High-fat and high-sucrose (western) diet induces steatohepatitis that is dependent on fructokinase

Abstract

Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with a high-fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild-type or fructokinase knockout mice were fed a low-fat (11%), high-fat (36%), or high-fat (36%) and high-sucrose (30%) diet for 15 weeks. Both wild-type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence of hepatic inflammation on a high-fat diet compared to a low-fat diet. In contrast, wild-type mice fed a high-fat and high-sucrose diet developed more severe hepatic steatosis with low-grade inflammation and fibrosis, as noted by increased CD68, tumor necrosis factor alpha, monocyte chemoattractant protein-1, alpha-smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice.

Conclusion: An additive effect of high-fat and high-sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high-fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis.

Figure 1

WT mice and KHK-A/C KO mice were given ad libitum LFD, HFD or HFHSD for 15 weeks (n = 7-8). (a) Growth curves of WT mice fed LFD, HFD or HFHSD. (b) Growth curves of KHK-A/C KO mice fed LFD, HFD or HFHSD. (c-e) Growth curves of WT mice and KHK-A/C KO mice fed LFD (c), HFD (d) or HFHSD (e). *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective LFD group. #P < 0.05 vs. WT fed HFD.

Figure 2

WT mice and KHK-A/C KO mice were given ad libitum LFD, HFD or HFHSD for 15 weeks (n = 7-8). (a) Representative images of H&E stained liver. Bar, 50 μm. CV, central vein. PT, portal triad. (b) Intrahepatic triglyceride levels (n =6). Data represent means (x000B1) s.e.m. *P < 0.05, ***P < 0.001 vs. respective LFD group. ##P < 0.01.

Figure 3

WT mice and KHK-A/C KO mice were given ad libitum LFD, HFD or HFHSD for 15 weeks (n = 7-8). (a) Serum aspartate aminotransferase (AST) levels (n = 7-8). (b) Serum alanine aminotransferase (ALT) levels (n = 7-8). (c-e) Quantitative real-time PCR for mouse tumor necrosis factor α (TNF-α, c), monocyte chemoattractant protein-1 (MCP-1, d) and CD68 (e) (n = 6-7). β-actin was used as a internal control. Data represent means (x000B1) s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective LFD group. #P < 0.05. ##P < 0.01. ### P < 0.001.

Figure 4

WT mice and KHK-A/C KO mice were given ad libitum LFD, HFD or HFHSD for 15 weeks (n = 7-8). (a) Representative images of trichrome stained liver. Bar, 50 μm. CV, central vein. PT, portal triad. (b-d) Quantitative real-time PCR for mouse collagen type I (COL1A1, b), mouse TIMP metallopeptidase inhibitor 1 (Timp1, c) and α-smooth muscle actin (αSMA, d) in liver (n = 6-7). β-actin was used as an internal control. Data represent means (x000B1) s.e.m. *P < 0.05, **P < 0.01 vs. respective LFD group. #P < 0.05. ##P < 0.01.

Figure 5

WT mice and KHK-A/C KO mice were given ad libitum LFD, HFD or HFHSD for 15 weeks (n = 7-8). (a) Left panel: Serum fructose concentration (n = 7-8). Right panel: Fructose content in liver (n = 6). (b) Quantitative real-time PCR for mouse KHK-C (left panel) and KHK-A (right panel) in liver (n = 6-7). (c-e) Western blot analysis of mouse fatty acid synthase (FAS, c), ATP citrate lyase (ACL, d) and mouse enoyl CoA-hydratase (ECH1, e) in liver (n = 4). β-actin was used as an internal control. (f) Left panel: Serum uric acid concentration (n = 7-8). Right panel: Uric acid content in liver (n = 6). Data represent means (x000B1) s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective LFD group. #P < 0.05. ##P < 0.01. ### P < 0.001. a, P < 0.05 by t-test. N.S., not significant.

Figure 6

WT mice and KHK-A/C KO mice were given ad libitum LFD, HFD or HFHSD for 15 weeks. (a, b) Dihydroethidium staining of the liver. (a) Representative images. Bar, 100 μm. (b) Quantification of fluorescence intensities (n = 3). (c) Western blot analysis of manganese superoxide dismutase (MnSOD) in liver (n = 4). β-actin was used as an internal control. (d) Western blot analysis of mitochondrial NADPH oxidase 4 (NOX4) in liver (n = 4). Voltage-dependent anion channel 1(VDAC1) was used as mitochondrial loading control. Data represent means (x000B1) s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective LFD group. #P < 0.05. ##P < 0.01. ### P < 0.001.