ADP-ribosylation Factor 1 and Its Activation of Phospholipase D Are Important for the Assembly of Very Low Density Lipoproteins

The in vivo effects of increased delivery of fatty acids (FA) to the liver are poorly defined. Therefore, we compared the effects of infusing either 6 mM oleic acid (OA) bound to albumin, 0.5-20% Intralipid, or saline for 3 or 6 h into male C57BL/6J mice. Infusions were followed by studies of triglyceride (TG) and apoB secretion. Although plasma FA levels increased similarly after either 20% Intralipid or 6 mM OA, TG secretion increased only after infusion of 4 -20% Intralipid; TG secretion was unchanged by 6 mM OA. By contrast, 6-h infusions of either 6 mM OA or 4 -20% Intralipid increased apoB secretion. 6 mM OA and 20% Intralipid each increased secretion of apoB from primary hepatocytes ex vivo. Importantly, 0.5-2% Intralipid, which delivered more FA to the liver than 6 mM OA, did not stimulate apoB secretion. Hepatic apoB mRNA levels were unaffected by either 6 mM OA or 20% Intralipid, but microsomal triglyceride transfer protein mRNA was significantly lower after 6-h infusions with 6 mM OA versus either saline or 20% Intralipid. Lower microsomal triglyceride transfer protein mRNA levels were associated with reduced hepatic TG mass after 6-h infusions of 6 mM OA. We conclude that 1) increased FA delivery to the liver in vivo increases secretion of apoBlipoproteins via post-transcriptional mechanisms, 2) OA-induced apoB-lipoprotein secretion occurred at least in part via mechanisms other than by providing substrate for TG synthesis, and 3) the route of delivery of FA is important for its effects on apoB secretion.Regulation of the assembly and secretion of apolipoprotein B (apoB)-lipoproteins and, in particular, very low density lipoprotein (VLDL), 1 by the liver has been studied intensively for many years in cultured hepatocytes (1-4). Many laboratories have also conducted in vivo studies of apoB-lipoprotein secretion in humans and animal models (5-7). However, despite the presence of a large body of work, several questions remain incompletely answered.First and foremost is the question of whether increased flux of plasma fatty acids (FA) to the liver can stimulate VLDL secretion. Studies in cultured liver cell lines, primary hepatocytes, and perfused livers have provided conflicting results relative to the role of FA delivery in VLDL secretion. Thus, several (8 -13), but not all (14 -16) studies in cultured liver cell lines support the proposal that apoB secretion is increased by increased FA uptake. Studies in primary hepatocytes from fed rats (16 -18) or hamsters (19,20) have failed to show that exogenous FA stimulate secretion of apoB. However, we were able recently to demonstrate oleic acid (OA)-induced apoB secretion in primary hepatocytes from fasted mice (21). Results from perfused rat livers have also been mixed; OA had no effect on apoB secretion in chow-fed rats (22) but increased apoB secretion in fasted (22) or high carbohydrate diet-fed (23) rats. In vivo studies in humans have also been inconclusive. Lewis et al. (24,25) demonstrate that, when they increased the plasma levels of FA by infusin...

Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Regulation of Hepatic Apolipoprotein B-lipoprotein Assembly and Secretion by the Availability of Fatty Acids

Zhang

Hernandez‐Ono

et al. 2004

“…This conclusion is based on several observations. First, release of the droplets was inhibited by 1-butanol but not by 2-butanol, an indication that a PLD and the formation of PA are essential to the process (21,32). Moreover, 2,3-diphosphoglycerate inhibited the release of the TG-containing structures.…”

Section: Or Tip 47 (Not Shown)mentioning

confidence: 89%

“…Although they exhibit about 50% identity, the two enzymes have different biochemical properties and depend on different cofactors (28 -31). Our studies (32) suggest that PLD is important for the second step in the assembly of very low density lipoproteins (reviewed in Refs. 33 and 34), which appears to involve the formation of a lipid droplet in the microsomal lumen (35).…”

mentioning

confidence: 99%

A Phospholipase D-dependent Process Forms Lipid Droplets Containing Caveolin, Adipocyte Differentiation-related Protein, and Vimentin in a Cell-free System

Marchesan

Rutberg

Andersson

et al. 2003

Self Cite

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 < 1.055 g/ml fraction by gradient ultracentrifugation and were similar in size and appearance to those isolated from rat adipocytes and 3T3-L1 cells. Caveolin 1 and 2, vimentin, adipocyte differentiation-related protein, and the 78-kDa glucose regulatory protein were identified on the droplets from the cell-free system. The caveolin was soluble in 1% Triton X-100, as was the caveolin on lipid droplets from 3T3-L1 cells. The lipid droplets from the cell-free system, like those from 3T3-L1 cells, contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The assembly of these TGcontaining structures was dependent on the rate of TG biosynthesis and required an activator present in the 160,000 ؋ g supernatant from homogenized rat adipocytes. The activator induced phospholipase D (PLD) activity, and its effect on the release of the TG-containing structures from the microsomes was inhibited by 1-butanol (but not 2-butanol) or 2,3-diphosphoglycerate. The activator could be replaced by a constitutively active PLD or phosphatidic acid. These results indicate that PLD and the formation of phosphatidic acid are important in the assembly of the TG-containing structures.Within the cell, triglycerides (TG) 1 are stored in cytosolic lipid droplets. Little is known about how these structures are assembled. Several proteins have been identified on their surface, including adipocyte differentiation-related protein (ADRP or adipophilin), perilipins (reviewed in Refs. 1 and 2), and caveolin (see Refs. 3-5 and reviewed in Refs. 2 and 6), but their roles in lipid droplet assembly are unknown.ADRP and perilipins are abundant on lipid droplets. ADRP is found mostly on smaller droplets; when overexpressed, it stimulates lipid droplet formation (7), suggesting a role in the assembly process. Perilipins are present on large lipid droplets (7) and have a central role in the turnover of TG within these structures (1, 8, 10), perhaps affording protection against hormone-sensitive lipase (11). Perilipin-null mice have smaller lipid droplets and a higher rate of basal lipolysis than wild-type mice and are resistant to diet-induced obesity (12).Caveolin is a 21-kDa membrane protein with a hairpin structure whose N and C termini face the cytosol (13, 14). In mammals, there are three caveolin genes. Caveolin 1 and 2 are expressed in adipocytes, whereas caveolin 3 is present in muscle. Caveolin plays a key role in intracellular lipid transport (15, 16), binding fatty acid (17), and transporting cholesterol (16) in a manner reminiscent of the way plasma apolipoproteins transport lipids in the blood (16,18).Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to phosphatidic acid (PA) and appears to be important in intracellular transport and sorting processes (19 -22), either as an intracellular messenger or as a cone-shaped l...

“…Recent studies suggest that transformation may require either lateral diffusion of apoB to a specialized compartment of the ER or vesicular transport of apoB from one secretory compartment to another (4,13,14), but a review of the literature indicates that significant uncertainty exists regarding this issue (2,5,(15)(16)(17)(18)(19)(20). In the studies presented here, using pharmacological and subcellular fractionation approaches we demonstrate that the oleic acid (OA)-induced conversion of apoB100 LDL/HL particles to VLDL in McA RH7777 cells occurs in the ER and not in the Golgi.…”

mentioning

confidence: 99%

The Conversion of ApoB100 Low Density Lipoprotein/High Density Lipoprotein Particles to ApoB100 Very Low Density Lipoproteins in Response to Oleic Acid Occurs in the Endoplasmic Reticulum and Not in the Golgi in McA RH7777 Cells

Yamaguchi

Gamble

Conlon

et al. 2003

The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35 S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.Although the early, crucial steps in apolipoprotein B100 (apoB100)-lipoprotein assembly, including translation, translocation, and targeting of apoB100 for the ubiquitin-proteasomal degradation pathway have been well studied (1), much less is known about how the mature, triglyceride (TG) 1 -enriched lipoprotein is assembled. Thus, the exact process of bulk lipid addition and, in particular, the site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) to form mature, TG-enriched very low density lipoproteins (VLDL) has not been identified definitively. Early work by Hamilton and co-workers using electron microscopy (2), suggests that a lipid-poor form of apoB present in the rough endoplasmic reticulum (ER) could fuse with lipid droplets present in the smooth ER. That work first raised the possibility that VLDL was assembled in a two-step process. This scheme has been supported by studies from several laboratories (3-5), including our own (6). The two-step model includes the transformation of an "HDL-like" apoB-lipoprotein to a TG-enriched lipoprotein in the presence of adequate cell ...