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Oral citrulline supplementation protects female mice from the development of non-alcoholic fatty liver disease (NAFLD)

Abstract

Purpose

Impairments of intestinal barrier function are discussed as risk factors for the development and progression of non-alcoholic fatty liver disease (NAFLD). Studies suggest an association between arginine/citrulline homeostasis and the development of liver damages. Here, the effect of an oral l-citrulline (Cit) supplement on the development of a Western-style diet (WSD)-induced NAFLD was determined in mice.

Methods

Female 6- to 8-week-old C57BL/6J mice were either pair-fed a liquid Western-style or control diet (C) ± 2.5 g/kg bodyweight Cit for 6 weeks (C + Cit or WSD + Cit). Indices of liver damage, glucose metabolism, intestinal barrier function and NO synthesis were measured.

Results

While bodyweight gain was similar between groups, markers of glucose metabolism like fasting blood glucose and HOMA index and markers of liver damage like hepatic triglyceride levels, number of neutrophils and plasminogen activator inhibitor-1 protein levels were significantly lower in WSD + Cit-fed mice when compared to WSD-fed mice only. Protein levels of the tight junction proteins occludin and zonula occludens-1 in duodenum were significantly lower in mice fed a WSD when compared to those fed a WSD + Cit (−~70 and −~60 %, respectively, P < 0.05), whereas portal endotoxin levels, concentration of 3-nitrotyrosine protein adducts in duodenum and toll-like receptor-4 mRNA expression in livers of WSD + Cit-fed mice were markedly lower than in WSD-fed mice (−~43 %, P = 0.056; −~80 and −~48 %, respectively, P < 0.05).

Conclusion

Our data suggest that the protective effects of supplementing Cit on the development of NAFLD in mice are associated with a decreased translocation of endotoxin into the portal vein.

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Abbreviations

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

C:

Control

Cit:

l-Citrulline

HOMA index:

Homeostasis model assessment index

iNOS:

Inducible nitric oxide synthase

MYD88:

Myeloid differentiation primary response gene 88

NAFLD:

Non-alcoholic fatty liver disease

NAS:

Non-alcoholic fatty liver disease activity score

NASH:

Non-alcoholic steatohepatitis

PAI-1:

Plasminogen activator inhibitor 1

TG:

Triglycerides

Tlr:

Toll-like receptor

TNFα:

Tumor necrosis factor alpha

WSD:

Western-style diet

ZO-1:

Zonula occludens 1

3-NT:

3-Nitrotyrosine

4-HNE:

4-Hydroxynonenal protein adducts

References

  1. Sass DA, Chang P, Chopra KB (2005) Nonalcoholic fatty liver disease: a clinical review. Dig Dis Sci 50:171–180

    Article  Google Scholar 

  2. Bedogni G, Miglioli L, Masutti F, Tiribelli C, Marchesini G, Bellentani S (2005) Prevalence of and risk factors for nonalcoholic fatty liver disease: the Dionysos nutrition and liver study. Hepatology 42:44–52

    Article  Google Scholar 

  3. Bellentani S, Scaglioni F, Marino M, Bedogni G (2010) Epidemiology of non-alcoholic fatty liver disease. Dig Dis 28:155–161

    Article  Google Scholar 

  4. Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F (2013) The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol 58:593–608

    Article  Google Scholar 

  5. Hashimoto E, Tokushige K, Ludwig J (2015) Diagnosis and classification of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis: current concepts and remaining challenges. Hepatol Res 45:20–28

    Article  Google Scholar 

  6. Jiang CM, Pu CW, Hou YH, Chen Z, Alanazy M, Hebbard L (2014) Non alcoholic steatohepatitis a precursor for hepatocellular carcinoma development. World J Gastroenterol 20:16464–16473

    CAS  Article  Google Scholar 

  7. Schnabl B, Brenner DA (2014) Interactions between the intestinal microbiome and liver diseases. Gastroenterology 146:1513–1524

    CAS  Article  Google Scholar 

  8. Volynets V, Kuper MA, Strahl S, Maier IB, Spruss A, Wagnerberger S, Konigsrainer A, Bischoff SC, Bergheim I (2012) Nutrition, intestinal permeability, and blood ethanol levels are altered in patients with nonalcoholic fatty liver disease (NAFLD). Dig Dis Sci 57:1932–1941

    CAS  Article  Google Scholar 

  9. 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

    CAS  Article  Google Scholar 

  10. Breuillard C, Cynober L, Moinard C (2015) Citrulline and nitrogen homeostasis: an overview. Amino Acids 47:685–691

    CAS  Article  Google Scholar 

  11. van de Poll MC, Ligthart-Melis GC, Boelens PG, Deutz NE, van Leeuwen PA, Dejong CH (2007) Intestinal and hepatic metabolism of glutamine and citrulline in humans. J Physiol 581:819–827

    Article  Google Scholar 

  12. Wijnands KA, Castermans TM, Hommen MP, Meesters DM, Poeze M (2015) Arginine and citrulline and the immune response in sepsis. Nutrients 7:1426–1463

    CAS  Article  Google Scholar 

  13. Akashi K, Miyake C, Yokota A (2001) Citrulline, a novel compatible solute in drought-tolerant wild watermelon leaves, is an efficient hydroxyl radical scavenger. FEBS Lett 508:438–442

    CAS  Article  Google Scholar 

  14. Chien SJ, Lin KM, Kuo HC, Huang CF, Lin YJ, Huang LT, Tain YL (2014) Two different approaches to restore renal nitric oxide and prevent hypertension in young spontaneously hypertensive rats: l-citrulline and nitrate. Transl Res 163:43–52

    CAS  Article  Google Scholar 

  15. Wu G, Collins JK, Perkins-Veazie P, Siddiq M, Dolan KD, Kelly KA, Heaps CL, Meininger CJ (2007) Dietary supplementation with watermelon pomace juice enhances arginine availability and ameliorates the metabolic syndrome in Zucker diabetic fatty rats. J Nutr 137:2680–2685

    CAS  Google Scholar 

  16. Antunes MM, Leocadio PC, Teixeira LG, Leonel AJ, Cara DC, Menezes GB, Generoso SV, Cardoso VN, Alvarez-Leite JI, Correia MI (2015) Pretreatment with l-citrulline positively affects the mucosal architecture and permeability of the small intestine in a murine mucositis model. JPEN J Parenter Enteral Nutr 40:279–286

    Article  Google Scholar 

  17. Spruss A, Henkel J, Kanuri G, Blank D, Puschel GP, Bischoff SC, Bergheim I (2012) Female mice are more susceptible to nonalcoholic fatty liver disease: sex-specific regulation of the hepatic AMP-activated protein kinase-plasminogen activator inhibitor 1 cascade, but not the hepatic endotoxin response. Mol Med 18:1346–1355

    CAS  Article  Google Scholar 

  18. Kanuri G, Wagnerberger S, Landmann M, Prigl E, Hellerbrand C, Bischoff SC, Bergheim I (2015) Effect of acute beer ingestion on the liver: studies in female mice. Eur J Nutr 54:465–474

    CAS  Article  Google Scholar 

  19. Sellmann C, Priebs J, Landmann M, Degen C, Engstler AJ, Jin CJ, Garttner S, Spruss A, Huber O, Bergheim I (2015) Diets rich in fructose, fat or fructose and fat alter intestinal barrier function and lead to the development of nonalcoholic fatty liver disease over time. J Nutr Biochem 26:1183–1192

    CAS  Article  Google Scholar 

  20. Jegatheesan P, Beutheu S, Ventura G, Sarfati G, Nubret E, Kapel N, Waligora-Dupriet AJ, Bergheim I, Cynober L, De-Bandt JP (2015) Effect of specific amino acids on hepatic lipid metabolism in fructose-induced non-alcoholic fatty liver disease. Clin Nutr 35:175–182

    Article  Google Scholar 

  21. Jegatheesan P, Beutheu S, Ventura G, Nubret E, Sarfati G, Bergheim I, De Bandt JP (2015) Citrulline and nonessential amino acids prevent fructose-induced nonalcoholic fatty liver disease in rats. J Nutr 145:2273–2279

    CAS  Article  Google Scholar 

  22. Demir M, Lang S, Steffen HM (2015) Nonalcoholic fatty liver disease—current status and future directions. J Dig Dis 16:541–557

    CAS  Article  Google Scholar 

  23. Portillo-Sanchez P, Bril F, Maximos M, Lomonaco R, Biernacki D, Orsak B, Subbarayan S, Webb A, Hecht J, Cusi K (2015) High prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus and normal plasma aminotransferase levels. J Clin Endocrinol Metab 100:2231–2238

    CAS  Article  Google Scholar 

  24. Maximos M, Bril F, Portillo SP, Lomonaco R, Orsak B, Biernacki D, Suman A, Weber M, Cusi K (2015) The role of liver fat and insulin resistance as determinants of plasma aminotransferase elevation in nonalcoholic fatty liver disease. Hepatology 61:153–160

    CAS  Article  Google Scholar 

  25. Singh SP, Misra B, Kar SK, Panigrahi MK, Misra D, Bhuyan P, Pattnaik K, Meher C, Agrawal O, Rout N, Swain M (2015) Nonalcoholic fatty liver disease (NAFLD) without insulin resistance: is it different? Clin Res Hepatol Gastroenterol 39:482–488

    CAS  Article  Google Scholar 

  26. Kirpich IA, Marsano LS, McClain CJ (2015) Gut-liver axis, nutrition, and non-alcoholic fatty liver disease. Clin Biochem 48:923–930

    CAS  Article  Google Scholar 

  27. Abdul-Hai A, Abdallah A, Malnick SD (2015) Influence of gut bacteria on development and progression of non-alcoholic fatty liver disease. World J Hepatol 7:1679–1684

    Article  Google Scholar 

  28. Batista MA, Nicoli JR, Martins FS, Machado JA, Arantes RM, Quirino IE, Correia MI, Cardoso VN (2012) Pretreatment with citrulline improves gut barrier after intestinal obstruction in mice. JPEN J Parenter Enteral Nutr 36:69–76

    CAS  Article  Google Scholar 

  29. Chapman JC, Liu Y, Zhu L, Rhoads JM (2012) Arginine and citrulline protect intestinal cell monolayer tight junctions from hypoxia-induced injury in piglets. Pediatr Res 72:576–582

    CAS  Article  Google Scholar 

  30. Gou L, Zhang L, Yin C, Jia G, Yin X, Zhuang X, Xu X, Liu Y (2011) Protective effect of l-citrulline against acute gastric mucosal lesions induced by ischemia–reperfusion in rats. Can J Physiol Pharmacol 89:317–327

    CAS  Article  Google Scholar 

  31. Lai CH, Lee CH, Hung CY, Lo HC (2015) Oral citrulline mitigates inflammation and jejunal damage via the inactivation of neuronal nitric oxide synthase and nuclear factor-kappaB in intestinal ischemia and reperfusion. JPEN J Parenter Enteral Nutr. doi:10.1177/0148607115590661

    Google Scholar 

  32. Fu X, Li S, Jia G, Gou L, Tian X, Sun L, Ling X, Lan N, Yin X, Ma R, Liu L, Liu Y (2013) Protective effect of the nitric oxide pathway in l-citrulline renal ischaemia–reperfusion injury in rats. Folia Biol (Praha) 59:225–232

    CAS  Google Scholar 

  33. Wijnands KA, Vink H, Briede JJ, van Faassen EE, Lamers WH, Buurman WA, Poeze M (2012) Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia. PLoS One 7:e37439

    CAS  Article  Google Scholar 

  34. Du PJ, Vanheel H, Janssen CE, Roos L, Slavik T, Stivaktas PI, Nieuwoudt M, van Wyk SG, Vieira W, Pretorius E, Beukes M, Farre R, Tack J, Laleman W, Fevery J, Nevens F, Roskams T, Van der Merwe SW (2013) Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function. J Hepatol 58:1125–1132

    Article  Google Scholar 

  35. Emami CN, Petrosyan M, Giuliani S, Williams M, Hunter C, Prasadarao NV, Ford HR (2009) Role of the host defense system and intestinal microbial flora in the pathogenesis of necrotizing enterocolitis. Surg Infect (Larchmt) 10:407–417

    Article  Google Scholar 

  36. Dai ZL, Li XL, Xi PB, Zhang J, Wu G, Zhu WY (2012) Regulatory role for l-arginine in the utilization of amino acids by pig small-intestinal bacteria. Amino Acids 43:233–244

    CAS  Article  Google Scholar 

  37. Kesar V, Odin JA (2014) Toll-like receptors and liver disease. Liver Int 34:184–196

    CAS  Article  Google Scholar 

  38. Kanuri G, Ladurner R, Skibovskaya J, Spruss A, Konigsrainer A, Bischoff SC, Bergheim I (2015) Expression of toll-like receptors 1–5 but not TLR 6–10 is elevated in livers of patients with non-alcoholic fatty liver disease. Liver Int 35:562–568

    CAS  Article  Google Scholar 

  39. Wagnerberger S, Spruss A, Kanuri G, Volynets V, Stahl C, Bischoff SC, Bergheim I (2012) Toll-like receptors 1–9 are elevated in livers with fructose-induced hepatic steatosis. Br J Nutr 107:1727–1738

    CAS  Article  Google Scholar 

  40. Chen BY, Lin DP, Su KC, Chen YL, Wu CY, Teng MC, Tsai YT, Sun CY, Wang SR, Chang HH (2011) Dietary zerumbone prevents against ultraviolet B-induced cataractogenesis in the mouse. Mol Vis 17:723–730

    CAS  Google Scholar 

  41. Locatelli I, Sutti S, Vacchiano M, Bozzola C, Albano E (2013) NF-kappaB1 deficiency stimulates the progression of non-alcoholic steatohepatitis (NASH) in mice by promoting NKT-cell-mediated responses. Clin Sci (Lond) 124:279–287

    CAS  Article  Google Scholar 

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Acknowledgments

The present study was funded by a grant from the German Research Foundation (DFG): BE 2376/6-1 (I. B.).

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Correspondence to Ina Bergheim.

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Conflict of interest

C. Sellmann, C.J. Jin, A.J. Engstler and I. Bergheim have no conflicts of interest. J-P De Bandt is a shareholder of Citrage company.

Ethical approval

Approval for all experiments was provided by the local Institutional Animal Care and Use Committee (IACUC).

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Sellmann, C., Jin, C.J., Engstler, A.J. et al. Oral citrulline supplementation protects female mice from the development of non-alcoholic fatty liver disease (NAFLD). Eur J Nutr 56, 2519–2527 (2017). https://doi.org/10.1007/s00394-016-1287-9

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  • DOI: https://doi.org/10.1007/s00394-016-1287-9

Keywords

  • Citrulline
  • Intestinal barrier function
  • Endotoxin
  • Occludin
  • Non-alcoholic fatty liver disease