CNX-011-67, a novel GPR40 agonist, enhances glucose responsiveness, insulin secretion and islet insulin content in n-STZ rats and in islets from type 2 diabetic patients.

BACKGROUND: GPR40 is a G-protein coupled receptor regulating free fatty acid induced and also glucose induced insulin secretion. We generated neonatally-streptozotocin-treated female rats (n-STZ) and treated them with CNX-011-67, a GPR40 agonist to examine the role of GPR40 in modulation of glucose metabolism, insulin secretion and content. METHODS: Female n-STZ animals were orally administered with CNX-011-67 (15 mg/kg body weight, twice daily) or with vehicle for 8 weeks (n = 8 per group). Glucose tolerance in treated animals and insulin secretion, islet insulin content and gene expression in isolated islets were determined. Islets from type 2 diabetic mellitus (T2DM) patients were treated with different concentrations of glucose in presence or absence of CNX-011-67 and insulin secretion was measured. RESULTS: Treatment of n-STZ rats with GPR40 agonist CNX-011-67 enhanced insulin secretion in response to oral glucose load on day 0 and this response persisted during the treatment period. The treatment also produced a 'memory effect' during which insulin secretion in response to oral glucose load remained enhanced, for a week, even in absence of the agonist. Activation of GPR40 enhanced responsiveness of islets to glucose and increased glucose induced insulin secretion and islet insulin content. An increase in islet mRNA expression of GCK, PDX1, insulin and PC was also observed. Acute treatment of islets from n-STZ rats with GPR40 agonist enhanced cellular ATP content. Activation of GPR40 enhanced mitochondrial calcium level in NIT-1 insulinoma cells. CNX-011-67 increased insulin secretion in islets from T2DM patients which were non-responsive to increased glucose concentration CONCLUSIONS: Our data provide evidence that activation of GPR40 with CNX-011-67 stimulates glucose metabolism, enhances glucose responsiveness, increases insulin secretion and content and that pharmacological activation of GPR40 will prove beneficial for treatment of T2DM.

CNX-012-570, a direct AMPK activator provides strong glycemic and lipid control along with significant reduction in body weight; studies from both diet-induced obese mice and db/db mice models.

OBJECTIVES: AMP activated protein kinase (AMPK) regulates the coordination of anabolic and catabolic processes and is an attractive therapeutic target for T2DM, obesity and metabolic syndrome. We report the anti-hyperglycemic and anti-hyperlipidemic effects of CNX-012-570 is an orally bioavailable small molecule (molecular weight of 530 Daltons) that directly activates AMPK in DIO and db/db animal models of diabetes. METHODS: Activity and efficacy of the compound was tested in cell based as well as cell free systems in vitro. Male C57BL/6 mice fed with high fat diet (HFD) were assigned to either vehicle or CNX-012-570 (3 mg/kg, orally once a day) for 8 weeks (n = 8). Genetically diabetic db/db mice on chow diet were dosed with vehicle control or CNX-012-570 (2.5 mg/kg, orally once a day) for 6 weeks (n = 8). RESULTS: CNX-012-570 is a highly potent and orally bioavailable compound activating AMPK in both cell and cell free systems. It inhibits lipolysis (33%) and gluconeogenesis (28%) in 3T3L1 cells and rat primary hepatocytes respectively. The efficacy of the molecule was translated to both DIO and db/db animal models of diabetes. CNX-012-570 has reduced fasting blood glucose levels by 14%, body weight by 24% and fasting serum triglycerides (TG) by 24%. CNX-012-570 showed a 22% reduction in fed serum cholesterol levels and 19% increase in HDL levels.In db/db mice model, CNX-012-570 has shown 18% decrease in fed glucose and 32% decrease in fasting glucose with a 2.57% reduction in absolute HbA1c. Decrease in serum insulin and glucose AUC indicates the increased insulin sensitivity. Body weight was reduced by 13% with increased browning of adipose tissue and decreased inguinal and mesenteric fat mass. There was significant reduction in liver TG and liver total cholesterol. CONCLUSIONS: CNX-012-570 has the potential to control hyperglycemia and hyperlipidemia. It also reduces body weight gain with an additional benefit of minimizing cardiovascular risks in diabetics.

Chronic glucolipotoxic conditions in pancreatic islets impair insulin secretion due to dysregulated calcium dynamics, glucose responsiveness and mitochondrial activity.

BACKGROUND: In the progression towards diabetes, glucolipotoxicity is one of the main causes of pancreatic beta cell pathology. The aim of this study was to examine the in vitro effects of chronic glucolipotoxic conditions on cellular responses in pancreatic islets, including glucose and fat metabolism, Calcium mobilization, insulin secretion and insulin content. RESULTS: Exposure of islets to chronic glucolipotoxic conditions decreased glucose stimulated insulin secretion in vitro. Reduced protein levels of Glut2/slc2a2, and decreased glucokinase and pyruvate carboxylase mRNA levels indicated a significant lowering in glucose sensing. Concomitantly, both fatty acid uptake and triglyceride accumulation increased significantly while fatty acid oxidation decreased. This general suppression in glucose metabolism correlated well with a decrease in mitochondrial number and activity, reduction in cellular ATP content and dampening of the TCA cycle. Further, we also observed a decrease in IP3 levels and lower Calcium mobilization in response to glucose. Importantly, chronic glucolipotoxic conditions in vitro decreased insulin gene expression, insulin content, insulin granule docking (to the plasma membrane) and insulin secretion. CONCLUSIONS: Our results present an integrated view of the effects of chronic glucolipotoxic conditions on known and novel signaling events, in vitro, that results in reduced glucose responsiveness and insulin secretion.

Treatment with CNX-011-67, a novel GPR40 agonist, delays onset and progression of diabetes and improves beta cell preservation and function in male ZDF rats.

BACKGROUND: The role of G protein-coupled receptor (GPR40), which is highly expressed in pancreatic beta cells, has been studied extensively in the amelioration of beta cell dysfunction in T2D using rat and mouse islets, beta cell lines and in animal models of diabetes. But its potential as a therapeutic target has not been fully explored. This aim of the study is to evaluate the therapeutic potential of CNX-011-67, a highly selective, potent and orally bioavailable GPR40 agonist, in controlling diabetes and other metabolic parameters. METHODS: Seven week old male ZDF rats were treated with either vehicle or CNX-011-67, 5 mg/kg twice daily, for seven weeks. The animals were subjected to oral glucose tolerance and insulin tolerance tests. Plasma glucose, insulin, triglyceride, HbA1c, fructosamine and free fatty acids were measured at selected time points. Pancreas from control and treated animals were subjected to insulin and pancreatic and duodenal homeobox 1 (PDX1) immunohistochemistry and were also evaluated by electron microscopy. Also the potential impact of CNX-011-67 on islet insulin secretion, content, ATP levels and markers of both glucose oxidation, beta cell health in rat islets under chronic glucolipotoxic conditions was evaluated. RESULTS: Treatment of male ZDF rats with CNX-011-67 for 7 weeks significantly enhanced insulin secretion in response to oral glucose load, delayed the onset of fasting hyperglycemia by 3 weeks, reduced nonfasting glucose excursions, fasting free fatty acids and triglyceride levels. A significant increase in PDX1 expression and insulin content and reduction in plasma fructosamine, HOMA-IR, and beta cell apoptosis were observed. CNX-011-67 improves glucose mediated insulin secretion, insulin gene transcription and islet insulin content in cultured rat islets under chronic glucolipotoxic condition. Also enhanced glucose oxidation in the form of increased islet ATP content and overall improvement in beta cell health in the form of reduced expression of stress markers (TXNIP and CHOP mRNA) were observed. CONCLUSIONS: These findings, suggest that long-term oral therapy with CNX-011-67 could be of clinical value to provide good glycemic control and improve islet beta cell function.

RacG regulates morphology, phagocytosis, and chemotaxis.

RacG is an unusual member of the complex family of Rho GTPases in Dictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 microM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPgammaS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or more Dictyostelium Rho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

Genomic organization and expression profile of the small GTPases of the RhoBTB family in human and mouse.

Members of the RhoBTB subfamily of Rho GTPases are present in vertebrates, Drosophila and Dictyostelium. RhoBTB proteins are characterized by a modular organization, consisting of a GTPase (guanosine triphosphatase) domain, a proline rich region, a tandem of two BTB (Broad-Complex, Tramtrack, and Bric à brac) domains and a C-terminal region of unknown function and might act as docking points for multiple components participating in signal transduction cascades. We have determined the genomic organization and the expression pattern of the three RHOBTB genes of human and mouse. The exon-intron organization of each gene is conserved in three vertebrate species (human, mouse and Fugu). RHOBTB1 and RHOBTB2 have a similar exon-intron organization and are closely related to the single gene encoding the RhoBTB orthologs of two insect species. By contrast, the exon-intron organization of RHOBTB3 differed substantially from that of the two other genes, indicating that this gene arose by a duplication event independent of the one that gave rise to RHOBTB1 and RHOBTB2. RHOBTB1 (located on chromosome 10) and RHOBTB3 (located on chromosome 5) appear ubiquitously expressed. However, they display a differential pattern of expression: RHOBTB1 showed high levels in stomach, skeletal muscle, placenta, kidney and testis, whereas RHOBTB3 was highly expressed in neural and cardiac tissues, pancreas, placenta and testis. RHOBTB2 (located on chromosome 8) showed much lower levels of expression than the other two human RHOBTB genes and it was most abundant in neural tissues. The expression patterns of the human and mouse genes were roughly comparable. All three genes were also detected in fetal tissues, and in a number of cell lines RHOBTB3 predominates. RHOBTB genes are upregulated in some cancer cell lines, suggesting that these proteins might participate in tumorigenesis.