DsbA-L alleviates endoplasmic reticulum stress-induced adiponectin downregulation

Abstract

Objective: Obesity impairs adiponectin expression, assembly, and secretion, yet the underlying mechanisms remain elusive. The aims of this study were 1) to determine the molecular mechanisms by which obesity impairs adiponectin multimerization and stability, and 2) to determine the potential role of disulfide-bond-A oxidoreductase-like protein (DsbA-L), a recently identified adiponectin interactive protein that promotes adiponectin multimerization and stability in obesity-induced endoplasmic reticulum (ER) stress and adiponectin downregulation.

Research design and methods: Tauroursodeoxycholic acid (TUDCA), a chemical chaperone that alleviates ER stress, was used to study the mechanism underlying obesity-induced adiponectin downregulation in db/db mice, high-fat diet-induced obese mice, and in ER-stressed 3T3-L1 adipocytes. The cellular levels of DsbA-L were altered by RNAi-mediated suppression or adenovirus-mediated overexpression. The protective role of DsbA-L in obesity- and ER stress-induced adiponectin downregulation was characterized.

Results: Treating db/db mice and diet-induced obese mice with TUDCA increased the cellular and serum levels of adiponectin. In addition, inducing ER stress is sufficient to downregulate adiponectin levels in 3T3-L1 adipocytes, which could be protected by treating cells with the autophagy inhibitor 3-methyladenine or by overexpression of DsbA-L.

Conclusions: ER stress plays a key role in obesity-induced adiponectin downregulation. In addition, DsbA-L facilitates adiponectin folding and assembly and provides a protective effect against ER stress-mediated adiponectin downregulation in obesity.

FIG. 1.

Suppression of ER stress improves adiponectin expression in db/db mice. A: The expression levels of adiponectin (Adpn), DsbA-L, and CHOP in white adipose tissue (WAT) and the serum adiponectin levels from saline- or TUDCA-treated lean or db/db mice were determined by Western blot with specific antibodies as indicated. β-actin or immunoglobulin (IgG) was used as a tissue or serum loading control, respectively. The relative protein levels of adiponectin in WAT (B), or in serum (C), and DsbA-L (D) were quantified. Data represent mean ± SEM. **P < 0.01, ***P < 0.001; n = 10 for the saline control mice, and n = 8 per treatment group for the db/db mice. E: Differentiated 3T3–L1 adipocytes were pretreated with or without TUDCA (1 mmol/l) for 1 h followed with TG (0.01 μmol/l) cotreatment for 24 h. The cells were lysed, and the expression levels of adiponectin, DsbA-L, CHOP, TNFα, resistin, and β-tubulin were determined by Western blot analysis with specific antibodies as indicated. Adiponectin levels in conditioned medium were determined with an antiadiponectin antibody. F: The mRNA levels of adiponectin were determined by quantitative reverse transcription PCR. The protein levels of adiponectin as shown in (E) were quantified (G). N = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns, no statistical difference; ctrl, control.

FIG. 2.

DsbA-L suppresses ER stress and improves adiponectin expression and multimerization. A: The expression levels of adiponectin, DsbA-L, and CHOP in DsbA-L–suppressed or scramble control 3T3–L1 adipocytes were determined by Western blot with specific antibodies. β-tubulin was used as a loading control. The data represent three independent experiments with similar results. B: The 3T3–L1-CAR adipocytes were infected with adenovirus encoding DsbA-L or GFP for 24 h. The cells were then treated with or without TG (0.01 μmol/l) for various times as indicated. The expression levels of adiponectin, CHOP, and myc-DsbA-L in cell lysates were determined by Western blot with specific antibodies as indicated. β-tubulin was used as a loading control. The data represent three independent experiments with similar results. C: The 3T3–L1-CAR adipocytes were infected with adenovirus encoding GFP, DsbA-L, or the S16A mutant of DsbA-L for 24 h. The cells were then treated with or without TG (0.01 μmol/l) for 24 h as indicated. Adiponectin multimers in cell lysates were separated by gel filtration and determined by Western blot with an antiadiponectin antibody. The percentages of HMW form in total adiponectin (D) were quantified (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). E: The 3T3–L1-CAR adipocytes were infected with adenoviruses encoding myc-tagged wild-type (WT) or the S16A mutant of DsbA-L for 24 h. The cells were then treated with or without TG (0.01 μmol/l) for 24 h. The adiponectin levels in conditioned medium (C.M.) and the expression levels of adiponectin, myc-DsbA-L or DsbA-L^S16A, and CHOP were determined by Western blot with specific antibodies as indicated. β-tubulin was used as a loading control. F: The mRNA levels of adiponectin were determined by quantitative reverse transcription PCR. N = 3, *P < 0.05; ns, no statistical difference.

FIG. 3.

ER stress or DsbA-L knockdown suppresses adiponectin levels by inducing autophagy-dependent degradation. A: DsbA-L-suppressed or scramble control 3T3–L1 adipocytes were treated with 3-MA (5 μmol/l) or vehicle for 24 h as indicated. The protein levels of adiponectin, DsbA-L, and LC3 in cell lysates were determined by Western blot using specific antibodies as indicated. β-tubulin was used as a loading control. Adiponectin levels in conditioned medium (C.M.) were determined with an antiadiponectin antibody. The data represent three independent experiments with similar results. B: The 3T3–L1 adipocytes were pretreated with (+) or without (−) 3-MA (5 μmol/l) for 1 h, followed by TG (0.01 μmol/l) for different times as indicated. The protein levels of adiponectin, LC3, and β-tubulin were determined by Western blot analysis with specific antibodies as indicated. Adiponectin levels in conditioned medium (C.M.) were determined with an antiadiponectin antibody. The mRNA levels of adiponectin (C) were determined by quantitative reverse transcription PCR; N = 3, *P < 0.05; ns, no statistical difference. D: Differentiated 3T3–L1 adipocytes were treated with TG (0.01 μmol/l) or vehicle for 36 h, and then fixed. The cellular localization of adiponectin (Adpn, red) was determined by immunofluorescence-staining with a specific antibody to the protein. The cellular localization of GFP-LC3 was observed by green fluorescent protein fluorescence. In each experiment, more than 100 cells from each group were checked, and ∼70% of the cells showed a similar pattern. The data represent three independent experiments with similar results. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Inhibition of ER stress improves adiponectin signaling in db/db mice. A: The expression levels and phosphorylation of AMPK (Thr¹⁷²) and ACC (Ser⁷⁹) in skeletal muscle of TUDCA- or saline-treated db/db mice were determined by Western blot with specific antibodies as indicated. The relative AMPK (B) and ACC (C) phosphorylation levels were quantified and analyzed (*P < 0.05, n = 4). D: The expression levels and phosphorylation of AMPK and ACC in the liver of TUDCA- or saline-treated db/db mice were determined by Western blot with specific antibodies as indicated. The relative AMPK (E) and ACC (F) phosphorylation levels were quantified and analyzed (*P < 0.05, ***P < 0.001, n = 4). G: 3T3–L1-CAR adipocytes were infected with adenoviruses encoding GFP or GFP plus myc-DsbA-L for 24 h. The cells were then pretreated with or without TG (0.01 μmol/l) for 24 h as indicated, followed with or without 10 nmol/l insulin stimulation for 5 min. The expression levels of myc-tagged DsbA-L, Akt, and phosphor-Akt (Thr³⁰⁸) in cell lysates were determined by Western blot with specific antibodies as indicated. β-tubulin was used as a loading control. The experiment represents three independent experiments with similar results.