The effect of knockdown of insulin receptor substrates 1 and 2 on glucose and lipid metabolism in human hepatoblastoma cells

Tianfei Yu, Ming Li, Huiying Dong, Pengyu Xie, Wanzhu Sun, Bo Meng, Yanshuang Mu


Paper description:

  • Hepatic insulin signaling mediated by insulin receptor substrates 1 and 2 plays a central role in the development of type 2 diabetes mellitus.
  • Our results show that knockdown of IRS-1 and IRS-2 results in PEPCK upregulation. Decreased IRS-1 was associated with a decrease in glucokinase expression. Knockdown of IRS-2 resulted in upregulation of lipogenic enzymes, SREBP-1c and Cyp7a1. Dual-knockdown of IRS-1 and IRS-2 was associated with defective Akt kinase activation.
  • Our results suggest that hepatic IRS-1 and IRS-2 play divergent roles in gene signaling pathways and have complementary roles hepatic metabolism control.

Abstract: Hepatic insulin signaling mediated by insulin receptor substrates 1 and 2 (IRS-1 and IRS-2, respectively) plays a central role in the development of type 2 diabetes mellitus. Although the functions of individual components in the signaling network have been extensively studied, our knowledge is still limited with regard to how the signals are integrated and coordinated in the complex network to render their functional roles. To investigate the specific functions of IRS-1 and IRS-2 in regulating liver function in humans, we developed a vector-mediated RNA interference (RNAi) technique in which short hairpin RNAs were used to knock down IRS-1, IRS-2, or both, by 50-60%, in cultured human hepatoblastoma (HepG2) cells. The knockdown of IRS-1 and IRS-2 resulted in upregulation of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Decreased IRS-1 was also associated with a decrease in glucokinase (GCK) expression, whereas the knockdown of IRS-2 resulted in the upregulation of lipogenic enzymes, sterol regulatory element-binding protein 1c (SREBP-1c) and cholesterol 7 alpha-hydroxylase (Cyp7a1). Dual-knockdown of IRS-1 and IRS-2 in HepG2 cells was associated with defective GTPase α serine/threonine-protein kinase (Akt) activation. Taken together, our results demonstrate that hepatic IRS-1 and IRS-2 play divergent roles in gene-signaling pathways and have complementary roles in the control of hepatic metabolism.

Received: July 5, 2018; Revised: September 15, 2018; Accepted: September 17, 2018; Published online: September 20, 2018

How to cite this article: Yu T, Li M, Dong H, Xie P, Sun W, Meng B, Mu Y. The effect of knockdown of insulin receptor substrates1 and 2 on glucose and lipid metabolism in human hepatoblastoma cells. Arch Biol Sci. 2018;70(4):757-64.


IRS-1; IRS-2; RNA interference (RNAi); hepatoblastoma cells; glucolipid metabolism

Full Text:



Fisher SJ and Kahn CR. Insulin signaling is required for insulin's direct and indirect action on hepatic glucose production. J Clin Invest. 2003;111:463-8.

Kim SP, Ellmerer M, Van Citters GW,Bergman RN. Primacy of hepatic insulin resistance in the development of the metabolic syndrome induced by an isocaloric moderate-fat diet in the dog. Diabetes. 2003;52:2453-60.

Tripathy D, Eriksson KF, Orho-Melander M, Fredriksson J, Ahlqvist G, Groop L. Parallel manifestation of insulin resistance and beta cell decompensation is compatible with a common defect in Type 2 diabetes. Diabetologia. 2004;47:782-93.

Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell. 2000;6:87-97.

Huang C, Wu M, Du J, Liu D, Chan C. Systematic modeling for the insulin signaling network mediated by IRS(1) and IRS(2). J Theor Biol. 2014;355:40-52.

Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell. 2000;6:77-86.

Patti ME and Kahn CR. The insulin receptor-a critical link in glucose homeostasis and insulin action. J Basic Clin Physiol Pharmacol. 1998;9:89-109.

Withers DJ, Gutierrez JS, Towery H, Burks DJ, Ren JM, Previs S, Zhang Y, Bernal D, Pons S, Shulman GI, Bonner-Weir S, White MF. Disruption of IRS-2 causes type 2 diabetes in mice. Nature. 1998;391:900-4.

Sun XJ, Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden PA, Cahill DA, Goldstein BJ, White MF. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature. 1991;352:73-7.

Anai M, Funaki M, Ogihara T, Terasaki J, Inukai K, Katagiri H, Fukushima Y, Yazaki Y, Kikuchi M, Oka Y, Asano T. Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes. 1998;47:13-23.

Aytug S, Reich D, Sapiro LE, Bernstein D, Begum N. Impaired IRS-1/PI3-kinase signaling in patients with HCV: a mechanism for increased prevalence of type 2 diabetes. Hepatology. 2003;38:1384-92.

Kerouz NJ, Horsch D, Pons S, Kahn CR. Differential regulation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) and phosphatidylinositol 3-kinase isoforms in liver and muscle of the obese diabetic (ob/ob) mouse. J Clin Invest. 1997;100:3164-72.

Kim YB, Peroni OD, Franke TF, Kahn BB. Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats. Diabetes. 2000;49:847-56.

Saad MJ, Araki E, Miralpeix M, Rothenberg PL, White MF, Kahn CR. Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance. J Clin Invest. 1992;90:1839-49.

Gual P, Le Marchand-Brustel Y, Tanti JF. Positive and negative regulation of insulin signaling through IRS-1 phosphorylation. Biochimie. 2005;87:99-109.

Zick Y. Uncoupling insulin signalling by serine/threonine phosphorylation: a molecular basis for insulin resistance. Biochem Soc Trans. 2004;32:812-6.

Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999;96:857-68.

Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, Kitamura Y, Altomonte J, Dong H, Accili D, Spiegelman BM. Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha interaction. Nature. 2003;423:550-5.

Fritsche L, Weigert C, Haring HU, Lehmann R. How insulin receptor substrate proteins regulate the metabolic capacity of the liver--implications for health and disease. Curr Med Chem. 2008;15:1316-29.

Haeusler RA and Accili D. The double life of Irs. Cell Metab. 2008;8:7-9.

Taniguchi CM, Ueki K, Kahn R. Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism. J Clin Invest. 2005;115:718-27.

Araki E, Lipes MA, Patti ME, Bruning JC, Haag B, Johnson RS, Kahn CR. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature. 1994;372:186-90.

Withers DJ, Burks DJ, Towery HH, Altamuro SL, Flint CL, White MF. Irs-2 coordinates Igf-1 receptor-mediated beta-cell development and peripheral insulin signalling. Nat Genet. 1999;23:32-40.

Yamauchi T, Tobe K, Tamemoto H, Ueki K, Kaburagi Y, Yamamoto-Honda R, Takahashi Y, Yoshizawa F, Aizawa S, Akanuma Y, Sonenberg N, Yazaki Y, Kadowaki T. Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice. Mol Cell Biol. 1996;16:3074-84.

Kubota N, Tobe K, Terauchi Y, Eto K, Yamauchi T, Suzuki R, Tsubamoto Y, Komeda K, Nakano R, Miki H, Satoh S, Sekihara H, Sciacchitano S, Lesniak M, Aizawa S, Nagai R, Kimura S, Akanuma Y, Taylor SI, Kadowaki T. Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory beta-cell hyperplasia. Diabetes. 2000;49:1880-9.

Kubota N, Kubota T, Itoh S, Kumagai H, Kozono H, Takamoto I, Mineyama T, Ogata H, Tokuyama K, Ohsugi M, Sasako T, Moroi M, Sugi K, Kakuta S, Iwakura Y, Noda T, Ohnishi S, Nagai R, Tobe K, Terauchi Y, Ueki K, Kadowaki T. Dynamic functional relay between insulin receptor substrate 1 and 2 in hepatic insulin signaling during fasting and feeding. Cell Metab. 2008;8:49-64.

Yang X, Nath A, Opperman MJ, Chan C. The double-stranded RNA-dependent protein kinase differentially regulates insulin receptor substrates 1 and 2 in HepG2 cells. Mol Biol Cell. 2010; 21: 3449-58.

Yang X and Chan C. Repression of PKR mediates palmitate-induced apoptosis in HepG2 cells through regulation of Bcl-2. Cell Res. 2009;19:469-86.

Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, Lindner J, Cherrington AD, Magnuson MA. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem. 1999;274:305-15.

Chen G, Liang G, Ou J, Goldstein JL, Brown MS. Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver. Proc Natl Acad Sci U S A. 2004;101:11245-50.

Shimomura I, Bashmakov Y, Ikemoto S, Horton JD, Brown MS, Goldstein JL. Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes. Proc Natl Acad Sci U S A. 1999;96:13656-61.

Ide T, Shimano H, Yahagi N, Matsuzaka T, Nakakuki M, Yamamoto T, Nakagawa Y, Takahashi A, Suzuki H, Sone H, Toyoshima H, Fukamizu A, Yamada N. SREBPs suppress IRS-2-mediated insulin signalling in the liver. Nat Cell Biol. 2004;6:351-7.

Tobe K, Suzuki R, Aoyama M, Yamauchi T, Kamon J, Kubota N, Terauchi Y, Matsui J, Akanuma Y, Kimura S, Tanaka J, Abe M, Ohsumi J, Nagai R, Kadowaki T. Increased expression of the sterol regulatory element-binding protein-1 gene in insulin receptor substrate-2(-/-) mouse liver. J Biol Chem. 2001;276:38337-40.

Hers I, Vincent EE, Tavare JM. Akt signalling in health and disease. Cell Signal. 2011;23:1515-27.

Stiles B, Wang Y, Stahl A, Bassilian S, Lee WP, Kim YJ, Sherwin R, Devaskar S, Lesche R, Magnuson MA, Wu H. Liver-specific deletion of negative regulator Pten results in fatty liver and insulin hypersensitivity [corrected]. Proc Natl Acad Sci U S A. 2004;101:2082-7.

Du K, Herzig S, Kulkarni RN, Montminy M. TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science. 2003;300:1574-7.

Valverde AM, Burks DJ, Fabregat I, Fisher TL, Carretero J, White MF, Benito M. Molecular mechanisms of insulin resistance in IRS-2-deficient hepatocytes. Diabetes. 2003;52:2239-48.


  • There are currently no refbacks.


Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.