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. 2012 Feb 24;287(9):6240-9.
doi: 10.1074/jbc.M111.299115. Epub 2012 Jan 5.

Lipolysis response to endoplasmic reticulum stress in adipose cells

Jingna Deng  1 , Shangxin Liu, Liangqiang Zou, Chong Xu, Bin Geng, Guoheng Xu
Affiliations

Lipolysis response to endoplasmic reticulum stress in adipose cells

Jingna Deng et al. J Biol Chem. .

Abstract

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.

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Figures

FIGURE 1.
FIGURE 1.
ER stress is induced by thapsigargin in rat adipocytes. Rat differentiated adipocytes were incubated for 0.5 to 8 h with or without 1 μm Thaps. Protein levels of GRP78, PERK, or its Thr-980 phosphorylation form, eIF2α, or its Ser-51 phosphorylation species, with actin as a loading control, were detected by immunoblotting analysis. The blots are representative of three separate experiments.
FIGURE 2.
FIGURE 2.
Lipolysis is activated in response to ER stress in rat adipose cells or tissues. Rat adipose cells or tissues were incubated in phenol red-free and serum-free DMEM. Glycerol content in the medium was assayed as an index of lipolysis. A, lipolysis responses to ER stress after rat differentiating adipocytes were incubated for 8 h with or without 1 μm Thaps, 5 μg/ml tunicamycin (Tun), or 0.5 μg/ml brefeldin A (BFA). Inset in A shows a positive stimulation of the lipolysis. After incubation with 1 μm Thaps or 100 nm Iso, an adrenergic lipolytic activator, 1-h glycerol release from the adipocytes, was assayed. B and C, time-dependent lipolysis in rat differentiated (B) and primary (C) adipocytes stimulated with 1 μm Thaps. D, ex vivo lipolysis. Minced rat epididymal adipose tissues were incubated with 1 μm Thaps for the indicated times and then 1-h glycerol release in freshly changed medium was determined. Lipolysis data are expressed as micromoles of glycerol/ml of packed cell volume (PCV) or milligram of cell proteins. Data are mean ± S.E. of at least three separate experiments performed in triplicate. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus control.
FIGURE 3.
FIGURE 3.
ER stress triggers lipolysis by elevating cellular cAMP and activating PKA. A, 125I radioimmunoassay of cellular cAMP level in rat differentiated adipocytes incubated with 1 μm Thaps for 1 and 8 h or with 1 μm Iso (as positive control) for 0.5 h. B, determination of PKA activity. Adipocytes were stimulated with 1 μm Thaps for the indicated times, and then whole cell lysates underwent immunoblot (IB) analysis with a primary antibody against PKA phosphosubstrate motif (RRX(S/T)). C, stimulation with 1 μm Thaps for 1 h or with 1 μm isoproterenol for 20 min promoted the level of PKA phosphosubstrates in adipocytes, which was attenuated by PKA inhibition with 20 μm H89. D, glycerol assay. Lipolysis in adipocytes stimulated with 1 μm Thaps for 8 h was inhibited by 1-h preincubation with 20 μm H89. The blots are representative of three separate experiments, and the glycerol data are mean ± S.E. of at least three separate experiments performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control; †††, p < 0.001 versus Thaps.
FIGURE 4.
FIGURE 4.
ERK1/2 activation accounts for lipolysis response to ER stress. A and B, immunoblot analysis of phosphorylation of Raf-1 and ERK1/2 in rat differentiated adipocytes incubated with or without 1 μm Thaps, 5 μg/ml tunicamycin (Tun), or 1 mm dithiothreitol (DTT). C, preincubation with 10 μm U0126 inhibited ERK1/2 phosphorylation induced by 1 μm Thaps or 50 ng/ml TNF-α as a test control. D–F, lipolysis assay. Adipocytes were preincubated with 10 μm U0126 (D), 50 μm PD98059 (E), or U0126 plus H89 each at 10 μm (F) and then stimulated for 8 h with 1 μm Thaps or 50 ng/ml TNF-α. Glycerol release in the medium was determined. Data are mean ± S.E. of three separate experiments performed in triplicate. **, p < 0.01 versus control; †, p < 0.05; ††, p < 0.01 versus Thaps. ***, p < 0.001 versus control.
FIGURE 5.
FIGURE 5.
JNK, p38 MAPK, and PKC do not regulate ER stress-stimulated lipolysis. A, immunoblot analysis of JNK phosphorylation in adipocytes stimulated with 1 μm Thaps. B–D, glycerol assay of rat differentiated adipocytes preincubated for 1 h with or without JNK inhibitor SP600125 at 12.5 μm (B), p38 inhibitor SB203580 at 10 μm (C), or PKC inhibitor Ro-31-8220 at 1 μm (D) and then stimulated with 1 μm Thaps for 8 h. Data are mean ± S.E. of three separate experiments in triplicate. ***, p < 0.001 versus control.
FIGURE 6.
FIGURE 6.
ER stress causes phosphorylation of perilipin. A, rat differentiated adipocytes were treated with 1 μm Thaps and then lysates underwent SDS-PAGE and immunoblot analysis with the anti-perilipin antibody. Electrophoresis was run for 7 h on a medium sized gel especially prepared with a low-bis concentration (10:0.07% acrylamide/bisacrylamide); these conditions allow for maximal resolution of perilipin proteins but may cause band distortion. The migration shift from 65 to 67 kDa indicates hyperphosphorylation of full-length perilipin A. The 46-kDa bands are the truncated perilipin B isoforms. B, top image, a control test for accessibility of phosphorylated perilipin with the anti-phospho-Ser/Thr antibody. The extracts of primary adipocytes stimulated with Iso were immunoprecipitated (IP) (with a monoclonal antibody against phospho-Ser/Thr PKA substrates), then immunoblotted (IB), and recognized by anti-perilipin antibodies. Bottom images, phosphorylation of perilipin A induced by Thaps was immunodetected with the anti-phospho-Ser/Thr antibody. Then the blot was stripped and reprobed with anti-perilipin antibodies to detect the native perilipins. C, adipocytes were stimulated with 1 μm Thaps, 5 μg/ml tunicamycin (Tun), or 1 mm DTT, and lysates underwent immunoblot analysis with antibodies against phospho-Ser/Thr or perilipin, respectively. D, H89 attenuated perilipin phosphorylation in adipocytes stimulated with 1 μm Thaps or Iso as a positive control for PKA activation. The blots represent data from three separate experiments. peri, perilipin; p-peri, phosphorylated perilipin.
FIGURE 7.
FIGURE 7.
Regulation of lipase activity in ER-stressed adipocytes. A, lipase activity was determined by measuring glycerol release from the hydrolysis of emulsified triolein substrates in the reaction containing extracts of primary adipocytes stimulated with 1 μm Thaps for 8 h. Stimulation with 1 μm Iso for 30 min served as a positive control. Data are mean ± S.E. of at least three separate experiments performed in triplicate. *, p < 0.05; **, p < 0.01 versus control. B, immunoblot analysis of protein levels of HSL and ATGL in differentiated adipocytes stimulated with 1 μm Thaps. C, HSL phosphorylation was detected with primary antibodies against the phospho-Ser-563 or -Ser-660 motif of HSL. D, immunofluorescence. Rat differentiated adipocytes were incubated with 1 μm Thaps for 8 h or with 1 μm Iso (as a positive control) for 30 min and then immunostained for HSL. The dark circles inside adipocytes indicate lipid droplets, and enhanced fluorescence at the lipid droplet surface indicates the translocation of HSL. Images are representative of three separate experiments.
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