PPARα Target Genes Research Articles

P465L-PPARγ mutation confers partial resistance to the hypolipidaemic action of fibrates.

AimsFamilial partial lipodystrophic syndrome 3 (FPLD3) is associated with mutations in the transcription factor PPARγ. One of these mutations, the P467L, confers a dominant negative effect. We and others have previously investigated the pathophysiology associated with this mutation using a humanized mouse model that recapitulates most of the clinical symptoms observed in patients who have been phenotyped under different experimental conditions. One of the key clinical manifestations observed, both in humans and mouse models, is the ectopic accumulation of fat in the liver. With this study we aim to dissect the molecular mechanisms that contribute to the excessive accumulation of lipids in the liver and characterize the negative effect of this PPARγ mutation on the activity of PPARα in vivo when activated by fibrates.Material and MethodsP465L‐PPAR mutant and wild‐type mice were divided into 8 experimental groups, 4 different conditions per genotype. Briefly, mice were fed a chow diet or a high‐fat diet (HFD 45% Kcal from fat) for a period of 28 days and treated with WY14643 or vehicle for five days before culling. At the end of the experiment, tissues and plasma were collected. We performed extensive gene expression, fatty acid composition and histological analysis in the livers. The serum collected was used to measure several metabolites and to perform basic lipoprotein profile.ResultsP465L mice showed increased levels of insulin and free fatty acids (FFA) as well as increased liver steatosis. They also exhibit decreased levels of very low density lipoproteins (VLDL) when fed an HFD. We also provide evidence of impaired expression of a number of well‐established PPARα target genes in the P465L mutant livers.ConclusionOur data demonstrate that P465L confers partial resistance to the hypolipidemic action of fibrates. These results show that the fatty liver phenotype observed in P465L mutant mice is not only the consequence of dysfunctional adipose tissue, but also involves defective liver metabolism. All in all, the deleterious effects of P465L‐PPARγ mutation may be magnified by their collateral negative effect on PPARα function.

Interaction of glucocorticoids with FXR/FGF19/FGF21-mediated ileum-liver crosstalk

At high doses, glucocorticoids (GC) have been associated with enhanced serum bile acids and liver injury. We have evaluated the effect of GC, in the absence of hepatotoxicity, on FXR/FGF91(Fgf15)/FGF21-mediated ileum-liver crosstalk. Rats and mice (wild type and Fxr−/−, Fgf15−/− and int-Gr−/− strains; the latter with GC receptor (Gr) knockout selective for intestinal epithelial cells), were treated (i.p.) with dexamethasone, prednisolone or budesonide. In both species, high doses of GC caused hepatotoxicity. At a non-hepatotoxic dose, GC induced ileal Fgf15 down-regulation and liver Fgf21 up-regulation, without affecting Fxr expression. Fgf21 mRNA levels correlated with those of several genes involved in glucose and bile acid metabolism. Surprisingly, liver Cyp7a1 was not up-regulated. The expression of factors involved in transcriptional modulation by Fxr and Gr (p300, Drip205, CBP and Smrt) was not affected. Pxr target genes Cyp3a11 and Mrp2 were not up-regulated in liver or intestine. In contrast, the expression of some Pparα target genes in liver (Fgf21, Cyp4a14 and Vanin-1) and intestine (Vanin-1 and Cyp3a11) was altered. In mice with experimental colitis, liver Fgf21 was up-regulated (4.4-fold). HepG2 cells transfection with FGF21 inhibited CYP7A1 promoter (prCYP7A1-Luc2). This was mimicked by pure human FGF21 protein or culture in medium previously conditioned by cells over-expressing FGF21. This response was not abolished by deletion of a putative response element for phosphorylated FGF21 effectors present in prCYP7A1. In conclusion, GC interfere with FXR/FGF19-mediated intestinal control of CYP7A1 expression by the liver and stimulate hepatic secretion of FGF21, which inhibits CYP7A1 promoter through an autocrine mechanism.

The olive constituent oleuropein, as a PPARα agonist, markedly reduces serum triglycerides

Oleuropein (OLE), a main constituent of olive, exhibits antioxidant and hypolipidemic effects, while it reduces the infarct size in chow- and cholesterol-fed rabbits. Peroxisome proliferator-activated receptor α (PPARα) has essential roles in the control of lipid metabolism and energy homeostasis. This study focused on the mechanisms underlying the hypolipidemic activity of OLE and, specifically, on the role of PPARα activation in the OLE-induced effect. Theoretical approach using Molecular Docking Simulations and luciferase reporter gene assay indicated that OLE is a ligand of PPARα. The effect of OLE (100 mg/kg, p.o., per day, ×6 weeks) on serum triglyceride (TG) and cholesterol levels was also assessed in adult male wild-type and Ppara-null mice. Molecular Docking Simulations, Luciferase reporter gene assay and gene expression analysis indicated that OLE is a PPARα agonist that up-regulates several PPARα target genes in the liver. This effect was associated with a significant reduction of serum TG and cholesterol levels. In contrast, OLE had no effect in Ppara-null mice, indicating a direct involvement of PPARα in the OLE-induced serum TG and cholesterol reduction. Activation of hormone-sensitive lipase in the white adipose tissue (WAT) and the liver of wild-type mice and up-regulation of several hepatic factors involved in TG uptake, transport, metabolism and clearance may also contribute in the OLE-induced TG reduction. In summary, OLE has a beneficial effect on TG homeostasis via PPARα activation. OLE also activates the hormone sensitive lipase in the WAT and liver and up-regulates several hepatic genes with essential roles in TG homeostasis.

Discovery of peroxisome proliferator–activated receptor α (PPARα) activators with a ligand-screening system using a human PPARα-expressing cell line

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator-responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both in vitro and in vivo Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Pharmacologic activation of peroxisome proliferator-activating receptor-α accelerates hepatic fatty acid oxidation in neonatal pigs

Up-regulation of peroxisome proliferator-activating receptor-α (PPARα) and increasing fatty acid oxidation are important for reducing pre-weaning mortality of pigs. We examined the time-dependent regulatory effects of PPARα activation via oral postnatal clofibrate administration (75 mg/(kg-BW·d) for up to 7 days) on mitochondrial and peroxisomal fatty acid oxidation in pigs, a species with limited hepatic fatty acid oxidative capacity due to low ketogenesis. Hepatic oxidation was increased by 44-147% (depending on fatty acid chain-length) and was attained after only 4 days of clofibrate treatment. Acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase I (CPTI) activities accelerated in parallel. The increase in CPTI activity was accompanied by a rapid reduction in the sensitivity of CPTI to malonyl-CoA inhibition. The mRNA abundance of CPTI and ACO, as well as peroxisomal keto-acyl-CoA thiolase (KetoACoA) and mitochondrial malonyl-CoA decarboxylase (MCD), also were augmented greatly. However, the increase in ACO activity and MCD expression were different from CPTI, and significant interactions were observed between postnatal age and clofibrate administration. Furthermore, the expression of acetyl-CoA carboxylase β (ACCβ) decreased with postnatal age and clofibrate had no effect on its expression. Collectively these results demonstrate that the expression of PPARα target genes and the increase in fatty acid oxidation induced by clofibrate are time- and age-dependent in the liver of neonatal pigs. Although the induction patterns of CPTI, MCD, ACO, KetoACoA, and ACCβ are different during the early postnatal period, 4 days of exposure to clofibrate were sufficient to robustly accelerate fatty acid oxidation.

Hepatic PPARα function is controlled by polyubiquitination and proteasome-mediated degradation through the coordinated actions of PAQR3 and HUWE1.

Peroxisome proliferator‐activated receptor α (PPARα) is a key transcriptional factor that regulates hepatic lipid catabolism by stimulating fatty acid oxidation and ketogenesis in an adaptive response to nutrient starvation. However, how PPARα is regulated by posttranslational modification is poorly understood. In this study, we identified that progestin and adipoQ receptor 3 (PAQR3) promotes PPARα ubiquitination through the E3 ubiquitin ligase HUWE1, thereby negatively modulating PPARα functions both in vitro and in vivo. Adenovirus‐mediated Paqr3 knockdown and liver‐specific deletion of the Paqr3 gene reduced hepatic triglyceride levels while increasing fatty acid oxidation and ketogenesis upon fasting. PAQR3 deficiency enhanced the fasting‐induced expression of PPARα target genes, including those involved in fatty acid oxidation and fibroblast growth factor 21, a key molecule that mediates the metabolism‐modulating effects of PPARα. PAQR3 directly interacted with PPARα and increased the polyubiquitination and proteasome‐mediated degradation of PPARα. Furthermore, the E3 ubiquitin ligase HUWE1 was identified to mediate PPARα polyubiquitination. Additionally, PAQR3 enhanced the interaction between HUWE1 and PPARα. Conclusion: Ubiquitination modification through the coordinated action of PAQR3 with HUWE1 plays a crucial role in regulating the activity of PPARα in response to starvation. (Hepatology 2018;68:289‐303).

Mogat1 is a fasting‐induced PPARα target gene that plays a role in coordinating the hepatic response to food deprivation

During prolonged fasting, the liver plays a central role in maintaining systemic energy homeostasis by producing glucose and ketones in processes fueled by oxidation of fatty acids liberated from adipose tissue. In mice, this is also accompanied by transient hepatic accumulation of complex lipids in the form of triglycerides. In mice fasted for 24 hours, the hepatic expression of Mogat1 was significantly increased compared to mice given ad libitum access to food. Mogat1 encodes an enzyme with monoacylglycerol (MAG) acyltransferase (MGAT) activity, which is an understudied step in glycerolipid synthesis. Basal and fasting‐induced expression of Mogat1 was markedly diminished in liver of mice lacking the transcription factor peroxisome proliferator‐activated receptor α (PPARα). Suppressing Mogat1 expression with antisense‐oligonucleotides (ASO) reduced hepatic MGAT activity and hepatic triglyceride content compared to fasted ASO control treated mice. Surprisingly, the expression of many other known PPARα target genes (Dgat1, Mgll, Cpt1α, Acadm, and Acadl) was also decreased in mice treated with Mogat1 ASO. We postulated that inhibiting Mogat1 suppressed PPARα activity, potentially by loss of a ligand for PPARα. Fasted mice treated with control or Mogat1 ASO were gavaged with the synthetic PPARα ligand WY‐14643, which reversed the effects of Mogat1 ASO on liver triglyceride content as well as PPARα target gene expression. In conclusion, Mogat1 is a fasting‐induced PPARα target gene and MGAT and may feed forward to regulate liver PPARα activity during fasting.Support or Funding InformationA.J.L. was supported by funds from NIH training grant T32 DK07120. K.S.M is supported by K99 HL136658 and A.M.H. is supported by 17‐IBS‐109 from the American Diabetes Association and R56 DK111735. This work was also funded by R01 DK078187 to B.N.F. The Core services of the Diabetes Research Center (P30 DK020579) and the Nutrition Obesity Research Center (P30 DK56341) at the Washington University School of Medicine also supported this work.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Phytol-Enriched Diet Activates PPAR-α in the Liver and Brown Adipose Tissue to Ameliorate Obesity-Induced Metabolic Abnormalities.

Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated transcription factor that regulates lipid and carbohydrate metabolism. We investigate the effects of naturally occurring PPAR-α agonists, phytol, and its metabolite phytanic acid, on obesity-induced metabolic disorders using a mouse model. A luciferase reporter assay shows that phytanic acid potently activates PPAR-α among PPAR subtypes. In high-fat-diet-induced, severely obese mice, a phytol-enriched diet increases phytanic acid levels in the liver and adipose tissue, where PPAR-α is abundantly expressed. A phytol-enriched diet ameliorates severe obesity and the related metabolic abnormalities of white adipose tissue. Moreover, the expression of PPAR-α target genes in the liver and brown adipose tissue is enhanced by a phytol-enriched diet, suggesting that phytol and phytanic acid activate PPAR-α in these organs. We confirm that phytanic acid treatment induced PPAR-α target gene expression in both primary hepatocytes and brown adipocytes from wild-type mice, but not in these cells from PPAR-α-deficient mice. A phytol-enriched diet may increase phytanic acid levels in the liver and brown adipocytes, thereby activating PPAR-α in these organs and ameliorating obesity-induced metabolic diseases.

Hepatic peroxisome proliferator-activated receptor alpha mediates the major metabolic effects of Wy-14643.

Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target of various fibrate drugs clinically used to lower serum lipids. However, the tissue-specific functions of PPARα remain to be elucidated. This study aimed to explore the tissue-specific functions of PPARα in response to Wy-14643. A hepatocyte-specific Ppara knockout mouse line was used to explore the impact of hepatic PPARα activity on the systemic response to treatment with the potent PPARα agonist Wy-14643. Wy-14643 mainly activated hepatic PPARα and regulated the expression of PPARα target genes in liver. Hepatic Ppara disruption abolished the triglyceride lowering effects of Wy-14643, prevented agonist-induced hypophagia, and ablated PPARα target gene response in the liver. These findings indicate that Wy-14643 treatment mainly activates hepatic PPARα, and the hypolipidemic and hypophagic effects of Wy-14643 are dependent on PPARα activation within hepatocytes.

Perilipin 3 Deficiency Stimulates Thermogenic Beige Adipocytes Through PPARα Activation.

Beige adipocytes can dissipate energy as heat. Elaborate communication between metabolism and gene expression is important in the regulation of beige adipocytes. Although lipid droplet (LD) binding proteins play important roles in adipose tissue biology, it remains unknown whether perilipin 3 (Plin3) is involved in the regulation of beige adipocyte formation and thermogenic activities. In this study, we demonstrate that Plin3 ablation stimulates beige adipocytes and thermogenic gene expression in inguinal white adipose tissue (iWAT). Compared with wild-type mice, Plin3 knockout mice were cold tolerant and displayed enhanced basal and stimulated lipolysis in iWAT, inducing peroxisome proliferator-activated receptor α (PPARα) activation. In adipocytes, Plin3 deficiency promoted PPARα target gene and uncoupling protein 1 expression and multilocular LD formation upon cold stimulus. Moreover, fibroblast growth factor 21 expression and secretion were upregulated, which was attributable to activated PPARα in Plin3-deficient adipocytes. These data suggest that Plin3 acts as an intrinsic protective factor preventing futile beige adipocyte formation by limiting lipid metabolism and thermogenic gene expression.

The Peroxisome Proliferator-Activated Receptor α is dispensable for cold-induced adipose tissue browning in mice

ObjectiveChronic cold exposure causes white adipose tissue (WAT) to adopt features of brown adipose tissue (BAT), a process known as browning. Previous studies have hinted at a possible role for the transcription factor Peroxisome Proliferator-Activated Receptor alpha (PPARα) in cold-induced browning. Here we aimed to investigate the importance of PPARα in driving transcriptional changes during cold-induced browning in mice. MethodsMale wildtype and PPARα−/− mice were housed at thermoneutrality (28 °C) or cold (5 °C) for 10 days. Whole genome expression analysis was performed on inguinal WAT. In addition, other analyses were carried out. Whole genome expression data of livers of wildtype and PPARα−/− mice fasted for 24 h served as positive control for PPARα-dependent gene regulation. ResultsCold exposure increased food intake and decreased weight of BAT and WAT to a similar extent in wildtype and PPARα−/− mice. Except for plasma non-esterified fatty acids, none of the cold-induced changes in plasma metabolites were dependent on PPARα genotype. Histological analysis of inguinal WAT showed clear browning upon cold exposure but did not reveal any morphological differences between wildtype and PPARα−/− mice. Transcriptomics analysis of inguinal WAT showed a marked effect of cold on overall gene expression, as revealed by principle component analysis and hierarchical clustering. However, wildtype and PPARα−/− mice clustered together, even after cold exposure, indicating a similar overall gene expression profile in the two genotypes. Pathway analysis revealed that cold upregulated pathways involved in energy usage, oxidative phosphorylation, and fatty acid β-oxidation to a similar extent in wildtype and PPARα−/− mice. Furthermore, cold-mediated induction of genes related to thermogenesis such as Ucp1, Elovl3, Cox7a1, Cox8, and Cidea, as well as many PPAR target genes, was similar in wildtype and PPARα−/− mice. Finally, pharmacological PPARα activation had a minimal effect on expression of cold-induced genes in murine WAT. ConclusionCold-induced changes in gene expression in inguinal WAT are unaltered in mice lacking PPARα, indicating that PPARα is dispensable for cold-induced browning.

PPARα-Target Gene Expression Requires TIS21/BTG2 Gene in Liver of the C57BL/6 Mice under Fasting Condition.

The TIS21/BTG2/PC3 gene belongs to the antiproliferative gene (APRO) family and exhibits tumor suppressive activity. However, here we report that TIS21 controls lipid metabolism, rather than cell proliferation, under fasting condition. Using microarray analysis, whole gene expression changes were investigated in liver of TIS21 knockout (TIS21-KO) mice after 20 h fasting and compared with wild type (WT). Peroxisome proliferator-activated receptor alpha (PPARα) target gene expression was almost absent in contrast to increased lipid synthesis in the TIS21-KO mice compared to WT mice. Immunohistochemistry with hematoxylin and eosin staining revealed that lipid deposition was focal in the TIS21-KO liver as opposed to the diffuse and homogeneous pattern in the WT liver after 24 h starvation. In addition, cathepsin E expression was over 10 times higher in the TIS21-KO liver than that in the WT, as opposed to the significant reduction of thioltransferase in both adult and fetal livers. At present, we cannot account for the role of cathepsin E. However, downregulation of glutaredoxin 2 thioltransferase expression might affect hypoxic damage in the TIS21-KO liver. We suggest that the TIS21/BTG2 gene might be essential to maintain energy metabolism and reducing power in the liver under fasting condition.

4′,6-Dimethoxyisoflavone-7-O-β-d-glucopyranoside (wistin) is a peroxisome proliferator-activated receptor α (PPARα) agonist in mouse hepatocytes

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that regulate lipid and glucose metabolism. PPARα mainly affects fatty acid metabolism, and its activation lowers lipid levels. PPARγ is involved in the regulation of adipogenesis, insulin sensitivity, energy balance, and lipid biosynthesis. We have previously reported that 4',6-dimethoxyisoflavone-7-O-β-D-glucopyranoside (wistin) can activate PPARγ. The purpose of the present study is to investigate the PPARα agonist activity of wistin. Using a luciferase reporter assay system of PPARα in monkey COS7 kidney cells, we showed that wistin could activate PPARα (P < 0.01at 10μg/mL) in a dose-dependent manner. Moreover, the addition of wistin upregulated the expression of PPARα (P < 0.01at 10μg/mL) and PPARα target genes including carnitine palmitoyltransferase 1a (P < 0.05at 10μg/mL), acyl-CoA oxidase (P < 0.01at 10μg/mL), acyl-CoA synthase (P < 0.05at 10μg/mL), PPARγ coactivator 1α (P < 0.05at 10μg/mL), uncoupling protein 2 (P < 0.05at 1μg/mL), and uncoupling protein 3 (P < 0.05at 10μg/mL), which are genes involved in lipid efflux and energy expenditure, in mouse primary hepatocytes. Furthermore, wistin inhibited cellular triglyceride accumulation in hepatocytes (P < 0.05at 10μg/mL) in a dose-dependent manner. These results indicate that wistin could suppress lipid accumulation through PPARα activation. The action of wistin on PPARα could be of interest for the amelioration of lipid metabolic disorders. To the best of our knowledge, wistin is the first reported isoflavonoid O-glycoside with PPARα agonist activity.

Targeting Peroxisome Proliferator Activated Receptor α (PPAR α) for the Prevention of Mitochondrial Impairment and Hypertrophy in Cardiomyocytes

Background/Aims: Morphological and biochemical maladaptation of cardiomyocytes are associated with mitochondrial dysfunction and dysregulation in hypertrophic conditions. Peroxisome proliferator activated receptor α (PPARα), a drug target for dyslipidemia, is known to be downregulated in cardiomyocytes in response to hypertrophic stimuli. The current study was undertaken to investigate the role of PPARα signaling in mitochondrial remodeling and thereby dysregulation of cardiomyocytes due to hypertrophy in vitro. Methods: Rat cardiomyocytes H9c2 (2-1) and neonatal rat ventricular myocytes (NRVMs) were cultured and treated with α₁-adrenergic agonist phenylephrine (PE, 100 µM, 24 hours) in the presence or absence of 10 µM fenofibrate or bezafibrate. Cellular hypertrophy was observed by atomic force microscopy and immunofluorescence with F-actin antibody. mRNA levels of hypertrophic marker genes and other genes were examined by quantitative real time PCR. Structural as well as functional remodeling of the mitochondria were evaluated by immunofluorescence (F-actin and COX-I), live cell imaging microscopy (JC-I, mitotracker), mitochondrial complex V activity, MPTP activity and ATP assay. Oxidative stress was measured by using sensitive fluorescent indicator probes. Cellular and mitochondrial calcium were measured by using fluorescent indicator probes Rhod-2 AM and X-rhod-1 AM, respectively. Targetscan prediction analysis was performed to find out miRNAs as putative regulators of VDAC. Luciferase assay was conducted to confirm binding of miR28 with VDAC. Results: Co-treatment of H9c2(2-1) cells with PE and fenofibrate restricted increase in cell size and expression of marker genes such as atrial-natriuretic peptide (ANP), brain-natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) compared to those with PE alone. Fenofibrate prevented PE-induced down regulation of PPARα-target genes like CPT-I and MCAD. Mitochondrial trans-membrane potential (Δψ_m) and motility were reduced by PE which were significantly checked by fenofibrate. Increased ROS production and calcium level in PE-treated cells were ameliorated by fenofibrate. Mitochondrial activity and ATP generation were reduced by PE which was rescued by fenofibrate. Fenofibrate also prevented PE-induced down regulation of mitochondrial genes like VDAC-I and COX-IV. Expression of several miRNAs was altered in hypertrophic cardiomyocytes which were restored when co-treated with fenofibrate. miR28 was found to target 3’ untranslated region of VDAC-I. Conclusion: Overall, the results demonstrate that PPARα signaling is critically involved in mitochondrial dysfunction in hypertrophic cardiomyocytes in which miR28 plays a pivotal role.

Small heterodimer partner (SHP) deficiency protects myocardia from lipid accumulation in high fat diet-fed mice.

The small heterodimer partner (SHP) regulates fatty acid oxidation and lipogenesis in the liver by regulating peroxisome proliferator-activated receptor (PPAR) γ expression. SHP is also abundantly expressed in the myocardium. We investigated the effect of SHP expression on myocardia assessing not only heart structure and function but also lipid metabolism and related gene expression in a SHP deletion animal model. Transcriptional profiling with a microarray revealed that genes participating in cell growth, cytokine signalling, phospholipid metabolism, and extracellular matrix are up-regulated in the myocardia of SHP knockout (KO) mice compared to those of wild-type (WT) mice (nominal p value < 0.05). Consistent with these gene expression changes, the left ventricular masses of SHP KO mice were significantly higher than WT mice (76.8 ± 20.5 mg vs. 52.8 ± 6.8 mg, P = 0.0093). After 12 weeks of high fat diet (HFD), SHP KO mice gained less weight and exhibited less elevation in serum-free fatty acid and less ectopic lipid accumulation in the myocardium than WT mice. According to microarray analysis, genes regulated by PPARγ1 and PPARα were down-regulated in myocardia of SHP KO mice compared to their expression in WT mice after HFD, suggesting that the reduction in lipid accumulation in the myocardium resulted from a decrease in lipogenesis regulated by PPARγ. We confirmed the reduced expression of PPARγ1 and PPARα target genes such as CD36, medium-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, and very long-chain acyl-CoA dehydrogenase by SHP KO after HFD.

Ketogenic Diet Reduces Midlife Mortality and Improves Memory in Aging Mice

Ketogenic diets recapitulate certain metabolic aspects of dietary restriction such as reliance on fattyacid metabolism and production of ketone bodies. We investigated whether an isoprotein ketogenic diet (KD) might, like dietary restriction, affect longevity and healthspan in C57BL/6 male mice. We find that Cyclic KD, KD alternated weekly with the Control diet to prevent obesity, reduces midlife mortality but does not affect maximum lifespan. A non-ketogenic high-fat diet (HF) fed similarly may have an intermediate effect on mortality. Cyclic KD improves memory performance in old age, while modestly improving composite healthspan measures. Gene expression analysis identifies downregulation of insulin, protein synthesis, and fatty acid synthesis pathways as mechanisms common to KD and HF. However, upregulation of PPARα target genes is unique to KD, consistent across tissues, and preserved in old age. In all, we show that a non-obesogenic ketogenic diet improves survival, memory, and healthspan in aging mice.

Abstract 276: The Role of Diacylglycerol Acetyltransferase 1 and 2 in Cardiac Metabolism and Function

Cardiac triglyceride (TG) plays an important role in myocardial metabolism. TG synthesis is catalyzed by diacylglycerol:acetyltransferase (DGAT). Enhancing cardiac TG synthesis and turnover, by means of overexpression DGAT1, has protected hearts against stresses while blocking the TG turnover causes cardiomyopathy. In the meantime DGAT inhibitors are being developed for lipid lowering therapy, raising concerns whether DGAT inhibition affects cardiac function. Here we determined the role of the two cardiac DGAT isoforms in TG synthesis and turnover in the heart and their contribution to cardiac fatty acid metabolism. Using an inducible cardiac specific DGAT1 deletion mouse (iKO) together with DGAT2-specific inhibitor, we were able to achieve graded inhibition of TG synthesis and turnover as determined by 13 C-NMR spectroscopy of isolated perfused mouse heart. The iKO heart has normal TG level (CON 5.7±1.2 vs. 6.9±0.7 μg/mg wwt) and perfusing hearts with glucose (5.5mM), fatty acids (0.4mM) and lactate (1.2mM) for 1hr did not change the TG content in control (CON, pre-perfusion 3.9 ± 0.6 vs. post-perfusion 4.5 ± 0.7 μg/mg wwt), iKO (3.8±0.6 vs. 4.2±0.5 μg/mg wwt) or iKO+DGAT2 inhibitor (3.8±0.6 vs. 5.0±0.8 μg/mg wwt). Relative to CON, the rate of 13 C labeled fatty acids incorporation into the TG pool decreased by 32% in iKO (AUC 5.170 of 7.547, p&lt;0.05) which was accompanied by an increase the oxidation of exogenous fatty acids (relative FAO: 48.5±5.3 for CON vs. 67.0±4.1% for iKO, p&lt;0.05). Cardiac function, assessed by echocardiography (FS: CON 47.5±1.3 vs. iKO 45.8±3.0%, p&gt;0.05) or by rate pressure product of the isolated perfused heart (CON 39149±1047 vs. iKO 40836±3424 bpm*mmHg, p&gt;0.05) is normal in iKO hearts and remained unchanged after treatment with the DGAT2 inhibitor (37083±8507 bpm*mmHg). Coinhibition of DGAT1 and 2 abrogated 13 C labeled fatty acids incorporation into the TG pool by 58% (AUC 5.042 of 11.82, p&lt;0.05) and suppressed expression of PPARα target genes relative to non-treated control hearts (p&lt;0.05). Taken together, our data show that both DGAT1 and 2 contribute TG synthesis in the heart. Inhibition of both isoforms abrogated TG synthesis and reduced PPARα activity in the heart but did not affect cardiac function in isolated perfused heart.

Citreoviridin induces triglyceride accumulation in hepatocytes through inhibiting PPAR-α in vivo and in vitro

Citreoviridin (CIT) is a mycotoxin produced by Penicillum citreonigrum, Aspergillus terreus and Eupenicillium ochrosalmoneum. CIT occurs naturally in moldy rice and corn. CIT is associated with the development of atherosclerosis in the general population. Alteration in hepatic lipid metabolism is a pathogenic factor in atherosclerosis. However the effect and the underlying mechanism of CIT on hepatic lipid metabolism are largely unknown. In this study, we reported that CIT induced triglyceride accumulation in mice liver and human liver HepG2 cells as shown in oil red O staining. CIT (0.1 mg/kg–0.3 mg/kg) for 6 weeks elevated liver triglyceride contents in mice. CIT inhibited the transactivation activity of peroxisome proliferator-activated receptor-α (PPAR-α) in hepatocyte in vivo and in vitro, as shown by the reduced mRNA levels of PPAR-α target genes which play key roles in lipid metabolism in various aspects. PPAR-α agonist fenofibrate attenuated CIT-induced triglyceride accumulation in HepG2 cells. Furthermore, CIT increased serum total cholesterol/high-density lipoprotein cholesterol ratio, a strong risk factor for cardiovascular disease. In summary, we reported that CIT induced PPAR-α−dependent hepatic triglyceride accumulation and dyslipidemia. Our data will provide new mechanistic insights into CIT-induced lipid alterations.

Acyl-CoA Thioesterase 1 (ACOT1) Regulates PPARα to Couple Fatty Acid Flux With Oxidative Capacity During Fasting.

Hepatic acyl-CoA thioesterase 1 (ACOT1) catalyzes the conversion of acyl-CoAs to fatty acids (FAs) and CoA. We sought to determine the role of ACOT1 in hepatic lipid metabolism in C57Bl/6J male mice 1 week after adenovirus-mediated Acot1 knockdown. Acot1 knockdown reduced liver triglyceride (TG) as a result of enhanced TG hydrolysis and subsequent FA oxidation. In vitro experiments demonstrated that Acot1 knockdown led to greater TG turnover and FA oxidation, suggesting that ACOT1 is important for controlling the rate of FA oxidation. Despite increased FA oxidation, Acot1 knockdown reduced the expression of peroxisome proliferator–activated receptor α (PPARα) target genes, whereas overexpression increased PPARα reporter activity, suggesting ACOT1 regulates PPARα by producing FA ligands. Moreover, ACOT1 exhibited partial nuclear localization during fasting and cAMP/cAMP-dependent protein kinase signaling, suggesting local regulation of PPARα. As a consequence of increased FA oxidation and reduced PPARα activity, Acot1 knockdown enhanced hepatic oxidative stress and inflammation. The effects of Acot1 knockdown on PPARα activity, oxidative stress, and inflammation were rescued by supplementation with Wy-14643, a synthetic PPARα ligand. We demonstrate through these results that ACOT1 regulates fasting hepatic FA metabolism by balancing oxidative flux and capacity.

Identification and characterization of a novel PPARα-regulated and 7α-hydroxyl bile acid-preferring cytosolic sulfotransferase mL-STL (Sult2a8)

PPARα has been known to play a pivotal role in orchestrating lipid, glucose, and amino acid metabolism via transcriptional regulation of its target gene expression during energy deprivation. Recent evidence has also suggested that PPARα is involved in bile acid metabolism, but how PPARα modulates the homeostasis of bile acids during fasting is still not clear. In a mechanistic study aiming to dissect the spectrum of PPARα target genes involved in metabolic response to fasting, we identified a novel mouse gene (herein named mL-STL for mouse liver-sulfotransferase-like) that shared extensive homology with the Sult2a subfamily of a superfamily of cytosolic sulfotransferases, implying its potential function in sulfonation. The mL-STL gene expressed predominantly in liver in fed state, but PPARα was required to sustain its expression during fasting, suggesting a critical role of PPARα in regulating the mL-STL-mediated sulfonation during fasting. Functional studies using recombinant His-tagged mL-STL protein revealed its narrow sulfonating activities toward 7α-hydroxyl primary bile acids, including cholic acid, chenodeoxycholic acid, and α-muricholic acid, and thus suggesting that mL-STL may be the major hepatic bile acid sulfonating enzyme in mice. Together, these studies identified a novel PPARα-dependent gene and uncovered a new role of PPARα as being an essential regulator in bile acid biotransformation via sulfonation during fasting.