Translocation Efficiency, Susceptibility to Proteasomal Degradation, and Lipid Responsiveness of Apolipoprotein B Are Determined by the Presence of β Sheet Domains

176

We previously showed that ⍀-3 fatty acids reduce secretion of apolipoprotein B (apoB) from cultured hepatocytes by stimulating post-translational degradation. In this report, we now characterize this process, particularly in regard to the two known processes that degrade newly synthesized apoB, endoplasmic reticulum (ER)-associated degradation and re-uptake from the cell surface. First, we found that ⍀-3-induced degradation preferentially reduces the secretion of large, assembled apoBlipoprotein particles, and apoB polypeptide length is not a determinant. Second, based on several experimental approaches, ER-associated degradation is not involved. Third, re-uptake, the only process known to destroy fully assembled nascent lipoproteins, was clearly active in primary hepatocytes, but ⍀-3-induced degradation of apoB continued even when re-uptake was blocked. Cell fractionation showed that ⍀-3 fatty acids induced a striking loss of apoB 100 from the Golgi, while sparing apoB 100 in the ER, indicating a post-ER process. To determine the signaling involved, we used wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which blocked most, if not all, of the ⍀-3 fatty acid effect. Therefore, nascent apoB is subject to ER-associated degradation, re-uptake, and a third distinct degradative pathway that appears to target lipoproteins after considerable assembly and involves a post-ER compartment and PI3K signaling. Physiologic, pathophysiologic, and pharmacologic regulation of net apoB secretion may involve alterations in any of these three degradative steps.Apolipoprotein B (apoB), 1 the major protein of atherogenic lipoproteins, is synthesized primarily by hepatic and intestinal cells. Most studies have focused on apoB metabolism in the liver, given the greater contribution to the plasma apoB pool made by that organ and the availability of relatively convenient primary and transformed hepatic cell models. The apoB message level and translational rate in hepatic cells are largely constitutive, and so secretory control is achieved primarily through co-and post-translational degradation of the protein (e.g. see Refs. 2-4 for recent reviews). Two specific mechanisms for the destruction of newly synthesized apoB in hepatic cells have been characterized. The first is endoplasmic reticulum-associated degradation (ERAD). Newly synthesized apoB in the endoplasmic reticulum (ER) is initially complexed with small amounts of lipid that are thought to be shuttled by the microsomal triglyceride transfer protein (MTP) (5). During severe lipid deprivation (6, 7) or MTP deficiency (8, 9), this initial lipidation fails, and the apoB becomes ubiquitinylated, which targets it for degradation by proteasomes (10 -14).The second mechanism for degradation of newly synthesized apoB is the re-uptake pathway. Re-uptake can occur after fully assembled apoB-containing particles have been exported across the plasma membrane but before they have diffused away from the vicinity of the cell by traversing the unstirred water layer that is adjacent...

“…Refs. 70,71). Furthermore, the apoC-III transgenic mouse provides a specific example of increased fatty acid availability in vivo, but without any increase in hepatic apoB secretion (72).…”

Section: Discussionmentioning

confidence: 99%

The Triple Threat to Nascent Apolipoprotein B

Fisher¹,

Pan²,

Chen³

et al. 2001

176

“…Our findings with the Apob knockout mice reveal that reduced amounts of apoB synthesis can also limit hepatic lipoprotein production. Other investigations have documented that the levels and intracellular availability of lipids can limit cellular lipoprotein secretion (27)(28)(29)(30)(31)(32). Thus, lipoprotein assembly and secretion require adequate levels of apoB, MTP, and lipids, and reductions in any one of these can limit the output of lipoproteins from cells.…”

Section: Effects Of Mtp Deficiency On Apob Secretionmentioning

confidence: 99%

A Deficiency of Microsomal Triglyceride Transfer Protein Reduces Apolipoprotein B Secretion

Leung¹,

Véniant²,

Kim³

et al. 2000

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of halfnormal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent (ϳ25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice. Microsomal triglyceride transfer protein (MTP)1 is expressed at high levels in the absorptive enterocytes of the intestine and hepatocytes, at moderate levels in the visceral endoderm of the yolk sac during embryonic development, and at relatively low levels in kidney and cardiac muscle (1-4). In each of these tissues, MTP plays a key role in the assembly and secretion of apoB-containing lipoproteins (1, 3, 5). MTP is located within the lumen of the endoplasmic reticulum (ER) and is thought to transfer lipids to apoB as that molecule is translated, allowing apoB to assume a proper conformation for forming a spherical lipoprotein with a neutral lipid core (6). In the absence of MTP, triglyceride-rich, apoB-containing lipoproteins cannot be assembled or secreted (3, 6, 7). The mechanism of MTP action has attracted considerable scrutiny. Of note, several lines of evidence have suggested that the assembly of lipoproteins involves a direct interaction between MTP and apoB. First, Wu et al. (8) demonstrated that apoB can be immunoprecipitated from the microsomes of cult...

“…The production of apolipoprotein B (apoB)-containing lipoproteins by the liver is mainly regulated by the relative amount of de novo synthesized apoB that is translocated into the endoplasmic reticulum and assembled into a secretion-competent lipoprotein particle (7)(8)(9)(10). MTP, an intraluminal protein in the endoplasmic reticulum (11), plays an important role in regulating the assembly and secretion of apoB-containing lipoproteins (12)(13)(14)(15)(16). In humans, the loss of MTP function blocks the production of apoB-containing lipoproteins by both the intestine and the liver (11).…”

mentioning

confidence: 99%

ARP-1/COUP-TF II Determines Hepatoma Phenotype by Acting as Both a Transcriptional Repressor of Microsomal Triglyceride Transfer Protein and an Inducer of CYP7A1

Kang

Spann

Hui

et al. 2003

Individual liver cells, distinguished by their anatomical localization within the portal triad and their relative expression of genes, allow the liver to provide diverse anabolic and catabolic functions. Hepatocytes localized near the portal vein express high levels of lipogenic (1) and gluconeogenic (2) enzymes, whereas those localized near the central vein express high levels of catabolic (CYP7A1) (3, 4) and glycolytic (2) enzymes. Environmental differences caused by hormonal, nutritional, and xenobiotic variations in portal versus central blood result in zonal distinctions in the gene expression of transcription factors within the hepatic acinus (5). The molecular mechanisms responsible for the distinct subsets of genes expressed by individual liver cells remain unknown. We have derived and characterized stable lines of rat hepatoma cells in order to identify the molecular mechanisms responsible for the phenotypic expression of the anabolic lipoprotein assembly pathway regulated by microsomal triglyceride transfer protein (MTP) 1 and the catabolic pathway through which cholesterol is converted into bile acids by the rate-limiting enzyme CYP7A1.The liver is the major site for both the production of plasma lipoproteins and their uptake from plasma and catabolism (reviewed in Ref. 6). The production of apolipoprotein B (apoB)-containing lipoproteins by the liver is mainly regulated by the relative amount of de novo synthesized apoB that is translocated into the endoplasmic reticulum and assembled into a secretion-competent lipoprotein particle (7-10). MTP, an intraluminal protein in the endoplasmic reticulum (11), plays an important role in regulating the assembly and secretion of apoB-containing lipoproteins (12-16). In humans, the loss of MTP function blocks the production of apoB-containing lipoproteins by both the intestine and the liver (11). On the other hand, the catabolic bile acid biosynthetic pathway, regulated by CYP7A1, is the major route through which hepatic (and total body) cholesterol homeostasis is maintained (17-20). Hepatic cholesterol levels indirectly regulate the expression of low density lipoprotein receptors via changes in serum response element (SRE)-binding proteins (21). Thus, the expression of CYP7A1 indirectly regulates the expression of low density lipoprotein receptors (22,23). Bile acids, which are the end products of the CYP7A1 reaction, stimulate the secretion of phospholipids and cholesterol into bile (24). Thus, the relative expression levels of MTP and CYP7A1 by individual hepatic parenchymal cells are important determinants of whether phospholipids and cholesterol are targeted into plasma (lipoproteins) or bile (biliary lipids).We have isolated stable cell clones from rat hepatoma cells as