Insulin resistance is a major defect underlying the development of type 2 diabetes and is a central component of the metabolic syndrome. It is characterized by complex interactions among genetic determinants, nutritional factors, and lifestyle. Multiple biochemical, metabolic, and signal transduction pathways contribute to insulin resistance. As the incidence of obesity and insulin resistance continues to rise in adolescents and adults at staggering levels, new approaches to tackling this world-wide epidemic are warranted. The overall focus of our laboratory is to explore the new mechanisms involved in induction of insulin resistance, and to identify and develop new molecules to counteract insulin resistance for the treatment of type 2 diabetes and associated secondary complications. We are investigating the role of innate immune components in inflammation-mediated insulin resistance. Simultaneously we are exploring the input of dietary fructose, with specific reference to extra hepatic metabolism, in induction of insulin resistance. At the same time we are involved in the discovery, development and validation of novel antidiabetic agents from natural as well as synthetic sources, using various in vitro and in vivo model systems.

Role of innate immune components in inflammation-induced insulin resistance The innate immune system provides the first line of defense against microbial pathogens and is imperative for the survival of all multicellular organisms. A critical step in the innate immune response is the identification of common motifs from invading organisms as foreign. This discrimination relies on a family of evolutionary conserved pattern recognition receptors (PRRs) that recognize a limited, but highly conserved, set of molecular structures inherent to microbial pathogens, which upon detection mediate inflammatory responses. Emerging evidences indicate that in addition to invading pathogens, PRRs can also be activated by endogenous molecules of non-microbial origin and may also participate in the induction of sterile inflammation. Both infection-induced inflammation and sterile inflammation can lead to chronic inflammation, which is now considered as one of the key etiological conditions leading to the development of many chronic diseases, including atherosclerosis, insulin resistance and cancer. Inflammation-mediated insulin resistance has been linked to macrophage and adipose tissue cross talk, which ultimately may impair insulin action in skeletal muscle and/or the liver leading to whole body insulin resistance. Since, the components of the innate immune system are ubiquitous expressed, a cell autonomous response is possible to external stimuli to induce inflammatory response. We are exploring the role of PRRs in the pathogenesis of tissue specific insulin resistance. We have shown the involvement of cytoplasmic PRRs, the Nucleotide Oligomerization Domain Protein 1/2 (NOD1 and NOD2) in inflammation-induced insulin resistance. Metabolic tissues harbor these components of the innate immune system and their activation though specific ligands confer insulin resistance in skeletal muscle cells in a cell autonomous manner (Endocrinology, 2010). We have identified the involvement of NOD proteins in diet-induced inflammation and insulin intolerance. Mice deficient in NOD1/2 were protected against high fat diet induced inflammation, lipid accumulation and peripheral insulin resistance, and acute activation of NOD proteins caused whole-body insulin resistance (Diabetes, 2011). We have further demonstrated the implication of oxidative stress in the development of mitochondrial dysfunction and insulin resistance in response to NOD2 activation in skeletal muscle cells (Free Redic Biol Med, 2015). Activation of innate immunity provides a new model for the pathogenesis of type-2 diabetes and the metabolic syndrome, which may explain some of these features, and points to research directions that might result in new therapeutic approaches for managing and predicting type-2 diabetes and its complications.

Search towards natural modulator of GLUT4 translocation for the treatment of insulin resistance Insulin resistance is the major defect underlying the development of type 2 diabetes and metabolic syndrome. Insulin resistance is characterized by impaired insulin-stimulated disposal of glucose in skeletal muscle, adipose and other peripheral tissues due to defect in translocation of insulin sensitive glucose transporter-4 (GLUT-4) from intracellular compartment to plasma membrane. There has been considerable interest in insulin-sensitizing agents to counteract insulin resistance and interventions with ability to stimulate GLUT-4 translocation might be useful for the treatment of the disease. Here, we are investigating plant derived molecule with ability to modulate GLUT-4 translocation leading to increased insulin sensitivity. We have proposed a targeted approach to investigate the active chemical molecule/s with ability to counteract insulin resistance from plants and optimize their biological efficacy by chemistry-based approaches and to investigate the underlying molecular mechanism of action and in vivo confirmation of biological efficacy of identified molecule/s.

Molecular cues towards insulin resistance under nutrient modification in skeletal muscle cells As the global incidence of type 2 diabetes and associated metabolic complications continues to increase, the search to identify the dietary components that contribute to this phenomenon is intense and ongoing. Fructose intake from added sugars correlates with the epidemic rise in obesity, type 2 diabetes and metabolic syndrome. Fructose is a highly lipogenic sugar that has profound metabolic effects in the liver and has been associated with the components of the metabolic syndrome. In addition to its well characterized hepatic effects, recent evidence has uncovered the effects of fructose to alter biological pathways in several extra-hepatic tissues including adipose tissue, skeletal muscle, brain, and the gastrointestinal system. These new areas of research have expanded the physiological role of high fructose diets in whole body metabolism. We are exploring the role of fructose in skeletal muscle carbohydrate metabolism. We demonstrated that exposure to fructose induces cell-autonomous oxidative response through ROS production leading to impaired insulin signaling and attenuated glucose utilization in skeletal muscle cells (Free Radic Res, 2015) and these effects of fructose are associated with oxidative stress that decreased mitochondrial DNA content and triggered mitochondrial dysfunction, which caused apoptosis in muscle cells (Apoptosis, 2015). We are interested in understanding the regulation of transport and metabolism of fructose in skeletal muscle, the major depot for carbohydrate utilization.

Discovery and development of novel antidiabetic molecules and natural products validation for the prevention of life style disorders Diabetes mellitus is a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both. Several classes of drugs are currently available for the management of disease; however they have certain limitations, such as adverse side effect and high rates of secondary failure. As such, there are increasing research efforts to identify novel targets and new chemical entities to combat this problem. In this direction, our research team is involved in the discovery, development and validation of new antidiabetic agents from natural as well as synthetic sources. We have established in vitro cell based assay system and various validated in vivo rodent models for the screening, validation and development of lead antidiabetic molecules.

Highlights of recent research

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes. (BBA - Molecular Basis of Disease 2019, 1865:136–146)

Fructose-induced AGEs-RAGE signaling in skeletal muscle contributes to impairment of glucose homeostasis Increased fructose intake has been linked to the development of dyslipidemia, obesity and impaired glucose tolerance. Due to its specific metabolic fate, fructose impairs normal lipid and carbohydrate metabolism and facilitates the non-enzymatic glycation reaction leading to enhanced accumulation of advanced glycation end products (AGEs). However, the formation of fructose-AGEs under in vivo setup and its tissue specific accumulation is less explored. Here, we investigated the impact of high fructose on AGEs accumulation in skeletal muscle and its causal role in impaired glucose homeostasis. In L6 rat skeletal muscle cells, chronic exposure to fructose induced AGEs accumulation and the cellular level of the receptor for AGEs (RAGE) and the effect was prevented by pharmacological inhibition of glycation. Under in vivo settings, Sprague Dawley rats exposed to 20% fructose in drinking water for 16 weeks, displayed increased fasting glycemia, impaired glucose tolerance, decreased skeletal muscle Akt (Ser-473) phosphorylation, and enhanced triglyceride levels in serum, liver and gastrocnemius muscle. We also observed a high level of AGEs in serum and gastrocnemius muscle of fructose-supplemented animals, associated with methylglyoxal accumulation and up regulated expression of RAGE in gastrocnemius muscle. Treatment with aminoguanidine inhibited fructose-induced AGEs accumulation and normalized the expression of RAGE and Dolichyl-Diphosphooligosaccharide-Protein Glycosyltransferase (DDOST) in gastrocnemius muscle. Inhibition of AGEs-RAGE axis counteracted fructose-mediated glucose intolerance without affecting energy metabolism. These data reveal diet-derived AGEs accumulation in skeletal muscle and the implication of tissue specific AGEs in metabolic derangement, that may open new perspectives in pathogenic mechanisms and management of metabolic diseases. (Journal of Nutritional Biochemistry 2019, 71: 35–44)

NOD1 activation induces oxidative stress via NOX1/4 in adipocytes Activation of innate immune components promotes cell autonomous inflammation in adipocytes. Oxidative stress links pattern recognition receptor-mediated detection of inflammatory ligands and the immune response. Reactive oxygen species (ROS) may mediate the effect of nucleotide-binding oligomerization domain protein-1 (NOD1) activation on inflammation in adipocytes. Here, we define the potential role of NADPH oxidase (NOX)-derived ROS in NOD1-mediated inflammatory response in adipocytes. Differentiated 3T3-L1 adipocytes were treated with NOD1 activating ligand D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) to evaluate the oxidative stress and contribution of NOX as source of intracellular ROS. NOD1 activation potently induced ROS generation in 3T3-L1 adipocytes. Of the NOX family members, expression of NOX1 and NOX4 was increased upon NOD1 activation, in a PKCδ-dependent manner. siRNA-mediated down-regulation of NOX1 or NOX4 inhibited NOD1-mediated ROS production and increased the expression of antioxidant defense enzyme catalase and superoxide dismutase (SOD). siRNA-mediated lowering of NOX1 or NOX4 also suppressed NOD1-mediated activation of JNK1/2 and NF-κB, and consequent activation of inflammatory response in 3T3-L1 adipocytes. In summary, our findings demonstrate that NOD1 activation provokes oxidative stress in adipocytes via NOX1/4 and that oxidative stress, at least in part, contributes to induction of inflammatory response. Defining the source of ROS after immune response engagement may lead to new therapeutic strategies for adipose tissue inflammation. (Free Radical Biology and Medicine 2021, 162: 118–128)