Jheem D. Medh

Lipoprotein Lipase Binds to Low Density Lipoprotein Receptors and Induces Receptor-mediated Catabolism of Very Low Density Lipoproteins in Vitro

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of plasma triglycerides, promotes binding and catabolism of triglyceride-rich lipoproteins by various cultured cells. Recent studies demonstrate that LPL binds to three members of the low density lipoprotein (LDL) receptor family, including the LDL receptor-related protein (LRP), GP330/LRP-2, and very low density lipoprotein (VLDL) receptors and induces receptor-mediated lipoprotein catabolism. We show here that LDL receptors also bind LPL and mediate LPL-dependent catabolism of large VLDL with Sf 100-400. Up-regulation of LDL receptors by lovastatin treatment of normal human foreskin fibroblasts (FSF cells) resulted in an increase in LPL-induced VLDL binding and catabolism to a level that was 10-15-fold greater than in LDL receptor-negative fibroblasts, despite similar LRP activity in both cell lines. This indicates that the contribution of LRP to LPL-dependent degradation of VLDL is small when LDL receptors are maximally up-regulated. Furthermore studies in LRP-deficient murine embryonic fibroblasts showed that the level of LPL-dependent degradation of VLDL was similar to that in normal murine embryonic fibroblasts. LPL also promoted the internalization of protein-free triglyceride emulsions; lovastatin-treatment resulted in 2-fold higher uptake in FSF cells, indicating that LPL itself could bind to LDL receptors. However, the lower induction of emulsion catabolism as compared with native VLDL suggests that LPL-induced catabolism via LDL receptors is only partially dependent on receptor binding by LPL and instead is primarily due to activation of apolipoproteins such as apoE. A fusion protein between glutathione S-transferase and the catalytically inactive carboxyl-terminal domain of LPL (GST-LPLC) also induced binding and catabolism of VLDL. However GST-LPLC was not as active as native LPL, indicating that lipolysis is required for a maximal LPL effect. Mutations of critical tryptophan residues in GST-LPLC that abolished binding to VLDL converted the protein to an inhibitor of lipoprotein binding to LDL receptors. In solid-phase assays using immobilized receptors, LDL receptors bound to LPL in a dose-dependent manner. Both LPL and GST-LPLC promoted binding of VLDL to LDL receptor-coated wells. These results indicate that LPL binds to LDL receptors and suggest that the carboxyl-terminal domain of LPL contributes to this interaction.

RECEPTOR-MEDIATED MECHANISMS OF LIPOPROTEIN REMNANT CATABOLISM

Chylomicron and VLDL are triglyceride-rich lipoprotein particles assembled by the intestine and liver respectively. These particles are not metabolized by the liver in their native form. However, upon entry into the plasma, their triglyceride component is rapidly hydrolyzed by lipoprotein lipase and they are converted to cholesterol-rich remnant particles. The remnant particles are recognized by the liver and rapidly cleared from the plasma. This process is believed to occur in two steps. (i) An initial sequestration of remnant particles on hepatic cell surface proteoglycans, and (ii) receptor-mediated endocytosis of remnants by hepatic parenchymal cells. The initial binding to proteoglycans may be facilitated by lipoprotein lipase and hepatic lipase which possess both lipid- and heparin-binding domains. The subsequent endocytic process may be mediated by LDL receptors and/or LRP. Both receptors have a high affinity for apoE, a major apolipoprotein component of remnant particles. The lipases may also serve as ligands for these receptors. An impairment of any component of this complex process may result in an accumulation of remnant particles in the plasma leading to atherosclerosis and coronary heart disease.

Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase.

We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTα protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTα degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTα degradation. The effects of Ox-LDL on CCTα breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTα isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTα primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTα generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTα amino terminus. Expression of a CCTα mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTα, resulting in impaired surfactant metabolism.

Down-regulation of lipoprotein lipase increases ABCA1-mediated cholesterol efflux in THP-1 macrophages.

The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of excess cholesterol from foam cells to lipid-poor apolipoprotein A-I, in a process called reverse cholesterol transport. Lipoprotein lipase (LPL) is a lipolytic enzyme expressed by macrophages within atherosclerotic lesions. Lentivirus-mediated RNA interference was used to genetically knock-down (KD) the expression of LPL in THP-1 macrophages. Silencing of the LPL gene was confirmed by end-point PCR, real time PCR, and protein analysis. Suppression of LPL expression correlated with a 1.6-fold up-regulation of ABCA1 mRNA levels, and resulted in a 4.5-fold increase in ABCA1-dependent cholesterol efflux. Replenishing LPL by addition of purified bovine LPL to the cell culture media resulted in down-regulation of ABCA1-mediated cholesterol efflux in both wild-type and LPL knockdown cells. These findings suggest an inverse correlation between macrophage LPL levels and ABCA1 cholesterol transport activity.

Simultaneous isolation of total cellular lipids and RNA from cultured cells

Lipid biochemists often need to analyze total cellular lipids as well as cellular messenger RNA (mRNA) levels. Conventionally, different cell samples are used for extraction of each class of biomolecule. Here, we describe a procedure for the simultaneous isolation of both total cellular lipids and total RNA from the same sample. The method is based on the fact that mild organic solvents efficiently extract lipids but do not disrupt/degrade cellular RNA. This procedure not only reduces the time and expense of analysis, but also allows a direct investigation of any correlation between lipid and transcript levels. While this method has only been applied in fibroblasts, prior delipidation of samples may be useful for extraction of nucleic acids from lipid-rich cells such as adipocytes. However, its application to other eukaryotic cell types needs to be tested. The method may not be useful in plant cells or bacterial cells, which are structurally quite different from eukaryotic cells.

Down-regulation of lipoprotein lipase increases glucose uptake in L6 muscle cells

Thiazolidinediones (TZDs) are synthetic hypoglycemic agents used to treat type 2 diabetes. TZDs target the peroxisome proliferator activated receptor-gamma (PPAR-gamma) and improve systemic insulin sensitivity. The contributions of specific tissues to TZD action, or the downstream effects of PPAR-gamma activation, are not very clear. We have used a rat skeletal muscle cell line (L6 cells) to demonstrate that TZDs directly target PPAR-gamma in muscle cells. TZD treatment resulted in a significant repression of lipoprotein lipase (LPL) expression in L6 cells. This repression correlated with an increase in glucose uptake. Down-regulation of LPL message and protein levels using siRNA resulted in a similar increase in insulin-dependent glucose uptake. Thus, LPL down-regulation improved insulin sensitivity independent of TZDs. This finding provides a novel method for the management of insulin resistance.

Phosphatidylcholine-Mediated Aqueous Diffusion of Cellular Cholesterol Down-Regulates the ABCA1 Transporter in Human Skin Fibroblasts

ATP-binding cassette protein A1 (ABCA1) is a cholesterol transporter that contributes to the active transport/removal of excess cellular cholesterol. ABCA1 expression is up-regulated when cells accumulate cholesterol. Aims The purpose of this study was to determine any correlation between extracellular phospholipid levels and ABCA1 expression and function. Methodology Human foreskin fibroblasts were incubated with cholesterol alone or cholesterol and phosphatidylcholine. Total RNA was isolated and subjected to end-point RT-PCR to compare ABCA1 transcript levels. Cell lysates were subjected to Western blot analysis to compare ABCA1 protein levels. Cells were loaded with radiolabeled cholesterol and cellular cholesterol efflux was measured in the presence and absence of apoE, a cholesterol acceptor. ApoE-dependent efflux was calculated as a measure of ABCA1-mediated efflux. Results Here we show that incubation of cholesterol-loaded human skin fibroblasts with L-α-phosphatidylcholine (PC) decreases ABCA1 mRNA and protein levels by 93% and 57%, respectively, compared to cells loaded with cholesterol alone. Similarly, PC treatment results in a 25% reduction in ABCG1 mRNA levels compared to cells treated with cholesterol alone, but there is no change in SR-BI transcript levels. Subsequent incubation of phospholipid-treated cells with a cholesterol acceptor such as apoE for 24 hours shows a 65% reduction in ABCA1-mediated cholesterol efflux compared to efflux in cells not treated with PC. During the lipid treatment itself, there is a 2.7-fold greater loss of cholesterol from PC treated cells compared to cells treated with cholesterol alone. Measurement of cholesterol in cellular lipid extracts reveals that cells incubated in the presence of phosphatidylcholine are significantly depleted of cholesterol having only 20% of the cholesterol compared to cells loaded with cholesterol alone. Conclusion Thus, phosphatidylcholine facilitates removal of cellular cholesterol, thereby negating the cholesterol-dependent induction of ABCA1 message, protein and function.

Genetics and molecular biology: a role for adipocyte lipid-binding protein in atherosclerosis.

Fatty acid binding proteins (FABPs) are members of a multigene family that consists of small (~15 kDa) cytoplasmic proteins that bind long-chain fatty acids, and facilitate their solubilization and transport within the cell [1–4]. Currently, at least nine FABPs are known, each expressed in a selective tissue-specific pattern. Of these, FABP4, or the adipocyte lipid-binding protein (ALBP; also known as aP2), has been extensively investigated for its role in the regulation of carbohydrate and lipid metabolism [5–9]. Targeted disruption of the Ap2 gene results in no obvious phenotypic changes in C57BL/6 mice [5]. However, in mice challenged with a high-fat diet a deficiency in ALBP is beneficial, as evidenced by decreased levels of fasting plasma glucose, insulin, cholesterol and triglycerides compared with wild-type mice [5,6]. In addition, Ap2−/− mice maintain insulin sensitivity, whereas wild-type mice develop insulin resistance after fat-feeding [5,7,8]. In spite of such overall metabolic changes, the Ap2−/− and wild-type mice have similar body mass and develop comparable diet-induced obesity [5,9]. Thus, deficiency in ALBP results in uncoupling of the insulin resistance syndrome from obesity [5]. This is an interesting finding and is worthy of consideration in the treatment of diabetes. A comparison of the levels of ALBP between diabetic and normal individuals should reveal any correlations between diabetes and ALBP. Apart from its metabolic functions, ALBP also participates in cell-signaling pathways [2,4]. In this role, ALBP targets fatty acid second messengers to various gene regulatory elements. It has been suggested that ALBP binds to 15-deoxy-Δ12,14-prostaglandin J2 and thiozolidinediones [natural and synthetic ligands, respectively, of peroxisome proliferator-activated receptor (PPAR)-γ, a nuclear hormone receptor and transcription factor [10,11•]. In turn, activation of PPAR-γ induces expression of Ap2 messenger RNA [12]. PPAR-γ is expressed in a differentiation-dependent manner in both adipocytes and macrophages, and has been implicated in atherosclerosis [13,14]. Until recently it was believed that ALBP is expressed exclusively in adipocytes [2,4]. Recent studies [15,16••] demonstrated that the 5.4 kilo-base enhancer/promoter element of the Ap2 gene drives the expression of ALBP in differentiated macrophages as well. The discovery that ALBP is expressed in macrophages and the interactions between PPAR-γ and ALBP have led to investigations into the role of macrophage ALBP in atherosclerosis [16••,17••]. Two recent reports support a proatherosclerotic role for ALBP. Perrella et al. [17••] demonstrated that ALBP messenger RNA is expressed in atherosclerotic lesions from hypercholesterolemic apolipoprotein E knockout mice, but not in normal mice. Similarly, ALBP protein was detected in macrophage foam cells from aortic lesions of Ap2−/− mice but not in Ap2+/+ mice [16••]. Macrophage ALBP expression is induced by oxidized LDL, a prominent contributor to the atherogenic process [17••]. Makowski et al. [16••] reported that macrophages from Ap2−/− mice accumulate significantly less cholesterol esters after exposure to oxidized LDL than do wild-type macrophages. Additional experiments in genetically engineered mouse models established that a deficiency of macrophage Ap2 protects against atherosclerosis [16••,17••]. Because mice do not develop significant size lesions in the presence of apoE, Ap2−/− mice were intercrossed with apolipoprotein-E-deficient mice. In two independent studies and in all diet and sex groups, mice that lacked Ap2 developed significantly smaller lesions than did Ap2+/+ mice [16••,17••]. The effect of ALBP on atherosclerosis appears to be independent of its metabolic functions, because in the apolipoprotein-E-knockout background both AP2−/− and Ap2+/+ mice had elevated but comparable levels of total plasma cholesterol and triglycerides, and the two groups did not differ in insulin sensitivity and glycemic control [16••]. Additionally, using the technique of bone marrow transplantation, Makowski et al. [16••] obtained mice whose adipocytes were Ap2+/+ but whose macrophages were Ap2−/−, also in the apolipoprotein-E-deficient background. Even in mice with macrophage-specific Ap2-deficiency, aortic lesions were significantly reduced as compared with mice with Ap2+/+ macrophages. Similarly, carotid arteries transplanted from another mouse strain into Ap2−/− mice developed transplant-associated atherosclerosis, presumably because of the presence of endogenous Ap2 in the transplanted arteries [17••]. ALBP may influence atherosclerosis by modulating the macrophage inflammatory response and cell-signaling functions. The expression of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6 and interleukin-1β, was negligible in Ap2−/− as compared with Ap2+/+ macrophages [16••]. Similarly, Ap2−/− macrophages secreted significantly lower levels of monocyte chemotactic factor-1 as compared with wild-type mice [16••]. Earlier studies indicated that PPAR-γ is a negative modulator of those cytokines in macrophages [18,19]. Given the ability of ALBP to activate PPAR-γ, it is unclear why ALBP deficiency leads to repression of cytokine secretion by macrophages. It is likely that regulatory mechanisms distinct from PPAR-γ also contribute to precipitating the proatherogenic effects of ALBP. These studies clearly indicate that the metabolic and inflammatory effects of ALBP may be independent of each other. This presents an opportunity to develop novel therapeutic approaches to combat selectively either the metabolic syndrome or atherosclerosis by targeted inactivation of adipocyte or macrophage ALBP. In any case, a deficiency in ALBP appears to be beneficial at both levels. However, further studies are warranted to investigate whether removal of ALBP may compromise the intracellular transport of long-chain fatty acids and adversely affect normal physiology at the cellular level.