- Developing an in vitro screening assay platform for evaluation of antifibrotic drugs using precision-cut liver slices
Fibrogenesis Tissue Repair 2015, Jan 9
Satish Kumar Sadasivan, Nethra Siddaraju, Khaiser Mehdi Khan, Balamuralikrishna Vasamsetti, Nimisha R Kumar, Vibha Haridas, Madhusudhan B Reddy, Somesh Baggavalli, Anup M Oommen and Raghavendra Pralhada Rao
Precision-cut liver slices present different cell types of liver in a physiological context, and they have been explored as effective in vitro model systems to study liver fibrosis. Inducing fibrosis in the liver slices using toxicants like carbon tetrachloride is of less relevance to human disease conditions. Our aim for this study was to establish physiologically relevant conditions in vitro to induce fibrotic phenotypes in the liver slices.
Precision-cut liver slices of 150 μm thickness were obtained from female C57BL/6 J mice. The slices were cultured for 24 hours in media containing a cocktail of 10 nM each of TGF-β, PDGF, 5 μM each of lysophosphatidic acid and sphingosine 1 phosphate and 0.2 μg/ml of lipopolysaccharide along with 500 μM of palmitate and were analyzed for triglyceride accumulation, stress and inflammation, myofibroblast activation and extracellular matrix (ECM) accumulation. Incubation with the cocktail resulted in increased triglyceride accumulation, a hallmark of steatosis. The levels of Acta2, a hallmark of myofibroblast activation and the levels of inflammatory genes (IL-6, TNF-α and C-reactive protein) were significantly elevated. In addition, this treatment resulted in increased levels of ECM markers - collagen, lumican and fibronectin.
This study reports the experimental conditions required to induce fibrosis associated with steatohepatitis using physiologically relevant inducers. The system presented here captures various aspects of the fibrosis process like steatosis, inflammation, stellate cell activation and ECM accumulation and serves as a platform to study the liver fibrosis in vitro and to screen small molecules for their antifibrotic activity.
- Investigating correlations in the altered metabolic profiles of obese and diabetic subjects in a South Indian Asian population using an NMR-based metabolomic approach
Molecular BioSystems 2014, Nov 25
Navdeep Gogna, Murahari Krishna, Anup Mammen Oommen, Kavita Dorai
It is well known that obesity/high body mass index (BMI) plays a key role in the evolution of insulin resistance and type-2 diabetes mellitus (T2DM). However, the exact mechanism underlying its contribution is still not fully understood. This work focuses on an NMR-based metabolomic investigation of the serum profiles of diabetic, obese South Indian Asian subjects. 1H 1D and 2D NMR experiments were performed to profile the altered metabolic patterns of obese diabetic subjects and multivariate statistical methods were used to identify metabolites that contributed significantly to the differences in the samples of four different subject groups: diabetic and non-diabetic with low and high BMIs. Our analysis revealed that the T2DM-high BMI group has higher concentrations of saturated fatty acids, certain amino acids (leucine, isoleucine, lysine, proline, threonine, valine, glutamine, phenylalanine, histidine), lactic acid, 3-hydroxybutyric acid, choline, 3,7-dimethyluric acid, pantothenic acid, myoinositol, sorbitol, glycerol, and glucose, as compared to the non-diabetic-low BMI (control) group. Of these 19 identified significant metabolites, the levels of saturated fatty acids, lactate, valine, isoleucine, and phenylalanine are also higher in obese non-diabetic subjects as compared to control subjects, implying that this set of metabolites could be identified as potential biomarkers for the onset of diabetes in subjects with a high BMI. Our work validates the utility of NMR-based metabolomics in conjunction with multivariate statistical analysis to provide insights into the underlying metabolic pathways that are perturbed in diabetic subjects with a high BMI.
- A novel GPR40 agonist, CNX-011-67, suppresses glucagon secretion in pancreatic islets under chronic glucolipotoxic conditions in vitro
BMC Research Notes 2014, Sep 3
Mahesh Kumar Verma, Sanghamitra Biswas, Bhawna Chandravanshi, Korrapati Neelima, Anup M Oommen, Madanahalli R Jagannath and Baggavalli P Somesh
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.
Activation of GPR40 by CNX-011-67 can reduce glucagon secretion from pancreatic islets.
- 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
Diabetology & Metabolic Syndrome 2014, Aug 12
Manoj Kumar Sadasivuni, Bobbili Madhusudhan Reddy, Jaideep Singh, Mammen O Anup, Venkategowda Sunil, Mudigere N Lakshmi, Sivakumaran Yogeshwari, Suni K Chacko, Talanki Lokesh Pooja, Anilkumar Dandu, Chandrashekaran Harish, Aralakuppe S Gopala, Shivakumar Pratibha, Baisani S Naveenkumar, Puttrevana M Pallavi, Mahesh Kumar Verma, Yoganand Moolemath, Baggavalli P Somesh, Marikunte V Venkataranganna and Madanahalli R Jagannath
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.
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.
CNX-013-B2 selectively activates PPARs- alpha, beta/delta and gamma 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.
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.
- 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
BMC Pharmacology and Toxicology 2014, Aug 7
Tharappel M Anil, Anilkumar Dandu, KrishnaReddy Harsha, Jaideep Singh, Nitya Shree, Venkatesh Satish Kumar, Mudigere N Lakshmi, Venkategowda Sunil, Chandrashekaran Harish, Gundalmandikal V Balamurali, Baisani S Naveen Kumar, Aralakuppe S Gopala, Shivakumar Pratibha, ManojKumar Sadasivuni, Mammen O Anup, Yoganand Moolemath, Marikunte V Venkataranganna, Madanahalli R Jagannath* and Baggavalli P Somesh
11ß–hydroxysteroid dehydrogenase type1 (11β-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 11β-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 11β-HSD1 inhibitor, for its potential to modulate multiple risk factors of the metabolic syndrome.
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.
CNX-010-49 a highly potent and selective pan tissue acting 11β-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.
These results indicate that inhibition of 11β-HSD1 with CNX-010-49 can give a potential benefit in the management of metabolic dysregulations that are seen in type 2 diabetes.
- Activation of GPR40 attenuates chronic inflammation induced impact on pancreatic beta-cells health and function
BMC Cell Biology 2014, Jun 30
Mahesh Kumar Verma, Manoj Kumar Sadasivuni, Aggunda N Yateesh, Korrapati Neelima, Srikanth Mrudula, Madhusudhan Reddy, Rachapalli Smitha, Sanghamitra Biswas, Bhawna Chandravanshi, Puttrevana M Pallavi, Anup M Oommen, Madanahalli R Jagannath and Baggavalli B Somesh
Chronic inflammation-mediated beta-cell apoptosis is known to decrease beta-cell mass in diabetes leading to reduced insulin secretion. Exposure to pro-inflammatory cytokines can stimulate apoptosis in pancreatic beta-cells. The G protein coupled receptor 40 (GPR40) is implicated for glucose induced insulin secretion. We hypothesized that GPR40 activation can protect beta-cells from inflammation-induced apoptosis and restore glucose stimulated insulin secretion.
By exposing NIT1 insulinoma cells and rat islets to a cocktail of pro-inflammatory cytokines (TNFalpha and IL1beta), we mimicked inflammatory signaling as seen by JNK and NFkappaB activation and increased mRNA levels of TNFalpha, IL1beta 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.
In this study, we provide evidences that CNX-011-67, a GPR40 agonist, reduces inflammatory signaling and apoptosis in pancreatic beta-cell while promoting insulin secretion and synthesis. Activation of GPR40 leads to attenuation of beta-cell dysfunction caused by chronic inflammation and thus could be of immense clinical value to improve insulin secretion and beta-cell survival.
- Inhibition of neutral sphingomyelinases in skeletal muscle attenuates fatty-acid induced defects in metabolism and stress
Springer Plus 2014, 20 May
Mahesh Kumar Verma, Aggunda Nagaraju Yateesh, Korrapati Neelima, Niketa Pawar, Kandoor Sandhya, Jayaram Poornima, Mudigere N Lakshmi, Sivakumaran Yogeshwari, Puttrevana M Pallavi, Anup M Oommen, Baggavalli P Somesh and Madanahalli R Jagannath
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.
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.
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
Springer Plus 2014, May 18
Mahesh Kumar Verma, Aggunda Nagaraju Yateesh, Rachapalli Smitha, Korrapati Neelima, Puttrevana M Pallavi, Madhusudhan Reddy, Jayaram Poornima, Anup M Oommen, Madanahalli R Jagannath and Baggavalli P Somesh
AbstractApart 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.
- Fast food diet with CCl4 micro-dose induced hepatic-fibrosis -a novel animal model
BMC Gastroenterology 2014, May 10
Tarak K Chheda, Pratibha Shivakumar, Satish Kumar Sadasivan, Harish Chanderasekharan, Yogananda Moolemath, Anup M Oommen, Jagannath R Madanahalli and Venkataranganna V Marikunte
Non-alcoholic fatty liver disease (NAFLD) is defined as a spectrum of conditions ranging from hepatocellular steatosis to steatohepatitis and fibrosis, progressing to cirrhosis, which occur in the absence of excessive alcohol use. Several animal models capture aspects of NAFLD but are limited either in their representation of the disease stages or use for development of therapeutics due to the extended periods of time required to develop full histological features.
Here, we report the development of a novel rat model for NAFLD that addresses some of these limitations. We used a fast food diet (FFD) and a CCl4 micro dose (0.5 ml/kg B.wt) for 8 weeks in Wistar rats. Serological analyses, gene expression profiling and liver histology studies were conducted to investigate the development of steatosis, steatohepatitis and fibrosis in the FFD-CCl4 model when compared to the individual effects of a FFD or a micro dose of CCl4 in rats.
The serum biochemical profile of the FFD-CCl4 model showed an increase in liver injury and fibrosis. This was also accompanied by a significant increase in liver triglycerides (TG), inflammation and oxidative stress. Importantly, we observed extensive fibrosis confirmed by: i) increased gene expression of fibrosis markers and, ii) moderate to severe collagen deposition seen as perisinusoidal and bridging fibrosis using H&E, Trichome and Sirius Red staining.
In summary, we find that the FFD-CCl4 rat model developed NAFLD histological features including, steatosis, inflammation and fibrosis in 8 weeks showing promise as a model that can be used to develop NAFLD therapeutics and liver anti-fibrotics.
- Modulation of de novo purine biosynthesis leads to activation of AMPK and
results in improved glucose handling and insulin sensitivity
Journal of Diabetes & Metabolic Disorders 2014, Apr 24
Satish Kumar Sadasivan, Balamuralikrishna Vasamsetti, Jaideep Singh,Nethra Siddaraju, Khaiser Mehdi Khan, Anup Mammen Oommen, Madanalli R Jagannath, Raghavendra Pralhada Rao
AMP activated protein kinase (AMPK) regulates key metabolic reactions and plays a major role in glucose homeostasis. Activating the AMPK is considered as one of the potential therapeutic strategies in treating type-2 diabetes. However, targeting AMPK by small molecule mediated approach can be challenging owing to diverse isoforms of the enzyme and their varied combination in different tissues. In the current study we employ a novel strategy of achieving AMPK activation through increasing the levels of cellular AMP (an allosteric activator of AMPK) levels by activating the enzyme involved in AMP biosynthesis namely Adenylosuccinate lyase (ADSL).
Rat primary hepatocytes were cultured under metabolic overload conditions (500 μM palmitate) to induce insulin resistance. ADSL was overexpressed in these hepatocytes and its effect on hepatic glucose output, and triglyceride accumulation was checked. In addition to this, ADSL was overexpressed in high fat diet induced obese mice by hydrodynamic tail vein injection and its effect on fasting glucose, glucose tolerance and pyruvate tolerance were checked.
Rat primary hepatocytes when cultured under metabolic overload conditions developed insulin resistance as measured in terms of failure of insulin to suppress the glucose output. Overexpressing the ADSL in these hepatocytes resulted in increased AMPK phosporylation and improved the insulin sensitivity and also resulted in reduced triglyceride accumulation and inflammatory cytokine levels. In addition to this, when ADSL was overexpressed in high fat diet induced obese mice, it resulted in reduced the fasting hyperglycemia (20% reduction), and increased glucose and pyruvate tolerance.
This study indicates that activating ADSL can be a potential mechanism to achieve the activation of AMPK in the cells. This leads to a novel idea of exploring the purine nucleotide metabolic pathway as a promising therapeutic target for diabetes and metabolic syndrome.
- 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
BMC Pharmacology & Toxicology 2014, Mar 25
Venkategowda Sunil, Mahesh Kumar Verma, Anup M Oommen, Manojkumar Sadasivuni, Jaideep Singh, Dasarahalli N. Vijayraghav, Bhawna Chandravanshi, Jayalaxmi Shetty, Sanghamitra Biswas, Anilkumar Dandu, Yoganand Moolemath1, Marikunte V. Venkataranganna, Baggavalli P. Somesh and Madanahalli R. Jagannath
GPR40 is a G-protein coupled receptor regulating free fatty acid induced as 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.
Female n-STZ animals were orally administered with CNX-011-67 (15mg/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.
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 nonresponsive to increased glucose concentration
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.
- Exogenous administration of spermine results in improved glucose utilization and decreases body weight in mice
European Journal of Pharmacology 2014, Apr 15
Satish K Sadasivan, Balamuralikrishna Vasamsetti, Jaideep Singh, Venkataranganna V Marikunte, Anup M Oommen, Jagannath M.R, Raghavendra Pralhada Rao
Abstract Polyamines are highly charged low molecular weight aliphatic polycations and are ubiquitously present in all living cells. In addition to their previously reported role in cell proliferation and cancer, recent studies support their role in energy homeostasis and glucose metabolism. In the present study we have evaluated a polyamine - spermine for its effect on glycemic, lipid and body weight parameters. High fat diet induced obese mice (6 week old male C57B6/J mice fed on high fat diet for 22 weeks) were dosed with spermine intraperitoneally at two different doses (5mg/kg and 10mg/kg body weight) for 4 weeks and its effect on body weight, glycemic and lipid parameters were monitored. We found that at a dose of 10mg/kg bodyweight, spermine treatment resulted in a 24% reduction in the body weight and 18% reduction in the fasting glucose compared to untreated controls.Besides, spermine treated mice exhibited improved glucose utilization associated with improved fat oxidation and loss of white adipose mass. Our study is promising in the direction of exploring the spermine and their analogues for treatment of metabolic syndrome.
- 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
Cardiovascular Diabetology 2014, Jan 25
Anil TM, Harish C, Lakshmi MN, Harsha K, Onkaramurthy M, Sathish Kumar V, Shree N, Geetha V, Balamurali GV, Gopala AS, Madhusudhan Reddy B, Govind MK, Anup MO, Moolemath Y, Venkataranganna MV, Jagannath MR, Somesh BP
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.
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).
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.
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.
- Sphingolipid metabolic pathway: an overview of major roles played in human diseases
Journal of Lipids Volume 2013, Article ID 178910
Pralhada Rao R, Vaidyanathan N, Rengasamy M, Mammen Oommen A, Somaiya N, Jagannath MR
Abstract Sphingolipids, a family of membrane lipids, are bioactive molecules that participate in diverse functions controlling fundamental cellular processes such as cell division, differentiation, and cell death. Given that most of these cellular processes form the basis for several pathologies, it is not surprising that sphingolipids are key players in several pathological processes. This review discusses the role of the sphingolipid metabolic pathway in diabetes, Alzheimer's disease, and hepatocellular carcinoma, with a special emphasis on the changes in gene expression pattern in these disease conditions. For convenience, the sphingolipid metabolic pathway is divided into hypothetical compartments (modules) with each compartment representing a physiological process and changes in gene expression pattern are mapped to each of these modules. It appears that alterations in the gene expression pattern in these disease conditions are biased to manipulate the system in order to result in a particular disease.
- Chronic glucolipotoxic conditions in pancreatic islets impair insulin secretion due to dysregulated calcium dynamics, glucose responsiveness and mitochondrial activity
BMC Cell Biology 2013, Jul 1
Somesh BP, Verma MK, Sadasivuni MK, Mammen-Oommen A, Biswas S, Shilpa PC, Reddy AK, Yateesh AN, Pallavi PM, Nethra S, Smitha R, Neelima K, Narayanan U, Jagannath MR
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.
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.
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
BMC Pharmacology & Toxicology 2013, May 21
Gowda N, Dandu A, Singh J, Biswas S, Raghav V, Lakshmi MN, Shilpa PC, Sunil V, Reddy A, Sadasivuni M, Aparna K, Verma MK, Moolemath Y, Anup MO, Venkataranganna MV, Somesh BP, Jagannath MR
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.
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.
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.
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.
- An Integrative Network Biology Approach to Evaluate the Role of Endoplasmic Reticulum Stress Response in Obese Type 2 Diabetes
ISRN Cell Biology Volume 2012, Article ID 278636 Anup Mammen Oommen, Usha Narayanan, and M. R. Jagannath
Abstract Extracellular/intracellular stimuli can influence eukaryotic cell function through organelles that regulate critical signaling pathways. The endoplasmic reticulum (ER), for example, impacts cellular processes including protein synthesis, folding and secretion; amino acid transport; apoptosis; cell proliferation; lipid synthesis across major cell types in response to stimuli such as accumulation of misfolded proteins and glucose deprivation. Dysregulated signaling pathways underlying the ER-mediated processes mentioned above have been linked to disease conditions such as diabetes, obesity, and Alzheimer's disease. Our current understanding, however, lacks a detailed network view that integrates organelle-mediated pathway dysregulation with cellular processes and disease pathogenesis. In this report, we introduce an integrative network biology approach that combines ER-stress response pathways with basic cellular processes using data from peer-reviewed literature. As an example, we apply our systems biology approach to study the role of ER stress in pancreatic β cells under obese diabetic conditions, generate testable hypotheses, and provide novel insights into β-cell pathogenesis.