Objective-To investigate the role of glucosamine-mediated endoplasmic reticulum (ER) stress and Grp78 (BiP) in the intracellular degradation of apolipoprotein B100 (apoB100) in cultured hepatocytes. Methods and Results-Glucosamine treatment (2.5 to 10 mmol/L) of HepG2 cells increased levels of the ER chaperones, 78-kDa glucose-regulated protein (Grp78) and Grp94, in a dose-dependent manner and led to significant decreases in both cellular and secreted apoB100 by up to 97% (PϽ0.01). In contrast, no changes were observed in ER resident (ER60, PTP-1B) or secretory (albumin, apoE) control proteins. Glucosamine-induced apoB degradation was similarly observed in primary hamster hepatocytes and McA-RH7777 cells. Glucosamine treatment led to reduced tranlocational efficiency of apoB100 in the ER and enhanced its ubiquitination and proteasomal degradation. Adenoviral overexpression of Grp78 also led to significantly decreased levels of newly synthesized apoB100 in a dose-dependent manner (PϽ0.01). Grp78-induced downregulation of apoB100 was sensitive to inhibition by the proteasome inhibitor, lactacystin, but not lysosomal protease inhibitors, E64 and leupeptin, suggesting that overexpression of Grp78 selectively induced proteasomal degradation of apoB100. Conclusion-These findings suggest that binding and retention by Grp78 may play a critical role in proteasomal targetingand the ER quality-control of misfolded apoB. Key Words: apolipoprotein B Ⅲ degradation Ⅲ glucosamine Ⅲ Grp78 Ⅲ proteasome H epatic biogenesis of apoB is a complex process involving regulation by multiple post-transcriptional control mechanisms. 1-3 Intracellular availability of core lipoprotein lipid substrates, particularly triglyceride, appears to dictate the intracellular fate of newly synthesized apoB protein. 4 In the absence of lipid, a significant proportion of newly synthesized apoB100 is degraded in cultured hepatoma cells, 5 as well as in primary hepatocytes from hamsters, 6 rats, 7 and rabbits. 8 The bulk of apoB degradation appears to be mediated by the ubiquitin-proteasome degradative system. 9 High exogenous free fatty acid flux (particularly in cultured hepatocytes) or SREBP1-mediated de novo lipogenesis appear to protect apoB from proteasomal degradation. 10 Ubiquitination and proteasomal degradation of apoB begins cotranslationally and involves the interaction of misfolded apoB with cytosolic chaperones, Hsp70 and Hsp90. 11 Association with these cytosolic chaperones may be important in unfolding and subsequent targeting of the apoB polypeptide to the ubiquitin-proteasome pathway. 11 Mechanisms that target misfolded apoB or luminal lipoprotein-associated apoB to either proteasomal or nonproteasomal degradative pathways are currently unknown. Ample evidence is available showing the association of newly synthesized apoB polypeptide with endoplasmic reticulum (ER) chaperones. Several laboratories have observed that apoB100 is tightly associated with the 78-kDa glucoseregulated protein/immunoglobulin heavy chain-binding protein (Grp78/BiP...

Hepatic lipoprotein overproduction in a fructose-fed hamster model of insulin resistance was previously shown to be associated with a significant elevation of intracellular mass of microsomal triglyceride transfer protein (MTP) and elevated plasma levels of free fatty acids (FFA). Here, we further establish that fructose feeding and development of an insulin resistant state result in higher levels of MTP mRNA, protein mass, and lipid transfer activity. MTP protein mass was increased in fructose-fed hamster hepatocytes to 161 +/- 35.8% of control (p < 0.05), while MTP mRNA levels and MTP lipid transfer activity were increased to 147.5 +/- 30.8% (p < 0.05) and 177.5 +/- 14.5% (p < 0.05) of control levels, respectively. To identify underlying mechanisms, we also investigated the potential link between enhanced FFA flux and hepatic MTP gene expression. Direct modulation of MTP gene transcription by fatty acids was investigated by transfecting HepG2 cells with a reporter (luciferase) construct containing various base pair regions of the human MTP promoter including pMTP124 (with the sterol response element (SRE)), pMTP116, pMTP109 and pMTP100 (no SRE), and pMTP124SREKO (SRE sequences mutated). Treatment of HepG2 cells with oleic acid (360 muM) significantly increased luciferase activities in cells transfected with pMTP124 (136.6 +/- 11.0%, p < 0.05) and pMTP124SREKO (153.9 +/- 11.1%, p < 0.01) compared with control. Luciferase activity was also increased in a time and dose-dependent manner in the presence of oleic acid when transfected with pMTP124SREKO but not pMTP109 and pMTP100. Furthermore, long-term oleic acid treatment of HepG2 cells (10 days) induced higher levels of MTP mRNA (p < 0.05) confirming transcriptional stimulation of the MTP gene by oleic acid. In contrast, palmitate, arachidonic acid or linoleic acid did not significantly stimulate pMTP124 or pMTP124SREKO luciferase activity (p > 0.05). These data demonstrate that (1) MTP gene transcription may be directly up-regulated by oleic acid; (2) up-regulation of MTP gene transcription by oleic acid is SRE sequence independent; and (3) the sequence -116 to -109 in the MTP promoter region is essential for oleic acid-mediated stimulation. Stimulation of MTP gene expression may be a novel mechanism by which certain FFAs can induce hepatic lipoprotein secretion in insulin resistant states.