Tumor necrosis factor alpha receptor 1 deficiency in hepatocytes does not protect from non-alcoholic steatohepatitis, but attenuates insulin resistance in mice

Abstract

Background: End-stage liver disease caused by non-alcoholic steatohepatitis (NASH) is the second leading indication for liver transplantation. To date, only moderately effective pharmacotherapies exist to treat NASH. Understanding the pathogenesis of NASH is therefore crucial for the development of new therapies. The inflammatory cytokine tumor necrosis factor alpha (TNF-α) is important for the progression of liver disease. TNF signaling via TNF receptor 1 (TNFR1) has been hypothesized to be important for the development of NASH and hepatocellular carcinoma in whole-body knockout animal models.

Aim: To investigate the role of TNFR1 signaling in hepatocytes for steatohepatitis development in a mouse model of diet-induced NASH.

Methods: NASH was induced by a western-style fast-food diet in mice deficient for TNFR1 in hepatocytes (TNFR1^ΔHEP) and their wild-type littermates (TNFR1^fl/fl). Glucose tolerance was assessed after 18 wk and insulin resistance after 19 wk of feeding. After 20 wk mice were assessed for features of NASH and the metabolic syndrome such as liver weight, liver steatosis, liver fibrosis and markers of liver inflammation.

Results: Obesity, liver injury, inflammation, steatosis and fibrosis was not different between TNFR1^ΔHEP and TNFR1^fl/fl mice. However, Tnfr1 deficiency in hepatocytes protected against glucose intolerance and insulin resistance.

Conclusion: Our results indicate that deficiency of TNFR1 signaling in hepatocytes does not protect from diet-induced NASH. However, improved insulin resistance in this model strengthens the role of the liver in glucose homeostasis.

Keywords: Glucose intolerance; Insulin resistance; Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis; Tumor necrosis factor alpha receptor 1; Type 2 diabetes.

Figure 1

Effect of tumor necrosis factor alpha receptor 1 deficiency in hepatocytes on body weight. A: Tumor necrosis factor alpha receptor 1 (Tnfr1) expression in liver samples (n = 4-5); B: Food intake per group over the course of the experiment, given in kilocalories (Kcal) and normalized to the mouse weight; C: Body weight and relative weights of epididymal (D) and brown fat tissues (E) after 20 wk of feeding; F: Example appearance of mice with a knockout of TNFR1 in hepatocytes (TNFR1^ΔHEP) and their TNFR1-expressing littermates (TNFR1^fl/fl) after 20 wk of fast food diet (FFD). Numbers of biological replicates: Ctrl/TNFR1^fl/fl n = 11; FFD/TNFR1^fl/fl n = 23; Ctrl/TNFR1^ΔHEP n = 12; FFD/TNFR1^ΔHEP n = 24. Line shows mean + standard error of the mean. Significant differences are marked with (a) if P < 0.05. Ctrl: Standard chow.

Figure 2

Effect of tumor necrosis factor alpha receptor 1 deficiency in hepatocytes on liver phenotype. A: Plasma levels of alanine amino transferase (ALT); B: Representative hematoxylin and eosin-stained liver sections; C: Relative liver weight; D: Relative amount of triglycerides normalized to liver weight. E: Total amount of hydroxyproline per liver; F: Representative Sirius Red-stained liver sections; G: Expression of the inflammatory genes interleukin 1-β (Il1-β), tumor necrosis factor alpha (Tnfα), and C-C motif chemokine ligand 2 (Ccl2). Scale bars: 200 µm. Number of biological replicates: Ctrl/tumor necrosis factor alpha receptor 1-expressing littermates (TNFR1^fl/fl) n = 9-11; fast food diet (FFD)/TNFR1^fl/fl n = 19-23; Ctrl/TNFR1^ΔHEP n = 11-12; FFD/TNFR1^ΔHEP n = 19-24. Line shows mean + standard error of the mean. Significant differences are marked with (a) if P < 0.05. Ctrl: Standard chow.

Figure 3

Effect of tumor necrosis factor alpha receptor 1 deficiency in hepatocytes on metabolic function. A: Glucose tolerance test performed after 18 wk of feeding; B: Insulin tolerance test performed after 19 wk of feeding. Numbers of biological replicates: Ctrl/tumor necrosis factor alpha receptor 1-expressing littermates (TNFR1^fl/fl) n = 11; fast food diet (FFD)/TNFR1^fl/fl n = 23-24; Ctrl/TNFR1^ΔHEP n = 12; FFD/TNFR1^ΔHEP n = 24. Line shows mean + standard error of the mean. Significant differences are marked with (a) if P < 0.05. Ctrl: Standard chow; GTT: Glucose tolerance test.