Activation of GPR40 attenuates chronic inflammation induced impact on pancreatic ß-cells health and function.

BACKGROUND: Chronic inflammation-mediated ß-cell apoptosis is known to decrease ß-cell mass in diabetes leading to reduced insulin secretion. Exposure to pro-inflammatory cytokines can stimulate apoptosis in pancreatic ß-cells. The G protein coupled receptor 40 (GPR40) is implicated for glucose induced insulin secretion. We hypothesized that GPR40 activation can protect ß-cells from inflammation-induced apoptosis and restore glucose stimulated insulin secretion. RESULTS: By exposing NIT1 insulinoma cells and rat islets to a cocktail of pro-inflammatory cytokines (TNFα and IL1ß), we mimicked inflammatory signaling as seen by JNK and NFκB activation and increased mRNA levels of TNFα, IL1ß and NOS2a. These changes were reversed by pharmacological activation of GPR40 by a specific, small molecule, CNX-011-67. Further, GPR40 activation reduced inflammation-mediated oxidative and endoplasmic reticulum (ER) stresses. Importantly, GPR40 activation decreased inflammation-induced apoptosis as measured by key markers. These impacts of GPR40 were mediated through activation of PLC, CaMKII, calcineurin and cAMP. Cell survival was also enhanced by GPR40 activation as seen from the increased phosphorylation of Akt/PKB and enhanced expression of BCL2 and PDX1 genes. Interestingly, GPR40 activation restored both, inflammation-mediated inhibition on insulin secretion and intracellular insulin content. CONCLUSIONS: In this study, we provide evidences that CNX-011-67, a GPR40 agonist, reduces inflammatory signaling and apoptosis in pancreatic ß-cells while promoting insulin secretion and synthesis. Activation of GPR40 leads to attenuation of ß-cell dysfunction caused by chronic inflammation and thus could be of immense clinical value to improve insulin secretion and ß-cell survival.

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.

A novel GPR40 agonist, CNX-011-67, suppresses glucagon secretion in pancreatic islets under chronic glucolipotoxic conditions in vitro.

BACKGROUND: Elevated glucose concentrations lead to increased insulin secretion and suppression of glucagon secretion. In fact, insulin is a physiological inhibitor of glucagon secretion. Type 2 diabetes mellitus (T2DM) patients have defects in insulin secretion. In addition to this, lack of suppression of glucagon secretion under elevated glucose concentrations is also observed in T2DM patients. We have earlier shown that GPR40 activation by CNX-011-67 stimulates glucose stimulated insulin secretion (GSIS). Here we extended our studies to examine the impact of GPR40 activation by CNX-011-67 on glucagon secretion from intact islets under both normal and glucolipotoxic conditions. FINDINGS: Glucagon secretion from intact rat islets was suppressed under elevated glucose concentration. Activation of GPR40 by CNX-011-67 further suppressed glucagon secretion. Culturing islets under chronic glucolipotoxic (GL) conditions, we have observed increased high glucose mediated glucagon secretion and content which were reduced with GPR40 activation by CNX-011-67. Interestingly, expression of pre-proglucagon gene (GCG) remained unchanged under glucolipotoxicity in the presence or absence of GPR40 activation. CONCLUSION: Activation of GPR40 by CNX-011-67 can reduce glucagon secretion from pancreatic islets.

Inhibition of neutral sphingomyelinases in skeletal muscle attenuates fatty-acid induced defects in metabolism and stress.

BACKGROUND: Chronic metabolic overload leads to insulin resistance in a variety of tissues. It has been shown that exposure to saturated fatty acid palmitate can cause insulin resistance in skeletal muscle cells. Fatty acid induced synthesis of ceramide is considered to be one of the major causes for insulin resistance. Both de novo synthesis and sphingomyelin hydrolysis by sphingomyelinase are implicated for ceramide generation. Aim of this study was to evaluate the impact of neutral sphingomyelinase (nSMase) inhibition on saturated fatty acid induced lipotoxicity and insulin resistance in skeletal muscle myotubes. RESULTS: Treatment of saturated fatty acid (palmitate) but not unsaturated fatty acid (oleate) caused an up-regulation in expression of various nSMase genes which are associated with ceramide synthesis through the salvage pathway. Inhibition of nSMase by a pharmacological inhibitor (GW4869) partially reverted the palmitate induced insulin resistance in C2C12 myotubes. Inhibition of nSMase improved metabolic functions of myotubes as measured by improved oxidative capacity in terms of increased mitochondrial number, PGC1α expression and ATP levels with concomitant decrease in intramyocellular triglyceride levels. Palmitate induced inflammatory response was also reduced by nSMase inhibitor. GW4869 treatment reduced palmitate induced oxidative and endoplasmic reticulum stress and improved cell survival. CONCLUSION: In this study, we provide evidences that inhibition of nSMase can protect skeletal muscles from saturated fatty acid induced insulin resistance, metabolic dysfunction, cellular stress and inflammation.

Integrated analysis of chronic lipotoxicity on muscle metabolism and stress and its reversal by antioxidants.

Apart from elevated glucose, triglyceride and cholesterol, elevated levels of serum free-fatty acid (FFA) are observed in diabetic patients. Increased FFA load can cause multiple dysregulation which are collectively known as lipotoxicity. Impacts of FFA induced lipotoxicity were evaluated on various cellular responses of metabolism and stress in skeletal muscle myotubes. Under lipotoxicity, oxidative capacity of C2C12 myotubes was reduced and decreased levels ATP and NAD were observed. Lipotoxicity augmented non-oxidative disposal of metabolites in terms of lactate release, IMTG and ceramide synthesis. Concomitantly, insulin resistance was also observed. These impacts were in conjunction with increased cellular stress, inflammation, proteolysis and apoptosis. Quenching of lipotoxicity mediated oxidative stress by antioxidant reverted its deleterious impacts and restored insulin stimulated glucose uptake. In conclusion, the in vitro lipotoxicity makes a system which resembles in vivo pathology of muscle as seen in diabetic patients and represents an integrated perspective of lipotoxicity on various parameters of metabolism and stress.

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.

A novel 11ß-hydroxysteroid dehydrogenase type1 inhibitor CNX-010-49 improves hyperglycemia, lipid profile and reduces body weight in diet induced obese C57B6/J mice with a potential to provide cardio protective benefits.

BACKGROUND: 11ß-hydroxysteroid dehydrogenase type1 (11ß-HSD1) converts inactive glucocorticoids to active glucocorticoids which, in excess, leads to development of the various risk factors of the metabolic syndrome. Recent studies clearly suggest that both increased expression and activity of 11ß-HSD1 in metabolically active tissues such as liver, muscle and adipose are implicated in tissue specific dysregulation which collectively contribute to the whole body pathology seen in metabolic syndrome. In the present study we have evaluated CNX-010-49, a highly potent, selective and 'pan tissue' acting 11ß-HSD1 inhibitor, for its potential to modulate multiple risk factors of the metabolic syndrome. METHODS: Male C57B6/J mice on high fat diet (DIO mice) were orally dosed with CNX-010-49 (30 mg/kg twice daily; n = 8) or vehicle for 10 weeks. Fasting glucose, triglycerides, glycerol, free fatty acids, body weight and feed intake were measured at selected time points. At the end of the treatment an OGTT and subsequently organ histology was performed. In vitro, CNX-010-49 was evaluated in 3T3-L1 preadipocytes to assess impact on adipocytes differentiation, hypertrophy and lipolysis whereas in fully differentiated C2C12 cells and in primary mouse hepatocytes to assess the impact on glucose metabolism and hepatic glucose output respectively. RESULTS: CNX-010-49 a highly potent and selective pan tissue acting 11ß-HSD1 inhibitor (EC50 = 6 nM) significantly inhibits glucocorticoids and isoproterenol mediated lipolysis in mature 3T3-L1 adipocytes, improves muscle glucose oxidation, reduces proteolysis and enhances mitochondrial biogenesis. Also a significant inhibition of gluconeogenesis in primary mouse hepatocytes was observed. The treatment with CNX-010-49 resulted in a significant decrease in fasting glucose, improved insulin sensitivity and glucose tolerance. Treatment also resulted in a significant decrease in serum triglycerides levels and a complete inhibition of body weight gain without affecting feed consumption. A significant reduction in the serum biomarkers like Plasminogen activator inhibitor-1 (PAI-1), interleukin 6 (IL-6) and Fetuin-A with CNX-010-49 treatment was observed indicating a potential to modulate processes implicated in cardiovascular benefits. CONCLUSIONS: These results indicate that inhibition of 11ß-HSD1 with CNX-010-49 can give a potential benefit in the management of metabolic dysregulations that are seen in type 2 diabetes.

CNX-013-B2, a unique pan tissue acting rexinoid, modulates several nuclear receptors and controls multiple risk factors of the metabolic syndrome without risk of hypertriglyceridemia, hepatomegaly and body weight gain in animal models.

BACKGROUND: In addition to their role in growth, cellular differentiation and homeostasis Retinoid X Receptors (RXR) regulate multiple physiological and metabolic pathways in various organs that have beneficial glucose and lipid (cholesterol) lowering, insulin sensitizing and anti-obesity effects. Rexinoids, compounds that specifically binds and activate RXR, are therefore considered as potential therapeutics for treating metabolic syndrome. Apparently many of the rexinoids developed in the past increased triglycerides, caused hepatomegaly and also suppressed the thyroid hormone axis. The aim of this study is to evaluate CNX-013-B2, a potent and highly selective rexinoid, for its potential to treat multiple risk factors of the metabolic syndrome. METHODS: CNX-013-B2 was selected in a screening system designed to identify compounds that selectively activated only a chosen sub-set of heterodimer partners of RXR of importance to treat insulin resistance. Male C57BL/6j mice (n = 10) on high fat diet (HFD) and 16 week old ob/ob mice (n = 8) were treated orally with CNX-013-B2 (10 mg/kg twice daily) or vehicle for 10 weeks and 4 weeks respectively. Measurement of plasma glucose, triglyceride, cholesterol including LDL-C, glycerol, free fatty acids, feed intake, body weight, oral glucose tolerance and non-shivering thermogenesis were performed at selected time points. After study termination such measurements as organ weight, triglyceride content, mRNA levels, protein phosphorylation along with histological analysis were performed. RESULTS: CNX-013-B2 selectively activates PPARs- α, ß/δ and γ and modulates activity of LXR, THR and FXR. In ob/ob mice a significant reduction of 25% in fed glucose (p < 0.001 ), a 14% (p < 0.05) reduction in serum total cholesterol and 18% decrease (p < 0.01) in LDL-C and in DIO mice a reduction of 12% (p < 0.01 ) in fasting glucose, 20% in fed triglyceride (p < 0.01) and total cholesterol (p < 0.001) levels, coupled with enhanced insulin sensitivity, cold induced thermogenesis and 7% reduction in body weight were observed. CONCLUSION: CNX-013-B2 is an orally bio available selective rexinoid that can be used as a novel therapeutic agent for management of multiple risk factors of the metabolic syndrome without the risk of side effects reported to be associated with rexinoids.

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.