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Aims/hypothesis: Orexin A (OXA) modulates body weight, food intake and energy expenditure. In vitro, OXA increases PPARγ (also known as PPARG) expression and inhibits lipolysis, suggesting direct regulation of lipid metabolism. Here, we characterise the metabolic effects and mechanisms of OXA action in adipocytes., Methods: Isolated rat adipocytes and differentiated murine 3T3-L1 adipocytes were exposed to OXA in the presence or absence of phosphoinositide 3-kinase (PI3K) inhibitors. Pparγ expression was silenced using small interfering RNA. Glucose uptake, GLUT4 translocation, phosphatidylinositol (3,4,5)-trisphosphate production, lipogenesis, lipolysis, and adiponectin secretion were measured. Adiponectin plasma levels were determined in rats treated with OXA for 4 weeks., Results: OXA PI3K-dependently stimulated active glucose uptake by translocating the glucose transporter GLUT4 from cytoplasm into the plasma membrane. OXA increased cellular triacylglycerol content via PI3K. Cellular triacylglycerol accumulation resulted from increased lipogenesis as well as from a decrease of lipolysis. Adiponectin levels in chow- and high-fat diet-fed rats treated chronically with OXA were increased. OXA stimulated adiponectin expression and secretion in adipocytes. Both pharmacological blockade of peroxisome proliferator-activated receptor γ (PPARγ) activity or silencing Pparγ expression prevented OXA from stimulating triacylglycerol accumulation and adiponectin production., Conclusions/interpretation: Our study demonstrates that OXA stimulates glucose uptake in adipocytes and that the evolved energy is stored as lipids. OXA increases lipogenesis, inhibits lipolysis and stimulates the secretion of adiponectin. These effects are conferred via PI3K and PPARγ2. Overall, OXA's effects on lipids and adiponectin secretion resemble that of insulin sensitisers, suggesting a potential relevance of this peptide in metabolic disorders.

The clinical manifestation of pheochromocytomas is highly variable and can closely resemble numerous clinical conditions. Here, we report on two cases of patients with pheochromocytoma, which manifested as sepsis or cardiomyopathy. The first patient initially presented with bacterial urosepsis due to klebsiella oxytoca. Despite effective antibiotic therapy, the patient developed recurring fever accompanied by hypertension. The inconsistency between therapy-refractory hypertension and fever indicated the possibility of excessive catecholamine production. In the second case, the patient presented with a suspected ST-segment elevation myocardial infarction accompanied by E. coli sepsis and a previously undiagnosed unilateral tumor mass of the adrenal gland. Severely impaired myocardial contraction of the apical anterior and inferior regions without significant coronary artery disease was consistent with the Takotsubo cardiomyopathy, a known transient functional myocardial complication associated with pheochromocytoma. Both patients were diagnosed with unilateral pheochromocytoma. Following pre-operative antihypertensive therapy, both patients were cured by surgery and still remain free of disease after two years of follow-up., (© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.)

The mitochondrial UCP2 mediates glucose-stimulated insulin secretion by decreasing intracellular ATP/ADP ratio. Insulin secretion is a tightly regulated process. Ghrelin, as well as obestatin, were intensively studied to determine their ability to modify insulin secretion. Ghrelin is considered to be an inhibitor of insulin release from pancreatic islets, however little is known about the effects of obestatin. In our study we demonstrate the stimulating effects of both peptides on insulin secretion in INS1 cells. Furthermore, we investigate the potential role of UCP2 in mediating the effects of both peptides on insulin secretion. UCP2 mRNA expression was down-regulated by ghrelin in the presence of 26.4 mM glucose, however it was unchanged after obestatin treatment. Our results confirm that UCP2 could be involved in the stimulating effect of ghrelin on insulin release from INS1 cells.

Background: Somatostatin inhibits gall bladder contraction. Impaired gall bladder emptying is associated with gall bladder stone formation. The incidence of cholecystolithiasis is high in patients treated with a somatostatin agonist octreotide, which predominantly interacts with somatostatin receptor subtype 2 (SSTR2). Therefore, it is believed that SSTR2 regulates gall bladder contraction; however, evidence has not been provided. Here, we evaluate the effects of SSTR1-SSTR5-selective agonists on egg yolk-induced gall bladder contraction in mice., Methods: Homozygous deletion of SSTR2 and SSTR5 was generated by cross-mating of SSTR2(-/-) with SSTR5(-/-) mice. Mice of different genotypes were injected with SSTR1-5-selective agonists or octreotide 15 min before induction of gall bladder emptying by egg yolk. One hour later, gall bladders were removed and weighed., Key Results: Egg yolk-reduced gall bladder weights in all mice, irrespective of their genotype. Octreotide was the most potent inhibitor of gall bladder emptying in wild-type mice. In contrast, agonists with high selectivity for SSTR2 or SSTR5 inhibited gall bladder emptying by approximately 50-60%, whereas SSTR1-, SSTR3- and SSTR4-selective agonists failed to influence gall bladder contraction. In SSTR2(-/-) mice, octreotide and an SSTR5-selective agonist inhibited gall bladder emptying by approximately 50%, whereas SSTR2-selective agonists were inactive. Octreotide inhibited gall bladder emptying in SSTR5(-/-) mice by approximately 50%, without any effect in SSTR2(-/-)/SSTR5(-/-) mice., Conclusions & Inferences: Our study provides evidence for the role of SSTR2 and SSTR5 in regulating gall bladder emptying in mice.

Cerebellin (CER) is a neuromodulatory hexadecapeptide that originates from the precursor protein precerebellin (Cbln1). Four highly homologous isoforms of Cbln are known (Cbln1-Cbln4), which are expressed in the central nervous system (CNS) and in peripheral tissues. CER modulates synaptic structure formation in the CNS, whereas in the peripheral tissues CER regulates catecholamine secretion. Cbln is also expressed in the pancreas; however, its function in the pancreas is unknown. Here, we demonstrate the role of CER in regulating insulin secretion in vivo and in vitro. We identified Cbln1 and Cbln3 transcripts in rat pancreatic islets and detected Cbln-immunoreactivity, predominantly located in the periphery of the rat endocrine pancreas. In vivo, CER reduced plasma insulin levels in rats after 1 and 2 h. CER decreased insulin secretion from isolated rat pancreatic islets at high (11 mM), but not at low (3.33 mM) glucose concentration. CER inhibited stimulated insulin secretion from clonal rat insulinoma (INS-1) cells, reduced forskolin-induced production of cAMP and intracellular calcium concentration. Our study demonstrates for the first time that Cbln1 and Cbln3 are expressed in the rat endocrine pancreas. Furthermore, we identify CER as an insulinostatic factor, which decreases intracellular cAMP production and calcium in INS-1 cells.

Objectives: Neuromedin U (NmU) is a neuropeptide with anorexigenic activity. Two receptor subtypes (NmUR1 and NmUR2) confer the effects of NmU on target cells. We have recently demonstrated that NmU reduces insulin secretion from isolated pancreatic islets. Aim of our current study is to investigate the role of somatostatin at mediating the effects of NmU on insulin secretion., Methods: Expression of NmU in the pancreas was detected by immunohistochemistry. Insulin and somatostatin secretion from in situ perfused rat pancreas and isolated pancreatic islets was measured by radioimmunoassay. The paracrine effects of somatostatin within pancreatic islets were blocked by cyclosomatostatin, a somatostatin receptor antagonist., Results: Receptor subtype NmUR1, but not NmUR2, was expressed in the endocrine pancreas, predominantly in the periphery. Neuromedin U reduced insulin secretion from in situ perfused rat pancreas and stimulated somatostatin secretion from isolated pancreatic islets. Neuromedin U stimulated somatostatin secretion at both physiological and supraphysiological glucose concentrations. Cyclosomatostatin increased insulin secretion and reduced NmU-induced inhibition of insulin secretion., Conclusions: Neuromedin U reduces insulin and increases somatostatin secretion. Blockade of somatostatin action abolishes the inhibition of insulin secretion by NmU. The results of the study suggest that somatostatin mediates the inhibitory action of NmU on insulin secretion.

Somatostatin (SST) inhibits Ca(2+) entry into pancreatic B-cells via voltage-operated Ca(2+) channels (VOCCs) of L-type, leading to the suppression of insulin secretion. Activation of R-type channels increases insulin secretion. However, the role of R-type Ca(2+) channels (Ca(V)2.3) in mediating the effects of SST on insulin secretion has not been so far investigated. Here, we identify the SST-receptor subtypes (SSTR) expressed on insulin-producing INS-1 cells by RT-PCR and by functional assays. The role of R-type channels in regulating [Ca(2+)](i) in response to SST-treatment was detected by cell fluorescence imaging and patch-clamp technique. INS-1 expressed SSTR2 and SSTR3 and agonists (ag.) selective for these receptors reduced 10 nM exendin-4/20 mM glucose-stimulated insulin secretion. Surprisingly, SST and SST2-ag. transiently increased [Ca(2+)](i). Subsequently, these agonists led to a decrease in [Ca(2+)](i) below the basal levels. In contrast, SST3-ag. failed to induce a transient peak of [Ca(2+)](i). Instead, a persistent minor suppression of [Ca(2+)](i) was detected from 25 min. R-type channel blocker SNX-482 altered [Ca(2+)](i) in SST- and SST2-ag.-treated cells. Notably, the inhibition of insulin secretion by SST and SST2-ag., but not SST3-ag. was attenuated by SNX-482. Taken together, SST and SSTR2 regulate [Ca(2+)](i) and insulin secretion in INS-1 cells via R-type channels. In contrast, the R-type calcium channel does not mediate the effects of SST3-ag. on insulin secretion. We conclude that R-type channels play a major role in the inhibition of insulin secretion by somatostatin in INS-1 cells.

Somatostatin (SST) regulates multiple biological processes via five genetically distinct, G-protein coupled receptors. Clinical interest in therapy for neuroendocrine and metabolic disorders has resulted in the development of new tools for exploring the function of somatostatin receptors (SSTRs). The development of highly SSTR-selective agonists and antagonists, animal models with the deletion of individual SSTRs, as well as SSTR-specific antibodies have all been utilized in delineating SSTR functions. In the pancreas, SST is a potent regulator of insulin and glucagon secretion. Indeed, the inappropriate regulation of pancreatic A- and B-cell function in metabolic diseases provides an impetus to evaluate the SSTRs as therapeutic targets. By combining the results obtained from molecular biology, pharmacology and immunochemical studies the current review provides a summary of important recent developments which have extended our knowledge of SST actions in the endocrine pancreas.

Orexin-A (OXA) regulates food intake and energy homeostasis. It increases insulin secretion in vivo and in vitro, although controversial effects of OXA on plasma glucagon are reported. We characterized the effects of OXA on glucagon secretion and identify intracellular target molecules in glucagon-producing cells. Glucagon secretion from in situ perfused rat pancreas, isolated rat pancreatic islets, and clonal pancreatic A-cells (InR1-G9) were measured by RIA. The expression of orexin receptor 1 (OXR1) was detected by Western blot and immunofluorescence. The effects of OXA on cAMP, adenylate-cyclase-kinase (AKT), phosphoinositide-dependent kinase (PDK)-1, forkhead box O-1 (Foxo1), and cAMP response element-binding protein were measured by ELISA and Western blot. Intracellular calcium (Ca(2+)(i)) concentration was detected by fura-2and glucagon expression by real-time PCR. Foxo1 was silenced in InR1-G9 cells by transfecting cells with short interfering RNA. OXR1 was expressed on pancreatic A and InR1-G9 cells. OXA reduced glucagon secretion from perfused rat pancreas, isolated rat pancreatic islets, and InR1-G9 cells. OXA inhibited proglucagon gene expression via the phosphatidylinositol 3-kinase-dependent pathway. OXA decreased cAMP and Ca(2+)(i) concentration and increased AKT, PDK-1, and Foxo1 phosphorylation. Silencing of Foxo1 caused a reversal of the inhibitory effect of OXA on proglucagon gene expression. Our study provides the first in vitro evidence for the interaction of OXA with pancreatic A cells. OXA inhibits glucagon secretion and reduces intracellular cAMP and Ca(2+)(i) concentration. OXA increases AKT/PDK-1 phosphorylation and inhibits proglucagon expression via phosphatidylinositol 3-kinase- and Foxo-1-dependent pathways. As a physiological inhibitor of glucagon secretion, OXA may have a therapeutic potential to reduce hyperglucagonemia in type 2 diabetes.

Hypersecretion of glucagon contributes to abnormally increased hepatic glucose output in type 2 diabetes. Somatostatin (SST) inhibits murine glucagon secretion from isolated pancreatic islets via somatostatin receptor subtype-2 (sst2). Here, we characterize the role of sst2 in controlling glucose homeostasis in mice with diet-induced obesity. Sst2-deficient (sst2(-/-)) and control mice were fed high-fat diet for 14 wk, and the parameters of glucose homeostasis were monitored. Hepatic glycogen and lipid contents were quantified enzymatically and visualized histomorphologically. Enzymes regulating glycogen and lipid synthesis and breakdown were measured by real-time PCR and/or Western blot. Gluconeogenesis and glycogenolysis were determined from isolated primary hepatocytes and glucagon or insulin secretion from isolated pancreatic islets. Nonfasting glucose, glucagon, and fasting nonesterified fatty acids of sst2(-/-) mice were increased. Inhibition of glucagon secretion from sst2-deficient pancreatic islets by glucose or somatostatin was impaired. Insulin less potently reduced blood glucose concentration in sst2-deficient mice as compared with wild-type mice. Sst2-deficient mice had decreased nonfasting hepatic glycogen and lipid content. The activity/expression of enzymes controlling hepatic glycogen synthesis of sst2(-/-) mice was decreased, whereas enzymes facilitating glycogenolysis and lipolysis were increased. Somatostatin and an sst2-selective agonist decreased glucagon-induced glycogenolysis, without influencing de novo glucose production using cultured primary hepatocytes. This study demonstrates that ablation of sst2 leads to hyperglucagonemia. Increased glucagon concentration is associated with impaired glucose control in sst2(-/-) mice, resulting from decreased hepatic glucose storage, increased glycogen breakdown, and reduced lipid accumulation. Sst2 may constitute a therapeutic target to lower hyperglucagonemia in type 2 diabetes.

Introduction: Pancreatic A- and B-cells express somatostatin receptors (SSTRs). Five pharmacologically distinct SSTR subtypes are known (SSTR1-SSTR5). In rodents, SSTR2 inhibits glucagon secretion, whereas SSTR5 suppresses the release of insulin. Human pancreatic A- and B-cells express SSTR1-3 and SSTR5; however, their contribution to the regulation of glucagon and insulin secretion is not well known., Aim of the Study: The goal of this study was to characterize the role of individual SSTR subtypes in regulating human glucagon and insulin secretion in vitro., Methods: Human pancreatic islets were isolated from healthy donors and incubated with somatostatin, SSTR1-3-selective and SSTR5-selective agonists, or an SSTR2-selective antagonist (DC-41-33). Stimulation of insulin secretion was induced by glucose (10, 20 mm) alone or in combination with 10 nm exendin-4 or 10 mm L-arginine. Glucagon secretion was induced by 20 mm L-arginine. Basal secretion of insulin and glucagon was measured at 2.8 or 3.3 mm glucose., Results: SSTR1-, SSTR2-, and SSTR5-selective agonists inhibited insulin secretion with the following order of potency: SSTR2 (EC50, 0.08 nm) > SSTR5 (EC50, 5.3 nm) > SSTR1 (EC50, 35 nm). Glucagon secretion was inhibited by SSTR-selective agonists with the following order of potency: SSTR2 (EC50, 0.05 nm) > SSTR1 (EC50, 1.8 nm) > SSTR5 (EC50, 28 nm). DC-41-33 dose-dependently reversed the effects of the SSTR2-selective agonist on insulin and glucagon secretion., Conclusion: Our study demonstrates that SSTR2-agonist is the most potent inhibitor of insulin and glucagon secretion from isolated human pancreatic islets. Furthermore, we identify SSTR1- and SSTR5-selective agonists as additional inhibitors of insulin and glucagon secretion from human pancreas.

TRPM8 is a member of the melastatin-type transient receptor potential ion channel family. Activation by cold or by agonists (menthol, icilin) induces a transient rise in intracellular free calcium concentration ([Ca(2+)](i)). Our previous study demonstrated that Ca(2+)-permeable cation channels play a role in IGF-1-induced secretion of chromogranin A in human neuroendocrine tumor (NET) cell line BON [Mergler et al.: Neuroendocrinology 2006;82:87-102]. Here, we extend our earlier study by investigating the expression of TRPM8 and characterizing its impact on [Ca(2+)](i) and the secretion of neurotensin (NT). We identified TRPM8 expression in NET BON cells by RT-PCR, Western blotting and immunofluorescence staining. Icilin increased [Ca(2+)](i) in TRPM8-transfected human embryonic kidney cells (HEK293) but not in mock-transfected cells. Icilin and menthol induced Ca(2+) transients in BON cells as well as in primary NET cell cultures of two different pancreatic NETs as detected by single cell fluorescence imaging. Icilin increased non-selective cation channel currents in BON cells as detected by patch-clamp recordings. This activation was associated with increased NT secretion. Taken together, this study demonstrates for the first time the expression TRPM8 in NET cells and its role in regulating [Ca(2+)](i) and NT secretion. The regulation of NT secretion in NETs by TRPM8 may have a potential clinical implication in diagnosis or therapy.

Somatostatin inhibits both glucagon and insulin secretion. Glucagon significantly contributes to hyperglycemia in type 2 diabetes. Despite its function in the inhibition of glucagon secretion, somatostatin fails to reduce hyperglycemia in type 2 diabetes, due to a parallel suppression of insulin secretion. Five pharmacologically distinct somatostatin receptor subtypes (sst(1)-sst(5)) mediate the effects of somatostatin on a cellular level. Pancreatic A cells express sst(2), whereas B cells express sst(5). In this study, we describe a novel approach to the treatment of type 2 diabetes using a highly sst(2)-selective, nonpeptide agonist (compound 1). Compound 1 effectively inhibited glucagon secretion from pancreatic islets isolated from wild-type mice, whereas glucagon secretion from sst(2)-deficient islets was not suppressed. Compound 1 did not influence nonfasted insulin concentration. In sst(2)-deficient mice, compound 1 did not have any effects on glucagon or glucose levels, confirming its sst(2) selectivity. In animal models of type 2 diabetes in the nonfasted state, circulating glucagon and glucose levels were decreased after treatment with compound 1. In the fasting state, compound 1 lowered blood glucose by approximately 25%. In summary, small-molecule sst(2)-selective agonists that suppress glucagon secretion offer a novel approach toward the development of orally bioavailable drugs for treatment of type 2 diabetes.

Orexins are recently identified neuropeptides that appear to play a role in the regulation of energy homeostasis and arousal. They bind to and activate two closely related G protein-coupled receptors (OXR1 and OXR2), previously described as orphans. In this study we examined involvement of orexins in regulation of insulin secretion from rat pancreatic islets utilizing an in situ perfused pancreas and isolated pancreatic islet models. By means of RT-PCR we found that both OXR1 and OXR2 are expressed in rat pancreatic islets. Furthermore, the expression levels of OXR1 were higher than OXR2. In both experimental models applied, orexins A and B (1, 10 and 100 nmol/l) concentration dependently stimulated insulin secretion at two different glucose concentrations (6.66 or 26.4 mmol/l), with orexin A being more potent than orexin B. This study demonstrates that orexins A and B modulate insulin secretion in vitro.

Adiposity positively correlates with insulin resistance and is a major risk factor of type 2 diabetes. Administration of exogenous insulin, which acts as an anabolic factor, facilitates adipogenesis. Recently nonpeptidal insulin receptor (IR) activators have been discovered. Here we evaluate the effects of the orally bioavailable small-molecule IR activator (Compound-2) on metabolic abnormalities associated with type 2 diabetes using a nongenetic mouse model in comparison with the effects of a novel non-thiazolidinedione (nTZD) peroxisome proliferator-activated receptor-gamma agonist. Both Compound-2 and nTZD alleviated fasting and postprandial hyperglycemia; accelerated glucose clearance rate; and normalized plasma levels of nonesterified fatty acids, triglycerides, and leptin. Unlike nTZD, which increased body weight gain, and total fat mass, which is a common feature for PPARgamma agonists, Compound-2 prevented body weight gain and hypertrophy of brown, and white adipose tissue depots and the development of hepatic steatosis in the mouse model of type 2 diabetes. The effect of the two compounds on proximal steps in insulin signal transduction pathway was analyzed in tissues. Compound-2 enhanced insulin-stimulated phosphorylation of IR tyrosine and/or Akt in the liver, skeletal muscle, and white adipose tissue, whereas nTZD potentiated the phosphorylation of IR and Akt in the adipose tissue only. In conclusion, small-molecule IR activators have unique features as insulin sensitizers and hold potential utility in the treatment of type 2 diabetes and obesity.

Somatostatin (somatotropin release inhibitory factor; SRIF) initiates its biological activity by interacting with a family of highly homologous integral membrane receptors (sst(1) -sst(5)). SRIF neuronal actions regulate protein phosphorylation levels, control second messenger production and modulate neuronal membrane potential. Recently, our understanding of SRIF neurobiology has been driven by new pharmacological and molecular biological tools. SRIF receptor subtype specific antibodies have identified a distinctive, yet overlapping, expression pattern for this receptor family, with multiple subtypes co-localizing in the central and peripheral nervous system. This complex expression profile has confounded efforts to establish each receptor's role in the nervous system in part by the possible homo- and heteroligomerization of the receptor proteins. However, the recent discovery of SRIF receptor subtype selective ligands, supplemented by in vitro and in vivo models with inactivated SRIF receptor genes, now provides opportunities to clearly delineate each receptor's neuronal role. The convergence of these pharmacologic, immunologic and molecular biologic approaches extend our understanding of SRIF neurobiology while promising new therapeutic avenues for SRIF research.

VEGF-A is a key regulator of inflammatory and tumor-associated angiogenesis. H. pylori plays a critical role in the pathogenesis of benign and malignant gastric diseases. It has been suggested that H. pylori infection is associated with activation of host angiogenesis, however, underlying mechanisms as well as angiogenic growth factors activated by the bacterium have not yet been identified. Therefore, we investigated the influence of the bacterium on VEGF-A as a candidate host target gene in vivo and in vitro. We show that H. pylori potently up-regulates production and release of VEGF-A protein as well as vegf-A mRNA levels, and we provide strong evidence that enhanced recruitment of Sp1 and Sp3 transcription factors to two proximal GC-rich vegf-A promoter elements mediates H. pylori-triggered vegf-A gene expression. In addition, H. pylori infection increased the transactivating capacity of both Sp1 and Sp3, which suggests additional mechanism(s) of vegf-A gene regulation by the bacterium. Signaling studies identified the MEK>ERK1/-2 kinase cascade as principal host signaling pathway mediating H. pylori-stimulated vegf-A transcription. By identifying H. pylori as potent activator of vegf-A gene expression and characterization of underlying molecular mechanisms, our results provide novel insights into pathways linking the bacterium to host angiogenesis and may help to develop strategies to influence vegf-A gene expression in the setting of H. pylori infection.

With an in vitro model using enclosed intrahepatic bile duct units (IBDUs) isolated from wild-type and somatostatin receptor (SSTR) subtype 2 knockout mice, we tested the effects of somatostatin, secretin, and a selective SSTR2 agonist (L-779976) on fluid movement across the bile duct epithelial cell layer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1, SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected in cholangiocytes in wild-type mice. In contrast, SSTR2A/2B were completely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 and SSTR4 were expressed in these cholangiocytes. Somatostatin induced a decrease of luminal area of IBDUs isolated from wild-type mice, reflecting net fluid absorption; L-779976 also induced a comparable decrease of luminal area. No significant decrease of luminal area by either somatostatin or L-779976 was observed in IBDUs from SSTR2 knockout mice. Secretin, a choleretic hormone, induced a significant increase of luminal area of IBDUs of wild-type mice, reflecting net fluid secretion; somatostatin and L-779976 inhibited (P < 0.01) secretin-induced fluid secretion. The inhibitory effect of both somatostatin and L-779976 on secretin-induced IBDU secretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibited secretin-stimulated cAMP synthesis in cholangiocytes; depletion of SSTR2 blocked these effects of somatostatin. These data suggest that somatostatin regulates ductal bile formation in mice not only by inhibition of ductal fluid secretion but also by stimulation of ductal fluid absorption via interacting with SSTR2 on cholangiocytes, a process involving the intracellular cAMP/cGMP second messengers.

Somatostatin (SRIF) regulates pancreatic insulin and glucagon secretion. In the present study we describe the generation of SRIF receptor subtype 5 knockout (sst(5) KO) mice to examine the role of SRIF receptor subtypes (sst) in regulating insulin secretion and glucose homeostasis. Mice deficient in sst(5) were viable, fertile, appeared healthy, and displayed no obvious phenotypic abnormalities. Pancreatic islets isolated from sst(5) KO mice displayed increased total insulin content as compared with islets obtained from wild-type (WT) mice. Somatostatin-28 (SRIF-28) and the sst(5)/sst(1)-selective agonist compound 5/1 potently inhibited glucose-stimulated insulin secretion from WT islets. SRIF-28 inhibited insulin secretion from sst(5) KO islets with 16-fold less potency while the maximal effect of compound 5/1 was markedly diminished when compared with its effects in WT islets. sst(5) KO mice exhibited decreased blood glucose and plasma insulin levels and increased leptin and glucagon concentrations compared with WT mice. Furthermore, sst(5) KO mice displayed decreased susceptibility to high fat diet-induced insulin resistance. The results of these studies suggest sst(5) mediates SRIF inhibition of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity. Our findings suggest a potential beneficial role of sst(5) antagonists for alleviating metabolic abnormalities associated with obesity and insulin resistance.

Somatostatin (SRIH) regulates pituitary adrenocorticotropin (ACTH) secretion by interacting with a family of homologous G protein-coupled membrane receptors. The SRIH receptor subtypes (sst(1)-sst(5)) that control ACTH release remain unknown. Using novel, subtype-selective SRIH analogs, we have identified the SRIH receptor subtypes involved in regulating ACTH release from AtT-20 cells, a model for cell line pituitary corticotropes. Radioligand-binding studies with (125)I-SRIH-14 and (125)I-SRIH-28 showed that SRIH-14 and SRIH-28 recognized specific, high-affinity and saturable membrane-binding sites. Nonpeptidyl agonists with selectivity for the sst(2) (L-779,976; compound 2) or sst(1)/sst(5)) (L-817,818; compound 5) receptor subtypes potently displaced (125)I-SRIH-28 from AtT-20 cell membranes, while agonists selective for the sst(1) (L-779,591; compound 1), sst(3) (L-796,778; compound 3) or sst(4) (L-803,087; compound 4) subtypes were inactive. Tyr(11)-SRIH-14, compound 2 (sst(2)) or compound 5 (sst(5)) inhibited forskolin and corticotropin-releasing hormone (CRH)-induced increases in intracellular cAMP. Furthermore, the sst(2) and sst(5) agonists potently inhibited CRH-induced ACTH release from AtT-20 cells. These results provide the first evidence that sst(2) and sst(5) receptor subtypes, but not sst(1), sst(3) or sst(4), inhibit cAMP accumulation and regulate ACTH secretion in the AtT-20 cell model of the rodent corticotrope., (Copyright 2002 S. Karger AG, Basel)

Obesity and insulin resistance are major risk factors for a number of metabolic disorders, such as type 2 diabetes mellitus. Insulin has been suggested to function as one of the adiposity signals to the brain for modulation of energy balance. Administration of insulin into the brain reduces food intake and body weight, and mice with a genetic deletion of neuronal insulin receptors are hyperphagic and obese. However, insulin is also an anabolic factor; when administered systemically, pharmacological levels of insulin are associated with body weight gain in patients. In this study, we investigated the efficacy and feasibility of small molecule insulin mimetic compounds to regulate key parameters of energy homeostasis. Central intracerebroventricular (i.c.v.) administration of an insulin mimetic resulted in a dose-dependent reduction of food intake and body weight in rats, and altered the expression of hypothalamic genes known to regulate food intake and body weight. Oral administration of a mimetic in a mouse model of high-fat diet-induced obesity reduced body weight gain, adiposity and insulin resistance. Thus, insulin mimetics have a unique advantage over insulin in the control of body weight and hold potential as a novel anti-obesity treatment.

Somatostatin (SST) potently inhibits insulin and glucagon release from pancreatic islets. Five distinct membrane receptors (SSTR1-5) for SST are known, and at least two (SSTR2 and SSTR5) have been proposed to regulate pancreatic endocrine function. Our current understanding of SST physiology is limited by the receptor subtype selectivity of peptidyl SST analogs, making it difficult to assign a physiological function to an identified SST receptor subtype. To better understand the physiology of SSTRs we studied the in vitro effects of potent subtype-selective nonpeptidyl SST analogs on the regulation of pancreatic glucagon and insulin secretion in wild-type (WT) and in somatostatin receptor 2 knockout (SSTR2KO) mice. There was no difference in basal glucagon and insulin secretion between islets isolated from SSTR2KO and WT mice; however, potassium/arginine-stimulated glucagon secretion was approximately 2-fold higher in islets isolated from SSTR2KO mice. Neither SST nor any SSTR-selective agonist inhibited basal glucagon or insulin release. SST-14 potently inhibited stimulated glucagon secretion in islets from WT mice and much less effectively in islets from SSTR2KO mice. The SSTR2 selective analog L-779,976 inhibited glucagon secretion in islets from WT, but was inactive in islets from SSTR2KO mice. L-817,818, an SSTR5 selective analog, slightly reduced glucagon release in both animal groups, whereas SSTR1, -3, and -4 selective analogs were inactive. SST and L-817,818 inhibited glucose stimulated insulin release in islets from WT and SSTR2KO mice. L-779,976 much less potently reduced insulin secretion from WT islets. In conclusion, our data demonstrate that SST inhibition of glucagon release in mouse islets is primarily mediated via SSTR2, whereas insulin secretion is regulated primarily via SSTR5.

We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.

Members of the Rab family of low molecular weight GTP-binding (LMWG) proteins regulate vesicular transport; distinct Rab proteins are located in specific vesicular compartments within the exocytotic and endocytotic pathway. Pancreatic zymogen granules possess multiple LMWG. Rab 3A, however, which has been suggested to regulate exocytosis of synaptic vesicles, is not present in rat pancreatic acinar cells or on zymogen granules (BBRC 1994,269:542-548). To look for homologues of Rab 3A we have screened a rat pancreatic cDNA library using Rab 3A as a probe. We now report the cDNA and deduced amino acid sequence of a new member of the Rab protein family. Rab 26 is a 190 AA protein with a predicted molecular mass of 21,459 Dalton. It is expressed in pancreas, kidney, brain, submandibular gland, and lung. It is most closely related to Rab 8. There is also close homology to Rab 15, Sec4 and Rab 3.

Low molecular weight GTP-binding (LMWG) proteins are known to be present on secretory organelles and a role for Rab 3A in exocytosis of synaptic vesicles has been postulated. Pancreatic zymogen granules possess multiple LMWG proteins which were shown to be on the outside of the granules by pronase susceptibility. Rab 3A, however, could not be identified in rat pancreatic acinar cells or on zymogen granules by immunoblots or by northern blots or PCR amplification although all three techniques demonstrated its presence in rat brain, RINm5F and AR42J cells. Immunoblots revealed expression of Rab 5, Rab 7, Rab 11 and ARF in acini. Of these, Rab 5 was identified in purified zymogen granule membranes where it may be involved in the endocytotic retrieval of granule membrane following exocytosis.