Abstract
Background: Elevation of plasma free fatty acids as a principal aspect of type 2 diabetes maintains etiologically insulin insensitivity in target cells. TNF-α inhibitory effects on key insulin signaling pathway elements remain to be verified in insulinresistant hepatic cells. Thus, TNF-α knockdown effects on the key elements of insulin signaling were investigated in the palmitate-induced insulin-resistant hepatocytes. The Akt serine kinase, a key protein of the insulin signaling pathway, phosphorylation was monitored to understand the TNF-α effect on probable enhancing of insulin resistance.
Methods: Insulin-resistant HepG2 cells were produced using 0.5 mM palmitate treatment and shRNA-mediated TNF-α gene knockdown and its down-regulation confirmed using ELISA technique. Western blotting analysis was used to assess the Akt protein phosphorylation status.
Results: Palmitate-induced insulin resistance caused TNF-α protein overexpression 1.2-, 2.78, and 2.25- fold as compared to the control cells at post-treatment times of 8 h, 16 h, and 24 h, respectively. In the presence of palmitate, TNF-α expression showed around 30% reduction in TNF-α knockdown cells as compared to normal cells. In the TNF-α down-regulated cell, Akt phosphorylation was approximately 62% more than control cells after treatment with 100 nM insulin in conjugation with 0.5 mM palmitate.
Conclusions: The obtained data demonstrated that TNF-α protein expression reduction improved insulin-stimulated Akt phosphorylation in the HepG2 cells and decreased lipidinduced insulin resistance of the diabetic hepatocytes.
Keywords: Insulin resistance, TNF-α, Akt kinase, Palmitate, HepG2 cell, diabetes.
[http://dx.doi.org/10.1016/j.abb.2014.12.009] [PMID: 25527164]
[http://dx.doi.org/10.2337/diabetes.52.3.581] [PMID: 12606496]
[http://dx.doi.org/10.1146/annurev.physiol.68.040104.124723] [PMID: 16460269]
[http://dx.doi.org/10.1210/jc.2003-030723] [PMID: 14764748]
[http://dx.doi.org/10.1146/annurev.biochem.66.1.581] [PMID: 9242918]
[http://dx.doi.org/10.3349/ymj.2005.46.1.42] [PMID: 15744804]
[http://dx.doi.org/10.1177/14746514050050040601]
[http://dx.doi.org/10.1111/j.1742-1241.2006.01128.x] [PMID: 16981971]
[http://dx.doi.org/10.2337/diabetes.54.4.976] [PMID: 15793235]
[http://dx.doi.org/10.1007/s13105-015-0440-x] [PMID: 26452501]
[http://dx.doi.org/10.1016/j.bbrc.2015.09.165] [PMID: 26449452]
[http://dx.doi.org/10.1210/endo.139.12.6336] [PMID: 9832430]
[http://dx.doi.org/10.1126/science.7678183] [PMID: 7678183]
[http://dx.doi.org/10.1172/JCI25151] [PMID: 16284649]
[http://dx.doi.org/10.1074/jbc.M109498200] [PMID: 11694522]
[http://dx.doi.org/10.2337/diabetes.54.10.2939] [PMID: 16186396]
[http://dx.doi.org/10.1042/bj3190179] [PMID: 8870666]
[http://dx.doi.org/10.1128/MCB.22.4.1016-1026.2002] [PMID: 11809794]
[http://dx.doi.org/10.1172/JCI6928] [PMID: 10491408]
[http://dx.doi.org/10.2337/diabetes.49.2.284] [PMID: 10868945]
[http://dx.doi.org/10.2337/diabetes.52.4.935] [PMID: 12663464]
[http://dx.doi.org/10.1007/s00125-007-0913-8] [PMID: 18204829]
[http://dx.doi.org/10.2337/db08-1539] [PMID: 19188436]
[http://dx.doi.org/10.1093/jn/135.8.1841] [PMID: 16046706]
[http://dx.doi.org/10.1016/j.atherosclerosis.2008.05.033] [PMID: 18599066]
[http://dx.doi.org/10.1210/en.2005-0440] [PMID: 16223857]
[http://dx.doi.org/10.1172/JCI117495] [PMID: 7523453]
[http://dx.doi.org/10.1016/j.bbrc.2015.03.137] [PMID: 25839650]
[http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051]
[http://dx.doi.org/10.1210/jc.84.1.272] [PMID: 9920095]
[http://dx.doi.org/10.1172/JCI117936] [PMID: 7738205]
[http://dx.doi.org/10.1073/pnas.91.11.4854] [PMID: 8197147]
[http://dx.doi.org/10.1074/jbc.272.2.971] [PMID: 8995390]
[http://dx.doi.org/10.1172/JCI118504] [PMID: 8613535]
[http://dx.doi.org/10.1046/j.1432-1327.1999.00809.x] [PMID: 10542046]
[http://dx.doi.org/10.1074/jbc.M212307200] [PMID: 12525490]
[http://dx.doi.org/10.1016/j.abb.2003.08.020] [PMID: 14592453]
[http://dx.doi.org/10.1074/jbc.274.34.24202] [PMID: 10446195]
[http://dx.doi.org/10.1007/s11745-010-3394-3] [PMID: 20177806]
[http://dx.doi.org/10.1016/j.freeradbiomed.2013.09.019] [PMID: 24120971]
[http://dx.doi.org/10.1016/j.diabres.2011.03.036] [PMID: 21514684]
[http://dx.doi.org/10.1152/ajpendo.00429.2001] [PMID: 12067837]
[http://dx.doi.org/10.1053/meta.2002.35202] [PMID: 12404200]
[http://dx.doi.org/10.1152/ajpendo.00033.2003] [PMID: 12676648]
[http://dx.doi.org/10.1016/j.cmet.2007.08.014] [PMID: 17908559]
[http://dx.doi.org/10.1016/j.bbrc.2010.04.118] [PMID: 20417620]
[http://dx.doi.org/10.1172/JCI115686] [PMID: 1556176]
[http://dx.doi.org/10.1074/jbc.M007231200] [PMID: 11063744]
[http://dx.doi.org/10.1074/jbc.M007281200] [PMID: 11278339]
[http://dx.doi.org/10.2337/db06-S002]
[http://dx.doi.org/10.1074/jbc.272.40.25267] [PMID: 9312143]
[http://dx.doi.org/10.1074/jbc.273.2.940] [PMID: 9422753]
[http://dx.doi.org/10.1038/39335] [PMID: 9335502]
[http://dx.doi.org/10.1016/j.cmet.2011.01.018] [PMID: 21356519]