Makiko Nishi

Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages

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.

Polyunsaturated fatty acids selectively suppress sterol regulatory element-binding protein-1 through proteolytic processing and autoloop regulatory circuit.

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 SREBP-binding 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.

Ablation of Neutral Cholesterol Ester Hydrolase 1 Accelerates Atherosclerosis

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.

Identification of a Novel Member of the Carboxylesterase Family That Hydrolyzes Triacylglycerol: A Potential Role in Adipocyte Lipolysis

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-fold more efficient than that of long-chain fatty acid triacylglycerol. Fasting increased the expression of both genes in white adipose tissue, whereas refeeding suppressed their expression. RNA silencing of TGH-2 reduced isoproterenol-stimulated glycerol release by 10% in 3T3-L1 adipocytes, while its overexpression increased the glycerol release by 20%. Thus, TGH-2 may make a contribution to adipocyte lipolysis during period of increased energy demand.

The Critical Role of Neutral Cholesterol Ester Hydrolase 1 in Cholesterol Removal From Human Macrophages

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, thereby stimulating cholesterol mobilization from THP-1 macrophages. Knockdown of NCEH1 specifically reduced the neutral CE hydrolase activity. Pharmacological inhibition of NCEH1 also increased the cellular CE in HMMs. In contrast, LIPE was barely detectable in HMMs, and its inhibition did not decrease neutral CE hydrolase activity. Neither overexpression nor knockdown of CES1 affected the neutral CE hydrolase activity. NCEH1 was expressed in CD68-positive macrophage foam cells of human atherosclerotic lesions. Conclusions: NCEH1 is expressed in human atheromatous lesions, where it plays a critical role in the hydrolysis of CE in human macrophage foam cells, thereby contributing to the initial part of reverse cholesterol transport in human atherosclerosis.

Glycogen shortage during fasting triggers liver–brain–adipose neurocircuitry to facilitate fat utilization

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 glycolysis 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.

Targeting of neutral cholesterol ester hydrolase to the endoplasmic reticulum via its N-terminal sequence.

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-membrane-spanning 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 (Asn270, Asn367, and Asn389) of NCEH are glycosylated. Glycosylation at Asn270, 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 efflux out of the cells.

Identification of human ELOVL5 enhancer regions controlled by SREBP

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.

Abrogation of neutral cholesterol ester hydrolytic activity causes adrenal enlargement.

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.

Hormone-sensitive lipase deficiency suppresses insulin secretion from pancreatic islets of Lepob/ob mice

It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis in pancreatic beta-cells can affect insulin secretion through the alteration of lipotoxicity. We generated mice lacking both leptin and HSL Lep(ob/ob)/HSL(-/-) and explored the role of HSL in pancreatic beta-cells in the setting of obesity. Lep(ob/ob)/HSL(-/-) 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.