Martin H. Kang

miR-33a Modulates ABCA1 Expression, Cholesterol Accumulation, and Insulin Secretion in Pancreatic Islets

Changes in cellular cholesterol affect insulin secretion, and β-cell–specific deletion or loss-of-function mutations in the cholesterol efflux transporter ATP-binding cassette transporter A1 (ABCA1) result in impaired glucose tolerance and β-cell dysfunction. Upregulation of ABCA1 expression may therefore be beneficial for the maintenance of normal islet function in diabetes. Studies suggest that microRNA-33a (miR-33a) expression inversely correlates with ABCA1 expression in hepatocytes and macrophages. We examined whether miR-33a regulates ABCA1 expression in pancreatic islets, thereby affecting cholesterol accumulation and insulin secretion. Adenoviral miR-33a overexpression in human or mouse islets reduced ABCA1 expression, decreased glucose-stimulated insulin secretion, and increased cholesterol levels. The miR-33a–induced reduction in insulin secretion was rescued by cholesterol depletion by methyl-β-cyclodextrin or mevastatin. Inhibition of miR-33a expression in apolipoprotein E knockout islets and ABCA1 overexpression in β-cell–specific ABCA1 knockout islets rescued normal insulin secretion and reduced islet cholesterol. These findings confirm the critical role of β-cell ABCA1 in islet cholesterol homeostasis and β-cell function and highlight modulation of β-cell miR-33a expression as a means to influence insulin secretion.

Both Hepatic and Extrahepatic ABCA1 Have Discrete and Essential Functions in the Maintenance of Plasma High-Density Lipoprotein Cholesterol Levels In Vivo

Background— Extrahepatic tissues have long been considered critical contributors of cholesterol to nascent HDL particles in the reverse cholesterol transport pathway, in which ABCA1 plays the crucial role. Recent studies, however, including both overexpression and deletion of ABCA1 selectively in the liver, have highlighted the primary role of the liver in the maintenance of HDL levels in vivo. Methods and Results— The availability of mice with complete deletion of ABCA1 (total knockout [TKO]) and with liver-specific deletion of ABCA1 (LSKO) has enabled us to dissect the discrete roles of hepatic relative to extrahepatic ABCA1 in HDL biogenesis. Delivery of adenoviral ABCA1 resulted in selective expression of physiological levels of ABCA1 in the livers of both LSKO and TKO mice, resulting in increased HDL cholesterol (HDL-C). Expression of ABCA1 in the liver of LSKO mice resulted in plasma HDL-C levels that were similar to those in wild-type mice and significantly above those seen in similarly treated TKO mice. HDL particles from ABCA1-expressing LSKO mice were larger and contained significantly increased cholesterol compared with TKO mice. Infusion of human apolipoprotein A-I/phospholipid reconstituted HDL particles normalized plasma HDL-C levels in LSKO mice but had no effect on HDL-C levels in TKO mice. Conclusions— Although hepatic ABCA1 appears crucial for phospholipid transport, extrahepatic tissues play an important role in cholesterol transfer to nascent HDL particles. These data highlight the discrete and specific roles of both liver and extrahepatic ABCA1 in HDL biogenesis in vivo and indicate that ABCA1 shows lipid cargo selectivity depending on its site of expression.

Tissue-specific roles of ABCA1 influence susceptibility to atherosclerosis.

Objective—The ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) plays a key role in HDL cholesterol metabolism. However, the role of ABCA1 in modulating susceptibility to atherosclerosis is controversial. Methods and Results—We investigated the role of ABCA1 in atherosclerosis using a combination of overexpression and selective deletion models. First, we examined the effect of transgenic overexpression of a full-length human ABCA1-containing bacterial artificial chromosome (BAC) in the presence or absence of the endogenous mouse Abca1 gene. ABCA1 overexpression in the atherosclerosis-susceptible Ldlr−/− background significantly reduced the development of atherosclerosis in both the presence and absence of mouse Abca1. Next, we used mice with tissue-specific inactivation of Abca1 to dissect the discrete roles of Abca1 in different tissues on susceptibility to atherosclerosis. On the Apoe−/− background, mice lacking hepatic Abca1 had significantly reduced HDL cholesterol and accelerated atherosclerosis, indicating that the liver is an important site at which Abca1 plays an antiatherogenic role. In contrast, mice with macrophage-specific inactivation of Abca1 on the Ldlr−/− background displayed no change in atherosclerotic lesion area. Conclusions—These data indicate that physiological expression of Abca1 modulates the susceptibility to atherosclerosis and establish hepatic Abca1 expression as an important site of atheroprotection. In contrast, we show that selective deletion of macrophage Abca1 does not significantly modulate atherogenesis.

Palmitoylation and function of glial glutamate transporter-1 is reduced in the YAC128 mouse model of Huntington disease

Excitotoxicity plays a key role in the selective vulnerability of striatal neurons in Huntington disease (HD). Decreased glutamate uptake by glial cells could account for the excess glutamate at the synapse in patients as well as animal models of HD. The major molecule responsible for clearing glutamate at the synapses is glial glutamate transporter GLT-1. In this study, we show that GLT-1 is palmitoylated at cysteine38 (C38) and further, that this palmitoylation is drastically reduced in HD models both in vitro and in vivo. Palmitoylation is required for normal GLT-1 function. Blocking palmitoylation either with the general palmitoylation inhibitor, 2-bromopalmitate, or with a GLT-1 C38S mutation, severely impairs glutamate uptake activity. In addition, GLT-1-mediated glutamate uptake is indeed impaired in the YAC128 HD mouse brain, with the defect in the striatum evident as early as 3 months prior to obvious neuropathological findings, and in both striatum and cortex at 12 months. These phenotypes are not a result of changes in GLT1 protein expression, suggesting a crucial role of palmitoylation in GLT-1 function. Thus, it appears that impaired GLT-1 palmitoylation is present early in the pathogenesis of HD, and may influence decreased glutamate uptake, excitotoxicity, and ultimately, neuronal cell death in HD.

Regulation of ABCA1 Protein Expression and Function in Hepatic and Pancreatic Islet Cells by miR-145

Objective—The ATP-binding cassette transporter A1 (ABCA1) protein maintains cellular cholesterol homeostasis in several different tissues. In the liver, ABCA1 is crucial for high-density lipoprotein biogenesis, and in the pancreas ABCA1 can regulate insulin secretion. In this study, our aim was to identify novel microRNAs that regulate ABCA1 expression in these tissues. Approach and Results—We combined multiple microRNA prediction programs to identify 8 microRNAs that potentially regulate ABCA1. A luciferase reporter assay demonstrated that 5 of these microRNAs (miR-148, miR-27, miR-144, miR-145, and miR-33a/33b) significantly repressed ABCA1 3′-untranslated region activity with miR-145 resulting in one of the larger decreases. In hepatic HepG2 cells, miR-145 can regulate both ABCA1 protein expression levels and cholesterol efflux function. In murine islets, an increase in miR-145 expression decreased ABCA1 protein expression, increased total islet cholesterol levels, and decreased glucose-stimulated insulin secretion. Inhibiting miR-145 produced the opposite effect of increasing ABCA1 protein levels and improving glucose-stimulated insulin secretion. Finally, increased glucose levels in media significantly decreased miR-145 levels in cultured pancreatic beta cells. These findings suggest that miR-145 is involved in glucose homeostasis and is regulated by glucose concentration. Conclusions—Our studies demonstrate that miR-145 regulates ABCA1 expression and function, and inhibiting this microRNA represents a novel strategy for increasing ABCA1 expression, promoting high-density lipoprotein biogenesis in the liver, and improving glucose-stimulated insulin secretion in islets.

Palmitoylation of ATP-Binding Cassette Transporter A1 Is Essential for Its Trafficking and Function

ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.

ABCA1 in adipocytes regulates adipose tissue lipid content, glucose tolerance, and insulin sensitivity

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1−ad/−ad). When fed a high-fat, high-cholesterol diet, ABCA1−ad/−ad mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1−ad/−ad mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.

Adenosine-Triphosphate-Binding Cassette Transporter-1 Trafficking and Function☆

Mutations in the adenosine-triphosphate-binding cassette transporter-1 (ABCA1) lead to Tangier disease, a genetic disorder characterized by an almost complete absence of plasma high-density lipoprotein cholesterol. Although the importance of ABCA1 localization to its cholesterol efflux function has been extensively characterized, the cellular itinerary of ABCA1 leading to the plasma membrane is not fully elucidated. This review will summarize the current knowledge of ABCA1 trafficking and its relationship to function. Understanding these crucial processes provides potential novel therapeutic targets to regulate high-density lipoprotein biogenesis through influencing pathways of ABCA1 trafficking.

Hepatic ATP-Binding Cassette Transporter A1 Is a Key Molecule in High-Density Lipoprotein Cholesteryl Ester Metabolism in Mice

Objective—Mutations in ATP-binding cassette transporter A1 (ABCA1), the cellular lipid transport molecule mutated in Tangier disease, result in the rapid turnover of high-density lipoprotein (HDL)–associated apolipoproteins that presumably are cleared by the kidneys. However, the role of ABCA1 in the liver for HDL apolipoprotein and cholesteryl ester (CE) catabolism in vivo is unknown. Methods and Results—Murine HDL was radiolabeled with 125I in its apolipoprotein and with [3H]cholesteryl oleyl ether in its CE moiety. HDL protein and lipid metabolism in plasma and HDL uptake by tissues were investigated in ABCA1-overexpressing bacterial artificial chromosome (BAC)–transgenic (BAC+) mice and in mice harboring deletions of total (ABCA1−/−) and liver-specific ABCA1 (ABCA1−L/−L). In BAC+ mice with elevated ABCA1 expression, fractional catabolic rates (FCRs) of both the protein and the lipid tracer were significantly decreased in plasma and in the liver, yielding a diminished hepatic selective CE uptake from HDL. In contrast, ABCA1−/− or ABCA1−L/−L mice had significantly increased plasma and liver FCRs for both HDL tracers. An ABCA1 deficiency was associated with increased selective HDL CE uptake by the liver under all experimental conditions. Conclusions—Hepatic ABCA1 has a critical role for HDL catabolism in plasma and for HDL selective CE uptake by the liver.

Clinical, Biochemical, and Molecular Characterization of Novel Mutations in ABCA1 in Families with Tangier Disease

Tangier disease is a rare, autosomal recessive disorder caused by mutations in the ABCA1 gene and is characterized by near absence of plasma high-density lipoprotein cholesterol, accumulation of cholesterol in multiple tissues, peripheral neuropathy, and accelerated atherosclerosis. Here we report three new kindreds with Tangier disease harboring both known and novel mutations in ABCA1. One patient was identified to be homozygous for a nonsense mutation, p.Gln1038*. In a remarkably large Tangier disease pedigree with four affected siblings, we identified compound heterozygosity for previously reported missense variants, p.Arg937Val and p.Thr940Met, and show that both of these mutations result in significantly impaired cholesterol efflux in transfected cells. In a third pedigree, the proband was identified to be compound heterozygous for two novel mutations, a frameshift (p.Ile1200Hisfs*4) and an intronic variant (c.4176-11T>G), that lead to the creation of a cryptic splice site acceptor and premature truncation, p.Ser1392Argfs*6. We demonstrate that this mutation arose de novo, the first demonstration of a pathogenic de novo mutation in ABCA1 associated with Tangier disease. We also report results of glucose tolerance testing in a Tangier disease kindred for the first time, showing a gene-dose relationship between ABCA1 activity and glucose tolerance and suggesting that Tangier disease patients may have substantially impaired islet function. Our findings provide insight into the diverse phenotypic manifestations of this rare disorder, expand the list of pathogenic mutations in ABCA1, and increase our understanding of how specific mutations in this gene lead to abnormal cellular and physiological phenotypes.