Identification of LRP16 as a Negative Regulator of Insulin Action and Adipogenesis in 3T3-L1 Adipocytes

Abstract

Leukemia related protein 16 (LRP16) was first cloned from acute myeloid leukemia cells in our laboratory. In the present study, we sought to investigate the role of LRP16 in insulin action and sensitivity, using LRP16-depleted and -overexpressing 3T3-L1 cells. LRP16 silencing resulted in a reduction of the expression and secretion of tumor necrosis factor-alpha (TNF-α) and a concomitant increase in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ). Moreover, LRP16 depletion promoted insulin-induced glucose uptake and adipocyte differentiation of 3T3-L1 cells. In contrast, LRP16 overexpression increased TNF-α secretion, suppressed glucose uptake, and attenuated 3T3-L1 cell differentiation. The phosphorylation levels of insulin receptor substrate 1 (IRS-1), phosphatidylinositide 3-kinase (PI3-K), and Akt were increased in LRP16-deficient 3T3-L1 cells, and conversely, diminished in LRP16-overexpressing 3T3-L1 cells, when compared to the corresponding control cells. Additionally, LRP16 overexpression raised the phosphorylation level of mammalian target of rapamycin (mTOR). The pretreatment with rapamycin, a specific inhibitor of mTOR, prevented the TNF-α elevation and PPAR-γ reduction and restored the phosphorylation of IRS-1, PI3-K, and Akt in LRP16-overexpressing cells. Our data collectively indicate that LRP16 acts as a negative regulator of insulin action and adipogenesis in 3T3-L1 adipocytes, which involves the activation of the mTOR signaling pathway.

Key words

leukemia related protein 16 (LRP16) - insulin receptor substrate 1 (IRS-1) - tumor necrosis factor-alpha (TNF-α) - peroxisome proliferator-activated receptor-gamma (PPAR-γ) - mammalian target of rapamycin (mTOR)

*

^* Both authors contributed equally to this work.

• References

• 1 Jeffery RW, Sherwood NE. Is the obesity epidemic exaggerated?. No MMJ 2008; 336: 245
• 2 Stumvoll M, Goldstein BJ, van Haeften TW. Type 2 diabetes: pathogenesis and treatment. Lancet 2008; 371: 2153-2156
• 3 Moller DE, Flier JS. Insulin resistance–mechanisms, syndromes, and implications. N Engl J Med 1991; 325: 938-948
• 4 Nishiumi S, Yoshida M, Azuma T, Yoshida K, Ashida H. 2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs an insulin signaling pathway through the induction of tumor necrosis factor-alpha in adipocytes. Toxicol Sci 2010; 115: 482-491
• 5 Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 1994; 14: 4902-4911
• 6 Shepherd PR, Navé BT, Siddle K. Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3-L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase. Biochem J 1995; 305: 25-28
• 7 Lazar DF, Wiese RJ, Brady MJ, Mastick CC, Waters SB, Yamauchi K, Pessin JE, Cuatrecasas P, Saltiel AR. Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin. J Biol Chem 1995; 270: 20801-20807
• 8 Sutherland C, Waltner-Law M, Gnudi L, Kahn BB, Granner DK. Activation of the Ras mitogen-activated protein kinase-ribosomal protein kinase pathway is not required for the repression of phosphoenolpyruvate carboxykinase gene transcription by insulin. J Biol Chem 1998; 273: 3198-3204
• 9 Ishiki M, Klip A. Minireview: recent developments in the regulation of glucose transporter-4 traffic: new signals, locations, and partners. Endocrinology 2005; 146: 5071-5078
• 10 Goossens GH. The role of adipose tissue dysfunction in the pathogenesis of obesity-related insulin resistance. Physiol Behav 2008; 94: 206-218
• 11 Kim JH, Bachmann RA, Chen J. Interleukin-6 and insulin resistance. Vitam Horm 2009; 80: 613-633
• 12 Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993; 259: 87-91
• 13 Jager J, Grémeaux T, Cormont M, Le Marchand-Brustel Y, Tanti JF. Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression. Endocrinology 2007; 148: 241-251
• 14 Han WD, Yu L, Lou FD, Wang QS, Zhao Y, Shi ZJ, Jin HJ. The Application of RACE Technique to Clone the Full-Length cDNA of A Novel Leukemia Associated Gene LRP16. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2001; 9: 18-21
• 15 Wu Z, Li Y, Li X, Ti D, Zhao Y, Si Y, Mei Q, Zhao P, Fu X, Han W. LRP16 integrates into NF-κB transcriptional complex and is required for its functional activation. PLoS One 2011; 6: e18157
• 16 Imagama S, Abe A, Suzuki M, Hayakawa F, Katsumi A, Emi N, Kiyoi H, Naoe T. LRP16 is fused to RUNX1 in monocytic leukemia cell line with t(11;21)(q13;q22). Eur J Haematol 2007; 79: 25-31
• 17 Yang B, Lu XC, Chi XH, Han WD, Yu L, Lou FD. Promotive effect of LRP16 gene on proliferation of K562 cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2009; 17: 1154-1158
• 18 Han WD, Si YL, Zhao YL, Li Q, Wu ZQ, Hao HJ, Song HJ. GC-rich promoter elements maximally confers estrogen-induced transactivation of LRP16 gene through ERalpha/Sp1 interaction in MCF-7 cells. J Steroid Biochem Mol Biol 2008; 109: 47-56
• 19 Yang J, Zhao YL, Wu ZQ, Si YL, Meng YG, Fu XB, Mu YM, Han WD. The single-macro domain protein LRP16 is an essential cofactor of androgen receptor. Endocr Relat Cancer 2009; 16: 139-153
• 20 Geer EB, Shen W. Gender differences in insulin resistance, body composition, and energy balance. Gend Med 2009; 6 (Suppl. 01) 60-75
• 21 Zang L, Mu YM, Lü ZH, Xue B, Ma XL, Yang GQ, Ba JM, Dou JT, Lu JM. LRP16 gene causes insulin resistance in C2-C12 cells by inhibiting the IRS-1 signaling and the transcriptional activity of peroxisome proliferator actived receptor γ. Zhonghua Yi Xue Za Zhi 2011; 91: 1408-1412
• 22 Han WD, Zhao YL, Meng YG, Zang L, Wu ZQ, Li Q, Si YL, Huang K, Ba JM, Morinaga H, Nomura M, Mu YM. Estrogenically regulated LRP16 interacts with estrogen receptor alpha and enhances the receptor’s transcriptional activity. Endocr Relat Cancer 2007; 14: 741-753
• 23 Han WD, Li Q, Zhao YL, Mu YM, Li X. Inhibition of Cell Proliferation by Small Interference RNA Against LRP16 Gene in Human Breast Cancer MCF-7 Cells. Chin J Biochem Mol Biol 2005; 23: 113-119
• 24 Muraki K, Okuya S, Tanizawa Y. Estrogen receptor alpha regulates insulin sensitivity through IRS-1 tyrosine phosphorylation in mature 3T3-L1 adipocytes. Endocr J 2006; 53: 841-851
• 25 Luo J, Field SJ, Lee JY, Engelman JA, Cantley LC. The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex. J Cell Biol 2005; 170: 455-464
• 26 Koshy S, Alizadeh P, Timchenko LT, Beeton C. Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry. J Vis Exp. 2010: e2429
• 27 Kim JE, Chen J. Regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 2004; 53: 2748-2756
• 28 Bloch-Damti A, Potashnik R, Gual P, Le Marchand-Brustel Y, Tanti JF, Rudich A, Bashan N.. Differential effects of IRS1 phosphorylated on Ser307 or Ser632 in the induction of insulin resistance by oxidative stress. Diabetologia 2006; 49: 2463-2473
• 29 Myers Jr MG. Backer JM, Sun XJ, Shoelson S, Hu P, Schlessinger J, Yoakim M, Schaffhausen B, White MF. IRS-1 activates phosphatidylinositol 3′-kinase by associating with src homology 2 domains of p85. Proc Natl Acad Sci USA 1992; 89: 10350-10354
• 30 Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB. NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999; 401: 82-85
• 31 Ozes ON, Akca H, Mayo LD, Gustin JA, Maehama T, Dixon JE, Donner DB. A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1. Proc Natl Acad Sci USA 2001; 98: 4640-4645
• 32 Kanai F, Ito K, Todaka M, Hayashi H, Kamohara S, Ishii K, Okada T, Hazeki O, Ui M, Ebina Y. Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase. Biochem Biophys Res Commun 1993; 195: 762-768
• 33 Khamzina L, Veilleux A, Bergeron S, Marette A. Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of obese rats: possible involvement in obesity-linked insulin resistance. Endocrinology 2005; 146: 1473-1481
• 34 Trembley F, Gagnon A, Veilleux A, Sorisky A, Marette A. Activation of the mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes. Endocrinology 2005; 146: 1328-1337
• 35 Scazzocchio B, Varì R, D’Archivio M, Santangelo C, Filesi C, Giovannini C, Masella R. Oxidized LDL impair adipocyte response to insulin by activating serine/threonine kinases. J Lipid Res 2009; 50: 832-845
• 36 Bulun SE, Yang S, Fang Z, Gurates B, Tamura M, Zhou J, Sebastian S. Role of aromatase in endometrial disease. J Steroid Biochem Mol Biol 2001; 79: 19-25
• 37 Van Pelt RE, Gozansky WS, Hickner RC, Schwartz RS, Kohrt WM. Acute modulation of adipose tissue lipolysis by intravenous estrogens. Obesity (Silver Spring) 2006; 14: 2163-2172
• 38 Stephens JM, Lee J, Pilch PF. Tumor necrosis factor-alpha-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: 971-976
• 39 Chen XL, Xia ZF, Ben DF, Duo W. mTOR partly mediates insulin resistance by phosphorylation of insulin receptor substrate-1 on serine(307) residues after burn. Burns 2011; 37: 86-93
• 40 Schoonjans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res 1996; 37: 907-925
• 41 Spiegelman BM. PPAR-γ: adipogenic regulator and thiazolinedione receptor. Diabetes 1998; 47: 507-514
• 42 Zhang B, Berger J, Hu E, Szalkowski D, White-Carrington S, Spiegelman BM, Moller DE. Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha. Mol Endocrinol 1996; 10: 1457-1466
• 43 Cho HJ, Park J, Lee HW, Lee YS, Kim JB. Regulation of adipocyte differentiation and insulin action with rapamycin. Biochem Biophys Res Commun 2004; 321: 942-948
• 44 El-Chaar D, Gagnon A, Sorisky A. Inhibition of insulin signaling and adipogenesis by rapamycin: effect on phosphorylation of p70 S6 kinase vs eIF4E-BP1. Int J Obes Relat Metab Disord 2004; 28: 191-198
• 45 Yu J, Henske EP. Estrogen-induced activation of mammalian target of rapamycin is mediated via tuberin and the small GTPase Ras homologue enriched in brain. Cancer Res 2006; 66: 9461-9466
• 46 Sabio G, Davis RJ. cJun NH2-terminal kinase 1 (JNK1): roles in metabolic regulation of insulin resistance. Trends Biochem Sci 2010; 35: 490-496