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Activation of Insulin-PI3K/Akt-p70S6K Pathway in Hepatic Stellate Cells Contributes to Fibrosis in Nonalcoholic Steatohepatitis

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Abstract

Background and Aims

Hyperinsulinemia and insulin resistance are hallmark features of nonalcoholic fatty liver disease and steatohepatitis (NASH). It remains unclear whether and how insulin contributes to the development of fibrosis in NASH. In this study, we explored insulin signaling in the regulation of hepatic stellate cell (HSC) activation and the progression of NASH-fibrosis.

Methods

Phosphorylation of Akt and p70S6K were examined in primary HSC and in a rat model of NASH-fibrosis induced by high-fat and high-cholesterol diet for 24 weeks. HSC activation was analyzed for the changes in cell morphology, intracellular lipid droplets, expression of α-SMA and cell proliferation. The serum markers and histology for NASH-fibrosis were also characterized in animals.

Results

Insulin enhanced the expression of smooth muscle actin-α in quiescent but not in activated HSC in culture. Insulin-mediated activation of the PI3K/Akt-p70S6K pathway was involved in the regulation of profibrogenic effects of insulin. Although insulin did not stimulate HSC proliferation directly, the insulin-PI3K/Akt-p70S6K pathway was necessary for serum-enhanced cell proliferation during initial HSC activation. In a rat model of NASH-fibrosis induced by high-fat and high-cholesterol diet, hyperinsulinemia is associated with the activation of p70S6K and enhanced fibrosis.

Conclusion

The insulin-PI3K/Akt-p70S6K pathway plays an important role in the early activation of HSC. The profibrogenic effect of insulin is dependent on the activation stage of HSC. Dysregulation of the insulin pathway likely correlates with the development of fibrosis in NASH, suggesting a potentially novel antifibrotic target of inhibiting insulin signaling in HSC.

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Abbreviations

NAFLD:

Nonalcoholic fatty liver disease

NASH:

Nonalcoholic steatohepatitis

HSC:

Hepatic stellate cells

ECM:

Extracellular matrix

α-SMA:

Smooth muscle actin-α

NAS:

NAFLD activity score

PI3K:

Phosphatidylinositol 3-kinase

TGFβ:

Transforming growth factor β

PDGF:

Platelet-derived growth factor

Akt:

Protein kinase B

MAPK:

Mitogen-activated protein kinase

ERK:

Extracellular signal-regulated kinase

IR:

Insulin receptor

IRS:

Insulin receptor substrate

mTOR:

Mammalian target of rapamycin

HFC diet:

High-fat and high-cholesterol diet

DMEM:

Dulbecco’s modified Eagle’s medium

FCS:

Fetal calf serum

PBS:

Phosphate-buffered saline

DMSO:

Dimethyl sulfoxide

ECL:

Enhanced chemiluminescence

H&E:

Hematoxylin and eosin

BrdU:

5-Bromo-2′-deoxyuridine

qPCR:

Quantitative polymerase chain reaction

Col1A1:

α1 chain of type 1 collagen

PPIA:

Peptidylprolyl isomerase A

SEM:

Standard error of mean

References

  1. Harrison SA, Neuschwander-Tetri BA. Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Clin Liver Dis. 2004;8:861–879. (ix).

    Article  PubMed  Google Scholar 

  2. Neuschwander-Tetri BA. Fatty liver and the metabolic syndrome. Curr Opin Gastroenterol. 2007;23:193–198.

    Article  CAS  PubMed  Google Scholar 

  3. Brunt EM, Neuschwander-Tetri BA, Oliver D, Wehmeier KR, Bacon BR. Nonalcoholic steatohepatitis: histologic features and clinical correlations with 30 blinded biopsy specimens. Hum Pathol. 2004;35:1070–1082.

    Article  CAS  PubMed  Google Scholar 

  4. Mehal WZ, Schuppan D. Antifibrotic therapies in the liver. Semin Liver Dis. 2015;35:184–198.

    Article  CAS  PubMed  Google Scholar 

  5. Wang P, Koyama Y, Liu X, et al. Promising therapy candidates for liver fibrosis. Front Physiol. 2016;7:47.

    PubMed  PubMed Central  Google Scholar 

  6. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–218.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev. 2008;88:125–172.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest. 2007;117:539–548.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Semin Liver Dis. 2001;21:397–416.

    Article  CAS  PubMed  Google Scholar 

  10. Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-beta in hepatic fibrosis. Front Biosci J Virtual Libr. 2002;7:d793–d807.

    Article  CAS  Google Scholar 

  11. Yoshida K, Matsuzaki K. Differential regulation of TGF-beta/Smad signaling in hepatic stellate cells between acute and chronic liver injuries. Front Physiol. 2012;3:53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wong L, Yamasaki G, Johnson RJ, Friedman SL. Induction of beta-platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture. J Clin Invest. 1994;94:1563–1569.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Britton RS, Bacon BR. Intracellular signaling pathways in stellate cell activation. Alcohol Clin Exp Res. 1999;23:922–925.

    Article  CAS  PubMed  Google Scholar 

  14. Bridle KR, Li L, O’Neill R, Britton RS, Bacon BR. Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells. J Lab Clin Med. 2006;147:234–241.

    Article  CAS  PubMed  Google Scholar 

  15. Liu C, Li J, Xiang X, et al. PDGF receptor-alpha promotes TGF-beta signaling in hepatic stellate cells via transcriptional and posttranscriptional regulation of TGF-beta receptors. Am J Physiol Gastrointest Liver Physiol. 2014;307:G749–G759.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. van der Poorten D, George J. Disease-specific mechanisms of fibrosis: hepatitis C virus and nonalcoholic steatohepatitis. Clin Liver Dis. 2008;12:805–824. (ix).

    Article  PubMed  Google Scholar 

  17. Rombouts K, Marra F. Molecular mechanisms of hepatic fibrosis in non-alcoholic steatohepatitis. Dig Dis. 2010;28:229–235.

    Article  PubMed  Google Scholar 

  18. Bian Z, Ma X. Liver fibrogenesis in non-alcoholic steatohepatitis. Front Physiol. 2012;3:248.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Angulo P, Machado MV, Diehl AM. Fibrosis in nonalcoholic fatty liver disease: mechanisms and clinical implications. Semin Liver Dis. 2015;35:132–145.

    Article  CAS  PubMed  Google Scholar 

  20. Tomita K, Teratani T, Suzuki T, et al. Free cholesterol accumulation in hepatic stellate cells: mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice. Hepatology. 2014;59:154–169.

    Article  CAS  PubMed  Google Scholar 

  21. Wallace MC, Friedman SL, Mann DA. Emerging and disease-specific mechanisms of hepatic stellate cell activation. Semin Liver Dis. 2015;35:107–118.

    Article  CAS  PubMed  Google Scholar 

  22. Leclercq IA, Da Silva Morais A, Schroyen B, Van Hul N, Geerts A. Insulin resistance in hepatocytes and sinusoidal liver cells: mechanisms and consequences. J Hepatol. 2007;47:142–156.

    Article  CAS  PubMed  Google Scholar 

  23. Utzschneider KM, Kahn SE. Review: the role of insulin resistance in nonalcoholic fatty liver disease. J Clin Endocrinol Metab. 2006;91:4753–4761.

    Article  CAS  PubMed  Google Scholar 

  24. Fartoux L, Poujol-Robert A, Guechot J, Wendum D, Poupon R, Serfaty L. Insulin resistance is a cause of steatosis and fibrosis progression in chronic hepatitis C. Gut. 2005;54:1003–1008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ota T, Takamura T, Kurita S, et al. Insulin resistance accelerates a dietary rat model of nonalcoholic steatohepatitis. Gastroenterology. 2007;132:282–293.

    Article  CAS  PubMed  Google Scholar 

  26. Bugianesi E, Manzini P, D’Antico S, et al. Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver. Hepatology. 2004;39:179–187.

    Article  CAS  PubMed  Google Scholar 

  27. Svegliati-Baroni G, Ridolfi F, Di Sario A, et al. Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways. Hepatology. 1999;29:1743–1751.

    Article  CAS  PubMed  Google Scholar 

  28. Lin J, Zheng S, Chen A. Curcumin attenuates the effects of insulin on stimulating hepatic stellate cell activation by interrupting insulin signaling and attenuating oxidative stress. Lab Invest. 2009;89:1397–1409.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Paradis V, Perlemuter G, Bonvoust F, et al. High glucose and hyperinsulinemia stimulate connective tissue growth factor expression: a potential mechanism involved in progression to fibrosis in nonalcoholic steatohepatitis. Hepatology. 2001;34:738–744.

    Article  CAS  PubMed  Google Scholar 

  30. Zhang F, Zhang Z, Kong D, et al. Tetramethylpyrazine reduces glucose and insulin-induced activation of hepatic stellate cells by inhibiting insulin receptor-mediated PI3K/AKT and ERK pathways. Mol Cell Endocrinol. 2014;382:197–204.

    Article  CAS  PubMed  Google Scholar 

  31. Yoneda A, Sakai-Sawada K, Niitsu Y, Tamura Y. Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence. Exp Cell Res. 2016;341:8–17.

    Article  CAS  PubMed  Google Scholar 

  32. Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol. 2006;7:85–96.

    Article  CAS  PubMed  Google Scholar 

  33. Gabele E, Reif S, Tsukada S, et al. The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation. J Biol Chem. 2005;280:13374–13382.

    Article  PubMed  Google Scholar 

  34. Farrar CT, DePeralta DK, Day H, et al. 3D molecular MR imaging of liver fibrosis and response to rapamycin therapy in a bile duct ligation rat model. J Hepatol. 2015;63:689–696.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kim YJ, Lee ES, Kim SH, et al. Inhibitory effects of rapamycin on the different stages of hepatic fibrosis. World J Gastroenterol. 2014;20:7452–7460.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Wang W, Yan J, Wang H, et al. Rapamycin ameliorates inflammation and fibrosis in the early phase of cirrhotic portal hypertension in rats through inhibition of mTORC1 but not mTORC2. PloS One. 2014;9:e83908.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Patsenker E, Schneider V, Ledermann M, et al. Potent antifibrotic activity of mTOR inhibitors sirolimus and everolimus but not of cyclosporine A and tacrolimus in experimental liver fibrosis. J Hepatol. 2011;55:388–398.

    Article  CAS  PubMed  Google Scholar 

  38. Bridle KR, Popa C, Morgan ML, et al. Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways. Liver Transpl. 2009;15:1315–1324.

    Article  PubMed  Google Scholar 

  39. Neef M, Ledermann M, Saegesser H, Schneider V, Reichen J. Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis. J Hepatol. 2006;45:786–796.

    Article  CAS  PubMed  Google Scholar 

  40. Biecker E, De Gottardi A, Neef M, et al. Long-term treatment of bile duct-ligated rats with rapamycin (sirolimus) significantly attenuates liver fibrosis: analysis of the underlying mechanisms. J Pharmacol Exp Ther. 2005;313:952–961.

    Article  CAS  PubMed  Google Scholar 

  41. Zhu J, Wu J, Frizell E, et al. Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis. Gastroenterology. 1999;117:1198–1204.

    Article  CAS  PubMed  Google Scholar 

  42. Xu ZJ, Fan JG, Ding XD, Qiao L, Wang GL. Characterization of high-fat, diet-induced, non-alcoholic steatohepatitis with fibrosis in rats. Dig Dis Sci. 2010;55:931–940. doi:10.1007/s10620-009-0815-3.

    Article  CAS  PubMed  Google Scholar 

  43. Wang W, Zhao C, Zhou J, Zhen Z, Wang Y, Shen C. Simvastatin ameliorates liver fibrosis via mediating nitric oxide synthase in rats with non-alcoholic steatohepatitis-related liver fibrosis. PloS One. 2013;8:e76538.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem. 1987;161:207–218.

    Article  CAS  PubMed  Google Scholar 

  45. Tura A, Pacini G, Kautzky-Willer A, Ludvik B, Prager R, Thomaseth K. Basal and dynamic proinsulin-insulin relationship to assess beta-cell function during OGTT in metabolic disorders. Am J Physiol Endocrinol Metab. 2003;285:E155–E162.

    Article  CAS  PubMed  Google Scholar 

  46. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol. 1999;94:2467–2474.

    Article  CAS  PubMed  Google Scholar 

  47. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–1321.

    Article  PubMed  Google Scholar 

  48. Kubrusly MS, Correa-Giannella ML, Bellodi-Privato M, et al. A role for mammalian target of rapamycin (mTOR) pathway in non alcoholic steatohepatitis related-cirrhosis. Histol Histopathol. 2010;25:1123–1131.

    CAS  PubMed  Google Scholar 

  49. Asai A, Chou PM, Bu HF, et al. Dissociation of hepatic insulin resistance from susceptibility of nonalcoholic fatty liver disease induced by a high-fat and high-carbohydrate diet in mice. Am J Physiol Gastrointest Liver Physiol. 2014;306:G496–G504.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Mei S, Yang X, Guo H, et al. A small amount of dietary carbohydrate can promote the HFD-induced insulin resistance to a maximal level. PloS One. 2014;9:e100875.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Ferreira DM, Castro RE, Machado MV, et al. Apoptosis and insulin resistance in liver and peripheral tissues of morbidly obese patients is associated with different stages of non-alcoholic fatty liver disease. Diabetologia. 2011;54:1788–1798.

    Article  CAS  PubMed  Google Scholar 

  52. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129:1261–1274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Adachi M, Osawa Y, Uchinami H, Kitamura T, Accili D, Brenner DA. The forkhead transcription factor FoxO1 regulates proliferation and transdifferentiation of hepatic stellate cells. Gastroenterology. 2007;132:1434–1446.

    Article  CAS  PubMed  Google Scholar 

  54. Xiao J, Ho CT, Liong EC, et al. Epigallocatechin gallate attenuates fibrosis, oxidative stress, and inflammation in non-alcoholic fatty liver disease rat model through TGF/SMAD, PI3K/Akt/FoxO1, and NF-kappa B pathways. Eur J Nutr. 2014;53:187–199.

    Article  CAS  PubMed  Google Scholar 

  55. Valenti L, Rametta R, Dongiovanni P, et al. Increased expression and activity of the transcription factor FOXO1 in nonalcoholic steatohepatitis. Diabetes. 2008;57:1355–1362.

    Article  CAS  PubMed  Google Scholar 

  56. Asgharpour A, Cazanave SC, Pacana T, et al. A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer. J Hepatol. 2016;65:579–588.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Neuschwander-Tetri BA, Clark JM, Bass NM, et al. Clinical, laboratory and histological associations in adults with nonalcoholic fatty liver disease. Hepatology. 2010;52:913–924.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. He L, Gubbins J, Peng Z, et al. Activation of hepatic stellate cell in Pten null liver injury model. Fibrogenesis Tissue Repair. 2016;9:8.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The study was supported by the Institutional Research Fund from VA Loma Linda Healthcare System/Loma Linda Veterans Association for Research and Education, and Saint Louis University Liver Center. The authors thank Drs. Subburaman Mohan, Donna Strong, Hidekazu Tsukamoto, David A. Brenner and Neil Kaplowitz for their helpful discussions on the project. We are grateful to Rosemary O’Neill, Vanita Talkad, Wei Zhao, Lucy Zhao, Wei-fen Hsu and Subhashri Das for technical assistances.

Financial support

VA Loma Linda Healthcare System/Loma Linda Veterans Association for Research and Education, Saint Louis University Liver Center.

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Authors and Affiliations

  1. Division of Gastroenterology and Hepatology, Department of Internal Medicine, VA Loma Linda Healthcare System, Loma Linda University, 11201 Benton Street, Loma Linda, CA, 92357, USA

    Cindy X. Cai & Hema Buddha

  2. Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saint Louis University Liver Center, Saint Louis University School of Medicine, 3635 Vista Avenue at Grand Blvd, St. Louis, MO, 63110, USA

    Cindy X. Cai, Hema Buddha, Robert S. Britton, Bruce R. Bacon & Brent A. Neuschwander-Tetri

  3. Department of Pathology, VA Loma Linda Healthcare System, Loma Linda University, 11201 Benton Street, Loma Linda, CA, 92357, USA

    Shobha Castelino-Prabhu

  4. Division of Nephrology, Department of Medicine, VA Loma Linda Healthcare System, Loma Linda University, 11201 Benton Street, Loma Linda, CA, 92357, USA

    Zhiwei Zhang

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  1. Cindy X. Cai
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Correspondence to Cindy X. Cai.

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Cai, C.X., Buddha, H., Castelino-Prabhu, S. et al. Activation of Insulin-PI3K/Akt-p70S6K Pathway in Hepatic Stellate Cells Contributes to Fibrosis in Nonalcoholic Steatohepatitis. Dig Dis Sci 62, 968–978 (2017). https://doi.org/10.1007/s10620-017-4470-9

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