Insulin is a peptide hormone that plays an important role in the regulation of carbohydrate, fat, and protein metabolism (Qaid and Abdelrahman, 2016). The effect of insulin varies depending on the target tissue. In liver, insulin inhibits gluconeogenesis and promotes glycogen synthesis, in muscle cells, it promotes glucose uptake and glycogen synthesis, whereas in adipose tissue, it enhances glucose uptake, lipogenesis and inhibits lipolysis (Petersen and Shulman, 2018a). In liver cells, insulin exerts its physiological action by activating insulin receptor tyrosine kinase, which phosphorylates and recruits different substrate adaptors such as the IRS family of proteins (Hurrle and Hsu, 2017). IRS1 then activates its downstream target Protein kinase B, also known as Akt, which suppresses the hepatic glucose production through two key mechanisms. Firstly, by decreasing the expression of gluconeogenic enzymes by phosphorylation and nuclear exclusion of the forkhead box protein O1 (FOXO1) and secondly by inactivation of glycogen synthase kinase-3 β (GSK3β) which in turn activates glycogen synthase (Huang et al., 2018). This action of insulin is lost when insulin resistance (IR) state develops (Hatting et al., 2018). In IR state, IRS1 gets phosphorylated at Ser-307 instead of Tyr-1222 which leads to its inactivation leading to failure of activation of downstream targets (Gao et al., 2010).

IR is associated with several chronic diseases such as, obesity, dyslipidemia, metabolic syndrome, cardiovascular diseases, nonalcoholic fatty liver disorder (NAFLD), cancer, and neurodegenerative diseases (Ormazabal et al., 2018; Hölscher, 2020). To understand molecular mechanisms involved in development of IR, it can be induced in vitro in different cell types using various agents such as high glucose, excess fatty acids, excess insulin, proinflammatory cytokines like TNF-α, IL-6 etc. (Lee et al., 2017; Holland et al., 2011). Saturated fatty acids such as palmitic acid (PA) and stearic acid in excess, which mimic physiological conditions, have been demonstrated as potent inducers of IR in liver, skeletal muscle cells and adipocytes (Sears and Perry, 2015). Different mechanisms have been proposed to explain PA induce IR. Recent studies have demonstrated that both fatty acid induced ROS and/or tissue accumulation of lipid metabolites namely diacylglycerol (DAG) and ceramides in the liver or skeletal muscle cause IR in humans and animal models (Boden, 2011; Ragheb and Medhat, 2011). Gao, et al. (2010) have demonstrated that the effect of PA on hepatic IR is mediated by NOX3-derived ROS through JNK (NH2-terminal Jun kinases/stress-activated protein kinases) activation. In liver, FFAs are taken up by the fatty acid transporters such as FATP2, FATP5, and CD36. Among theseFATP2 and FATP5 increase fatty acid uptake by conversion of fatty acid to fatty Acyl-CoA (Chabowski et al., 2013), whereas CD36 enhances intracellular esterification of fatty acid, and thereby increases the rate of fatty acid uptake (Xu et al., 2013). Accumulation of esterified lipids metabolites such as DAG and ceramides are shown to activate PKCε which in turn enhances Ser-307 phosphorylation of IRS1 and inhibits its tyrosine kinase activity (Birkenfeld and Shulman, 2014).

Thus, it is well established that both the mechanism namely ROS activated JNK and lipid activated protein kinase C (PKCε) are involved in induction of IR. However, there are few open questions which need to be addressed, such as whether these two mechanisms act alone or in consortium. In addition, each cell type has a threshold for tolerating stress after which it succumbs to IR. It is necessary to understand when this threshold is reached i.e. at which concentration of PA and/or time of exposure cell would develop IR which will help in management of the disease progression. In this study, we report that both the mechanisms are triggered simultaneously and work in consortium to induce an IR. We further show that IR is developed only when HepG2 cells are treated with higher concentration of PA for longer duration whereas same concentrations for shorter duration are well tolerated by the defense mechanism of the cell.