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...

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Receptor-dependent productive uptake of GLP1-conjugated antisense oligonucleotides occurs selectively in pancreatic β-cells.

In this study, we tested the hypothesis that two separate pathways, the two-step process and an apolipoprotein B (apoB) size-dependent lipidation process, give rise to different lipoproteins. Expression of apoB-100 and C-terminally truncated forms of apoB-100 in McA-RH7777 cells demonstrated that VLDL particles can be assembled by apoB size-dependent linear lipidation, resulting in particles whose density is inversely related to the size of apoB. This lipidation results in a LDL-VLDL 2 particle containing apoB-100. VLDL 1 is assembled by the two-step process by apoB-48 and larger forms of apoB but not to any significant amount by apoB-41. The major amount of intracellular apoB-80 and apoB-100 banded with a mean density of 1.10 g/ml. Its formation was dependent on the sequence between apoB-72 and apoB-90. This dense particle, which is retained in the cell, possibly by chaperones or association with the microsomal membrane, is a precursor of secreted VLDL 1. The intracellular LDL-VLDL 2 particles formed during size-dependent lipidation appear to be the precursors of intracellular VLDL 1. We propose that the dense apoB-100 intracellular particle is converted to LDL-VLDL 2 by size-dependent lipidation. LDL-VLDL 2 is secreted or converted to VLDL 1 by the uptake of the major amount of triglycerides. -Stillemark-Billton, P., C. Beck, J. Immunoelectron microscopy (1) and kinetic studies (2, 3) indicate that VLDLs are assembled in two major steps (4, 5). The first step occurs during the translation of apolipoprotein B (apoB) and gives rise to a premature particle (2, 6) we refer to as a primordial lipoprotein. Major amounts of lipid are added in the second step, resulting in bona fide VLDL (2, 7, 8); a second precursor of VLDL, an apoB-free "lipid droplet" in the smooth endoplasmic reticulum (1, 9) whose assembly requires microsomal triglyceride transfer protein (MTP), may also be involved (10). VLDLs are secreted in two forms: large, triglyceride-rich VLDL 1 and smaller, triglyceride-poor VLDL 2. Overproduction of VLDL 1 is linked to conditions such as insulin resistance and type II diabetes (11).The lengths of C-terminally truncated forms of apoB-100 are inversely related to the amount of lipid in the lipoproteins they assemble; assembly with apoB-100 results in VLDL (12). This size-dependent lipidation of apoB does not fit the two-step model; in particular, it cannot explain why apoB-48 has the ability to assemble VLDL but apoB-40 lacks this ability (8).In this study, we tested the hypothesis that two separate pathways, the two-step process and an apoB size-dependent lipidation process, give rise to different lipoproteins. Our results indicate that although apoB can assemble VLDL 1 once it reaches the size of apoB-48, the size-dependent process gives rise to LDL-VLDL 2 particles first when apoB-100 is reached. In contrast to apoB-48 (2), the major intracellular form of apoB-100 is much denser than expected from the size/density relation, and it is retained in the secretory pathway. The sequence between apoB-7...

Macrophages are prominent in hypoxic areas of atherosclerotic lesions, and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia-inducible factor (HIF-1alpha and HIF-2alpha). We found that versican co-localized with HIF-1alpha in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O(2) (hypoxia) compared with 21% O(2) (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1alpha and HIF-2alpha in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1alpha and HIF-2alpha but expression of perlecan was influenced only by HIF-1alpha and not by HIF-2alpha knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.

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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.-Li, L., P. Stillemark-Billton, C. Accumulation of triglycerides, particularly in the liver and skeletal muscle, is associated with metabolic disorders such as insulin resistance and type 2 diabetes (1, 2), diseases that are strong risk factors for cardiovascular diseases. Accumulation of triglycerides in the liver is also the landmark of nonalcoholic fatty liver disease, which results in inflammation and liver damage (3). Moreover, the stores of triglyceride in the liver fuel the assembly and secretion of VLDLs (3-6). Thus, it is obvious that elucidation of the mechanism of the storage of lipid in the cytosol is important for our understanding of the pathogenesis of major metabolic diseases.Triglycerides are stored in the cytosol in the form of lipid droplets (also referred to as lipid bodies) (for reviews, see 7, 8). These droplets are formed from microsomes (9; see also 7, 8 for reviews) as a primordial droplet with a diameter of z0.1 Am It has been demonstrated that the fatty acids used for the biosynthesis of VLDL triglycerides are derived from triglycerides stored in the liver (3). The triglycerides are hydrolyzed and reesterified into new triglycerides before being assembled into VLDL (4-6). VLDLs are assembled in a complex series of events (for reviews, see 11-16), starting with the formation of a primordial particle (pre-VLDL) during the translation and translocation of apolipoprotein B-100 (apoB-100) to the lumen of the endoplasmic reticulum. This step is catalyzed by the microsomal triglyceride transfer protein (MTP). The pre-VLDL par-

Intermediate filaments (nanofilaments) have many functions, especially in response to cellular stress. Mice lacking vimentin (Vim−/−) display phenotypes reflecting reduced levels of cell activation and ability to counteract stress, for example, decreased reactivity of astrocytes after neurotrauma, decreased migration of astrocytes and fibroblasts, attenuated inflammation and fibrosis in lung injury, delayed wound healing, impaired vascular adaptation to nephrectomy, impaired transendothelial migration of lymphocytes and attenuated atherosclerosis. To address the role of vimentin in fat accumulation, we assessed the body weight and fat by dual-energy X-ray absorptiometry (DEXA) in Vim−/− and matched wildtype (WT) mice. While the weight of 1.5-month-old Vim−/− and WT mice was comparable, Vim−/− mice showed decreased body weight at 3.5, 5.5 and 8.5 months (males by 19–22%, females by 18–29%). At 8.5 months, Vim−/− males and females had less body fat compared to WT mice (a decrease by 24%, p < 0.05, and 33%, p < 0.0001, respectively). The body mass index in 8.5 months old Vim−/− mice was lower in males (6.8 vs. 7.8, p < 0.005) and females (6.0 vs. 7.7, p < 0.0001) despite the slightly lower body length of Vim−/− mice. Increased mortality was observed in adult Vim−/− males. We conclude that vimentin is required for the normal accumulation of body fat.