Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents (original) (raw)
Incubation of McA cells with OA or IL results in dose- and duration-dependent increases in cell TG content. To develop an in vitro model of steatosis, we incubated OA or IL at concentrations ranging from 0.1 to 2.0 mM for OA or 10 to 1,000 mg/dl for IL for 6 hours in McA cells. Incubation with OA resulted in an increase in cell TGs from control levels of 45.0 ± 13.0 μg/mg cell protein in the absence of OA to 358.7 ± 24.3 μg/mg cell protein in the presence of 2.0 mM OA; incubation with IL resulted in an increase in cell TGs from control levels of 54.3 ± 1.7 μg/mg cell protein in the absence of IL to 453.2 ± 43.2 μg/mg cell protein in the presence of 1,000 mg/dl IL (Figure 1A). Effects of duration of incubations were determined using 1.0 mM OA and 500 mg/dl IL, with fresh medium provided every 12 hours (Figure 1B). At these concentrations, OA and IL increased total cellular TG levels in McA cells in a duration-dependent manner.
Incubation of McA cells with OA or IL results in dose- and duration-dependent increases in cell TG content. (A) Incubation of McA cells with OA (0.1–2.0 mM; black bars) or IL (10–1,000 mg/dl; gray bars) for 6 hours resulted in dose-dependent increases in cellular TGs. (B) Incubation of McA cells with 1.0 mM OA or 500 mg/dl IL for 1–48 hours increased cellular TG content in a duration-dependent manner. The incubation medium was changed every 12 hours. TG levels are presented as microgram per milligram cell protein. Data are mean ± SD (n = 3 for each condition). LpL, lipoprotein lipase.
Incubation of McA cells with OA or IL increases GRP78 expression in McA cells. We next sought to demonstrate a link between lipid loading and ER stress. Incubations of McA cells with increasing concentrations of either OA or IL for increasing durations also led to dose- and duration-dependent increases in mRNA levels of GRP78, an ER chaperone that is a central indicator of ER stress (27) (Figure 2A). There were no effects of 3-hour incubations at any dose of OA or IL. However, after 6-hour incubations, significant increases in GRP78 mRNA began to be observed with higher doses of each source of FAs, and marked elevations of GRP78 mRNA were seen at most concentrations of OA and IL after 16 hours of incubation. In this experiment, the 6-hour incubations with 0.4 mM OA were associated with cell TG levels of 128.5 ± 10.3 (0.4 mM) μg/mg cell protein, whereas the 16-hour incubations with 0.4 mM OA resulted in TG levels of 166.5 ± 17.6 μg/mg cell protein. Incubations of cells with 500 mg/dl IL for 6 hours resulted in TG levels of 448.9 ± 30.3 μg/mg cell protein, whereas 16-hour incubations with 500 mg/dl IL produced TG levels of 628.5 ± 64.4 μg/mg cell protein. These conditions were used to look at several other markers of ER stress.
Incubation of McA cells with OA or IL increases GRP78 mRNA and protein levels. (A). Incubation of McA cells with either OA (0.4–1.6 mM) or IL (100–1,000 mg/dl) for 3 hours did not affect mRNA levels of GRP78. However, incubation for 6 or 16 hours with OA or IL increased GRP78 mRNA in a dose-dependent manner. Data are mean ± SD normalized to cells incubated without OA or IL (n = 6 for each condition); *P < 0.05, **P < 0.001 versus control incubations. (B) Incubation of McA cells for 3 or 6 hours with OA (0.4 mM) or IL (500 mg/dl) did not significantly affect GRP78 protein levels. (C) GRP78 protein was significantly increased by incubation with either OA (0.4 mM) or IL (500 mg/dl) for 16 hours. ER stress, as indicated by increased GRP78, was also induced by a 3-hour treatment with tunicamycin (5 μg/ml) as a positive control. Protein data are mean ± SD normalized to cells incubated without OA or IL (n = 3 for each condition); *P < 0.001 versus control incubations.
To determine whether the increases in GRP78 mRNA were associated with elevations in the protein levels, we incubated McA cells with 0.4 mM OA or 500 mg/dl IL for 3, 6, or 16 hours. GRP78 protein expression was not significantly changed by incubation with either OA or IL after 3 or 6 hours of incubation (Figure 2B). However, both lipids increased levels of GRP78 protein after a 16-hour treatment (2.9- and 2.7-fold, respectively; P < 0.001 for both vs. control incubations) (Figure 2C). These changes correlated with the dose- and time-dependent responses of TG loading of the cells (see above and Figure 1).
Incubation of McA cells with OA or IL induces phosphorylation of eIF2α in McA cells. To determine whether lipid-loading induces hepatic ER stress downstream of GRP78, we examined its effects on components of the UPR. One component of the UPR involves phosphorylation of cytosolic eIF2α on Ser51 by the ER-localized kinase PERK (4). Phosphorylated eIF2α mediates both a transient decrease in global translation and the translational upregulation of stress-induced mRNAs (5). We determined the phosphorylation status of eIF2α (Ser51) using phospho-specific antibodies during lipid loading of McA cells. Incubation of McA cells for 3 or 6 hours with OA (0.4 mM) or IL (500 mg/dl) had no significant effect on the levels of total or phosphorylated eIF2α (Figure 3A). However, incubation at these concentrations for 16 hours induced significant phosphorylation of eIF2α (7.5- and 6.7-fold, respectively; P < 0.01 vs. control incubations) without changing the total eIF2α protein levels (Figure 3B).
OA or IL induces phosphorylation of eIF2α in McA cells. (A) Incubation of McA cells for 3 or 6 hours with OA (0.4 mM) or IL (500 mg/dl) did not significantly induce phosphorylation of eIF2α. (B) Phosphorylation of eIF2α was significantly increased by incubation with either OA (0.4 mM) or IL (500 mg/dl) for 16 hours without a change in total eIF2α protein level. ER stress was also induced by a 3-hour treatment with tunicamycin (5 μg/ml) as a positive control. Lanes were run on the same gel but were noncontiguous. Data are mean ± SD normalized to cells incubated without OA or IL (n = 3 for each condition); *P < 0.01 versus control incubations.
Incubation of McA cells with OA or IL induces XBP1 mRNA alternative splicing and the nuclear translocation of the alternative form of the protein. Another branch of the UPR is mediated by activation of x-box–binding protein–1 (XBP1) by inositol requiring ER-to-nucleus signaling protein–1α (IRE-1α), which is an ER-localized protein. IRE-1α activates XBP1 by splicing its mRNA in response to ER stress (28, 29). The splicing results in the excision of a 26-bp fragment and a frameshift that can be identified by the loss of a Pst1 restriction site and a longer PCR product (28). McA cells were exposed for 3, 6, or 16 hours to OA (0.4 mM) or IL (500 mg/dl), and the extent of XBP1 splicing was characterized by determining the relative amounts of 300-bp (Pst1+ derived from native, unspliced mRNA) and 601-bp (Pst1– derived from activated, spliced mRNA) PCR products after Pst1 digestion. Incubation of cells with 5 μg/ml tunicamycin, which was used as a positive control for ER stress, resulted in complete processing of XBP1 mRNA, and only larger 601-bp products were seen after Pst1 digestion (Figure 4, A and B, far right lane). In the control condition, the majority of the XBP1 PCR product was 300 bp (Pst1+), indicating predominance of the native, unspliced XBP1 mRNA (Figure 4, A, lanes 1 and 4, and B, lanes 1–3 from left). There was no effect, compared with control, of 3-hour (Figure 4A, lanes 2 and 3) or 6-hour incubations (Figure 4A, lanes 5 and 6) with either OA or IL on XBP1 mRNA splicing. However, in cells exposed to OA or IL for 16 hours, a larger proportion of the XBP1 PCR product was the 601-bp length (OA 43% ± 6%; IL 38% ± 4%) (Pst1–), indicating greater XBP1 mRNA splicing in the presence of ER stress (Figure 4B, lanes 4–9).
OA or IL induces alternative splicing of XBP1 mRNA and translocation of the alternative form of the protein into the nucleus in McA cells. McA cells were exposed for 3 or 6 hours (A) or for 16 hours (B) to OA (0.4 mM) or IL (500 mg/dl). XBP1 cDNA was amplified by PCR followed by incubation with Pst1. The products of the incubation with Pst1 are shown on the left; the bar graphs show the percentage of total XBP1 mRNA that was resistant to Pst1 and was, therefore, already spliced and activated. After 3 or 6 hours of incubation with OA or IL, most of the XBP1 PCR products were cut by Pst1 (Pst1+), producing a 300-bp amplification product, indicating a predominance of the native, unspliced form of XBP1 mRNA; less than 20% of total XBP1 was detected as the Pst1–, 601-bp amplification product, indicative of spliced XBP1 mRNA. By contrast, after a 16-hour incubation with either OA or IL, a larger proportion of the XBP1 PCR product was Pst1– and kept its full 601-bp length (OA 43% ± 6%; IL 38% ± 4%), indicating partial XBP1 mRNA splicing and presence of ER stress. Complete XBP1 activation and splicing were induced by a 3-hour treatment with tunicamycin (5 μg/ml) as a positive control. Data are mean ± SD (n = 6 for each condition); *P < 0.05, **P < 0.01 versus control incubations (without OA or IL). The nuclear content of XBP1 was not increased after a 6-hour incubation with OA or IL (C) but was increased after incubation with either lipid source for 16 hours (D). Nucleophosmin is shown as a control for the efficiency of the nuclear extraction. ER stress was also induced by a 3-hour treatment with 5 μg/ml tunicamycin as a positive control. Lanes were run on the same gel but were noncontiguous. Data are mean ± SD normalized to cells incubated without OA or IL (n = 3 for each condition); *P < 0.05, **P < 0.01 versus control incubations.
The spliced XBP1 mRNA produces a protein that is a potent basic leucine zipper transcription factor after translocation into the nucleus (29). Although the nuclear content of XBP1 was not increased after 3 or 6 hours of incubation of McA cells with either OA or IL (Figure 4C), nuclear translocation of XBP1 protein was clearly increased after 16 hours of incubation of either OA (Figure 4D, lanes 5 and 6) or IL (Figure 4D, lanes 7 and 8) compared with control cells incubated without either lipid (Figure 4D, lanes 3 and 4) (P < 0.05 and P < 0.01 vs. control incubations, respectively). Thus, prolonged lipid loading by either OA or IL activated XBP1 in McA cells, as evidenced by splicing of mRNA and nuclear translocation of XBP1.
There is a parabolic relationship between lipid loading–mediated ER stress and apoB100 secretion in McA cells. The above results demonstrated that we could induce hepatic ER stress by loading the cells with FAs and/or TGs. Because delivery of FAs or TGs to cultured liver cells is typically associated with increased apoB secretion (18), we wanted to characterize the relationship between FA/TG-induced hepatic ER stress and the assembly and secretion of apoB-Lps. Cells were treated with varying doses of OA for 3, 6, or 16 hours and then labeled with [35S]methionine. Media were collected, and apoB100, apoB48, apoA-I, and albumin were immunoprecipitated with specific antibodies. Incubation of McA cells with 0.4, 0.8, or 1.2 mM OA for 3 hours induced progressive, dose-related increases in apoB100 secretion compared with incubations in the absence of OA (P < 0.01) (Figure 5A). Both 0.4 mM and 0.8 mM OA continued to stimulate apoB100 secretion after 6 hours of incubation, but the effect of lipid loading on apoB100 secretion was lost after 6-hour incubations at 1.2 mM OA. Furthermore, while 0.4 mM OA still stimulated apoB100 secretion after 16 hours, 0.8 mM no longer had an effect, and 1.2 mM OA for 16 hours actually inhibited apoB100 secretion compared with control cells. These complex dose-related relationships between OA-induced lipid loading and apoB100 secretion at 16 hours were not observed with apoB48, apoA-I, or albumin (Figure 5B). However, when we incubated McA cells for 16 hours with 1.6 mM OA, which was associated with the highest levels of GRP78 gene expression (Figure 2A), there were significant reductions in the secretion of all 4 proteins (Figure 5C). When IL was used as a means of delivering FAs to the cells, there was a dose-dependent increase in ER stress, indicated by rising levels of GRP78 protein (Figure 5D). Concomitantly, we observed a parabolic response pattern for apoB100 (Figure 5E): compared with incubation of McA cells without any IL, incubation with 100 mg/dl of IL for 16 hours increased apoB100 secretion; incubation with 500 mg/dl IL resulted in apoB100 secretion at the same level as control; and incubation with 1,000 mg/dl IL (which caused the greatest rise in GRP78; Figure 5D) caused a reduction in apoB100 secretion. IL had no effect on apoB48, apoA-I, or albumin secretion at any of the concentrations used.
Lipid loading–induced ER stress has a parabolic effect on apoB100 secretion in McA cells. (A). McA cells were preincubated with 0.4, 0.8, or 1.2 mM of OA for 3, 6, or 16 hours, incubated for 2 hours in methionine/cysteine-free DMEM, and then labeled with [35S]methionine for 2 hours, the latter 2 incubations still in the presence of OA. There was a time and dose-dependent change from stimulation to inhibition of apoB100 secretion. (B) At the same doses, there were no effects of OA on the secretion of apoB48, apoA-I, or albumin at any duration of preincubation. (C) Using the same preincubation and incubation protocol, 1.6 mM OA decreased apoB100, apoB48, albumin, and apoA-I secretion. (D) McA cells were preincubated with 100, 500, or 1,000 mg/dl IL for 16 hours, followed by the protocol described in A with IL present during the last 2 steps. There was a dose-dependent increase in ER stress, as indicated by increasing levels of GRP78 protein. (E) 100 mg/dl IL stimulated apoB100 secretion, whereas 500 mg/dl IL did not. Incubation with 1,000 mg/dl IL actually inhibited apoB100 secretion. Intralipid, at the doses used, had no effects on apoB48, albumin, or apoA-I secretion. (F) TCA precipitable radioactivity was unaffected at concentrations of 0.4, 0.8, or 1.2 mM OA. However, a 16-hour preincubation with 1.6 mM OA did reduce TCA-precipitable radioactivity by about 30%. None of the doses of IL that were used affected TCA-precipitable radioactivity. All data are mean ± SD normalized to cells incubated without OA or IL (n = 3 for each condition); *P < 0.05, **P < 0.01 versus control incubations.
OA, at 0.4, 0.8, and 1.2 mM, had no effect on total protein synthesis in the McA cells, as determined by total TCA-precipitable radioactivity; the 30% reduction in TCA-precipitable radioactivity seen with 1.6 mM OA indicated an effect of this dose of OA on global protein synthesis and secretion, consistent with the reduction in secretion of all 4 proteins tested (Figure 5F). IL, at the doses tested, had no effect on TCA-precipitable radioactivity. Normal trypan blue staining confirmed a lack of toxicity of OA and IL at any concentration and duration of treatment (data not shown). These results indicate a complex, parabolic relationship between the degree of lipid-induced ER stress and apoB100 secretion.
ER stress–induced inhibition of apoB100 secretion is not associated with altered gene expression of either Apob or microsomal TG transfer protein. Since ER stress and the UPR significantly alter gene expression, we next determined the effects of ER stress on the expression of 2 key genes: Apob and microsomal TG transfer protein (Mttp). There was no effect of incubating McA cells with increasing doses of either OA (0.4–1.6 mM) or IL (100–1,000 mg/dl), for varying periods, on the levels of Apob or Mttp mRNA (Figure 6).
Incubation of McA cells with OA or IL does not affect either Apob or Mttp mRNA levels. The effect of varying doses of OA (0.4–1.6 mM) or IL (100–1,000 mg/dl) on Apob (A) and Mttp (B) expression in McA cells was assessed using quantitative real-time RT-PCR. Apob and Mttp mRNA levels were normalized with GAPDH mRNA. Apob and Mttp mRNA expression was unchanged by incubation with increasing doses of either OA or IL for 3–16 hours. Data are mean ± SD (n = 6 for each condition).
ER stress induces cotranslational proteasomal degradation of apoB. With no evidence for effects of ER stress on apoB gene expression, we turned our attention to posttranscriptional pathways that regulate apoB secretion (18). When puromycin was used to synchronize translation of McA cells, the accumulation of newly synthesized, full-length apoB100 was dramatically reduced in cells preincubated with 1.2 mM OA for 16 hours (Figure 7 top right, third lane for each time point) compared with cells incubated with either no OA or with 0.4 mM OA for 16 hours. In contrast, pretreatment of the cells for 16 hours with 0.4 mM OA moderately increased the appearance of full-length apoB100 (second lane for each time point) compared with cells incubated without OA. Importantly, cotreatment of the cells with lactacystin, a specific inhibitor of proteasomal degradation (30), substantially, although not completely, corrected the abnormality in the appearance of apoB100 seen with 1.2 mM OA. (Figure 7, bottom right). There appears to have been a similar, but much less striking, abnormality in the appearance of apoB48 that was also reversed by lactacystin treatment. There was no effect of OA on the rate of accumulation of new synthesized albumin (data not shown).
Prolonged incubation of McA cells with high-dose OA reduces the accumulation of full-length apoB100; this is reversed by cotreatment with lactacystin. McA cells were incubated for 16 hours with serum-free DMEM containing 1.5% BSA (lane 1), 1.5% BSA plus 0.4 mM OA (lane 2), 1.5% BSA plus 1.2 mM OA (lane 3); this was followed by an additional 10 minutes incubation, under the same 3 conditions, without (top left) or with 100 μM puromycin. The puromycin-treated cells were also incubated in the absence (top right) or presence (bottom right) of 10 μM lactacystin (Lacta). The cells were then put on ice and the puromycin removed. The cells were transferred to a 37°C water bath and labeled with serum-free, methionine/cysteine-free DMEM containing [35S]methionine for 15, 20, or 25 minutes. (The 2 latter steps were also performed with or without lactacystin.) 1.2 mM OA inhibited the accumulation of full-length apoB100; lactacystin treatment (bottom right) significantly increased the accumulation of full-length apoB100 after incubation with 1.2 mM OA for 16 hours (compare with top right). The data shown are representative of 2 experiments.
ER stress also induces nonproteasomal degradation of apoB100. In addition to cotranslational proteasomal degradation, there are a number of posttranslational pathways for degradation of apoB (18, 31). To further characterize the effects of FA-induced ER stress on intracellular degradation of apoB, pulse-chase studies were performed after preincubation of McA cells in the presence or absence of OA for 16 hours (Figure 8). The quantity of radiolabeled protein in the cell lysates at the 15-minute chase time point was taken as 100% of newly synthesized apoB100, apoB48, or albumin. Relative to incubation in the absence of OA, preincubation with 0.4 mM OA caused increased apoB100 secretion (Figure 8A, bottom left) in association with decreased intracellular disappearance (Figure 8A, middle) and greater total recovery (Figure 8A, right) of newly synthesized [35S]methionine-labeled apoB100 over a 90-minute pulse-chase protocol. This OA-mediated increase in apoB100 secretion and the rescue of apoB100 from intracellular degradation were lost after incubation with 0.8 mM OA. Furthermore, preincubation with 1.2 mM OA for 16 hours resulted in decreased apoB100 secretion associated with increased disappearance of intracellular apoB100. In contrast, there was no significant effect of preincubation of McA cells with as much as 1.2 mM OA on either the secretion or intracellular degradation of apoB48 (Figure 8B) or albumin (Figure 8C). In view of the results with lactacystin in puromycin-synchronized cells, we next preincubated McA cells with varying amounts of OA for 16 hours and then radiolabeled the cells for 2 hours, with all incubations performed in the absence and presence of the specific proteasome inhibitor lactacystin. Surprisingly, inhibition of proteasomal degradation in this prolonged labeling study had no effect on ER stress–mediated inhibition of apoB100 secretion (Figure 8D). Overall, the results presented in Figures 7 and 8 indicate that the parabolic relationship between FA-induced ER stress and apoB100 secretion is associated with both proteasomal and nonproteasomal degradation of apoB100. The latter pathway appears, based on these experiments, to be the dominant one.
Incubation of McA cells with OA results in dose-related increases in intracellular degradation of apoB100 by nonproteasomal pathways. McA cells were incubated in the presence or absence of OA for 16 hours; cells were pulse labeled with [35S]methionine for 20 minutes and chased for 90 minutes. Samples were taken every 15 minutes and processed by immunoprecipitation and 4% SDS-PAGE. The quantity of protein in the cell lysates at the 15-minute chase time point was taken as 100% of newly synthesized apoB100 (A), apoB48 (B), or albumin (C). Data are mean ± SD normalized to cells incubated without OA. *P < 0.05 vs. control; n = 3 for each condition. Incubations with increasing concentrations of OA were associated with a parabolic effect on intracellular degradation and secretion of apoB100; there were no effects of increasing OA on degradation or secretion of apoB48 or albumin. (D). McA cells were incubated for 16 hours with no OA, 0.4 mM OA, or 1.2 mM OA in the absence or presence of lactacystin and then radiolabeled for 2 hours Lactacystin did not alter the parabolic effect of increasing concentrations of OA on apoB100 secretion.
As noted above, there are several posttranslational pathways for apoB degradation in hepatocytes, including a recently described pathway that is stimulated by increased availability of polyunsaturated FAs. This latter degradation is inhibited by vitamin E or desferrioxamine (32). We cotreated McA cells with either vitamin E or desferrioxamine during incubations with varying concentrations of OA for 16 hours. Neither treatment affected the parabolic relationship between OA and apoB secretion, suggesting that lipid peroxidation and/or reactive oxygen species were not playing roles in the ER stress–induced degradation of apoB100 (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI32752DS1).
4-Phenyl butyric acid inhibits lipid-induced ER stress and reverses the inhibition of apoB100 secretion by high concentrations of OA. Based on the data to this point, we wished to determine apoB100 secretion when lipid loading was dissociated from ER stress. 4-Phenyl butyric acid (PBA) is a low-molecular-weight chaperone known to stabilize protein conformation and improve ER folding capacity (33, 34). Treatment of McA cells with PBA suppressed OA-induced increases of GRP78 expression as well as the phosphorylation of eIF2α (Figure 9A). Importantly, PBA reduced lipid-induced ER stress without affecting the rise in cellular TG content after incubations with OA (Figure 9B). PBA treatment reversed the parabolic effects of OA on apoB100 secretion. Thus, secretion of apoB100 doubled after incubations of McA cells with 0.4 mM OA for 16 hours compared with cells incubated without OA; similar increases were seen after incubations with 0.8 mM and 1.2 mM OA for 16 hours (Figure 9C). These results indicated that PBA prevented the inhibition of apoB100 secretion previously observed in association with OA-induced ER stress, without changing TG content in vitro. Furthermore, under these experimental conditions, the effects of PBA were selective for apoB100: there were no significant effects of PBA on apoB48, apoA-I, or albumin secretion (data not shown). Of note, pretreatment of McA cells with PBA reversed the abnormality in the appearance of full-length apoB100 seen with 1.2 mM OA (Figure 9D).
Treatment of McA cells with PBA inhibits ER stress and restores OA stimulation of apoB100 secretion. McA cells were incubated with or without PBA (1 mM) for 6 hours and then with or without PBA plus 0–1.2 mM OA for an additional 16 hours. (A) Incubations with OA increased GRP78 protein levels and the levels of phosphorylated eIF2α, as analyzed by immunoblot; activation of these markers of ER stress was completely blocked by PBA. Lanes were run on the same gel but were noncontiguous. (B) Under these same conditions, coincubation of OA with PBA did not alter the ability of OA to increase cell TG content. (C) McA cells were incubated with or without PBA (1 mM) for 6 hours and were then incubated with or without PBA plus 0–1.2 mM OA for 16 hours, followed by methionine/cysteine-free DMEM for 2 hours and then [35S]methionine for 2 hours Increasing OA concentrations caused a rise and then a fall in apoB100 secretion (left); PBA treatment was associated with similar increases in apoB100 at all doses of OA (right). Data are mean ± SD; n = 3 per group. *P < 0.05 vs. incubations in the absence of both OA and PBA; †P < 0.05 vs. incubations with the same concentration of OA but without PBA. (D) McA cells were preincubated with serum-free DMEM with or without 1 mM PBA for 6 hours and then incubated with serum-free DMEM containing no OA, 0.4 mM OA, or 1.2 mM OA for 16 hours, followed by an additional 10-minute incubation, under the same 3 conditions, with 100 μM puromycin. After washing the cells free of puromycin while on ice, they were radiolabeled with [35S]methionine/cysteine in the DMEM for 15, 20, 25 minutes. PBA treatment (right) reversed the delayed appearance of full-length apoB100 translation seen after incubation with 1.2 mM OA for 16 hours (left; also Figure 7, top right).
Tunicamycin-induced ER stress inhibits apoB100 secretion. Tunicamycin, an inhibitor of glycosylation, is widely used to induce ER stress. In order to determine whether ER stress induced in the absence of increased delivery of FAs also affected apoB secretion, we treated McA cells with tunicamycin. Supplemental Figure 2A shows that tunicamycin, as expected, caused a dose-dependent rise in GRP78 and phospho-eIF2α levels. These effects of tunicamycin were significantly inhibited by cotreatment with PBA. Concomitantly, tunicamycin dose-dependently inhibited apoB100 secretion (Supplemental Figure 2B), and this was also reversed by cotreatment with PBA at all except the highest dose of tunicamycin; PBA only partially reversed the effects of 5.0 μg/ml tunicamycin. Importantly, 0.2 μg/ml and 1.0 μg/ml of tunicamycin, which significantly inhibited apoB100 secretion, did not have any effect on secretion of apoB48, albumin, or apoA-I (Supplemental Figure 2C). However, 5.0 μg/ml of tunicamycin inhibited secretion of all of these proteins, an effect that was reversed by PBA. Finally, total TCA-precipitable radioactivity was unaffected by 0.2 or 1.0 μg/ml of tunicamycin but was reduced about 30% by the 5.0-μg/ml dose. The latter effect was reversed by PBA. Trypan blue staining was normal at all doses of tunicamycin (data not shown).
Intravenous infusions of 6 mM OA for 6 and 9 hours increase hepatic markers of ER stress. To assess the relationship between FA-induced ER stress and apoB secretion in vivo, we infused 6 mM OA bound to albumin for either 6 or 9 hours into C57BL/6J mice to increase the amount of FAs delivered to the liver. OA infusions raised plasma FA levels to 0.87 ± 0.33 mmol/l and to 1.20 ± 0.75 mmol/l after 6 and 9 hours, respectively. These levels were significantly greater (P < 0.001) than those present during saline infusions (0.39 ± 0.12 mmol/l and 0.29 ± 0.09 mmol/l, respectively). At the end of 6- and 9-hour OA infusions, liver TG levels tended to be higher compared with saline infusions, but there were no significant differences observed (6-hour: 187 ± 64 vs. 138 ± 46 μg TG/mg protein; 9-hour: 275 ± 169 vs. 192 ± 82 μg TG/mg protein; both NS) (Figure 10A).
Intravenous infusions of 6 mM OA increase markers of hepatic ER stress in normal mice. C57BL/6J mice were infused intravenously with saline (white bars) or 6 mM OA bound to albumin (gray bars) for 6 hours (n = 9 and 11 mice, respectively) or 9 hours (n = 4 mice/group). (A) At the end of 6- or 9-hour infusions, mice infused with 6 mM OA and saline were sacrificed, and the livers were collected for the measurement of liver TG content. The mean values of liver TG content are expressed as micrograms of TG per milligram of liver total protein. After either 6- or 9-hour OA infusions, liver TG content tended to be higher compared with the saline infusion, but no significant differences were observed. Data are mean ± SD. (B) The level of ER stress marker GRP78 was significantly increased by infusion of OA for either 6 hours or 9 hours compared with the levels present after infusion of saline. Lanes were run on the same gel but were noncontiguous. (C) Phosphorylation of eIF2α was significantly increased by infusion of OA for either 6 or 9 hours compared with levels present after infusion of saline. (D) Either 6- or 9-hour infusion of 6 mM OA induced partial XBP1 mRNA splicing, indicative of the presence of ER stress in OA-infused compared with saline-infused mice. Lanes were run on the same gel but were noncontiguous. Data for B–D are mean ± SD normalized to saline-infused mice. *P < 0.05, **P < 0.01 versus saline.
We next determined the levels of ER stress markers in the liver after 6- and 9-hour infusions of OA. After infusion of 6 mM OA for 6 hours, GRP78 mRNA expression tended to increase compared with saline infusion (151% ± 85% vs. 100% ± 30%; NS); after 9 hours, however, we observed a significant, 1.8-fold increase in GRP78 mRNA expression (180% ± 52% vs. 100% ± 26%; P < 0.05). After 6-hour infusions of OA, GRP78 protein (Figure 10B) increased about 2-fold compared with saline infusions (192% ± 104% vs. 100% ± 17%; P < 0.05); GRP78 protein increased by almost 3-fold after 9-hour infusions (288% ± 126% vs. 100% ± 49%; P < 0.01). Compared with saline infusion, levels of phospho-eIF2α increased about 2- to 2.5-fold after both 6-hour (209% ± 73% vs. 100% ± 51%; P < 0.01) and 9-hour infusions of OA (231% ± 83% vs. 100% ± 68%; P < 0.05) (Figure 10C). Activation of XBP1 was determined by measuring the degree of processing of its mRNA (28, 29). XBP1 processing was increased at the end of both 6-hour (123% ± 11% vs. saline; P < 0.05) and 9-hour (152% ± 31% vs. saline; P < 0.01) infusions, but the degree of processing tended to be greater after 9 hours (Figure 10D).
Intravenous infusions of 6 mM OA for 6 hours increases apoB secretion, but this effect is lost after 9-hour infusions. We previously reported that intravenous infusions of OA led to increased secretion of apoB without changes in TG secretion (35). In the present studies, we confirmed that finding: although (as noted above) there were significant elevations of plasma FAs by infusion of OA for either 6 or 9 hours, there were no increases in TG secretion after either infusion relative to infusions with saline (data not shown). We also confirmed our prior result that 6-hour infusions of OA increased secretion of newly synthesized apoB100 and apoB48 compared with saline infusion (185% ± 99% vs. 100% ± 19% and 231% ± 78% vs. 100% ± 32%, respectively; both P < 0.05) (Figure 11A). By contrast, the stimulation of apoB secretion by 6-hour infusions of OA was not seen after 9-hour infusions of OA (Figure 11B). Indeed, secretion of newly synthesized apoB100 and apoB48 was essentially unchanged after 9-hour infusions of OA compared with saline. Based on our in vitro results and published data (34), we next pretreated mice for 7 days with PBA before infusing OA for 9 hours. PBA treatment was associated with significantly less OA-mediated ER stress, as indicated by reduced responses of GRP78 and phospho-eIF2α (data not shown). PBA treatment was also associated with increased secretion of apoB100; PBA-treated mice secreted twice as much apoB100 in response to OA as mice not treated with PBA (203% ± 36% vs. 116% ± 43%, respectively; P < 0.01) (Figure 11C). We did not see any effect of PBA on secretion of apoB48 (132% ± 39% vs. 117% ± 40%, PBA-treated mice vs. non-treated mice, respectively). In these experiments, secretion of apoB100 and apoB48 in non–PBA-treated, saline-infused mice, was set at 100% (Figure 11C).
Stimulation of the secretion of apoB100 by 6-hour infusions of 6 mM OA is lost after infusion of OA for 9 hours but rescued after pretreatment of mice with PBA. C57BL/6J mice were infused with saline (white bars) or 6 mM OA in albumin (gray bars) intravenously for 6 or 9 hours, and both Triton WR1339 and [35S]methionine were injected into the mice at the end of each infusion to measure the secretion of newly synthesized apoB48 and apoB100. (A) Infusion of OA for 6 hours significantly increased the secretion of both apoB100 and apoB48 secretion into the bloodstream 1 hour after Triton injection compared with saline. (B) Infusion of OA for 9 hours did not increase either apoB100 or apoB48 secretion compared with saline. (C) PBA or water was administered orally to C57BL/6J mice for 7 days. After 9 hours of OA infusion, the PBA-treated mice showed an increase in apoB100 secretion compared with the nontreated mice. Data are mean ± SD normalized to saline-infused livers (for 6-hour infusions: n = 9 and 11 for saline and OA, respectively; for 9-hour infusions: n = 4/group; for 9-hour infusion with or without PBA: n = 6/group). *P < 0.05 versus saline; **P < 0.01 versus without PBA treatment.
Intravenous infusions of 20% Intralipid for 9 hours increases hepatic TG but does not stimulate either apoB or TG secretion. The quantity of FAs delivered to the liver during either 6- or 9-hour infusions of OA is very small (about 1.5 mg/6 hours and 2.3 mg/9 hours, respectively; ref. 35). Therefore, we conducted experiments in which 20% Intralipid was infused for 9 hours. In previous studies (35), infusions of 20% Intralipid for 6 hours (which delivered about 45 mg FAs to the liver) did not increase hepatic TG mass significantly but did stimulate secretion of both TGs and apoB by about 100% (35). The results (Supplemental Figure 3) show that a 9-hour infusion of 20% Intralipid (which would deliver about 60 mg FAs to the liver) increased hepatic TG mass by approximately 70% (Supplemental Figure 3A) but did not increase the secretion of TGs (Supplemental Figure 3B) or either apoB100 or apoB48 (Supplemental Figure 3C).