, Volume 36, Issue 1, pp 60–69 | Cite as

Resistin induces insulin resistance by both AMPK-dependent and AMPK-independent mechanisms in HepG2 cells

  • Zhaofan Luo
  • Ying Zhang
  • Fangping Li
  • Juan He
  • Helin Ding
  • Li Yan
  • Hua Cheng
Original Article


Resistin is a 12.5-KDa cysteine-rich peptide that has been implicated in the impairment of glucose homeostasis via the AMP-activated protein kinase (AMPK) pathway in a rodent model. However, the role resistin plays in humans is controversial. This study investigated the effect of resistin on glucose metabolism and insulin signaling using human recombinant resistin and small interfering RNA (siRNA) against AMPKα2 to treat the human liver HepG2 cells. The mRNA of key genes involved in glucose metabolism and the insulin-signaling pathway were detected by real-time RT-PCR. Phosphorylation levels of Akt and AMPK were measured by western blot. The incorporation of D-[U–14C] glucose into glycogen was quantitated by liquid scintillation counting. The results demonstrate that resistin stimulated expressions of glucose-6-phosphatase (G6Pase), phosphoenolypyruvate carboxykinase (PEPCK), and suppressor of cytokine signaling 3 (SOCS-3), repressed the expressions of insulin receptor substrate 2(IRS-2) and glucose transporter 2(GLUT2). In addition, resistin inhibited the insulin-induced phosphorylation of Akt independent of AMPK. In conclusion, our findings suggest that resistin induces insulin resistance in HepG2 cells at least partly via induction of SOCS-3 expression and reduction of Akt phosphorylation through an AMPK-independent mechanism. Resistin also increases glucose production via AMPK-mediated upregulated expression of the genes encoding hepatic gluconeogenic enzymes, G6Pase, and PEPCK.


AMPK Diabetes Insulin Resistin siRNA HepG2 


  1. 1.
    B.B. Kahn, J.S. Flier, Obesity and insulin resistance. J. Clin. Invest. 106, 473–481 (2000)CrossRefPubMedGoogle Scholar
  2. 2.
    J.S. Flier, Obesity wars: molecular progress confronts an expanding epidemic. Cell 116, 337–350 (2004)CrossRefPubMedGoogle Scholar
  3. 3.
    V. Mohamed-Ali, J.H. Pinkney, S.W. Coppack, Adipose tissue as an endocrine and paracrine organ. Int. J. Obes. Relat. Metab. Disord. 22, 1145–1158 (1998)CrossRefPubMedGoogle Scholar
  4. 4.
    P. Trayhurn, J.H. Beattie, Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proc. Nutr. Soc. 60, 329–339 (2001)CrossRefPubMedGoogle Scholar
  5. 5.
    I.N. Holcomb, R.C. Kabakoff, B. Chan, T.W. Baker, A. Gurney, W. Henzel, C. Nelson, H.B. Lowman, B.D. Wright, N.J. Skelton, G.D. Frantz, D.B. Tumas, F.V. Peale Jr., D.L. Shelton, C.C. Hebert, FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family. EMBO J. 19, 4046–4055 (2000)CrossRefPubMedGoogle Scholar
  6. 6.
    K.H. Kim, K. Lee, Y.S. Moon, H.S. Sul, A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J. Biol. Chem. 276, 11252–11256 (2001)CrossRefPubMedGoogle Scholar
  7. 7.
    C.M. Steppan, S.T. Bailey, S. Bhat, E.J. Brown, R.R. Banerjee, C.M. Wright, H.R. Patel, R.S. Ahima, M.A. Lazar, The hormone resistin links obesity to diabetes. Nature 409, 307–312 (2001)CrossRefPubMedGoogle Scholar
  8. 8.
    E.D. Muse, S. Obici, S. Bhanot, B.P. Monia, R.A. McKay, M.W. Rajala, P.E. Scherer, L. Rossetti, Role of resistin in diet-induced hepatic insulin resistance. J. Clin. Invest. 114, 232–239 (2004)PubMedGoogle Scholar
  9. 9.
    R.R. Banerjee, S.M. Rangwala, J.S. Shapiro, A.S. Rich, B. Rhoades, Y. Qi, J. Wang, M.W. Rajala, A. Pocai, P.E. Scherer, C.M. Steppan, R.S. Ahima, S. Obici, L. Rossetti, M.A. Lazar, Regulation of fasted blood glucose by resistin. Science 303, 1195–1198 (2004)CrossRefPubMedGoogle Scholar
  10. 10.
    M. Gawa-Yamauchi, J.E. Bovenkerk, B.E. Juliar, W. Watson, K. Kerr, R. Jones, Q. Zhu, R.V. Considine, Serum resistin (FIZZ3) protein is increased in obese humans. J. Clin. Endocrinol. Metab. 88, 5452–5455 (2003)CrossRefGoogle Scholar
  11. 11.
    S.R. Smith, F. Bai, C. Charbonneau, L. Janderova, G. Argyropoulos, A promoter genotype and oxidative stress potentially link resistin to human insulin resistance. Diabetes 52, 1611–1618 (2003)CrossRefPubMedGoogle Scholar
  12. 12.
    J.H. Lee, J.L. Chan, N. Yiannakouris, M. Kontogianni, E. Estrada, R. Seip, C. Orlova, C.S. Mantzoros, Circulating resistin levels are not associated with obesity or insulin resistance in humans and are not regulated by fasting or leptin administration: cross-sectional and interventional studies in normal, insulin-resistant, and diabetic subjects. J. Clin. Endocrinol. Metab. 88, 4848–4856 (2003)CrossRefPubMedGoogle Scholar
  13. 13.
    L.K. Heilbronn, J. Rood, L. Janderova, J.B. Albu, D.E. Kelley, E. Ravussin, S.R. Smith, Relationship between serum resistin concentrations and insulin resistance in nonobese, obese, and obese diabetic subjects. J. Clin. Endocrinol. Metab. 89, 1844–1848 (2004)CrossRefPubMedGoogle Scholar
  14. 14.
    S. Ghosh, A.K. Singh, B. Aruna, S. Mukhopadhyay, N.Z. Ehtesham, The genomic organization of mouse resistin reveals major differences from the human resistin: functional implications. Gene. 305, 27–34 (2003)CrossRefPubMedGoogle Scholar
  15. 15.
    I. Nagaev, U. Smith, Insulin resistance and type 2 diabetes are not related to resistin expression in human fat cells or skeletal muscle. Biochem. Biophys. Res. Commun. 285, 561–564 (2001)CrossRefPubMedGoogle Scholar
  16. 16.
    J.N. Fain, P.S. Cheema, S.W. Bahouth, H.M. Lloyd, Resistin release by human adipose tissue explants in primary culture. Biochem. Biophys. Res. Commun. 300, 674–678 (2003)CrossRefPubMedGoogle Scholar
  17. 17.
    D.G. Hardie, The AMP-activated protein kinase pathway—new players upstream and downstream. J. Cell Sci. 117, 5479–5487 (2004)CrossRefPubMedGoogle Scholar
  18. 18.
    D. Carling, The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem. Sci. 29, 18–24 (2004)CrossRefPubMedGoogle Scholar
  19. 19.
    B.E. Kemp, D. Stapleton, D.J. Campbell, Z.P. Chen, S. Murthy, M. Walter, A. Gupta, J.J. Adams, F. Katsis, D.B. van, I.G. Jennings, T. Iseli, B.J. Michell, L.A. Witters, AMP-activated protein kinase, super metabolic regulator. Biochem. Soc. Trans. 31, 162–168 (2003)CrossRefPubMedGoogle Scholar
  20. 20.
    D. Stapleton, E. Woollatt, K.I. Mitchelhill, J.K. Nicholl, C.S. Fernandez, B.J. Michell, L.A. Witters, D.A. Power, G.R. Sutherland, B.E. Kemp, AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location. FEBS Lett. 409, 452–456 (1997)CrossRefPubMedGoogle Scholar
  21. 21.
    M.S. Kim, Y.K. Pak, P.G. Jang, C. Namkoong, Y.S. Choi, J.C. Won, K.S. Kim, S.W. Kim, H.S. Kim, J.Y. Park, Y.B. Kim, K.U. Lee, Role of hypothalamic Foxo1 in the regulation of food intake and energy homeostasis. Nat. Neurosci. 9, 901–906 (2006)CrossRefPubMedGoogle Scholar
  22. 22.
    B. Viollet, F. Andreelli, S.B. Jorgensen, C. Perrin, A. Geloen, D. Flamez, J. Mu, C. Lenzner, O. Baud, M. Bennoun, E. Gomas, G. Nicolas, J.F. Wojtaszewski, A. Kahn, D. Carling, F.C. Schuit, M.J. Birnbaum, E.A. Richter, R. Burcelin, S. Vaulont, The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity. J. Clin. Invest. 111, 91–98 (2003)PubMedGoogle Scholar
  23. 23.
    Y.C. Long, J.R. Zierath, AMP-activated protein kinase signaling in metabolic regulation. J. Clin. Invest. 116, 1776–1783 (2006)CrossRefPubMedGoogle Scholar
  24. 24.
    K. Ravnskjaer, M. Boergesen, L.T. Dalgaard, S. Mandrup, Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation. J. Mol. Endocrinol. 36, 289–299 (2006)CrossRefPubMedGoogle Scholar
  25. 25.
    D.R. Alessi, C.P. Downes, The role of PI 3-kinase in insulin action. Biochim. Biophys. Acta 1436, 151–164 (1998)PubMedGoogle Scholar
  26. 26.
    P.J. Coffer, J. Jin, J.R. Woodgett, Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem. J. 335(Pt 1), 1–13 (1998)PubMedGoogle Scholar
  27. 27.
    L.C. Cantley, The phosphoinositide 3-kinase pathway. Science 296, 1655–1657 (2002)CrossRefPubMedGoogle Scholar
  28. 28.
    P. Cohen, D.R. Alessi, D.A. Cross, PDK1, one of the missing links in insulin signal transduction? FEBS Lett. 410, 3–10 (1997)CrossRefPubMedGoogle Scholar
  29. 29.
    S.R. Datta, A. Brunet, M.E. Greenberg, Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927 (1999)CrossRefPubMedGoogle Scholar
  30. 30.
    E.L. Whiteman, H. Cho, M.J. Birnbaum, Role of Akt/protein kinase B in metabolism. Trends Endocrinol. Metab. 13, 444–451 (2002)CrossRefPubMedGoogle Scholar
  31. 31.
    B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream. Cell 129, 1261–1274 (2007)CrossRefPubMedGoogle Scholar
  32. 32.
    P.G. McTernan, F.M. Fisher, G. Valsamakis, R. Chetty, A. Harte, C.L. McTernan, P.M. Clark, S.A. Smith, A.H. Barnett, S. Kumar, Resistin and type 2 diabetes: regulation of resistin expression by insulin and rosiglitazone and the effects of recombinant resistin on lipid and glucose metabolism in human differentiated adipocytes. J. Clin. Endocrinol. Metab. 88, 6098–6106 (2003)CrossRefPubMedGoogle Scholar
  33. 33.
    P.G. McTernan, C.M. Kusminski, S. Kumar, Resistin. Curr. Opin. Lipidol. 17, 170–175 (2006)CrossRefPubMedGoogle Scholar
  34. 34.
    L. Zhou, H. Sell, K. Eckardt, Z. Yang, J. Eckel, Conditioned medium obtained from in vitro differentiated adipocytes and resistin induce insulin resistance in human hepatocytes. FEBS Lett. 581, 4303–4308 (2007)CrossRefPubMedGoogle Scholar
  35. 35.
    C.M. Taniguchi, K. Ueki, R. Kahn, Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism. J. Clin. Invest. 115, 718–727 (2005)PubMedGoogle Scholar
  36. 36.
    K. Nakamaru, K. Matsumoto, T. Taguchi, M. Suefuji, Y. Murata, M. Igata, J. Kawashima, T. Kondo, H. Motoshima, K. Tsuruzoe, N. Miyamura, T. Toyonaga, E. Araki, AICAR, an activator of AMP-activated protein kinase, down-regulates the insulin receptor expression in HepG2 cells. Biochem. Biophys. Res. Commun. 328, 449–454 (2005)CrossRefPubMedGoogle Scholar
  37. 37.
    A. Woods, D. Zzout-Marniche, M. Foretz, S.C. Stein, P. Lemarchand, P. Ferre, F. Foufelle, D. Carling, Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase. Mol. Cell. Biol. 20, 6704–6711 (2000)CrossRefPubMedGoogle Scholar
  38. 38.
    M. Zang, A. Zuccollo, X. Hou, D. Nagata, K. Walsh, H. Herscovitz, P. Brecher, N.B. Ruderman, R.A. Cohen, AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells. J. Biol. Chem. 279, 47898–47905 (2004)CrossRefPubMedGoogle Scholar
  39. 39.
    S.M. Rangwala, A.S. Rich, B. Rhoades, J.S. Shapiro, S. Obici, L. Rossetti, M.A. Lazar, Abnormal glucose homeostasis due to chronic hyperresistinemia. Diabetes 53, 1937–1941 (2004)CrossRefPubMedGoogle Scholar
  40. 40.
    C.M. Steppan, J. Wang, E.L. Whiteman, M.J. Birnbaum, M.A. Lazar, Activation of SOCS-3 by resistin. Mol. Cell. Biol. 25, 1569–1575 (2005)CrossRefPubMedGoogle Scholar
  41. 41.
    L. Patel, A.C. Buckels, I.J. Kinghorn, P.R. Murdock, J.D. Holbrook, C. Plumpton, C.H. Macphee, S.A. Smith, Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators. Biochem. Biophys. Res. Commun. 300, 472–476 (2003)CrossRefPubMedGoogle Scholar
  42. 42.
    N. Silswal, A.K. Singh, B. Aruna, S. Mukhopadhyay, S. Ghosh, N.Z. Ehtesham, Human resistin stimulates the pro-inflammatory cytokines TNF-alpha and IL-12 in macrophages by NF-kappaB-dependent pathway. Biochem. Biophys. Res. Commun. 334, 1092–1101 (2005)CrossRefPubMedGoogle Scholar
  43. 43.
    A. Niederwanger, M. Kranebitter, C. Ciardi, T. Tatarczyk, J.R. Patsch, M.T. Pedrini, Resistin impairs basal and insulin-induced glycogen synthesis by different mechanisms. Mol. Cell. Endocrinol. 263, 112–119 (2007)CrossRefPubMedGoogle Scholar
  44. 44.
    B. Viollet, M. Foretz, B. Guigas, S. Horman, R. Dentin, L. Bertrand, L. Hue, F. Andreelli, Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders. J. Physiol. 574, 41–53 (2006)CrossRefPubMedGoogle Scholar
  45. 45.
    M.C. Towler, D.G. Hardie, AMP-activated protein kinase in metabolic control and insulin signaling. Circ. Res. 100, 328–341 (2007)CrossRefPubMedGoogle Scholar
  46. 46.
    A.Y. Viana, H. Sakoda, M. Anai, M. Fujishiro, H. Ono, A. Kushiyama, Y. Fukushima, Y. Sato, Y. Oshida, Y. Uchijima, H. Kurihara, T. Asano, Role of hepatic AMPK activation in glucose metabolism and dexamethasone-induced regulation of AMPK expression. Diabetes Res. Clin. Pract. 73, 135–142 (2006)CrossRefPubMedGoogle Scholar
  47. 47.
    P.A. Lochhead, I.P. Salt, K.S. Walker, D.G. Hardie, C. Sutherland, 5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase. Diabetes 49, 896–903 (2000)CrossRefPubMedGoogle Scholar
  48. 48.
    J.J. Senn, P.J. Klover, I.A. Nowak, T.A. Zimmers, L.G. Koniaris, R.W. Furlanetto, R.A. Mooney, Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes. J. Biol. Chem. 278, 13740–13746 (2003)CrossRefPubMedGoogle Scholar
  49. 49.
    B. Emanuelli, P. Peraldi, C. Filloux, C. Chavey, K. Freidinger, D.J. Hilton, G.S. Hotamisligil, O.E. Van, SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice. J. Biol. Chem. 276, 47944–47949 (2001)PubMedGoogle Scholar
  50. 50.
    L. Rui, M. Yuan, D. Frantz, S. Shoelson, M.F. White, SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J. Biol. Chem. 277, 42394–42398 (2002)CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Zhaofan Luo
    • 1
  • Ying Zhang
    • 1
  • Fangping Li
    • 1
    • 2
  • Juan He
    • 1
  • Helin Ding
    • 1
  • Li Yan
    • 1
  • Hua Cheng
    • 1
  1. 1.Department of Endocrinology, The Second Affiliated HospitalSun Yat-Sen UniversityGuangzhouPeople’s Republic of China
  2. 2.Endocrinology and Metabolism Department, The Second Affiliated HospitalSun Yat-sen UniversityGuangzhouPeople’s Republic of China

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