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Palmitate induces endoplasmic reticulum stress and autophagy in mature adipocytes: implications for apoptosis and inflammation.

Yin J, Wang Y, Gu L, Fan N, Ma Y, Peng Y - Int. J. Mol. Med. (2015)

Bottom Line: In conclusion, our data indicate that PA elicits a ER stress-JNK-autophagy axis, and that this confers a pro-survival effect against PA-induced cell death and stress in hypertrophied adipocytes.The JNK-dependent activation of autophagy diminishes PA-induced inflammation.Therefore, the stimulation of autophagy may become a method with which to attenuate adipocyte dysfunction and inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Shanghai First People's Hospital, Shanghai Jiao Tong University, Shanghai 200080, P.R. China.

ABSTRACT
Endoplasmic reticulum (ER) stress and inflammation induced by obesity lead to adipocyte dysfunction, with the impairment of the insulin pathway. Recent studies have indicated that understanding the physiological role of autophagy is of great significance. In the present study, an in vitro model was used in which 3T3-L1 adipocytes were pre-loaded with palmitate (PA) to generate artificially hypertrophied mature adipocytes. PA induced an autophagic flux, determined by an increased microtubule-associated protein 1 light chain 3 (LC3)-II formation, as shown by western blot analysis and fluorescence microscopy, and was confirmed using transmission electron microscopy (TEM). Using TEM and western blot analysis, we observed increased ER stress in response to PA, as indicated by the increased levels of the ER stress markers, BiP, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and the phosphoralytion of eukaryotic translation initiation factor 2α and c-Jun N-terminal kinase (JNK). Of note, we observed that the PA-induced ER stress occurred prior to the activation of autophagy. We confirmed that autophagy was induced in response to JNK-dependent ER stress, as autophagy was suppressed by treatment with the ER stress inhibitor, 4-phenyl butyrate (4-PBA), and the JNK inhibitor, SP600125. Upon the inhibition of autophagy using chloroquine (CQ), we observed exacerbated ER stress and an increased level of cell death. Importantly, to determine whether autophagy is linked to inflammation, the autophagy inhibitor, 3-methyladenine (3-MA) was used. The inhibition of autophagy led to a further increase in the PA-induced expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). Consistently, such an increase was also observed following treatment with SP600125. In conclusion, our data indicate that PA elicits a ER stress-JNK-autophagy axis, and that this confers a pro-survival effect against PA-induced cell death and stress in hypertrophied adipocytes. The JNK-dependent activation of autophagy diminishes PA-induced inflammation. Therefore, the stimulation of autophagy may become a method with which to attenuate adipocyte dysfunction and inflammation.

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Autophagy limits palmitate (PA)-induced inflammatory cytokine expression. Mature 3T3-L1 adipocytes were pre-treated with or without 3-methyladenine (3-MA) (10 mM) or SP600125 [c-Jun N-terminal kinase (JNK) inhibitor, 10 μM] for 1 h, followed by treatment with PA (0.5 mM) for 12 h. (A-D) mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were analyzed by qPCR. (E and F) Western blot analysis and quantification of protein expression was performed using LC3 antibodies. Data are presented as the means ± SEM of 3 to 5 separate experiments. *P<0.05 vs. control group (0.5% BSA); #P<0.05 vs. PA-treated group.
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f6-ijmm-35-04-0932: Autophagy limits palmitate (PA)-induced inflammatory cytokine expression. Mature 3T3-L1 adipocytes were pre-treated with or without 3-methyladenine (3-MA) (10 mM) or SP600125 [c-Jun N-terminal kinase (JNK) inhibitor, 10 μM] for 1 h, followed by treatment with PA (0.5 mM) for 12 h. (A-D) mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were analyzed by qPCR. (E and F) Western blot analysis and quantification of protein expression was performed using LC3 antibodies. Data are presented as the means ± SEM of 3 to 5 separate experiments. *P<0.05 vs. control group (0.5% BSA); #P<0.05 vs. PA-treated group.

Mentions: Pre-loading of the cells with 0.5 mM/l of the saturated fatty acid, PA, for 12 h resulted in an increase in the mRNA expression levels of the inflammatory cytokines, IL-6 and MCP-1, in the mature adipocytes (Fig. 6A and C). In order to determine the role of autophagy in PA-induced inflammatory cytokine expression, we pre-treated the adipocytes with the autophagy inhibitor, 3-MA, for 1 h followed by treatment with PA for 12 h. 3-MA effectively inhibited the induction of autophagy by PA, as evidenced by the decrease in LC3-II accumulation (Fig. 6E). RT-qPCR revealed that treatment with 3-MA further increased the mRNA expression levels of MCP-1 and IL-6 compared to treatment with PA alone (Fig. 6A and C). Since the induction of autophagy by ER stress is in part mediated by JNK activation (20), we then examined the role of JNK in PA-induced autophagy in mature adipocytes using the specific JNK inhibitor, SP600125. As anticipated, the enhanced accumulation of LC3-II was decreased by pre-treatment with SP600125 compared to treatment with PA alone (Fig. 6F). Importantly, the expression of IL-6 and MCP-1 was further increased by SP600125 in the PA-treated adipocytes (Fig. 6B and D). These results demonstrate that PA-induced autophagy is partially JNK-dependent and that the activation of JNK-dependent autophagy plays a role in limiting inflammatory cytokine expression.


Palmitate induces endoplasmic reticulum stress and autophagy in mature adipocytes: implications for apoptosis and inflammation.

Yin J, Wang Y, Gu L, Fan N, Ma Y, Peng Y - Int. J. Mol. Med. (2015)

Autophagy limits palmitate (PA)-induced inflammatory cytokine expression. Mature 3T3-L1 adipocytes were pre-treated with or without 3-methyladenine (3-MA) (10 mM) or SP600125 [c-Jun N-terminal kinase (JNK) inhibitor, 10 μM] for 1 h, followed by treatment with PA (0.5 mM) for 12 h. (A-D) mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were analyzed by qPCR. (E and F) Western blot analysis and quantification of protein expression was performed using LC3 antibodies. Data are presented as the means ± SEM of 3 to 5 separate experiments. *P<0.05 vs. control group (0.5% BSA); #P<0.05 vs. PA-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356450&req=5

f6-ijmm-35-04-0932: Autophagy limits palmitate (PA)-induced inflammatory cytokine expression. Mature 3T3-L1 adipocytes were pre-treated with or without 3-methyladenine (3-MA) (10 mM) or SP600125 [c-Jun N-terminal kinase (JNK) inhibitor, 10 μM] for 1 h, followed by treatment with PA (0.5 mM) for 12 h. (A-D) mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were analyzed by qPCR. (E and F) Western blot analysis and quantification of protein expression was performed using LC3 antibodies. Data are presented as the means ± SEM of 3 to 5 separate experiments. *P<0.05 vs. control group (0.5% BSA); #P<0.05 vs. PA-treated group.
Mentions: Pre-loading of the cells with 0.5 mM/l of the saturated fatty acid, PA, for 12 h resulted in an increase in the mRNA expression levels of the inflammatory cytokines, IL-6 and MCP-1, in the mature adipocytes (Fig. 6A and C). In order to determine the role of autophagy in PA-induced inflammatory cytokine expression, we pre-treated the adipocytes with the autophagy inhibitor, 3-MA, for 1 h followed by treatment with PA for 12 h. 3-MA effectively inhibited the induction of autophagy by PA, as evidenced by the decrease in LC3-II accumulation (Fig. 6E). RT-qPCR revealed that treatment with 3-MA further increased the mRNA expression levels of MCP-1 and IL-6 compared to treatment with PA alone (Fig. 6A and C). Since the induction of autophagy by ER stress is in part mediated by JNK activation (20), we then examined the role of JNK in PA-induced autophagy in mature adipocytes using the specific JNK inhibitor, SP600125. As anticipated, the enhanced accumulation of LC3-II was decreased by pre-treatment with SP600125 compared to treatment with PA alone (Fig. 6F). Importantly, the expression of IL-6 and MCP-1 was further increased by SP600125 in the PA-treated adipocytes (Fig. 6B and D). These results demonstrate that PA-induced autophagy is partially JNK-dependent and that the activation of JNK-dependent autophagy plays a role in limiting inflammatory cytokine expression.

Bottom Line: In conclusion, our data indicate that PA elicits a ER stress-JNK-autophagy axis, and that this confers a pro-survival effect against PA-induced cell death and stress in hypertrophied adipocytes.The JNK-dependent activation of autophagy diminishes PA-induced inflammation.Therefore, the stimulation of autophagy may become a method with which to attenuate adipocyte dysfunction and inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Shanghai First People's Hospital, Shanghai Jiao Tong University, Shanghai 200080, P.R. China.

ABSTRACT
Endoplasmic reticulum (ER) stress and inflammation induced by obesity lead to adipocyte dysfunction, with the impairment of the insulin pathway. Recent studies have indicated that understanding the physiological role of autophagy is of great significance. In the present study, an in vitro model was used in which 3T3-L1 adipocytes were pre-loaded with palmitate (PA) to generate artificially hypertrophied mature adipocytes. PA induced an autophagic flux, determined by an increased microtubule-associated protein 1 light chain 3 (LC3)-II formation, as shown by western blot analysis and fluorescence microscopy, and was confirmed using transmission electron microscopy (TEM). Using TEM and western blot analysis, we observed increased ER stress in response to PA, as indicated by the increased levels of the ER stress markers, BiP, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and the phosphoralytion of eukaryotic translation initiation factor 2α and c-Jun N-terminal kinase (JNK). Of note, we observed that the PA-induced ER stress occurred prior to the activation of autophagy. We confirmed that autophagy was induced in response to JNK-dependent ER stress, as autophagy was suppressed by treatment with the ER stress inhibitor, 4-phenyl butyrate (4-PBA), and the JNK inhibitor, SP600125. Upon the inhibition of autophagy using chloroquine (CQ), we observed exacerbated ER stress and an increased level of cell death. Importantly, to determine whether autophagy is linked to inflammation, the autophagy inhibitor, 3-methyladenine (3-MA) was used. The inhibition of autophagy led to a further increase in the PA-induced expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). Consistently, such an increase was also observed following treatment with SP600125. In conclusion, our data indicate that PA elicits a ER stress-JNK-autophagy axis, and that this confers a pro-survival effect against PA-induced cell death and stress in hypertrophied adipocytes. The JNK-dependent activation of autophagy diminishes PA-induced inflammation. Therefore, the stimulation of autophagy may become a method with which to attenuate adipocyte dysfunction and inflammation.

Show MeSH
Related in: MedlinePlus