Dietary trans-fatty acid induced NASH is normalized following loss of trans-fatty acids from hepatic lipid pools

Abstract

Previous experiments in mice showed that dietary trans-fats could play a role in non-alcoholic steatohepatitis (NASH) yet little is known about the accumulation trans-fats in hepatic lipid pools in relationship to liver injury. NASH is also associated with obesity yet improves with only modest weight loss. To distinguish the role of obesity versus sustained consumption of a trans-fat containing diet in causing NASH, mice with obesity and NASH induced by consuming a high trans-fat diet for 16 weeks were subsequently fed standard chow or maintained on trans-fat chow for another 8 weeks. The accumulation, partitioning and loss of trans-fats in the major hepatic lipid pools during and after trans-fat consumption were determined. Obese mice switched to standard chow remained obese but steatohepatitis improved. trans-fats were differentially incorporated into the major hepatic lipid pools and the loss of trans-fats after crossover to control chow was greatest in the cholesteryl ester pool. In summary, dietary changes can improve the biochemical and histopathological changes of NASH despite persistent obesity in mice. Analysis of hepatic lipids confirmed that dietary trans-fats accumulate in the major lipid pools and are released differentially with diet normalization. The substantial loss of trans-fats from the cholesteryl ester pool in parallel with improvement in NASH suggests that this pool of trans-fats could play a role in the pathogenesis of NASH.

Fig. 1

Mean body weight after crossover from ALIOS conditions to control conditions (standard chow and water, no activity restrictions) for another 8 weeks compared to the weight of mice that remained on ALIOS conditions or were treated with control conditions for the entire 24 weeks. When feeding began at week 0, all mice weighed the same (18.4 ± 0.9 g). By week 16, the ALIOS mice weighed substantially more than control mice [37.2 ± 3.2 g (n = 30) vs. 28.4 ± 3.2 g (n = 20), P < 0.001]. Crossover at week 16 did not result in subsequent weight loss although there was no further weight gain (n = 10). A trend towards continued weight gain was evident in mice that remained on ALIOS conditions (n = 10). The weights at the final time point tended to be reduced compared to the previous week because of overnight fasting before sacrifice. The mean weights of ALIOS mice and crossover mice were significantly greater than control mice (n = 10) at all time points whereas there was not significant differences between ALIOS and crossover mice at any of these time points (P < 0.05; n = 10 mice in each group, error bars denote SD)

Fig. 2

Liver weight (a) and triglyceride content (b) at 24 weeks. Crossover to control conditions from weeks 16 to 24 (XO) caused reduction in liver weight and triglyceride content approaching normal levels (*P < 0.05 compared to control and crossover, **P < 0.05 compared to control and ALIOS; n = 10 mice per group; error bars denote SD)

Fig. 3

Plasma ALT (a) and AST (b) levels were increased after 24 weeks of ALIOS conditions but returned to near normal in crossover mice (*P < 0.05 compared to control and crossover;n = 10 mice per group; error bars denote SD)

Fig. 4

Plasma total cholesterol levels (a) were increased by 59 % in ALIOS mice at 24 weeks and were near normal in crossover mice (*P < 0.05 compared to control and crossover by ANOVA; n = 10 mice per group; error bars denote SD). In contrast, plasma triglyceride levels (b) were unchanged in ALIOS mice but were increased by 36 % after 8 weeks of crossover conditions (*P < 0.05 compared to control and ALIOS; n = 5 mice per group; error bars denote SD)

Fig. 5

Plasma leptin (a) and resistin (b) levels were increased in ALIOS mice at 16 and 24 weeks compared to control levels at the same time point (denoted by asterisks). Levels did not normalize with crossover to control chow. Resistin levels at 24 weeks were lower than at 16 weeks in both the control mice and ALIOS mice (P < 0.05). *P < 0.05 compared to control at the same time point; n = 5 mice per group; error bars denote SD

Fig. 6

Representative liver histology from control (a), ALIOS (b) and crossover (c) mice at 24 weeks. ALIOS livers at 24 weeks are characterized by extensive zone 1 macrovesicular steatosis around the portal venules (pv) and microvesicular steatosis are the central venules (cv) similar to that described previously at 16 weeks. Most of the macrovesicular fat was gone after 8 weeks of crossover to control chow leaving a ring of macrovesicular fat in zone 2 and residual microvesicular fat in zone 3. Hematoxylin and eosin stain, ×20 original magnification

Fig. 7

Incorporation of trans-fats into the free fatty acid, triglyceride, polar lipid and cholesteryl ester pools of fatty acid containing hepatic lipids after trans-fat feeding for 24 weeks or after trans-fat feeding for 16 weeks followed by crossover to control chow for a further 8 weeks. No trans-fats were detectable in any of the pools in control mice fed standard chow for the 24 week period (not shown). The polar lipid pool, mostly phosphatidylcholine, was the most trans-fat enriched pool in the liver and retained the greatest amount after crossover to control chow. The cholesteryl ester pool by comparison was mostly devoid of trans-fats after crossover to control chow, suggesting rapid turnover. The data represents the mean of analyses of 5–9 mice per group, error bars indicate standard deviation; the percent trans-fat incorporation into the fatty acids pools was significantly less in the crossover group compared to the ALIOS group for each lipid pool (P < 0.001 for all groups)

Fig. 8

Saturated and cis-unsaturated C16 fatty acids incorporation into triglyceride, polar lipids, cholesteryl esters or present as free fatty acids in the liver in control, ALIOS and crossover (XO) mice at 24 weeks. The identities of species represented by segments of the stacked bars are shown on the left panel. The monounsaturated pool (MUFA) was expanded in the ALIOS mice, especially in the cholesteryl ester pool. The polar lipid pool, representing membrane lipids was relatively unchanged. All pools contracted proportionally after crossover to control chow for 8 weeks. The essential fatty acid linoleic acid (18:2) was increased in the triglyceride and cholesteryl ester pools and remained increased in the triglyceride pool after crossover to control chow. The desaturation index (the ratio of 16:1c/16:0) was increased in the cholesteryl ester and free fatty acid pools under ALIOS conditions. The data represents the mean of analyses of 5–9 mice per group; a the ratio is significantly different from control; b TF-HC and XO ratios are significantly different; non-significance is indicated by no letter; significance is defined as P < 0.05 by t test