Dietary fat intake promotes the development of hepatic steatosis independently from excess caloric consumption in a murine model

Abstract

Nonalcoholic fatty liver disease results from overconsumption and is a significant and increasing cause of liver failure. The type of diet that is conducive to the development of this disease has not been established, and evidence-based treatment options are currently lacking. We hypothesized that the onset of hepatic steatosis is linked to the consumption of a diet with a high fat content, rather than related to excess caloric intake. In addition, we also hypothesized that fully manifested hepatic steatosis could be reversed by reducing the fat percentage in the diet of obese mice. C57BL/6J male mice were fed either a purified rodent diet containing 10% fat or a diet with 60% of calories derived from fat. A pair-feeding design was used to distinguish the effects of dietary fat content and caloric intake on dietary-induced hepatic lipid accumulation and associated injury. Livers were analyzed by quantitative reverse transcriptase polymerase chain reaction for lipid metabolism-related gene expression. After 9 weeks, mice on the 60%-fat diet exhibited more weight gain, insulin resistance, and hepatic steatosis compared with mice on a 10%-fat diet with equal caloric intake. Furthermore, mice with established metabolic syndrome at 9 weeks showed reversal of hepatic steatosis, insulin resistance, and obesity when switched to a 10%-fat diet for an additional 9 weeks, independent of caloric intake. Quantitative reverse transcriptase polymerase chain reaction revealed that transcripts related to both de novo lipogenesis and increased uptake of free fatty acids were significantly up-regulated in mice pair-fed a 60%-fat diet compared with 10%-fat-fed animals. Dietary fat content, independent from caloric intake, is a crucial factor in the development of hepatic steatosis, obesity, and insulin resistance in the C57BL/6J diet-induced obesity model caused by increased uptake of free fatty acids and de novo lipogenesis. In addition, once established, all these features of the metabolic syndrome can be successfully reversed after switching obese mice to a diet low in fat. Low-fat diets deserve attention in the investigation of a potential treatment of patients with nonalcoholic fatty liver disease.

Figure 1

Absolute mean caloric intake per mouse per week (A) was calculated per group on a daily base. Body weight gain (B) was calculated relative to the weight of each individual animal before initiation of the experiment. Plasma alanine aminotransferase (ALT; C), total cholesterol (D) and triglyceride (E) levels in the different groups. Values represent the mean ± SEM. Statistical significance is calculated between the HF-P animals and the difference between LF animals and HF animals (*, P<0.05; **, P<0.01).

Figure 2

Representative liver sections stained with hematoxylin and eosin (H&E; left panels; original magnification 400x), and Oil Red O (right panels; original magnification 200x). LF livers exhibited normal hepatic architecture, whereas HF-P livers revealed moderate, microvesicular steatosis, and HF livers revealed extensive, microvesicular and macrovesicular steatosis. P indicates portal tract; CV, central vein.

Figure 3

Magnetic resonance spectra for LF (A), HF-P (B) and HF (C) livers. Percent fat content was determined relative to water (100%) by numerical integration of the areas under the lipid and water peaks. Mean hepatic fat fraction as measured by magnetic resonance spectroscopy (D). Statistical significance is calculated between the HF-P animals and the difference between LF animals and HF animals (*, P<0.05; **, P<0.01). Variance statistic is SEM.

Figure 4

Body weight gain (A) was calculated relative to the weight of each individual animal before initiation of the experiment. After 9 weeks, blood was collected via retro-orbital puncture. Mice were allowed one week to recover, before groups were randomly assigned to the study diets. Absolute mean caloric intake per mouse per week (B) was calculated per group on a daily base. Plasma alanine aminotransferase (ALT; C), total cholesterol (D) and triglyceride (E) levels in the different groups. Values represent the mean ± SEM. Statistical significance is calculated between the RHF-P animals and the difference between RLF animals and RHF animals, as well as repeated measurements within groups (*, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant).

Figure 5

Representative liver sections stained with hematoxylin and eosin (H&E; left panels), and Oil Red O (right panels). RLF livers exhibited normal hepatic architecture, with occasional fat droplets. RHF-P livers revealed moderate macrovesicular steatosis around the portal tract, and moderate microvesicular steatosis around the central vein. RHF livers revealed extensive microvesicular steatosis around the central vein, and extensive macrovesicular steatosis around the portal tract. Original magnification 20x. P indicates portal tract; CV, central vein.

Figure 6

Magnetic resonance spectra for RLF (A), RHF-P (B) and RHF (C) livers. Percent fat content was determined relative to water (100%) by numerical integration of the areas under the lipid and water peaks. Mean hepatic fat fraction as measured by magnetic resonance spectroscopy (D). Statistical significance is calculated between the RHF-P animals and the difference between RLF animals and RHF animals (*, P<0.05; **, P<0.01). Variance statistic is SEM.

Figure 7

Upregulation of genes related to lipogenesis in livers of mice pair-fed a HF diet and in mice that were fed a HF diet ad libitum, compared to LF-fed controls. Hepatic expression of (A) SREBP1c, (B) SREBP2, (C) Elovl6, (D) Fasn, (E) SCD1 and (F) PPAR-α. Relative transcript levels were quantified by TaqMan quantitative RTPCR, normalized by β2MG and expressed in arbitrary units versus LF (9 weeks experiment) or RLF (19 weeks experiment) controls (*, P<0.05; **, P<0.01; ***, P<0.001). Variance statistic is SEM.