[Show abstract][Hide abstract] ABSTRACT: Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.
[Show abstract][Hide abstract] ABSTRACT: Hyperlipidemia is a common feature of diabetes and is related to cardiovascular disease. The very low-density lipoprotein receptor (VLDL-R) is a member of the low-density lipoprotein receptor (LDL-R) family. It binds and internalizes triglyceride-rich lipoproteins with high specificity. We examined the etiology of hyperlipidemia in the insulin-deficient state. VLDL-R expression in heart and skeletal muscle were measured in rats with streptozotocin (STZ)-induced diabetes. STZ rats showed severe hyperlipidemia on d 21 and 28, with a dramatic decline in VLDL-R protein in skeletal muscle (>90%), heart (approximately 50%) and a loss of adipose tissues itself on d 28. The reduction of VLDL-R protein in skeletal muscle could not be explained simply by a decrease at the transcriptional level, because a dissociation between VLDL-R protein and mRNA expression was observed. The expression of LDL-R and LDL-R-related protein in liver showed no consistent changes. Furthermore, no effect on VLDL-triglyceride production in liver was observed in STZ rats. A decrease in postheparin plasma lipoprotein lipase activity started on d 7 and continued to d 28 at the 50% level even though severe hyperlipidemia was detected only on d 21 and 28. In rat myoblast cells, serum deprivation for 24 h induced a reduction in VLDL-R proteins. Insulin (10(-6) m), but not IGF-I (10 ng/ml), restored the decreased VLDL-R proteins by serum deprivation. These results suggest that the combination of VLDL-R deficiency and reduced plasma lipoprotein lipase activity may be responsible for severe hyperlipidemia in insulin-deficient diabetes.
[Show abstract][Hide abstract] ABSTRACT: Acetyl-CoA synthetase 2 (AceCS2) produces acetyl-CoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5'-flanking region of the mouse AceCS2 gene demonstrates that Krüppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.
Journal of Biological Chemistry 05/2004; 279(17):16954-62. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH2 terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized beta VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease.
Journal of Human Genetics 02/2004; 49(11):622-8. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor family. In vitro and in vivo studies have shown that VLDL receptor binds triglyceride (TG)-rich lipoproteins but not LDL, and functions as a peripheral remnant lipoprotein receptor. VLDL receptor is expressed abundantly in fatty acid-active tissues (heart, skeletal muscle and fat), the brain and macrophages. It is likely that VLDL receptor functions in concert with lipoprotein lipase (LPL), which hydrolyses TG in VLDL and chylomicron. In contrast to the LDL receptor, VLDL receptor binds apolipoprotein (apo) E2/2 VLDL particles as well as apoE3/3 VLDL, and the expression is not down-regulated by intracellular lipoproteins. Recently, various functions of the VLDL receptor have been reported in lipoprotein metabolism, metabolic syndrome/atherosclerosis, cardiac fatty acid metabolism, neuronal migration and angiogenesis/tumor growth. Gene therapy of VLDL receptor into the liver showed a benefit effect for lipoprotein metabolism in both LDL receptor knockout and apoE mutant mice. Beyond its function as a peripheral lipoprotein receptor, possibilities of its physiological function have been extended to include signal transduction, angiogenesis and tumor growth.
Journal of atherosclerosis and thrombosis 02/2004; 11(4):200-8. · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acetyl-CoA synthetase (AceCS) catalyzes the production of acetyl-CoA from acetate, CoA and ATP. There are two types of AceCS in mammals with different functions. One designated AceCS1 is a cytosolic enzyme expressed in the liver and plays a role in the production of acetyl-CoA for the synthesis of fatty acids and cholesterol. The other enzyme AceCS2 is a mitochondrial matrix enzyme that produces acetyl-CoAs mainly utilized for oxidation. Consistent with its function, the transcription of AceCS1 is regulated by SREBPs. In contrast, the expression of AceCS2 is upregulated during starvation and ketogenesis via unknown mechanisms. Specific inhibitors of AceCS may provide therapeutic agents for the treatment of obesity, cardiovascular diseases and type 2 diabetes.
[Show abstract][Hide abstract] ABSTRACT: The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.
Molecular and Cellular Biochemistry 07/2003; 248(1-2):121-7. · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.
Journal of Biological Chemistry 04/2003; 278(13):11331-6. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: By expression cloning using fluorescent-labeled high density lipoprotein (HDL), we isolated two clones that conferred the cell surface binding of HDL. Nucleotide sequence of the two clones revealed that one corresponds to scavenger receptor class B, type 1 (SRBI) and the other encoded a novel protein with 228 amino acids. The primary structure of the newly identified HDL-binding protein resembles GPI-anchored proteins consisting of an N-terminal signal sequence, an acidic region with a cluster of aspartate and glutamate residues, an Ly-6 motif highly conserved among the lymphocyte antigen family, and a C-terminal hydrophobic region. This newly identified HDL-binding protein designated GPI-anchored HDL-binding protein 1 (GPI-HBP1), was susceptible to phosphatidylinositol-specific phospholipase C treatment and binds HDL with high affinity (calculated K(d) = 2-3 microg/ml). Similar to SRBI, GPI-HBP1 mediates selective lipid uptake but not the protein component of HDL. Among various ligands for SRBI, HDL was most preferentially bound to GPI-HBP1. In contrast to SRBI, GPI-HBP1 lacked HDL-dependent cholesterol efflux. The GPI-HBP1 transcripts were detected with the highest levels in heart and, to a much lesser extent, in lung and liver. In situ hybridization revealed the accumulation of GPI-HBP1 transcripts in cardiac muscle cells, hepatic Kupffer cells and sinusoidal endothelium, and bronchial epithelium and alveolar macrophages in the lung.
Journal of Biological Chemistry 03/2003; 278(9):7344-9. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca(2+) in response to glucose, and thereby glucose-induced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5-- islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5-- islets. Furthermore, exposure of LRP5++ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that WntLRP5 signaling contributes to the glucose-induced insulin secretion in the islets.
Proceedings of the National Academy of Sciences 02/2003; 100(1):229-34. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The VLDL (very low-density lipoprotein) receptor is a peripheral lipoprotein receptor expressing in fatty acid active tissues abundantly. In the Balb/c fasting mice, VLDL receptor as well as LPL (lipoprotein lipase), FAT (fatty acid translocase)/CD36, H-FABP (heart-type fatty acid-binding protein), ACS (acyl-CoA synthetase) and LCAD (long-chain acyl-CoA dehydrogenase) expressions increased. An electron microscopic examination indicated the lipid droplets that accumulated in the hearts of fasting Balb/c mice. During the development of SD (Sprague-Dawley) rats, VLDL receptor, LPL, FAT/CD36, H-FABP, ACS, and LCAD mRNAs concomitantly increased with growth. However, PK (pyruvate kinase) mRNA expression was negligible. In cultured neonatal rat cardiomyocytes, VLDL receptor expression increased with days in culture. Oil red-O staining showed that cardiomyocytes after 7 days in culture (when the VLDL receptor protein is present) accumulated beta-migrating VLDL. Thereby, we showed that the cardiac VLDL receptor pathway for delivery of remnant lipoprotein particles might be part of a cardiac fatty acid metabolism.
Biochemical and Biophysical Research Communications 06/2002; 293(3):1007-13. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acyl-CoA synthetases (ACSs) play an essential role in fatty acid metabolism. ACS3 is an arachidonate-preferring enzyme expressed in a wide range of human tissues including brain, heart, placenta, prostate, skeletal muscle, testis and thymus. As an initial step to understanding the transcriptional regulation of the human ACS3 gene, we analyzed the genomic organization and transcription units of the human ACS3 gene. Sequence analysis of genomic clones demonstrates that the human ACS3 gene spans at least 80.6 kb and contains 17 exons. The human ACS3 gene was mapped between the sequence-tagged site markers D2S360 and WI-21901. Sequence inspection of the 5'-flanking region revealed potential DNA elements including CCAAT, AP-1, Oct-1, GATAs, SRY, CdxA, Nkx-2.5, c-Myb, HSF2, NF-AT, AP-2, NF-Y, and p300. A minimal promoter region required for the expression of the human ACS3 gene in melanoma G361 cells was determined.
[Show abstract][Hide abstract] ABSTRACT: In this study, we identified and characterized two murine cDNAs encoding medium-chain acyl-CoA synthetase (MACS). One, designated MACS1, is a novel protein and the other the product of the Sa gene (Sa protein), which is preferentially expressed in spontaneously hypertensive rats. Based on the murine MACS1 sequence, we also identified the location and organization of the human MACS1 gene, showing that the human MACS1 and Sa genes are located in the opposite transcriptional direction within a 150-kilobase region on chromosome 16p13.1. Murine MACS1 and Sa protein were overexpressed in COS cells, purified to homogeneity, and characterized. Among C4-C16 fatty acids, MACS1 preferentially utilizes octanoate, whereas isobutyrate is the most preferred fatty acid among C2-C6 fatty acids for Sa protein. Like Sa gene transcript, MACS1 mRNA was detected mainly in the liver and kidney. Subcellular fractionation revealed that both MACS1 and Sa protein are localized in the mitochondrial matrix. (14)C-Fatty acid incorporation studies indicated that acyl-CoAs produced by MACS1 and Sa protein are utilized mainly for oxidation.
Journal of Biological Chemistry 10/2001; 276(38):35961-6. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5'-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (approximately 2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility "supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.
Journal of Biological Chemistry 10/2001; 276(36):34259-69. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring isozyme of ACS family predominantly expressed in steroidogenic tissues. Isolation and characterization of genomic clones encoding human ACS4 revealed that the genomic organization of the gene. The human ACS4 gene spans approximately 90 kb and consists of 16 exons. Sequence inspection of the 5'-flanking region revealed potential DNA elements including GATAs, p300, AP-4, SRY, CREB and MyoD. A minimal promoter region required for the expression of ACS4 in HeLa S3 cells was determined. The human ACS4 gene was mapped between the STS markers, WI-17685 and CHLC.GATA81B07 on Xq22-23 region.
Biochemical and Biophysical Research Communications 09/2001; 286(1):80-6. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arachidonate released by various stimuli is rapidly reesterified into membrane phospholipids initiated by acyl-CoA synthetase (ACS) and subsequent acyl-transfer reactions. ACS4 is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. Female mice heterozygous for ACS4 deficiency become pregnant less frequently and produce small litters with extremely low transmission of the disrupted alleles. Striking morphological changes, including extremely enlarged uteri and lumina filled with numerous proliferative cysts of various sizes, were detected in ACS4+/- females. Furthermore, marked accumulation of prostaglandins was seen in the uterus of the heterozygous females. These results indicate that ACS4 modulates female fertility and uterine prostaglandin production.
Biochemical and Biophysical Research Communications 07/2001; 284(4):993-7. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using peptide sequences derived from bovine cardiac acetyl-CoA synthetase (AceCS), we isolated and characterized cDNAs for a bovine and murine cardiac enzyme designated AceCS2. We also isolated a murine cDNA encoding a hepatic type enzyme, designated AceCS1, identical to one reported recently (Luong, A., Hannah, V. C., Brown, M. S., and Goldstein, J. L. (2000) J. Biol. Chem. 275, 26458-26466). Murine AceCS1 and AceCS2 were purified to homogeneity and characterized. Among C2-C5 short and medium chain fatty acids, both enzymes preferentially utilize acetate with similar affinity. The AceCS2 transcripts are expressed in a wide range of tissues, with the highest levels in heart, and are apparently absent from the liver. The levels of AceCS2 mRNA in skeletal muscle were increased markedly under ketogenic conditions. Subcellular fractionation revealed that AceCS2 is a mitochondrial matrix enzyme. [(14)C]Acetate incorporation indicated that acetyl-CoAs produced by AceCS2 are utilized mainly for oxidation.
Journal of Biological Chemistry 05/2001; 276(14):11420-6. · 4.60 Impact Factor