Peroxisome proliferator–activated receptor α mediates the adaptive response to fasting (original) (raw)
A high fat diet induces a fatty liver in PPARα-null mice. The high fat diet used in the experiments was based on either coconut oil (saturated fat diet) or safflower oil (unsaturated fat diet), and contained about 40 en% fat, which is more than 3-fold higher than the normal diet. In our studies, feeding mice a high fat diet visually increased fat deposition in the adipose tissue of both wild-type and PPARα-null mice. Elevated adipose tissue stores promote the development of a fatty liver, as has been observed in obese human patients (24). Feeding a high fat (saturated or unsaturated) diet for several weeks resulted in an accumulation of triacylglycerols in the livers of wild-type mice, as indicated by a slight discoloration of the liver (Figure 1a). This was confirmed by oil red O staining of liver sections, which revealed numerous little lipid droplets that were not observed in mice fed the normal diet (Figure 1b). Strikingly, the appearance of lipid droplets was much more pronounced in PPARα-null mice fed the high fat diet; as a result, the liver had a pale appearance (Figure 1a). Oil red O staining also revealed large empty white spaces, which were probably large fat droplets that had their contents washed away during the staining procedure. Liver weights of PPARα-null mice fed the high saturated fat diet were 22% higher than the liver weights of wild-type mice fed the same diet (6.0% of total body weight for PPARα-null mice vs. 4.9% for wild-type mice; P < 0.01). These differences were not observed in PPARα-null and wild-type mice fed a normal diet.
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.
Combined, these results show that PPARα-null mice are particularly sensitive to a high fat diet and develop a fatty liver in response. However, because the metabolic responses to a high fat diet are relatively ill-defined, and because different mouse strains display a marked disparity in their reactions to a high fat diet (25, 26), we decided to focus our efforts on better characterizing the response to fasting.
Fasting induces a fatty liver in PPARα-null mice. Paradoxically, not only obesity but also chronic undernutrition is associated with a fatty liver (27). In mice and other animals, an overnight fast leads to significant lipid accumulation in the liver (28, 29, and see below). This can be explained by the high rate of fatty acid uptake by the liver, which under fasting conditions can exceed the capacity of the liver to secrete triacylglycerols (29). To test the effect of deletion of PPARα on lipid accumulation, PPARα-null and wild-type mice were subjected to 24 hours of fasting. Visual inspection of livers of fasting PPARα-null mice showed that they were distinctly paler than the livers of fasting wild-type mice. Staining for lipids with oil red O demonstrated that the pale color is due to the presence of numerous lipid droplets (Figure 1b), which were observed only to a small extent in fasted wild-type mice. Control staining by hematoxylin and eosin revealed these lipid droplets as small unstained vacuoles in liver sections of fasted PPARα-null mice but not wild-type mice (data not shown). These findings show that deletion of PPARα leads to a marked increase in lipid accumulation in the liver during fasting.
Diminished hepatic uptake of fatty acids in fasted PPARα-null mice. Once inside the liver cell, fatty acids can enter the mitochondria for subsequent oxidation, or they can be re-esterified to triacylglycerols, followed by secretion of the triacylglycerols in the form of VLDL. The capacity for secretion of VLDL is limited, which may lead to accumulation of triacylglycerols under conditions of an enlarged intracellular pool of fatty acids. Theoretically, the greater increase in lipid accumulation in fasted PPARα-null mice compared with fasted wild-type mice may be due to 3 potential, nonexclusive mechanisms: increased uptake of fatty acids, decreased secretion of VLDL, and/or decreased oxidation of fatty acids. To determine whether fatty acid uptake is increased in fasted PPARα-null mice, the concentration of circulating FFAs was measured. Plasma FFAs were significantly elevated in fasted mice compared with fed mice (Figure 2a), consistent with the increased rate of lipolysis occurring in the adipose tissue. Interestingly, plasma FFAs were more than 2-fold higher in the fasted PPARα-null mice compared with fasted wild-type mice. Because the liver represents the major sink of fatty acids under fasting conditions, this result suggests that uptake of fatty acids into the liver is decreased rather than increased. This is in agreement with the known stimulatory effect of PPARα on transcription of fatty acid transporter genes (19, 30).
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.
Expression of genes involved in VLDL secretion is not altered in PPARα-null mice. Another possible explanation for the increased triacylglycerol accumulation in livers of fasted PPARα-null mice is that VLDL secretion is defective. To test whether some of the genes involved in VLDL secretion or that encode constituents of VLDL are controlled by PPARα, the expression of 3 genes coding for apo E, apo B, and MTP were determined in PPARα-null and wild-type mice. The expression of all 3 genes was identical between the 2 sets of mice, in both the fed and fasted states (Figure 3, left). Expression of apo C-III, another constituent of VLDL, has been reported to be identical between PPARα-null and wild-type mice (18). Although this does not completely rule out the possibility that secretion of triacylglycerols is altered in PPARα-null mice, these results indicate that VLDL secretion is unlikely to be a PPARα target.
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.
Hepatic fatty acid oxidation is dramatically impaired in fasted PPARα-null mice. The third possibility that may explain the accumulation of lipid in the livers of fasted PPARα-null mice is impairment of fatty acid oxidation. To examine whether fatty acid oxidation is affected in PPARα-null mice, the concentration of plasma β-hydroxybutyrate, a ketone body that represents an important intermediate in the fatty acid oxidation pathway, was assessed. As expected, β-hydroxybutyrate levels were greatly increased in fasted wild-type mice (Figure 2b) but only slightly increased in fasted PPARα-null mice, resulting in a concentration that was about 7-fold less than in fasted wild-type mice. This low β-hydroxybutyrate level most likely reflects a markedly reduced rate of fatty acid oxidation, as well as a potential diminution in the rate of the reaction catalyzed by HMG-CoA synthase, a PPARα target gene in the liver (4).
An increased rate of hepatic fatty acid oxidation during fasting is not only responsible for the enhanced production of ketone bodies; it also assures efficient operation of gluconeogenesis. Indeed, gluconeogenesis is driven by fatty acid oxidation, which supplies ATP and reducing equivalents and directs pyruvate and lactate away from oxidation toward glucose synthesis. To examine whether deletion of PPARα may have an effect on gluconeogenesis, we measured the concentration of glucose in the blood during fasting. Remarkably, it was observed that PPARα-null mice suffer from hypoglycemia just several hours after food has been withdrawn (Figure 4a). Because blood glucose at this time is mostly maintained by glycogenolysis, the steeper drop in blood glucose in PPARα-null mice probably reflects the reduced concentration of glycogen in the livers of fed PPARα-null mice (Figure 2d). After prolonged fasting, when blood glucose is exclusively maintained by gluconeogenesis, the glucose concentration continued to drop more sharply in PPARα-null mice, being less than half the value of that in the wild-type animals. These data suggest that gluconeogenesis is impaired in PPARα-null mice.
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).
Intraperitoneal injection of glucose into PPARα-null mice fasted for 6 hours resulted in rapid normalization of their blood glucose concentration (Figure 4b). This intraperitoneal glucose tolerance test also showed that PPARα-null mice do not suffer from oversensitivity (or insensitivity) to insulin, because the glucose response curves were similar in PPARα-null and wild-type mice. This important result indicates that deletion of PPARα has no effect on the sensitivity of peripheral tissues to insulin.
Finally, plasma lactate, which reflects the rate of glycolysis in muscle, decreased during fasting, but no differences in plasma lactate levels were observed between wild-type and PPARα-null mice (Figure 2c). Taken together, these data demonstrate that hepatic fatty acid oxidation becomes severely impaired during fasting in the absence of PPARα, resulting in hypoglycemia, hypoketonemia, elevated plasma levels of FFAs, and a fatty liver.
Fasted PPARα-null mice are hypothermic and have a lower metabolic rate. When PPARα-null mice were subjected to fasting, we noticed that they appeared to be very cold. Rectal temperature measurements revealed that the mice suffered from severe hypothermia (Figure 5a). In wild-type SV129 mice, a 24-hour fast caused a drop in rectal temperature of 1.6°C, whereas in the PPARα-null mice, mean rectal temperature fell by 9.1°C, indicating that PPARα plays a major role in temperature homeostasis during fasting. This was also evident by measurement of the whole-body metabolic rate by indirect calorimetry. The data show that in fed mice of both genotypes, the metabolic rate was similar and fasting caused a significant rate decrease (Figure 5b). However, consistent with the lowered body temperature, the metabolic rate in fasted PPARα-null mice was significantly (about 30%) lower than in fasted wild-type mice.
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.
The lowered body temperature and associated lowered metabolic rate in fasted PPARα-null mice indicate that their temperature regulation was adjusted so that their metabolic needs would match the low, fasting-induced energy supply. In fact, when mice were exposed to the cold (5°C), no differences in rectal temperature were observed between PPARα-null and wild-type mice after 3 hours (Figure 6a), suggesting that cold-induced thermogenesis is not affected in PPARα-null mice. Similarly, expression levels of UCPs, which are postulated to be involved in thermogenesis and are probable target genes of PPAR, were similar in PPARα-null and wild-type mice in several tissues (Figure 6b). This was true despite a fasting-induced increase in expression levels of UCP2 in liver, gastrocnemius, and BAT, and of UCP3 in gastrocnemius. These data indicate that the hypothermia in fasted PPARα-null mice is not due to a defect in overall thermogenic capacity caused by reduced expression levels of UCPs.
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.
A more likely explanation for the hypothermia in fasted PPARα-null mice is the lack of fuel available for energy generation, forcing the animals into a state of torpor to reduce energy expenditure (31). Both plasma ketone bodies and glucose levels are dramatically reduced in fasted PPARα-null mice, as a result of inhibition of fatty acid oxidation in the liver. The data thus lead to the argument that the severe hypothermia in fasted PPARα-null mice is essentially caused by a defect in hepatic metabolism.
Lack of obvious phenotype in fed PPARα-null mice is partially due to the low rate of hepatic fatty acid oxidation. An important question is why PPARα-null mice need to be subjected to fasting to elicit a strong phenotype. One possible explanation is that the process regulated by PPARα only becomes important during fasting. Hepatic fatty acid oxidation operates at a relatively modest level in fed mice, whereas it is strongly stimulated during long periods of food deprivation. Hence, it is possible that deletion of PPARα affects gene expression levels in fed mice, but because of the low rate of fatty acid oxidation, the effects do not clearly manifest themselves. Northern blot analysis of liver mRNA revealed that under fed conditions, deletion of PPARα did in fact decrease expression of both CPTI, which catalyzes the rate-limiting step in fatty acid oxidation (32), and SCAD (Figure 3, center, first 4 lanes), both of which are PPARα target genes. This effect was not observed for all PPARα targets, e.g., L-FABP. The expression of L-FABP is dependent upon PPARα during fasting, but not in the fed state. The data indicate that, in the fed state, the marked differences in expression levels of important PPARα target genes between wild-type and PPARα-null mice have little apparent effect at the metabolic level. Thus, the lack of a strong phenotype in fed PPARα-null mice must be partially due to the relatively low rate of hepatic fatty acid oxidation under those conditions.
PPARα mRNA is induced during fasting. Another possible explanation of why PPARα-null mice need to be subjected to fasting to elicit a recognizable phenotype is that PPARα is suppressed under normal conditions and becomes activated during fasting. Accordingly, deletion of the PPARα gene will have a more pronounced effect under fasting conditions. Activation of PPARα during fasting can be effected by induction of PPARα mRNA and a resulting increase in PPARα protein, and/or by an increase in PPARα ligand activation. With respect to the first mechanism, fasting did induce PPARα mRNA expression (Figure 3, right), an effect probably mediated by glucocorticoids (33). Because PPARα protein level is strongly correlated with the expression of its mRNA (33), it can be expected that PPARα protein is similarly induced under fasting conditions. Regarding the second mechanism, it is important to note that fasting is associated with a marked increase in the plasma concentration of FFAs (Figure 2a). Since fatty acids are bona fide ligands for the PPAR receptors, it is conceivable that the elevated levels of FFAs activate PPARα.
Both in vitro and in vivo studies have demonstrated that PPARα preferentially binds polyunsaturated fatty acids (PUFAs) (7–9, 34). Fat tissue rich in PUFAs thus constitutes a source of higher-affinity ligands than does normal fat tissue, possibly resulting in stronger ligand activation of PPARα during fasting. Because the fatty acid composition of adipose tissue directly reflects the long-term diet (35), to enrich fat tissue with PUFAs, wild-type mice were fed a diet high in polyunsaturated fat. L-FABP gene was selected as a marker to assess the effect of PPARα activation, because during fasting, the expression of L-FABP is highly dependent upon PPARα (Figure 3, right) and is increased by fatty acids that act via PPARα (36, 37).
In fasted wild-type mice that were fed a diet high in linoleic acid for 7 weeks, the expression of L-FABP was about 4-fold higher than in fasted mice fed the normal diet (Figure 7). Surprisingly, the increase in L-FABP mRNA was accompanied by a similar increase in PPARα mRNA (about 3-fold). Thus, although PUFAs were able to increase expression of the PPARα target gene L-FABP, this effect could be almost entirely accounted for by an induction of PPARα mRNA itself. PUFAs seemed to have little or no additional effect on L-FABP mRNA beyond that mediated by increased PPARα expression, indicating that no increased ligand activation of PPARα took place. It cannot be completely ruled out that PUFAs increased L-FABP mRNA via a mechanism not involving PPARα. However, because PPARα has an exclusive role in maintaining L-FABP expression during fasting (Figure 3), we consider this explanation unlikely. Unfortunately, these data do not tell us whether PPARα is saturated with ligand in the fed state, and thus whether the elevation in plasma FFA concentration during the initial stage of fasting might contribute to PPARα activation.
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.
Transcriptional regulation of PPARα target genes during fasting is complicated by other hormonal signaling pathways. Because PPARα activity is enhanced by increased expression, and perhaps by increased ligand activation, it can be expected that some PPARα target genes are upregulated during fasting in a PPARα-dependent manner. Northern blot analysis showed, however, that the situation is considerably more complex. It was found that, in the fasted state, despite a strong increase in expression of CPTI and SCAD, the PPARα dependence was lost, suggesting that other signaling pathways activated during fasting override the effect of PPARα.
The expression of L-FABP displayed a completely different pattern. In the fed state, the expression of L-FABP was identical between wild-type and PPARα-null mice (Figure 3). In sharp contrast to CPTI and SCAD, in the fasted state, the expression of L-FABP was not upregulated in wild-type mice but was reduced more than 30-fold in PPARα-null mice. Although it may be tempting to correlate the emergence of a strong phenotype during fasting with this markedly reduced expression of L-FABP, it should be noted that L-FABP is only one of many genes whose expression is likely to be decreased in fasted PPARα-null mice. These data indicate that although PPARα mRNA is induced during fasting, the effects on PPARα target genes are complicated by the additional influence of other hormonal signaling pathways.
PEPCK is not a PPARα target gene in liver. Lastly, the expression of PEPCK, a PPARγ target gene in adipose tissue that catalyzes the rate-limiting step in gluconeogenesis (38), was unchanged in wild-type and PPARα-null mice in both the fed and fasted states (Figure 3), suggesting that PEPCK is not a PPARα target gene in liver.