A new way for punicalagin to alleviate insulin resistance: regulating gut microbiota and autophagy

  • Yuan Cao
  • Guofeng Ren
  • Yahui Zhang
  • Hong Qin
  • Xin An
  • Yi Long
  • Jihua Chen
  • Lina Yang
Keywords: Punicalagin, Insulin resistance, Liver, Gut microbiota, IKKβ/NF-κB, Autophagy


Background: Insulin resistance, defined as a diminished ability to respond to the stimulation of insulin, is the main line for a variety of metabolic-related diseases. Punicalagin (PU), a hydrolyzable tannin of pomegranate juice, exhibits multiple biological properties, including anti-oxidant, anti-cancer and anti-inflammatory activities.

Objective: This research study aimed at determining the protective effect of PU on insulin resistance and to uncover the underlying mechanism based on the gut microbiota, IKKβ/NF-κB pathway, and autophagy.

Design: An insulin resistance animal model was established using C57BL/6 mice fed with a high-fat diet (HFD) for 8 weeks. The model included two groups continuing a HFD for 12 weeks with or without administering via gavage with PU 20 mg/kg/day. Changes in fasting plasma glucose levels, fasting serum insulin levels, glucose and insulin tolerance, glycolipid metabolism, gut microbiota composition (16S rRNA gene sequencing), inflammatory responses, and autophagy in the liver were evaluated. Body weight gain, glycolipid metabolic disorder, liver injury, as well as systemic and hepatic insulin sensitivity, were significantly attenuated after supplementing with PU.

Results: This research study revealed that PU alleviated HFD-induced glucose and lipid disorders, liver injury and insulin resistance; decreased the Firmicutes/Bacteroides ratio, decreased the abundance of Coprococcus and Anaerotruncus, and increased Rikenellaceae; and decreased serum and liver tumor necrosis factor-alpha and interleukin-1β levels, inhibited liver IKKβ and NF-κB phosphorylation; and increased liver autophagy-related proteins LC3-II, P62, and Beclin1, and increased the number of liver autophagosomes.

Conclusion: PU can improve HFD-induced insulin resistance, improved liver glucose and lipid metabolism disorder and liver injury, and the potential mechanism is that PU inhibited the IKKβ/NF-κB inflammatory pathway by regulating gut microbiota homeostasis and up-regulating liver autophagy


Download data is not yet available.


  1. Lebovitz HE. Insulin resistance: definition and consequences. Exp Clin Endocrinol Diabetes 2001; 109 Suppl 2: S135–48. doi: 10.1055/s-2001-18576

  2. Titchenell PM, Lazar MA, Birnbaum MJ. Unraveling the regulation of hepatic metabolism by insulin. Trends Endocrinol Metab 2017; 28(7): 497–505. doi: 10.1016/j.tem.2017.03.003

  3. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001; 414(6865): 799–806. doi: 10.1038/414799a

  4. Perry RJ, Camporez JG, Kursawe R, Titchenell PM, Zhang D, Perry CJ, et al. Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes. Cell 2015; 160(4): 745–58. doi: 10.1016/j.cell.2015.01.012

  5. Shoelson SE, Lee J, Yuan M. Inflammation and the IKK beta/I kappa B/NF-kappa B axis in obesity- and diet-induced insulin resistance. Int J Obes Relat Metab Disord 2003; 27 Suppl 3: S49–52. doi: 10.1038/sj.ijo.0802501

  6. Gilmore TD. Introduction to NF-kappaB: players, pathways, perspectives. Oncogene 2006; 25(51): 6680–4. doi: 10.1038/sj.onc.1209954

  7. Samuel VT, Shulman GI. The pathogenesis of insulin resistance: integrating signaling pathways and substrate flux. J Clin Invest 2016; 126(1): 12–22. doi: 10.1172/jci77812

  8. Glass CK, Olefsky JM. Inflammation and lipid signaling in the etiology of insulin resistance. Cell Metab 2012; 15(5): 635–45. doi: 10.1016/j.cmet.2012.04.001

  9. Winer DA, Winer S, Shen L, Wadia PP, Yantha J, Paltser G, et al. B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies. Nat Med 2011; 17(5): 610–7. doi: 10.1038/nm.2353

  10. Saad MJ, Santos A, Prada PO. Linking gut microbiota and inflammation to obesity and insulin resistance. Physiology (Bethesda) 2016; 31(4): 283–93. doi: 10.1152/physiol.00041.2015

  11. Marra F, Svegliati-Baroni G. Lipotoxicity and the gut-liver axis in NASH pathogenesis. J Hepatol 2018; 68(2): 280–95. doi: 10.1016/j.jhep.2017.11.014

  12. Medzhitov R, Horng T. Transcriptional control of the inflammatory response. Nat Rev Immunol 2009; 9(10): 692–703. doi: 10.1038/nri2634

  13. Kim KH, Lee MS. Autophagy – a key player in cellular and body metabolism. Nat Rev Endocrinol 2014; 10(6): 322–37. doi: 10.1038/nrendo.2014.35

  14. Lapaquette P, Guzzo J, Bretillon L, Bringer MA. Cellular and molecular connections between autophagy and inflammation. Mediators Inflamm 2015; 2015: 398483. doi: 10.1155/2015/398483

  15. Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 2008; 456(7219): 264–8. doi: 10.1038/nature07383

  16. Bernini R, Gilardini Montani MS, Merendino N, Romani A, Velotti F. Hydroxytyrosol-derived compounds: a basis for the creation of new pharmacological agents for cancer prevention and therapy. J Med Chem 2015; 58(23): 9089–107. doi: 10.1021/acs.jmedchem.5b00669

  17. Serino A, Salazar G. Protective role of polyphenols against vascular inflammation, aging and cardiovascular disease. Nutrients 2018; 11(1): 53–65. doi: 10.3390/nu11010053

  18. Altieri F, Cairone F, Giamogante F, Carradori S, Locatelli M, Chichiarelli S, et al. Influence of ellagitannins extracted by pomegranate fruit on disulfide isomerase PDIA3 activity. Nutrients 2019; 11(1): 186–200. doi: 10.3390/nu11010186

  19. Zhou BH, Qiu ZP, Yi HL, Zhou DS, Wang J, Wu Y. [Research progress of ellagitannin intestinal metabolite urolithins]. Zhongguo Zhong Yao Za Zhi 2016; 41(16): 2968–74. doi: 10.4268/cjcmm20161604

  20. González-Sarrías A, Larrosa M, Tomás-Barberán FA, Dolara P, Espín JC. NF-kappaB-dependent anti-inflammatory activity of urolithins, gut microbiota ellagic acid-derived metabolites, in human colonic fibroblasts. Br J Nutr 2010; 104(4): 503–12. doi: 10.1017/s0007114510000826

  21. Anhe FF, Roy D, Pilon G, Dudonne S, Matamoros S, Varin TV, et al. A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. population in the gut microbiota of mice. Gut 2015; 64(6): 872–83. doi: 10.1136/gutjnl-2014-307142

  22. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28(7): 412–9. doi: 10.1007/bf00280883

  23. Zhao R, Long X, Yang J, Du L, Zhang X, Li J, et al. Pomegranate peel polyphenols reduce chronic low-grade inflammatory responses by modulating gut microbiota and decreasing colonic tissue damage in rats fed a high-fat diet. Food Funct 2019; 10(12): 8273–85. doi: 10.1039/c9fo02077b

  24. Mokáň M, Galajda P. Primary and secondary insulin resistance. Vnitr Lek 2019; 65(4): 264–72.

  25. Peng J, Wei D, Fu Z, Li D, Tan Y, Xu T, et al. Punicalagin ameliorates lipopolysaccharide-induced acute respiratory distress syndrome in mice. Inflammation 2015 Apr; 38(2): 493–9. doi: 10.1007/s10753-014-9955-5

  26. Yaidikar L, Thakur S. Punicalagin attenuated cerebral ischemia-reperfusion insult via inhibition of proinflammatory cytokines, up-regulation of Bcl-2, down-regulation of Bax, and caspase-3. Mol Cell Biochem 2015 Apr; 402(1–2): 141–8. doi: 10.1007/s11010-014-2321-y

  27. Chen Y, Qian Q, Yu J. Carbenoxolone ameliorates insulin sensitivity in obese mice induced by high fat diet via regulating the IκB-α/NF-κB pathway and NLRP3 inflammasome. Biomed Pharmacother 2019; 115: 108868. doi: 10.1016/j.biopha.2019.108868

  28. Xu H, Zhou Y, Liu Y, Ping J, Shou Q, Chen F, et al. Metformin improves hepatic IRS2/PI3K/Akt signaling in insulin-resistant rats of NASH and cirrhosis. J Endocrinol 2016; 229(2): 133–44. doi: 10.1530/joe-15-0409

  29. Bonora E, Targher G, Alberiche M, Bonadonna RC, Saggiani F, Zenere MB, et al. Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity: studies in subjects with various degrees of glucose tolerance and insulin sensitivity. Diabetes Care 2000; 23(1): 57–63. doi: 10.2337/diacare.23.1.57

  30. Guo S. Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms. J Endocrinol 2014; 220(2): T1–T23. doi: 10.1530/joe-13-0327

  31. Rehman K, Akash MS. Mechanisms of inflammatory responses and development of insulin resistance: how are they interlinked? J Biomed Sci 2016; 23(1): 87. doi: 10.1186/s12929-016-0303-y

  32. Akash MSH, Rehman K, Liaqat A. Tumor necrosis factor-alpha: role in development of insulin resistance and pathogenesis of type 2 diabetes mellitus. J Cell Biochem 2018; 119(1): 105–10. doi: 10.1002/jcb.26174

  33. Ballak DB, Stienstra R, Tack CJ, Dinarello CA, van Diepen JA. IL-1 family members in the pathogenesis and treatment of metabolic disease: focus on adipose tissue inflammation and insulin resistance. Cytokine 2015; 75(2): 280–90. doi: 10.1016/j.cyto.2015.05.005

  34. Boulangé CL, Neves AL, Chilloux J, Nicholson JK, Dumas ME. Impact of the gut microbiota on inflammation, obesity, and metabolic disease. Genome Med 2016; 8(1): 42. doi: 10.1186/s13073-016-0303-2

  35. Santos-Marcos JA, Perez-Jimenez F, Camargo A. The role of diet and intestinal microbiota in the development of metabolic syndrome. J Nutr Biochem 2019; 70: 1–27. doi: 10.1016/j.jnutbio.2019.03.017

  36. Ochoa-Repáraz J, Mielcarz DW, Wang Y, Begum-Haque S, Dasgupta S, Kasper DL, et al. A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease. Mucosal Immunol 2010; 3(5): 487–95. doi: 10.1038/mi.2010.29

  37. Velázquez KT, Enos RT, Bader JE, Sougiannis AT, Carson MS, Chatzistamou I, et al. Prolonged high-fat-diet feeding promotes non-alcoholic fatty liver disease and alters gut microbiota in mice. World J Hepatol 2019; 11(8): 619–37. doi: 10.4254/wjh.v11.i8.619

  38. Di Luccia B, Crescenzo R, Mazzoli A, Cigliano L, Venditti P, Walser JC, et al. Rescue of fructose-induced metabolic syndrome by antibiotics or faecal transplantation in a rat model of obesity. PLoS One 2015; 10(8): e0134893. doi: 10.1371/journal.pone.0134893

  39. Gurung M, Li Z, You H, Rodrigues R, Jump DB, Morgun A, et al. Role of gut microbiota in type 2 diabetes pathophysiology. EBioMedicine 2020; 51: 102590. doi: 10.1016/j.ebiom.2019.11.051

  40. Conley MN, Wong CP, Duyck KM, Hord N, Ho E, Sharpton TJ. Aging and serum MCP-1 are associated with gut microbiome composition in a murine model. PeerJ 2016; 4: e1854. doi: 10.7717/peerj.1854

  41. Konkol Y, Keskitalo A, Vuorikoski H, Pietilä S, Elo LL, Munukka E, et al. Chronic nonbacterial prostate inflammation in a rat model is associated with changes of gut microbiota that can be modified with a galactoglucomannan-rich hemicellulose extract in the diet. BJU Int 2019; 123(5): 899–908. doi: 10.1111/bju.14553

  42. Monk JM, Lepp D, Zhang CP, Wu W, Zarepoor L, Lu JT, et al. Diets enriched with cranberry beans alter the microbiota and mitigate colitis severity and associated inflammation. J Nutr Biochem 2016; 28: 129–39. doi: 10.1016/j.jnutbio.2015.10.014

  43. Alard J, Lehrter V, Rhimi M, Mangin I, Peucelle V, Abraham AL, et al. Beneficial metabolic effects of selected probiotics on diet-induced obesity and insulin resistance in mice are associated with improvement of dysbiotic gut microbiota. Environ Microbiol 2016; 18(5): 1484–97. doi: 10.1111/1462-2920.13181

  44. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 2009; 43: 67–93. doi: 10.1146/annurev-genet-102808-114910

  45. Nunes RD, Ventura-Martins G, Moretti DM, Medeiros-Castro P, Rocha-Santos C, Daumas-Filho CR, et al. Polyphenol-rich diets exacerbate AMPK-mediated autophagy, decreasing proliferation of mosquito midgut microbiota, and extending vector lifespan. PLoS Negl Trop Dis 2016; 10(10): e0005034. doi: 10.1371/journal.pntd.0005034

  46. Netea-Maier RT, Plantinga TS, van de Veerdonk FL, Smit JW, Netea MG. Modulation of inflammation by autophagy: consequences for human disease. Autophagy 2016; 12(2): 245–60. doi: 10.1080/15548627.2015.1071759

  47. Chung KW, Kim KM, Choi YJ, An HJ, Lee B, Kim DH, et al. The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis. Autophagy 2017; 13(7): 1113–29. doi: 10.1080/15548627.2017.1319040

  48. Katsuyama Y, Taira N, Yoshioka M, Okano Y, Masaki H. Disruption of melanosome transport in melanocytes treated with theophylline causes their degradation by autophagy. Biochem Biophys Res Commun 2017; 485(1): 126–30. doi: 10.1016/j.bbrc.2017.02.033

  49. Fujita K, Srinivasula SM. TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS). Autophagy 2011; 7(5): 552–4. doi: 10.4161/auto.7.5.15101

  50. Xu C, Wang W, Zhong J, Lei F, Xu N, Zhang Y, et al. Canagliflozin exerts anti-inflammatory effects by inhibiting intracellular glucose metabolism and promoting autophagy in immune cells. Biochem Pharmacol 2018; 152: 45–59. doi: 10.1016/j.bcp.2018.03.013

  51. Lee S, Keirsey KI, Kirkland R, Grunewald ZI, Fischer JG, de La Serre CB. Blueberry supplementation influences the gut microbiota, inflammation, and insulin resistance in high-fat-diet-fed rats. J Nutr 2018 Feb 1; 148(2): 209–19. doi: 10.1093/jn/nxx027

  52. Chaplin A, Carpéné C, Mercader J. Resveratrol, metabolic syndrome, and gut microbiota. Nutrients 2018 Nov 3; 10(11): 1651. doi: 10.3390/nu10111651

  53. Zhan X, Yan C, Chen Y, Wei X, Xiao J, Deng L, et al. Celastrol antagonizes high glucose-evoked podocyte injury, inflammation and insulin resistance by restoring the HO-1-mediated autophagy pathway. Mol Immunol 2018 Dec; 104: 61–8. doi: 10.1016/j.molimm.2018.10.021

  54. Zhou W, Ye S. Rapamycin improves insulin resistance and hepatic steatosis in type 2 diabetes rats through activation of autophagy. Cell Biol Int 2018 Sep; 42(10): 1282–91. doi: 10.1002/cbin.11015

How to Cite
Cao, Y., Ren, G., Zhang, Y., Qin, H., An, X., Long, Y., Chen, J., & Yang, L. (2021). A new way for punicalagin to alleviate insulin resistance: regulating gut microbiota and autophagy. Food & Nutrition Research, 65. https://doi.org/10.29219/fnr.v65.5689
Original Articles