Cardiomyocyte-specific PCSK9 deficiency compromises mitochondrial bioenergetics and heart function.

AIMS: Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9-/- mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. METHODS AND RESULTS: Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9-/- mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9-/- hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9-/- mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9-/- mice. Circulating lipid levels were unchanged in CM-Pcsk9-/- mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9-/- mice had an increased number of mitochondria-endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. CONCLUSION: PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

pH-Dependent Protonation of Histidine Residues Is Critical for Electrostatic Binding of Low-Density Lipoproteins to Human Coronary Arteries.

BACKGROUND: The initiating step in atherogenesis is the electrostatic binding of LDL (low-density lipoprotein) to proteoglycan glycosaminoglycans in the arterial intima. However, although proteoglycans are widespread throughout the intima of most coronary artery segments, LDL is not evenly distributed, indicating that LDL retention is not merely dependent on the presence of proteoglycans. We aim to identify factors that promote the interaction between LDL and the vessel wall of human coronary arteries. METHODS: We developed an ex vivo model to investigate binding of labeled human LDL to human coronary artery sections without the interference of cellular processes. RESULTS: By staining consecutive sections of human coronary arteries, we found strong staining of sulfated glycosaminoglycans throughout the arterial intima, whereas endogenous LDL deposits were focally distributed. Ex vivo binding of LDL was uniform at all intimal areas with sulfated glycosaminoglycans. However, lowering the pH from 7.4 to 6.5 triggered a 35-fold increase in LDL binding. The pH-dependent binding was abolished by pretreating LDL with diethyl-pyrocarbonate, which blocks the protonation of histidine residues, or cyclohexanedione, which inhibits the positive charge of site B on LDL. Thus, both histidine protonation and site B are required for strong electrostatic LDL binding to the intima. CONCLUSIONS: This study identifies histidine protonation as an important component for electrostatic LDL binding to human coronary arteries. Our findings show that the local pH will have a profound impact on LDL's affinity for sulfated glycosaminoglycans, which may influence the retention and accumulation pattern of LDL in the arterial vasculature.

Vimentin deficiency in macrophages induces increased oxidative stress and vascular inflammation but attenuates atherosclerosis in mice.

The aim was to clarify the role of vimentin, an intermediate filament protein abundantly expressed in activated macrophages and foam cells, in macrophages during atherogenesis. Global gene expression, lipid uptake, ROS, and inflammation were analyzed in bone-marrow derived macrophages from vimentin-deficient (Vim-/-) and wild-type (Vim+/+) mice. Atherosclerosis was induced in Ldlr-/- mice transplanted with Vim-/- and Vim+/+ bone marrow, and in Vim-/- and Vim+/+ mice injected with a PCSK9 gain-of-function virus. The mice were fed an atherogenic diet for 12-15 weeks. We observed impaired uptake of native LDL but increased uptake of oxLDL in Vim-/- macrophages. FACS analysis revealed increased surface expression of the scavenger receptor CD36 on Vim-/- macrophages. Vim-/- macrophages also displayed increased markers of oxidative stress, activity of the transcription factor NF-κB, secretion of proinflammatory cytokines and GLUT1-mediated glucose uptake. Vim-/- mice displayed decreased atherogenesis despite increased vascular inflammation and increased CD36 expression on macrophages in two mouse models of atherosclerosis. We demonstrate that vimentin has a strong suppressive effect on oxidative stress and that Vim-/- mice display increased vascular inflammation with increased CD36 expression on macrophages despite decreased subendothelial lipid accumulation. Thus, vimentin has a key role in regulating inflammation in macrophages during atherogenesis.

Glucosylceramide modifies the LPS-induced inflammatory response in macrophages and the orientation of the LPS/TLR4 complex in silico.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol- and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.

ARAP2 promotes GLUT1-mediated basal glucose uptake through regulation of sphingolipid metabolism.

Lipid droplet formation, which is driven by triglyceride synthesis, requires several droplet-associated proteins. We identified ARAP2 (an ADP-ribosylation factor 6 GTPase-activating protein) in the lipid droplet proteome of NIH-3T3 cells and showed that knockdown of ARAP2 resulted in decreased lipid droplet formation and triglyceride synthesis. We also showed that ARAP2 knockdown did not affect fatty acid uptake but reduced basal glucose uptake, total levels of the glucose transporter GLUT1, and GLUT1 levels in the plasma membrane and the lipid micro-domain fraction (a specialized plasma membrane domain enriched in sphingolipids). Microarray analysis showed that ARAP2 knockdown altered expression of genes involved in sphingolipid metabolism. Because sphingolipids are known to play a key role in cell signaling, we performed lipidomics to further investigate the relationship between ARAP2 and sphingolipids and potentially identify a link with glucose uptake. We found that ARAP2 knockdown increased glucosylceramide and lactosylceramide levels without affecting ceramide levels, and thus speculated that the rate-limiting enzyme in glycosphingolipid synthesis, namely glucosylceramide synthase (GCS), could be modified by ARAP2. In agreement with our hypothesis, we showed that the activity of GCS was increased by ARAP2 knockdown and reduced by ARAP2 overexpression. Furthermore, pharmacological inhibition of GCS resulted in increases in basal glucose uptake, total GLUT1 levels, triglyceride biosynthesis from glucose, and lipid droplet formation, indicating that the effects of GCS inhibition are the opposite to those resulting from ARAP2 knockdown. Taken together, our data suggest that ARAP2 promotes lipid droplet formation by modifying sphingolipid metabolism through GCS.

ARF6 regulates neuron differentiation through glucosylceramide synthase.

The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels.

The formation of lipid droplets: possible role in the development of insulin resistance/type 2 diabetes.

Neutral lipids are stored in so-called lipid droplets, which are formed as small primordial droplets at microsomal membranes and increase in size by a fusion process. The fusion is catalyzed by the SNARE proteins SNAP23, syntaxin-5 and VAMP4. SNAP23 is involved in the insulin dependent translocation of GLUT4 to the plasma membrane, and has an important role in the development of insulin resistance. Thus fatty acids relocalize SNAP23 from the plasma membrane (and the translocation of GLUT 4) to the interior of the cell giving rise to insulin resistance. Moreover this relocalization is seen in skeletal muscles biopsies from patients with type 2 diabetes compared to matched control. Thus a missorting of SNAP23 is essential for the development of insulin resistance.

The SNARE protein SNAP23 and the SNARE-interacting protein Munc18c in human skeletal muscle are implicated in insulin resistance/type 2 diabetes.

OBJECTIVE: Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS: We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp. Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6. RESULTS: We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects. Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS: We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23.

Lipid droplets as dynamic organelles connecting storage and efflux of lipids.

Neutral lipids are stored in the cytosol in so-called lipid droplets. These are dynamic organelles with neutral lipids as the core surrounded by a monolayer of amphipathic lipids (phospholipids and cholesterol) and specific proteins (PAT proteins and proteins involved in the turnover of lipids and in the formation and trafficking of the droplets). Lipid droplets are formed at microsomal membranes as primordial droplets with a diameter of 0.1-0.4 microm and increase in size by fusion. In this article, we review the assembly and fusion of lipid droplets, and the processes involved in the secretion of triglycerides. Triglycerides are secreted from cells by two principally different processes. In the mammary gland, lipid droplets interact with specific regions of the plasma membrane and bud off with an envelope consisting of the membrane, to form milk globules. In the liver and intestine, very low-density lipoproteins (VLDL) and chylomicrons are secreted by using the secretory pathway of the cell. Finally, we briefly review the importance of lipid droplets in the development of insulin resistance and atherosclerosis.

Triglyceride containing lipid droplets and lipid droplet-associated proteins.

PURPOSE OF REVIEW: Cytosolic lipid droplets are now recognized as dynamic organelles. This review summarizes our current understanding of the mechanisms involved in the formation of lipid droplets, the importance of lipid droplet-associated proteins and the link between lipid droplet accumulation and development of insulin resistance. RECENT FINDINGS: Lipid droplets are formed as primordial droplets and they increase in size by fusion. This fusion process requires the alpha-soluble N-ethylmaleimide-sensitive factor adaptor protein receptor SNAP23, which is also involved in the insulin-dependent translocation of a glucose transporter to the plasma membrane. Recent data suggest that SNAP23 is the link between increased lipid droplet accumulation and development of insulin resistance. Lipid droplets also form tight interactions with other organelles. Furthermore, additional lipid droplet-associated proteins have been identified and shown to play a role in droplet assembly and turnover, and in sorting and trafficking events. SUMMARY: Recent studies have identified a number of key proteins that are involved in the formation and turnover of lipid droplets, and SNAP23 has been identified as a link between accumulation of lipid droplets and development of insulin resistance. Further understanding of lipid droplet biology could indicate potential therapeutic targets to prevent accumulation of lipid droplets and associated complications.

SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity.

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.

PLD1 and ERK2 regulate cytosolic lipid droplet formation.

We have previously uncovered roles for phospholipase D (PLD) and an unknown cytosolic protein in the formation of cytosolic lipid droplets using a cell-free system. In this report, PLD1 has been identified as the relevant isoform, and extracellular signal-regulated kinase 2 (ERK2) as the cytosolic protein. Increased expression of PLD1 increased lipid droplet formation whereas knockdown of PLD1 using siRNA was inhibitory. A role for ERK2 in basal lipid droplet formation was revealed by overexpression or microinjection, and ablation by siRNA knockdown or pharmacological inhibition. Similar manipulations of other Map kinases such as ERK1, JNK1 or JNK2 and p38alpha or p38beta were without effect. Insulin stimulated the formation of lipid droplets and this stimulation was inhibited by knockdown of PLD1 (by siRNA) and by inhibition or knockdown (by siRNA) of ERK2. Inhibition of ERK2 eliminated the effect of PLD1 on lipid droplet formation without affecting PLD1 activity, suggesting that PLD1 functions upstream of ERK2. ERK2 increased the phosphorylation of dynein which increased the amount of the protein on ADRP-containing lipid droplets. Microinjection of antibodies to dynein strongly inhibited the formation of lipid droplets, demonstrating that dynein has a central role in this formation. Thus dynein is a possible target for ERK2.

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL.

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.

Cytosolic lipid droplets increase in size by microtubule-dependent complex formation.

OBJECTIVE: Adipocyte differentiation-related protein (ADRP)-containing lipid droplets have an essential role in the development of insulin resistance and atherosclerosis. Such droplets form in a cell-free system with a diameter of 0.1 to 0.4 microm, while the droplets present in cells vary in size, from small to very large, suggesting that the droplets can increase in size after being assembled. We have addressed this possibility. METHODS AND RESULTS: Experiments in NIH 3T3 cells demonstrated that the lipid droplets could increase in size independently of triglyceride biosynthesis. NIH 3T3 cells were either microinjected with ADRP-GFP (green fluorescent protein) or stained with Nile Red and followed by confocal microscopy and time-lapse recordings. The results showed that lipid droplets formed complexes with each other, with a volume equal to the sum of the merging particles. The formation of complexes could be inhibited by the nocodazole-induced depolymerization of the microtubules; thus, the process is dependent on microtubules. The presence of dynein on ADRP-containing droplets supports a role for this motor protein. CONCLUSIONS: Lipid droplets can grow after they have been assembled. This increase in size is independent of triglyceride biosynthesis and involves formation of complexes, which requires intact microtubules.

Role of ADP ribosylation factor 1 in the assembly and secretion of ApoB-100-containing lipoproteins.

OBJECTIVE: We investigated the role of ADP ribosylation factor 1 (ARF1) in the assembly of very-low-density lipoproteins (VLDLs). METHODS AND RESULTS: The dominant-negative ARF1 mutant, T31N, decreased the assembly of apoB-100 VLDL 1 (Svedberg floatation units [Sf] 60 to 400) by 80%. The decrease coincided with loss of coatamer I (COPI) from the Golgi apparatus and inhibition of anterograde transport, as demonstrated by time-lapse studies of the vesicular stomatitis virus G protein. The VLDL 1 assembly was also completely inhibited at 15 degrees C. Thus, the antegrade transport is essential for the assembly of VLDL 1. Intracellular localization of N-acetylgalactosaminyl transferase 2 indicated that the Golgi apparatus was at least partly intact when the VLDL assembly was inhibited. Transient transfection with phospholipase D 1 increased the assembly of VLDL 1 and VLDL 2 (Sf 20 to 60). Overexpression of ARF1 in stably transfected McA-RH7777 cells increased the secretion of VLDL 2 but not of VLDL 1, which was dependent on the availability of oleic acid. Secretion of VLDL 1 increased with increasing amounts of oleic acid, and VLDL 2 secretion decreased simultaneously. CONCLUSIONS: Overexpression of ARF1 increased the assembly of VLDL 2 but not of VLDL 1, whose production was dependent on both anterograde transport and the availability of fatty acids.

A phospholipase D-dependent process forms lipid droplets containing caveolin, adipocyte differentiation-related protein, and vimentin in a cell-free system.

We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d