12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response - PubMed (original) (raw)
12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response
Banumathi K Cole et al. Am J Physiol Endocrinol Metab. 2012.
Abstract
Central obesity is associated with chronic inflammation, insulin resistance, β-cell dysfunction, and endoplasmic reticulum (ER) stress. The 12/15-lipoxygenase enzyme (12/15-LO) promotes inflammation and insulin resistance in adipose and peripheral tissues. Given that obesity is associated with ER stress and 12/15-LO is expressed in adipose tissue, we determined whether 12/15-LO could mediate ER stress signals. Addition of 12/15-LO lipid products 12(S)-HETE and 12(S)-HPETE to differentiated 3T3-L1 adipocytes induced expression and activation of ER stress markers, including BiP, XBP-1, p-PERK, and p-IRE1α. The ER stress inducer, tunicamycin, upregulated ER stress markers in adipocytes with concomitant 12/15-LO activation. Addition of a 12/15-LO inhibitor, CDC, to tunicamycin-treated adipocytes attenuated the ER stress response. Furthermore, 12/15-LO-deficient adipocytes exhibited significantly decreased tunicamycin-induced ER stress. 12/15-LO action involves upregulation of interleukin-12 (IL-12) expression. Tunicamycin significantly upregulated IL-12p40 expression in adipocytes, and IL-12 addition increased ER stress gene expression; conversely, LSF, an IL-12 signaling inhibitor, and an IL-12p40-neutralizing antibody attenuated tunicamycin-induced ER stress. Isolated adipocytes and liver from 12/15-LO-deficient mice fed a high-fat diet revealed a decrease in spliced XBP-1 expression compared with wild-type C57BL/6 mice on a high-fat diet. Furthermore, pancreatic islets from 12/15-LO-deficient mice showed reduced high-fat diet-induced ER stress genes compared with wild-type mice. These data suggest that 12/15-LO activity participates in ER stress in adipocytes, pancreatic islets, and liver. Therefore, reduction of 12/15-LO activity or expression could provide a new therapeutic target to reduce ER stress and downstream inflammation linked to obesity.
Figures
Fig. 1.
12-Hydroxyeicosatetranoic acid [12(S)-HETE] and 12-hydroperoxyeicosatetraenoic acid [12(S)-HPETE] induce expression of endoplasmic reticulum (ER) stress markers. 3T3-L1 adipocytes were treated with 10 nM 12(S)-HETE, 1 nM 12(S)-HPETE, or ethanol (EtOH) solvent control for 24 h, and mRNA (A) and protein measurements (B and C) of ER stress markers were examined. The mRNA measurements were done by RT-PCR and protein measurements done by Western blot analysis. Representative Western blots are shown, and separate panels for each antibody represent the same exposure from the same gel (B and C). All data were normalized to total actin, and the fold changes in expression were calculated relative to EtOH control. All data represent means ± SE; n = 3. *P < 0.05, †P < 0.02, and #P < 0.001 vs. control. BiP, binding immunoglobulin protein; CHOP, CCAAT/enhancer-binding protein homologous protein; WFS1, Wolfram syndrome 1; XBP-1, X-box protein-1; P, phosphorylated; PERK, RNA-dependent protein kinase-like ER-regulated kinase; eIF2α, eukaryotic translation initiation factor 2α; ATF4 and -6, activating transcription factor 4 and 6, respectively; IRE1α, inositol-requiring enzyme 1α.
Fig. 2.
Inhibition of 12/15-lipoxygenase (12/15-LO) activity attenuates tunicamycin-induced ER stress and insulin resistance. 3T3-L1 adipocytes were treated with 5 μM tunicamycin or DMSO solvent control for 24 h (A_–_C) with or without a 2-h pretreatment and coincubation with 0.1 μM of the 12/15-LO inhibitor cynnamyl 1–3,4-dihydroxy-α-cyanocinnamate (CDC) or 10 mM of the chemical chaperone 4-phenylbutyric acid (PBA). mRNA (A) and protein measurements (B) of ER stress markers and 12/15-LO were examined. C: 12(S)-HETE was measured in medium cultured with treated 3T3-L1 adipocytes by ELISA. D: 3T3-L1 adipocytes were treated with 5 μM tunicamycin or DMSO solvent control for 4 h and stimulated with 10 nM insulin for 10 min, and protein measurements of key insulin signaling markers were examined. The mRNA measurements were done by RT-PCR and protein measurements done by Western blot analysis; the data were normalized to total actin, and the fold changes in expression were calculated relative to DMSO control. Western blots are shown, and separate panels for each antibody represent the same exposure from the same gel (B and D). All data represent means ± SE; n = 3. Statistics were performed comparing each treatment with control as well as tunicamycin with tunicamycin + CDC/PBA; only statistically significant results are shown. *P < 0.05, †P < 0.02, and #P < 0.001 vs. control unless otherwise indicated.
Fig. 3.
ER stress signals through IL-12 and is attenuated by the anti-inflammatory agent lysofylline (LSF). A_–_E: 3T3-L1 adipocytes were treated with 5 μM tunicamycin or DMSO solvent control for 24 h, pretreated for 2 h, and coincubated with the anti-inflammatory compound LSF (10 μM) or with 2 μg/ml IL-12p40 antibody (Ab). F: 3T3-L1 adipocytes were treated with 2.5 ng/ml mouse IL-12 cytokine for 24 h. mRNA (A, B, E, and F) and protein measurements (C) of IL-12p40, ER stress markers, and 12/15-LO were examined. IL-12p40 (A) and 12(S)-HETE (D) were measured in media cultured with treated 3T3-L1 adipocytes by ELISA. The mRNA measurements were done by RT-PCR and protein measurements done by Western blot analysis; the data were normalized to total actin, and the fold changes in expression were calculated relative to DMSO control. Representative Western blots are shown, and separate panels for each antibody represent the same exposure; however, samples for BiP and corresponding actin were run on different gels and clearly demarcated (C) (see
materials and methods
for quantitation). All data represent means ± SE; n = 3. Statistics were performed, comparing each treatment with control as well as tunicamycin with tunicamycin + LSF/Ab; only statistically significant results are shown. *P < 0.05, †P < 0.02, and #P < 0.001 vs. control unless otherwise indicated.
Fig. 4.
Effect of 12/15-LO and ER stress on adiponectin, CCAAT/enhancer-binding protein-α (C/EBPα), and ATF3 expression. 3T3-L1 adipocytes were treated with 5 μM tunicamycin (or DMSO solvent control) and/or 10 nM 12(S)-HETE (or EtOH solvent control) for 24 h with or without a 2-h pretreatment and coincubation with the 12/15-LO inhibitor CDC (0.1 μM). Protein (A) and mRNA measurements (B and C) were performed for genes indicated by Western blot analysis and RT-PCR, respectively. All data were normalized to tubulin (A) or actin (B and C), and fold changes in expression were calculated relative to indicated solvent control. Representative Western blots are shown, and separate panels for each antibody represent the same exposure; however, samples were run on different gels and clearly demarcated (see
materials and methods
for quantitation). Furthermore, tubulin samples are shown for both adiponectin and C/EBPα because the same sample is not displayed for all experimental conditions (A). All data represent means ± SE; n = 3. Statistics were performed, comparing each treatment with control as well as tunicamycin with tunicamycin + CDC and tunicamycin + CDC with tunicamycin + CDC + 12(S)-HETE; only statistically significant results are shown. *P < 0.05, †P < 0.02, and #P < 0.001 vs. control unless otherwise indicated.
Fig. 5.
12/15-LO deficiency protects mouse adipocytes, pancreatic islets, and liver from several aspects of the high-fat diet-induced ER stress response. Eight-week-old C57BL/6 (BL6) and 12/15-LO-deficient (12-LO KO) male mice were placed on a chow or high-fat diet (HFD) for 16 wk. Epididymal adipocytes (A), pancreatic islets (B), and liver (C) were isolated, and mRNA measurements by RT-PCR were performed for ER stress genes. All data were normalized to actin, and fold changes in expression were calculated relative to BL6-CHOW control. All data represent means ± SE; n = 5 mice/group. Statistics were performed, comparing each group with BL6-CHOW control as well as 12-LO KO-CHOW with 12-LO KO-HFD and BL6-HFD with 12-LO KO-HFD; only statistically significant results are shown. *P < 0.05, †P < 0.02, and #P < 0.001 vs. control unless otherwise indicated.
Fig. 6.
12/15-LO deficiency attenuates tunicamycin-induced ER stress in isolated adipocytes and pancreatic islets. Adipocytes and pancreatic islets were isolated from 8-wk-old BL6 and 12-LO KO male mice and treated with 5 μM tunicamycin or DMSO solvent control for 4 or 24 h. A_–_C: mRNA measurements of ER stress markers were examined and done by RT-PCR; the data were normalized to total actin. Each bar indicates the tunicamycin-induced fold change in expression relative to DMSO control of the same genotype. All data represent means ± SE; n = 4 mice/group. *P < 0.05, †P < 0.02, and #P < 0.001 vs. BL6 + tunicamycin control.
Fig. 7.
12/15-LO deficiency leads to decreased IL-12p40 expression. A: body weights of BL6 and 12-LO KO male mice fed a chow or HFD for 16 wk are shown. IL-12p40 mRNA expression was measured in isolated adipocytes and pancreatic islets from BL6 and 12-LO KO mice fed a HFD for 16 wk (B) or from isolated adipocytes and pancreatic islets from normal 8-wk-old BL6 and 12-LO KO mice that were treated with 5 μM tunicamycin or DMSO solvent control for 24 h (D); mRNA measurements were performed by RT-PCR, and the data were normalized to total actin. In B, fold changes in expression are calculated relative to BL6-HFD; in D, each bar indicates the tunicamycin-induced fold change in expression relative to DMSO control of the same genotype. C: IL-12p40 protein level was measured in serum from BL6 and 12-LO KO mice fed a chow diet or HFD for 16 wk by ELISA. All data represent means ± SE; n = 3–5. *P < 0.05 and †P < 0.02 vs. control unless otherwise indicated.
Fig. 8.
12/15-LO and ER stress. A schematic describing the proposed hypothetical complex dynamic interplay between arms of 12/15-LO activation, inflammation, and ER stress induction when cells are exposed to a pathological excess of nutrients. ER stress can activate 12/15-LO to generate 12(S)-HETE, and 12(S)-HETE can induce ER stress in an IL-12-dependent manner. Activation of each arm creates a vicious cycle that predisposes individuals to the development of insulin resistance (IR), type 2 diabetes (T2D), and cardiovascular disease (CVD).
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