Long term highly saturated fat diet does not induce NASH in Wistar rats

Abstract

Background: Understanding of nonalcoholic steatohepatitis (NASH) is hampered by the lack of a suitable model. Our aim was to investigate whether long term high saturated-fat feeding would induce NASH in rats.

Methods: 21 day-old rats fed high fat diets for 14 weeks, with either coconut oil or butter, and were compared with rats feeding a standard diet or a methionine choline-deficient (MCD) diet, a non physiological model of NASH.

Results: MCDD fed rats rapidly lost weight and showed NASH features. Rats fed coconut (86% of saturated fatty acid) or butter (51% of saturated fatty acid) had an increased caloric intake (+143% and +30%). At the end of the study period, total lipid ingestion in term of percentage of energy intake was higher in both coconut (45%) and butter (42%) groups than in the standard (7%) diet group. No change in body mass was observed as compared with standard rats at the end of the experiment. However, high fat fed rats were fattier with enlarged white and brown adipose tissue (BAT) depots, but they showed no liver steatosis and no difference in triglyceride content in hepatocytes, as compared with standard rats. Absence of hepatic lipid accumulation with high fat diets was not related to a higher lipid oxidation by isolated hepatocytes (unchanged ketogenesis and oxygen consumption) or hepatic mitochondrial respiration but was rather associated with a rise in BAT uncoupling protein UCP1 (+25-28% vs standard).

Conclusion: Long term high saturated fat feeding led to increased "peripheral" fat storage and BAT thermogenesis but did not induce hepatic steatosis and NASH.

Figure 1

Estimated daily caloric intake (A and C) and body mass (B and D) in different diet groups over time. Rats received either a standard diet (white triangles), a coconut diet (black circles), a butter diet (grey circles) or a methionine-choline deficient diet (grey squares). Values are mean ± SEM from 4 to 10 animals per group, P < 0.05: different from standard.

Figure 2

Relative weight of retroperitoneal white adipose tissue (WAT) in rats fed either a standard (Std), coconut (Coco), butter (Butter) or methionine-choline deficient diet (MCD). Data are means ± SEM; n = 10 for standard, coco and MCD groups, and n = 4 for butter group. P < 0.01 vs standard diet.

Figure 3

Diameter and volume of isolated adipocytes from the retroperitoneal white adipose tissue of rats fed standard diet or coconut diet. Measures were realised in triplicate on 3 rats per group; values are means ± SEM, P < 0.001 vs standard diet.

Figure 4

H&E and reticulin stained sections from rats fed standard diet (A), coconut diet (B), butter diet (C) for 14 weeks or MCD diet (D) for 6 weeks (magnification × 10), lipid vesicles are shown by a black arrow and inflammation point by a white arrow. (E) Liver triacylglycerol content (mg/g liver) in standard diet (std), coconut diet (Coco), butter diet (Butter) or MCD diet (MCDD). Results are means ± SEM, P < 0.05 vs standard.

Figure 5

Liver capacity to oxidize fatty acids was assessed by (A) ketone body production and (B) oxygen uptake. Hepatocytes were isolated from 24 h starved Wistar rats. Cell viability was checked by a measure of (C) gluconeogenesis, in the presence of dihydroxyacetone and medium (octanoate) or long (oleate) chain fatty acids. (D) Rat liver mitochondria control state respiration was obtained with 5 Mm succinate/0.5 mM malate/1.25 μM rotenone as substrates. State 3 respirations were initiated with 1 mM ADP. Each determination was performed in triplicate from at least ten preparations for the three different diets: standard diet (white), coconut diet (black), butter diet (hatched). Results are means ± SEM. No significant difference between diets was observed.

Figure 6

(A) Relative interscapular brown adipose tissue (BAT) mass in rats fed either a standard diet (Std), coconut diet (Coco) or butter diet (Butter). Data are means ± SEM; n = 10 for standard group, coco group, and n = 4 for butter group. P < 0.05. (B) Western blot analysis of UCP1 content in brown adipose tissue from rat fed standard diet, coconut diet or butter diet. Detections were performed with 40 μg of BAT mitochondrial proteins. Negative control was liver from standard rats. (C) 3-hydroxyacyl-CoA dehydrogenase (HAD) activity is measured spectrophotometrically and expressed in micromoles per minute per gram protein. Data are means ± SEM; n = 8 for standard group, coco group, and n = 4 for butter group. P < 0.05.