A novel homozygous nonsense variant of LMF1 in pregnancy-induced hypertriglyceridemia with acute pancreatitis.

Hypertriglyceridemia (HTG)-induced pancreatitis during pregnancy could lead to maternal and fetal death. However, its genetic bases are not fully understood, and its treatment strategies are yet to be established. Here we report a case with a novel homozygous nonsense variant of LMF1 in pregnancy-associated HTG with acute pancreatitis. Our patient had childhood-onset severe HTG that had been well-controlled by dietary management in the non-pregnant period with plasma triglyceride (TG) levels at around 200 mg/dL. Milky plasma was noted at the first-trimester pregnancy checkup, followed by a severe increase in plasma TG (10,500 mg/dL) that resulted in pancreatitis in the last trimester. The implementation of strict dietary fat restriction (less than 4 grams per day) reduced plasma TG levels and led to successful delivery. Exome sequencing revealed a novel homozygous nonsense variant in LMF1 (c.697C>T, p.Arg233Ter). The activities of lipoprotein lipase (LPL) and hepatic lipase in post-heparin plasma were not abolished but reduced. The use of pemafibrate decreased plasma TG levels with a concomitant increase in LPL activity. HTG in childhood or early pregnancy is commonly assumed to be polygenic in origin but should be regarded as a feature suggestive of monogenic hyperchylomicronemia. Adequate TG monitoring and dietary fat restriction should be implemented to prevent potentially lethal events of pancreatitis.

A case of NASH with genetic predisposition successfully treated with an SGLT2 inhibitor: a possible involvement of mitochondrial dysfunction.

Summary: In this study, we herein describe a 47-year-old Japanese woman who manifested inheritable non-alcoholic steatohepatitis (NASH) and severe dyslipidemia. Interestingly, her NASH progression was ameliorated by treatment with a sodium-glucose co-transporter 2 (SGLT2) inhibitor. This inheritability prompted us to comprehensively decode her genomic information using whole-exome sequencing. We found the well-established I148M mutation in PNPLA3 as well as mutations in LGALS3 and PEMT for her NASH. Mutations in GCKR may contribute to both NASH and dyslipidemia. We further mined gene mutations potentially responsible for her manifestations that led to the identification of a novel M188fs mutation in MUL1 that may be causally associated with her mitochondrial dysfunction. Our case may provide some clues to better understand this spectrum of disease as well as the rationale for selecting medications. Learning points: While the PNPLA3 I148M mutation is well-established, accumulation of other mutations may accelerate susceptibility to non-alcoholic steatohepatitis (NASH). NASH and dyslipidemia may be intertwined biochemically and genetically through several key genes. SGLT2 inhibitors emerge as promising treatment for NASH albeit with interindividual variation in efficacy. Genetic background may explain the mechanisms behind the variation. A novel dysfunctional mutation in MUL1 may lead to metabolic inflexibilities through impaired mitochondrial dynamics and function.

Critical Role of SREBP-1c Large-VLDL Pathway in Environment-Induced Hypertriglyceridemia of Apo AV Deficiency.

Objective- APOA5 variants are strongly associated with hypertriglyceridemia, as well as increased risks of cardiovascular disease and acute pancreatitis. Hypertriglyceridemia in apo AV dysfunction often aggravates by environmental factors such as high-carbohydrate diets or aging. To date, the molecular mechanisms by which these environmental factors induce hypertriglyceridemia are poorly defined, leaving the high-risk hypertriglyceridemia condition undertreated. Previously, we reported that LXR (liver X receptor)-SREBP (sterol regulatory element-binding protein)-1c pathway regulates large-VLDL (very low-density lipoprotein) production induced by LXR agonist. However, the pathophysiological relevance of the finding remains unknown. Approach and Results- Here, we reconstitute the environment-induced hypertriglyceridemia phenotype of human APOA5 deficiency in Apoa5-/- mice and delineate the role of SREBP-1c in vivo by generating Apoa5-/- ;Srebp-1c-/- mice. The Apoa5-/- mice, which showed moderate hypertriglyceridemia on a chow diet, developed severe hypertriglyceridemia on high-carbohydrate feeding or aging as seen in patients with human apo AV deficiency. These responses were nearly completely abolished in the Apoa5-/- ;Srebp-1c-/- mice. Further mechanistic studies revealed that in response to these environmental factors, SREBP-1c was activated to increase triglyceride synthesis and to permit the incorporation of triglyceride into abnormally large-VLDL particles, which require apo AV for efficient clearance. Conclusions- Severe hypertriglyceridemia develops only when genetic factors (apo AV deficiency) and environmental effects (SREBP-1c activation) coexist. We demonstrate that the regulated production of large-sized VLDL particles via SREBP-1c determines plasma triglyceride levels in apo AV deficiency. Our findings explain the long-standing enigma of the late-onset hypertriglyceridemia phenotype of apo AV deficiency and suggest a new approach to treat hypertriglyceridemia by targeting genes that mediate environmental effects.

GPIHBP1 autoantibody syndrome during interferon ß1a treatment.

BACKGROUND: Autoantibodies against glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) cause chylomicronemia by blocking the ability of GPIHBP1 to bind lipoprotein lipase (LPL) and transport the enzyme to its site of action in the capillary lumen. OBJECTIVE: A patient with multiple sclerosis developed chylomicronemia during interferon (IFN) ß1a therapy. The chylomicronemia resolved when the IFN ß1a therapy was discontinued. Here, we sought to determine whether the drug-induced chylomicronemia was caused by GPIHBP1 autoantibodies. METHODS: We tested plasma samples collected during and after IFN ß1a therapy for GPIHBP1 autoantibodies (by western blotting and with enzyme-linked immunosorbent assays). We also tested whether the patient's plasma blocked the binding of LPL to GPIHBP1 on GPIHBP1-expressing cells. RESULTS: During IFN ß1a therapy, the plasma contained GPIHBP1 autoantibodies, and those autoantibodies blocked GPIHBP1's ability to bind LPL. Thus, the chylomicronemia was because of the GPIHBP1 autoantibody syndrome. Consistent with that diagnosis, the plasma levels of GPIHBP1 and LPL were very low. After IFN ß1a therapy was stopped, the plasma triglyceride levels returned to normal, and GPIHBP1 autoantibodies were undetectable. CONCLUSION: The appearance of GPIHBP1 autoantibodies during IFN ß1a therapy caused chylomicronemia. The GPIHBP1 autoantibodies disappeared when the IFN ß1a therapy was stopped, and the plasma triglyceride levels fell within the normal range.

Role of Hormone-sensitive Lipase in Leptin-Promoted Fat Loss and Glucose Lowering.

AIM: Myriad biological effects of leptin may lead to broad therapeutic applications for various metabolic diseases, including diabetes and its complications; however, in contrast to its anorexic effect, the molecular mechanisms underlying adipopenic and glucose-lowering effects of leptin have not been fully understood. Here we aim to clarify the role of hormone-sensitive lipase (HSL) in leptin's action. METHODS: Wild-type (WT) and HSL-deficient (HSLKO) mice were made hyperleptinemic by two commonly-used methods: adenovirus-mediated overexpression of leptin and continuous subcutaneous infusion of leptin by osmotic pumps. The amount of food intake, body weights, organ weights, and parameters of glucose and lipid metabolism were measured. RESULTS: Hyperleptinemia equally suppressed the food intake in WT and HSLKO mice. On the other hand, leptin-mediated fat loss and glucose-lowering were significantly blunted in the absence of HSL when leptin was overexpressed by recombinant adenovirus carrying leptin. By osmotic pumps, the fat-losing and glucose-lowering effects of leptin were milder due to lower levels of hyperleptinemia; although the difference between WT and HSLKO mice did not reach statistical significance, HSLKO mice had a tendency to retain more fat than WT mice in the face of hyperleptinemia. CONCLUSIONS: We clarify for the first time the role of HSL in leptin's effect using a genetic model: leptin-promoted fat loss and glucose-lowering are at least in part mediated via HSL-mediated lipolysis. Further studies to define the pathophysiological role of adipocyte lipases in leptin action may lead to a new therapeutic approach to circumvent leptin resistance.

Apolipoprotein C-II deficiency with no rare variant in the APOC2 gene.

AIM: 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.

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.

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.

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 (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 efflux out of the cells.

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.

Hormone-sensitive lipase deficiency suppresses insulin secretion from pancreatic islets of Lep ob/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.

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.

Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis.

Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lep(ob/ob)/HSL(-/-)) showed massive accumulation of CE in the liver compared with Lep(ob/ob)/HSL(+/+) mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL(-/-) mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL(-/-) mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL(-/-) mice showed reduced activity of CE hydrolase and contained more CE than those from HSL(+/+) mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes.