Glucosamine-Induced Endoplasmic Reticulum Stress Promotes ApoB100 Degradation

Abstract

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...

Increased hexosamine biosynthetic pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway

“…For example, an increased HBP flux causes hyper-O-glucosamine acylation (GlcNAc) of nuclear and cytoplasmic proteins [10], oxidative stress [11] and ER stress. This last mechanism has been demonstrated in cells other than beta cells [12].…”

“…Under physiological conditions, only 1-3% of intracellular glucose enters the hexosamine pathway; however, the flux increases with glucose concentration [9]. Increased HBP flux, in turn, causes hyper-O-GlcNAc of proteins [10], oxidative stress [11] and ER stress, although this last mechanism has been demonstrated in cells other than beta cells [12,29].…”

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“…14,[17][18][19][20] ER stress signaling pathways, collectively named the unfolded protein response (UPR), regulate ER homeostasis. [21][22][23][24] The UPR is important for hepatocyte ER adaptation to excessive lipid input in conditions associated with ER stress.…”

“…[21][22][23] Indeed, the genetic ablation of either branch of the UPR leads to hepatic steatosis in acute ER stress conditions, 25,26 whereas enforced maintenance of ER homeostasis increases apoB100 secretion, prevents SREBP-1c activation, and reduces hepatic steatosis in mice or cell culture models. 9,14,[18][19][20] Beyond their conventional role in monitoring ER homeostasis in acute ER stress, UPR effectors are activated under physiological conditions and regulate glucose and lipid metabolic pathways, thus contributing to basal cellular homeostasis. 27,28 Importantly, UPR signaling intersects with other signaling cascades, rendering the former amenable to regulation by signals other than directly resulting from ER stress.…”

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“…The expression of GRP78 clearly increases during ER stress, and therefore is used as an ER stress marker (4). In addition, GRP78 is involved in the integrity and the regulation of the ER, and it prevents the aggregation of misfolded proteins, including apoB100, by targeting them to the degradation by the 26S proteasome (5). When ER stress occurs, GRP78 is separated from inositol-requiring enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase and/or activating transcription factor 6, thus enabling them to be activated, which leads to the unfolded protein response, cell survival or apoptosis (4).…”