Impaired hepatic apolipoprotein B and E translation in streptozotocin diabetic rats.

Sparks, Janet D.; Zolfaghari, Reza; Sparks, Charles E.; Smith, Harold C.; Fisher, Edward A.

doi:10.1172/jci115731

Cited by 75 publications

(39 citation statements)

References 75 publications

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“…ApoB mRNA abundance is not elevated in livers of 10-wk ZDF rats, supporting the constitutive nature of apoB gene expression (34). Therefore, the increased hepatic apoB output that occurs with hyperinsulinemia and insulin resistance of the 10-wk ZDF rat cannot be explained by transcriptional changes in apoB mRNA.…”

Section: Discussionmentioning

confidence: 71%

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Altered triglyceride-rich lipoprotein production in Zucker diabetic fatty rats

Chirieac¹,

Collins²,

Cianci³

et al. 2004

American Journal of Physiology-Endocrinology and Metabolism

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diabetic fatty (ZDF) rats, a model of insulin-resistant type 2 diabetes progression. TRL production was measured in vivo by blocking catabolism with Triton WR-1339. Ten-week ZDF rats are hyperinsulinemic with increased TRL production [both triglyceride and apolipoprotein B (apoB)]. Twenty-week ZDF rats are insulinopenic, and TRL production is similar to lean controls. Insulin infusion suppresses glucose and free fatty acids in 10-and 20-wk ZDF rats. Increased TRL production is not reduced by insulin in 10-wk rats; however, at 20 wk, TRL production is suppressed by insulin. In vitro studies with hepatocytes derived from 10-wk ZDF rats showed minimal insulin dose effects on apoB secretion compared with the response and sensitivity of hepatocytes derived from 20-wk ZDF and control lean rats. Hepatic sterol regulatory-binding protein (SREBP)-1c mRNA levels are increased at 10 wk but return to control levels at 20 wk. ApoB mRNA levels are similar to lean controls at 10 and 20 wk. The following two mechanisms for hypertriglyceridemia associated with hyperinsulinemia are suggested: increased TRL synthesis and loss of TRL suppression. Increased triglyceride production in hyperinsulinemic rats likely relates to increased expression of SREBP-1c, whereas increased apoB production involves posttranscriptional processes. hyperinsulinemic diabetes; insulinopenic diabetes; apolipoprotein b metabolism; triglyceride-rich lipoprotein production HYPERTRIGLYCERIDEMIA IS A key component of metabolic syndrome and type 2 diabetes and is strongly correlated with increased risk of cardiovascular disease (12). Insulin increases the intracellular availability of triglyceride (TG) for very low density lipoprotein (VLDL) production (1), possibly through enhanced lipogenesis (23). Pancreatic availability of insulin is diminished as disease progresses, with late-stage diabetes being characterized by insulinopenic hyperglycemia. Previous in vitro studies performed in cultured hepatocytes isolated from fructose-fed, insulin-resistant rats demonstrate blunting of the acute suppressive effects of insulin on VLDL secretion (37). Similar results are observed in hepatocytes from insulin-resistant obese rats (31). Furthermore, insulin fails to suppress hepatic VLDL apolipoprotein B (apoB) production in diabetic patients (16) and in hyperinsulinemic individuals during euglycemic clamp experiments (13,21). It is known that, during late-stage diabetes, pancreatic insulin release is progressively diminished; however, the effect of insulin on liver VLDL production during this transition from hyperinsulinemia to insulinopenia is not known.Hepatic expression of key enzymes involved in lipogenesis and cholesterogenesis is regulated by a family of transcription factors known as sterol regulatory element-binding proteins (SREBP; see Ref. 9). SREBP-1c is mainly involved in regulation of TG-synthesizing genes, whereas SREBP-2 has its main effect on cholesterol-synthesizing genes. SREBP-1c has been implicated as a mediator of insulin action in hepatic lipogenesis...

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Section: Discussionmentioning

confidence: 71%

“…This suggests that there is a catabolic defect in VLDL metabolism in 20-wk ZDF rats, since they become insulinopenic. In severely hypoinsulinemic streptozotocin-induced diabetic rats, hypertriglyceridemia results mainly from defective TRL catabolism (34).…”

Section: Discussionmentioning

confidence: 99%

Altered triglyceride-rich lipoprotein production in Zucker diabetic fatty rats

Chirieac¹,

Collins²,

Cianci³

et al. 2004

American Journal of Physiology-Endocrinology and Metabolism

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“…It seems unlikely, therefore, that pause transfer sequences play significant roles in the translocation of apoB100 in cultured cells. However, it is clear that "stuttering" does occur during translation of apoB (41)(42) including during translation of apoB in puromycin-treated cells (43). Because we would have expected decreasing intensity of the 70-kDa band and increasing intensity of the 120-and 220-kDa bands as translocation progressed, the finding in Fig.…”

Section: Resultsmentioning

confidence: 99%

The Amino-terminal Domain of Apolipoprotein B Does Not Undergo Retrograde Translocation from the Endoplasmic Reticulum to the Cytosol

Liang¹,

Wu²,

Fisher³

et al. 2000

Journal of Biological Chemistry

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We studied the sequential topology of the NH 2 and COOH termini of apoB during translocation by expressing, in Chinese hamster ovary (CHO) and HepG2 cells, an apoB42 construct with c-Myc and hemagglutinin (HA) tags at 2 and 41% (relative to apoB100) of its amino acid sequence. We conducted similar studies using monoclonal antibodies against the NH 2 and COOH termini of apoB100 in HepG2 cells. After radiolabeling, microsomes were immunoisolated from transfected CHO cells using anti-c-Myc or anti-HA antibodies. Throughout a 60-min chase in the presence of N-acetyl-leucylnorleucinal, more than 90% of microsomes were isolated by anti-HA antibodies, whereas less than 10% were isolated by anti-c-Myc antibodies. Proteinase K digestion of total microsomes consistently generated two fragments (ϳ70 and ϳ120 kDa) of apoB42 containing the NH 2 terminus throughout the chase; no fragments containing the COOH terminus were detected. Immunofluorescent studies of transfected CHO cells were consistent with results from the labeling studies. Essentially identical results were obtained from pulse-chase studies in both native and apoB42-transfected HepG2 cells. The present studies support a model in which, in the absence of adequate core lipid synthesis, there is partial translocation of apoB leading to cytosolic exposure, ubiquitination, and proteasomal degradation directly from the original translocation channel. The endoplasmic reticulum (ER)1 is the site of synthesis and maturation of proteins destined for secretion (1-3). It has also been recognized that the ER is able to target misfolded or otherwise defective proteins for degradation. This degradation was thought to occur in the ER. Studies in the past few years have, however, provided many examples of "ER degradation" that actually occur via the cytosolic ubiquitin-proteasome degradation pathway (3-7). Thus, a variety of proteins, which were believed to undergo ER degradation, have been identified as substrates for the cytosolic proteasome, including mutant carboxypeptidase ysc Y (CPY*) (8), ␣-antitrypsin variant piZ (9), and the T cell receptor ␣ subunit (10). The precise mechanism whereby these ER proteins make their way to the cytosolic proteasome has remained elusive. A post-translational retrograde translocation model, in which unfolded or misfolded proteins move from the ER lumen into the cytosol, has been proposed (4, 7, 12). The Sec61p translocation complex (or translocon) (6, 13), Ubc6p (14), a ubiquitin-conjugating enzyme that catalyzes the covalent attachment of ubiquitin to specific proteolytic substrates, and Cue1p (15), an ER membrane protein, have each been reported to participate in this retrograde translocation pathway.Apolipoprotein B100 (apoB) is the major protein of atherogenic very low density and low density lipoproteins (16). Extensive studies of cultured primary hepatocyte and hepatoma cell lines have established that significant control over apoB secretion can be achieved at co-and post-translational levels by degradation of newly synthesized apoB an...

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“…All reagents, unless otherwise specified, were purchased from Sigma Chem- . Rabbit polyclonal antisera to rat apoB or apoE and mouse monoclonal antibody to rat apoB were developed in the authors' laboratories and were previously described (29,35,36).…”

Section: General-malementioning

confidence: 99%

The Triple Threat to Nascent Apolipoprotein B

Fisher¹,

Pan²,

Chen³

et al. 2001

Journal of Biological Chemistry

176

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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...

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Impaired hepatic apolipoprotein B and E translation in streptozotocin diabetic rats.

Cited by 75 publications

References 75 publications

Altered triglyceride-rich lipoprotein production in Zucker diabetic fatty rats

Altered triglyceride-rich lipoprotein production in Zucker diabetic fatty rats

The Amino-terminal Domain of Apolipoprotein B Does Not Undergo Retrograde Translocation from the Endoplasmic Reticulum to the Cytosol

The Triple Threat to Nascent Apolipoprotein B

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