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 p38α or p38β 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.

Objectives-Atherosclerotic lesions have regions that are hypoxic. Because the lesion contains macrophages that are loaded with lipid, we investigated whether hypoxia can influence the accumulation of lipids in these cells. Methods and Results-Exposure of human macrophages to hypoxia for 24 hours resulted in an increased formation of cytosolic lipid droplets and an increased accumulation of triglycerides. Exposure of the macrophages to oxidized low-density lipoprotein (oxLDL) increased the accumulation of cytosolic lipid droplets because of an increase in cellular cholesterol esters. The accumulation of lipid droplets in oxLDL-treated cells was further increased after hypoxia, caused by an increased level of triglycerides. Expression analyses combined with immunoblot or RT-PCR demonstrated that hypoxia increased the expression of several genes that could promote the accumulation of lipid droplets. Hypoxia increased the mRNA and protein levels of adipocyte differentiation-related protein (ADRP). It is well known that an increased expression of ADRP increases the formation of lipid droplets. Hypoxia decreased the expression of enzymes involved in ␤-oxidation (acyl-coenzyme A synthetase and acyl-coenzyme A dehydrogenase) and increased the expression of stearoyl-coenzyme A desaturase, an important enzyme in the fatty acid biosynthesis. Moreover, exposure to hypoxia decreased the rate of ␤-oxidation, whereas the accumulation of triglycerides increased. The lipids are stored in cytosolic lipid droplets, 1 which have been suggested to consist of a core of neutral lipids (cholesterol esters or triglycerides) surrounded by a monolayer of amphipathic structures such as phospholipids and proteins. 2 The most well known of these proteins are the PAT proteins perilipin, adipocyte differentiation-related protein (ADRP), and tailinteracting protein 47. 2 ADRP is the predominant PAT protein of the lipid droplets in macrophages 3 and is present on newly formed droplets. 4 It been shown to strongly influence the formation of lipid droplets. 2,5 Phospholipase D1 (PLD1), which catalyzes the formation of phosphatidic acid, has been shown to have an important role in the assembly of lipid droplets. 6 The atherosclerotic lesion is characterized by regions of hypoxia. 7 The role of hypoxia in the development of the lesion is unknown. However, hypoxia has been shown to reduce macrophage migration. 8 Moreover, our previous results indicated that hypoxia resulted in an increased expression of 15-lipoxygenase-2 in macrophages, which correlated with an increased ability of the macrophage to participate in the oxidation of low-density lipoprotein (LDL). 9 Furthermore, hypoxia caused an increase in the secretion of interleukins. 10,11 Together, these observations suggest that the influence of hypoxia on macrophages is of fundamental importance for the inflammation that characterizes the atherosclerotic lesion. It was demonstrated recently that leukocytes respond to an inflammatory stimulus by the accumulation of cytosolic lipid droplets,...

Objective-We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes. Methods and Results-An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into ␤-oxidation. Conclusions-ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to ␤-oxidation and promotes the secretion of apoB-48 VLDL1. Key Words: adipose differentiation-related protein Ⅲ cytosolic lipid droplets Ⅲ apolipoproteins B Ⅲ ␤-oxidation Ⅲ small interfering RNA C ytosolic lipid droplets are ubiquitous organelles involved in the storage and turnover of neutral lipids such as triglycerides. Several proteins have been identified on these droplets, the most well known being the PAT domain proteins, 1-3 including the perilipins, adipocyte differentiationrelated protein (ADRP or adipophilin) and Tip 47. ADRP, which is ubiquitously expressed, 4 has a central role in the formation of lipid droplets. 5 These droplets are assembled at the microsomal membrane by an insulin-dependent process 6 that requires phospholipase D1, extracellular signal regulated kinase 2, and the motor protein dynein. 6,7 The assembly process involves the formation of small primordial droplets, 7 which grow in size by a fusion process that is dependent on intact microtubules 8 and dynein. 6 The assembly of very-low density lipoproteins (VLDLs) 9 -12 starts with the cotranslational lipidation of apolipoprotein B-100 (apoB-100), forming a pre-VLDL particle. VLDL2 (Svedberg flotation [sf] units 20 to 60) is formed from pre-VLDL by additional lipidation, 13 whereas VLDL1 (sf 60 to 80) is formed from VLDL2 by a mechanism that is dependent on an ADP ribosylation factor 1-controlled sorting/transport process 14 and involves the addition of a bulk load of lipids to the particle. 12,13 The triglycerides used in this assembly process are largely derived from triglycerides in cytosolic lipid droplets. 15,16 In this article, we demonstrate that an increase in ADRP promotes the storage of triglycerides in cytosolic lipid dropl...

SUMMARYTwo phospholipase enzymes have been identified in toxic preparations from Staphylococcus aureus, each having a mode of action like that of phospholipase C. One enzyme hydrolysed phosphatidyl inositol and lysophosphatidyl inositol, while the other hydrolysed sphingomyelin and lysophosphatidyl choline. The latter enzyme was always associated with /I-haemolysin activity and it is concluded that P-haemolysin, sphingomyelinase and lysophospholipase are activities of one protein. A phospholipase A was also detected in a toxic preparation from an ap strain.

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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°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. Key Words: ADP ribosylation factor 1 Ⅲ apolipoprotein B Ⅲ coatamer 1 Ⅲ intracellular transport Ⅲ very-low-density lipoproteins A polipoprotein B (apoB)-containing very-low-density lipoproteins (VLDLs) are formed in a 2-step process. 1-3 In the first step, apoB forms a partially lipidated particle (a primordial lipoprotein or pre-VLDL) during its cotranslational translocation to the lumen of the endoplasmic reticulum (ER). This step involves the transfer of lipids to apoB catalyzed by the microsomal triglyceride transfer protein. In the absence of lipids or microsomal triglyceride transfer protein, the translocation of apoB is halted, and the protein is retracted through the translocon and degraded by proteasomes. 4 -8 The second step, in which the major amount of triglycerides is added to pre-VLDL, is less well-characterized. This step involves the formation of apoB-free lipid droplets in the smooth ER 9 that associate with apoB-containing pre-VLDL in a compartment that is separate from the rough ER. 9,10 Thus, transport and sorting processes involved in the transfer of proteins out of the rough ER may be important for the assembly of VLDL. Consistent with this possibility, the second step can be inhibited by brefeldin A 11 and is dependent on ADP ribosylation factor 1 (ARF1) and phospholipase D (PLD) activity. 12 ARF1, a member of the Ras superfamily of GTP-binding proteins, participates in the formation of coatamer I (COPI) secretory vesicles, which perform retrograde transport from the Golgi to the ER and within the Golgi stacks. 13 ARF1 and COPI are important for the anterograde transport from the ER to the Golgi apparatus 14 -16 , and ARF1 activates PLD1. [17][18][19] In...

With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.