Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88.

BACKGROUND: GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions. METHODS: In this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader. RESULTS: The CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z'-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays. CONCLUSIONS: We have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.

Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of ß-cell mass.

Insufficient plasma insulin levels caused by deficits in both pancreatic ß-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed ß-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating ß-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric G(z) protein, Gα(z), impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gα(z)-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gα(z) is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gα(z) are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in ß-cell proliferation is correlated with significantly increased ß-cell mass. Further, an endogenous Gα(z) signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gα(z) to both major aspects of ß-cell decompensation: insufficient ß-cell function and mass.

Hyperinsulinemia predicts survival in a hyperglycemic mouse model of critical illness.

OBJECTIVES: : The mechanisms by which correcting hyperglycemia with exogenous insulin improves mortality and morbidity in critically ill patients remain unclear. We designed this study to test the hypothesis that relative endogenous insulin deficiency is associated with adverse outcomes in critical illness related to hyperglycemia. DESIGN: : Prospective controlled animal study. SETTING: : University research laboratory. SUBJECTS: : Male C57BL/6J mice, 8-12 wks old. INTERVENTIONS: : Spontaneously breathing mice were instrumented with chronic indwelling arterial and venous catheters. After a postoperative recovery period, endotoxemia was initiated with intra-arterial lipopolysaccharide (1 mg/kg) in the presence of dextrose infusion (100 microL/hr). Insulin secretion was blocked with diazoxide (2.5-30 mg/kg/day). Mice were monitored continuously for 48 hrs with blood sampled serially for blood glucose and plasma insulin determinations. MEASUREMENTS AND MAIN RESULTS: : In both saline- and glucose-infused mice, lipopolysaccharide administration induced transient hemodynamic instability without significant impact on mortality. In the saline-infused group, lipopolysaccharide administration caused a transient reduction in blood glucose and in circulating insulin. However, in glucose-infused mice, lipopolysaccharide induced a large and unexpected increase in circulating insulin without significant alteration in blood glucose. Blockade of insulin secretion in response to lipopolysaccharide in the presence of exogenous glucose precipitated marked hyperglycemia and resulted in >90% mortality. In a subanalysis of animals matched for the degree of hyperglycemia, nonsurvivors had markedly lower insulin levels compared with survivors (3.5 +/- 0.8 ng/dL vs. 9.3 +/- 1.4 ng/dL; p < .004). CONCLUSIONS: : Endogenous insulin deficiency in the face of hyperglycemia is associated with mortality in a mouse model of lipopolysaccharide-induced critical illness.

Intermittent hypoxia reverses the diurnal glucose rhythm and causes pancreatic beta-cell replication in mice.

Obstructive sleep apnoea (OSA) and type 2 diabetes frequently co-exist and potentially interact haemodynamically and metabolically. However, the confounding effects of obesity have obscured the examination of any independent or interactive effects of the hypoxic stress of OSA and the hyperglycaemia of type 2 diabetes on haemodynamic and metabolic outcomes. We have developed a chronically catheterized, unhandled, lean murine model to examine the effects of intermittent hypoxic (IH) exposure and exogenous glucose infusion on the diurnal pattern of arterial blood pressure and blood glucose, as well as pancreatic beta-cell growth and function. Four experimental groups of adult male C57BL/J mice were exposed to 80 h of (1) either IH (nadir of inspired oxygen 5-6% at 60 cycles h(-1) for 12 h during light period) or intermittent air (IA; control) and (2) continuous infusion of either 50% dextrose or saline (control). IH exposure during saline infusion caused a sustained increase in arterial blood pressure of 10 mmHg (P < 0.0001), reversed the normal diurnal rhythm of blood glucose (P < 0.03), doubled corticosterone levels (P < 0.0001), and increased replication of pancreatic beta-cells from 1.5 +/- 0.3 to 4.0 +/- 0.8% bromodeoxyuridine (BrdU)-positive) beta-cells. The combined stimulus of IH exposure and glucose infusion attenuated the hypertension, exacerbated the reversed diurnal glucose rhythm, and produced the highest rates of apoptosis in beta-cells, without any additive effects on beta-cell replication. We conclude that, in contrast to the development of sustained hypertension, IH impaired glucose homeostasis only during periods of hypoxic exposure. IH acted as a stimulus to pancreatic beta-cell replication, but the presence of hyperglycaemia may increase the hypoxic susceptibility of beta-cells. This model will provide a basis for future mechanistic studies as well as assessing the metabolic impact of common comorbities in OSA, including obesity, insulin resistance and type 2 diabetes.

Hepatocyte growth factor ameliorates hyperglycemia and corrects ß-cell mass in IRS2-deficient mice.

Insulin resistance, when combined with decreased ß-cell mass and relative insufficient insulin secretion, leads to type 2 diabetes. Mice lacking the IRS2 gene (IRS2(-/-) mice) develop diabetes due to uncompensated insulin resistance and ß-cell failure. Hepatocyte growth factor (HGF) activates the phosphatidylinositol 3-kinase/Akt signaling pathway in ß-cells without recruitment of IRS1 or IRS2 and increases ß-cell proliferation, survival, mass, and function when overexpressed in ß-cells of transgenic (TG) mice. We therefore hypothesized that HGF may protect against ß-cell failure in IRS2 deficiency. For that purpose, we cross-bred TG mice overexpressing HGF in ß-cells with IRS2 knockout (KO) mice. Glucose homeostasis analysis revealed significantly reduced hyperglycemia, compensatory hyperinsulinemia, and improved glucose tolerance in TG/KO mice compared with those in KO mice in the context of similar insulin resistance. HGF overexpression also increased glucose-stimulated insulin secretion in IRS2(-/-) islets. To determine whether this glucose homeostasis improvement correlated with alterations in ß-cells, we measured ß-cell mass, proliferation, and death in these mice. ß-Cell proliferation was increased and death was decreased in TG/KO mice compared with those in KO mice. As a result, ß-cell mass was significantly increased in TG/KO mice compared with that in KO mice, reaching levels similar to those in wild-type mice. Analysis of the intracellular targets involved in ß-cell failure in IRS2 deficiency showed Pdx-1 up-regulation, Akt/FoxO1 phosphorylation, and p27 down-regulation in TG/KO mouse islets. Taken together, these results indicate that HGF can compensate for IRS2 deficiency and subsequent insulin resistance by normalizing ß-cell mass and increasing circulating insulin. HGF may be of value as a therapeutic agent against ß-cell failure.

Activation of protein kinase C-ζ in pancreatic ß-cells in vivo improves glucose tolerance and induces ß-cell expansion via mTOR activation.

OBJECTIVE: PKC-ζ activation is a key signaling event for growth factor-induced ß-cell replication in vitro. However, the effect of direct PKC-ζ activation in the ß-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in ß-cell expansion and function in vivo in mice and the mechanisms associated with these effects. RESEARCH DESIGN AND METHODS: We characterized glucose homeostasis and ß-cell phenotype of transgenic (TG) mice with constitutive activation of PKC-ζ in the ß-cell. We also analyzed the expression and regulation of signaling pathways, G1/S cell cycle molecules, and ß-cell functional markers in TG and wild-type mouse islets. RESULTS: TG mice displayed increased plasma insulin, improved glucose tolerance, and enhanced insulin secretion with concomitant upregulation of islet insulin and glucokinase expression. In addition, TG mice displayed increased ß-cell proliferation, size, and mass compared with wild-type littermates. The increase in ß-cell proliferation was associated with upregulation of cyclins D1, D2, D3, and A and downregulation of p21. Phosphorylation of D-cyclins, known to initiate their rapid degradation, was reduced in TG mouse islets. Phosphorylation/inactivation of GSK-3ß and phosphorylation/activation of mTOR, critical regulators of D-cyclin expression and ß-cell proliferation, were enhanced in TG mouse islets, without changes in Akt phosphorylation status. Rapamycin treatment in vivo eliminated the increases in ß-cell proliferation, size, and mass; the upregulation of cyclins Ds and A in TG mice; and the improvement in glucose tolerance-identifying mTOR as a novel downstream mediator of PKC-ζ-induced ß-cell replication and expansion in vivo. CONCLUSIONS PKC:-ζ, through mTOR activation, modifies the expression pattern of ß-cell cycle molecules leading to increased ß-cell replication and mass with a concomitant enhancement in ß-cell function. Approaches to enhance PKC-ζ activity may be of value as a therapeutic strategy for the treatment of diabetes.

Disruption of hepatocyte growth factor/c-Met signaling enhances pancreatic beta-cell death and accelerates the onset of diabetes.

OBJECTIVE: To determine the role of hepatocyte growth factor (HGF)/c-Met on ß-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro. RESEARCH DESIGN AND METHODS: We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced ß-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB. RESULTS: Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in ß-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and ß-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for ß-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced ß-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and ß-cell apoptosis. c-Met-null ß-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human ß-cells against cytokines. CONCLUSIONS: These results show that HGF/c-Met is critical for ß-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing ß-cell protection.

Novel proapoptotic effect of hepatocyte growth factor: synergy with palmitate to cause pancreatic {beta}-cell apoptosis.

Increasing evidence suggests that elevation of plasma fatty acids that often accompanies insulin resistance contributes to beta-cell insufficiency in obesity-related type 2 diabetes. Circulating levels of hepatocyte growth factor (HGF) are increased in humans with metabolic syndrome and obesity. HGF is known to protect beta-cells against streptozotocin and during islet engraftment. However, whether HGF is a beta-cell prosurvival factor in situations of excessive lipid supply has not been deciphered. Mice overexpressing HGF in the beta-cell [rat insulin type II promoter (RIP)-HGF transgenic mice] fed with standard chow display improved glucose homeostasis and increased beta-cell mass and proliferation compared with normal littermates. However, after 15 wk of high-fat feeding, glucose homeostasis and beta-cell expansion and proliferation are indistinguishable between normal and transgenic mice. Interestingly, RIP-HGF transgenic mouse beta-cells and normal beta-cells treated with HGF display increased sensitivity to palmitate-mediated apoptosis in vitro. Palmitate completely eliminates Akt and Bad phosphorylation in RIP-HGF transgenic mouse islets. HGF-overexpressing islets also show significantly decreased AMP-activated protein kinase-alpha and acetyl-coenzyme A carboxylase phosphorylation, diminished fatty acid oxidation, increased serine palmitoyltransferase expression, and enhanced ceramide formation compared with normal islets. Importantly, human islets overexpressing HGF also display increased beta-cell apoptosis in the presence of palmitate. Treatment of both mouse and human islet cells with the de novo ceramide synthesis inhibitors myriocin and fumonisin B1 abrogates beta-cell apoptosis induced by HGF and palmitate. Collectively, these studies indicate that HGF can be detrimental for beta-cell survival in an environment with excessive fatty acid supply.

Protein kinase C-zeta activation markedly enhances beta-cell proliferation: an essential role in growth factor mediated beta-cell mitogenesis.

OBJECTIVE: Diabetes results from a deficiency of functional beta-cells. Previous studies have identified hepatocyte growth factor (HGF) and parathyroid hormone-related protein (PTHrP) as two potent beta-cell mitogens. The objective of this study is to determine 1) whether HGF and PTHrP have additive/synergistic effects on beta-cell growth and proliferation; 2) the signaling pathways through which these growth factors mediate beta-cell mitogenesis; and 3) whether activation of this/these signaling pathway(s) enhances human beta-cell replication. RESEARCH DESIGN AND METHODS: We generated and phenotypically analyzed doubly transgenic mice overexpressing PTHrP and HGF in the beta-cell. INS-1 and primary mouse and human islet cells were used to identify mitogenic signaling pathways activated by HGF and/or PTHrP. RESULTS: Combined overexpression of HGF and PTHrP in the beta-cell of doubly transgenic mice did not result in additive/synergistic effects on beta-cell growth and proliferation, suggesting potential cross-talk between signaling pathways activated by both growth factors. Examination of these signaling pathways in INS-1 cells revealed atypical protein kinase C (PKC) as a novel intracellular target activated by both HGF and PTHrP in beta-cells. Knockdown of PKC zeta, but not PKC iota/lambda, expression using specific small-interfering RNAs blocked growth factor-induced INS-1 cell proliferation. Furthermore, adenovirus-mediated delivery of kinase-dead PKC zeta completely inhibited beta-cell proliferation in primary islet cells overexpressing PTHrP and/or HGF. Finally, adenovirus-mediated delivery of constitutively active PKC zeta in mouse and human primary islet cells significantly enhanced beta-cell proliferation. CONCLUSIONS: PKC zeta is essential for PTHrP- and HGF-induced beta-cell proliferation. PKC zeta activation could be useful in therapeutic strategies for expanding beta-cell mass in vitro and in vivo.