Our goal in the present studies was to reconcile 2 well-described associations that we found inconsistent. First, it has been well demonstrated that IR is associated with increased assembly and secretion of both VLDL apoB and TGs in animal models and humans (36
). The link between IR and increased VLDL secretion has been thought to derive from increased delivery of FAs to the liver secondary to increased lipolysis in adipose tissue; increased hepatic lipogenesis; increased levels and activity of MTP; and loss of insulin’s ability to direct apoB toward degradation. Second, it is also clear that obesity, IR, and dyslipidemia frequently coexist with nonalcoholic steatosis (7
). In view of the extensive body of evidence indicating that FAs and TGs can stimulate the assembly and secretion of VLDL (18
), one could question why the liver cannot maintain lipid homeostasis by increasing both the number of VLDL particles secreted and the quantity of TGs on each particle.
Recent studies implicating hepatic ER stress as a central abnormality linking obesity, hepatic IR, and hepatic steatosis have added a new level of complexity to this issue (13
). For example, ER stress has also been linked to increased hepatic lipogenesis (23
). Additionally, a large, complex secretory protein such as apoB might be a prime target for UPR-mediated downregulation. It is well known that apoB is regulated at translational and posttranslational steps, including a number of pathways for intracellular degradation (18
). Thus, ER stress–mediated reductions in apoB secretion could be a link between ER stress and hepatic steatosis. On the other hand, Ozcan et al. demonstrated that obesity-induced ER stress led to hepatic IR through activation of JNK activity via inositol-requiring enzyme–1 (IRE-1), with subsequent inhibition of insulin receptor signaling (13
). Although insulin has been shown to acutely inhibit hepatic secretion of apoB in both in vitro and in vivo studies (42
), this effect is lost in animal models of hepatic IR (45
) and in obese subjects with chronic hyperinsulinemia (43
). Thus, ER stress–induced IR could actually increase the assembly and secretion of VLDL, thereby reducing the risk for steatosis.
Based on these complex and potentially discordant findings, we hypothesized that modest increases in the delivery of FAs to the liver and/or modest accumulation of hepatic lipids would increase apoB secretion despite some stimulation of ER stress, while more substantial accumulation of hepatic lipids would further increase ER stress, leading to reduced apoB secretion; i.e., there would be a parabolic relationship between lipid-induced ER stress and apoB secretion. We tested this hypothesis in both in vitro and in vivo systems and demonstrated that (a) increased FA delivery and/or accumulation of liver TGs (or intermediary metabolites) caused ER stress; and (b) although mild ER stress secondary to increased FA delivery was associated with increased secretion of apoB100, greater ER stress and/or the presence of ER stress for a longer period of time in response to increased FA delivery and/or TG accumulation resulted in reduced apoB100 secretion and greater hepatic steatosis. We discuss each of these findings below.
In mammals, the UPR is mediated through 3 distinct pathways that are under the control of PERK, activating transcription factor–6 (ATF-6), and IRE-1α (3
). To decrease the accumulation of proteins in the ER, the UPR attenuates translation, particularly of secretory proteins, via activation of PERK and subsequent phosphorylation of eIF2α at Ser51. Additionally, the UPR increases the folding capacity of the ER by upregulating ER chaperones, such as GRP78, via the transcription factors XBP1 and ATF-6. Our data demonstrate that increased delivery of OA alone or FAs generated from Intralipid — which consists mainly of linoleic acid (54%), OA (22%), and palmitic acid (11%) — to McA cells in vitro and to mouse liver in vivo triggered increases in the levels of GRP78 and activation of at least 2 branches of the UPR, PERK-eIF2α and IRE1-XBP1. Previous studies in pancreatic β cells (48
) and cardiac myoblasts (51
) suggested that the ER stress was particularly sensitive to saturated FAs. Indeed, it has been reported that unsaturated FAs (OA or linoleate) reduce palmitate-induced upregulation of GRP78, CHOP, and GADD34, as well as apoptosis, in H4IIE liver cells (52
). It has also been reported that hepatic steatosis caused by diets enriched in saturated FAs, but not polyunsaturated FAs, was associated with ER stress and liver injury despite similar accumulation of TGs with each diet (53
). In the present studies, both OA and Intralipid were similarly effective in causing ER stress, both in vitro and in vivo (data for Intralipid in vivo not shown). We have conducted preliminary experiments with palmitic acid in McA cells and observed a similar ER stress response (data not shown). Further studies will be required to determine whether there are differential effects of FAs with varying degrees of saturation on apoB100 secretion in response to ER stress. It should be noted, however, that our incubations (16 hours) were longer than those in most other studies.
Identification of the molecular links between increased delivery of FAs to the liver and induction of ER stress will also require further study. Clearly, the stimulation of an ER stress response correlated closely with increases in the TG content of the McA cells in our in vitro incubation studies and, to a lesser degree, with increases in hepatic TG content in our in vivo studies. However, a previous study showed that loss of MTP in mouse hepatocytes (by either gene disruption or chemical inactivation), with concomitant inhibition of apoB secretion and marked accumulation of hepatic TGs, was not associated with the induction of typical markers of ER stress (54
), suggesting that TG was not the offending molecular species. Furthermore, when we used an antisense oligonucleotide to inhibit the expression of acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2), a key enzyme that catalyzes the last step in mammalian TG synthesis, TG accumulation was reduced by 50%, but we observed the same degree of OA-induced GRP78 expression and eIF2α phosphorylation in McA cells (data not shown). These data provide support for the view that TG, per se, does not cause ER stress.
Many studies by our group and others have demonstrated increased secretion of apoB and TG in response to increased hepatic FAs and/or TGs (18
). The effects of FAs/TGs are posttranscriptional; few physiologic perturbations alter Apob
mRNA levels, although translation can be affected under certain circumstances (18
). Overall, the availability of the core lipid ligands of apoB determines whether the protein is targeted for co- and posttranslational degradation or for secretion (19
). MTP clearly plays a key role in this process, probably by both transferring core lipids from the ER membrane to the amino-terminal of apoB and acting as a kinetic anchor after interacting directly with the amino-terminal of the nascent apoB as it enters the ER lumen (58
). In the present experiments, we confirmed many prior studies that have demonstrated increased apoB100 secretion when McA cells are incubated with moderate concentrations of OA for short periods of time. In fact, with only 3-hour exposures, OA stimulated the secretion of apoB100 across a wide dose range. However, when we incubated the cells with 1.2 mM OA for 6 hours, there was a loss of stimulation of apoB100 secretion, and this was paralleled by increased GRP78
under those same conditions. Finally, when we incubated McA cells for 16 hours, we observed a parabolic relationship between OA concentrations and apoB100 secretion. Of note, 16-hour incubations were associated with increases in ER stress markers at all concentrations of OA (data shown for GRP78), including 0.4 mM OA (shown for eIF2α and XBP1). The parabolic relationship between FA-induced ER stress and apoB100 secretion was also observed with IL. Of note, with both approaches to lipid delivery, inhibition of apoB100 occurred without any changes in the secretion of apoB48, albumin, or apoA-I from McA cells. It was only when the cells were exposed to OA at a concentration of 1.6 mM for 16 hours that we observed inhibition of the secretion of all 4 proteins tested (we did not test IL at concentrations greater than 1,000 mg/dl). These data suggest that at doses of OA below 1.6 mM, and at doses of IL up to 1,000 mg/dl, the degree of ER stress was relatively mild and the global effects of ER stress on gene expression and translation of mRNA had not yet occurred. This view is supported by our finding that total TCA-precipitable radioactivity was reduced only when cells were exposed to 1.6 mM OA for 16 hours, a perturbation that inhibited secretion of all 4 proteins tested. On the other hand, trypan blue staining was unchanged at all doses of OA and IL, indicating that lipid loading did not, in these experiments, induce generalized cell toxicity. It is clear from these data that apoB100, a protein that is extensively regulated by intracellular degradation (19
), is particularly sensitive to FA-induced ER stress. However, when the level of ER stress was further increased, the expected generalized effects of the UPR were demonstrated in our studies. Sparks et al. (59
) showed inhibition of apoB secretion from McA cells incubated for 8 hours with 0.75 mM OA. Furthermore, they demonstrated that the reduced apoB secretion was not associated with changes in Apob
gene expression. They did not, however, look at other doses of OA, and there were no measurements of ER stress.
As reviewed above, the UPR includes pathways that increase the transcription of chaperone proteins and decrease the translation of secretory proteins (3
). Therefore, we first looked at the expression of 2 genes, Apob
, that obviously can affect apoB secretion: We saw no effect of lipid-induced ER stress on the mRNA levels of either Apob
. However, when we looked at posttranscriptional steps regulating apoB synthesis and secretion, we found clear evidence for complexity.
First, our studies with puromycin-synchronized cells, with and without concomitant lactacystin, indicated that both reduced rates of elongation of nascent apoB and cotranslational proteasomal degradation were affecting the appearance of full-length apoB100. The ability of lactacystin to significantly increase the appearance of apoB100 suggests that cotranslational ubiquitinylation and proteasomal degradation of apoB was the more significant process affecting the appearance of newly synthesized full-length apoB100 (60
). Importantly, cotranslational ubiquitinylation of apoB100 does not become significant until about 40%–50% of the nascent protein has been translated (60
). Thus, apoB48 would be much less likely to be polyubiquitinylated and targeted for proteasomal degradation. Similarly, small proteins such as albumin and apoA-I would likely escape ubiquitinylation. Future studies, using constructs of apoB of varying size and amino acid sequence, should provide further insights into the link between mild ER stress and cotranslational degradation of apoB100. In addition, despite the absence of change in Mttp
mRNA levels, further studies will be needed to determine whether MTP function, which is key to the assembly and secretion of apoB-Lps, is altered by ER stress.
Lactacystin, however, did not completely reverse the reduced appearance of apoB100 in cells incubated with high concentrations of OA, suggesting reduced rates of translation. We had previously seen reduced translation of apoB100 when cotranslational lipidation was inhibited (61
). Furthermore, a recent study by Borradaile et al. (51
) implicated eukaryotic elongation factor (eEF1A) in FA-induced lipotoxicity; disruption of the gene protected cells. Although those authors did not demonstrate reduced protein synthesis in cells with disrupted eEF1A, those results raise the possibility that reduced elongation might be a response to FA-induced ER stress. Further studies are planned to address this question directly.
In addition to the cotranslational, proteasomal degradation we observed, we also demonstrated nonproteasomal, likely posttranslocational intracellular degradation of apoB100 in association with ER stress. Importantly, the pattern of increased degradation mimicked exactly the parabolic secretory pattern we observed. Qiu et al. reported that treatment of HepG2 cells with glucosamine resulted in elevated levels of GRP78 and reduced rates of secretion of apoB100, the latter due to increases in both proteasomal and nonproteasomal degradation of apoB100 (26
). Similar results were observed by that group when GRP78 was overexpressed by adenovirus, suggesting a direct effect of an increase quantity of this chaperone in targeting of apoB100 for proteasomal degradation (26
). Oyadomari et al. found that P58IPK
, an ER-associated protein whose synthesis is stimulated by ER stress, is a mediator of proteasomal degradation of newly synthesized apoB100 (63
). Those authors found that the increase in proteasomal degradation of apoB100 induced by inhibition of MTP was attenuated in P58IPK–/–
hepatocytes, suggesting that P58IPK
is required for degradation of misfolded apoB associated with impaired lipidation. Further studies will be required to determine the exact roles, if any, of increased GRP78 and P58IPK
in the stimulation of apoB100 degradation we have observed. Of note, lactacystin did not alter ER stress–induced inhibition of apoB100 secretion when studied over a 2-hour period of radiolabeling. This was in contrast to the reversal of cotranslational degradation we observed in very short radiolabeling studies in puromycin-synchronized cells. We believe that together, these results indicate that nonproteasomal, posttranslocational pathways of degradation play a greater role in the overall response to ER stress than does cotranslational degradation. A recent study demonstrating that proteasomal degradation is transiently inhibited during the UPR adds further complexity to any interpretation of these findings (64
). The exact site of the intracellular degradation of apoB100 during ER stress remains to be determined (31
). However, the absence of any effects of either vitamin E or desferrioxamine on ER stress–associated inhibition of apoB100 secretion (32
) indicates that lipid peroxidation and/or reactive oxygen species did not play a significant role. Finally, it is also noteworthy that we demonstrated increased intracellular degradation of nascent apoB100 despite finding evidence for activation of JNK and subsequent serine phosphorylation of IRS-1, indicative of ER stress–induced hepatic IR (data not shown). As noted above, diminished insulin signaling would be expected to lead to reduced degradation of apoB100 and increased secretion (43
PBA has been shown to be a chemical chaperone that stabilizes protein folding (33
). Recently, Ozcan et al., reported that treatment with the chemical chaperones PBA and taurine-conjugated ursodeoxycholic acid alleviated ER stress in cultured liver cells and ob/ob
). Those authors showed that PBA improved insulin action, leading to normalization of both glucose intolerance and steatosis in genetic and diet-induced mouse models of obesity. In that study (34
), PBA attenuated the induction of ER stress by tunicamycin in Fao rat hepatoma cells. PBA has also been shown to improve the trafficking of mutant proteins in cystic fibrosis (CFTRΔ508) (66
) and to enhance the secretion of α1-AT in α1-AT deficiency (67
). In our study, PBA blocked lipid-induced ER stress in McA cells and specifically reversed the inhibition of apoB100 secretion by higher concentrations of OA, at least in part by normalizing the translation of apoB100 and/or protecting it from cotranslational degradation.
FA loading causes many cellular responses, including ER stress, raising the question of whether there is a direct link between FA-associated ER stress and inhibition of apoB100 secretion. Indeed, PBA, which we used to support such a direct link, has been shown to stimulate FA oxidation and have additional effects on cellular lipid metabolism (68
). However, in our studies, PBA did not affect cellular TG content, suggesting that this chemical chaperone directly prevents the decrease in apoB100 secretion by inhibiting lipid-induced ER stress. More importantly, we have recapitulated nearly all the effects of FA loading on apoB100 secretion by using tunicamycin, a well-established inducer of ER stress. Thus, although tunicamycin did not, at any dose, stimulate apoB100 secretion, it did inhibit apoB100 secretion in a dose-dependent manner that paralleled its effects on ER stress markers. Furthermore, tunicamycin showed the same “relative selectivity” for apoB100 that we observed with OA and IL; it was only at the 5-μg/ml dose of tunicamycin that secretion of apoB48, albumin, and apoA-I were inhibited. Finally, PBA reversed not only (as expected) tunicamycin-mediated ER stress, but also the inhibition of apoB100 secretion.
We had previously reported that in vivo exposure of mouse livers to high concentrations of FA for 6 hours resulted in significant increases in the secretion of apoB100 and apoB48 (35
). In the present study, we confirmed that finding and further demonstrated that longer infusions of OA (9 hours) were associated with the loss of OA-stimulated apoB secretion; this loss of FA-stimulated apoB secretion was paralleled by increased and/or prolonged hepatic ER stress. Importantly, when we treated mice with PBA for 7 days, we reduced OA-mediated ER stress and rescued apoB100 from the effects of 9 hours of elevated FA levels. However, neither 6- nor 9-hour infusions of OA significantly increased liver TGs, so we also infused 20% Intralipid. Although our previous studies showed that 6-hour infusions of Intralipid stimulated both TG and apoB secretion without significantly increasing hepatic TG content (35
), we found, in the present studies, that 9-hour infusions of 20% Intralipid did not stimulate TG and apoB secretion despite significantly increasing hepatic TG mass. These in vivo results confirm our in vitro studies and provide strong evidence directly implicating ER stress as the basis for the inhibition of apoB100 secretion during prolonged exposure of livers to high concentrations of FAs. Although 9-hour infusions of OA also led to the loss of FA-stimulated apoB48 secretion (Figure B), that abnormality was not reversed by PBA treatment. Further experiments will be needed to determine the differences we have observed between the in vitro and in vivo effects of ER stress and PBA on apoB48 metabolism.
The results of these studies provide new and novel insights into the pathophysiology of steatosis and hypertriglyceridemia, a combination of abnormalities characteristic of animal models and humans with IR. As noted above, a substantial proportion of individuals with IR have both hepatic steatosis and hyperlipidemia. Furthermore, the severity of hepatic steatosis correlates with secretion of increased numbers of large, TG-rich VLDL (69
). Finally, it has been repeatedly demonstrated, both in vitro and in vivo (18
), that increased FA delivery to hepatocytes stimulates VLDL apoB and TG secretion. Together, those results raise a key question: why can’t lipid-loaded livers, which are typically also insulin resistant, secrete as much VLDL as is necessary to avoid steatosis. The results we present demonstrate that lipid-induced hepatic ER stress can explain why steatosis develops despite increased VLDL secretion. The parabolic relationship between the degree of lipid-induced ER stress and apoB100 secretion suggests that in most animals and people with lipid-induced steatosis, VLDL secretion may be increased above normal, consistent with existing literature, but secretion will be less than maximal (on the downside of the parabola) due to excessive ER stress. Further studies will be required to determine both the molecular basis for FA-induced ER stress and for the increase in intracellular degradation of apoB. Of interest, our findings suggest that pharmacologic approaches directly targeting hepatic ER stress could be a therapy for steatosis, albeit at the expense of increased secretion of atherogenic apoB-Lps.