TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.     

Interleukin-18 mRNA, but not interleukin-18 receptor mRNA, is constitutively expressed in islet beta-cells and up-regulated by interferon-gamma

Interleukin-18 (IL-18) mRNA is expressed in islets of NOD mice during the early stages of insulitis and IL-18 has therefore been implicated as a contributing factor in immune-mediated beta-cell destruction. However, a recent study failed to show any effect of human IL-18 on the function of isolated rat islets. Since species differences have been shown between human and murine IL-18, the aims of this study were to investigate 1) if species homologous IL-18 alone or following IL-12 pre-exposure affected rat islet function, 2) if IL-18 dose-dependently modulated IL-1 beta or interferon-gamma (IFN-gamma) + tumor necrosis factor-alpha (TNF-alpha) actions on islet function, and 3) if IL-18 and IL-18 receptor (IL-18R) were expressed in rat islet beta-cells. Insulin release and nitric oxide (NO) production from isolated rat islets were measured after incubation with or without cytokines. RT-PCR was used to quantitate mRNA expression of IL-18 and the IL-18R signaling chain (IL-18R beta). There were no significant effects of 0.625-10 nM recombinant murine (rm) IL-18 alone on accumulated or glucose-challenged insulin release or NO production after 24 hours. Fifteen pg/ml of recombinant human (rh) IL-1 beta as well as 200 U/ml recombinant rat (rr) IFN-gamma + 250 U/ml rhTNF-alpha significantly increased islet NO production and inhibited both accumulated and glucose-challenged islet insulin release. However, rmIL-18 failed to modulate these effects of IL-1 beta or IFN-gamma + TNF-alpha. Although IL-12 induces IL-18R expression in Th1 and B lymphocytes, 24-hours rmIL-12 preincubation neither sensitized islets to effects of 10 nM of rm or rrIL-18 alone nor primed the islets to IL-1 beta actions on insulin release and NO production. IL-18R beta mRNA, which was expressed in human peripheral blood mononuclear cells (PBMC), was not expressed in rat insulinoma (RIN) cells or in isolated rat islets, even after exposure to IL-1 beta and/or IFN-gamma + TNF-alpha or IL-12. IL-18 mRNA was constitutively expressed in RIN cells, in FACS-purified rat beta-cells and in intact rat and mouse islets, and was up-regulated by IFN-gamma in an interferon regulatory factor-1- IRF-1) and NO - independent manner. However, IL-18 protein was undetectable in lysates and supernates of RIN cells by ECL, Western blotting and immunoprecipitation. In conclusion, we show for the first time that IL-18 but not IL-18R is expressed in rodent islet beta-cells. The physiological importance and pathological role of IL-18 originating from islet beta-cells deserve further investigation.     

Glucagon decreases cytokine induction of nitric oxide synthase and action on insulin secretion in RIN5F cells and rat and human islets of Langerhans.

Nitric oxide synthase, induced by cytokines in insulin-containing cells, produces nitric oxide which inhibits function and may promote cell killing. Since glucagon was shown to prevent inducible nitric oxide synthase (iNOS) expression in rat hepatocytes it was of interest to examine the action of glucagon (and cyclic AMP) on iNOS induction in insulin-producing cells. Cultured RIN5F cells and primary rat and human islets of Langerhans were treated with interleukin 1beta (IL-1beta) or a combination of cytokines, and were co-treated or pre-treated with glucagon. In RIN5F cells, the activity of iNOS induced by IL-1beta (10 pM, 24 h), was significantly reduced by glucagon (1000 nM), which raises cyclic AMP, and by forskolin (1-10 microM), a non specific activator of adenylate cyclase. Glucagon and forskolin also decreased iNOS expression in RIN5F cells, and rat and human islets, as shown by Western blotting. The inhibitory action of IL-1beta (100 pM, 24 h) on rat islet insulin secretion was partially reversed by 1-h pre-treatment with glucagon (10-1000 nM), while the contrasting stimulatory effect of 48-h treatment with cytokines on insulin secretion from human islets was similarly prevented by glucagon (1000 nM) pre-treatment. These results suggest that glucagon inhibits iNOS expression in insulin-containing cells and imply that glucagon could modulate the inhibitory effects of cytokines.     

Neurotensin is a regulator of insulin secretion in pancreatic beta-cells

Neurotensin (NT) is secreted from neurons and gastrointestinal endocrine cells. We previously reported that the three NT receptors (NTSRs) are expressed in pancreatic islets and beta cell lines on which we observed a protective effect of NT against cytotoxic agents. In this study, we explored the role of NT on insulin secretion in the endocrine pancreatic beta cells. We observed that NT stimulates insulin secretion at low glucose level and has a small inhibiting effect on stimulated insulin secretion from isolated islets or INS-1E cells. We studied the mechanisms by which NT elicited calcium concentration changes using fura-2 loaded islets or INS-1E cells. NT increases calcium influx through the opening of cationic channels. Similar calcium influxes were observed after treatment with NTSR selective ligands. NT-evoked calcium regulation involves PKC and the translocation of PKCα and PKC? to the plasma membrane. Part of NT effects appears to be also mediated by PKA but not via the Erk pathway. Taken together, these data provide evidence for an important endocrine role of NT in the regulation of the secretory function of beta cells.     

Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells.

Ghrelin is produced mainly by endocrine cells in the stomach and is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). It also influences feeding behavior, metabolic regulation, and energy balance. It affects islet hormone secretion, and expression of ghrelin and GHS-R in the pancreas has been reported. In human islets, ghrelin expression is highest pre- and neonatally. We examined ghrelin and GHS-R in rat islets during development with immunocytochemistry and in situ hybridization. We also studied the effect of ghrelin on insulin secretion from INS-1 (832/13) cells and the expression of GHS-R in these cells. We found ghrelin expression in rat islet endocrine cells from mid-gestation to 1 month postnatally. Islet expression of GHS-R mRNA was detected from late fetal stages to adult. The onset of islet ghrelin expression preceded that of gastric ghrelin. Islet ghrelin cells constitute a separate and novel islet cell population throughout development. However, during a short perinatal period a minor subpopulation of the ghrelin cells co-expressed glucagon or pancreatic polypeptide. Markers for cell lineage, proliferation, and duct cells revealed that the ghrelin cells proliferate, originate from duct cells, and share lineage with glucagon cells. Ghrelin dose-dependently inhibited glucose-stimulated insulin secretion from INS-1 (832/13) cells, and GHS-R was detected in the cells. We conclude that ghrelin is expressed in a novel developmentally regulated endocrine islet cell type in the rat pancreas and that ghrelin inhibits glucose-stimulated insulin secretion via a direct effect on the beta-cell.     

Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.     

SNAP-25 is expressed in islets of Langerhans and is involved in insulin release

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP- 25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.     

Persistent expression of HNF6 in islet endocrine cells causes disrupted islet architecture and loss of beta cell function

We used transgenesis to explore the requirement for downregulation of hepatocyte nuclear factor 6 (HNF6) expression in the assembly, differentiation, and function of pancreatic islets. In vivo, HNF6 expression becomes downregulated in pancreatic endocrine cells at 18. 5 days post coitum (d.p.c.), when definitive islets first begin to organize. We used an islet-specific regulatory element (pdx1(PB)) from pancreatic/duodenal homeobox (pdx1) gene to maintain HNF6 expression in endocrine cells beyond 18.5 d.p.c. Transgenic animals were diabetic. HNF6-overexpressing islets were hyperplastic and remained very close to the pancreatic ducts. Strikingly, alpha, delta, and PP cells were increased in number and abnormally intermingled with islet beta cells. Although several mature beta cell markers were expressed in beta cells of transgenic islets, the glucose transporter GLUT2 was absent or severely reduced. As glucose uptake/metabolism is essential for insulin secretion, decreased GLUT2 may contribute to the etiology of diabetes in pdx1(PB)-HNF6 transgenics. Concordantly, blood insulin was not raised by glucose challenge, suggesting profound beta cell dysfunction. Thus, we have shown that HNF6 downregulation during islet ontogeny is critical to normal pancreas formation and function: continued expression impairs the clustering of endocrine cells and their separation from the ductal epithelium, disrupts the spatial organization of endocrine cell types within the islet, and severely compromises beta cell physiology, leading to overt diabetes.     

Role for the TRPV1 Channel in Insulin Secretion from Pancreatic Beta Cells

Transient receptor potential channels have been put forward as regulators of insulin secretion. A role for the TRPV1 ion channel in insulin secretion has been suggested in pancreatic beta cell lines. We explored whether TRPV1 is functionally expressed in RINm5F and primary beta cells from neonate and adult rats. We examined if capsaicin could activate cationic non-selective currents. Our results show that TRPV1 channels are not functional in insulin-secreting cells, since capsaicin did not produce current activation, not even under culture conditions known to induce the expression of other ion channels in these cells. Although TRPV1 channels seem to be irrelevant for the physiology of isolated beta cells, they may play a role in glucose homeostasis acting through the nerve fibers that regulate islet function. At the physiological level, we observed that Trpv1 ^?/? mice presented lower fasting insulin levels than their wild-type littermates, however, we did not find differences between these experimental groups nor in the glucose tolerance test or in the insulin secretion. However, we did find that the Trpv1 ^?/? mice exhibited a higher insulin sensitivity compared to their wild-type counterparts. Our results demonstrate that TRPV1 does not contribute to glucose-induced insulin secretion in beta cells as was previously thought, but it is possible that it may control insulin sensitivity.     

CART is a novel islet regulatory peptide

CART peptides have emerged as important islet regulators. CART is expressed both in islet endocrine cells and in parasympathetic and sensory nerves innervating the islets. In adult rats the intra-islet expression of CART is limited to the somatostatin producing delta-cells, while in adult mice CART is mainly expressed in nerve fibers. During development islet CART is upregulated; in rats in almost all types of islet endocrine cells, including the insulin-producing beta-cells, and in mice mainly in the alpha-cells. This pattern of expression peaks around birth. CART is also expressed in human pancreatic nerves and in islet tumours where the expression level of CART may be related to the degree of differentiation of the tumour. Interestingly, in several rat models of type 2 diabetes CART expression is robustly upregulated in the beta-cells, and is prominent during the phase of beta cell proliferation and hypertrophy. While CART inhibits glucose stimulated insulin secretion from rat islets it augments insulin secretion amplified by cAMP. Mice lacking CART, on the other hand, have islet dysfunction, and humans with a missense mutation in the cart gene are prone to develop type 2 diabetes. These data favor a role of CART in normal islet function and in the pathophysiology of type 2 diabetes.     

Phoenixin-14 stimulates proliferation and insulin secretion in insulin producing INS-1E cells

Phoenixin (PNX) is a recently discovered neuropeptide which modulates appetite, pain sensation and neurons of the reproductive system in the central nervous system. PNX is also detectable in the circulation and in peripheral tissues. Recent data suggested that PNX blood levels positively correlate with body weight as well as nutritional status suggesting a potential role of this peptide in controlling energy homeostasis. PNX is detectable in endocrine pancreas, however it is unknown whether PNX regulates insulin biosynthesis or secretion. Using insulin producing INS-1E cells and isolated rat pancreatic islets we evaluated therefore, whether PNX controls insulin expression, secretion and cell proliferation. We identified PNX in pancreatic alpha as well as in beta cells. Secretion of PNX from pancreatic islets was stimulated by high glucose. PNX stimulated insulin mRNA expression in INS-1E cells. Furthermore, PNX enhanced glucose-stimulated insulin secretion in INS-1E cells and pancreatic islets in a time-dependent manner. Stimulation of insulin secretion by PNX was dependent upon cAMP/Epac signalling, while potentiation of cell growth and insulin mRNA expression was mediated via ERK1/2- and AKT-pathway. These results indicate that PNX may play a role in controlling glycemia by interacting with pancreatic beta cells.     

Selective Expression of Huntingtin-associated Protein 1 in ��-Cells of the Rat Pancreatic Islets

Huntingtin-associated protein-1 (HAP1) was initially identified as a binding partner of huntingtin, the Huntington''s disease protein. Based on its preferred distribution among neurons and endocrine cells, HAP1 has been suggested to play roles in vesicular transportation in neurons and hormonal secretion of endocrine cells. Given that HAP1 is selectively expressed in the islets of rat pancreas, in this study, we analyzed the expression pattern of HAP1 in the islets. In rats injected intraperitoneally with streptozotocin, which can selectively destroy β-cells of the pancreatic islets, the number of HAP1 immunoreactive cells was dramatically decreased and was accompanied by a parallel decrease in the number of insulin-immunoreactive cells. Immunofluorescent double staining of pancreas sections showed that, in rat islets, HAP1 is selectively expressed in the insulin-immunoreactive β-cells but not in the glucagon-immunoreactive α-cells and somatostatin immunoreactive δ-cells. In isolated rat pancreatic islets, ∼80% of cells expressed both HAP1 and insulin. Expression of HAP1 in the INS-1 rat insulinoma cell line was also demonstrated by immunofluorescent staining. Western blotting further revealed that HAP1 in both the isolated rat pancreatic islets and the INS-1 cells also has two isoforms, HAP1A and HAP1B, which are the same as those in the hypothalamus. These results demonstrated that HAP1 is selectively expressed in β-cells of rat pancreatic islets, suggesting the involvement of HAP1 in the regulation of cellular trafficking and secretion of insulin. (J Histochem Cytochem 58:255–263, 2010)     

The insulin-secreting and proliferative activity of established islet cells in the presence of sulfonylurea]

Exponentially growing rat islet cells (RINr) and hamster islet cells (HIT T-15) were incubated in presence of tolbutamide (10-1000 microM), gliclazide (0.1-10 microM) or glibenclamide (0.01-10 microM) for 15 hrs. Accumulation of insulin in culture medium was estimated by RIA. Effects of sulfonylureas (SU) on cell proliferation were assessed by 3H-thymidine (3H-T) incorporation into cellular DNA. All of SUs used stimulated insulin production in RIN and HIT cell cultures (with an exception of tolbutamide, which markedly suppressed insulin secretion in HIT cells at 1000 microM). 3H-T incorporation into RIN cells was elevated only in presence of gliclazide (10 microM), whereas tolbutamide at 1000 M significantly inhibited RIN cell proliferation. Gliclazide (0.1 microM) and glibenclamide (0.01-10 microM) enhanced 3H-T incorporation into HIT cells. Further detailed investigations of mechanisms of SU effects on islet cell reproduction will be of use for designing optimal strategy of hypoglycemizing therapy of diabetes mellitus.     

Co-expression and regulation of connexins 36 and 43 in cultured neonatal rat pancreatic islets

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)