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.

Patatin-like phospholipase domain-containing 3 (PNPLA3) I148M (rs738409) affects hepatic VLDL secretion in humans and in vitro.

BACKGROUND & AIMS: The robust association between non-alcoholic fatty liver disease (NAFLD) and the genetic variant I148M (rs738409) in PNPLA3 has been widely replicated. The aim of this study was to investigate the effect of the PNPLA3 I148M mutation on: (1) hepatic secretion of very low density lipoproteins (VLDL) in humans; and (2) secretion of apolipoprotein B (apoB) from McA-RH 7777 cells, which secrete VLDL-sized apoB-containing lipoproteins. METHODS: VLDL kinetics was analyzed after a bolus infusion of stable isotopes in 55 overweight/obese men genotyped for the PNPLA3 I148M variant. Intracellular lipid content, apoB secretion and glycerolipid metabolism were studied in McA-RH 7777 cells overexpressing the human 148I wild type or 148M mutant PNPLA3 protein. RESULTS: In humans, carriers of the PNPLA3 148M allele had increased liver fat compared to 148I homozygotes, and kinetic analysis showed a relatively lower secretion of the large, triglyceride-rich VLDL (VLDL(1)) in 148M carriers vs. 148I homozygotes for the same amount of liver fat. McA-RH 7777 cells overexpressing the 148M mutant protein showed a higher intracellular triglyceride content with a lower apoB secretion and fatty acid efflux, compared to cells overexpressing the 148I wild type protein. The responses with 148M matched those observed in cells expressing the empty vector, indicating that the mutation results in loss of function. CONCLUSIONS: We have shown that PNPLA3 affects the secretion of apoB-containing lipoproteins both in humans and in vitro and that the 148M protein is a loss-of-function mutation. We propose that PNPLA3 148M promotes intracellular lipid accumulation in the liver by reducing the lipidation of VLDL.

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.

Retention of low-density lipoprotein in atherosclerotic lesions of the mouse: evidence for a role of lipoprotein lipase.

Direct binding of apolipoprotein (apo)B-containing lipoproteins to proteoglycans is the initiating event in atherosclerosis, but the processes involved at later stages of development are unclear. Here, we investigated the importance of the apoB-proteoglycan interaction in the development of atherosclerosis over time and investigated the role of lipoprotein lipase (LPL) to facilitate low-density lipoprotein (LDL) retention at later stages of development. Atherosclerosis was analyzed in apoB transgenic mice expressing LDL with normal (control LDL) or reduced proteoglycan-binding (RK3359-3369SA LDL) activity after an atherogenic diet for 0 to 40 weeks. The initiation of atherosclerosis was delayed in mice expressing RK3359-3369SA LDL, but they eventually developed the same level of atherosclerosis as mice expressing control LDL. Retention studies in vivo showed that although higher levels of 131I-tyramine cellobiose-labeled control LDL (131I-TC-LDL) were retained in nonatherosclerotic aortae compared with RK3359-3369SA 131I-TC-LDL, the retention was significantly higher and there was no difference between the groups in atherosclerotic aortae. Lower levels of control 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL were retained in atherosclerotic aortae from ldlr-/- mice transplanted with lpl-/- compared with lpl+/+ bone marrow. Uptake of control LDL or RK3359-3369SA LDL into macrophages with specific expression of human catalytically active or inactive LPL was increased compared with control macrophages. Furthermore, transgenic mice expressing catalytically active or inactive LPL developed the same extent of atherosclerosis. Thus, retention of LDL in the artery wall is initiated by direct LDL-proteoglycan binding but shifts to indirect binding with bridging molecules such as LPL.

The VLDL receptor promotes lipotoxicity and increases mortality in mice following an acute myocardial infarction.

Impaired cardiac function is associated with myocardial triglyceride accumulation, but it is not clear how the lipids accumulate or whether this accumulation is detrimental. Here we show that hypoxia/ischemia-induced accumulation of lipids in HL-1 cardiomyocytes and mouse hearts is dependent on expression of the VLDL receptor (VLDLR). Hypoxia-induced VLDLR expression in HL-1 cells was dependent on HIF-1α through its interaction with a hypoxia-responsive element in the Vldlr promoter, and VLDLR promoted the endocytosis of lipoproteins. Furthermore, VLDLR expression was higher in ischemic compared with nonischemic left ventricles from human hearts and was correlated with the total lipid droplet area in the cardiomyocytes. Importantly, Vldlr-/- mice showed improved survival and decreased infarct area following an induced myocardial infarction. ER stress, which leads to apoptosis, is known to be involved in ischemic heart disease. We found that ischemia-induced ER stress and apoptosis in mouse hearts were reduced in Vldlr-/- mice and in mice treated with antibodies specific for VLDLR. These findings suggest that VLDLR-induced lipid accumulation in the ischemic heart worsens survival by increasing ER stress and apoptosis.

High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry.

Technological advances in mass spectrometry and meticulous method development have produced several shotgun lipidomic approaches capable of characterizing lipid species by direct analysis of total lipid extracts. Shotgun lipidomics by hybrid quadrupole time-of-flight mass spectrometry allows the absolute quantification of hundreds of molecular glycerophospholipid species, glycerolipid species, sphingolipid species and sterol lipids. Future applications in clinical cohort studies demand detailed lipid molecule information and the application of high-throughput lipidomics platforms. In this review we describe a novel high-throughput shotgun lipidomic platform based on 96-well robot-assisted lipid extraction, automated sample infusion by mircofluidic-based nanoelectrospray ionization, and quantitative multiple precursor ion scanning analysis on a quadrupole time-of-flight mass spectrometer. Using this platform to compile comprehensive lipid arrays associated with metabolic dysfunctions is a powerful strategy for pinpointing the mechanistic details by which alterations in tissue-specific lipid metabolism are directly linked to the etiology of many lipid-mediated disorders.

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.