Activation of peroxisome proliferator-activated receptor-α (PPARα) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes

Activation of peroxisome proliferator-activated receptor (PPAR)-α which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPARα activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPARα activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPARα agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO₂ and acid soluble metabolites in enterocytes. Moreover, bezafibrate treatment suppressed postprandial lipidemia after oral administration of olive oil to the mice. These findings indicate that PPARα activation suppresses postprandial lipidemia through enhancement of fatty acid oxidation in enterocytes, suggesting that intestinal lipid metabolism regulated by PPARα activity is a novel target of PPARα agonist for decreasing circulating levels of lipids under postprandial conditions.     

Metabolic Hormone FGF21 Is Induced in Ground Squirrels during Hibernation but Its Overexpression Is Not Sufficient to Cause Torpor

Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3–10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor–all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.     

Torpor patterns,arousal rates,and temporal organization of torpor entry in wildtype and UCP1-ablated mice

In eutherian mammals, uncoupling protein 1 (UCP1) mediated non-shivering thermogenesis from brown adipose tissue (BAT) provides a mechanism through which arousal from torpor and hibernation is facilitated. In order to directly assess the magnitude by which the presence or absence of UCP1 affects torpor patterns, rewarming and arousal rates within one species we compared fasting induced torpor in wildtype (UCP1^+/+) and UCP1-ablated mice (UCP^−/−). Torpor was induced by depriving mice of food for up to 48 h and by a reduction of ambient temperature (T _a) from 30 to 18°C at four different time points after 18, 24, 30 and 36 h of food deprivation. In most cases, torpor bouts occurred within 20 min after the switch in ambient temperature (30–18°C). Torpor bouts expressed during the light phase lasted 3–6 h while significantly longer bouts (up to 16 h) were observed when mice entered torpor during the dark phase. The degree of hypometabolism (5–22 ml h⁻¹) and hypothermia (19.5–26.7°C) was comparable in wildtype and UCP1-ablated mice, and both genotypes were able to regain normothermia. In contrast to wildtype mice, UCP1-ablated mice did not display multiple torpor bouts per day and their peak rewarming rates from torpor were reduced by 50% (UCP1^+/+: 0.24 ± 0.08°C min⁻¹; UCP1^−/−: 0.12 ± 0.04°C min⁻¹). UCP1-ablated mice therefore took significantly longer to rewarm from 25 to 32°C (39 vs. 70 min) and required 60% more energy for this process. Our results demonstrate the energetic benefit of functional BAT for rapid arousal from torpor. They also suggest that torpor entry and maintenance may be dependent on endogenous rhythms.     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.     

PPARα deficiency augments a ketogenic diet-induced circadian PAI-1 expression possibly through PPARγ activation in the liver

An increased level of plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular diseases, and PAI-1 gene expression is under the control of molecular circadian clocks in mammals. We recently showed that PAI-1 expression is augmented in a phase-advanced circadian manner in mice fed with a ketogenic diet (KD). To determine whether peroxisome proliferator-activated receptor α (PPARα) is involved in hypofibrinolytic status induced by a KD, we examined the expression profiles of PAI-1 and circadian clock genes in PPARα-null KD mice. Chronic administration of bezafibrate induced the PAI-1 gene expression in a PPARα-dependent manner. Feeding with a KD augmented the circadian expression of PAI-1 mRNA in the hearts and livers of wild-type (WT) mice as previously described. The KD-induced mRNA expression of typical PPARα target genes such as Cyp4A10 and FGF21 was damped in PPARα-null mice. However, plasma PAI-1 concentrations were significantly more elevated in PPARα-null KD mice in accordance with hepatic mRNA levels. These observations suggest that PPARα activation is dispensable for KD-induced PAI-1 expression. We also found that hyperlipidemia, fatty liver, and the hepatic expressions of PPARγ and its coactivator PCG-1α were more effectively induced in PPARα-null, than in WT mice on a KD. Furthermore, KD-induced hepatic PAI-1 expression was significantly suppressed by supplementation with bisphenol A diglycidyl ether, a PPARγ antagonist, in both WT and PPARα-null mice. PPARγ activation seems to be involved in KD-induced hypofibrinolysis by augmenting PAI-1 gene expression in the fatty liver.     

Norepinephrine Controls Both Torpor Initiation and Emergence via Distinct Mechanisms in the Mouse

Some mammals, including laboratory mice, enter torpor in response to food deprivation, and leptin can attenuate these bouts of torpor. We previously showed that dopamine β-hydroxylase knockout (Dbh −/−) mice, which lack norepinephrine (NE), do not reduce circulating leptin upon fasting nor do they enter torpor. To test whether the onset of torpor in mice during a fast requires a NE-mediated reduction in circulating leptin, double mutant mice deficient in both leptin (ob/ob) and DBH (DBL MUT) were generated. Upon fasting, control and ob/ob mice entered torpor as assessed by telemetric core T_b acquisition. While fasting failed to induce torpor in Dbh −/− mice, leptin deficiency bypassed the requirement for NE, as DBL MUT mice readily entered torpor upon fasting. These data indicate that sympathetic activation of white fat and suppression of leptin is required for the onset of torpor in the mouse. Emergence from torpor was severely retarded in DBL MUT mice, revealing a novel, leptin-independent role for NE in torpor recovery. This phenotype was mimicked by administration of a β₃ adrenergic receptor antagonist to control mice during a torpor bout. Hence, NE signaling via β₃ adrenergic receptors presumably in brown fat is the first neurotransmitter-receptor system identified that is required for normal recovery from torpor.     

Monocarboxylate transporter (MCT)-1 is up-regulated by PPARα

Peroxisome proliferator-activated receptor (PPAR)-α mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARα in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARα agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARγ agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARα we treated wild-type and PPARα-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARα-null mice.     

PPARα-activation results in enhanced carnitine biosynthesis and OCTN2-mediated hepatic carnitine accumulation

In fasted rodents hepatic carnitine concentration increases considerably which is not observed in PPARα−/− mice, indicating that PPARα is involved in carnitine homeostasis. To investigate the mechanisms underlying the PPARα-dependent hepatic carnitine accumulation we measured carnitine biosynthesis enzyme activities, levels of carnitine biosynthesis intermediates, acyl-carnitines and OCTN2 mRNA levels in tissues of untreated, fasted or Wy-14643-treated wild type and PPARα−/− mice. Here we show that both enhancement of carnitine biosynthesis (due to increased γ-butyrobetaine dioxygenase activity), extra-hepatic γ-butyrobetaine synthesis and increased hepatic carnitine import (OCTN2 expression) contributes to the increased hepatic carnitine levels after fasting and that these processes are PPARα-dependent.     

Circadian expression of FGF21 is induced by PPARalpha activation in the mouse liver

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that regulates the expression of genes associated with lipid metabolism. Recent studies have suggested that the expression of PPARalpha-dependent fibroblast growth factor 21 (FGF21) plays important roles in adaptation to fasting, such as lipolysis and ketogenesis. We found that a nighttime injection of bezafibrate, a ligand of PPARalpha, effectively induced FGF21 expression, whereas a daytime injection did not affect it. Furthermore, bezafibrate-induced circadian FGF21 expression was abolished in PPARalpha-deficient mice. These observations suggest that bezafibrate-induced circadian FGF21 expression is due to circadian variations in the responsiveness of the PPARalpha system in the liver.     

Effect of low-quality diet on torpor frequency and depth in the pichi Zaedyus pichiy (Xenarthra,Dasypodidae), a South American armadillo

Daily torpor is a physiological adaptation that allows mammals to cope with energetic challenges associated with unpredictable periods of food shortage. We experimentally tested whether food quality influences torpor frequency and depth in the pichi (Zaedyus pichiy), a small, opportunistically omnivorous armadillo endemic to arid and semi-arid habitats of southern South America. We recorded body temperature (T_sc) changes in 10 semi-captive, adult female pichis using dataloggers implanted subcutaneously during periods of 21 days. All individuals entered spontaneous daily torpor, but those receiving a low-quality diet had significantly lower daily mean and minimum T_sc, spent more time at T_sc below their individual lower limit of normothermia, and had a higher Heterothermy Index than controls. Five individuals entered prolonged torpor bouts lasting more than 24 h, two of them repeatedly. Nine out of ten prolonged torpor bouts occurred in individuals feeding on a low-quality diet, suggesting that pichis are able to enter prolonged periods of torpor during severe environmental stress. In combination with their ability to hibernate and to respond to a reduced insect abundance by ingesting other food items, this physiological adaptation allows pichis to better cope with food shortages and a more extreme climate than other armadillos. It may explain why Z. pichiy naturally occurs farther south than any other armadillo species.     

Hepatosteatosis in peroxisome deficient liver despite increased β-oxidation capacity and impaired lipogenesis

Peroxisome deficiency in liver causes hepatosteatosis both in patients and in mice. Here, we studied the mechanisms that contribute to this lipid accumulation and to activation of peroxisome proliferator activated receptor α (PPARα) by using liver-specific Pex5^−/− mice (L-Pex5^−/− mice). Surprisingly, steatosis was accompanied both by increased mitochondrial β-oxidation capacity, confirming previous observations, and by impaired de novo lipid synthesis mediated by reduced expression of sterol regulatory element binding protein 1c and its targets. As a consequence, when challenged with a high fat diet, L-Pex5^−/− mice were protected from adiposity. Hepatic fatty acid uptake was strongly increased whereas the expression of apolipoproteins and the lipoprotein assembly factor microsomal triglyceride transfer protein were markedly reduced resulting in reduced secretion of very low density lipoproteins. Most of these changes seemed to be orchestrated by the endogenous activation of PPARα, challenging the assumption that PPARα activation in hepatocytes requires fatty acid synthase dependent de novo fatty acid synthesis. Expression of cholesterol synthesizing enzymes and cholesterol levels were not affected in peroxisome deficient liver. In conclusion, increased fatty acid uptake driven by endogenous PPARα activation and reduced fatty acid secretion cause hepatosteatosis in peroxisome deficient livers.     

The liver-enriched transcription factor CREBH is nutritionally regulated and activated by fatty acids and PPARα

To elucidate the physiological role of CREBH, the hepatic mRNA and protein levels of CREBH were estimated in various feeding states of wild and obesity mice. In the fast state, the expression of CREBH mRNA and nuclear protein were high and profoundly suppressed by refeeding in the wild-type mice. In ob/ob mice, the refeeding suppression was impaired. The diet studies suggested that CREBH expression was activated by fatty acids. CREBH mRNA levels in the mouse primary hepatocytes were elevated by addition of the palmitate, oleate and eicosapenonate. It was also induced by PPARα agonist and repressed by PPARα antagonist. Luciferase reporter gene assays indicated that the CREBH promoter activity was induced by fatty acids and co-expression of PPARα. Deletion studies identified the PPRE for PPARα activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay confirmed that PPARα directly binds to the PPRE. Activation of CREBH at fasting through fatty acids and PPARα suggest that CREBH is involved in nutritional regulation.     

The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man

FGF21 is a critical metabolic regulator, pivotal for fasting adaptation and directly regulated by PPARα in rodents. However, the physiological role of FGF21 in man is not yet defined and was investigated in our study. Serum FGF21 varied 250-fold among 76 healthy individuals and did not relate to age, gender, body mass index (BMI), serum lipids, or plasma glucose. FGF21 levels had no diurnal variation and were unrelated to bile acid or cholesterol synthesis. Ketosis induced by a 2 day fast or feeding a ketogenic diet (KD) did not influence FGF21 levels, whereas a 74% increase occurred after 7 days of fasting. Hypertriglyceridemic nondiabetic patients had 2-fold elevated FGF21 levels, which were further increased by 28% during fenofibrate treatment. FGF21 circulates in human plasma and increases by extreme fasting and PPARα activation. The wide interindividual variation and the induction of ketogenesis independent of FGF21 levels indicate that the physiological role of FGF21 in humans may differ from that in mice.     

A pan-PPAR ligand induces hepatic fatty acid oxidation in PPARα−/− mice possibly through PGC-1 mediated PPARδ coactivation

Tetradecylthioacetic acid (TTA) is a hypolipidemic modified fatty acid and a peroxisome proliferator-activated receptor (PPAR) ligand. The mechanisms of TTA-mediated effects seem to involve the PPARs, but the effects have not been assigned to any specific PPAR subtype. PPARα−/− mice were employed to study the role of PPARα after TTA treatment. We also performed in vitro transfection assays to obtain mechanistic knowledge of how TTA affected PPAR activation in the presence of PPARγ coactivator (PGC)-1 and steroid receptor coactivators (SRC)-1 and SRC-2, which are associated with energy balance and mitochondrial biogenesis. We show that TTA increases hepatic fatty acid β-oxidation in PPARα−/− mice. TTA acts as a pan-PPAR ligand in vitro, and PGC-1, SRC-1 and SRC-2 have cell type and PPAR-specific effects together with TTA. In the absence of exogenous ligands, SRC-1 did not induce PPAR activity, while PGC-1 was the most potent PPAR coactivator. When the coactivators were overexpressed, pronounced effects of TTA were observed especially for PPARδ and PPARγ. We conclude that PPARα is involved in, but not required for, the hypolipidemic mechanisms of TTA. It appears that the activity of PPARδ, with substantial contribution of nuclear receptor coactivators, PGC-1 in special, is conducive to TTA's mechanism of action.