, Volume 26, Issue 4, pp 1103–1115 | Cite as

Short-term treatment with metformin reduces hepatic lipid accumulation but induces liver inflammation in obese mice

  • Alexandre Abilio de Souza Teixeira
  • Camila O. Souza
  • Luana A. Biondo
  • Loreana Sanches Silveira
  • Edson A. Lima
  • Helena A. Batatinha
  • Adriane Pereira Araujo
  • Michele Joana Alves
  • Sandro Massao Hirabara
  • Rui Curi
  • José Cesar Rosa Neto
Original Article


The study aimed to evaluate the metabolic and inflammatory effects of short-term treatments (10 days) with metformin (MET) on the NAFLD caused by a high-fat diet (HFD) in C57BL/6 mice. After the treatment, histological liver slices were obtained, hepatocytes and macrophages were extracted and cultured with phosphate buffered saline, LPS (2.5 µg/mL) and MET (1 µM) for 24 h. Cytokine levels were determined by ELISA. NAFLD caused by the HFD was partially reduced by MET. The lipid accumulation induced by the HFD was not associated with liver inflammation; however, MET seemed to promote pro-inflammatory effects in liver, since it increased hepatic concentration of IL-1β, TNF-α, IL-6, MCP-1 and IFN-γ. Similarly, MET increased the concentration of IL-1β, IL-6 in hepatocyte cultures. However, in macrophages culture, MET lowered levels of IL-1β, IL-6 and TNF-α stimulated by LPS. Overall, MET reduced liver NAFLD but promoted hepatocyte increase in pro-inflammatory cytokines, thus, leading to liver inflammation.


Obesity Metformin Inflammation Liver 



To the funding sources, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) process number 2013/09367-4, 2015/16777-0 and 2016/01409-8, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this paper.

Supplementary material

10787_2018_443_MOESM1_ESM.docx (123 kb)
Supplementary material 1 (DOCX 122 kb)


  1. Adams LA, Angulo P, Lindor KD (2005) Nonalcoholic fatty liver disease. CMAJ 172:899–905. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bergmeyer H, Bernet E (1974) Determination of glucose with glucoseoxidase and peroxidase. In: Bergmeyer H (ed) Methods of enzymatic analysis. Academic Press, New York, pp 1205–1215Google Scholar
  3. Berlanga A, Guiu-Jurado E, Porras JA, Auguet T (2014) Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol 7:221–239. PubMedPubMedCentralGoogle Scholar
  4. Bi Y et al (2013) The beneficial effect of metformin on beta-cell function in non-obese Chinese subjects with newly diagnosed type 2 diabetes. Diabetes Metab Res Rev 29:664–672. CrossRefPubMedGoogle Scholar
  5. Bogachus LD, Turcotte LP (2010) Genetic downregulation of AMPK-alpha isoforms uncovers the mechanism by which metformin decreases FA uptake and oxidation in skeletal muscle cells. Am J Physiol Cell Physiol 299:C1549–1561. CrossRefPubMedGoogle Scholar
  6. Brestoff JR, Artis D (2015) Immune regulation of metabolic homeostasis in health and disease. Cell 161:146–160. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Casas R, Sacanella E, Estruch R (2014) The immune protective effect of the mediterranean diet against chronic low-grade inflammatory diseases. Endocr Metab Immune Disord Drug Targets 14:245–254CrossRefPubMedGoogle Scholar
  8. Challiss RA, Espinal J, Newsholme EA (1983) Insulin sensitivity of rates of glycolysis and glycogen synthesis in soleus, stripped soleus, epitrochlearis, and hemi-diaphragm muscles isolated from sedentary rats. Biosci Rep 3:675–679CrossRefPubMedGoogle Scholar
  9. Chen M, Zhang J, Liu S, Zhou Z (2015) Effects of metformin on the polarization and Notch 1 expression of RAW264.7 macrophages. Zhonghua Yi Xue Za Zhi 95:1258–1261PubMedGoogle Scholar
  10. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. CrossRefPubMedGoogle Scholar
  11. de Morais H, Cassola P, Moreira CC, Bôas SK, Borba-Murad GR, Bazotte RB, de Souza HM (2012) Decreased response to cAMP in the glucose and glycogen catabolism in perfused livers of Walker-256 tumor-bearing rats. Mol Cell Biochem 368:9–16. CrossRefPubMedGoogle Scholar
  12. Edwards M, Houseman L, Phillips IR, Shephard EA (2013) Isolation of mouse hepatocytes. Methods Mol Biol 987:283–293. CrossRefPubMedGoogle Scholar
  13. Espinal J, Challiss RA, Newsholme EA (1983) Effect of adenosine deaminase and an adenosine analogue on insulin sensitivity in soleus muscle of the rat. FEBS Lett 158:103–106CrossRefPubMedGoogle Scholar
  14. Fader KA et al (2015) 2,3,7,8-Tetrachlorodibenzo-p-Dioxin alters lipid metabolism and depletes immune cell populations in the Jejunum of C57BL/6 Mice. Toxicol Sci 148:567–580. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Flannery C, Dufour S, Rabol R, Shulman GI, Petersen KF (2012) Skeletal muscle insulin resistance promotes increased hepatic de novo lipogenesis, hyperlipidemia, and hepatic steatosis in the elderly. Diabetes 61:2711–2717. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Foretz M, Viollet B (2011) Regulation of hepatic metabolism by AMPK. J Hepatol 54:827–829. CrossRefPubMedGoogle Scholar
  17. Hirabara SM et al (2006) Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle. Biochim Biophys Acta 1757:57–66. CrossRefPubMedGoogle Scholar
  18. Kelly B, Tannahill GM, Murphy MP, O’Neill LA (2015) Metformin inhibits the production of reactive oxygen species from NADH: ubiquinone oxidoreductase to limit induction of interleukin-1beta (IL-1beta) and boosts Interleukin-10 (IL-10) in Lipopolysaccharide (LPS)-activated Macrophages. J Biol Chem 290:20348–20359. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kim J, Kwak HJ, Cha JY, Jeong YS, Rhee SD, Kim KR, Cheon HG (2014) Metformin suppresses lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages via activating transcription factor-3 (ATF-3) induction. J Biol Chem 289:23246–23255. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kitajima Y et al (2013) Severity of non-alcoholic steatohepatitis is associated with substitution of adipose tissue in skeletal muscle. J Gastroenterol Hepatol 28:1507–1514. CrossRefPubMedGoogle Scholar
  21. Kitzmann M, Lantier L, Hebrard S, Mercier J, Foretz M, Aguer C (2011) Abnormal metabolism flexibility in response to high palmitate concentrations in myotubes derived from obese type 2 diabetic patients. Biochim Biophys Acta 1812:423–430. CrossRefPubMedGoogle Scholar
  22. Koh SJ, Kim JM, Kim IK, Ko SH, Kim JS (2014) Anti-inflammatory mechanism of metformin and its effects in intestinal inflammation and colitis-associated colon cancer. J Gastroenterol Hepatol 29:502–510CrossRefPubMedGoogle Scholar
  23. Leighton B, Budohoski L, Lozeman FJ, Challiss RA, Newsholme EA (1985) The effect of prostaglandins E1, E2 and F2 alpha and indomethacin on the sensitivity of glycolysis and glycogen synthesis to insulin in stripped soleus muscles of the rat. Biochem J 227:337–340CrossRefPubMedPubMedCentralGoogle Scholar
  24. Liu C et al (2016) Targeting arginase-II protects mice from high-fat-diet-induced hepatic steatosis through suppression of macrophage inflammation. Sci Rep 6:20405. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. CrossRefPubMedGoogle Scholar
  26. Love-Osborne KA, Nadeau KJ, Sheeder J, Fenton LZ, Zeitler P (2008) Presence of the metabolic syndrome in obese adolescents predicts impaired glucose tolerance and nonalcoholic fatty liver disease. J Adolesc Health 42:543–548. CrossRefPubMedPubMedCentralGoogle Scholar
  27. McCullough AJ (2004) The clinical features, diagnosis and natural history of nonalcoholic fatty liver disease. Clin Liver Dis 8:521–533. CrossRefPubMedGoogle Scholar
  28. McNelis JC, Olefsky JM (2014) Macrophages, immunity, and metabolic disease. Immunity 41:36–48. CrossRefPubMedGoogle Scholar
  29. Mikami Y et al (2014) Macrophages and dendritic cells emerge in the liver during intestinal inflammation and predispose the liver to inflammation. PLoS ONE 9:e84619. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Moon JS, Yoon JS, Won KC, Lee HW (2013) The role of skeletal muscle in development of nonalcoholic Fatty liver disease. Diabetes Metab J 37:278–285. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nasri H, Rafieian-Kopaei M (2014) Metformin: current knowledge. J Res Med Sci 19:658–664PubMedPubMedCentralGoogle Scholar
  32. Nati M, Haddad D, Birkenfeld AL, Koch CA, Chavakis T, Chatzigeorgiou A (2016) The role of immune cells in metabolism-related liver inflammation and development of non-alcoholic steatohepatitis (NASH). Rev Endocr Metab Disord. PubMedGoogle Scholar
  33. Patane G, Piro S, Rabuazzo AM, Anello M, Vigneri R, Purrello F (2000) Metformin restores insulin secretion altered by chronic exposure to free fatty acids or high glucose: a direct metformin effect on pancreatic beta-cells. Diabetes 49:735–740CrossRefPubMedGoogle Scholar
  34. Reeves PG, Nielsen FH, Fahey GC Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123:1939–1951CrossRefPubMedGoogle Scholar
  35. Said A, Akhter A (2017) Meta-analysis of randomized controlled trials of pharmacologic agents in non-alcoholic steatohepatitis. Ann Hepatol 16:538–547. CrossRefPubMedGoogle Scholar
  36. Saisho Y (2015) Metformin and inflammation: its potential beyond glucose-lowering effect. Endocr Metab Immune Disord Drug Targets 15:196–205CrossRefPubMedGoogle Scholar
  37. Salminen A, Hyttinen JM, Kaarniranta K (2011) AMP-activated protein kinase inhibits NF-kappaB signaling and inflammation: impact on healthspan and lifespan. J Mol Med (Berl) 89:667–676. CrossRefGoogle Scholar
  38. Souza-Mello V, Gregorio BM, Cardoso-de-Lemos FS, de Carvalho L, Aguila MB, Mandarim-de-Lacerda CA (2010) Comparative effects of telmisartan, sitagliptin and metformin alone or in combination on obesity, insulin resistance, and liver and pancreas remodelling in C57BL/6 mice fed on a very high-fat diet. Clin Sci (Lond) 119:239–250. CrossRefGoogle Scholar
  39. Spruss A, Kanuri G, Stahl C, Bischoff SC, Bergheim I (2012) Metformin protects against the development of fructose-induced steatosis in mice: role of the intestinal barrier function. Lab Invest 92:1020–1032. CrossRefPubMedGoogle Scholar
  40. Teixeira AA et al (2016) Aerobic exercise modulates the free fatty acids and inflammatory response during obesity and cancer cachexia. Crit Rev Eukaryot Gene Expr 26:187–198. CrossRefPubMedGoogle Scholar
  41. Tiniakos DG, Vos MB, Brunt EM (2010) Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol 5:145–171. CrossRefPubMedGoogle Scholar
  42. VanSaun MN, Lee IK, Washington MK, Matrisian L, Gorden DL (2009) High fat diet induced hepatic steatosis establishes a permissive microenvironment for colorectal metastases and promotes primary dysplasia in a murine model. Am J Pathol 175(1):355–364CrossRefPubMedPubMedCentralGoogle Scholar
  43. Vasamsetti SB, Karnewar S, Kanugula AK, Thatipalli AR, Kumar JM, Kotamraju S (2015) Metformin inhibits monocyte-to-macrophage differentiation via AMPK-mediated inhibition of STAT3 activation: potential role in atherosclerosis. Diabetes 64:2028–2041. CrossRefPubMedGoogle Scholar
  44. Viollet B et al (2009) Targeting the AMPK pathway for the treatment of Type 2 diabetes. Front Biosci (Landmark Ed) 14:3380–3400CrossRefGoogle Scholar
  45. Wan X, Xu C, Yu C, Li Y (2016) Role of NLRP3 inflammasome in the progression of NAFLD to NASH. Can J Gastroenterol Hepatol 2016:6489012. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Wang C et al (2014) Metformin suppresses lipid accumulation in skeletal muscle by promoting fatty acid oxidation. Clin Lab 60:887–896PubMedGoogle Scholar
  47. Woo SL et al (2014) Metformin ameliorates hepatic steatosis and inflammation without altering adipose phenotype in diet-induced obesity. PLoS ONE 9:e91111. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Xu L, Kitade H, Ni Y, Ota T (2015a) Roles of chemokines and chemokine receptors in obesity-associated insulin resistance and nonalcoholic fatty liver disease. Biomolecules 5:1563–1579. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Xu W et al (2015b) Metformin ameliorates the proinflammatory state in patients with carotid artery atherosclerosis through sirtuin 1 induction. Transl Res 166:451–458. CrossRefPubMedGoogle Scholar
  50. Yadav UC, Ramana KV (2013) Regulation of NF-kappaB-induced inflammatory signaling by lipid peroxidation-derived aldehydes. Oxid Med Cell Longev 2013:690545. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Alexandre Abilio de Souza Teixeira
    • 1
  • Camila O. Souza
    • 1
  • Luana A. Biondo
    • 1
  • Loreana Sanches Silveira
    • 2
  • Edson A. Lima
    • 1
  • Helena A. Batatinha
    • 1
  • Adriane Pereira Araujo
    • 1
  • Michele Joana Alves
    • 1
  • Sandro Massao Hirabara
    • 4
  • Rui Curi
    • 3
    • 4
  • José Cesar Rosa Neto
    • 1
  1. 1.Immunometabolism Research Group, Department of Cell Biology and Development, Institute of Biomedical SciencesUniversity of São PauloSão PauloBrazil
  2. 2.Exercise and Immunometabolism Research Group, Department of Physical EducationUNESPPresidente PrudenteBrazil
  3. 3.Department of Physiology and Biophysics, Institute of Biomedical SciencesUniversity of São PauloSão PauloBrazil
  4. 4.Cruzeiro do Sul UniversitySão PauloBrazil

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