Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulate... more Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulated lipogenesis, upstream mechanisms governing Srebf1 expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1 promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1 and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link betw...
Biochemical and Biophysical Research Communications, 2015
Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty ac... more Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis.
International Journal of Radiation Applications and Instrumentation. Part C. Radiation Physics and Chemistry, 1991
Abstract Gas evolution, oxygen consumption, and change of mechanical property induced by the γ-ir... more Abstract Gas evolution, oxygen consumption, and change of mechanical property induced by the γ-irradiation of ultra-high molecular weight polyethylene (UHMW-PE) fiber were studied, and compared with those of high density PE (HDPE). Samples were irradiated in oxygen under pressure from 0 to 60 kPa by 60Co γ-ray up to 1.0 MGy at room temperature. For irradiation under vacuum, G(H2) is 2.4 and G(CH4) is 2.0 × 10-3. In the presence of oxygen, G(H2) was about the same, and the G-values for other hydrocarbons were more than 20 times those under vacuum. The tensile strength of the fiber was decreased greatly by irradiation in oxygen.
Familial apolipoprotein C-Ⅱ (apoC-Ⅱ) deficiency is a rare autosomal recessive disorder with marke... more Familial apolipoprotein C-Ⅱ (apoC-Ⅱ) deficiency is a rare autosomal recessive disorder with marked hypertriglyceridemia resulting from impaired activation of lipoprotein lipase. In most cases of apoC-Ⅱ deficiency, causative mutations have been found in the protein-coding region of APOC2; however, several atypical cases of apoC-Ⅱ deficiency were reported to have markedly reduced, but detectable levels of plasma apoC-Ⅱ protein (hereafter referred to as hypoapoC-Ⅱ), which resulted from decreased promoter activity or improper splicing of apoC-Ⅱ mRNA due to homozygous mutations in APOC2. Here we aim to dissect the molecular bases of a new case of hypoapoC-Ⅱ. Methods: We performed detailed biochemical/genetic analyses of our new case of hypoapoC-Ⅱ, manifesting severe hypertriglyceridemia (plasma triglycerides, 3235 mg • dL −1) with markedly reduced levels of plasma apoC-Ⅱ (0.6 mg • dL −1). Results: We took advantage of a monocyte/macrophage culture system to prove that transcription of apoC-Ⅱ mRNA was decreased in the patient's cells, which is compatible with the reported features of hypoapoC-Ⅱ. Concomitantly, transcriptional activity of the minigene reporter construct of the patient's APOC2 gene was decreased; however, no rare variant was detected in the patient's APOC2 gene. Fifty single nucleotide variants were detected in the patient's APOC2, but all were common variants (allele frequencies ＞35%) that are supposedly not causative. Conclusions: A case of apoC-Ⅱ deficiency was found that is phenotypically identical to hypoapoC-Ⅱ but with no causative mutations in APOC2, implying that other genes regulate apoC-Ⅱ levels. The clinical entity of hypoapoC-Ⅱ is discussed.
During fasting, animals maintain their energy balance by shifting their energy source from carboh... more During fasting, animals maintain their energy balance by shifting their energy source from carbohydrates to triglycerides. However, the trigger for this switch has not yet been entirely elucidated. Here we show that a selective hepatic vagotomy slows the speed of fat consumption by attenuating sympathetic nerve-mediated lipolysis in adipose tissue. Hepatic glycogen pre-loading by the adenoviral overexpression of glycogen synthase or the transcription factor TFE3 abolished this liver-brain-adipose axis activation. Moreover, the blockade of glycogenolysis through the knockdown of the glycogen phosphorylase gene and the resulting elevation in the glycogen content abolished the lipolytic signal from the liver, indicating that glycogen is the key to triggering this neurocircuitry. These results demonstrate that liver glycogen shortage activates a liver-brain-adipose neural axis that has an important role in switching the fuel source from glycogen to triglycerides under prolonged fasting conditions.
cells (2-5), a hallmark of atherosclerosis, it is important to clarify the mechanisms that mediat... more cells (2-5), a hallmark of atherosclerosis, it is important to clarify the mechanisms that mediate the hydrolysis of CE in foam cells (2-5). As the hydrolysis of CE preceding reverse cholesterol transport takes place at neutral pH, the enzymes catalyzing it have been collectively called neutral CE hydrolases (nCEHs) to distinguish them from lysosomal acid lipase, the CE hydrolase in lysosome whose optimal pH is acidic. Since carboxyl ester lipase (also called cholesterol esterase or bile salt-dependent lipase) is a secreted protein, it is not involved in the hydrolysis of intracellular CE (6). To date, three enzymes have been proposed to serve as nCEHs in macrophages: hormone-sensitive lipase (HSL) (7, 8); cholesterol ester hydrolase (CEH) (9), which is identical to a human liver carboxylesterase 1 (hCE-1) (10) or macrophage serine esterase1 (11), also known as a human ortholog of porcine triacylglycerol hydrolase (TGH) (12); and neutral cholesterol ester hydrolase (NCEH) (13), which is also known as KIAA1363 or arylacetamide deacetylase-like 1 (14). However, it is not clear which enzyme is of primary importance in the hydrolysis of CE in macrophages. In mice, the ablation of HSL did not signifi cantly reduce nCEH activity in murine peritoneal macrophages (MPMs) (15, 16). Hence, HSL is not the major enzyme mediating nCEH activity in MPMs. Ghosh reported CEH as a promising candidate for an nCEH because its macrophage-specifi c overexpression under the promoter of scavenger receptor Abstract Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membranespanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn 270 , Asn 367 , and Asn 389) of NCEH are glycosylated. Glycosylation at Asn 270 , which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for effl ux out of the cells.
Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulat... more Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for PUFA suppression of SREBP-1 is at the proteolytic processing level and that this suppression in turn decreases the mRNA transcription through lowering SREBP-1 binding to the SREBPbinding element on the promoter ("autoloop regulatory circuit"), although liver X receptor, an activator for Srebf1c transcription, is not involved in this regulation by PUFA. The mechanisms for PUFA suppression of SREBP-1 confirm that the autoloop regulation for transcription is crucial for the nutritional regulation of triglyceride synthesis.
Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophag... more Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.
Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose... more Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose tissue, are not fully understood. A database search for enzymes with α/β hydrolase folds, the GXSXG motif for serine esterase and the His-Gly dipeptide motif, has provided a previously unannotated gene that is induced during 3T3-L1 adipocytic differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4% identity, we have tentatively designated this enzyme as TGH-2 and the original TGH as TGH-1. TGH-2 is also similar to TGH-1 in terms of tissue distribution, subcellular localization, substrate specificity, and regulation. Both enzymes are predominantly expressed in liver, adipose tissue, and kidney. In adipocytes, they are localized in microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein, cholesteryl oleate, or phospholipids; hydrolysis of short-chain fatty acid ester was 30,000-fol...
Rationale: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse chol... more Rationale: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse cholesterol transport in macrophage foam cells. We have recently shown that neutral cholesterol ester hydrolase (Nceh)1 and hormone-sensitive lipase (Lipe) are key regulators of this process in mouse macrophages. However, it remains unknown which enzyme is critical in human macrophages and atherosclerosis. Objective: We aimed to identify the enzyme responsible for the CE hydrolysis in human macrophages and to determine its expression in human atherosclerosis. Methods and Results: We compared the expression of NCEH1, LIPE, and cholesterol ester hydrolase (CES1) in human monocyte-derived macrophages (HMMs) and examined the effects of inhibition or overexpression of each enzyme in the cholesterol trafficking. The pattern of expression of NCEH1 was similar to that of neutral CE hydrolase activity during the differentiation of HMMs. Overexpression of human NCEH1 increased the hydrolysis of CE, th...
Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the h... more Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the hydrolysis of intracellular CE is one of the key steps in foam cell formation. Although hormone-sensitive lipase (LIPE) and cholesterol ester hydrolase (CEH), which is identical to carboxylsterase 1 (CES1, hCE1), were proposed to mediate the neutral CE hydrolase (nCEH) activity in macrophages, recent evidences have suggested the involvement of other enzymes. We have recently reported the identification of a candidate, neutral cholesterol ester hydrolase 1 (Nceh1). Here we demonstrate that genetic ablation of Nceh1 promotes foam cell formation and the development of atherosclerosis in mice. We further demonstrate that Nceh1 and Lipe mediate a comparable degree of nCEH activity in macrophages and together account for most of the activity. Mice lacking both Nceh1 and Lipe aggravated atherosclerosis in an additive manner. Thus, Nceh1 is a promising target for the treatment of atherosclerosis.
Biochemical and Biophysical Research Communications, 2011
We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam... more We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement.
Biochemical and Biophysical Research Communications, 2009
It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis... more It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis in pancreatic b-cells can affect insulin secretion through the alteration of lipotoxicity. We generated mice lacking both leptin and HSL (Lep ob/ob /HSL À/À) and explored the role of HSL in pancreatic b-cells in the setting of obesity. Lep ob/ob /HSL À/À developed elevated blood glucose levels and reduced plasma insulin levels compared with Lep ob/ob /HSL +/+ in a fed state, while the deficiency of HSL did not affect glucose homeostasis in Lep +/+ background. The deficiency of HSL exacerbated the accumulation of triglycerides in Lep ob/ob islets, leading to reduced glucose-stimulated insulin secretion. The deficiency of HSL also diminished the islet mass in Lep ob/ob mice due to decreased cell proliferation. In conclusion, HSL affects insulin secretary capacity especially in the setting of obesity.
Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by... more Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipocytes. Using the in vivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around -100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepatocytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes.
Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by... more Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipo-cytes. Using the in vivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around À100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepato-cytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes.
Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulate... more Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulated lipogenesis, upstream mechanisms governing Srebf1 expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1 promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1 and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link betw...
Biochemical and Biophysical Research Communications, 2015
Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty ac... more Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis.
International Journal of Radiation Applications and Instrumentation. Part C. Radiation Physics and Chemistry, 1991
Abstract Gas evolution, oxygen consumption, and change of mechanical property induced by the γ-ir... more Abstract Gas evolution, oxygen consumption, and change of mechanical property induced by the γ-irradiation of ultra-high molecular weight polyethylene (UHMW-PE) fiber were studied, and compared with those of high density PE (HDPE). Samples were irradiated in oxygen under pressure from 0 to 60 kPa by 60Co γ-ray up to 1.0 MGy at room temperature. For irradiation under vacuum, G(H2) is 2.4 and G(CH4) is 2.0 × 10-3. In the presence of oxygen, G(H2) was about the same, and the G-values for other hydrocarbons were more than 20 times those under vacuum. The tensile strength of the fiber was decreased greatly by irradiation in oxygen.
Familial apolipoprotein C-Ⅱ (apoC-Ⅱ) deficiency is a rare autosomal recessive disorder with marke... more Familial apolipoprotein C-Ⅱ (apoC-Ⅱ) deficiency is a rare autosomal recessive disorder with marked hypertriglyceridemia resulting from impaired activation of lipoprotein lipase. In most cases of apoC-Ⅱ deficiency, causative mutations have been found in the protein-coding region of APOC2; however, several atypical cases of apoC-Ⅱ deficiency were reported to have markedly reduced, but detectable levels of plasma apoC-Ⅱ protein (hereafter referred to as hypoapoC-Ⅱ), which resulted from decreased promoter activity or improper splicing of apoC-Ⅱ mRNA due to homozygous mutations in APOC2. Here we aim to dissect the molecular bases of a new case of hypoapoC-Ⅱ. Methods: We performed detailed biochemical/genetic analyses of our new case of hypoapoC-Ⅱ, manifesting severe hypertriglyceridemia (plasma triglycerides, 3235 mg • dL −1) with markedly reduced levels of plasma apoC-Ⅱ (0.6 mg • dL −1). Results: We took advantage of a monocyte/macrophage culture system to prove that transcription of apoC-Ⅱ mRNA was decreased in the patient's cells, which is compatible with the reported features of hypoapoC-Ⅱ. Concomitantly, transcriptional activity of the minigene reporter construct of the patient's APOC2 gene was decreased; however, no rare variant was detected in the patient's APOC2 gene. Fifty single nucleotide variants were detected in the patient's APOC2, but all were common variants (allele frequencies ＞35%) that are supposedly not causative. Conclusions: A case of apoC-Ⅱ deficiency was found that is phenotypically identical to hypoapoC-Ⅱ but with no causative mutations in APOC2, implying that other genes regulate apoC-Ⅱ levels. The clinical entity of hypoapoC-Ⅱ is discussed.
During fasting, animals maintain their energy balance by shifting their energy source from carboh... more During fasting, animals maintain their energy balance by shifting their energy source from carbohydrates to triglycerides. However, the trigger for this switch has not yet been entirely elucidated. Here we show that a selective hepatic vagotomy slows the speed of fat consumption by attenuating sympathetic nerve-mediated lipolysis in adipose tissue. Hepatic glycogen pre-loading by the adenoviral overexpression of glycogen synthase or the transcription factor TFE3 abolished this liver-brain-adipose axis activation. Moreover, the blockade of glycogenolysis through the knockdown of the glycogen phosphorylase gene and the resulting elevation in the glycogen content abolished the lipolytic signal from the liver, indicating that glycogen is the key to triggering this neurocircuitry. These results demonstrate that liver glycogen shortage activates a liver-brain-adipose neural axis that has an important role in switching the fuel source from glycogen to triglycerides under prolonged fasting conditions.
cells (2-5), a hallmark of atherosclerosis, it is important to clarify the mechanisms that mediat... more cells (2-5), a hallmark of atherosclerosis, it is important to clarify the mechanisms that mediate the hydrolysis of CE in foam cells (2-5). As the hydrolysis of CE preceding reverse cholesterol transport takes place at neutral pH, the enzymes catalyzing it have been collectively called neutral CE hydrolases (nCEHs) to distinguish them from lysosomal acid lipase, the CE hydrolase in lysosome whose optimal pH is acidic. Since carboxyl ester lipase (also called cholesterol esterase or bile salt-dependent lipase) is a secreted protein, it is not involved in the hydrolysis of intracellular CE (6). To date, three enzymes have been proposed to serve as nCEHs in macrophages: hormone-sensitive lipase (HSL) (7, 8); cholesterol ester hydrolase (CEH) (9), which is identical to a human liver carboxylesterase 1 (hCE-1) (10) or macrophage serine esterase1 (11), also known as a human ortholog of porcine triacylglycerol hydrolase (TGH) (12); and neutral cholesterol ester hydrolase (NCEH) (13), which is also known as KIAA1363 or arylacetamide deacetylase-like 1 (14). However, it is not clear which enzyme is of primary importance in the hydrolysis of CE in macrophages. In mice, the ablation of HSL did not signifi cantly reduce nCEH activity in murine peritoneal macrophages (MPMs) (15, 16). Hence, HSL is not the major enzyme mediating nCEH activity in MPMs. Ghosh reported CEH as a promising candidate for an nCEH because its macrophage-specifi c overexpression under the promoter of scavenger receptor Abstract Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membranespanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn 270 , Asn 367 , and Asn 389) of NCEH are glycosylated. Glycosylation at Asn 270 , which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for effl ux out of the cells.
Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulat... more Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for PUFA suppression of SREBP-1 is at the proteolytic processing level and that this suppression in turn decreases the mRNA transcription through lowering SREBP-1 binding to the SREBPbinding element on the promoter ("autoloop regulatory circuit"), although liver X receptor, an activator for Srebf1c transcription, is not involved in this regulation by PUFA. The mechanisms for PUFA suppression of SREBP-1 confirm that the autoloop regulation for transcription is crucial for the nutritional regulation of triglyceride synthesis.
Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophag... more Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.
Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose... more Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose tissue, are not fully understood. A database search for enzymes with α/β hydrolase folds, the GXSXG motif for serine esterase and the His-Gly dipeptide motif, has provided a previously unannotated gene that is induced during 3T3-L1 adipocytic differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4% identity, we have tentatively designated this enzyme as TGH-2 and the original TGH as TGH-1. TGH-2 is also similar to TGH-1 in terms of tissue distribution, subcellular localization, substrate specificity, and regulation. Both enzymes are predominantly expressed in liver, adipose tissue, and kidney. In adipocytes, they are localized in microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein, cholesteryl oleate, or phospholipids; hydrolysis of short-chain fatty acid ester was 30,000-fol...
Rationale: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse chol... more Rationale: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse cholesterol transport in macrophage foam cells. We have recently shown that neutral cholesterol ester hydrolase (Nceh)1 and hormone-sensitive lipase (Lipe) are key regulators of this process in mouse macrophages. However, it remains unknown which enzyme is critical in human macrophages and atherosclerosis. Objective: We aimed to identify the enzyme responsible for the CE hydrolysis in human macrophages and to determine its expression in human atherosclerosis. Methods and Results: We compared the expression of NCEH1, LIPE, and cholesterol ester hydrolase (CES1) in human monocyte-derived macrophages (HMMs) and examined the effects of inhibition or overexpression of each enzyme in the cholesterol trafficking. The pattern of expression of NCEH1 was similar to that of neutral CE hydrolase activity during the differentiation of HMMs. Overexpression of human NCEH1 increased the hydrolysis of CE, th...
Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the h... more Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the hydrolysis of intracellular CE is one of the key steps in foam cell formation. Although hormone-sensitive lipase (LIPE) and cholesterol ester hydrolase (CEH), which is identical to carboxylsterase 1 (CES1, hCE1), were proposed to mediate the neutral CE hydrolase (nCEH) activity in macrophages, recent evidences have suggested the involvement of other enzymes. We have recently reported the identification of a candidate, neutral cholesterol ester hydrolase 1 (Nceh1). Here we demonstrate that genetic ablation of Nceh1 promotes foam cell formation and the development of atherosclerosis in mice. We further demonstrate that Nceh1 and Lipe mediate a comparable degree of nCEH activity in macrophages and together account for most of the activity. Mice lacking both Nceh1 and Lipe aggravated atherosclerosis in an additive manner. Thus, Nceh1 is a promising target for the treatment of atherosclerosis.
Biochemical and Biophysical Research Communications, 2011
We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam... more We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement.
Biochemical and Biophysical Research Communications, 2009
It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis... more It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis in pancreatic b-cells can affect insulin secretion through the alteration of lipotoxicity. We generated mice lacking both leptin and HSL (Lep ob/ob /HSL À/À) and explored the role of HSL in pancreatic b-cells in the setting of obesity. Lep ob/ob /HSL À/À developed elevated blood glucose levels and reduced plasma insulin levels compared with Lep ob/ob /HSL +/+ in a fed state, while the deficiency of HSL did not affect glucose homeostasis in Lep +/+ background. The deficiency of HSL exacerbated the accumulation of triglycerides in Lep ob/ob islets, leading to reduced glucose-stimulated insulin secretion. The deficiency of HSL also diminished the islet mass in Lep ob/ob mice due to decreased cell proliferation. In conclusion, HSL affects insulin secretary capacity especially in the setting of obesity.
Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by... more Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipocytes. Using the in vivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around -100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepatocytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes.
Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by... more Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipo-cytes. Using the in vivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around À100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepato-cytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes.
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Papers by Makiko Nishi