Skip to main content
Log in

Vitamin B5 and N-Acetylcysteine in Nonalcoholic Steatohepatitis: A Preclinical Study in a Dietary Mouse Model

  • Original Article
  • Published:
Digestive Diseases and Sciences Aims and scope Submit manuscript

Abstract

Background

Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation.

Objectives

We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH.

Methods

CoA metabolism was evaluated in methionine–choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH.

Results

Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity and correlated with worse liver cell apoptosis, inflammation, and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice.

Conclusion

In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

NAFLD:

Nonalcoholic fatty liver disease

NASH:

Nonalcoholic steatohepatitis

CoA:

Coenzyme A

MCD:

Methionine–choline deficient

PANK:

Pantothenate kinase

WT:

Wild type

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

TBARS:

Thiobarbituric acid-reactive substances

α-SMA:

Alpha-smooth muscle actin

SOD:

Superoxide dismutase

GPX:

Glutathione peroxidase

4-HNE:

4-Hydroxynonenal

TNF-α:

Tumor necrosis factor alpha

References

  1. Loomba R, Sanyal AJ. The global NAFLD epidemic. Nat Rev Gastroenterol Hepatol. 2013;10:686–690.

    Article  PubMed  CAS  Google Scholar 

  2. Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther. 2011;34:274–285.

    Article  PubMed  CAS  Google Scholar 

  3. Angulo P, Bugianesi E, Bjornsson ES, et al. Simple noninvasive systems predict long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology. 2013;145:782 e784–789 e784.

    Article  Google Scholar 

  4. Ekstedt M, Hagstrom H, Nasr P, et al. Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up. Hepatology. 2015;61:1547–1554.

  5. Wong RJ, Aguilar M, Cheung R, et al. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology. 2015;148:547–555.

    Article  PubMed  Google Scholar 

  6. Sanyal AJ, Chalasani N, Kowdley KV, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 2010;362:1675–1685.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Neuschwander-Tetri BA, Loomba R, Sanyal AJ, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet. 2015;385:956–965.

    Article  PubMed  CAS  Google Scholar 

  8. Johnson ES, Lindblom KR, Robeson A, et al. Metabolomic profiling reveals a role for caspase-2 in lipoapoptosis. J Biol Chem. 2013;288:14463–14475.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Machado MV, Michelotti GA, Pereira TD, et al. Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis. Gut. 2015;64:1148–1157.

    Article  PubMed  CAS  Google Scholar 

  10. McCoy F, Darbandi R, Lee HC, et al. Metabolic activation of CaMKII by coenzyme A. Mol Cell. 2013;52:325–339.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Horie S, Isobe M, Suga T. Changes in CoA pools in hepatic peroxisomes of the rat under various conditions. J Biochem. 1986;99:1345–1352.

    PubMed  CAS  Google Scholar 

  12. Leonardi R, Zhang YM, Rock CO, Jackowski S. Coenzyme A: back in action. Prog Lipid Res. 2005;44:125–153.

    Article  PubMed  CAS  Google Scholar 

  13. Robishaw JD, Neely JR. Coenzyme A metabolism. Am J Physiol. 1985;248:E1–E9.

    PubMed  CAS  Google Scholar 

  14. Spry C, Kirk K, Saliba KJ. Coenzyme A biosynthesis: an antimicrobial drug target. FEMS Microbiol Rev. 2008;32:56–106.

    Article  PubMed  CAS  Google Scholar 

  15. Daugherty M, Polanuyer B, Farrell M, et al. Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics. J Biol Chem. 2002;277:21431–21439.

    Article  PubMed  CAS  Google Scholar 

  16. Hodges RE, Ohlson MA, Bean WB. Pantothenic acid deficiency in man. J Clin Invest. 1958;37:1642–1657.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. Wittwer CT, Beck S, Peterson M, Davidson R, Wilson DE, Hansen RG. Mild pantothenate deficiency in rats elevates serum triglyceride and free fatty acid levels. J Nutr. 1990;120:719–725.

    PubMed  CAS  Google Scholar 

  18. Ohsuga S, Ohsuga H, Takeoka T, Ikeda A, Shinohara Y. Metabolic acidosis and hypoglycemia during calcium hopantenate administration—report on 5 patients. Rinsho Shinkeigaku. 1989;29:741–746.

    PubMed  CAS  Google Scholar 

  19. Noda S, Haratake J, Sasaki A, Ishii N, Umezaki H, Horie A. Acute encephalopathy with hepatic steatosis induced by pantothenic acid antagonist, calcium hopantenate, in dogs. Liver. 1991;11:134–142.

    Article  PubMed  CAS  Google Scholar 

  20. Zhang YM, Chohnan S, Virga KG, et al. Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis. Chem Biol. 2007;14:291–302.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Syn WK, Jung Y, Omenetti A, et al. Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease. Gastroenterology. 2009;137:1478 e1478–1488 e1478.

    Article  Google Scholar 

  22. Michelotti GA, Xie G, Swiderska M, et al. Smoothened is a master regulator of adult liver repair. J Clin Invest. 2013;123:2380–2394.

    PubMed  CAS  PubMed Central  Google Scholar 

  23. Machado MV, Diehl AM. Animal models of NAFLD. In: Chalasani N, Szabo G, eds. Alcoholic and nonalcoholic fatty liver disease. Powell: Springer; 2015.

    Google Scholar 

  24. Karasawa T, Yoshida K, Furukawa K, Hosoki K. Feedback inhibition of pantothenate kinase by coenzyme A and possible role of the enzyme for the regulation of cellular coenzyme A level. J Biochem. 1972;71:1065–1067.

    PubMed  CAS  Google Scholar 

  25. Zhang YM, Rock CO, Jackowski S. Feedback regulation of murine pantothenate kinase 3 by coenzyme A and coenzyme A thioesters. J Biol Chem. 2005;280:32594–32601.

    Article  PubMed  CAS  Google Scholar 

  26. Rock CO, Karim MA, Zhang YM, Jackowski S. The murine pantothenate kinase (Pank1) gene encodes two differentially regulated pantothenate kinase isozymes. Gene. 2002;291:35–43.

    Article  PubMed  CAS  Google Scholar 

  27. Machado MV, Michelotti GA, Xie G, et al. Mouse models of diet-induced nonalcoholic steatohepatitis reproduce the heterogeneity of the human disease. PLoS One. 2015;10:e0127991.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Moylan CA, Pang H, Dellinger A, et al. Hepatic gene expression profiles differentiate presymptomatic patients with mild versus severe nonalcoholic fatty liver disease. Hepatology. 2014;59:471–482.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Angulo P, Machado MV, Diehl AM. Fatty liver disease and fibrosis: mechanisms and clinical implications. Semin. Liver Dis. 2015;35:132–145.

  30. Richardson MM, Jonsson JR, Powell EE, et al. Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology. 2007;133:80–90.

    Article  PubMed  Google Scholar 

  31. Ucar F, Sezer S, Erdogan S, Akyol S, Armutcu F, Akyol O. The relationship between oxidative stress and nonalcoholic fatty liver disease: its effects on the development of nonalcoholic steatohepatitis. Redox Rep. 2013;18:127–133.

    Article  PubMed  CAS  Google Scholar 

  32. Alkhouri N, Berk M, Yerian L, et al. OxNASH score correlates with histologic features and severity of nonalcoholic fatty liver disease. Dig Dis Sci. 2014;59:1617–1624.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. Pereira-Filho G, Ferreira C, Schwengber A, Marroni C, Zettler C, Marroni N. Role of N-acetylcysteine on fibrosis and oxidative stress in cirrhotic rats. Arq Gastroenterol. 2008;45:156–162.

    Article  PubMed  Google Scholar 

  34. Thong-Ngam D, Samuhasaneeto S, Kulaputana O, Klaikeaw N. N-acetylcysteine attenuates oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis. World J Gastroenterol. 2007;13:5127–5132.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  35. Choi AM, Alam J. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol. 1996;15:9–19.

    Article  PubMed  CAS  Google Scholar 

  36. Cotter DG, Ercal B, Huang X, et al. Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia. J Clin Invest. 2014;124:5175–5190.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Naruta E, Buko V. Hypolipidemic effect of pantothenic acid derivatives in mice with hypothalamic obesity induced by aurothioglucose. Exp Toxicol Pathol. 2001;53:393–398.

    Article  PubMed  CAS  Google Scholar 

  38. Shibata K, Takahashi C, Fukuwatari T, Sasaki R. Effects of excess pantothenic acid administration on the other water-soluble vitamin metabolisms in rats. J Nutr Sci Vitaminol. 2005;51:385–391.

    Article  PubMed  CAS  Google Scholar 

  39. Wang G, Wang J, Ma H, Ansari GA, Khan MF. N-Acetylcysteine protects against trichloroethene-mediated autoimmunity by attenuating oxidative stress. Toxicol Appl Pharmacol. 2013;273:189–195.

    Article  PubMed  CAS  Google Scholar 

  40. Palekar A. Effect of pantothenic acid on hippurate formation in sodium benzoate-treated HepG2 cells. Pediatr Res. 2000;48:357–359.

    Article  PubMed  CAS  Google Scholar 

  41. Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, Other B Vitamins, and Choline. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academies Press (US); 1998.

  42. Tahiliani AG, Beinlich CJ. Pantothenic acid in health and disease. Vitam Horm. 1991;46:165–228.

    Article  PubMed  CAS  Google Scholar 

  43. Leonardi R, Zhang YM, Lykidis A, Rock CO, Jackowski S. Localization and regulation of mouse pantothenate kinase 2. FEBS Lett. 2007;581:4639–4644.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  44. Leonardi R, Rock CO, Jackowski S, Zhang YM. Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine. Proc Natl Acad Sci USA. 2007;104:1494–1499.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  45. Rommelaere S, Millet V, Gensollen T, et al. PPARalpha regulates the production of serum Vanin-1 by liver. FEBS Lett. 2013;587:3742–3748.

    Article  PubMed  CAS  Google Scholar 

  46. van Diepen JA, Jansen PA, Ballak DB, et al. PPAR-alpha dependent regulation of vanin-1 mediates hepatic lipid metabolism. J Hepatol. 2014;61:366–372.

    Article  PubMed  Google Scholar 

  47. Zhang B, Lo C, Shen L, et al. The role of vanin-1 and oxidative stress-related pathways in distinguishing acute and chronic pediatric ITP. Blood. 2011;117:4569–4579.

    Article  PubMed  CAS  Google Scholar 

  48. Wilson MJ, Jeyasuria P, Parker KL, Koopman P. The transcription factors steroidogenic factor-1 and SOX9 regulate expression of Vanin-1 during mouse testis development. J Biol Chem. 2005;280:5917–5923.

    Article  PubMed  CAS  Google Scholar 

  49. Thurston JH, Hauhart RE. Amelioration of adverse effects of valproic acid on ketogenesis and liver coenzyme A metabolism by cotreatment with pantothenate and carnitine in developing mice: possible clinical significance. Pediatr Res. 1992;31:419–423.

    Article  PubMed  CAS  Google Scholar 

  50. Mitchell GA, Gauthier N, Lesimple A, Wang SP, Mamer O, Qureshi I. Hereditary and acquired diseases of acyl-coenzyme A metabolism. Mol Genet Metab. 2008;94:4–15.

    Article  PubMed  CAS  Google Scholar 

  51. Rana A, Seinen E, Siudeja K, et al. Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration. Proc Natl Acad Sci USA. 2010;107:6988–6993.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  52. van Gijsel-Bonnello M, Acar N, Molino Y, et al. Pantethine alters lipid composition and cholesterol content of membrane rafts, with down-regulation of CXCL12-induced T cell migration. J Cell Physiol. 2015;230:2415–2425.

    Article  PubMed  Google Scholar 

Download references

Financial Support

This research is supported by NIH R01 DK077794-08, R37 AA010154-19 and R56 DK106633-01 (Diehl AM), and Duke Endowment: The Florence McAlister Professorship (Diehl AM). MVM is the recipient of a PhD grant from Fundação para a Ciência e Tecnologia, FCT, Portugal.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Anna Mae Diehl.

Ethics declarations

Conflict of interest

None.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Machado, M.V., Kruger, L., Jewell, M.L. et al. Vitamin B5 and N-Acetylcysteine in Nonalcoholic Steatohepatitis: A Preclinical Study in a Dietary Mouse Model. Dig Dis Sci 61, 137–148 (2016). https://doi.org/10.1007/s10620-015-3871-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10620-015-3871-x

Keywords

Navigation