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

Abstract

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 ...

A fatty acid-dependent hypothalamic–DVC neurocircuitry that regulates hepatic secretion of triglyceride-rich lipoproteins

“…Scd-1 catalyses the desaturation of saturated fatty acyl-CoAs (for example, stearoyl-CoA) to monounsaturated fatty acyl-CoAs (for example, oleyl-CoA). Given that oleic acids promote the final steps of lipid-rich VLDL assembly and potentiate the secretion of VLDL-TG 49 , we postulate that an inhibition of scd-1, and the consequent reduction of oleyl-CoA availability and bulk lipid transfer to apoB-lipoproteins in the late stages of their assembly, underlies the MBH oleic acid-induced lowering of hepatic VLDL-TG secretion. Also, it remains a possibility that converging neuronal and hepatic metabolic pathways may mediate the ability of MBH fatty acid sensing to regulate hepatic lipid and glucose homeostasis.…”

apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB

“…Our studies raise the possibility that apoA-IV may function as a modulatory cofactor to reduce apoB's rate of intracellular transport, thereby increasing its residence time within a lipoprotein expansion compartment. The exact intracellular compartments involved in second step expansion are under active investigation but appear to involve the ER (25,46) and/or Golgi (47)(48)(49). Because the capacity to enlarge nascent lipoproteins is the predominant means by which the intestine accommodates increased lipid flux, apoA-IV, via its trafficking effects on apoB, may play an important role in this process, particularly under conditions of high dietary fat intake.…”

ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich lipoproteins

“…Using this system, we observed that although transfection of apoA-IV-KDEL and native apoA-IV both reduced the secretory effi ciency of apoB relative to control cells, apoA-IV-KDEL markedly inhibited cellular TG secretion, whereas native apoA-IV increased TG secretion into media, the association of apoB with d < 1.006 g/ml lipoproteins, and the diameter of VLDL 1 particles. Considered together, these data suggest that apoA-IV may act as a chaperone that modulates the traffi cking of apoB through the secretory pathway, thereby prolonging the residence time of nascent apoB-containing particles in cellular compartments where lipidation occurs ( 13,14,16,46 ), enhancing TG loading and, ultimately, increasing TG secretion.…”