Generic placeholder image

Current Molecular Medicine

Editor-in-Chief

ISSN (Print): 1566-5240
ISSN (Online): 1875-5666

Research Article

The Key Role of Akt Protein Kinase in Metabolic-Inflammatory Pathways Cross-Talk: TNF-α Down-Regulation and Improving of Insulin Resistance in HepG2 Cell Line

Author(s): Iraj Alipourfard, Salar Bakhtiyari*, Ali Gheysarzadeh, Laura Di Renzo, Antonio De Lorenzo, David Mikeladze and Atefeh Khamoushi

Volume 21, Issue 3, 2021

Published on: 27 April, 2020

Page: [257 - 264] Pages: 8

DOI: 10.2174/1566524020666200427102209

Price: $65

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.

« Previous
[1]
Ishii M, Maeda A, Tani S, Akagawa M. Palmitate induces insulin resistance in human HepG2 hepatocytes by enhancing ubiquitination and proteasomal degradation of key insulin signaling molecules. Arch Biochem Biophys 2015; 566: 26-35.
[http://dx.doi.org/10.1016/j.abb.2014.12.009] [PMID: 25527164]
[2]
Robertson RP, Harmon J, Tran PO, Tanaka Y, Takahashi H. Glucose toxicity in β-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection. Diabetes 2003; 52(3): 581-7.
[http://dx.doi.org/10.2337/diabetes.52.3.581] [PMID: 12606496]
[3]
Biddinger SB, Kahn CR. From mice to men: insights into the insulin resistance syndromes. Annu Rev Physiol 2006; 68: 123-58.
[http://dx.doi.org/10.1146/annurev.physiol.68.040104.124723] [PMID: 16460269]
[4]
Bays H, Mandarino L, DeFronzo RA. Role of the adipocyte, free fatty acids, and ectopic fat in pathogenesis of type 2 diabetes mellitus: peroxisomal proliferator-activated receptor agonists provide a rational therapeutic approach. J Clin Endocrinol Metab 2004; 89(2): 463-78.
[http://dx.doi.org/10.1210/jc.2003-030723] [PMID: 14764748]
[5]
Hanson RW, Reshef L. Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu Rev Biochem 1997; 66(1): 581-611.
[http://dx.doi.org/10.1146/annurev.biochem.66.1.581] [PMID: 9242918]
[6]
Kim M-J, Yoo KH, Park HS, et al. Plasma adiponectin and insulin resistance in Korean type 2 diabetes mellitus. Yonsei Med J 2005; 46(1): 42-50.
[http://dx.doi.org/10.3349/ymj.2005.46.1.42] [PMID: 15744804]
[7]
Campbell IW. Pioglitazone-an oral antidiabetic agent and metabolic syndrome modulator. Can theory translate into practice? Br J Diabetes Vasc Dis 2005; 5(4): 209-16.
[http://dx.doi.org/10.1177/14746514050050040601]
[8]
Wilding J. Thiazolidinediones, insulin resistance and obesity: Finding a balance. Int J Clin Pract 2006; 60(10): 1272-80.
[http://dx.doi.org/10.1111/j.1742-1241.2006.01128.x] [PMID: 16981971]
[9]
Shao J, Qiao L, Janssen RC, Pagliassotti M, Friedman JE. Chronic hyperglycemia enhances PEPCK gene expression and hepatocellular glucose production via elevated liver activating protein/liver inhibitory protein ratio. Diabetes 2005; 54(4): 976-84.
[http://dx.doi.org/10.2337/diabetes.54.4.976] [PMID: 15793235]
[10]
Wang S, Zhao Y, Xia N, et al. KPNβ1 promotes palmitate-induced insulin resistance via NF-κB signaling in hepatocytes. J Physiol Biochem 2015; 71(4): 763-72.
[http://dx.doi.org/10.1007/s13105-015-0440-x] [PMID: 26452501]
[11]
Zhang W, Tang Z, Zhu X, et al. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway. Biochem Biophys Res Commun 2015; 467(3): 527-33.
[http://dx.doi.org/10.1016/j.bbrc.2015.09.165] [PMID: 26449452]
[12]
Cheung AT, Ree D, Kolls JK, Fuselier J, Coy DH, Bryer-Ash M. An in vivo model for elucidation of the mechanism of tumor necrosis factor-α (TNF-α)-induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-α. Endocrinology 1998; 139(12): 4928-35.
[http://dx.doi.org/10.1210/endo.139.12.6336] [PMID: 9832430]
[13]
Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993; 259(5091): 87-91.
[http://dx.doi.org/10.1126/science.7678183] [PMID: 7678183]
[14]
Morino K, Petersen KF, Dufour S, et al. Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. J Clin Invest 2005; 115(12): 3587-93.
[http://dx.doi.org/10.1172/JCI25151] [PMID: 16284649]
[15]
Rydén M, Dicker A, van Harmelen V, et al. Mapping of early signaling events in tumor necrosis factor-α -mediated lipolysis in human fat cells. J Biol Chem 2002; 277(2): 1085-91.
[http://dx.doi.org/10.1074/jbc.M109498200] [PMID: 11694522]
[16]
Yang X, Xiadong Z, Bradlee LH. Relative contribution of adipose triglyceride lipase and hormone-sensitive lipase to TNF-α-induced lipolysis in adipocytes. J Biol Chem 2011; M1(11): 257-923.
[17]
Plomgaard P, Bouzakri K, Krogh-Madsen R, Mittendorfer B, Zierath JR, Pedersen BK. Tumor necrosis factor-α induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes 2005; 54(10): 2939-45.
[http://dx.doi.org/10.2337/diabetes.54.10.2939] [PMID: 16186396]
[18]
Long SD, Pekala PH. Lipid mediators of insulin resistance: ceramide signalling down-regulates GLUT4 gene transcription in 3T3-L1 adipocytes. Biochem J 1996; 319(Pt 1): 179-84.
[http://dx.doi.org/10.1042/bj3190179] [PMID: 8870666]
[19]
Zhande R, Mitchell JJ, Wu J, Sun XJ. Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1. Mol Cell Biol 2002; 22(4): 1016-26.
[http://dx.doi.org/10.1128/MCB.22.4.1016-1026.2002] [PMID: 11809794]
[20]
Kim Y-B, Nikoulina SE, Ciaraldi TP, Henry RR, Kahn BB. Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest 1999; 104(6): 733-41.
[http://dx.doi.org/10.1172/JCI6928] [PMID: 10491408]
[21]
Krook A, Björnholm M, Galuska D, et al. Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes 2000; 49(2): 284-92.
[http://dx.doi.org/10.2337/diabetes.49.2.284] [PMID: 10868945]
[22]
Brozinick JT Jr, Roberts BR, Dohm GL. Defective signaling through Akt-2 and -3 but not Akt-1 in insulin-resistant human skeletal muscle: potential role in insulin resistance. Diabetes 2003; 52(4): 935-41.
[http://dx.doi.org/10.2337/diabetes.52.4.935] [PMID: 12663464]
[23]
Cozzone D, Fröjdö S, Disse E, et al. Isoform-specific defects of insulin stimulation of Akt/protein kinase B (PKB) in skeletal muscle cells from type 2 diabetic patients. Diabetologia 2008; 51(3): 512-21.
[http://dx.doi.org/10.1007/s00125-007-0913-8] [PMID: 18204829]
[24]
Karlsson HK, Chibalin AV, Koistinen HA, et al. Kinetics of GLUT4 trafficking in rat and human skeletal muscle. Diabetes 2009; 58(4): 847-54.
[http://dx.doi.org/10.2337/db08-1539] [PMID: 19188436]
[25]
Ajuwon KM, Spurlock ME. Palmitate activates the NF-kappaB transcription factor and induces IL-6 and TNFalpha expression in 3T3-L1 adipocytes. J Nutr 2005; 135(8): 1841-6.
[http://dx.doi.org/10.1093/jn/135.8.1841] [PMID: 16046706]
[26]
Håversen L, Danielsson KN, Fogelstrand L, Wiklund O. Induction of proinflammatory cytokines by long-chain saturated fatty acids in human macrophages. Atherosclerosis 2009; 202(2): 382-93.
[http://dx.doi.org/10.1016/j.atherosclerosis.2008.05.033] [PMID: 18599066]
[27]
Jové M, Planavila A, Sánchez RM, Merlos M, Laguna JC, Vázquez-Carrera M. Palmitate induces tumor necrosis factor-α expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation. Endocrinology 2006; 147(1): 552-61.
[http://dx.doi.org/10.1210/en.2005-0440] [PMID: 16223857]
[28]
Hotamisligil GS, Budavari A, Murray D, Spiegelman BM. Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha. J Clin Invest 1994; 94(4): 1543-9.
[http://dx.doi.org/10.1172/JCI117495] [PMID: 7523453]
[29]
Haghani K, Pashaei S, Vakili S, Taheripak G, Bakhtiyari S. TNF-α knockdown alleviates palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Biochem Biophys Res Commun 2015; 460(4): 977-82.
[http://dx.doi.org/10.1016/j.bbrc.2015.03.137] [PMID: 25839650]
[30]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72(1-2): 248-54.
[http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051]
[31]
Zinman B, Hanley AJ, Harris SB, Kwan J, Fantus IG. Circulating tumor necrosis factor-α concentrations in a native Canadian population with high rates of type 2 diabetes mellitus. J Clin Endocrinol Metab 1999; 84(1): 272-8.
[http://dx.doi.org/10.1210/jc.84.1.272] [PMID: 9920095]
[32]
Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 1995; 95(5): 2409-15.
[http://dx.doi.org/10.1172/JCI117936] [PMID: 7738205]
[33]
Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM. Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 1994; 91(11): 4854-8.
[http://dx.doi.org/10.1073/pnas.91.11.4854] [PMID: 8197147]
[34]
Stephens JM, Lee J, Pilch PF. Tumor necrosis factor-α-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction. J Biol Chem 1997; 272(2): 971-6.
[http://dx.doi.org/10.1074/jbc.272.2.971] [PMID: 8995390]
[35]
Saghizadeh M, Ong JM, Garvey WT, Henry RR, Kern PA. The expression of TNF alpha by human muscle. Relationship to insulin resistance. J Clin Invest 1996; 97(4): 1111-6.
[http://dx.doi.org/10.1172/JCI118504] [PMID: 8613535]
[36]
Storz P, Döppler H, Wernig A, Pfizenmaier K, Müller G. Cross-talk mechanisms in the development of insulin resistance of skeletal muscle cells palmitate rather than tumour necrosis factor inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake. Eur J Biochem 1999; 266(1): 17-25.
[http://dx.doi.org/10.1046/j.1432-1327.1999.00809.x] [PMID: 10542046]
[37]
Chavez JA, Knotts TA, Wang LP, et al. A role for ceramide, but not diacylglycerol, in the antagonism of insulin signal transduction by saturated fatty acids. J Biol Chem 2003; 278(12): 10297-303.
[http://dx.doi.org/10.1074/jbc.M212307200] [PMID: 12525490]
[38]
Chavez JA, Summers SA. Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 2003; 419(2): 101-9.
[http://dx.doi.org/10.1016/j.abb.2003.08.020] [PMID: 14592453]
[39]
Schmitz-Peiffer C, Craig DL, Biden TJ. Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate. J Biol Chem 1999; 274(34): 24202-10.
[http://dx.doi.org/10.1074/jbc.274.34.24202] [PMID: 10446195]
[40]
Bakhtiyari S, Meshkani R, Taghikhani M, Larijani B, Adeli K. Protein tyrosine phosphatase-1B (PTP-1B) knockdown improves palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Lipids 2010; 45(3): 237-44.
[http://dx.doi.org/10.1007/s11745-010-3394-3] [PMID: 20177806]
[41]
Taheripak G, Bakhtiyari S, Rajabibazl M, Pasalar P, Meshkani R. Protein tyrosine phosphatase 1B inhibition ameliorates palmitate-induced mitochondrial dysfunction and apoptosis in skeletal muscle cells. Free Radic Biol Med 2013; 65: 1435-46.
[http://dx.doi.org/10.1016/j.freeradbiomed.2013.09.019] [PMID: 24120971]
[42]
Li Y, Zhao S, Zhang W, et al. Epigallocatechin-3-O-gallate (EGCG) attenuates FFAs-induced peripheral insulin resistance through AMPK pathway and insulin signaling pathway in vivo. Diabetes Res Clin Pract 2011; 93(2): 205-14.
[http://dx.doi.org/10.1016/j.diabres.2011.03.036] [PMID: 21514684]
[43]
Boden G, Cheung P, Stein TP, Kresge K, Mozzoli M. FFA cause hepatic insulin resistance by inhibiting insulin suppression of glycogenolysis. Am J Physiol Endocrinol Metab 2002; 283(1): E12-9.
[http://dx.doi.org/10.1152/ajpendo.00429.2001] [PMID: 12067837]
[44]
Clore JN, Allred J, White D, Li J, Stillman J. The role of plasma fatty acid composition in endogenous glucose production in patients with type 2 diabetes mellitus. Metabolism 2002; 51(11): 1471-7.
[http://dx.doi.org/10.1053/meta.2002.35202] [PMID: 12404200]
[45]
Lam TK, Carpentier A, Lewis GF, van de Werve G, Fantus IG, Giacca A. Mechanisms of the free fatty acid-induced increase in hepatic glucose production. Am J Physiol Endocrinol Metab 2003; 284(5): E863-73.
[http://dx.doi.org/10.1152/ajpendo.00033.2003] [PMID: 12676648]
[46]
Lorenzo M, Fernandez-Veledo S, Vila-Bedmar R. Insulin resistance induced by tumor necrosis factor-α in myocytes and brown adipocytes Insulin resistance induced by tumor necrosis factor-α in myocytes and brown adipocytes. Journal of animal Science 2008; 86(suppl_14): E94-E104.
[47]
Sun C, Zhang F, Ge X, et al. SIRT1 improves insulin sensitivity under insulin-resistant conditions by repressing PTP1B. Cell Metab 2007; 6(4): 307-19.
[http://dx.doi.org/10.1016/j.cmet.2007.08.014] [PMID: 17908559]
[48]
Parvaneh L, Meshkani R, Bakhtiyari S, et al. Palmitate and inflammatory state additively induce the expression of PTP1B in muscle cells. Biochem Biophys Res Commun 2010; 396(2): 467-71.
[http://dx.doi.org/10.1016/j.bbrc.2010.04.118] [PMID: 20417620]
[49]
Rothman DL, Shulman RG, Shulman GI. 31P nuclear magnetic resonance measurements of muscle glucose-6-phosphate. Evidence for reduced insulin-dependent muscle glucose transport or phosphorylation activity in non-insulin-dependent diabetes mellitus. J Clin Invest 1992; 89(4): 1069-75.
[http://dx.doi.org/10.1172/JCI115686] [PMID: 1556176]
[50]
Ravichandran LV, Esposito DL, Chen J, Quon MJ. Protein kinase C-ζ phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin. J Biol Chem 2001; 276(5): 3543-9.
[http://dx.doi.org/10.1074/jbc.M007231200] [PMID: 11063744]
[51]
Liu Y-F, Paz K, Herschkovitz A, et al. Insulin stimulates PKCzeta -mediated phosphorylation of insulin receptor substrate-1 (IRS-1). A self-attenuated mechanism to negatively regulate the function of IRS proteins. J Biol Chem 2001; 276(17): 14459-65.
[http://dx.doi.org/10.1074/jbc.M007281200] [PMID: 11278339]
[52]
Morino K, Petersen KF, Shulman GI. Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction 2006; 55(Suppl 2): S9-S15.
[http://dx.doi.org/10.2337/db06-S002]
[53]
Ogihara T, Isobe T, Ichimura T, et al. 14-3-3 protein binds to insulin receptor substrate-1, one of the binding sites of which is in the phosphotyrosine binding domain. J Biol Chem 1997; 272(40): 25267-74.
[http://dx.doi.org/10.1074/jbc.272.40.25267] [PMID: 9312143]
[54]
Kosaki A, Yamada K, Suga J, Otaka A, Kuzuya H. 14-3-3β protein associates with insulin receptor substrate 1 and decreases insulin-stimulated phosphatidylinositol 3′-kinase activity in 3T3L1 adipocytes. J Biol Chem 1998; 273(2): 940-4.
[http://dx.doi.org/10.1074/jbc.273.2.940] [PMID: 9422753]
[55]
Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS. Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function. Nature 1997; 389(6651): 610-4.
[http://dx.doi.org/10.1038/39335] [PMID: 9335502]
[56]
Kubota T, Kubota N, Kumagai H, et al. Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle. Cell Metab 2011; 13(3): 294-307.
[http://dx.doi.org/10.1016/j.cmet.2011.01.018] [PMID: 21356519]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy