A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders

Abstract

We hypothesized that the lipid-activated transcription factor, the peroxisome proliferator-activated receptor alpha (PPARalpha), plays a pivotal role in the cellular metabolic response to fasting. Short-term starvation caused hepatic steatosis, myocardial lipid accumulation, and hypoglycemia, with an inadequate ketogenic response in adult mice lacking PPARalpha (PPARalpha-/-), a phenotype that bears remarkable similarity to that of humans with genetic defects in mitochondrial fatty acid oxidation enzymes. In PPARalpha+/+ mice, fasting induced the hepatic and cardiac expression of PPARalpha target genes encoding key mitochondrial (medium-chain acyl-CoA dehydrogenase, carnitine palmitoyltransferase I) and extramitochondrial (acyl-CoA oxidase, cytochrome P450 4A3) enzymes. In striking contrast, the hepatic and cardiac expression of most PPARalpha target genes was not induced by fasting in PPARalpha-/- mice. These results define a critical role for PPARalpha in a transcriptional regulatory response to fasting and identify the PPARalpha-/- mouse as a potentially useful murine model of inborn and acquired abnormalities of human fatty acid utilization.

Figure 1

Accumulation of intracellular lipid in the liver of fasted PPARα^−/− mice. (A) Representative photograph demonstrating the appearance of livers from fasted PPARα^+/+ (Left) and PPARα^−/− (Right) mice. The livers were harvested rapidly from age-matched PPARα^−/− and PPARα^+/+ mice after a 24-h fast. The left hepatic lobes are shown. (B) Photomicrographs demonstrating the histologic appearance of the livers of PPARα^−/− mice under fed and fasted conditions. Frozen tissue sections were prepared from the livers of PPARα^−/− mice after a 24-h fast (FASTED) and age-matched PPARα^−/− controls fed ad libitum (FED). The sections were stained with oil red O. The red droplets indicate positive staining for neutral lipid.

Figure 2

PPARα^−/− mice exhibit a hypoglycemic response to fasting. Mean blood glucose levels (ordinate) of age-matched PPARα^−/− and PPARα^+/+ mice at various time points during a 48-h fast. Mean values were based on 21 PPARα^+/+ mice (6 male; 15 female) and 30 PPARα^−/− mice (12 male; 18 female). ∗ indicates a statistically significant difference (P < 0.001; Fisher’s test) compared with the corresponding value obtained in age-matched PPARα^+/+ animals.

Figure 3

The ketogenic response to fasting is blunted in PPARα^−/− mice. Bars = mean ± SE serum β-hydroxybutyrate (β-HBA) levels determined in age-matched PPARα^−/− and PPARα^+/+ mice after a 48-h fast compared with the fed ad libitum state. ∗ denotes a significant difference (P < 0.05) compared with the value of the fed PPARα^+/+ group. Values are based on at least six mice per group and two independent experiments.

Figure 4

The fasting-induced hepatic expression of most PPARα target genes encoding cellular FAO enzymes is abolished in PPARα^−/− mice. (A) Representative autoradiographs of Northern blot analyses performed with total RNA (15 μg/lane) isolated from the livers of fed control (C) and 24-h-fasted (F) littermate PPARα^+/+ (+/+) and PPARα^−/− (−/−) mice. The cDNA probes are denoted on the left (abbreviations defined in the text). The signal for GAPDH is shown as a control for loading and RNA integrity. (B) Bars represent mean fasting fold induction compared with the values of fed littermate controls based on phosphorimager analysis of Northern blots described in A. All values were normalized to the signal for GAPDH. ∗ denotes a significantly (P < 0.05) higher mean signal intensity compared with the corresponding fed control values. The values are based on a minimum of nine animals per group and four independent experiments.

Figure 5

The fasting-induced cardiac expression of PPARα target genes is altered in PPARα^−/− mice. (A) Representative autoradiographs of Northern blot analyses performed with total RNA (15 μg/lane) isolated from the hearts of fed control (C) and 24-h-fasted (F) littermate PPARα^+/+ (+/+) and PPARα^−/− (−/−) mice. The cDNA probes are denoted on the left. The signal for GAPDH is shown as a control for loading and RNA integrity. (B) Bars represent mean fasting fold induction compared with fed littermate controls after normalization to the GAPDH signal based on phosphorimager analysis of Northern blots described in A. Asterisks represent a significantly (P < 0.05) higher mean signal intensity compared with corresponding fed control values. The values are based on a minimum of six animals per group and three independent experiments.