Impact of serine protease inhibitor alpha1-antitrypsin on expression of endoplasmic reticulum stress-induced proinflammatory factors in adipocytes

Abstract

Obesity-induced endoplasmic reticulum (ER) stress contributes to low-grade chronic inflammation in adipose tissue and may cause metabolic disorders such as diabetes mellitus and dyslipidemia. Identification of high serpina A1 (alpha-1 antitrypsin, A1AT) expression in mouse adipose tissue and adipocytes prompted us to explore the role of A1AT in the inflammatory response of adipocytes under ER stress. We aimed to determine the role of A1AT expression in adipocytes with ER stress during regulation of adipocyte homeostasis and inflammation. To this end, we chemically induced ER stress in A1AT small interfering RNA-transfected differentiating adipocytes using thapsigargin. Induction of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker, by thapsigargin was lower in A1AT-deficient SW872 adipocytes. Thapsigargin or the proinflammatory cytokine tumor necrosis factor (TNF)α increased basal expression of cytokines such as interleukin (IL)-1β and IL-8 in both SW872 and primary omental adipocytes. This thapsigargin- or TNFα-induced expression of proinflammatory genes was increased by A1AT deficiency. These findings indicate that adipose A1AT may suppress the ER stress response to block excessive expression of proinflammatory factors, which suggests that A1AT protects against adipose tissue dysfunction associated with ER stress activation.

Keywords: Adipocyte; Alpha-1 antitrypsin; Endoplasmic reticulum stress; Proinflammatory factor.

Fig. 1

Effect of the differentiation media on lipid accumulation in adipocyte SW872. SW872 cells (2×10⁴ cells) were cultured for 4, 7, or 10 days in normal media or differentiation media. Accumulation of lipid droplets during adipocyte differentiation was visualized with Oil-Red O staining. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Effects of alpha-1 antitrypsin (A1AT) knockdown on expression of ER stress markers, Bip, and CHOP in SW872 cells. SW872 cells (2×10⁴ cells) were transfected for 24 h with A1AT siRNA (20 pmol) and then treated for 24 h with thapsigargin (Thap: 10 or 100 nM). The protein levels of glucose- regulated protein 78 (Bip), CCAAT-enhancer-binding protein homologous protein (CHOP), and alpha-1 antitrypsin (A1AT) were measured by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Representative results (A) and densitometric analyses of n = 3 independent experiments (B) are shown.

Fig. 3

Effect of alpha-1 antitrypsin (A1AT) knockdown on expression of ER stressor-induced proinflammatory factors in SW872 cells. SW872 cells (2×10⁴) were transfected for 24 h with A1AT siRNA (20 pmol) and then treated for 24 h with thapsigargin (Thap: 10 or 100 nM). Expression of mRNA encoding proinflammatory factors IL1B, IL6, IL8, and NLRP3 was quantified by real-time RT-PCR (A). A1AT-deficient SW872 cells were stimulated for 6 h, 12 h, or 24 h with Thap (100 nM) (B). The culture medium was collected and the concentration of IL-6 was measured by ELISA. Three independent experiments were performed. Results are expressed as the mean ± S.E.M. and compared using the Tuker-Kramer test. *p<0.05, **p<0.01 vs. Thap (0) in control siRNA (−)-transfected cells; ^#p<0.05, ^##p<0.001 vs. control siRNA-transfected cells.

Fig. 4

Effect of alpha-1 antitrypsin (A1AT) knockdown on TNFα-induced proinflammatory factor expression in SW872 cells. SW872 cells (2×10⁴ cells) were transfected for 24 h with A1AT siRNA (20 pmol) and then treated for 24 h with TNFα (100 ng/ml). Expression of mRNA expression encoding proinflammatory factors IL1B, IL6, CXCL8, and NLRP3 was quantified by real-time RT-PCR (A). A1AT-deficient SW872 cells were treated for 6 h, 12 h, and 24 h with TNFα (100 ng/ml) (B). The culture medium was collected and the concentration of IL-6 was measured by ELISA. Three independent experiments were performed. Results are expressed as the mean ± S.E.M. *p<0.05, **p<0.01 vs. TNFα (0) in control siRNA (−)-transfected cells; ^#p<0.05, ^##p<0.001 vs. control siRNA-transfected cells.

Fig. 5

Effect of alpha-1 antitrypsin (A1AT) knockdown on expression of ER stressor- and TNFα-induced proinflammatory factors in primary omental adipocytes. Omental adipocytes (2×10⁴ cells) were transfected for 24 h with A1AT siRNA and then treated for 24 h with thapsigargin (Thap: 10 or 100 nM) (A, B) and TNFα (100 ng/ml) (C). A, C: Expression of mRNA encoding IL1B, IL6, IL8, NLRP3, and PTGS2 was quantified by real-time RT-PCR. B: The concentration of IL-6 in the culture medium was measured by ELISA. Three independent experiments were performed. Results are expressed as the mean ± S.E.M. *p<0.05, **p<0.01 vs. Thap (0) or TNFα (0) in control siRNA-transfected cells; ^#p<0.05, ^##p<0.001 vs. control siRNA-transfected cells.