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Feeding of rapeseed (canola) oil with a high erucic acid concentration is known to cause hepatic steatosis in animals. Mitochondrial fatty acid oxidation plays a central role in liver lipid homeostasis, so it is possible that hepatic metabolism of erucic acid might decrease mitochondrial fatty acid oxidation. However, the precise mechanistic relationship between erucic acid levels and mitochondrial fatty acid oxidation is unclear. Using male Sprague–Dawley rats, along with biochemical and molecular biology approaches, we report here that peroxisomal β-oxidation of erucic acid stimulates malonyl-CoA formation in the liver and thereby suppresses mitochondrial fatty acid oxidation. Excessive hepatic uptake and peroxisomal β-oxidation of erucic acid resulted in appreciable peroxisomal release of free acetate, which was then used in the synthesis of cytosolic acetyl-CoA. Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD(+) ratio, suppressed sirtuin 1 (SIRT1) activity, and thereby activated acetyl-CoA carboxylase, which stimulated malonyl-CoA biosynthesis from acetyl-CoA. Chronic feeding of a diet including high-erucic-acid rapeseed oil diminished mitochondrial fatty acid oxidation and caused hepatic steatosis and insulin resistance in the rats. Of note, administration of a specific peroxisomal β-oxidation inhibitor attenuated these effects. Our findings establish a cross-talk between peroxisomal and mitochondrial fatty acid oxidation. They suggest that peroxisomal oxidation of long-chain fatty acids suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation, which might play a role in fatty acid–induced hepatic steatosis and related metabolic disorders.

Beige fat is a potential therapeutic target for obesity and other metabolic diseases due to its inducible brown fat-like functions. Inguinal white adipose tissue (iWAT) can undergo robust brown remodeling with appropriate stimuli and is therefore widely considered as a representative beige fat depot. However, adipose tissues residing in different anatomic depots exhibit a broad range of plasticity, raising the possibility that better beige fat depots with greater plasticity may exist. Here we identified and characterized a novel, naturally-existing beige fat depot, thigh adipose tissue (tAT). Unlike classic WATs, tAT maintains beige fat morphology at room temperature, whereas high-fat diet (HFD) feeding or aging promotes the development of typical WAT features, namely unilocular adipocytes. The brown adipocyte gene expression in tAT is consistently higher than in iWAT under cold exposure, HFD feeding, and rosiglitazone treatment conditions. Our molecular profiling by RNA-Seq revealed up-regulation of energy expenditure pathways and repressed inflammation in tAT relative to eWAT and iWAT. Furthermore, we demonstrated that the master fatty acid oxidation regulator peroxisome proliferator-activated receptor α is dispensable for maintaining and activating the beige character of tAT. Therefore, we have identified tAT as a natural beige adipose depot in mice with a unique molecular profile that does not require peroxisome proliferator-activated receptor α.

Fatty acid channeling into oxidation or storage modes depends on physiological conditions and hormonal signaling. However, the directionality of this channeling may also depend on the association of each of the five acyl-CoA synthetase isoforms with specific protein partners. Long-chain acyl-CoA synthetases (ACSLs) catalyze the conversion of long-chain fatty acids to fatty acyl-CoAs, which are then either oxidized or used in esterification reactions. In highly oxidative tissues, ACSL1 is located on the outer mitochondrial membrane (OMM) and directs fatty acids into mitochondria for β-oxidation. In the liver, however, about 50% of ACSL1 is located on the endoplasmic reticulum (ER) where its metabolic function is unclear. Because hepatic fatty acid partitioning is likely to require the interaction of ACSL1 with other specific proteins, we used an unbiased protein interaction technique, BioID, to discover ACSL1-binding partners in hepatocytes. We targeted ACSL1 either to the ER or to the OMM of Hepa 1–6 cells as a fusion protein with the Escherichia coli biotin ligase, BirA*. Proteomic analysis identified 98 proteins that specifically interacted with ACSL1 at the ER, 55 at the OMM, and 43 common to both subcellular locations. We found subsets of peroxisomal and lipid droplet proteins, tethering proteins, and vesicle proteins, uncovering a dynamic role for ACSL1 in organelle and lipid droplet interactions. Proteins involved in lipid metabolism were also identified, including acyl-CoA–binding proteins and ceramide synthase isoforms 2 and 5. Our results provide fundamental and detailed insights into protein interaction networks that control fatty acid metabolism.

The receptor cycle of type I peroxisomal matrix protein import is completed by ubiquitination of the membrane-bound peroxisome biogenesis factor 5 (Pex5p) and its subsequent export back to the cytosol. The receptor export is the only ATP-dependent step of the whole process and is facilitated by two members of the AAA family of proteins (ATPases associated with various cellular activities), namely Pex1p and Pex6p. To gain further insight into substrate recognition by the AAA complex, we generated an N-terminally linked ubiquitin–Pex5p fusion protein. This fusion protein displayed biological activity because it is able to functionally complement a PEX5-deletion in Saccharomyces cerevisiae. In vitro assays revealed its interaction at WT level with the native cargo protein Pcs60p and Pex14p, a constituent of the receptor docking complex. We also demonstrate in vitro deubiquitination by the deubiquitinating enzyme Ubp15p. In vitro pulldown assays and cross-linking studies demonstrate that Pex5p recognition by the AAA complex depends on the presence of the ubiquitin moiety and is mediated by Pex1p.

Analogous to the c-Myc (Myc)/Max family of bHLH-ZIP transcription factors, there exists a parallel regulatory network of structurally and functionally related proteins with Myc-like functions. Two related Myc-like paralogs, termed MondoA and MondoB/carbohydrate response element–binding protein (ChREBP), up-regulate gene expression in heterodimeric association with the bHLH-ZIP Max-like factor Mlx. Myc is necessary to support liver cancer growth, but not for normal hepatocyte proliferation. Here, we investigated ChREBP's role in these processes and its relationship to Myc. Unlike Myc loss, ChREBP loss conferred a proliferative disadvantage to normal murine hepatocytes, as did the combined loss of ChREBP and Myc. Moreover, hepatoblastomas (HBs) originating in myc(−/−), chrebp(−/−), or myc(−/−)/chrebp(−/−) backgrounds grew significantly more slowly. Metabolic studies on livers and HBs in all three genetic backgrounds revealed marked differences in oxidative phosphorylation, fatty acid β-oxidation (FAO), and pyruvate dehydrogenase activity. RNA-Seq of livers and HBs suggested seven distinct mechanisms of Myc–ChREBP target gene regulation. Gene ontology analysis indicated that many transcripts deregulated in the chrebp(−/−) background encode enzymes functioning in glycolysis, the TCA cycle, and β- and ω-FAO, whereas those dysregulated in the myc(−/−) background encode enzymes functioning in glycolysis, glutaminolysis, and sterol biosynthesis. In the myc(−/−)/chrebp(−/−) background, additional deregulated transcripts included those involved in peroxisomal β- and α-FAO. Finally, we observed that Myc and ChREBP cooperatively up-regulated virtually all ribosomal protein genes. Our findings define the individual and cooperative proliferative, metabolic, and transcriptional roles for the “Extended Myc Network” under both normal and neoplastic conditions.

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.

Xanthine oxidase (XOD), also known as xanthine dehydrogenase, is a rate-limiting enzyme in purine nucleotide degradation, which produces uric acid. Uric acid concentrations in the blood and liver exhibit circadian oscillations in both humans and rodents; however, the underlying mechanisms remain unclear. Here, we demonstrate that XOD expression and enzymatic activity exhibit circadian oscillations in the mouse liver. We found that the orphan nuclear receptor peroxisome proliferator–activated receptor-α (PPARα) transcriptionally activated the mouse XOD gene and that bile acids suppressed XOD transactivation. The synthesis of bile acids is known to be under the control of the circadian clock, and we observed that the time-dependent accumulation of bile acids in hepatic cells interfered with the recruitment of the co-transcriptional activator p300 to PPARα, thereby repressing XOD expression. This time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the hepatic expression of XOD, which, in turn, led to circadian alterations in uric acid production. Finally, we also demonstrated that the anti-hyperuricemic effect of the XOD inhibitor febuxostat was enhanced by administering it at the time of day before hepatic XOD activity increased. These results suggest an underlying mechanism for the circadian alterations in uric acid production and also underscore the importance of selecting an appropriate time of day for administering XOD inhibitors.

The transcription factors Msn2 and Msn4 (multicopy suppressor of SNF1 mutation proteins 2 and 4) bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae. However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid β-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4-binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wild-type cells. Of note, the double mutant msn2Δmsn4Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major β-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of β-oxidation genes in glucose-depleted conditions, and in vivo reporter gene analysis indicated reduced expression of these genes in msn2Δmsn4Δ cells. Moreover, mobility shift assays revealed that Msn4 binds β-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2Δmsn4Δ strain had fewer peroxisomes than the wild type, and lipid analysis indicated that the msn2Δmsn4Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. Because glucose sensing, signaling, and fatty acid β-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2Δmsn4Δ strain could therefore be a good model system for further study of these critical processes.

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei. When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30–60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

International audience; ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.

Acyl-CoA binding domain-containing 5 (ACBD5) is a peroxisomal protein that carries an acyl-CoA binding domain (ACBD) at its N-terminal region. The recent identification of a mutation in the ACBD5 gene in patients with a syndromic form of retinal dystrophy highlights the physiological importance of ACBD5 in humans. However, the underlying pathogenic mechanisms and the precise function of ACBD5 remain unclear. We herein report that ACBD5 is a peroxisomal tail-anchored membrane protein exposing its ACBD to the cytosol. Using patient-derived fibroblasts and ACBD5 knock-out HeLa cells generated via genome editing, we demonstrate that ACBD5 deficiency causes a moderate but significant defect in peroxisomal β-oxidation of very-long-chain fatty acids (VLCFAs) and elevates the level of cellular phospholipids containing VLCFAs without affecting peroxisome biogenesis, including the import of membrane and matrix proteins. Both the N-terminal ACBD and peroxisomal localization of ACBD5 are prerequisite for efficient VLCFA β-oxidation in peroxisomes. Furthermore, ACBD5 preferentially binds very-long-chain fatty acyl-CoAs (VLC-CoAs). Together, these results suggest a direct role of ACBD5 in peroxisomal VLCFA β-oxidation. Based on our findings, we propose that ACBD5 captures VLC-CoAs on the cytosolic side of the peroxisomal membrane so that the transport of VLC-CoAs into peroxisomes and subsequent β-oxidation thereof can proceed efficiently. Our study reclassifies ACBD5-related phenotype as a novel peroxisomal disorder.

Fasting induces lipid accumulation in the liver, while the mechanisms by which fasting dysregulates liver fatty acid oxidation are not clear. Fatty acid ω-oxidation is induced in the fasting state, and administration of dicarboxylic acids to fasting animals decreases plasma ketone bodies. We hypothesized that endogenous dicarboxylic acids might play a role in controlling mitochondrial β-oxidation in fasting animals. A peroxisome proliferator-activated receptor-alpha agonist and an inhibitor for peroxisomal β-oxidation were administered to the fasting rats to investigate the role of dicarboxylic acids in liver fatty acid oxidation and lipid homeostasis. We observed that excessive β-oxidation of endogenous dicarboxylic acids by peroxisomes generated considerable levels of succinate in the liver. Excessive succinate oxidation subsequently increased the mitochondrial NADH/NAD+ ratio and led to an accumulation of 3-OH-CoA and 2-enoyl-CoA intermediates in the liver. This further induced feedback suppression of mitochondrial β-oxidation and promoted hepatic lipid deposition and steatosis. Specific inhibition of peroxisomal β-oxidation attenuated fasting-induced lipid deposition in the liver by reducing succinate production and enhancing mitochondrial fatty acid oxidation. We conclude that suppression of mitochondrial β-oxidation by oxidation of dicarboxylic acids serves as a mechanism for fasting-induced hepatic lipid accumulation and identifies cross talk between peroxisomal and mitochondrial fatty acid oxidation.

Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. Capitalizing on this therapeutically, however, has been only moderately successful because of the relatively small magnitude of these differences and because cancers may further adapt their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bifunctional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate the toxic metabolite dodecanedioic acid (DDDA), which causes acute hepatocyte necrosis and liver failure. We noted that, in a murine model of pediatric hepatoblastoma (HB) and in primary human HBs, downregulation of Ehhadh occurs in association with the suppression of mitochondrial β- and endosomal/peroxisomal ω-fatty acid oxidation pathways. This suggested that HBs might be more susceptible than normal liver tissue to C12 dietary intervention. Indeed, HB-bearing mice provided with C12- and/or DDDA-supplemented diets survived significantly longer than those on standard diets. In addition, larger tumors developed massive necrosis following short-term DDDA administration. In some HBs, the eventual development of DDDA resistance was associated with 129 transcript differences, ∼90% of which were downregulated, and approximately two-thirds of which correlated with survival in numerous human cancers. These transcripts often encoded extracellular matrix components, suggesting that DDDA resistance arises from reduced Ehhadh uptake. Lower Ehhadh expression was also noted in murine hepatocellular carcinomas and in subsets of certain human cancers, supporting the likely generality of these results. Our results demonstrate the feasibility of C12 or DDDA dietary supplementation that is nontoxic, inexpensive, and likely compatible with more standard chemotherapies.

Physical interaction between organelles is a flexible event and essential for cells to adapt rapidly to environmental stimuli. Germinating plants utilize oil bodies and peroxisomes to mobilize storage lipids for the generation of sucrose as the main energy source. Although membrane interaction between oil bodies and peroxisomes has been widely observed, its underlying molecular mechanism is largely unknown. Here we present genetic evidence for control of the physical interaction between oil bodies and peroxisomes. We identified alleles of the sdp1 mutant altered in oil body morphology. This mutant accumulates bigger and more oil body aggregates compared with the wild type and showed defects in lipid mobilization during germination. SUGAR DEPENDENT 1 (SDP1) encodes major triacylglycerol lipase in Arabidopsis. Interestingly, sdp1 seedlings show enhanced physical interaction between oil bodies and peroxisomes compared with the wild type, whereas exogenous sucrose supplementation greatly suppresses the interaction. The same phenomenon occurs in the peroxisomal defective 1 (ped1) mutant, defective in lipid mobilization because of impaired peroxisomal β-oxidation, indicating that sucrose production is a key factor for oil body-peroxisomal dissociation. Peroxisomal dissociation and subsequent release from oil bodies is dependent on actin filaments. We also show that a peroxisomal ATP binding cassette transporter, PED3, is the potential anchor protein to the membranes of these organelles. Our results provide novel components linking lipid metabolism and oil body-peroxisome interaction whereby sucrose may act as a negative signal for the interaction of oil bodies and peroxisomes to fine-tune lipolysis.

Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and β-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor α, which plays a dominant role in the expression of β-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor α is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced β-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced β-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis.

Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

Phospholipase A/acyltransferase (PLA/AT)-3 (also known as H-rev107 or AdPLA) was originally isolated as a tumor suppressor and was later shown to have phospholipase A1/A2 activity. We have also found that the overexpression of PLA/AT-3 in mammalian cells results in specific disappearance of peroxisomes. However, its molecular mechanism remained unclear. In the present study, we first established a HEK293 cell line, which stably expresses a fluorescent peroxisome marker protein (DsRed2-Peroxi) and expresses PLA/AT-3 in a tetracycline-dependent manner. The treatment with tetracycline, as expected, caused disappearance of peroxisomes within 24 h, as revealed by diffuse signals of DsRed2-Peroxi and a remarkable decrease in a peroxisomal membrane protein, PMP70. A time-dependent decrease in ether-type lipid levels was also seen. Because the activation of LC3, a marker of autophagy, was not observed, the involvement of autophagy was unlikely. Among various peroxins responsible for peroxisome biogenesis, Pex19p functions as a chaperone protein for the transportation of peroxisomal membrane proteins. Immunoprecipitation analysis showed that PLA/AT-3 binds to Pex19p through its N-terminal proline-rich and C-terminal hydrophobic domains. The protein level and enzyme activity of PLA/AT-3 were increased by its coexpression with Pex19p. Moreover, PLA/AT-3 inhibited the binding of Pex19 to peroxisomal membrane proteins, such as Pex3p and Pex11βp. A catalytically inactive point mutant of PLA/AT-3 could bind to Pex19p but did not inhibit the chaperone activity of Pex19p. Altogether, these results suggest a novel regulatory mechanism for peroxisome biogenesis through the interaction between Pex19p and PLA/AT-3. Background: The overexpression of phospholipase A/acyltransferase-3 (PLA/AT-3) in mammalian cells causes specific disappearance of peroxisomes. Results: PLA/AT-3 bound to Pex19p, one of peroxins, and inhibited the binding of Pex19p to peroxisomal membrane proteins. Conclusion: The interaction between Pex19p and PLA/AT-3 may be related to the down-regulation of peroxisomes by PLA/AT-3. Significance: PLA/AT-3 may be involved in a novel regulatory mechanism for peroxisome biogenesis.

We investigated the compartmentation of the catabolism of dodecanedioate (DODA), azelate, and glutarate in perfused rat livers, using a combination of metabolomics and mass isotopomer analyses. Livers were perfused with recirculating or nonrecirculating buffer containing one fully 13C-labeled dicarboxylate. Information on the peroxisomal versus mitochondrial catabolism was gathered from the labeling patterns of acetyl-CoA proxies, i.e. total acetyl-CoA, the acetyl moiety of citrate, C-1 + 2 of β-hydroxybutyrate, malonyl-CoA, and acetylcarnitine. Additional information was obtained from the labeling patterns of citric acid cycle intermediates and related compounds. The data characterize the partial oxidation of DODA and azelate in peroxisomes, with terminal oxidation in mitochondria. We did not find evidence of peroxisomal oxidation of glutarate. Unexpectedly, DODA contributes a substantial fraction to anaplerosis of the citric acid cycle. This opens the possibility to use water-soluble DODA in nutritional or pharmacological anaplerotic therapy when other anaplerotic substrates are impractical or contraindicated, e.g. in propionic acidemia and methylmalonic acidemia.

Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4(-/-) mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4(-/-) mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4(-/-) mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4(-/-) BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4(-/-) brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.

The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have been characterized as peroxisomal targeting signals (PTS) residing either at the C terminus (PTS1) or close to the N terminus (PTS2). PTS2-carrying proteins interact with their cognate receptor protein PEX7 that mediates their transport to peroxisomes by a concerted action with a co-receptor protein, which in mammals is the PTS1 receptor PEX5L. Using a modified version of the mammalian two-hybrid assay, we demonstrate that the interaction strength between cargo and PEX7 is drastically increased in the presence of the co-receptor PEX5L. In addition, cargo binding is a prerequisite for the interaction between PEX7 and PEX5L and ectopic overexpression of PTS2-carrying cargo protein drastically increases the formation of PEX7-PEX5L complexes in this assay. Consistently, we find that the peroxisomal transfer of PEX7 depends on cargo binding and that ectopic overexpression of cargo protein stimulates this process. Thus, the sequential formation of a highly stable trimeric complex involving cargo protein, PEX7 and PEX5L stabilizes cargo binding and is a prerequisite for PTS2-mediated peroxisomal import.

In the n-alkane assimilating yeast Yarrowia lipolytica, n-alkanes are oxidized to fatty acids via fatty alcohols and fatty aldehydes, after which they are utilized as carbon sources. Here, we show that four genes (HFD1-HFD4) encoding fatty aldehyde dehydrogenases (FALDHs) are involved in the metabolism of n-alkanes in Y. lipolytica. A mutant, in which all of four HFD genes are deleted (Δhfd1-4 strain), could not grow on n-alkanes of 12-18 carbons; however, the expression of one of those HFD genes restored its growth on n-alkanes. Production of Hfd2Ap or Hfd2Bp, translation products of transcript variants generated from HFD2 by the absence or presence of splicing, also supported the growth of the Δhfd1-4 strain on n-alkanes. The FALDH activity in the extract of the wild-type strain was increased when cells were incubated in the presence of n-decane, whereas this elevation in FALDH activity by n-decane was not observed in Δhfd1-4 strain extract. Substantial FALDH activities were detected in the extracts of Escherichia coli cells expressing the HFD genes. Fluorescent microscopic observation suggests that Hfd3p and Hfd2Bp are localized predominantly in the peroxisome, whereas Hfd1p and Hfd2Ap are localized in both the endoplasmic reticulum and the peroxisome. These results suggest that the HFD multigene family is responsible for the oxidation of fatty aldehydes to fatty acids in the metabolism of n-alkanes, and raise the possibility that Hfd proteins have diversified by gene multiplication and RNA splicing to efficiently assimilate or detoxify fatty aldehydes in Y. lipolytica.

Pex1p and Pex6p are required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We herein show that mammalian Pex26p directly binds to Pex14p, the initial docking receptor of Pex5p, and interacts with Pex5p via Pex14p. The binding affinity of Pex26p to Pex14p is altered by Pex5p. Further evidence suggests that the N-terminal region in Pex26p acts as a scaffold protein to recruit Pex14p·Pex5p complex together with Pex1p·Pex6p complexes on peroxisomes. Pex26p binding to Pex14p was suppressed by overexpression of Pex1p and Pex6p in an ATP-dependent manner, whereas Pex14p was not competed out by Pex1p and Pex6p from Pex26p mutant defective in peroxisomal matrix protein import. These results suggested that peroxisome biogenesis requires Pex1p- and Pex6p-regulated dissociation of Pex14p from Pex26p. Pex1p homo-oligomer directly binds to Pex5p as assessed by a surface plasmon resonance-based assay. Moreover, cytosolic Pex1p is likely to maintain the functional oligomer of Pex5p. Taken together, in the peroxisomal protein import, AAA peroxins modulate the interaction between Pex26p and Pex14p on peroxisome membrane as well as Pex5p oligomer in the cytosol.

The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1Δ gat2Δ. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.

Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.

In Pichia pastoris, the peroxisomal targeting signal 2 (PTS2)-dependent peroxisomal matrix protein import pathway requires the receptor, Pex7, and its co-receptor Pex20. A conserved lysine (Lys(19)) near the N terminus of Pex20 is required for its polyubiquitination and proteasomal degradation, whereas a conserved cysteine (Cys(8)) is essential for its recycling. In this study, we found that Cys(8) is required for the DTT-sensitive mono- and diubiquitination of Pex20. We also show that the PTS2 cargo receptor, Pex7, is required for Pex20 polyubiquitination. Pex4, the E2 ubiquitin-conjugation enzyme, is required for monoubiquitination of Pex20. However, it is also necessary for polyubiquitination of Pex20, making its behavior distinct from the ubiquitination described for other PTS receptors. Unlike the roles of specific RING peroxins in Pex5 ubiquitination, we found that all the RING peroxins (Pex2, Pex10, and Pex12) are required as E3 ubiquitin ligases for Pex20 mono- and polyubiquitination. A model for Pex20 ubiquitination is proposed based on these observations. This is the first description of the complete ubiquitination pathway of Pex20, which provides a better understanding of the recycling and degradation of this PTS2 cargo co-receptor.

Fatty acids are essential components of the dynamic lipid metabolism in cells. Fatty acids can also signal to intracellular pathways to trigger a broad range of cellular responses. Oleic acid is an abundant monounsaturated omega-9 fatty acid that impinges on different biological processes, but the mechanisms of action are not completely understood. Here, we report that oleic acid stimulates the cAMP/protein kinase A pathway and activates the SIRT1-PGC1α transcriptional complex to modulate rates of fatty acid oxidation. In skeletal muscle cells, oleic acid treatment increased intracellular levels of cyclic adenosine monophosphate (cAMP) that turned on protein kinase A activity. This resulted in SIRT1 phosphorylation at Ser-434 and elevation of its catalytic deacetylase activity. A direct SIRT1 substrate is the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), which became deacetylated and hyperactive after oleic acid treatment. Importantly, oleic acid, but not other long chain fatty acids such as palmitate, increased the expression of genes linked to fatty acid oxidation pathway in a SIRT1-PGC1α-dependent mechanism. As a result, oleic acid potently accelerated rates of complete fatty acid oxidation in skeletal muscle cells. These results illustrate how a single long chain fatty acid specifically controls lipid oxidation through a signaling/transcriptional pathway. Pharmacological manipulation of this lipid signaling pathway might provide therapeutic possibilities to treat metabolic diseases associated with lipid dysregulation.

Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific up-regulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H2O2 production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H2O2 produced by increased fat metabolism. To determine whether the catalase-specific response is a common feature of physiological conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fasted versus fed mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24 h. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H2O2-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization. Background: High dietary fat is linked to cardiac oxidative stress, however, little is known about the endogenous antioxidant response. Results: High fat feeding and fasting rapidly up-regulate catalase. Conclusion: Up-regulation of catalase is designed to protect mitochondria from oxidative damage while not perturbing H2O2-mediated signaling. Significance: Coupling fatty acid oxidation to H2O2 production creates a mechanism for sensing and communicating diet composition.

Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the K(m) values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.

Peroxisome proliferator-activated receptors (PPARα, -β/δ, and -γ) are a subfamily of nuclear receptors that plays key roles in glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione class of antidiabetic drugs that has many side effects. PPARγ is also activated by long chain unsaturated or oxidized/nitrated fatty acids, but its relationship with the medium chain fatty acids remains unclear even though the medium chain triglyceride oils have been used to control weight gain and glycemic index. Here, we show that decanoic acid (DA), a 10-carbon fatty acid and a major component of medium chain triglyceride oils, is a direct ligand of PPARγ. DA binds and partially activates PPARγ without leading to adipogenesis. Crystal structure reveals that DA occupies a novel binding site and only partially stabilizes the AF-2 helix. DA also binds weakly to PPARα and PPARβ/δ. Treatments with DA and its triglyceride form improve glucose sensitivity and lipid profiles without weight gain in diabetic mice. Together, these results suggest that DA is a modulating ligand for PPARs, and the structure can aid in designing better and safer PPARγ-based drugs.

H-rev107 is a mammalian protein belonging to the HRAS-like suppressor family. Although the protein was originally found as a tumor suppressor, currently it is receiving considerable attention as a regulator of adipocyte lipolysis. We recently revealed that purified recombinant H-rev107 has phospholipase A(1/2) activity, releasing free fatty acids from glycerophospholipids with a preference for esterolysis at the sn-1 position. In the present study, we constitutively expressed H-rev107 in cloned HEK293 cells to examine its biological function in living cells. Initially, the cells accumulated free fatty acids. We also found a remarkable decrease in the levels of ether-type lipids, including plasmalogen and ether-type triglyceride, with a concomitant increase in fatty alcohols, substrates for the biosynthesis of ether-type lipids. Considering that peroxisomes are involved in the ether-type lipid biosynthesis, we next focused on peroxisomes and found that the peroxisomal markers 70-kDa peroxisomal membrane protein and catalase were abnormally distributed in the transfected cells. These biochemical and morphological abnormalities were not seen in HEK293 cells stably expressing a catalytically inactive mutant of H-rev107. When H-rev107 or its fusion protein with enhanced green fluorescence protein was transiently expressed in mammalian cells, both proteins were associated with peroxisomes in some of the observed cells. These results suggest that H-rev107 interferes with the biosynthesis of ether-type lipids and is responsible for the dysfunction of peroxisomes in H-rev107-expressing cells.

The molecular mechanisms underlying protein turnover and enzyme regulation in the peroxisomal matrix remain largely unknown. Trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon) are newly identified peroxisomal matrix proteins that harbor both a serine protease-like domain and a peroxisome-targeting signal 1 (PTS1) sequence. Tysnd1 processes several PTS1-containing proteins and cleaves N-terminal presequences from PTS2-containing protein precursors. Here we report that knockdown of Tysnd1, but not PsLon, resulted in accumulation of endogenous β-oxidation enzymes in their premature form. The protease activity of Tysnd1 was inactivated by intermolecular self-conversion of the 60-kDa form to 15- and 45-kDa chains, which were preferentially degraded by PsLon. Peroxisomal β-oxidation of a very long fatty acid was significantly decreased by knockdown of Tysnd1 and partially lowered by PsLon knockdown. Taken together, these data suggest that Tysnd1 is a key regulator of the peroxisomal β-oxidation pathway via proteolytic processing of β-oxidation enzymes. The proteolytic activity of oligomeric Tysnd1 is in turn controlled by self-cleavage of Tysnd1 and degradation of Tysnd1 cleavage products by PsLon.

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.

Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5. PEX5 has a dual role in this process. First, it acts as a soluble receptor recognizing these proteins in the cytosol. Subsequently, at the peroxisomal docking/translocation machinery, PEX5 promotes their translocation across the organelle membrane. Despite significant advances made in recent years, several aspects of this pathway remain unclear. Two important ones regard the formation and disruption of the PEX5-cargo protein interaction in the cytosol and at the docking/translocation machinery, respectively. Here, we provide data on the interaction of PEX5 with catalase, a homotetrameric enzyme in its native state. We found that PEX5 interacts with monomeric catalase yielding a stable protein complex; no such complex was detected with tetrameric catalase. Binding of PEX5 to monomeric catalase potently inhibits its tetramerization, a property that depends on domains present in both the N- and C-terminal halves of PEX5. Interestingly, the PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, a component of the docking/translocation machinery. One or two of the seven PEX14-binding diaromatic motifs present in the N-terminal half of PEX5 are probably involved in this phenomenon. These results suggest the following: 1) catalase domain(s) involved in the interaction with PEX5 are no longer accessible upon tetramerization of the enzyme; 2) the catalase-binding interface in PEX5 is not restricted to its C-terminal peroxisomal targeting sequence type 1-binding domain and also involves PEX5 N-terminal domain(s); and 3) PEX14 participates in the cargo protein release step.

Septic shock results from bacterial infection and is associated with multi-organ failure, high mortality, and cardiac dysfunction. Sepsis causes both myocardial inflammation and energy depletion. We hypothesized that reduced cardiac energy production is a primary cause of ventricular dysfunction in sepsis. The JNK pathway is activated in sepsis and has also been implicated in impaired fatty acid oxidation in several tissues. Therefore, we tested whether JNK activation inhibits cardiac fatty acid oxidation and whether blocking JNK would restore fatty acid oxidation during LPS treatment. LPS treatment of C57BL/6 mice and adenovirus-mediated activation of the JNK pathway in cardiomyocytes inhibited peroxisome proliferator-activated receptor α expression and fatty acid oxidation. Surprisingly, none of the adaptive responses that have been described in other types of heart failure, such as increased glucose utilization, reduced αMHC:βMHC ratio or induction of certain microRNAs, occurred in LPS-treated mice. Treatment of C57BL/6 mice with a general JNK inhibitor (SP600125) increased fatty acid oxidation in mice and a cardiomyocyte-derived cell line. JNK inhibition also prevented LPS-mediated reduction in fatty acid oxidation and cardiac dysfunction. Inflammation was not alleviated in LPS-treated mice that received the JNK inhibitor. We conclude that activation of JNK signaling reduces fatty acid oxidation and prevents the peroxisome proliferator-activated receptor α down-regulation that occurs with LPS.

Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.

HMGCS2 (hydroxymethylglutaryl CoA synthase 2), the gene that regulates ketone body production, is barely expressed in cultured cell lines. In this study, we restored HMGCS2 expression and activity in HepG2 cells, thus showing that the wild type enzyme can induce fatty acid β-oxidation (FAO) and ketogenesis, whereas a catalytically inactive mutant C166A did not generate either process. Peroxisome proliferator-activated receptor (PPAR) α expression also induces fatty acid β-oxidation and endogenous HMGCS2 expression. Interestingly, PPARα-mediated induction was abolished when HMGCS2 expression was down-regulated by RNAi. These results indicate that HMGCS2 expression is both sufficient and necessary to the control of fatty acid oxidation in these cells. Next, we examined the expression pattern of several PPARα target genes in this now “ketogenic” HepG2 cell line. FGF21 (fibroblast growth factor 21) expression was specifically induced by HMGCS2 activity or by the inclusion of the oxidized form of ketone bodies (acetoacetate) in the culture medium. This effect was blunted by SirT1 (sirtuin 1) RNAi, so we propose a SirT1-dependent mechanism for FGF21 induction by acetoacetate. These data suggest a novel feed-forward mechanism by which HMGCS2 could regulate adaptive metabolic responses during fasting. This mechanism could be physiologically relevant, because fasting-mediated induction of liver FGF21 was dependent on SirT1 activity in vivo.