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. 2015:2015:272313.
doi: 10.1155/2015/272313. Epub 2015 Jan 14.

Rapamycin improves palmitate-induced ER stress/NF κ B pathways associated with stimulating autophagy in adipocytes

Jiajing Yin  1 , Liping Gu  1 , Yufan Wang  1 , Nengguang Fan  1 , Yuhang Ma  1 , Yongde Peng  1
Affiliations

Rapamycin improves palmitate-induced ER stress/NF κ B pathways associated with stimulating autophagy in adipocytes

Jiajing Yin et al. Mediators Inflamm. 2015.

Abstract

Obesity-induced endoplasmic reticulum (ER) stress and inflammation lead to adipocytes dysfunction. Autophagy helps to adapt to cellular stress and involves in regulating innate inflammatory response. In present study, we examined the activity of rapamycin, a mTOR kinase inhibitor, against endoplasmic reticulum stress and inflammation in adipocytes. An in vitro model was used in which 3T3-L1 adipocytes were preloaded with palmitate (PA) to generate artificial hypertrophy mature adipocytes. Elevated autophagy flux and increased number of autophagosomes were observed in response to PA and rapamycin treatment. Rapamycin attenuated PA-induced PERK and IRE1-associated UPR pathways, evidenced by decreased protein levels of eIF2α phosphorylation, ATF4, CHOP, and JNK phosphorylation. Inhibiting autophagy with chloroquine (CQ) exacerbated these ER stress markers, indicating the role of autophagy in ameliorating ER stress. In addition, cotreatment of CQ abolished the anti-ER stress effects of rapamycin, which confirms the effect of rapamycin on ERs is autophagy-dependent. Furthermore, rapamycin decreased PA-induced nuclear translocation of NFκB P65 subunit, thereby NFκB-dependent inflammatory cytokines MCP-1 and IL-6 expression and secretion. In conclusion, rapamycin attenuated PA-induced ER stress/NFκB pathways to counterbalance adipocytes stress and inflammation. The beneficial of rapamycin in this context partly depends on autophagy. Stimulating autophagy may become a way to attenuate adipocytes dysfunction.

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Figures

Figure 1
Figure 1
ER stress is induced by palmitate and contributes to increase MCP-1 and IL-6 expression. (a) Mature 3T3-L1 adipocytes were treated with 0.5% BSA or BSA-conjugated PA (0.5 mM) for 12 h. mRNA of ATF4 and CHOP measured by qPCR. (b) Western blot of indicated ER stress markers with or without PA (0.5 or 1.0 mM) treatment. Representative blots and quantifications are shown. (c) mRNA levels of MCP-1 and IL-6 with or without the ER stressors tunicamycin (TM, 1 μg/mL) or thapsigargin (TG, 1 μM) measured by qPCR. Results are mean ± SEM of three to five separate experiments. * P < 0.05 versus nontreatment group (0.5% BSA).
Figure 2
Figure 2
Autophagy is activated in response to palmitate and rapamycin. (a) Adipocytes were treated with or without palmitate (0.5 or 1.0 mM) and rapamycin (50 or 100 nM) for 12 h, followed by LC3 Western blotting analysis. (b) Preincubated with or without CQ (10 μM) for 1 h and with BSA or BSA-conjugated PA for 12 h, followed by LC3 Western blotting analysis. (c) Electron microscopy and MDC staining of mature adipocytes expose to 0.5 mM PA or 100 nM RAP for 12 h. Lipid droplets (white arrow); autophagosomes and autolysosome of double-membrane, single-membrane, and multivesicular body-like vesicles (blue arrows); endoplasmic reticulum (red arrow); mitochondria (yellow arrow). Scale bars are indicated at bottom. (d) Pretreat with or without 4-PBA (10 mM) for 1 h and with PA (0.5 mM) for 12 h followed by LC3 Western blotting analysis. The proteins are quantified and normalized to GAPDH. Results are mean ± SEM of three to five separate experiments. * P < 0.05 versus nontreatment group (0.5% BSA); # P < 0.05 versus PA 0.5 mM group.
Figure 3
Figure 3
Rapamycin decreases PA-induced inflammatory cytokines and ER stress pathways. Mature 3T3-L1 adipocytes were pretreated with or without RAP (100 nM) for 1 h and with 0.5% BSA or BSA-conjugated PA (0.5 mM) for 12 h. (a, b) mRNA levels of MCP-1 and IL-6 were analyzed by qPCR. (c) Western blot and quantification for the protein expression with the indicated antibodies. (d) IL-6 secretion in mature 3T3-L1 adipocytes medium. Data are presented as mean ± SEM of three to five separate experiments. * P < 0.05 versus nontreatment group (0.5% BSA); # P < 0.05 versus PA 0.5 mM group.
Figure 4
Figure 4
Rapamycin decreases PA-induced ER stress via stimulating autophagy. Mature 3T3-L1 adipocytes were pretreated with or without CQ (10 μM) for 1 h and followed with PA (0.5 mM) and/or RAP (100 nM) for 12 h. Western blot and quantification for the protein expression with the indicated antibodies. Data are presented as mean ± SEM of three to five separate experiments. * P < 0.05 versus nontreatment group (0.5% BSA); # P < 0.05 versus PA 0.5 mM group; § P < 0.05 versus PA 0.5 mM with RAP 100 nM group.
Figure 5
Figure 5
Rapamycin inhibits PA-induced inflammatory gene expression via depressing NFκB pathways. (a) Mature 3T3-L1 adipocytes were pretreated with or without RAP (100 nM) and with 0.5% BSA or BSA-conjugated PA (0.5 mM) for 12 h. (a) Expression of nuclear and cytoplasmic p65 and p S6 are determined by Western blotting. Lamin/C and β-actin are included as loading controls. (b) Mature adipocytes were pretreatment with NKi (NF-κB inhibitor, BAY11-7082 10 μM) followed by PA treatment for 12 h. mRNA levels of cytokines and chemokines were determined by qPCR. Data are presented as mean ± SEM of three to five separate experiments. * P < 0.05 versus nontreatment group (0.5% BSA); # P < 0.05 versus PA 0.5 mM group.
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