Protective effect of protein hydrolysates from Litopenaeus vannamei waste on oxidative status, glucose regulation, and autophagy genes in non-alcoholic fatty liver disease in Wistar rats

Document Type : Original Article


1 Department of Biology, Faculty of Science, Urmia University, Urmia, Iran

2 Department of Pathobiology and Quality Control, Artemia & Aquaculture Research institute, Urmia University, Urmia, Iran


Objective(s): The effects of protein hydrolysates (FP) from Litopenaeus vannamei on oxidative stress, and autophagy gene expression was investigated in the NAFLD-induced rats.  
Materials and Methods: For this purpose, twenty-four male rats were divided into four groups: Control, High-fat diet (HFD), FP20+HFD, and FP300+HFD (20 and 300 mg FP /kg rat body weight) and fed for 70 days. 
Results: The results indicated that the rat body and relative weight of the liver were not affected by experimental treatments (P>0.05) although the highest relative weight of the liver was observed in HFD treatment. The highest and lowest values for antioxidant enzymes and MDA concentration were observed in FP treatments (P<0.05). Also, the results showed that FP significantly decreased liver enzymes (ALT, AST) in the liver in comparison with HFD treatment (P<0.05). Plasma biochemical indices were investigated and the lowest amylase, ALP, fasting glucose, insulin, HOMA-IR, triglycerides, cholesterol, and inflammation cytokines (TNF-α, IL-6) were seen in the FP treatments which had a significant difference with HFD (P<0.05). Autophagy gene expression in the liver cells was affected by experimental diets and the lowest expression of Beclin-1 and Atg7 was observed in HFD and FP300 treatments. Interestingly, the highest expression of LC3-ɪ and P62 was seen in HFD and FP treatments, not in the control. 
Conclusion: Overall, the results of this experiment indicated that FPs extracted from Whiteleg shrimp at 50 °C improve the oxidative status, glucose metabolism, and autophagy gene expression and could be used as a useful nutritional strategy in fatty liver prevention.


1. Fabbrini E, Sullivan S, Klein S. Obesity and nonalcoholic fatty liver disease: Biochemical, metabolic, and clinical implications. Hepatology 2010; 51: 679-689.
2. Carabelli J, Burgueño AL, Rosselli MS, Gianotti TF, Lago NR, Pirola CJ, et al. High fat diet‐induced liver steatosis promotes an increase in liver mitochondrial biogenesis in response to hypoxia. J Cell Mol Med 2011; 15: 1329-1338.
3. Erdmann K, Grosser N, Schipporeit K, Schröder H. The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via induction of heme oxygenase-1 and ferritin. J Nutr 2006; 136: 2148-2152.
4. Karlas T, Wiegand J, Berg T. Gastrointestinal complications of obesity: non-alcoholic fatty liver disease (NAFLD) and its sequelae. Best Pract Res Clin Endocrinol Metab 2013; 27: 195-208.
5. Gao J, Song J, Du M, Mao X. Bovine α-lactalbumin hydrolysates (α-LAH) attenuate high-fat diet induced nonalcoholic fatty liver disease by modulating hepatic lipid metabolism in C57BL/6J mice. JFF 2019; 54: 254-262.
6. Giri A, Ohshima T. Bioactive marine peptides: nutraceutical value and novel approaches. Adv Food Nutr Res 2012; 65:73-105.
7. Agyei D, Danquah MK. Rethinking food-derived bioactive peptides for antimicrobial and immunomodulatory activities. Trends Food Sci Technol 2012; 23: 62-69.
8. Tang W, Zhang H, Wang L, Qian H, Qi X. Targeted separation of antibacterial peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis. Food Chemist 2015; 168: 115-123.
9. Athmani N, Dehiba F, Allaoui A, Barkia A, Bougatef A, Lamri-Senhadji MY, et al. Sardina pilchardus and Sardinella aurita protein hydrolysates reduce cholesterolemia and oxidative stress in rat fed high cholesterol diet. J Exp Integr Med 2015; 5: 47-54.
10. Chiang W-D, Huang CY, Paul CR, Lee Z-Y, Lin W-T. Lipolysis stimulating peptides of potato protein hydrolysate effectively suppresses high-fat-diet-induced hepatocyte apoptosis and fibrosis in aging rats. Food Nutr Res 2016; 60: 31417-31425.
11. Vik R, Tillander V, Skorve J, Vihervaara T, Ekroos K, Alexson SE, et al. Three differently generated salmon protein hydrolysates reveal opposite effects on hepatic lipid metabolism in mice fed a high-fat diet. Food Chemist 2015; 183: 101-110.
12. Huang F, Wang J, Yu F, Tang Y, Ding G, Yang Z, et al. Protective effect of meretrix meretrix oligopeptides on high-fat-diet-induced non-alcoholic fatty liver disease in mice. Mar Drugs 2018; 16: 39-55.
13. Abbate JM, Macrì F, Capparucci F, Iaria C, Briguglio G, Cicero L, et al. Administration of protein hydrolysates from anchovy (engraulis encrasicolus) waste for twelve weeks decreases metabolic dysfunction-associated fatty liver disease severity in ApoE–/–Mice. Animals 2020; 10: 2303-2317.
14. FAO. Global aquaculture production. United Nations Fisheries and Aquaculture Department 2018.
15. Sachindra N, Bhaskar N, Mahendrakar N. Recovery of carotenoids from shrimp waste in organic solvents. Waste Manag 2006; 26: 1092-1098.
16. Nikoo M, Xu X, Regenstein JM, Noori F. Autolysis of pacific white shrimp (Litopenaeus vannamei) processing by-products: enzymatic activities, lipid and protein oxidation, and anti-oxidant activity of hydrolysates. Food Biosci 2021; 39: 100844-100854.
17. Chen H, Tsai T, Tsai Y, Liao J, Yen C, Chen C. Kefir peptides prevent high-fructose corn syrup-induced non-alcoholic fatty liver disease in a murine model by modulation of inflammation and the JAK2 signaling pathway. Nutr Diabetes 2016; 6: 237-237.
18. Nasri R, Abdelhedi O, Jemil I, Daoued I, Hamden K, Kallel C, et al. Ameliorating effects of goby fish protein hydrolysates on high-fat-high-fructose diet-induced hyperglycemia, oxidative stress and deterioration of kidney function in rats. Chem Biol Interact 2015; 242: 71-80.
19. Pakfetrat A, Dalirsani Z, Hashemy SI, Ghazi A, Mostaan LV, Anvari K, et al. Evaluation of serum levels of oxidized and reduced glutathione and total anti-oxidant capacity in patients with head and neck squamous cell carcinoma. J Cancer Res Ther 2018; 14: 428-431.
20. Zeb A, Ullah F. A simple spectrophotometric method for the determination of thiobarbituric acid reactive substances in fried fast foods. J Anal Methods Chem 2016; 2016: 9412767-9412772.
21. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497-500.
22. Classics Lowry O, Rosebrough N, Farr A, Randall R. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275.
23. Boonloh K, Kukongviriyapan V, Kongyingyoes B, Kukongviriyapan U, Thawornchinsombut S, Pannangpetch P. Rice bran protein hydrolysates improve insulin resistance and decrease pro-inflammatory cytokine gene expression in rats fed a high carbohydrate-high fat diet. Nutrients 2015; 7: 6313-6329.
24. Matthews DR, Hosker J, Rudenski A, Naylor B, Treacher D, Turner R. Homeostasis model assessment: insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: 412-419.
25. Gharehbagh SA, Azar JT, Razi M. ROS and metabolomics-mediated autophagy in rat’s testicular tissue alter after exercise training; evidence for exercise intensity and outcomes. Life Sci 2021; 277: 119585-119599.
26. 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.
27. Adams L, Angulo P. Treatment of non-alcoholic fatty liver disease. Postgrad Med J 2006; 82: 315-322.
28. Ipsen DH, Lykkesfeldt J, Tveden-Nyborg P. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cell Mol Life Sci 2018; 75: 3313-3327.
29. Rao MS, Reddy JK, editors. Peroxisomal β-oxidation and steatohepatitis. Seminars in liver disease; 2001: Copyright© 2001 by Thieme Medical Publishers, Inc.
30. Matsuzawa N, Takamura T, Kurita S, Misu H, Ota T, Ando H, et al. Lipid‐induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet. Hepatology 2007; 46: 1392-1403.
31. Chen C, Su X, Hu Z. Immune promotive effect of bioactive peptides may be mediated by regulating the expression of SOCS1/miR155. Exp Ther Med 2019; 18: 1850-1862.
32. Aloysius TA, Carvajal AK, Slizyte R, Skorve J, Berge RK, Bjørndal B. Chicken protein hydrolysates have anti-inflammatory effects on high-fat diet induced obesity in mice. Medicines 2019; 6: 5-20.
33. Lemus-Conejo A, Millan-Linares MdC, Toscano R, Millan F, Pedroche J, Muriana FJ, et al. GPETAFLR, a peptide from Lupinus angustifolius L. prevents inflammation in microglial cells and confers neuroprotection in brain. Nutr Neurosci 2020; 25: 472-484.
34. Lemus-Conejo A, Grao-Cruces E, Toscano R, Varela LM, Claro C, Pedroche J, et al. A lupine (Lupinus angustifolious L.) peptide prevents non-alcoholic fatty liver disease in high-fat-diet-induced obese mice. Food Funct 2020; 11: 2943-2952.
35. Bujanda L, Hijona E, Larzabal M, Beraza M, Aldazabal P, García-Urkia N, et al. Resveratrol inhibits nonalcoholic fatty liver disease in rats. BMC Gastroenterol 2008; 8: 1-8.
36. Martin K, Pritchett J, Llewellyn J, Mullan AF, Athwal VS, Dobie R, et al. PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis. Nat Commun 2016; 7: 1-11.
37. Chitapanarux T, Tienboon P, Pojchamarnwiputh S, Leelarungrayub D. Open‐labeled pilot study of cysteine‐rich whey protein isolate supplementation for nonalcoholic steatohepatitis patients. J Gastroenterol Hepatol 2009; 24: 1045-1050.
38. Hamad EM, Taha SH, Abou Dawood A-GI, Sitohy MZ, Abdel-Hamid M. Protective effect of whey proteins against nonalcoholic fatty liver in rats. Lipids Health Dis 2011; 10: 1-7.
39. Marnett LJ. Lipid peroxidation—DNA damage by malondialdehyde. Mutat Res 1999; 424: 83-95.
40. Sarmadi BH, Ismail A. Anti-oxidative peptides from food proteins: a review. Peptides 2010; 31: 1949-1956.
41. Xiong J-P, Long J-Y, Xu W-Y, Bian J, Huang H-C, Bai Y, et al. Albumin-to-alkaline phosphatase ratio: a novel prognostic index of overall survival in cholangiocarcinoma patients after surgery. World J Gastrointest Oncol 2019; 11: 39-47.
42. Khodadoostan M, Shariatifar B, Motamedi N, Abdolahi H. Comparison of liver enzymes level and sonographic findings value with liver biopsy findings in nonalcoholic fatty liver disease patients. Adv Biomed Res 2016; 5: 40-45.
43. Caúla A, Lira‐Junior R, Tinoco E, Fischer R. Serum creatinine and alkaline phosphatase levels are associated with severe chronic periodontitis. J Periodontal Res 2015; 50: 793-797.
44. Burski K, Ueland T, Maciejewski R. Serum amylase activity disorders in the course of experimental diabetes in rabbits. Veterinarni Medicina 2004; 49: 197-200.
45. Huang Y-L, Ma M-F, Chow C-J, Tsai Y-H. Angiotensin I-converting enzyme inhibitory and hypocholesterolemic activities: effects of protein hydrolysates prepared from Achatina fulica snail foot muscle. Int J Food Prop 2017; 20: 3102-3111.
46. Mohamed RS, Marrez DA, Salem SH, Zaghloul AH, Ashoush IS, Farrag ARH, et al. Hypoglycemic, hypolipidemic and anti-oxidant effects of green sprouts juice and functional dairy micronutrients against streptozotocin-induced oxidative stress and diabetes in rats. Heliyon 2019; 5: 1-24. 
47. Liu M, Wang Y, Liu Y, Ruan R. Bioactive peptides derived from traditional chinese medicine and traditional Chinese food: a review. Food Res Int 2016; 89: 63-73.
48. Drotningsvik A, Mjøs SA, Pampanin DM, Slizyte R, Carvajal A, Remman T, et al. Dietary fish protein hydrolysates containing bioactive motifs affect serum and adipose tissue fatty acid compositions, serum lipids, postprandial glucose regulation and growth in obese Zucker fa/fa rats. Br J Nutr 2016; 116: 1336-1345.
49. Drotningsvik A, Mjøs SA, Høgøy I, Remman T, Gudbrandsen OA. A low dietary intake of cod protein is sufficient to increase growth, improve serum and tissue fatty acid compositions, and lower serum postprandial glucose and fasting non-esterified fatty acid concentrations in obese Zucker fa/fa rats. Eur J Nutr 2015; 54: 1151-1160.
50. Sarteshnizi RA, Sahari MA, Gavlighi HA, Regenstein JM, Nikoo M, Udenigwe CC. Influence of fish protein hydrolysate-pistachio green hull extract interactions on anti-oxidant activity and inhibition of α-glucosidase, α-amylase, and DPP-IV enzymes. LWT 2021; 142: 111019-111028.
51. Das J, Ghosh S, Sil PC. Taurine and cardiac oxidative stress in diabetes. Diabetes 2020; 36: 361-372.
52. Kulakowski EC, Maturo J. Hypoglycemic properties of taurine: not mediated by enhanced insulin release. Biochem Pharmacol 1984; 33: 2835-2838.
53. Sampath W, Rathnayake R, Yang M, Zhang W, Mai K. Roles of dietary taurine in fish nutrition. Marine Life Sci Technol 2020; 2: 360-375.
54. Yang L, Li P, Fu S, Calay ES, Hotamisligil GS. Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell Metab 2010; 11: 467-478.
55. Madrigal-Matute J, Cuervo AM. Regulation of liver metabolism by autophagy. Gastroenterology 2016; 150: 328-339.
56. Czaja MJ. Autophagy in health and disease. 2. regulation of lipid metabolism and storage by autophagy: pathophysiological implications. Am J Physiol Cell Physiol 2010; 298: 973-978.
57. Ryter SW, Mizumura K, Choi AM. The impact of autophagy on cell death modalities. Int J Cell Biol 2014; 2014: 502676-502688.
58. Liu WJ, Ye L, Huang WF, Guo LJ, Xu ZG, Wu HL, et al. P62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett 2016; 21: 1-14.
59. Koga H, Kaushik S, Cuervo AM. Altered lipid content inhibits autophagic vesicular fusion. FASEB J 2010; 24:3052-3065.
60. Gonzalez-Rodriguez A, Mayoral R, Agra N, Valdecantos M, Pardo V, Miquilena-Colina M, et al. Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD. Cell Death Dis 2014; 5: 1179.