William R. Lagor

Proteomic Analysis of Defined HDL Subpopulations Reveals Particle-Specific Protein Clusters Relevance to Antioxidative Function

Objective—Recent proteomic studies have identified multiple proteins that coisolate with human HDL. We hypothesized that distinct clusters of protein components may distinguish between physicochemically-defined subpopulations of HDL particles, and that such clusters may exert specific biological function(s). Methods and Results—We investigated the distribution of proteins across 5 physicochemically-defined particle subpopulations of normolipidemic human HDL (HDL2b, 2a, 3a, 3b, 3c) fractionated by isopycnic density gradient ultracentrifugation. Liquid chromatography/electrospray mass spectrometry identified a total of 28 distinct HDL-associated proteins. Using an abundance pattern analysis of peptide counts across the HDL subfractions, these proteins could be grouped into 5 distinct classes. A more in-depth correlational network analysis suggested the existence of distinct protein clusters, particularly in the dense HDL3 particles. Levels of specific HDL proteins, primarily apoL-I, PON1, and PON3, correlated with the potent capacity of HDL3 to protect LDL from oxidation. Conclusions—These findings suggest that HDL is composed of distinct particles containing unique (apolipo)protein complements. Such subspeciation forms a potential basis for understanding the numerous observed functions of HDL. Further work using additional separation techniques will be required to define these species in more detail.

Trib1 is a lipid- and myocardial infarction–associated gene that regulates hepatic lipogenesis and VLDL production in mice

Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic-specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans.

Impact of Combined Deficiency of Hepatic Lipase and Endothelial Lipase on the Metabolism of Both High-Density Lipoproteins and Apolipoprotein B–Containing Lipoproteins

Rationale: Hepatic lipase (HL) and endothelial lipase (EL) are extracellular lipases that both hydrolyze triglycerides and phospholipids and display potentially overlapping or complementary roles in lipoprotein metabolism. Objective: We sought to dissect the overlapping roles of HL and EL by generating mice deficient in both HL and EL (HL/EL-dko) for comparison with single HL-knockout (ko) and EL-ko mice, as well as wild-type mice. Methods and Results: Reproduction and viability of the HL/EL-dko mice were impaired compared with the single-knockout mice. The plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, non–HDL cholesterol, and phospholipids in the HL/EL-dko mice were markedly higher than those in the single-knockout mice. Most notably, the HL/EL-dko mice exhibited an unexpected substantial increase in small low-density lipoproteins. Kinetic studies with [3H]cholesteryl ether–labeled very-low-density lipoproteins demonstrated that the HL/EL-dko mice accumulated counts in the smallest low-density lipoprotein–sized fractions, as assessed by size exclusion chromatography, suggesting that it arises from lipolysis of very-low-density lipoproteins. HDL from all 3 lipase knockout models had an increased cholesterol efflux capacity but reduced clearance of HDL cholesteryl esters versus control mice. Despite their higher HDL cholesterol levels, neither HL-ko, EL-ko, nor HL/EL-dko mice demonstrated an increased rate of macrophage reverse cholesterol transport in vivo. Conclusions: These studies reveal an additive effect of HL and EL on HDL metabolism but not macrophage reverse cholesterol transport in mice and an unexpected redundant role of HL and EL in apolipoprotein B lipoprotein metabolism.

Overexpression of apolipoprotein F reduces HDL cholesterol levels in vivo.

Objective—Apolipoprotein F (ApoF) is a protein component of several lipoprotein classes including HDL. It is also known as lipid transfer inhibitor protein (LTIP) based on its ability to inhibit lipid transfer between lipoproteins ex vivo. We sought to investigate the role of ApoF in HDL metabolism. Methods and Results—Adeno-associated viruses (AAV) based on serotype 8, were used to overexpress either murine or human ApoF in mice. Overexpression of murine ApoF significantly reduced total cholesterol levels by 28% (P<0.001), HDL by 27% (P<0.001), and phospholipid levels by 19% (P<0.001). Overexpression of human ApoF had similar effects. Human ApoF was nearly exclusively HDL-associated in mice. In agreement with this finding, greater than 90% of the ApoF in human plasma was found on HDL3, with only a small amount on LDL. Overexpression of mouse ApoF accelerated the plasma clearance of [3H]-cholesteryl ether labeled HDL. Plasma from mice overexpressing ApoF showed improved macrophage cholesterol efflux on a per HDL-C basis. Conclusions—ApoF overexpression reduces HDL cholesterol levels in mice by increasing clearance of HDL-CE. ApoF may be an important determinant of HDL metabolism and reverse cholesterol transport.

Overexpression of apolipoprotein O does not impact on plasma HDL levels or functionality in human apolipoprotein A-I transgenic mice.

Apolipoprotein (apo) O is a newly discovered apolipoprotein preferentially contained within HDL; however, currently, no data are available on the (patho)physiological effects of apoO. Therefore, the present study assessed the impact of apoO overexpression on (i) plasma lipids and lipoproteins as well as on (ii) HDL functionality. Human apoO was overexpressed by means of recombinant adenovirus (AdhapoO) in human apoA-I transgenic mice, a humanized mouse model of HDL metabolism. AdhapoO substantially increased apoO in plasma and within HDL. However, plasma triglycerides, phospholipids, total cholesterol and HDL cholesterol did not change. HDL size distribution, lipid composition and the apoA-I and the apoO distribution over the different HDL fractions separated by FPLC remained unaltered. Furthermore, enrichment of HDL with apoO did not impact on HDL functionality assessed in four independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells, (ii) protection against LDL oxidation, (iii) anti-inflammatory activity on endothelial cells, and (iv) induction of vasodilation in isolated aortic rings ex vivo as a measure of stimulating vascular NO production. These results demonstrate that although overexpression of apoO results in a substantial enrichment of HDL particles with this novel apolipoprotein, apoO does not impact the plasma lipoprotein profile or HDL functionality.

Diabetes alters the occupancy of the hepatic 3-hydroxy-3-methylglutaryl-CoA reductase promoter.

Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) protein and mRNA are substantially decreased in diabetic animals and rapidly restored by the administration of insulin. To begin to examine the underlying molecular mechanisms, measurements of transcription by nuclear run-on assays and an investigation of occupancy of the promoter were performed. The rate of transcription was substantially reduced in the diabetic rats and fully restored within 2 h after insulin treatment. In vivo footprinting revealed several areas of protein binding as shown by dimethyl sulfate protection or enhancement. The cAMP-response element was heavily protected in all conditions, including diabetes, feeding of dietary cholesterol, or statin treatment. Striking enhancements in footprints from diabetic animals were visible at –142 and at –161 (in the sterol-response element). Protections at a newly identified NF-Y site at –70/–71 were observed in normal animals and not in diabetics. This NF-Y site was found to be required for efficient HMGR transcription in luciferase assays. CREB-1 was able to bind the HMGR cAMP-response element in vitro and the promoter in vivo. This evidence supports an essential role for cAMP-response element-binding protein in transcription of hepatic HMGR and identifies at least two sites where in vivo occupancy is regulated by insulin.

Overview of the LDL receptor: relevance to cholesterol metabolism and future approaches for the treatment of coronary heart disease

Since the discovery of the low-density lipoprotein (LDL) receptor over 30 years ago manipulation of its activity has been pursued through dietary and pharmacological means with the goal of reducing LDL cholesterol levels. This has proven to be one of the most widely implemented and beneficial interventions to reduce LDL cholesterol levels and the incidence of cardiovascular death. This review provides an overview of the LDL receptor and its role in regulating whole body cholesterol homeostasis and LDL cholesterol levels. New findings on regulation of the LDL receptor are discussed and how they can be used to develop novel therapeutics to reduce cardiovascular risk.

Thyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter

OBJECTIVE The goal of this study was to examine the effects of thyroid hormone status on the ability of serum to accept cellular cholesterol. METHODS AND RESULTS Sera from hypophysectomized rats treated ± T(3) was used to evaluate the role of thyroid hormone on serum efflux capacity. 2D-DIGE analysis of serum proteins showed that T(3) treated rats had increased ApoA-I, ApoA-IV and fetuin A levels with decreased Apo E levels. Microarray and real-time RT-PCR analysis of rat liver revealed large increases in ApoA-I, ApoA-IV, ABCG5, and ABCG8 in response to T(3). J774 macrophages, BHK cells, and Fu5AH rat hepatoma cells were used to measure cholesterol efflux mediated by ABCA1, ABCG1 transporters or SR-BI. Sera from T(3)-treated rats stimulated efflux via ABCA1 but not by ABCG1 or SR-BI. Gel filtration chromatography revealed that T(3) treatment caused a decrease in HDL particle size accompanied by higher levels of lipid-poor ApoA-I. CONCLUSIONS Thyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter. This effect is most likely attributable to increases in small HDL and lipid poor ApoA-I in response to T(3).

Functional Analysis of the Hepatic HMG-CoA Reductase Promoter by In Vivo Electroporation

HMG-CoA reductase (HMGR) catalyzes the rate-controlling step in cholesterol production. This enzyme is highly expressed in the liver, where it is subject to extensive hormonal and dietary regulation. Although much is known about the regulation of the HMGR promoter in cultured cells, this issue has not been directly addressed in liver. The technique of in vivo electroporation was utilized to perform the first functional analysis of the HMGR promoter in live animals. Analysis of a series of deletion constructs showed that deletion of the region containing the cyclic AMP response element (CRE) at −104 to −96 and an NF-Y site at −70 to −65 resulted in marked reduction of promoter activity. Sterol regulation of this promoter was investigated by raising tissue cholesterol levels by feeding cholesterol and by decreasing them through administration of a statin (lovastatin). Using this approach, we found that HMGR promoter constructs were sterol responsive in live animals, adding in vivo relevance to previous findings in cultured cells. We also conclude that in vivo electroporation is a convenient and powerful technique for the analysis of promoter elements in the livers of live animals.

Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism

Background: Ca2+ binding and/or permeation via CaV1.1 in skeletal muscle activates CaMKII. Results: Mice with a Ca2+ binding/permeation defect in CaV1.1 have increased body fat, reduced fatty acid metabolism, and altered CD36 distribution. Conclusion: CaV1.1 regulates CD36 distribution and fatty acid metabolism. Significance: New therapeutic targets are identified to increase skeletal muscle energy expenditure. Ca2+ permeation and/or binding to the skeletal muscle L-type Ca2+ channel (CaV1.1) facilitates activation of Ca2+/calmodulin kinase type II (CaMKII) and Ca2+ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca2+ binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle.