| dc.description.abstract | Obesity has become a growing epidemic globally and a burden to the health care system due to its association or contribution to the development of other chronic diseases. The excessive accumulation of fat in adipocytes or expansion of the adipocytes results in dysfunctional adipocytes which leads to the expression and secretion of cytokines, specifically pro-inflammatory cytokines such tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) which cause inflammation. Moreover, chronic inflammation has been linked with the development of insulin resistance which is a hallmark for type 2 diabetes. Rutin is a naturally occurring flavonoid found in various plants species with known therapeutic effects such as antioxidants, anti-diabetic and anti-inflammatory. This study aims to investigate whether rutin can ameliorate TNF-α-induced insulin resistance and inflammation in 3T3-L1 adipocytes.
In this study, 3T3-L1 preadipocytes were cultured, differentiated into mature adipocytes using cocktail media containing 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and rosiglitazone. Fully differentiated adipocytes were cultured in DMEM with or without 10 ng/mL TNF-α for 24h under tissue culture conditions. Thereafter, 3T3-L1 adipocytes were cultured with 1 μM insulin (added 15 minutes prior to termination), while 1 μM CL 316,243, and 10 μM rutin were kept for 6h. Cell viability was assessed using MTT assay. Lipid accumulation and lipolysis were evaluated using Oil Red O and glycerol release assay kit, respectively. Inflammation was evaluated using ELISA. Quantitative real-time polymerase chain reaction (qPCR) and Western blot were used to investigate mRNA and protein expression associated with inflammation, lipid metabolism, fat browning, and glucose metabolism.
MTT assay showed that apart from insulin which improved cell viability, culturing cells with TNF-α, CL 316,243, and rutin had no significant effect after 6h of treatment. A significant decrease in lipid accumulation by isoproterenol and TNF-α, and an increase by insulin, CL 316,243, and rutin after culturing with TNF-α for 24h was observed. There was an increase in lipolysis by CL 316,243, isoproterenol and TNF-α. Interestingly, all the treatments were able to supress TNF-α mediated lipolysis and rutin was most significant. TNF-α induced insulin resistance and inflammation after 24h, this was evident as pro-inflammatory markers TNF-α and IL-6 were increased and AKT phosphorylation was reduced. Also, positive controls insulin and CL 316,243, and rutin were able to abolish TNF-α- induced insulin resistance and inflammation. Furthermore, results showed that TNF-α affected glucose metabolism by blocking AKT phosphorylation. However, insulin and rutin reversed this effect on AKT. Subsequently, to elucidate the mechanism by which rutin could exert its beneficial effect, we investigated gene and protein markers associated with fat browning. Our results showed that fat browning markers PR-domain
containing 16 (PRDM16) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) were decreased by TNF-α. All treatments were able to upregulate PGC1-α protein expression and only insulin and rutin restore Prdm16 expression. In terms of lipid metabolism, culturing 3T3-L1 adipocytes with Tnf-α greatly reduced peroxisome proliferator-activated receptor (Pparγ), and carnitine palmitoyl transferase I (Cpt1) gene expression. Treating with insulin, CL 316,243, and rutin reversed the inhibition of Pparγ. Only CL316,243 and rutin were able to upregulate Cpt1 gene expression. Lastly, rutin improved mitochondrial energy expenditure comparable to thepositive control CL 316,243 by increasing ATP production and mitochondrial spare capacity. Results demonstrated that rutin is a potential therapeutic compound for the treatment of TNF-α-induced inflammation and insulin resistance through inhibiting the expression of inflammatory cytokines and activating the insulin mediated insulin signaling pathway by activating AKT. Moreover, we observed that rutin upregulated Prdm16 and PGC1-α which are markers found in brown fat, suggesting rutin induces white adipocyte browning and protected against TNF-α mitochondrial dysfunction. This could be a possible mechanism through which rutin ameliorates obesity-induced inflammation and associated disorders. Additional experiments, especially in vivo are required to confirm the role of AMPK in rutin response against inflammation. | en_US |
