Diabetes Metab J.  2024 Jul;48(4):802-815. 10.4093/dmj.2023.0124.

Deficiency of ASGR1 Alleviates Diet-Induced Systemic Insulin Resistance via Improved Hepatic Insulin Sensitivity

• Yu X
• Tao J
• Wu Y
• Chen Y
• Li P
• Yang F
• Tang M
• Sammad A
• Tao Y
• Xu Y
• Li YX

• KMID: 2558047
• DOI: http://doi.org/10.4093/dmj.2023.0124

Abstract

Background
Insulin resistance (IR) is the key pathological basis of many metabolic disorders. Lack of asialoglycoprotein receptor 1 (ASGR1) decreased the serum lipid levels and reduced the risk of coronary artery disease. However, whether ASGR1 also participates in the regulatory network of insulin sensitivity and glucose metabolism remains unknown.
Methods
The constructed ASGR1 knockout mice and ASGR1^-/- HepG2 cell lines were used to establish the animal model of metabolic syndrome and the IR cell model by high-fat diet (HFD) or drug induction, respectively. Then we evaluated the glucose metabolism and insulin signaling in vivo and in vitro.
Results
ASGR1 deficiency ameliorated systemic IR in mice fed with HFD, evidenced by improved insulin intolerance, serum insulin, and homeostasis model assessment of IR index, mainly contributed from increased insulin signaling in the liver, but not in muscle or adipose tissues. Meanwhile, the insulin signal transduction was significantly enhanced in ASGR1^-/- HepG2 cells. By transcriptome analyses and comparison, those differentially expressed genes between ASGR1 null and wild type were enriched in the insulin signal pathway, particularly in phosphoinositide 3-kinase-AKT signaling. Notably, ASGR1 deficiency significantly reduced hepatic gluconeogenesis and glycogenolysis.
Conclusion
The ASGR1 deficiency was consequentially linked with improved hepatic insulin sensitivity under metabolic stress, hepatic IR was the core factor of systemic IR, and overcoming hepatic IR significantly relieved the systemic IR. It suggests that ASGR1 is a potential intervention target for improving systemic IR in metabolic disorders.

Keyword

Asialoglycoprotein receptor; Gluconeogenesis; Glycogen; Insulin resistance; Liver

Figure

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  Fig. 1. Deficiency of asialoglycoprotein receptor 1 (ASGR1) protected against diet-induced insulin resistance (IR). (A, B) Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed on mice fed with high-fat diet (HFD) for 6 weeks (wild type [WT], n=8; Asgr1+/-, n=7; Asgr1-/-, n=7) and 7 weeks (WT, n=5; Asgr1+/-, n=5; Asgr1-/-, n=5). The results quantified by the area under the curve (AUC) or area above the curve (AAC) according to the line chart is on the right side. (C, D) Fasting serum insulin content and homeostasis model assessment-insulin resistance (HOMA-IR) of mice fed with HFD for 8 weeks were determined and calculated (WT, n=17; Asgr1+/-, n=14; Asgr1-/-, n=14). All data are shown as the mean±standard deviation. aP<0.05, bP<0.01, cP<0.001, dP<0.0001, as compared to the indicated WT by one-way analysis of variance (ANOVA).

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  Fig. 2. Deficiency of asialoglycoprotein receptor 1 (ASGR1) improved hepatic and hepatocellular insulin sensitivity. (A) Western blot of key proteins in insulin signal pathway in liver. Representative images of the band and grey intensity of each band with insulin stimulation were shown (n=3). (B) Western blot of insulin-protein kinase B (AKT) signal pathway in the adipose and muscle tissues. Representative images of the band and grey intensity of phosphorylation of protein kinase B (pS-473) (pAKT) with insulin stimulation were shown (n=3). (C) Western blot analysis for insulin-induced phosphorylation of insulin receptor β (pInsRβ) and AKT in HepG2 and ASGR1-/- HepG2 cells. Representative images of the band and grey intensity of pAKT with insulin stimulation were shown (n=3). All data are shown as the mean±standard deviation. GLN, glucosamine (18 mM). aP<0.05, bP<0.01, as compared to the indicated wild type (WT) by one-way analysis of variance (ANOVA) among three groups. Statistical significance between two groups was assessed with an unpaired, 2-tailed t-test.

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  Fig. 3. Differentially expressed genes in asialoglycoprotein receptor 1 (ASGR1) knockout cells were enriched in the insulin signal pathway. (A) Heatmap shows hierarchical clustering of Pearson’s correlation coefficient based on sequencing. The score of 1 (red) denotes perfect correlation. (B) Heatmap shows hierarchical clustering of significant differentially expressed genes (DEGs) in ASGR1-/- HepG2 cells compared to the HepG2 cells. Values are column-scaled to show expression level. (C) Gene Ontology (GO) analysis identified molecular functions and biological processes of significant DEGs. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significant DEGs. The enrichment score indicates the degree of enrichment of DEGs in the biological process or pathway. PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; MAPK, mitogen-activated protein kinase; PPAR, peroxisome proliferator-activated receptor.

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  Fig. 4. Deficiency of asialoglycoprotein receptor 1 (ASGR1) represented decreased gluconeogenesis and glycogenolysis under metabolic stress conditions. (A) Glycogen content in liver tissues (wild type [WT], n=7; Asgr1+/-, n=5; Asgr1-/-, n=6). (B) The relative mRNA expression level of glycogen phosphorylase (Pygl) in liver tissues (n=4). (C) The relative mRNA expression levels of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) in liver tissues (n=4). (D) Glycogen content (n=3). (E) The relative mRNA expression level of PYGL (n=3). (F) The relative mRNA expression level of PCK1 and G6PC (n=3). (G) Western blot of PEPCK1 and glucose-6-phosphatase (G6Pase). Representative images of the band and grey intensity of each band relative to β-actin were shown (n=3). All data are shown as the mean±standard deviation. GLN, glucosamine. aP<0.05, bP<0.01, cP<0.001, as compared to the indicated WT by one-way analysis of variance (ANOVA) among three groups. Statistical significance between two groups was assessed with an unpaired, 2-tailed t-test.

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  Fig. 5. Asialoglycoprotein receptor 1 (ASGR1) deficiency affected hepatic glucose metabolism under standard diet-fed condition. (A) Western blot of insulin signal pathway in the liver from chow-fed mice. Representative images of the band and the grey intensity of each band were shown. (B) liver glycogen content. (C) The relative mRNA expression levels of glycogen synthase 2 (Gys2), glycogen phosphorylase (Pygl), and glucokinase (Gck) in liver tissues. (D) The relative mRNA expression levels of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) in liver tissues. (E) Western blot of glucose-6-phosphatase (G6Pase) and PEPCK1 in liver tissues. Representative images of the band and the grey intensity of each band relative to β-actin were shown (n=4). All data are shown as the mean±standard deviation. AKT, protein kinase B; WT, wild type; pInsRβ, phosphorylation of insulin receptor β. aP<0.05, bP<0.01, cP<0.001, as compared to the indicated WT by one-way analysis of variance (ANOVA).

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  Fig. 6. Knockout asialoglycoprotein receptor 1 (ASGR1) led to decreased gluconeogenesis and increased glycogen content in HepG2 cells under normal culture condition. (A) Western blot analysis of insulin signal pathway in HepG2 and ASGR1-/- HepG2 cells. Representative images of the band and grey intensity of phosphorylation of protein kinase B (pS-473) (pAKT) with insulin stimulation were shown, the remaining two electrophoresis are shown in Supplementary Fig. 6. (B) Glycogen content. (C) The relative mRNA expression level of glycogen phosphorylase (PYGL). (D) The relative mRNA expression levels of phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6-phosphatase catalytic subunit (G6PC). (E) Western blot of PEPCK1 and glucose6-phosphatase (G6Pase). Representative images of the band and the grey intensity of each band relative to β-actin were shown (n=3). All data are shown as the mean±standard deviation. pInsRβ, phosphorylation of insulin receptor β. aP<0.05, unpaired 2-tailed Student’s t-test.

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Reference

1. James DE, Stockli J, Birnbaum MJ. The aetiology and molecular landscape of insulin resistance. Nat Rev Mol Cell Biol. 2021; 22:751–71.

2. Lee SH, Park SY, Choi CS. Insulin resistance: from mechanisms to therapeutic strategies. Diabetes Metab J. 2022; 46:15–37.

3. 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; 160:745–58.

4. Santoleri D, Titchenell PM. Resolving the paradox of hepatic insulin resistance. Cell Mol Gastroenterol Hepatol. 2019; 7:447–56.

5. Patel BM, Goyal RK. Liver and insulin resistance: new wine in old bottle!!! Eur J Pharmacol. 2019; 862:172657.

6. Foretz M, Guigas B, Viollet B. Understanding the glucoregulatory mechanisms of metformin in type 2 diabetes mellitus. Nat Rev Endocrinol. 2019; 15:569–89.

7. Cariou B, Charbonnel B, Staels B. Thiazolidinediones and PPARγ agonists: time for a reassessment. Trends Endocrinol Metab. 2012; 23:205–15.

8. Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982; 51:531–54.

9. Nioi P, Sigurdsson A, Thorleifsson G, Helgason H, Agustsdottir AB, Norddahl GL, et al. Variant ASGR1 associated with a reduced risk of coronary artery disease. N Engl J Med. 2016; 374:2131–41.

10. Wang JQ, Li LL, Hu A, Deng G, Wei J, Li YF, et al. Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion. Nature. 2022; 608:413–20.

11. Xie B, Shi X, Li Y, Xia B, Zhou J, Du M, et al. Deficiency of ASGR1 in pigs recapitulates reduced risk factor for cardiovascular disease in humans. PLoS Genet. 2021; 17:e1009891.

12. Xu Y, Tao J, Yu X, Wu Y, Chen Y, You K, et al. Hypomorphic ASGR1 modulates lipid homeostasis via INSIG1-mediated SREBP signaling suppression. JCI Insight. 2021; 6:e147038.

13. Parhofer KG. Interaction between glucose and lipid metabolism: more than diabetic dyslipidemia. Diabetes Metab J. 2015; 39:353–62.

14. Diamond RH, Du K, Lee VM, Mohn KL, Haber BA, Tewari DS, et al. Novel delayed-early and highly insulin-induced growth response genes. Identification of HRS, a potential regulator of alternative pre-mRNA splicing. J Biol Chem. 1993; 268:15185–92.

15. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, et al. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell. 2000; 6:87–97.

16. Ekberg K, Landau BR, Wajngot A, Chandramouli V, Efendic S, Brunengraber H, et al. Contributions by kidney and liver to glucose production in the postabsorptive state and after 60 h of fasting. Diabetes. 1999; 48:292–8.

17. Nordlie RC, Foster JD, Lange AJ. Regulation of glucose production by the liver. Annu Rev Nutr. 1999; 19:379–406.

18. Han HS, Kang G, Kim JS, Choi BH, Koo SH. Regulation of glucose metabolism from a liver-centric perspective. Exp Mol Med. 2016; 48:e218.

19. Petersen MC, Shulman GI. Mechanisms of insulin action and insulin resistance. Physiol Rev. 2018; 98:2133–223.

20. Lin HV, Accili D. Hormonal regulation of hepatic glucose production in health and disease. Cell Metab. 2011; 14:9–19.

21. Barthel A, Schmoll D. Novel concepts in insulin regulation of hepatic gluconeogenesis. Am J Physiol Endocrinol Metab. 2003; 285:E685–92.

22. Magnusson I, Rothman DL, Katz LD, Shulman RG, Shulman GI. Increased rate of gluconeogenesis in type II diabetes mellitus: a 13C nuclear magnetic resonance study. J Clin Invest. 1992; 90:1323–7.

23. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995; 378:785–9.

24. Saltiel AR. Insulin signaling in health and disease. J Clin Invest. 2021; 131:e142241.

25. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 2001; 414:799–806.

26. Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol. 2006; 7:85–96.

27. Sakai K, Clemmons DR. Glucosamine induces resistance to insulin-like growth factor I (IGF-I) and insulin in Hep G2 cell cultures: biological significance of IGF-I/insulin hybrid receptors. Endocrinology. 2003; 144:2388–95.

28. Sun C, Zhang F, Ge X, Yan T, Chen X, Shi X, et al. SIRT1 improves insulin sensitivity under insulin-resistant conditions by repressing PTP1B. Cell Metab. 2007; 6:307–19.

29. Gao D, Nong S, Huang X, Lu Y, Zhao H, Lin Y, et al. The effects of palmitate on hepatic insulin resistance are mediated by NADPH oxidase 3-derived reactive oxygen species through JNK and p38MAPK pathways. J Biol Chem. 2010; 285:29965–73.

30. Yang Z, Huang X, Zhang J, You K, Xiong Y, Fang J, et al. Hepatic DKK1-driven steatosis is CD36 dependent. Life Sci Alliance. 2022; 6:e202201665.

31. Xiong Y, Chen Y, Huang X, Yang Z, Zhang J, Yu X, et al. Icaritin ameliorates hepatic steatosis via promoting fatty acid β-oxidation and insulin sensitivity. Life Sci. 2021; 268:119000.

32. Virtue S, Vidal-Puig A. GTTs and ITTs in mice: simple tests, complex answers. Nat Metab. 2021; 3:883–6.

33. Chaour M, Theroux P, Gilfix BM, Campeau L, Lesperance J, Ghitescu M, et al. ‘True’ fasting serum insulin level, insulin resistance syndrome and coronary artery disease. Coron Artery Dis. 1997; 8:683–8.

34. Rewers M, Zaccaro D, D’Agostino R, Haffner S, Saad MF, Selby JV, et al. Insulin sensitivity, insulinemia, and coronary artery disease: the Insulin Resistance Atherosclerosis Study. Diabetes Care. 2004; 27:781–7.

35. Houstis N, Rosen ED, Lander ES. Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature. 2006; 440:944–8.

36. Wang J, Gao WJ, Deng SL, Liu X, Jia H, Ma WZ. High temperature suppressed SSC self-renewal through S phase cell cycle arrest but not apoptosis. Stem Cell Res Ther. 2019; 10:227.

37. Rizza RA. Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications for therapy. Diabetes. 2010; 59:2697–707.

38. Biddinger SB, Kahn CR. From mice to men: insights into the insulin resistance syndromes. Annu Rev Physiol. 2006; 68:123–58.

39. Wu C, Okar DA, Kang J, Lange AJ. Reduction of hepatic glucose production as a therapeutic target in the treatment of diabetes. Curr Drug Targets Immune Endocr Metabol Disord. 2005; 5:51–9.

40. Rines AK, Sharabi K, Tavares CD, Puigserver P. Targeting hepatic glucose metabolism in the treatment of type 2 diabetes. Nat Rev Drug Discov. 2016; 15:786–804.

41. Corssmit EP, Romijn JA, Sauerwein HP. Review article: regulation of glucose production with special attention to nonclassical regulatory mechanisms: a review. Metabolism. 2001; 50:742–55.

42. Petersen MC, Vatner DF, Shulman GI. Regulation of hepatic glucose metabolism in health and disease. Nat Rev Endocrinol. 2017; 13:572–87.

43. Gerich JE, Meyer C, Woerle HJ, Stumvoll M. Renal gluconeogenesis: its importance in human glucose homeostasis. Diabetes Care. 2001; 24:382–91.

44. Grewal PK. The Ashwell-Morell receptor. Methods Enzymol. 2010; 479:223–41.

45. Ishibashi S, Hammer RE, Herz J. Asialoglycoprotein receptor deficiency in mice lacking the minor receptor subunit. J Biol Chem. 1994; 269:27803–6.

46. Samuel VT, Shulman GI. Mechanisms for insulin resistance: common threads and missing links. Cell. 2012; 148:852–71.

47. O-Sullivan I, Zhang W, Wasserman DH, Liew CW, Liu J, Paik J, et al. FoxO1 integrates direct and indirect effects of insulin on hepatic glucose production and glucose utilization. Nat Commun. 2015; 6:7079.