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Comparative Study
. 2015 Mar;26(3):285-92.
doi: 10.1016/j.jnutbio.2014.11.002. Epub 2014 Dec 6.

Saturated fat and cholesterol are critical to inducing murine metabolic syndrome with robust nonalcoholic steatohepatitis

Jamie E Mells  1 , Ping P Fu  2 , Pradeep Kumar  2 , Tekla Smith  2 , Saul J Karpen  1 , Frank A Anania  3
Affiliations
Comparative Study

Saturated fat and cholesterol are critical to inducing murine metabolic syndrome with robust nonalcoholic steatohepatitis

Jamie E Mells et al. J Nutr Biochem. 2015 Mar.

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome (MetS). Up to a third of NAFLD subjects are at risk for developing nonalcoholic steatohepatitis (NASH). Many rodent models fail to replicate both MetS and NASH. The purpose of this study was to develop a reliable mouse model of NASH and MetS using a diet containing cholesterol, saturated fat and carbohydrate that is reflective of Western diets of North Americans.

Experimental design: We used adult male C57BL/6 J 4- to 5-week-old mice and administered a solid diet containing 0.2% cholesterol, 45% of its calories from fat, with 30% of the fat in the form of partially hydrogenated vegetable oil. We also provided carbohydrate largely as high-fructose corn syrup equivalent in water. In a separate cohort, we gave the identical diet in the absence of cholesterol. Glucose and insulin tolerance testing was conducted throughout the feeding period. The feeding was conducted for 16 weeks, and the mice were sacrificed for histological analysis, markers of MetS, liver inflammation, circulating lipids, as well as liver staining for fibrosis and alpha smooth muscle actin (α-SMA).

Results: We found that cholesterol significantly increased serum leptin, interleukin-6, liver weight and liver weight/body weight ratio, fibrosis and liver α-SMA.

Conclusions: Mice administered a diet accurately reflecting patterns associated with humans afflicted with MetS can reliably replicate features of MetS, NASH and significant liver fibrosis. The model we describe significantly reduces the time by several months for development of stage 3 hepatic fibrosis.

Keywords: Adipocytokine; Cholesterol; Fibrosis; Fructose; NASH.

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Figures

Fig. 1
Fig. 1
Body weight, glucose and insulin tolerance tests after 16 weeks of feeding. (A) Weights at 16 weeks. Mice were fed control chow, HFTF or HFTFX. (B–C) Glucose and insulin tolerance tests were performed at 16 weeks. Both the HFTF and HFTFX groups have significantly larger AUCs than control. “*” denotes significant difference (P<.05, n=8 per group).
Fig. 2
Fig. 2
(A–C) Hematoxylin and eosin staining of liver sections (magnification, 100×). Black arrows denote Mallory body formation and hepatocyte ballooning. (D–F) Oil red O staining of liver sections. Red staining indicates lipid accumulation (magnification, 100×; insert, 200×).
Fig. 3
Fig. 3
(A–C) Siruis red staining of liver sections (magnification, 40×; inset, 100×). (D–F) Masson Trichome staining (magnification, 100×). (G) Quantification of Sirius red staining. “*” indicates significant difference from control (P<.05); “╪” indicates a significant difference between HFTF and HFTFX (P<.05).
Fig. 4
Fig. 4
(A–C) Liver sections stained for α-SMA activation, a marker for stellatecell activation. Black arrows indicateactivated stellatecells. (D) Serum levels of cholesterol and triglycerides. (F) Serum levels of ALT and AST. “*” or “╪” indicates significant difference from control (P<.0001); “**” or “╪╪” indicates a significant difference between HFTF and HFTFX (P<.001).
Fig. 5
Fig. 5
Hydroxyproline content of livers from mouse cohorts; “*” indicates significant difference from control (P<.05).
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