Farnesol, an isoprenoid, improves metabolic abnormalities in mice via both PPARα-dependent and -independent pathways

Peroxisome proliferator-activated receptors (PPARs) control energy homeostasis. In this study, we showed that farnesol, a naturally occurring ligand of PPARs, could ameliorate metabolic diseases. Obese KK-Ay mice fed a high-fat diet (HFD) containing 0.5% farnesol showed significantly decreased serum glucose level, glucosuria incidence, and hepatic triglyceride contents. Farnesol-containing HFD upregulated the mRNA expressions of PPARα target genes involved in fatty acid oxidation in the liver. On the other hand, farnesol was not effective in upregulating the mRNA expressions of PPARγ target genes in white adipose tissues. Experiments using PPARα-deficient [(-/-)] mice revealed that the upregulation of fatty acid oxidation-related genes required PPARα function, but the suppression of hepatic triglyceride accumulation was partially PPARα-dependent. In hepatocytes isolated from the wild-type and PPARα (-/-) mice, farnesol suppressed triglyceride synthesis. In luciferase assay, farnesol activated both PPARα and the farnesoid X receptor (FXR) at similar concentrations. Moreover, farnesol increased the mRNA expression level of a small heterodimer partner known as one of the FXR target genes and decreased those of sterol regulatory element-binding protein-1c and fatty acid synthase in both the wild-type and PPARα (-/-) hepatocytes. These findings suggest that farnesol could improve metabolic abnormalities in mice via both PPARα-dependent and -independent pathways and that the activation of FXR by farnesol might contribute partially to the PPARα-independent hepatic triglyceride content-lowering effect. To our knowledge, this is the first study on the effect of the dual activators of PPARα and FXR on obesity-induced metabolic disorders.     

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.     

Dietary β-conglycinin prevents fatty liver induced by a high-fat diet by a decrease in peroxisome proliferator-activated receptor γ2 protein

Diets high in sucrose/fructose or fat can result in hepatic steatosis (fatty liver). Mice fed a high-fat diet, especially that of saturated-fat-rich oil, develop fatty liver with an increase in peroxisome proliferator-activated receptor (PPAR) γ2 protein in liver. The fatty liver induced by a high-fat diet is improved by knockdown of liver PPARγ2. In this study, we investigated whether β-conglycinin (a major protein of soy protein) could reduce PPARγ2 protein and prevent high-fat-diet-induced fatty liver in ddY mice. Mice were fed a high-starch diet (70 energy% [en%] starch) plus 20% (wt/wt) sucrose in their drinking water or a high-safflower-oil diet (60 en%) or a high-butter diet (60 en%) for 11 weeks, by which fatty liver is developed. As a control, mice were fed a high-starch diet with drinking water. Either β-conglycinin or casein (control) was given as dietary protein. β-Conglycinin supplementation completely prevented fatty liver induced by each type of diet, along with a reduction in adipose tissue weight. β-Conglycinin decreased sterol regulatory element-binding protein (SREBP)-1c and carbohydrate response element-binding protein (ChREBP) messenger RNAs (mRNAs) in sucrose-supplemented mice, whereas it decreased PPARγ2 mRNA (and its target genes CD36 and FSP27), but did not decrease SREBP-1c and ChREBP mRNAs, in mice fed a high-fat diet. β-Conglycinin decreased PPARγ2 protein and liver triglyceride (TG) concentration in a dose-dependent manner in mice fed a high-butter diet; a significant decrease in liver TG concentration was observed at a concentration of 15 en%. In conclusion, β-conglycinin effectively prevents fatty liver induced by a high-fat diet through a decrease in liver PPARγ2 protein.     

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.     

Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis

Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression.     

Dietary α-tocopherol and atorvastatin reduce high-fat-induced lipid accumulation and down-regulate CD36 protein in the liver of guinea pigs

The increased uptake and storage of lipids in the liver are important features of steatotic liver diseases. The fatty acid translocase/scavenger receptor cluster of differentiation (CD)36 facilitates the hepatic uptake of lipids. We investigated if RRR-α-tocopherol (αT) alone or in combination with atorvastatin (ATV) is capable of preventing hepatic lipid accumulation via down-regulation of CD36. To this end, Dunkin Hartley guinea pigs were fed a control diet (5% fat); or a high-fat control diet (21% fat, 0.15% cholesterol); or a high-fat control diet fortified with αT (250 mg/kg diet), ATV (300 mg/kg diet) or both ATV+αT for 6 weeks. Hepatic triacylglycerols, hepatic protein and mRNA expression of CD36 as well as the mRNA expression of the controlling nuclear receptors LXRα, PXR and PPARγ were determined. Animals fed the high-fat control diet accumulated significantly more triacylglycerols in the liver than control animals. This was significantly reduced by ATV and numerically by αT and ATV+αT. Hepatic CD36 protein concentrations were significantly higher in the high-fat than in the control group, and both αT and ATV reduced CD36 expression to the level observed in the control group. However, no synergistic effect of the combined treatment was observed. Neither CD36 mRNA nor that of the nuclear receptors (LXRα, PXR and PPARγ) differed between groups, suggesting a posttranslational regulatory mechanism. Our results indicate that orally administered ATV and αT individually, but not synergistically, prevent diet-induced lipid accumulation in the liver of guinea pigs by down-regulation of hepatic CD36 protein.     

Proxisome proliferator-activated receptor-α mediates high-fat, diet-enhanced daily oscillation of plasminogen activator inhibitor-1 activity in mice

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitation showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning.     

Rosiglitazone and fenofibrate improve insulin sensitivity of pre-diabetic OLETF rats by reducing malonyl-CoA levels in the liver and skeletal muscle

AimsRosiglitazone and fenofibrate, specific agonists of the peroxisome proliferator activated receptors-γ (PPARγ) and -α (PPARα), respectively, improve insulin sensitivity in diabetic animals and in patients with type 2 diabetes. Here we investigated how pre-diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats fed with normal and high-fat diets respond to these PPAR agonists.Main methodsPre-diabetic OLETF rats were subjected to high-fat or standard diets with or without rosiglitazone or fenofibrate for 2 weeks. The metabolism of the rats and the levels of malonyl-CoA and activities of malonyl-CoA decarboxylase (MCD), acetyl-CoA carboxylase (ACC), and AMP-activated protein kinase (AMPK) in metabolic tissues were assessed.Key findingsRosiglitazone and fenofibrate significantly improved insulin sensitivity and reduced the levels of plasma triglycerides and free fatty acids in OLETF rats fed with a high-fat diet. Fenofibrate particularly reduced the body weight, fat, and total cholesterol in high fat diet OLETF rats. The highly elevated malonyl-CoA levels in the skeletal muscle and liver of OLETF rat were significantly reduced by rosiglitazone or fenofibrate due to, in part, the increased MCD activities and expression. On the other hand, ACC activities were unchanged in skeletal muscle and decreased in liver in high fat diet group. AMPK activities were dramatically decreased in OLETF rats and not affected by these agonists.SignificanceThese results demonstrate that treatment of pre-diabetic OLETF rats–particularly those fed a high-fat diet–with rosiglitazone and fenofibrate significantly improves insulin sensitivity and fatty acid metabolism by increasing the activity of MCD and reducing malonyl-CoA levels in the liver and skeletal muscle.     

Molecular characterisation of tumour necrosis factor alpha and its potential connection with lipoprotein lipase and peroxisome proliferator-activated receptors in blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>)

Tumour necrosis factor alpha (TNF-α) is one kind of cytokines which is related to inflammation and lipid metabolism. TNF-α cDNA was cloned from the liver of blunt snout bream (Megalobrama amblycephala) through real-time polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. The full-length cDNA of TNF-α covered 1467 bp, with an open reading frame (ORF) of 723 bp, which encodes 240 amino acids. It possessed the TNF family signature IIIPDDGIYFVYSQ. After the lipopolysaccharide (LPS) challenge test, a graded tissue-specific expression pattern of TNF-α was observed and there was high expression abundance in the kidney, brain and liver. After 8 weeks feeding trial, liver samples, two groups fed with 6% and 11% lipid levels, were collected. The results showed that, for fish fed with high-fat diet, the triglyceride of serum and lipid content of liver were elevated. Furthermore, TNF-α and peroxisome proliferator-activated receptors (PPARα, β) mRNA expression of fish fed 11% lipid diet were significantly up-regulated (p?<?0.05). Lipoprotein lipase (LPL) and PPARγ mRNA expression of fish fed 11% lipid lever diet were significantly decreased compared to those of fish fed 6% (p?<?0.05). The differences between the various expression of related genes in the high and low fat groups demonstrated that TNF-α played a key role in lipid metabolism, which may have an influence on fat metabolism through reducing fat synthesis and strengthening the β-oxidation of fatty acid. These discrepancies warrant further research.     

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.     

Dissociation of diabetes and obesity in mice lacking orphan nuclear receptor small heterodimer partner

Mixed background SHP(-/-) mice are resistant to diet-induced obesity due to increased energy expenditure caused by enhanced PGC-1α expression in brown adipocytes. However, congenic SHP(-/-) mice on the C57BL/6 background showed normal expression of PGC-1α and other genes involved in brown adipose tissue thermogenesis. Thus, we reinvestigated the impact of small heterodimer partner (SHP) deletion on diet-induced obesity and insulin resistance using congenic SHP(-/-) mice. Compared with their C57BL/6 wild-type counterparts, SHP(-/-) mice subjected to a 6 month challenge with a Western diet (WestD) were leaner but more glucose intolerant, showed hepatic insulin resistance despite decreased triglyceride accumulation and increased β-oxidation, exhibited alterations in peripheral tissue uptake of dietary lipids, maintained a higher respiratory quotient, which did not decrease even after WestD feeding, and displayed islet dysfunction. Hepatic mRNA expression analysis revealed that many genes expressed higher in SHP(-/-) mice fed WestD were direct peroxisome proliferator-activated receptor alpha (PPARα) targets. Indeed, transient transfection and chromatin immunoprecipitation verified that SHP strongly repressed PPARα-mediated transactivation. SHP is a pivotal metabolic sensor controlling lipid homeostasis in response to an energy-laden diet through regulating PPARα-mediated transactivation. The resultant hepatic fatty acid oxidation enhancement and dietary fat redistribution protect the mice from diet-induced obesity and hepatic steatosis but accelerate development of type 2 diabetes.     

Regulation of MTP expression in developing swine

To define the developmental expression of microsomal triglyceride transfer protein (MTP) large subunit mRNA and protein, samples of small intestine and liver were collected from 40-day gestation fetal, 2-day-old newborn, 3-week-old suckling, and 2-month-old weanling swine. In fetal animals, MTP mRNA expression was high in intestine and liver. Postnatally, jejunal expression paralleled the intake of a high-fat breast milk diet and declined after weaning. Ileal expression was comparable with that of jejunum in 2-day-old animals, but declined to low levels afterward. Hepatic expression declined postnatally and remained low. MTP protein expression generally paralleled mRNA expression, except in fetal intestine in which no 97 kDa protein was detected. In 2-day-old piglets, a high-triacylglycerol diet increased jejunal and ileal MTP mRNA levels, as compared to a low-triacylglycerol diet. To test the roles of glucocorticoids and fatty acids in MTP regulation, a newborn swine enterocyte cell line (IPEC-1) was used. Except at day 2 of differentiation, dexamethasone did not influence MTP expression. Fatty acids either up-regulated or down-regulated MTP expression, depending on the specific fatty acid and duration of exposure. Although programmed genetic cues regulate MTP expression during development, clearly the amount and fatty acid composition of dietary lipid also play regulatory roles.