High fat diet feeding results in gender specific steatohepatitis and inflammasome activation

Abstract

Aim: To develop an animal model that encompasses the different facets of non-alcoholic steatohepatitis (NASH), which has been a challenge.

Methods: In this study, we used a high fat diet (HFD) feeding supplemented with fructose and sucrose in the water mimicking the high-fructose corn syrup that is abundant in the diet in the United States. We used C57Bl/6 wild-type mice for short and long-term feedings of 6 and 16 wk respectively, and evaluated the extent of liver damage, steatosis, and inflammasome activation. Our methods included histopathological analysis to assess liver damage and steatosis, which involved H and E and oil-red-o staining; biochemical studies to look at ALT and triglyceride levels; RNA analysis using quantitative polymerase chain reaction; and cytokine analysis, which included the enzyme-linked immunosorbent assay method to look at interleukin (IL)-1β and tumor necrosis factor-α (TNFα) levels. Furthermore, at each length of feeding we also looked at insulin resistance and glucose tolerance using insulin tolerance tests (ITT) and glucose tolerance tests.

Results: There was no insulin resistance, steatosis, or inflammasome activation at 6 wk. In contrast, at 16 wk we found significant insulin resistance demonstrated by impaired glucose and ITT in male, but not female mice. In males, elevated alanine aminotransferase and triglyceride levels, indicated liver damage and steatosis, respectively. Increased liver TNFα and monocyte chemoattractant protein-1 mRNA and protein, correlated with steatohepatitis. The inflammasome components, adaptor molecule, Aim2, and NOD-like receptor 4, increased at the mRNA level, and functional inflammasome activation was indicated by increased caspase-1 activity and IL-1β protein levels in male mice fed a long-term HFD. Male mice on HFD had increased α-smooth muscle actin and pro-collagen-1 mRNA indicating evolving fibrosis. In contrast, female mice displayed only elevated triglyceride levels, steatosis, and no fibrosis.

Conclusion: Our data indicate gender differences in NASH. Male mice fed a long-term HFD display steatohepatitis and inflammasome activation, whereas female mice have steatosis without inflammation.

Keywords: Gender differences; High fat diet; Inflammasome; Non-alcoholic steatohepatitis.

Figure 1

Effect of high fat diet feeding on body weight. Male and female C57Bl/6 mice were fed with either a high fat diet (HFD) supplemented with sucrose and fructose in the drinking water, or with a control chow diet for 6 or 16 wk. Body weights were recorded during both the short-term (6 wk, A), and long-term (16 wk, B) feedings. ^bP < 0.01 vs chow fed controls, n = 10/group.

Figure 2

High fat diet results in insulin resistance in male mice after 16 wk high fat diet feeding. Male and female mice were fed with either high fat diet (HFD) or chow diet for 6 or 16 wk. Glucose tolerance test (GTT) was performed: glucose levels were measured 0, 30, 75, 120 and 180 min after ip glucose injection in male (A) and female mice (B) fed with HFD for 16 wk. Insulin tolerance test (ITT) was performed: glucose levels were measured 0, 30, 75 and 120 min after ip insulin injection in male and female mice fed with HFD for 6 wk (C and D, respectively) or 16 wk (E and F, respectively). ^aP < 0.05, ^bP < 0.01 vs chow fed controls, n = 5/group, P = 0.03 vs chow fed controls in GTT-males, 16 wk, n = 5/group.

Figure 3

Elevated insulin levels in male mice after long-term high fat diet feeding. Insulin levels were measured using enzyme-linked immunosorbent assay prior to and 30 min after glucose stimulation in males (A) and females (B), following a short-term high fat diet (HFD) feeding, and males (C) and females (D), after a long-term HFD feeding. ^aP < 0.05 vs chow fed controls, n = 5/group.

Figure 4

Long-term high fat diet feeding results in steatohepatitis in male mice, and steatosis in female mice. Male and female mice were fed with either high fat diet (HFD) or control chow diet for 6 or 16 wk. A, B: Liver tissue was subjected to HE of male (A) and Oil-Red-O staining (B) (× 100). Dark arrow: Macrovesicular steatosis, white arrow: Microvesicular steatosis. One representative slide from n = 5/group is shown; C, D: Liver triglyceride levels (C) were evaluated in mice fed with HFD for 16 wk. Serum alanine aminotransferase (ALT) levels were measured after long-term HFD feeding (D).

Figure 5

Long-term high fat diet results in hepatic inflammation in male mice. A-C: Liver tumor necrosis factor-α (TNFα) protein levels (A) were evaluated in the short-term feeding levels of TNFα in the liver were analyzed at the mRNA (B) and protein (C) level after a long-term feeding; D-F: Monocyte chemoattractant protein 1 (MCP-1) protein levels (D) were evaluated in the short-term feeding. Levels of MCP-1 in the liver were analyzed at the mRNA (E) and protein (F) level after a long-term feeding. ^bP < 0.01 vs chow fed controls, n = 10/group.

Figure 6

Long-term high fat diet induced inflammasome upregulation and activation in male mice. Hepatic mRNA expression of the inflammasome components NOD-like receptors3 (NLRP3), caspase-1, adaptor molecule, Aim2, and NLRC4 were measured using real-time quantitative polymerase chain reaction in male (A) and female (B) mice after a long-term high fat diet. The functional activity of the inflammasome was evaluated by measurements of caspase-1 activity (C) and total nterleukin (IL)-1β protein (D) levels, in the liver. ^aP < 0.05, ^bP < 0.01 vs chow fed controls, n = 10/group.

Figure 7

Long-term male mice fed a high fat diet display increase in fibrotic markers. Hepatic mRNA expression of α-smooth muscle actin (αSMA) (A) and pro-collagen-1 (B), were measured using real-time quantitative polymerase chain reaction in male mice following a long-term high fat diet feeding. ^aP < 0.05, ^bP < 0.01 vs chow fed controls, n = 10/group.