Modulation of fatty acid and bile acid metabolism by peroxisome proliferator-activated receptor α protects against alcoholic liver disease

Abstract

Background: Chronic alcohol intake affects liver function and causes hepatic pathological changes. It has been shown that peroxisome proliferator-activated receptor α (PPARα)-null mice developed more pronounced hepatic changes than wild-type (WT) mice after chronic exposure to a diet containing 4% alcohol. The remarkable similarity between the histopathology of alcoholic liver disease (ALD) in Ppara-null model and in humans, and the fact that PPARα expression and activity in human liver are less than one-tenth of those in WT mouse liver make Ppara-null a good system to investigate ALD.

Methods: In this study, the Ppara-null model was used to elucidate the dynamic regulation of PPARα activity during chronic alcohol intake. Hepatic transcriptomic and metabolomic analyses were used to examine alterations of gene expression and metabolites associated with pathological changes. The changes triggered by alcohol consumption on gene expression and metabolites in Ppara-null mice were compared with those in WT mice.

Results: The results showed that in the presence of PPARα, 3 major metabolic pathways in mitochondria, namely the fatty acid β-oxidation, the tricarboxylic acid cycle, and the electron transfer chain, were induced in response to a 2-month alcohol feeding, while these responses were greatly reduced in the absence of PPARα. In line with the transcriptional modulations of these metabolic pathways, a progressive accumulation of triglycerides, a robust increase in hepatic cholic acid and its derivatives, and a strong induction of fibrogenesis genes were observed exclusively in alcohol-fed Ppara-null mice.

Conclusions: These observations indicate that PPARα plays a protective role to enhance mitochondrial function in response to chronic alcohol consumption by adaptive transcriptional activation and suggest that activation of this nuclear receptor may be of therapeutic value in the treatment for ALD.

Keywords: Fibrogenesis; Metabolic Pathways; Metabolomics; Ppara-Null Mice; Transcriptomics.

Figure 1

Fibrosis and inflammatory changes in alcohol-fed Ppara-null mice. Panels A to D: Histologic changes with Sirius red staining in liver of WT and Ppara-null mice fed control or 4% ethanol diet for six months. Red staining indicate pericellular collagen fibrils. WT mice fed a 4% ethanol diet (B) showed no fibrosis. In contrast, Ppara-null mice fed alcohol for the same period (D) displayed pericellular fibrosis. (E) Hematoxylin and eosin staining of liver section from Ppara-null mice fed alcohol for six months indicates inflammatory cell infiltration. (F) Alterations in hepatic mRNA levels of Col1a1 and Thbs1, two genes associated with fibrogenesis, after two-months of alcohol feeding. “C” and “E” represents control and ethanol feeding respectively. qRT-PCR results were normalized with internal control Gapdh. *, **, and *** indicate statistically significant difference with a p value less than 0.05, 0.01, and 0.001 between two groups respectively .

Figure 2

Statistical analysis of metabolomic profiling results from control and alcohol-fed Ppara-null mice in ESI negative mode. (A) Volcano plot displays t-test results. Ions labeled in red have significant difference in intensity between control and alcohol-fed Ppara-null mice. (B) KEGG pathway analysis results of the differential ions (red points in panel A).

Figure 3

Measurements of bile acids and genes involved in bile acids’ synthesis and transport. (A) Levels of hepatic cholic acid and its derivatives in control- and alcohol-fed WT and Ppara-null mice after two-month alcohol feeding. Y-axis represents the UPLC-QTOFMS intensity data with arbitrary units. (B) Hepatic Abcb11, Cyp7a1 and Cyp27a1 mRNA levels after two-month alcohol feeding. “C” and “E” represents control and ethanol feeding respectively. qRT-PCR results were normalized with internal control Gapdh mRNA * p value<0.05, ** p value < 0.01.

Figure 4

Multivariate data analysis of hepatic lipidomics profiles from control- and alcohol-fed wt and Ppara-null mice. Principle components analysis (PCA) unsupervised clustering plots for one-month (1M), two-month (2M), and four-month (4M) treatment are shown. In each panel, WC, WE, PC, and PE designate WT control, WT alcohol-fed, Ppara-null control, and Ppara-null alcohol-fed respectively.

Figure 5

Levels of indicated triglyceride ions after alcohol ingestion. Panels from top to bottom show results from one-month, two-month, and four-month treatment respectively for four groups defined in the Fig. 4 legend. Y-axis represents the ratio of measurement of the specified triglyceride ion to that of the spike-in standard ion, trinonadecenoin (TG(57:3)).

Figure 6

Alteration of expression of genes involved in fatty acid β-oxidation. (A) The ordinate represents the expression after two months of alcohol relative to control, log₂ratio (alcohol-fed/control). The symbols “*” and “◆” indicate statistically significant differences observed in WT and Ppara-null mice respectively. (B) A schematic diagram of central steps of the fatty acid α-oxidation pathway, the gene names for these enzymes are in italic and the ones in red are those showing statistically significant induction (P value < 0.05) in panel A.

Figure 7

Alteration of expression of genes involved in the TCA cycle (A) and electron transfer chain (B) after 2 m of alcohol exposure. (A) In the upper panel, Y-axis represents Log₂Ratio (alcohol-fed/control). Asterisks (*) designate statistically significant differences observed in wt mice, while none of the changes in and Ppara-null mice were significant. (B) The heatmaps of gene intensity of the corresponding electron transfer chain complexes from four experimental groups after 2 m of alcohol: Ppara-null control, Ppara-null alcohol-fed, WT control, and WT alcohol-fed,. The raw intensity data were pre-processed by log-transformed and autoscaled to zero mean with Partek Genomics Suite. Processed intensity data was visualized by Genesis.

Figure 8

Proposed mechanisms for the protective role of PPARα in ALD progression. Arrows in red indicate the changes caused by alcohol consumption.