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. 2009 Aug;58(8):1879-86.
doi: 10.2337/db08-1706. Epub 2009 Jun 2.

Endoplasmic reticulum stress regulates adipocyte resistin expression

Martina I Lefterova  1 , Shannon E Mullican, Takuya Tomaru, Mohammed Qatanani, Michael Schupp, Mitchell A Lazar
Affiliations

Endoplasmic reticulum stress regulates adipocyte resistin expression

Martina I Lefterova et al. Diabetes. 2009 Aug.

Abstract

Objective: Resistin is a secreted polypeptide that impairs glucose metabolism and, in rodents, is derived exclusively from adipocytes. In murine obesity, resistin circulates at elevated levels but its gene expression in adipose tissue is paradoxically reduced. The mechanism behind the downregulation of resistin mRNA is poorly understood. We investigated whether endoplasmic reticulum (ER) stress, which is characteristic of obese adipose tissue, regulates resistin expression in cultured mouse adipocytes.

Research design and methods: The effects of endoplasmic stress inducers on resistin mRNA and secreted protein levels were examined in differentiated 3T3-L1 adipocytes, focusing on the expression and genomic binding of transcriptional regulators of resistin. The association between downregulated resistin mRNA and induction of ER stress was also investigated in the adipose tissue of mice fed a high-fat diet.

Results: ER stress reduced resistin mRNA in 3T3-L1 adipocytes in a time- and dose-dependent manner. The effects of ER stress were transcriptional because of downregulation of CAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma transcriptional activators and upregulation of the transcriptional repressor CAAT/enhancer binding protein homologous protein-10 (CHOP10). Resistin protein was also substantially downregulated, showing a close correspondence with mRNA levels in 3T3-L1 adipocytes as well as in the fat pads of obese mice.

Conclusions: ER stress is a potent regulator of resistin, suggesting that ER stress may underlie the local downregulation of resistin mRNA and protein in fat in murine obesity. The paradoxical increase in plasma may be because of various systemic abnormalities associated with obesity and insulin resistance.

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Figures

FIG. 1.
FIG. 1.
Endoplasmic reticulum stress activation downregulates resistin expression in vitro in 3T3-L1 adipocytes. A: Downregulation of resistin mRNA levels following treatment with 50 nmol/l thapsigargin for 24 h. B: Time course of resistin and ATF3 mRNA gene expression upon induction of ER stress with 5 μg/ml tunicamycin, presented as fold change over the levels at 0 h. C: Resistin gene expression in response to vehicle and various doses of tunicamycin for 24 h, presented as fold change over vehicle alone. D: Resistin protein concentration in tissue culture media and whole cell lysates presented as nanogram per milliliter per milligram of total cell protein. Mature adipocytes were electroporated with resistin (siResistin) or nontarget control (NTC) siRNA oligos and treated with vehicle or 5 μg/ml tunicamycin. Twenty-four hours later the cells were washed and treated with vehicle or 5 μg/m tunicamycin for 24 h and resistin protein levels were assayed with ELISA. Data are mean ± SE, n = 3. E: Gene expression validation that siResistin and tunicamycin treatment reduced resistin mRNA to similar levels, and only tunicamycin induced markers of ER stress such as BiP. Data in A–C and E were normalized to the house-keeping gene Arbp/36b4 and are shown as mean ± SE, n = 3. ***P < 0.001.
FIG. 2.
FIG. 2.
Reduced resistin levels in EWAT of obese mice are associated with markers of ER stress. All data are from male C57Bl/6 mice fed NC (n = 5) or HFD (n = 5) for 30 weeks to induce obesity. A: Resistin mRNA levels in EWAT. Data were normalized to the house-keeping gene Arbp/36b4 and presented as mean ± SE. B: Serum resistin levels after 30 weeks of diet. C: Resistin protein levels in EWAT, measured with ELISA in tissue homogenates, and normalized to the total protein concentration of each homogenate. Data are presented as nanogram resistin per milligram total protein, mean ± SE. D: Western blot demonstrating the phosphorylation status of Eukaryotic translation initiation factor 2a (eIF2α) in representative animals selected at random from the NC and HFD groups. Total eIF2α and Ran were used as loading controls. E: mRNA levels of the ER chaperone BiP measured in EWAT. Data were normalized to the house-keeping gene Arbp/36b4 and are presented as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.
FIG. 3
FIG. 3
Endoplasmic reticulum stress downregulates resistin in adipocytes by a transcriptional mechanism. A: Changes in resistin mRNA levels over 48 h in response to vehicle or 5 μg/ml tunicamycin in the presence of 5 μg/ml Actinomycin D. Data are presented as percent of the mRNA level at time 0, mean ± SD of a triplicate experiment. B: Normalized luciferase activity of resistin-luc (−13,580 bp to +243 bp) or empty vector (pGL3 basic) in the presence of vehicle or 5 μg/ml tunicamycin for 24 h. Data are presented as mean ± SE, n = 3. **P = 0.003 for pGL3 basic versus resistin-luc activity in vehicle-treated cells. ##P = 0.005 for resistin-luc activity in vehicle- versus tunicamycin-treated cells.
FIG. 4
FIG. 4
Downregulation of C/EBPα by ER stress decreases resistin expression. A and B: Endoplasmic reticulum stress induced by treatment with 5 μg/ml tunicamycin for 24 h leads to downregulation of C/EBPα mRNA and protein compared with vehicle control. Gene expression data are presented as mean ± SE, n = 3. C/EBPα protein levels were assayed by Western blotting, and HDAC2 served as a loading control. C: Endoplasmic reticulum stress reduces C/EBPα recruitment to resistin at a downstream site at the known C/EBPα binding site at −50 bp relative to the transcription start site (TSS), and at a site located ∼9 kb upstream of the TSS. Mature adipocytes were treated as in A, and recruitment was measured by ChIP-QPCR. A region at the TSS of albumin was used as negative control for C/EBPα recruitment. Enrichment was normalized to a site on the Arbp/36b4 gene were C/EBPα does not bind. Data are presented as mean ± SE of three independent ChIP experiments. D: C/EBPα knockdown (siC/EBPα) mimics the effects of ER stress on resistin expression. Mature adipocytes, electroporated with C/EBPα or nontarget control (NTC) siRNA oligos, were treated with vehicle or 5 μg/m tunicamycin for 24 h. Efficiency of siC/EBPα was assayed by Western blot and compared with NTC in the presence or absence of 5 μg/m tunicamycin. HDAC2 was used as a loading control. E: Resistin mRNA levels were measured in vehicle-treated treated C/EBPα kd or NTC cells by QPCR and normalized to Arbp/36b4. Data are presented as mean ± SE, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.
FIG. 5
FIG. 5
C/EBPα is downregulated in EWAT of obese mice. All data were derived from EWAT of the same animals as in Fig. 1, NC (n = 5) or HFD (n = 5). A: C/EBPα mRNA levels normalized to Arbp/36b4 and are presented as mean ± SE. B: C/EBPα protein levels were measured by Western blotting in EWAT of two randomly selected animals per group. HDAC2 was used as a loading control. ***P < 0.001.
FIG. 6
FIG. 6
Downregulation of PPARγ contributes to the effects of ER stress on resistin expression. A: PPARγ protein levels were measured by Western blotting in mature adipocytes treated with vehicle or 5 μg/ml tunicamycin for 24 h. Ran was used as a loading control. B: Endoplasmic reticulum stress decreases recruitment of PPARγ at a binding site near the resitin gene. ChIP-QPCR was performed as in Fig. 4C. A region at the insulin gene was used as negative control for PPARγ recruitment. Data are presented as mean ± SE of three independent ChIP experiments. C: PPARγ knockdown (siPPARγ) can mimic the effects of ER stress on resistin. Mature adipocytes were electroporated with PPARγ (siPPARγ) or NTC siRNA oligos. Resistin mRNA levels were measured 48 h later by QPCR and normalized to Arbp/36b4. D: Efficiency of PPARγ knockdown was assayed by QPCR. C and D: Data are presented as mean + SE of three replicates. ***P < 0.001.
FIG. 7
FIG. 7
Upregulation of CHOP10 by ER stress induction contributes to reduced resistin expression. A: CHOP10 is upregulated by ER stress. CHOP10 mRNA levels were measured by QPCR in mature adipocytes treated with vehicle or 5 μg/ml tunicamycin for 24 h. B–D: CHOP10 knockdown (siCHOP10) partially rescues the effects of ER stress on resistin and C/EBPα expression. B: Efficiency of CHOP10 knockdown was assayed by QPCR. C: Effects of siCHOP10 or NTC on resistin mRNA levels in the presence or absence of 5 μg/ml tunicamycin for 24 h. D: Effects of siCHOP10 on C/EBPα mRNA levels. All QPCR data were normalized to Arbp/36b4 and presented as mean ± SE of three replicates. nontarget control, NTC. **P < 0.01, ***P < 0.001.
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