Reversal of TNF-alpha induced insulin resistance in adipocytes: Inhibition of stress activated protein kinase (p-SAPK/JNK) using purified natural compounds

Epidemiological and experimental studies have shown that obesity-induced insulin resistance is a major factor in the etiology of type II diabetes mellitus. The inflammatory molecule TNF-alpha expressed in adipose tissues has been found to be a potent regulator for the induction of insulin resistance in both cultured cells and animal models. TNF-alpha activates c-Jun NH2-terminal kinase (JNK) and the activation of SAPK/JNK (Stress activates protein kinase) has been shown to increase insulin resistance in both in-vitro and in-vivo diabetic models. Recently, natural compounds, were shown to be beneficial in preventing insulin resistance and useful in reducing the risk of diabetes and metabolic syndrome. The present study involves the screening of over 20 natural compounds. Four of these exhibited positive results in-vitro in reversing TNF-alpha induced insulin resistance in 3T3-L1 adipocytes by blocking the phosphorylation of SAPK/JNK (Stress activates protein kinase). Based on the preliminary studies, 1Ong/m1 of TNF-alpha for 72 hr treatment was used to induce insulin resistance in 3T3-L1 adipocytes. All four natural compounds were screened at three different concentrations - 1microM, 10 microM and 100 microM. It was found that Chlorogenic acid, Naringenin, Catechin hydrate and Kaempferol provided significant protection against TNF-a-induced insulin resistance in 3T3-L1 adipocytes. They all significantly increased insulin stimulated glucose uptake in 72 hr TNF-alpha pretreated adipocytes compared to TNF-alpha pretreated group. All the potential compounds showed no cytotoxic effects at any tested concentrations using MTT (3-[4,5-dimethylthiazol-2-y1]-2,5 diphenyl tetrazolium bromide) assay.;All in-vitro tested potential compounds were further evaluated in-vivo using Zucker diabetic rats, an insulin resistance model. These studies found that Catechin hydrate (50mg/kg) and kaempferol (50mg/kg) significantly reduced insulin resistance by lowering fasting blood glucose level, plasma insulin level, and plasma TNF-alpha level.;Chlorogenic acid and Naringenin in these studies did not show significant in-vivo results. In addition, western blot analysis confirmed that Catechin hydrate and kaempferol significantly blocked the expression of p-SAPK (Phospho - Stress activated protein kinase). Moreover, the Catechin hydrate and kaempferol treated groups significantly reduced GLUT-4 content in cytoplasm which further support the reduction of insulin resistance by these compounds. Based on our results, it can be concluded that Catechin hydrate and kaempferol significantly reversed insulin resistance both in-vitro and in-vivo and may be considered as potential therapeutic agents to increase insulin sensitivity in type II diabetes mellitus.