Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting

Abstract

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

Figure 1

PPARα-null mice fed a high fat diet or subjected to fasting develop a fatty liver. Wild-type SV129 and PPARα-null mice were fed a high saturated fat diet for 10 weeks or a high unsaturated fat diet for 7 weeks. Fasted mice were deprived of food for 24 hours. (a) Gross morphology and color of livers of mice fed the high saturated fat diet or the normal diet. (b) Oil red O staining of liver sections of mice fed the normal diet, a high unsaturated fat diet, a high saturated fat diet (sections taken during the light cycle), or fasted for 24 hours.

Figure 2

Fasting-induced gross disturbances in the levels of several plasma metabolite levels in PPARα-null mice. SV129 wild-type or PPARα-null mice were sacrificed at the end of the dark cycle (fed state) or after a 24-hour fast that was started at the beginning of the light cycle (fasted state). (a) Plasma FFA concentrations. (b) Plasma β-hydroxybutyrate concentrations. (c) Plasma lactate concentrations. (d) Glycogen concentrations in liver. Error bars represent SEM. For the data in a–c, ANOVA yielded a significant effect for fasting vs. feeding (P < 0.01). For the data in a and b, the same was true for genotype and for the interaction between fasting/feeding and genotype (P < 0.01). ^§Significantly different from fed wild-type mice (P < 0.05). *Significantly different from all other values (P < 0.01). ^†Significantly different from fed mice (P < 0.01). All analyses by post hoc t test.

Figure 3

Fasting/feeding has dramatic effects on the expression of several PPAR target genes in a PPARα-dependent manner. Northern blot analysis of RNA from livers of fed and fasted SV129 wild-type or PPARα-null mice. Total RNA was isolated from livers of SV129 wild-type or PPARα-null mice sacrificed at the end of the dark cycle (fed state) or after a 24-hour fast started at the beginning of the light cycle (fasted state). Probes used were as indicated.

Figure 4

PPARα-null mice subjected to fasting become severely hypoglycemic. (a) Time course of blood glucose after removal of food. Blood glucose was measured at the end of the dark cycle when the animals were in the fully fed state. Food was subsequently withdrawn and blood glucose measured at several time points. Values at different time points are not necessarily from the same group of animals. Open squares, SV129 wild-type mice; open circles, PPARα-null mice. Error bars represent SEM. ANOVA showed a significant effect for genotype (P < 0.01), for time after removal of food (P < 0.01), and for interaction between these 2 parameters (P < 0.01). *Significantly different from wild-type mice (P < 0.01 by post hoc t test). (b) Intraperitoneal glucose tolerance test. Food was withdrawn for 6 hours starting at the beginning of the light cycle. At time 0, blood glucose was measured. Immediately thereafter, 2 g glucose/kg body weight was injected intraperitoneally by means of a sterile 20% glucose solution. Blood glucose was subsequently measured at several time points. Open squares, SV129 wild-type mice; open circles, PPARα-null mice. Error bars represent SEM. *Significantly different from wild-type mice (P < 0.01 by t test).

Figure 5

Fasted PPARα-null mice suffer from hypothermia and have a lower metabolic rate than fasted wild-type mice. Only female mice were used for these measurements. (a) Rectal temperature. Measurements were taken at the beginning of the light cycle (fed state) or after a 24-hour fast that was started at the beginning of the light cycle (fasted state). Note that the y axis starts at 20°C. Error bars represent SEM. ANOVA showed a significant difference between fasting and feeding (P < 0.05), genotype (P < 0.01), and interaction between fasting/feeding and genotype (P < 0.01). *Significantly different from fasted wild-type mice (P < 0.01). ^†Significantly different from fed mice (P < 0.05 [+/+] or P < 0.01 [–/–]). All analyses by post hoc t test. (b) Metabolic rate. For each mouse, mean metabolic rate was calculated for a 23-hour period with free access to food and water (fed state), or during the last 3 hours of a 24-hour fast (fasted state). Error bars represent SEM. ANOVA yielded significant effects for fasting vs. feeding (P < 0.01) and genotype (P < 0.05). *Significantly different from fasted wild-type mice (P < 0.05). ^†Significantly different from fed mice (P < 0.01). All analyses by post hoc t test.

Figure 6

PPARα-null mice can activate cold-induced thermogenesis. (a) Rectal temperature of wild-type and PPARα-null mice exposed to the cold. Mice were placed in individual precooled cages in a cold room maintained at 5°C. Rectal temperature was subsequently measured at various intervals. Open squares, SV129 wild-type mice; open circles, PPARα-null mice. (b) Analysis of UCP expression in various tissues by Northern blot. Total RNA was isolated from tissues of SV129 wild-type or PPARα-null mice sacrificed at the end of the dark cycle (fed state) or after a 24-hour fast started at the beginning of the light cycle (fasted state). Probes used were as indicated.

Figure 7

Prolonged feeding of a high unsaturated fat diet more strongly induces expression of PPARα and L-FABP genes after a 24-hour fast than does feeding of a normal diet. (a) Northern blot analysis of RNA from livers of fasted SV129 wild-type mice that had been fed a normal diet or a diet high in unsaturated fat (>70% linoleic acid) for 7 weeks. Total RNA was isolated from livers of SV129 wild-type mice sacrificed after a 24-hour fast, started at the beginning of the light cycle. (b) Quantitation of the intensity of the autoradiography signal corrected for control probe L27 . Means of 2 identical experiments are shown. Error bars have no statistical meaning but connect the 2 individual values.

Figure 8

Hepatic fatty acid oxidation is crucial during fasting to ensure an adequate supply of substrates that can be metabolized by other tissues. During fasting, lipolysis of stored triglycerides (TG) in adipose tissue is strongly activated under the influence of changes in hormonal status. The fatty acids (FA) released are delivered to the liver, where they are either re-esterified and secreted (not shown) or oxidized in the mitochondria. Partial oxidation of fatty acids yields acetyl-CoA, which condenses with itself to form ketone bodies. The ketone bodies are subsequently used as an important substrate for energy production by the brain, muscles, and kidney. Oxidation of fatty acids in the liver is also tightly coupled to glucose synthesis. The glucose produced by the liver serves as an important fuel for the brain. The important role of PPARα in these processes is illustrated. Although the figure is drawn with human tissues shown, important differences in lipid metabolism exist between mice and humans, including the function of PPARα. Thus, the validity of this figure with regard to human metabolism remains to be demonstrated.