Abstract: The liver is not a storage site of excess energy as triacylglycerides but a major site of carbohydrate storage, playing a vital role in glucose homeostasis, and the hepatic lipid droplets (LDs) should have a distinct physiologic role from those in lipid-storing tissues. Most studies so far have been limited to characterization of the LDs in cultured cells or of the liver of animals maintained on a normal laboratory diet, and little is known about the properties of the LDs in the liver responding to dietary excess, irregular fats, and potentially toxic compounds contained in a natural food diet. We started to characterize the hepatic LDs in wild-type and peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice fed various natural diets by identifying the liver-enriched LD-associated proteins and the changes in lipid compositions. Based on the currently available data, we propose the hypothesis that hepatic LDs play vital protective roles against diet-derived excess fatty acids and potentially toxic hydrophobic compounds by temporarily storing them as neutral lipids or compounds until completion of the remodeling of fatty acids and detoxification of the compounds in a PPARalpha-dependent manner.
Abstract: Fatty aldehyde dehydrogenase (FALDH; also known as ALDH3A2 or ALDH10) oxidizes medium- or long-chain aliphatic aldehydes. FALDH deficiency in humans is known to be the cause of Sjögren-Larsson syndrome, in which individuals display neurological symptoms and cutaneous abnormality. FALDH-V, a splice isoform of FALDH, is localized in the peroxisome and contributes to the oxidization of pristanal, an intermediate of the alpha-oxidation pathway. FALDH-N, another splice isoform of FALDH, is induced by peroxisomal proliferator-activated receptor alpha ligands, although its activation mechanism has not been clarified. In the present study, we show that transcriptional activation of FALDH is directly regulated by peroxisomal proliferator-activated receptor alpha through a direct repeat-1 site located in the FALDH promoter. In addition, FALDH is efficiently induced by linoleic acid in rat hepatoma Fao cells through transcriptional activation by peroxisomal proliferator-activated receptor alpha. Furthermore, ectopic expression of endoplasmic reticulum-localizing FALDH-N, but not peroxisome-localizing FALDH-V, suppresses endoplasmic reticulum stress caused by linoleic acid in HEK293 cells. These results suggest the autocatalytic nature of the FALDH-N system against endoplasmic reticulum stress that is induced by polyunsaturated fatty acid; polyunsaturated fatty acid binds to peroxisomal proliferator-activated receptor alpha to activate the expression of FALDH-N, which then detoxifies polyunsaturated fatty acid-derived fatty aldehydes and protects cells from endoplasmic reticulum stress.
Abstract: Primary human hepatocytes are extensively used to study the potential of drugs to induce cytochrome P450 (CYP). However, the activities of these enzymes decrease rapidly during culture. Previously we reported that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various CYP genes were significantly increased compared with the monolayer cultured HepG2 cells. Here, we examined the induction of CYP gene expression by an inducer by examining the effect of phenobarbital treatment on CYP gene expression in the co-culture system. In the layered co-cultured HepG2, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment. We also detected CYP3A4 enzyme induction using this co-culture system. Moreover, the induction of hepatic drug transporters by phenobarbital was detected. These results suggest that functional regulation of the CYP and transporter gene pathway is retained in these layered co-cultured cells. Thus, this system may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of transport and metabolism.
Abstract: We previously reported that using thermo-responsive culture surfaces, a layered co-culture was achieved by placing an endothelial cell sheet onto a layer of human hepatoma cell line HepG2 in order to develop a culture model that mimics hepatic lobules. In the layered co-culture cells, the expression levels of liver-specific genes gradually increased. A cross-sectional view of the layered co-culture cells showed that the thickness of the layer slowly increased after layering, as did extracellular matrix (ECM) deposition around HepG2 cells. In this study, we report that the molecular compositions of ECM and cell-adhesion molecules changed in the layered co-culture cells. Gene expression of integrin alpha4 and decorin gradually increased after layering, and the time-course pattern of these genes was correlated with that of liver-specific genes. Moreover, the layered co-culture system has the ability to assemble a branching network of fibronectin fibrils. These results suggest that a vastly different extracellular environment in layered co-culture cells may induce an increase in liver-specific functions.
Abstract: We previously demonstrated that Nrf2 regulates oxidized LDL-mediated CD36 expression in macrophages. The current study aimed to determine the mechanism of Nrf2-mediated macrophage CD36 induction. Treatment with the Nrf2 activator diethylmaleate, but not PPARgamma specific ligands, caused marked upregulation of CD36 in mouse macrophage RAW264.7 cells. Similarly, Nrf2 activators induced CD36 expression in bone marrow-derived macrophages in a Nrf2-dependent manner. Induced expression of the three alternative first exons of mouse CD36, deemed 1A, 1B, and 1C, occurred upon Nrf2 activation with exon1A mainly contributing to the CD36 expression. Four antioxidant response elements (AREs) lie within close proximity to these three exons, and chromatin immunoprecipitation assays demonstrated that two AREs upstream of exon1A, the distal 1A-ARE1, and the proximal 1A-ARE2, were Nrf2-responsive. Luciferase reporter assays conclusively demonstrated that 1A-ARE2 is the critical regulatory element for the Nrf2-mediated gene expression. Thus Nrf2 directly regulates CD36 gene expression by binding to 1A-ARE2.
Abstract: Primary human hepatocytes are used extensively to study drug-metabolizing enzymes such as the cytochrome P450 (CYP) enzymes. However, the activities of these enzymes decrease rapidly during culture. In the present study, using a thermo-responsive culture dish, layered co-culture was achieved by placing a bovine pulmonary artery endothelial cell (BPAEC) sheet onto the human hepatoma cell line HepG2. In the BPAEC/HepG2 layered co-culture system, real-time polymerase chain reaction analysis showed that the expression levels of various CYP enzymes were more than 10 times greater 21 days after layering than with a HepG2 monolayer. The expression levels of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 were up-regulated in a time-dependent manner, gradually increasing from day 10 after layering, and continuing to increase until at least day 21. The gene expression levels of the various CYP enzymes were almost identical to that of human liver. These results suggest that our layered co-culture system enhances the function of HepG2 cells and that our BPAEC/HepG2 layered co-culture system can serve as a useful model for the in vitro evaluation of CYP regulation.
Abstract: 17beta-Hydroxysteroid dehydrogenase type 11 (17betaHSD11) is mostly localized on the endoplasmic reticulum (ER) membrane under normal conditions and redistributes to lipid droplets (LDs) when the formation of LDs is induced. In this study, confocal microscopy analyses of the subcellular localization of the mutated 17betaHSD11 proteins in cells with or without LDs revealed that both an N-terminal hydrophobic sequence and an adjacent sequence that has a weak homology with the PAT motif are independently necessary and both parts together (28 amino acid residues in total) are sufficient for the dual localization of 17betaHSD11. Mutation analyses suggest that the PAT-like motif in 17betaHSD11 will not be functionally similar to the canonical PAT motif. Hsp60 was identified as a possibly interacting protein with the PAT-like motif, and biochemical and microscopic analyses suggest that Hsp60 may be partly, but not necessarily involved in recognition of the PAT-like part of the targeting sequence of 17betaHSD11.
Abstract: The tissue distribution and disposition of carnitine, which plays an important role in the transport of long-chain fatty acids across the mitochondrial inner membrane for beta-oxidation, are well controlled by carnitine transporter organic cation/carnitine transporter 2 (OCTN2). Since little information is available on regulation of the expression of the OCTN2 gene, we examined the factors that affect the expression level of rat Octn2 (rOctn2), focusing on nuclear receptor peroxisome proliferator activated receptor alpha (PPARalpha), which regulates expression of genes associated with beta-oxidation of fatty acids. mRNA of rOctn2 was induced by the PPARalpha ligand fenofibrate in primary-cultured rat hepatocytes. Further, the PPARalpha ligand Wy14643 increased the expression of Octn2 in wild-type mice, but not in PPARalpha knockout mice. Analysis of the rOctn2 promoter region suggested the presence of putative cis elements of PPARalpha. Wistar rats treated with intraperitoneal fenofibrate administration showed increased expression of rOctn2 mRNA in liver, and uptake of [3H]carnitine by freshly isolated hepatocytes derived from those rats was also increased. In conclusion, our results indicate that the nuclear receptor PPARalpha directly up-regulates the expression of rOctn2 and increases the hepatic uptake of carnitine via rOctn2.
Abstract: 17beta-Hydroxysteroid dehydrogenase (17betaHSD) type 13 is identified as a new lipid droplet-associated protein. 17betaHSD type 13 has an N-terminal sequence similar to that of 17betaHSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17betaHSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17betaHSD type 11, however, expression of 17betaHSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor alpha and its ligand. Instead the expression level of 17betaHSD type 13 in the receptor-null mice was increased several-fold. 17betaHSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.
Abstract: We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.
Abstract: Statins are widely used to treat hypercholesterolemia, but they are associated with muscle-related adverse events, by as yet, inadequately resolved mechanisms. In this study, we report that statins induced autophagy in cultured human rhabdomyosarcoma A204 cells. Potency differed widely among the statins: cerivastatin induced autophagy at 0.1muM, simvastatin at 10muM but none was induced by pravastatin. Addition of mevalonate, but not cholesterol, blocked induction of autophagy by cerivastatin, suggesting that this induction is dependent on modulation of isoprenoid metabolic pathways. The statin-induced autophagy was not observed in other types of cells, such as human hepatoma HepG2 or embryonic kidney HEK293 cells. Muscle-specific abortive induction of autophagy by hydrophobic statins is a possible mechanism for statin-induced muscle-related side effects.
Abstract: Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.
Abstract: A systematic analysis to examine the effects of peroxisome proliferator-activated receptor (PPAR)alpha agonists on the expression levels of all the nutrient/drug plasma-membrane transporters in the mouse small intestine was performed. Transporter mRNAs that were induced or repressed by two independent PPARalpha-specific agonists were identified by a genome-wide microarray method, and the changes were confirmed by real-time PCR using RNA isolated from the intestines and livers of wild-type and PPARalpha-null mice. Expression levels of seven nutrient/drug transporters (Abcd3, Octn2/Slc22a5, FATP2/Slc27a2, Slc22a21, Mct13/Slc16a13, Slc23a1 and Bcrp/Abcg2) in the intestine were up-regulated and the expression level of one (Mrp1/Abcc1) was down-regulated by PPARalpha; although the previously report that the H(+)/peptide co-transporter 1 (Pept1) is up-regulated by PPARalpha was not replicated in our study. We propose that the transport processes can be coordinately regulated with intracellular metabolism by nutrient nuclear receptors.
Abstract: 17beta-Hydroxysteroid dehydrogenase type 11 (17beta-HSD11) is a member of the short-chain dehydrogenase/reductase family involved in the activation and inactivation of sex steroid hormones. We recently identified 17beta-HSD11 as a gene that is efficiently regulated by peroxisome proliferator-activated receptor-alpha PPARalpha in the intestine and the liver [Motojima K (2004) Eur J Biochem271, 4141-4146]. In this study, we characterized 17beta-HSD11 at the protein level to obtain information about its physiologic role in the intestine and liver. For this purpose, specific antibodies against 17beta-HSD11 were obtained. Western blotting analysis showed that administration of a peroxisome proliferator-activated receptor-alpha agonist induced 17beta-HSD11 protein in the jejunum but not in the colon, and to a much higher extent than in the liver of mice. A subcellular localization study using Chinese hamster ovary cells and green fluorescent protein-tagged 17beta-HSD11 showed that it was mostly localized in the endoplasmic reticulum under normal conditions, whereas it was concentrated on lipid droplets when they were induced. A pulse-chase experiment suggested that 17beta-HSD11 was redistributed to the lipid droplets via the endoplasmic reticulum. Immunohistochemical analysis using tissue sections showed that 17beta-HSD11 was induced mostly in intestinal epithelia and hepatocytes, with heterogeneous localization both in the cytoplasm and in vesicular structures. A subcellular fractionation study of liver homogenates confirmed that 17beta-HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endoplasmic reticulum and the lipid droplets of which formation was induced by feeding a diet containing a proliferator-activated receptor-alpha agonist. Taken together, these data indicate that 17beta-HSD11 localizes both in the endoplasmic reticulum and in lipid droplets, depending on physiologic conditions, and that lipid droplet 17beta-HSD11 is not merely an endoplasmic reticulum contaminant or a nonphysiologically associated protein in the cultured cells, but a bona fide protein component of the membranes of both intracellular compartments.
Abstract: Pyruvate dehydrogenase kinase 4 (PDK4) phosphorylates and inactivates the pyruvate dehydrogenase complex to respond to physiologic conditions. This response switches the energy source from glucose to fatty acids to maintain blood glucose levels. Transcription of the PDK4 gene is activated by fasting or by the administration of a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand in a tissue-specific manner. However, the two mechanisms to induce PDK4 mRNA as well as the relationship between the two have not been studied in detail. In this study, we show that the two mechanisms are independent, at least in the mouse skeletal muscle, and that estrogen-related receptor alpha (ERRalpha) is directly involved in the PPARalpha-independent transcriptional activation of the PDK4 gene with peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) as a specific partner. The latter conclusion is based on the following evidence: 1) Deletion and point mutation analyses of the cloned mouse PDK4 gene promoter sequence identified an exact possible ERRalpha-binding motif as the PGC-1alpha responsive element. 2) The overexpression of ERRalpha by cotransfection enhanced, and the knocking down of it by specific shRNAs diminished, the PGC-1alpha-dependent activation. 3) Specific binding of ERRalpha to the identified PGC-1alpha-responsive sequence of the mouse PDK4 promoter was confirmed in the electrophoresis mobility shift assay using anti-ERRalpha antibodies. These results suggest that PGC-1alpha plays an essential role not only in regulating the amounts of energy creating enzymes, but also at the step of metabolic switching with unevenly distributed tissue transcription factors such as ERRalpha in the skeletal muscle, thus harmonizing tissue-specific functions and energy metabolism.
Abstract: Fatty acid translocase (FAT)/CD36 is a multi-functional membrane protein participating in uptake of long-chain fatty acids and oxidized low density lipoprotein, playing an important role in lipid homeostasis. We reported the dual promoter structures of the mouse and human FAT/CD36 genes in relation to the tissue-specific expression and regulation by peroxisome proliferator-activated receptors (PPARs) (Sato et.al. J. Biol. Chem. 277, 15703-15711, 2002). However, these structures alone could not explain a large induction of the mRNA by a PPAR ligand in the mouse intestine. To learn more about the tissue-specific transcriptional regulation of the FAT/CD36 gene for lipid homeostasis at the whole body level, we cloned the cDNA for the induced mRNA in the mouse intestine and identified third promoter close to the first common exon of the gene. The human gene also has essentially the same gene organization. RT-PCR and Northern blot analyses showed that the three promoters are differentially used in various mouse tissues and the cultured human cells. The physiological significance of these complex promoter structures was suggested by the differential responsiveness to a peroxisome proliferator-activated receptor ligand and the differences in translational efficiencies or stabilities of the mRNA among the isoforms.
Abstract: Cytosolic lipid droplets (LDs) are multifunctional organelles that exist in all types of eukaryotic cells and control lipid homeostasis. In mammalian cells LDs contain a class of proteins in their surface layers that share a homologous sequence called the PAT domain, including perilipin, adipose differentiation-related protein (ADRP), a tail-interacting protein of 47 kDa (TIP47), and S3-12, which are distributed tissue- or cell type-selectively. Expression in some cases is regulated by peroxisome proliferator-activated receptors (PPARs). In this study we identified a new PAT family member named MLDP (myocardial LD protein) in a murine cDNA data base and showed the mRNA and protein to be highly enriched in the heart and also expressed at lower levels in the liver and adrenals. Upon subcellular fractionation, a substantial amount of MLDP was detected in the top fraction enriched with LDs. Furthermore, overexpressed MLDP tagged with green fluorescent protein accumulated at the surfaces of LDs and co-localized with perilipin and ADRP. Deletion analysis demonstrated the N-terminal region containing a PAT-1 domain and the following 33-mer domain to be required for targeting of MLDP to LDs. MLDP was found to be up-regulated at both mRNA and protein levels in the heart and liver by a selective ligand for PPARalpha, Wy14,643, but not in PPARalpha knock-out mice. MLDP expression was also increased upon fasting in parallel with ADRP. These results indicate that MLDP is a bona fide new PAT family member localized in LDs. Its expression depends on the physiological conditions and the action of PPARalpha.
Abstract: Peroxisome proliferator-activated receptor alpha (PPARalpha) is thought to play an important role in lipid metabolism in the liver. To clarify the extra-hepatic and/or unknown function of PPARalpha, we previously performed a proteome analysis of the intestinal proteins and identified 17beta-hydroxysteroid dehydrogenase type 11 as a mostly induced protein by a PPARalpha ligand [Motojima, K. (2004) Eur. J. Biochem. 271, 4141-4146]. Because of its supposed wide substrate specificity, we examined the possibility that PPARalpha plays an important role in inducing detoxification systems for some natural foods by feeding mice with various plant seeds and grains. Feeding with sesame but not others often killed PPARalpha knockout mice but not wild-type mice. A microarray analysis of the sesame-induced mRNAs in the intestine revealed that PPARalpha plays a vital role in inducing various xenobiotic metabolizing enzymes in the mouse intestine and liver. A PPARalpha ligand alone could not induce most of these enzymes, suggesting that there is an essential crosstalk among PPARalpha and other xenobiotic nuclear receptors to induce a detoxification system for plant compounds.
Abstract: Pyruvate dehydrogenase kinase 4 (PDK4) is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions. Transcription of the PDK4 gene is activated by fasting or by the administration of a PPARalpha ligand in a tissue-specific manner. Here, we show that the two mechanisms are independent, and that ERRalpha is directly involved in PPARalpha-independent transcriptional activation of the PDK4 gene with PGC-1alpha as a specific partner. This conclusion is based on the following evidence. First, detailed mutation analyses of the cloned PDK4 gene promoter sequence identified a possible ERRalpha-binding motif as the PGC-1alpha responsive element. Second, overexpression of ERRalpha by cotransfection enhanced, and the knockout of it by shRNAs diminished, PGC-1alpha-dependent activation. Third, specific binding of ERRalpha to the identified PGC-1alpha responsive sequence was confirmed by the electrophoresis mobility shift assay. Finally, cell-type-specific responsiveness to PGC-1alpha was observed and this could be explained by differences in the expression levels of ERRalpha, however, ectopic expression of ERRalpha in poorly responsive cells did not restore PGC-1alpha responsiveness, indicating that ERRalpha is necessary, but not sufficient for the response.
Abstract: Many mammalian genes are clustered on the genomes, and hence the genes in the same cluster can be regulated through a common regulatory element. We indeed showed previously that the perilipin/PEX11alpha gene pair is transactivated tissue-selectively by PPARgamma and PPARalpha, respectively, through a common binding site. In the present study, we identified a gene, named GSPA, neighboring a canonical PPAR target, acyl-CoA oxidase (AOX) gene. GSPA expression was induced by a peroxisome proliferator, Wy14,643, in the liver of wild-type mice, but not PPARalpha-null mice. GSPA and AOX share the promoter and two peroxisome proliferator-response elements. GSPA mRNA was also found in the heart and skeletal muscle, as well as 3T3-L1 cells. GSPA encodes a protein of 161 amino acids that is enriched in 3T3-L1 cells. Even other gene pairs might be regulated through common sequence elements, and conversely it would be interesting how each gene is aptly regulated in clusters.
Abstract: Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesterol and cholesterol secretion into bile in the liver. Previously, we identified an SR-BI-associated protein, termed PDZK1, from rat liver membrane extracts. PDZK1 contains four PSD-95/Dlg/ZO-1 (PDZ) domains, the first of which in the N-terminal region is responsible for the association with SR-BI. PDZK1 controls hepatic SR-BI expression in a posttranscriptional fashion both in cell culture and in vivo. In this study, we demonstrated that the C-terminal region of PDZK1 is crucial for up-regulating SR-BI protein expression. Metabolic labeling experiments and phosphoamino acid analysis revealed that PDZK1 is phosphorylated at Ser residues within this region. Point-mutation analysis demonstrated that PDZK1 is phosphorylated at Ser-509. Interestingly, a mutant PDZK1, in which Ser-509 was replaced with Ala, lost the ability to up-regulate SR-BI protein. We identified Ser-509 of PDZK1 as the residue that is phosphorylated by the cAMP-dependent PKA in vitro as well as in cell culture. Ser-509 of PDZK1 in rat liver was also phosphorylated, as shown by an Ab that specifically detects phosphorylated Ser-509. Administration of glucagon to Wistar rats increased PDZK1 phosphorylation as well as hepatic SR-BI and PDZK1 expression while it decreased plasma HDL levels, indicating that PDZK1 phosphorylation is hormonally regulated. These findings suggest that phosphorylation of PDZK1 has an important role in the regulation of hepatic SR-BI expression and, thus, influences plasma HDL levels.
Abstract: Expression of heart-type fatty acid binding protein is restricted mainly to the skeletal and cardiac muscles and further regulated by peroxisome proliferator-activated receptor alpha. The molecular basis for the muscle-restricted peroxisome proliferator-activated receptor alpha action on the fatty acid binding gene was analyzed using normal and the receptor-null mice and the cultured cells. Two possible peroxisome proliferator-response elements were found in the promoter region of the mouse gene. A gel shift assay showed that both elements were functional. However, neither the tandem repeats of the elements nor the cloned promoter sequence could be activated by peroxisome proliferator-activated receptor alpha and its ligand in the reporter gene assay using cultured cells. The cloned promoter responded to the ligand only in the muscle when the reporter gene was introduced into the mouse muscle. Using a chimeric receptor with the activation domain of herpes virus VP16 protein and the tandem repeats of the elements with or without mutation, the upstream element was finally demonstrated to be potentially involved in the receptor-dependent transcriptional activation. These results suggest that the peroxisome proliferator-response element of the mouse gene is atypical and there is a muscle-specific mechanism to enhance the weak binding of the receptor to the response element to ensure the muscle-specific action of peroxisome proliferator-activated receptor alpha on the heart-type fatty acid binding protein gene promoter.
Abstract: In order to study the role of peroxisome proliferator-activated receptor alpha in mouse intestine, its agonist-induced proteins were identified by peptide mass fingerprinting followed by Northern blot analysis using their cDNAs. One of the most remarkably induced proteins was identified as 17beta-hydroxysterol dehydrogenase type 11. Its very rapid induction by various agonists was most efficient in intestine and then in liver. These findings together with recently reported results showing the enzyme family's wide substrate spectrum, including not only glucocorticoids and sex steroids but also bile acids, fatty acids and branched chain amino acids, suggest new roles for both peroxisome proliferator-activated receptor alpha and 17beta-hydroxysterol dehydrogenase type 11 in lipid metabolism and/or detoxification in the intestine.
Abstract: We previously identified pyruvate dehydrogenase kinase 4 (PDK4) mRNA as a most rapidly induced mRNA by fibrates and suggested the possibility that the coupled induction of PDK4 and reduction of serum triglyceride and fatty acid levels can cause protein degradation in muscles. To investigate whether the drugs that are known to have a risk of rhabdomyolysis induce PDK4 mRNA in skeletal muscle, the effects of statins and new quinolon anti-bacterial drugs on the expression levels of the mRNA were examined using mice and cultured cells. Several statins and new quinolon anti-bacterial drugs solely induced PDK4 mRNA in the muscle as efficiently as fibrates and at least some combinations were synergistic. The present results suggest that induction of PDK4 mRNA is involved in the drug induced acute rhabdomyolysis when the muscle is restricted to use fatty acids as a major energy source.
Abstract: Peroxisome proliferator (PPAR)alpha ligand Wy14,643 induces liver-fatty acid binding protein (FABP) spontaneously and heart-FABP gradually, but not intestine-FABP mRNA expression in the mouse liver. These strict regulations have not been reproduced in cultured cell systems. We applied a DNA electroporation method to directly introduce reporter gene constructs into the livers of mice. This system reproduced the in vivo responses of the above three FABP gene promoters to the PPARalpha ligand but not that of a promoter containing the typical three PPAR binding sites in tandem. Deletion and mutation analyses of the mouse L-FABP gene suggested that, in addition to the binding site for PPARalpha, a far upstream sequence is required for PPAR-dependent transactivation in the liver. In contrast to the cultured cell systems, our in vivo DNA electroporation method showed that PPARalpha binding to the promoter is necessary but not sufficient for PPARalpha ligand-dependent transcriptional activation of the L-FABP gene in vivo.
Abstract: Fatty acid translocase (FAT)/CD36 is a glycoprotein involved in multiple membrane functions including uptake of long-chain fatty acids and oxidized low density lipoprotein. In mice, expression of the gene is regulated by peroxisome proliferator-activated receptor (PPAR) alpha in the liver and by PPAR gamma in the adipose tissues (Motojima, K., Passilly, P. P., Peters, J. M., Gonzalez, F. J., and Latruffe, N. (1998) J. Biol. Chem. 273, 16710-16714). However, the time course of PPAR alpha ligand-induced expression of FAT/CD36 in the liver, and also in the cultured hepatoma cells, is significantly slower than those of other PPAR alpha target genes. To study the molecular mechanism of the slow transcriptional activation of the gene by a PPAR ligand, we first cloned the 5' ends of the mRNA and then the mouse gene promoter region from a genomic bacterial artificial chromosome library. Sequencing analyses showed that transcription of the gene starts at two initiation sites 16 kb apart and splicing occurs alternatively, producing at least three mRNA species with different 5'-noncoding regions. The PPAR alpha ligand-responsive promoter in the liver was identified as the new upstream promoter where we found several possible binding sites for lipid metabolism-related transcriptional factors but not for PPAR. Neither promoter responded to a PPAR alpha ligand in the in vitro or in vivo reporter assays using cultured hepatoma cells and the liver of living mice. We also have cloned the human FAT/CD36 gene from a bacterial artificial chromosome library and identified a new independent promoter that is located 13 kb upstream of the previously reported promoter. Only the upstream promoter responded to PPAR alpha and PPAR gamma ligands in a cell type-specific manner. The absence of PPRE in the responding upstream promoter region, the delayed activation by the ligand, and the results of the reporter assays all suggested that transcriptional activation of the FAT/CD36 gene by PPAR ligands is indirectly dependent on PPAR.
Abstract: FAT/CD36 is involved in various processes including uptake of fatty acid into the heart and of oxidized low density lipoprotein (LDL) into macrophages. Expression of the FAT/CD36 gene is regulated in a tissue-specific manner, and loss or inadequately regulated expression of FAT/CD36 is thought to be one of the causes of some diseases such as cardiomyopathy and atherosclerosis. We recently found that the mouse and human FAT/CD36 genes have two independent promoters. To elucidate the physiological significance of the two promoters, we characterized the peroxisome proliferator-activated receptor ligand-responsive new promoter that is located 14 kb upstream of the previously reported promoter of the human gene. We found several SNPs in this region some of which were found only when analyzing DNA samples from the patients lacking FAT/CD36 totally or in a cell-type-specific manner. However, we could not detect any negative effect of these SNPs on the transcription by transient transfection analysis, suggesting that the identified SNPs alone are not directly linked to low transcriptional activities.
Abstract: AIM: Resistin has been suggested to link obesity to diabetes by antagonizing insulin action. However, this model is based on limited observations and how resistin links the two complex processes is not known. In this study, we investigated the effects of various factors on the expression of resistin and examined the generality of the proposal. METHODS: Total RNA was isolated from the adipose tissues of lean, obese and peroxisome proliferator-activated receptor (PPAR)alpha-null mice fed a control diet or that contained a PPAR ligand, and analysed by Northern blotting using cDNAs for resistin, leptin, aP2 and other mRNAs as probes. For quantitative analysis, an image analyser was used. RESULTS: Basal expression of resistin mRNA was suppressed by obesity, but the extent of suppression differed significantly among the mouse strains and types of adipose tissue examined. Anti-diabetic thiazolidinediones induced resistin expression in the lean mice and showed smaller effects in obese mice. Furthermore, PPARalpha was shown to play an important role in constitutive expression of resistin in the adipose tissue. CONCLUSION: Our results indicated that diverse factors modulate the expression of resistin in the adipose tissues of mice, and suggested that resistin is not a master hormone linking obesity to diabetes.
Abstract: Pyruvate dehydrogenase kinase 4 (PDK4) mRNA was identified as a transcript that was rapidly and generally induced by fibrates in various tissues of the mouse. The time course of induction was much faster than those of typical peroxisome proliferator-activated receptor alpha (PPAR alpha)-regulated peroxisomal mRNAs. Rapid and efficient induction of PDK4 mRNA in various tissues would inactivate pyruvate dehydrogenase and stimulate fatty acid oxidation at the whole body level, leading to enhanced utilization of serum fatty acids and triglycerides. This metabolic switching mechanism can explain the early phase of the fibrate-induced decrease in the serum levels of triglycerides. Furthermore, the muscle in the fibrate-induced early state with limited activity of pyruvate dehydrogenase and low levels of fatty acids will utilize proteins as an energy source and extensive degradation of proteins may lead to myopathy or rhabdomyolysis.
Abstract: The present study was addressed on the effect of 3,3',4,4',5-pentachlorobiphenyl (PenCB) to the expression of molecular chaperon proteins, glucose regulated protein (GRP) 78, GRP94, calreticulin and calnexin in liver endoplasmic reticulum of rat by treatment with acute exposure. Male Wistar rats received PenCB in corn oil at once a dose of 10 mg/kg i.p., then at 5 days after treatment the microsomes were prepared. Free-fed and pair-fed control groups were given the vehicle. The microsomal proteins were separated on SDS-PAGE, transferred to membrane and blotted using antibody towards respective chaperone proteins. The protein levels of GRP78, GRP94, calreticulin and calnexin were significantly decreased with the acute exposure. In addition, albumin level in the microsomes was also significantly reduced by the PenCB treatment. The transferrin level was significantly higher than pair-fed but not from free-fed group. These chaperone proteins have important physiological functions against synthesized and/or denatured proteins, which include assembling, folding of proteins. PenCB-treatment may alter the extent of biosynthesis of secretory protein such as albumin through the decreasing levels of chaperone proteins in endoplasmic reticulum. Interestingly, reduced GRP78 protein level by PenCB was not due to decreased mRNA level. Our results suggested that a part of the toxicity of PenCB is associated to significant decrease of the chaperone proteins in the endoplasmic reticulum.
Abstract: PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.
Abstract: Peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARgamma and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARgamma by PPARgamma agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARgamma activity by heterozygous PPARgamma deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARgamma deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARgamma deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor alpha, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARgamma deficiency and PPARgamma agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy.
Abstract: TAPL is a half-type ABC transporter with sequence similarity to TAP1 and TAP2 that is transcribed in various rat tissues [Yamaguchi, Y., Kasano, M., Terada, T., Sato, R., and Maeda, M. (1999) FEBS Lett. 457, 231-236]. Primary structures of the human and mouse orthologous counterparts were deduced from cDNAs cloned by means of polymerase chain reaction, and they were compared with that of the rat. The mammalian TAPLs (rat, mouse, and human) are highly conserved, since about 95% of the amino acid residues are identical between rodents and man. Phylogenetic analysis demonstrated that the evolutional rate of TAPL is much slower than those of TAP1 and TAP2, although TAPL could have diverged from an ancestor of TAP1 or that of TAP1 and TAP2. The TAPL-GFP fusion protein transiently expressed in Cos-1 cells was co-localized with PDI, suggesting that TAPL is inserted into endoplasmic reticulum membrane. The conservation of the peptide-binding motifs of TAP proteins in TAPL raises the possibility that the TAPL might be a peptide transporter. The gene for human TAPL is assigned to chromosome 12q24.31-q24.32, while those for TAP1 and TAP2 are located at the MHC locus of chromosome 6p21.!3. Furthermore, the transcription of TAPL gene is not responsive to interferon-gamma, in contrast to TAP1 and TAP2. These results indicate that the gene regulation of TAPL is different from those of TAP1 and TAP2.
Abstract: Peroxisome proliferator-activated receptors (PPARs) play the essential role in transcriptional modulations of the genes involved in lipid metabolism. In vitro studies have shown that all the mechanisms of the modulations are similarly mediated by PPAR and the response elements through heterodimerization with retinoid X receptors (RXRs). However, PPARs mediate the diverse effects induced in various tissues of mouse by their activators. First, PPAR alpha also plays an obligatory role in the activator-induced transcriptional modulations of various genes, some of which are not related to lipid metabolism. Second, responsiveness of various genes to several activators varies considerably. Third, some of the activator-induced transcriptional activation of several genes is strictly tissue-specific. Fourth, the time courses of the activator-induced transcriptional modulations are diverse. Following brief review of these diverse responses, emphasis is laid on the need for studies of these diversely regulated genes to understand the species-general and coordinated interplay between PPARs and other factors to maintain lipid homeostasis at the body level.
Abstract: The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of 32P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, alpha isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.
Abstract: Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes.
Abstract: The effect of a potent peroxisome proliferator-activated receptor (PPAR) alpha activator Wy14,643 on tissue-specific expression of fatty acid binding (FABP) genes was studied. Wy14,643 immediately induced liver-, intestine- and FABP but not PPARgamma-regulated adipose-FABP (or aP2) mRNAs in respective mouse tissues. Moreover, it gradually induced ectopic expression of heart- and adipose-FABP mRNAs to significant levels in the liver. However, ectopic expression was not induced in the liver of PPARalpha-null mouse, indicating an obligatory role of the receptor in the modulated expression. Among the four PPARalpha activators examined, only Wy14,643 induced ectopical expression of heart-FABP in the liver. Thus, tissue-specificity of the FABP gene expression is not absolute and, with a potent activator, can be distorted by PPARalpha.
Abstract: The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 microM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 microM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the beta isoform of phosphatidylinositol 3-kinase regulatory subunit.
Abstract: Thiazolidinediones (TZDs) reduce insulin resistance in type 2 diabetes by increasing peripheral uptake of glucose, and they bind to and activate the transcriptional factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Studies have suggested that TZD-induced activation of PPAR-gamma correlates with antidiabetic action, but the mechanism by which the activated PPAR-gamma is involved in reducing insulin resistance is not known. To examine whether activation of PPAR-gamma directly correlates with antidiabetic activities, we compared the effects of 4 TZDs (troglitazone, pioglitazone, BRL-49653, and a new derivative, NC-2100) on the activation of PPAR-gamma in a reporter assay, transcription of the target genes, adipogenesis, plasma glucose and triglyceride levels, and body weight using obese KKAy mice. There were 10- to 30-fold higher concentrations of NC-2100 required for maximal activation of PPAR-gamma in a reporter assay system, and only high concentrations of NC-2100 weakly induced transcription of the PPAR-gamma but not PPAR-alpha target genes in a whole mouse and adipogenesis of cultured 3T3L1 cells, which indicates that NC-2100 is a weak PPAR-gamma activator. However, low concentrations of NC-2100 efficiently lowered plasma glucose levels in KKAy obese mice. These results strongly suggest that TZD-induced activation of PPAR-gamma does not directly correlate with antidiabetic (glucose-lowering) action. Furthermore, NC-2100 caused the smallest body weight increase of the 4 TZDs, which may be partly explained by the finding that NC-2100 efficiently induces uncoupling protein (UCP)-2 mRNA and significantly induces UCP1 mRNA in white adipose tissue (WAT). NC-2100 induced UCP1 efficiently in mesenteric WAT and less efficiently in subcutaneous WAT, although pioglitazone and troglitazone also slightly induced UCP1 only in mesenteric WAT. These characteristics of NC-2100 should be beneficial for humans with limited amounts of brown adipose tissue.
Abstract: Regulation of gene expression of three putative long-chain fatty acid transport proteins, fatty acid translocase (FAT), mitochondrial aspartate aminotransferase (mAspAT), and fatty acid transport protein (FATP), by drugs that activate peroxisome proliferator-activated receptor (PPAR) alpha and gamma were studied using normal and obese mice and rat hepatoma cells. FAT mRNA was induced in liver and intestine of normal mice and in hepatoma cells to various extents only by PPARalpha-activating drugs. FATP mRNA was similarly induced in liver, but to a lesser extent in intestine. The induction time course in the liver was slower for FAT and FATP mRNA than that of an mRNA encoding a peroxisomal enzyme. An obligatory role of PPARalpha in hepatic FAT and FATP induction was demonstrated, since an increase in these mRNAs was not observed in PPARalpha-null mice. Levels of mAspAT mRNA were higher in liver and intestine of mice treated with peroxisome proliferators, while levels in hepatoma cells were similar regardless of treatment. In white adipose tissue of KKAy obese mice, thiazolidinedione PPARgamma activators (pioglitazone and troglitazone) induced FAT and FATP more efficiently than the PPARalpha activator, clofibrate. This effect was absent in brown adipose tissue. Under the same conditions, levels of mAspAT mRNA did not change significantly in these tissues. In conclusion, tissue-specific expression of FAT and FATP genes involves both PPARalpha and -gamma. Our data suggest that among the three putative long-chain fatty acid transporters, FAT and FATP appear to have physiological roles. Thus, peroxisome proliferators not only influence the metabolism of intracellular fatty acids but also cellular uptake, which is likely to be an important regulatory step in lipid homeostasis.
Abstract: To better characterize peroxisome proliferator-induced non-peroxisomal responses, we searched the mRNAs of which the levels were modulated by proliferators. We used the PCR-based methods including differential display. The mRNAs were divided into at least four groups by their time-courses of induction and repression: group 1 very rapidly increased then decreased; group 2 increased after a time lag (well-characterized peroxisomal mRNAs belonged to this group); group 3 decreased reciprocally compared with group 2 mRNAs; group 4 increased after group 2 mRNAs, with a much longer lag period. All of these modulations cannot be explained by peroxisome proliferator action through PPAR and RXR dimerization on the target genes to activate transcription. Another unidentified transcription factor may be involved in some of these modulations. It will also be important to consider PPAR-independent pathways when studying the diverse effects of peroxisome proliferators.
Abstract: The strain difference, peroxisome proliferator specificity and role of PPAR alpha in peroxisome proliferator-induced transcriptional repression of nonperoxisomal transthyretin and alpha2u-globulin genes were examined. The genes were repressed by four peroxisome proliferators in all seven mouse strains tested. The extent of repression was strongly dependent on both the mouse strains and type of proliferator, although the mRNA levels of PPAR alpha and its partner in heterodimerization, RXR alpha were not different. The role of PPAR alpha in repression was confirmed by the finding that PPAR alpha-null mice were not responsive to transcriptional repression. These results indicate that PPAR alpha plays an obligatory role in transcription of various genes, some of which are not related to lipid metabolism.
Abstract: The effects of peroxisome proliferators on the levels of rat serum proteins were examined. The proliferators reduced the levels of several proteins in various ways with respect to proliferator-specificity and the time courses of the changes. The identification of three proteins by amino acid sequencing showed that they were functionally unrelated. This diversity suggests that several primary and secondary effects of the peroxisome proliferators caused the various changes in the levels of several serum proteins. Among these, apolipoprotein E was the most but transiently down-regulated by the two proliferators tested. Northern blots of the rat liver mRNA suggested that the primary step of the down-regulation was at pre-translation.
Abstract: Protein histidine kinase (Motojima, K., and Goto, S. (1993) FEBS Lett. 319, 75-79) and phosphatase in rat liver extract were characterized. The histidine kinase was recovered mostly in the membrane and the phosphatase in the soluble fraction. The kinase and its substrate 36-kDa protein (P36) were co-solubilized from the membrane under conditions in which most of the other kinases, and their substrate proteins were not solubilized. The solubilized kinase and P36 were co-eluted after high pressure liquid chromatography gel filtration, showing an apparent molecular mass of 70-75 kDa. They were also co-eluted after ion exchange chromatography. These characteristics, together with its complete resistance to genistein, indicate that the rat liver histidine kinase is not cognate to the yeast enzyme (Huang, J., Nasr, M., Kim, Y., and Matthews, H.R. (1992) J. Biol. Chem. 267, 15511-15515). The phosphatase that dephosphorylates histidyl-phosphorylated P36 was also studied using rat liver subcellular fractions and in vitro phosphorylated P36 as the substrate. The characteristics of the phosphatase, that is, 1) Mg2+ requirement for activity, 2) apparent molecular mass of 45 kDa by high performance liquid chromatography gel filtration, and 3) resistance to 100 microM okadaic acid, suggest that the primary phosphatase active in vitro is protein phosphatase 2C.
Abstract: Removal of a peroxisome proliferator from the diet triggered the degradation of peroxisomes and induced the transient expression of a 220 kDa soluble protein in rat liver. The 220 kDa protein was purified by conventional methods and analyzed by amino acid sequencing. A total of 99 amino acid residues in 4 lysylendopeptidase-digested peptides completely matched those in rat fatty acid synthase. The transient induction of fatty acid synthase mRNA during peroxisome degradation was confirmed by Northern blotting.
Abstract: We have previously shown that a membrane-associated P36 from rat liver was in vitro phosphorylated at His residue(s) with a phosphoric amide bond (FEBS Lett., 319:75-79, 1993), and the activity was solubilized and partially purified (J. Biol. Chem., 269:9030-9037, 1994). The present study demonstrates that the P36 histidyl phosphorylation occurs in rat hepatoma cells under normal conditions. Phosphorylation and dephosphorylation of histidine as well as those of serine, threonine and tyrosine residues may also play an important role in animal cells.
Abstract: The structurally diverse xenobiotic peroxisome proliferators (PPs) increase the number of peroxisomes per cell and the levels of several enzymes, and cause hepatomegaly, often leading to hepatocarcinogenesis in a species- and tissue-specific manner. The deadlocked problems of the molecular mechanism of PP action and its physiological meanings have begun to be understood through cDNA cloning of a PP-activated receptor (PPAR). PPAR, a member of the steroid/thyroid/vitamin superfamily of nuclear receptors, has isoforms and differentially heterodimerizes with other nuclear receptors, providing potential mechanisms not only for species- and tissue-specific actions but also for diverse actions of PPs. Recent findings related to PPAR are summarized, and its possible role in lipid metabolism and involvement in PP-induced hepatocarcinogenesis are discussed.
Abstract: The changes in protein phosphorylation during induction and deinduction of peroxisome proliferation in rat liver by three types of proliferators were studied by in vitro phosphorylation assay. Among the variously phosphorylated proteins, an increase during induction and a decrease during deinduction in phosphorylation of P100, a cytosolic protein having a molecular weight of 100 kDa, was most remarkable. The time course of enhancement of phosphorylation by the administration of the proliferators, however, was not parallel with proliferation of peroxisome but with increase in the liver DNA content. Amino acid sequencing of the protein indicated the identity of its N-terminal 17 amino acid residues with those of elongation factor 2 (EF2). Increase in the amount of EF2 by peroxisome proliferators was confirmed by immunoblotting and this was almost parallel with peroxisome proliferation, suggesting that both increase in the amount of EF2 and some changes in phosphorylation activities account for a large increase in in vitro phosphorylation of EF2 by the administration of peroxisome proliferators.
Abstract: A novel protein kinase is induced in rat liver plasma membrane by the administration of peroxisome proliferators. A 36 kDa protein (P36) on the membrane was rapidly phosphorylated in vitro by the kinase and the phosphorylated amino acid was identified as phosphohistidine. Histidine phosphorylation of P36 was activated in vitro by recombinant Ras protein and GTP; both decreased Michaelis constant (Km) for ATP from 1.25 to 0.25 microM. The novel histidine kinase, products of which have been overlooked due to their acid lability, may participate in cellular signaling and peroxisome proliferators may perturb the pathway.
Abstract: The cDNA for subunit d of rat mitochondrial H(+)-ATP synthase was cloned from a brain cDNA library. The protein contains internally repeat structures that have sequences similar to those of other ATP-related proteins. The antibodies raised against the protein A-subunit d fusion protein expressed in E. coli specifically recognized only the protein in the mitochondria. The Na2 CO3 fractionation followed by immunoblotting analyses suggest that at least a part of the protein is inserted into the membrane.
Abstract: The effect of clofibrate, a hypolipidemic drug and a potent peroxisome proliferator, on expression of a nonperoxisomal transthyretin (prealbumin) gene was studied using rats fed clofibrate for various periods. Upon feeding a diet containing clofibrate, the level of transthyretin mRNA was down-regulated, reaching 20% of the control level, in an almost reciprocal time course to that of increases in the levels of peroxisomal mRNAs. Studies on expression of other steroid hormone regulated genes suggest that clofibrate may down-regulate several but not all types of steroid hormone regulated mRNA expression.
Abstract: Administration of clofibrate in rat results in down-regulation of several liver proteins and a vast induction of peroxisomal proteins. One protein was identified as BiP/GRP78 using antibodies and cDNA cloning. The level of mRNA was reduced by the drug.
Abstract: A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 155 amino acids with a molecular mass of 15,807 Da (pI = 9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H(+)-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed.
Abstract: A cDNA clone encoding ribonuclease inhibitor was isolated from a rat lung cDNA library. The deduced amino acid sequence revealed a high degree of conservation of a repeated structure. The mRNA was detected in all seven tissues of rat examined, the amount being highest in the lung and lowest in the heart.
Abstract: The rat gastric H+,K(+)-ATPase alpha subunit gene was cloned and the nucleotide sequence of its 5'-upstream region was determined. Sequence comparison with the corresponding part of the human gene indicated the presence of highly conserved regions which may be important for specific transcription of the alpha subunit in gastric parietal cells. The amino-terminal sequence (Met-Gly-Lys-Ala-Glu-) of the rat enzyme was similar to those of the pig and human enzymes. The gene organization of the rat enzyme was also similar to that of the human gene: introns 1, 2 and 9 were located in exactly the same positions as those in the human gene, and, as in the latter, exon 6 was not separated by an intron. The sequences of introns 1 and 2 were highly conserved among the rat, human and pig genes, but were entirely different from those of Na+,K(+)-ATPase catalytic subunit genes. Northern blot hybridization indicated that the gene was transcribed only in gastric mucosa.
Abstract: The rat chromosomal gene for uricase was cloned. The gene spans more than 20 kilobases and the coding region is divided into 8 exons by 7 introns. The organization of the rat uricase gene is so greatly different from that of the soybean gene that the difference may not have been caused only by the removal of some ancestral introns during the period of widely separated evolution.
Abstract: Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.
Abstract: The rat chromosomal gene for transthyretin was cloned and its dual promoter region was analyzed by primer extension. Semi-quantitative studies by primer extension analysis to determine the transcriptional start sites and their usage in the three tissues have suggested that the rat transthyetin gene has dual promoters: a major proximal promoter which is used in the liver and in the brain but not at all in the kidney, and a minor distal promoter which is totally used in the kidney and significantly in the brain.
Abstract: Presence of two uricase mRNA species in rabbit was revealed by cDNA cloning. They were different only in the 5'-non-coding region. Their hepatic and extra-hepatic expression in a tissue-specific manner was demonstrated by Northern blot and primer extension analyses.
Abstract: cDNAs for rat transthyretin mRNA were cloned from a brain cDNA library. Sequencing analyses showed the presence of an additional 5' sequence that had not been reported for the liver mRNA corresponding to the flanking promoter region of the gene. This additional sequence was expressed only in the brain, suggesting the presence of a brain-specific promoter.
Abstract: cDNA clones encoding uricase have been isolated from a rabbit liver cDNA library. The nucleotide sequences of the cDNAs have been determined and those of the rat uricase cDNA have been revised. In all three uricases, the carboxy-terminal tripeptides are Ser-Arg/Lys-Leu sequences, which have recently been suggested as an essential element of peroxisomal targetting signals for many but not all peroxisomal proteins.
Abstract: We have isolated cDNA clones for rat liver uricase using an oligonucleotide corresponding to the N-terminal sequence of 8 amino acids. The nucleotide sequences of the cDNAs have been determined, and the amino acid sequence of the protein deduced. A 867-base open reading frame coding for 289 amino acids, corresponding to a molecular mass of 33,274 daltons, was confirmed by matching eight sequences of a total of 53 amino acids from peptide sequence analyses of the fragments generated by lysyl endopeptidase digestion of purified rat liver uricase. The deduced amino acid sequence of rat liver uricase shares 40% homology with that of soybean nodulin-specific uricase and has an N-terminal extension of 7 amino acids. In contrast, soybean uricase has a C-terminal extension of 12 amino acids, which is presumably the result of local gene duplication. Completely different N- and C-terminal structures of the two uricases suggest that the signals for targeting the proteins to the peroxisome are not located on the terminal continuous stretches of amino acids.
Abstract: A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.
Abstract: The mRNA sequence of whey acidic protein, a major mouse milk protein, was cloned in full length. The sequence analysis of the cDNA clones revealed that the mRNA contains a 28 nucleotide-long 5'-noncoding region. Three different portions of this region were identified as possible sites of interaction with the 3'-end of 18S rRNA that may facilitate efficient rate of translation of the mRNA.
Abstract: A previous report [Motojima, K., Yamato, I. & Anraku, Y. (1978) J. Bacteriol. 136, 5-9) described the characteristics of mutants (proT-) of Escherichia coli K-12 that are defective in proline transport carrier activity. Two hybrid plasmids from the Clarke and Carbon colony bank were found to complement the mutation by conjugation and transformation. Analysis with restriction endonucleases showed that both plasmid DNAs carried the same fragment of the E. coli chromosome. A polypeptide with a molecular weight of 24,000, specifically coded for by the proT+ plasmid, was identified in the cytoplasmic membrane by using a double-isotope labeling method in a minicell system. The strain harboring the proT+ plasmid has 6 times as much proline transport carrier as the wild strain. This amplification of the carrier makes it possible to measure proline binding to the carrier by microequilibrium dialysis. Detailed analysis of binding indicated that the maximal amount of proline bound to the carrier is 0.70 nmol/mg of protein of the cytoplasmic membrane of the amplified strain. From this value and the assumption that the carrier has one binding site per minimal molecular weight of 24,000, the content of the proline transport carrier in the cytoplasmic membrane was estimated to be 1.7%.
Abstract: A series of mutants of Escherichia coli K-12 requiring a high concentration of L-proline for growth were isolated from a proline auxotroph strain, JE2133. Genetic studies of the mutants, PT19, PT21, and PT22, showed that all the mutations (proT) were point mutations, and these were mapped at 82 min on the E. coli genetic map. Intact cells and cytoplasmic membrane vesicles of these mutants were specifically defective in L-proline transport activity. Strain PT21 had no detectable activity of the L-proline transport carrier at all, and strains PT19 and PT22 had only 1/35 and 1/70, respectively, of the transport activity of the parental strain. The mutants were also shown to have a defect in proline-binding function of the carrier by measuring specific binding of proline to sonically disrupted membranes. These results indicate that the gene proT determines the function of proline carrier in the cytoplasmic membrane.
