iNOS expression was found in control islets. Addition of PACAP27 to incubated islets from lipid-infused rats resulted in loss of iNOS protein expression, increased cyclic AMP, decreased cyclic GMP, and suppression of the activities of neuronal constitutive (nc)NOS and iNOS and increased glucose-stimulated insulin response. These effects were reversed by the PKA inhibitor H-89. The suppression of islet iNOS expression induced by PACAP27 was not affected by the proteasome inhibitor MG-132, which by itself induced the loss of iNOS protein, making a direct proteasomal involvement less likely. Our results suggest that PACAP27 through its cyclic AMP- and PKA-stimulating capacity strongly suppresses not only ncNOS but, importantly, also the lipid-induced stimulation of iNOS expression, possibly by a nonproteasomal mechanism. Thus PACAP27 restores the impairment of glucose-stimulated insulin release and additionally might induce cytoprotection against deleterious actions of iNOS-derived NO in beta-cells.     

Interleukin-1beta-induced nitric oxide production and inhibition of insulin secretion in rat islets of langerhans is dependent upon the nitric oxide synthase cofactor tetrahidrobiopterin

Interleukin (IL)-1 beta-induced inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans is mediated in part by nitric oxide (NO). The NO synthase cofactor 5,6,7,8-tetrahydrobiopterin (BH(4)) supports NO synthesis in many cell types and IL-1 beta-induced NO generation and inhibition of insulin secretion have been previously correlated with intracellular BH(4 )levels in rat insulinoma cells. Using rat islets and the beta cell line BRIN-BD11, we have investigated whether synthesis of BH(4) limits IL-1beta-induced NO generation and inhibition of glucose-induced insulin secretion. IL-1 beta-induced NO generation by BRIN cells and islets was reduced by 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of de novo BH(4) synthesis. Sepiapterin, the substrate for salvage pathway BH(4) synthesis, reversed this inhibitory effect of DAHP in islets but not BRIN cells. DAHP reversed IL-1 beta-induced inhibition of islet insulin secretion, an effect prevented by sepiapterin. We conclude that BH(4) generation is necessary for IL-1 beta-induced NO generation in rat islets and BRIN cells. While a contribution of non-NO mediators cannot be excluded, our results support the proposal that IL-1 beta-induced, NO-mediated inhibition of insulin secretion in rat islets is dependent on the NOS cofactor BH(4).     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

The microtubule associated protein syntabulin is required for glucose-stimulated and cAMP-potentiated insulin secretion

Syntabulin is a microtubule-associated protein that mediates anterograde transport of vesicles to neuronal processes. Here, we found that syntabulin was expressed in mouse pancreas and insulin-secreting β-cells, and that it partially co-localized with microtubule and insulin-containing granules. The association of syntabulin with these organelles increased upon glucose stimulation. Knock-down of syntabulin by shRNA reduced both basal and glucose-stimulated insulin secretion, and diminished cAMP-Epac2 and cAMP-PKA potentiated insulin secretion. Additionally, syntabulin was preferentially phosphorylated by the Epac2 agonist 8-pCPT-2′-O-Me-cAMP, suggesting that syntabulin could be a novel effector of Epac2 and play a critical role in cAMP-enhanced insulin secretion.     

Nitric oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis

Nitric oxide (NO) has emerged as a key molecule involved in many physiological processes in plants. To characterize roles of NO in tolerance of Arabidopsis (Arabidopsis thaliana) to salt stress, effect of NaCl on Arabidopsis wild-type and mutant (Atnoa1) plants with an impaired in vivo NO synthase (NOS) activity and a reduced endogenous NO level was investigated. Atnoa1 mutant plants displayed a greater Na+ to K+ ratio in shoots than wild-type plants due to enhanced accumulation of Na+ and reduced accumulation of K+ when exposed to NaCl. Germination of Atnoa1 seeds was more sensitive to NaCl than that of wild-type seeds, and wild-type plants exhibited higher survival rates than Atnoa1 plants when grown under salt stress. Atnoa1 plants had higher levels of hydrogen peroxide than wild-type plants under both control and salt stress, suggesting that Atnoa1 is more vulnerable to salt and oxidative stress than wild-type plants. Treatments of wild-type plants with NOS inhibitor and NO scavenger reduced endogenous NO levels and enhanced NaCl-induced increase in Na+ to K+ ratio. Exposure of wild-type plants to NaCl inhibited NOS activity and reduced quantity of NOA1 protein, leading to a decrease in endogenous NO levels measured by NO-specific fluorescent probe. Treatment of Atnoa1 plants with NO donor sodium nitroprusside attenuated the NaCl-induced increase in Na+ to K+ ratio. Therefore, these findings provide direct evidence to support that disruption of NOS-dependent NO production is associated with salt tolerance in Arabidopsis.     

Biotin enhances glucose-stimulated insulin secretion in the isolated perfused pancreas of the rat

The effects of biotin on insulin secretion in pair-fed control rats and biotin-deficient rats were investigated using the method of isolated pancreas perfusion. Isolated pancreas perfusion was performed using 20 mM glucose, 10 mM arginine, and 20 mM glucose plus various concentrations of biotin (20 mM glucose + biotin solution) as stimulants of insulin secretion. The insulin response to 20 mM glucose in biotin-deficient rats was approximately 22% of that seen in control rats. The level of the insulin response to 10 mM arginine was also significantly lower in biotin-deficient rats than in control rats. These results indicate that insulin release from the pancreas was disturbed in biotin-deficient rats. The insulin responses to 20 mM glucose + 1 mM biotin in biotin-deficient and control rats increased to 165% and 185%, respectively, of that to 20 mM glucose. These biotin-induced increases in glucose-stimulated insulin release were evident within the first few minutes of the infusion. An enhancement of the arginine-induced insulin response in control rats was not found when arginine and biotin was administered. These results suggest that biotin may play an important role in the mechanism by which glucose stimulates insulin secretion from the beta cells of the pancreatic islets.     

Compensatory responses to pyruvate carboxylase suppression in islet beta-cells. Preservation of glucose-stimulated insulin secretion

We have previously reported that glucose-stimulated insulin secretion (GSIS) is tightly correlated with pyruvate carboxylase (PC)-catalyzed anaplerotic flux into the tricarboxylic acid cycle and stimulation of pyruvate cycling activity. To further evaluate the role of PC in beta-cell function, we constructed a recombinant adenovirus containing a small interfering RNA (siRNA) specific to PC (Ad-siPC). Ad-siPC reduced PC mRNA levels by 83 and 64% and PC protein by 56 and 35% in INS-1-derived 832/13 cells and primary rat islets, respectively. Surprisingly, this manipulation did not impair GSIS in rat islets. In Ad-siPC-treated 832/13 cells, GSIS was slightly increased, whereas glycolytic rate and glucose oxidation were unaffected. Flux through PC at high glucose was decreased by only 20%, suggesting an increase in PC-specific activity. Acetyl carnitine, a surrogate for acetyl-CoA, an allosteric activator of PC, was increased by 36% in Ad-siPC-treated cells, suggesting a mechanism by which PC enzymatic activity is maintained with suppressed PC protein levels. In addition, the NADPH:NADP ratio, a proposed coupling factor for GSIS, was unaffected in Ad-siPC-treated cells. We conclude that beta-cells activate compensatory mechanisms in response to suppression of PC expression that prevent impairment of anaplerosis, pyruvate cycling, NAPDH production, and GSIS.     

Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements

Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.     

Stimulation of endogenous nitric oxide production is involved in the inhibitory effect of adrenomedullin on aldosterone secretion in the rat

Adrenomedullin (AM) (10(-8) M) partially suppressed aldosterone response of dispersed rat zona glomerulosa (ZG) cells to 10 mM K+, and the nitric oxide (NO) synthase inhibitors L-NAME (10(-3) M) and 1400W (10(-4) M) effectively counteracted this effect of AM. The NO donor L-Arginine (L-Arg) (10(-5) M) decreased both basal and K+ -stimulated aldosterone secretion. The guanylate-cyclase inhibitor Ly-83583, at a concentration (10(-4) M) abolishing either the guanylate-cyclase activator guanylin- or L-Arg-induced cGMP release from dispersed ZG cells, did not affect the aldosterone antisecretagogue action of AM and L-Arg. AM (10(-8) M) evoked a moderate increase in cGMP release by dispersed ZG cells, and the effect was blocked by both 10(-4) M Ly-83583 and 10(-3) M L-NAME. Collectively, these findings allow us (1) to confirm that NO inhibits aldosterone secretion through a cGMP-independent mechanism; and (2) to suggest that stimulation of endogenous NO synthesis plays a role in the mechanisms underlying the inhibitory effect of AM on K+ -stimulated aldosterone secretion from rat ZG cells.     

Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets

Islet amyloid polypeptide has 37 amino acids and is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes mellitus. To determine whether the peptide is involved in the impaired insulin secretion in this type of diabetes mellitus, we synthesized islet amyloid polypeptide and its fragments and examined its effect on insulin secretion. Islet amyloid polypeptide inhibited the glucose-stimulated insulin secretion from isolated rat pancreatic islets, as calcitonin gene-related peptide did, but the fragments failed to inhibit the secretion. Thus, we propose that amyloid deposition may be an important factor in the impairment of insulin secretion in type 2 diabetes mellitus.     

Metabolic cycling in control of glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) is central to normal control of metabolic fuel homeostasis, and its impairment is a key element of beta-cell failure in type 2 diabetes. Glucose exerts its effects on insulin secretion via its metabolism in beta-cells to generate stimulus/secretion coupling factors, including a rise in the ATP/ADP ratio, which serves to suppress ATP-sensitive K(+) (K(ATP)) channels and activate voltage-gated Ca(2+) channels, leading to stimulation of insulin granule exocytosis. Whereas this K(ATP) channel-dependent mechanism of GSIS has been broadly accepted for more than 30 years, it has become increasingly apparent that it does not fully describe the effects of glucose on insulin secretion. More recent studies have demonstrated an important role for cyclic pathways of pyruvate metabolism in control of insulin secretion. Three cycles occur in islet beta-cells: the pyruvate/malate, pyruvate/citrate, and pyruvate/isocitrate cycles. This review discusses recent work on the role of each of these pathways in control of insulin secretion and builds a case for the particular relevance of byproducts of the pyruvate/isocitrate cycle, NADPH and alpha-ketoglutarate, in control of GSIS.     

DOC2b is a SNARE regulator of glucose-stimulated delayed insulin secretion

Insulin secretion is precisely regulated by blood glucose with unique biphasic pattern. The regulatory mechanism of the second-phase insulin release is unclear. In this study, we report that DOC2b (double C2 domain protein isoform b), a SNARE related protein, was associated with insulin vesicles and translocated to plasma membrane within several minutes upon high-glucose stimulation followed by an interaction with syntaxin4, but not syntaxin1. This binding specificity and the time course of DOC2b translocation were suitable for the regulation of second-phase insulin release. Increased DOC2b expression enhanced glucose-stimulated insulin secretion. In contrast, silencing DOC2b inhibited delayed release of insulin, without affecting rapid (∼7 min) phase secretion. Interestingly, DOC2b had no effects on KCl-triggered insulin release. These data suggest that DOC2b may be a regulator for delayed (second-phase) insulin secretion in MIN6 cells.     

Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in beta-cells

Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in beta-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 +/- 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 +/- 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 +/- 311; WT, 425 +/- 231 pg x islet(-1) x 35 min(-1); P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 +/- 0.8 mM glucose, compared with 11.8 +/- 0.7 mM in WT (P < 0.05). Older Tg mice secreted less insulin in response to 20 mM glucose (Tg, 2,256 +/- 342; WT, 3,493 +/- 367 pg x islet(-1) x 35 min(-1); P < 0.05). Secretion in response to carbachol was similar in WT and Tg at both ages. Glucose oxidation was blunted in older Tg islets. At 5 mM glucose, islet CO2 production was comparable between Tg and WT. However, WT mice increased islet CO2 production 2.7 +/- 0.4-fold in 20 mM glucose, compared with only 1.4 +/- 0.1-fold in Tg (P < 0.02). Results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, acting at least in part by modulating glucose oxidation pathways. Prolonged excess hexosamine flux results in glucose desensitization and mimics glucose toxicity.     

Induction of insulin secretion in engineered liver cells by nitric oxide

Background  

Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway.