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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. pancreatic islets; insulin secretion; isoforms of nitric oxide synthase; pituitary adenylate cyclase-activating polypeptide 27 doi:10.1152/ajpendo.00172.2006

Lundquist, Ingmar, Per Alm, Albert Salehi, Ragnar Henningsson, Eva Grapengiesser, and Bo Hellman. Carbon monoxide stimulates insulin release and propagates [Ca.sup.2.sup.+] signals between pancreatic [beta]-cells. Am J Physiol Endocrinol Metab 285: E1055-E1063, 2003; 10.1152/ ajpendo.00498.2002.--A key question for understanding the mechanisms of pulsatile insulin release is how the underlying [beta]-cell oscillations of the cytoplasmic [Ca.sup.2+] concentration ([[Ca.sup.2.sup.+].sub.i]) are synchronized within and among the islets in the pancreas. Nitric oxide has been proposed to coordinate the activity of the [beta]-cells by precipitating transients of [[Ca.sup.2.sup.+].sub.i]. Comparing ob/ob mice and lean controls, we have now studied the action of carbon monoxide (CO), another neurotransmitter with stimulatory effects on cGMP production. A strong immunoreactivity for the CO-producing constitutive heme oxygenase (HO-2) was found in ganglionic cells located in the periphery of the islets and in almost all islet endocrine cells. Islets from ob/ob mice had sixfold higher generation of CO (1 nmol*[min.sup.-1]*mg [protein.sup.-1]) than the lean controls. This is 100-fold the rate for their constitutive production of NO. Moreover, islets from ob/ob mice showed a threefold increase in HO-2 expression and expressed inducible HO (HO-1). The presence of an excessive islet production of CO in the ob/ob mouse had its counterpart in a pronounced suppression of the glucose-stimulated insulin release from islets exposed to the HO inhibitor Zn-protoporhyrin (10 [micro]M) and in a 16 times higher frequency of [[Ca.sup.2.sup.+].sub.i] transients in their [beta]-cells. Hemin (0.1 and 1.0 [micro]M), the natural substrate for HO, promoted the appearance of [[Ca.sup.2.sup.+].sub.i] transients, and 10 [micro]M of the HO inhibitors Zn-protoporphyrin and Cr-mesoporphyrin had a suppressive action both on the firing of transients and their synchronization. It is concluded that the increased islet production of CO contributes to the hyperinsulinemia in ob/ob mice. In addition to serving as a positive modulator of glucose-stimulated insulin release, CO acts as a messenger propagating [Ca.sup.2.sup.+] signals with coordinating effects on the [beta]-cell rhythmicity. calcium ion; carbon monoxide

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of ~80 fF that plateaued within ~200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the [K.sub.d] values were 6 and 29 [micro]M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice ([SUR1.sup.-/-] mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from [SUR1.sup.-/-] mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of [C1.sup.-] into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane [K.sub.ATP]-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of [Ca.sup.2+]-induced exocytosis. KEY WORDS: insulin * [Ca.sup.2+] * cAMP * cAMP-GEFII * SUR1

Role of nitric oxide synthase isoforms in glucose stimulatedinsulin release. Am J Physiol Cell Physiol 283: C296-C304, 2002. First published March 20, 2002; 10.1152/ ajpcell.00537.2001.--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.sup+]-depolarized islets in the presence of the ATP-sensitive [K.sup.+] 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.sup.G]-monomethyl-Larginine (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 mmolfl 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. islets of Langerhans; nitric oxide synthase isoforms; nitric oxide synthase inhibition

The important role of nitric oxide (NO) within the islets of Langerhans and its action on L-arginine-induced hormone release were examined by comparing the effects of L-arginine and L-homoarginine in islet NO production with regards to the potency of the amino acids to induce insulin and glucagon secretion. Results showed that while both amino acids have a dual action on insulin release, L-arginine is a better substrate than L-homoarginine for islet cNOS. The amino acids' ability to release oxygen is enhanced by the relative rates of their NO production.

Experiment was conducted to show that the constitutive isoform (HO-2) is also expressed in the islets of Langerhans and that activation and inhibition of HO-2 activity markedly influence insulin and glucagon release and implicate CO as a novel physiological modulator of islet hormone release. Most insulin and glucagon cells in the rat endocrine pancreas expressed strong immunoreactivity for HO-2. Scattered HO-2-positive ganglionic cell bodies were also occasionally observed in the exocrine parenchyma.

The presence of constitutive nitric oxide synthase (NOS) activity in pancreatic islets suppresses insulin secretion and enhances glucagon secretion as a result of negative feedback mechanism by beta cell NO on glucose-induced insulin release. Enantiomerically specific NOS inhibitor, N(super G)-nitro-L-arginine methyl ester, L-NAME suppresses the activity of NOS islet. The activity of NOS islet depends upon Ca2+/calmodulin and the presence of L-arginine in the substrate.

Insulin secretion and islet glycogenolytic alpha-glycosidehydrolases were correlated. Insulin was secreted due to the presence of 3-isobutyl-1-methylxanthine (IBMX) and glucose. Islet alpha-glucosidehydrolase activity was lessened by calcium ion deficiency and the presence of miglitol. Insulin secretion due to IBMX was adversely affected by miglitol in a dose dependent manner. Glucose-induced insulin release is affected by acid amyloglucosidase, unlike IBMX-induced insulin production.

TPN-evoked dysfunction of islet lysosomal activity mediates impairment of glucose-stimulated insulin release. Am J Physiol Endocrinol Metab 281: E171-E179, 2001.--We examined the relation between nutrient-stimulated insulin secretion and the islet lysosome acid glucan-1,4-[Alpha]-glucosidase system in rats undergoing total parenteral nutrition (TPN). During TPN treatment, serum glucose was normal, but free fatty acids, triglycerides, and cholesterol were elevated. Islets from TPN-infused rats showed increased basal insulin release, a normal insulin response to cholinergic stimulation but a greatly impaired response when stimulated by glucose or [Alpha]-ketoisocaproic acid. This impairment of glucose-stimulated insulin release was only slightly ameliorated by the carnitine palmitoyltransferase 1 inhibitor etomoxir. However, in parallel with the impaired insulin response to glucose, islets from TPN-infused animals displayed reduced activities of islet lysosomal enzymes including the acid glucan-1,4-[Alpha]-glucosidase, a putative key enzyme in nutrient-stimulated insulin release. By comparison, the same lysosomal enzymes were increased in liver tissue. Furthermore, in intact control islets, the pseudotetrasaccharide acarbose, a selective inhibitor of acid [Alpha]-glucosidehydrolases, dose dependently suppressed islet acid glucan-1,4-[Alpha]-glucosidase and acid a-glucosidase activities in parallel with an inhibitory action on glucose-stimulated insulin secretion. By contrast, when incubated with intact TPN islets, acarbose had no effect on either enzyme activity or glucose-induced insulin release. Moreover, when acarbose was added directly to TPN islet homogenates, the dose-response effect on the catalytic activity of the acid [Alpha]-glucosidehydrolases was shifted to the right compared with control homogenates. We suggest that a general dysfunction of the islet lysosomal/vacuolar system and reduced catalytic activities of acid glucan-1,4-[Alpha]-glucosidase and acid [Alpha]-glucosidase may be important defects behind the impairment of the transduction mechanisms for nutrient-stimulated insulin release in islets from TPN-infused rats. total parenteral nutrition; insulin secretion; islet acid glucan1,4-[Alpha]-glucosidase activity; lysosomal/vacuolar system; plasma lipids; etomoxir; acarbose

Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia. Am J Physiol Cell Physiol 280: C1242-C1254, 2001.--We investigated, by a combined in vivo and in vitro approach, the temporal changes of islet nitric oxide synthase (NOS)-derived nitric oxide (NO) and heme oxygenase (HO)-derived carbon monoxide (CO) production in relation to insulin and glucagon secretion during acute endotoxemia induced by lipopolysaccharide (LPS) in mice. Basal plasma glucagon, islet cAMP and cGMP content after in vitro incubation, the insulin response to glucose in vivo and in vitro, and the insulin and glucagon responses to the adenylate cyclase activator forskolin were greatly increased after LPS. Immunoblots demonstrated expression of inducible NOS (iNOS), inducible HO (HO-1), and an increased expression of constitutive HO (HO-2) in islet tissue. Immunocytochemistry revealed a marked expression of iNOS in many [Beta]-cells, but only in single [Alpha]-cells after LPS. Moreover, biochemical analysis showed a time dependent and markedly increased production of NO and CO in these islets. Addition of a NOS inhibitor to such islets evoked a marked potentiation of glucose-stimulated insulin release. Finally, after incubation in vitro, a marked suppression of NO production by both exogenous CO and glucagon was observed in control islets. This effect occurred independently of a concomitant inhibition of guanylyl cyclase. We suggest that the impairing effect of increased production of islet NO on insulin secretion during acute endotoxemia is antagonized by increased activities of the islet cAMP and HO-CO systems, constituting important compensatory mechanisms against the noxious and diabetogenic actions of NO in endocrine pancreas. lipopolysaccharide; pancreatic islets; nitric oxide; carbon monoxide; cyclic nucleotides; insulin; glucagon secretion

Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release. Am J Physiol Endocrinol Metab 279: E95-E107, 2000.--Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems. chronic nitric oxide synthase blockade; nitric oxide and carbon monoxide; insulin and glucagon secretion; isolated islets; cyclic nucleotides