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Endoplasmic reticulum stress regulates adipocyte resistin expression.

Lefterova MI, Mullican SE, Tomaru T, Qatanani M, Schupp M, Lazar MA - Diabetes (2009)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.

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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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.
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Figure 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.

Mentions: One candidate transcription factor that could explain the effects of ER stress on resistin expression is C/EBPα. C/EBPα is critical for adipocyte differentiation (28) and contributes to activation of adipocyte-specific genes such as adiponectin (29), PPARγ (30), and resistin (31). A binding site for C/EBPα on the resistin promoter has been characterized, and it has been shown that ectopic C/EBPα expression in nonadipogenic cells could drive luciferase expression from the resistin promoter (31). Furthermore, C/EBPα was recently reported to be downregulated on the mRNA level by the inducers of ER stress tunicamycin and thapsigargin (32), and indeed we confirmed that C/EBPα mRNA and protein are reduced in tunicamycin-treated adipocytes (Fig. 4A and B). This treatment also abolished C/EBPα occupancy at the resistin gene as measured by ChIP both at the previously characterized C/EBPα binding site (31) as well as an additional upstream site identified in a recent genome-wide ChIP-chip study (33) (Fig. 4C). Similar decreases in occupancy were noted on the known C/EBPα binding sites on the promoters of PPARγ and adiponectin (data not shown).


Endoplasmic reticulum stress regulates adipocyte resistin expression.

Lefterova MI, Mullican SE, Tomaru T, Qatanani M, Schupp M, Lazar MA - Diabetes (2009)

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.
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Figure 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.
Mentions: One candidate transcription factor that could explain the effects of ER stress on resistin expression is C/EBPα. C/EBPα is critical for adipocyte differentiation (28) and contributes to activation of adipocyte-specific genes such as adiponectin (29), PPARγ (30), and resistin (31). A binding site for C/EBPα on the resistin promoter has been characterized, and it has been shown that ectopic C/EBPα expression in nonadipogenic cells could drive luciferase expression from the resistin promoter (31). Furthermore, C/EBPα was recently reported to be downregulated on the mRNA level by the inducers of ER stress tunicamycin and thapsigargin (32), and indeed we confirmed that C/EBPα mRNA and protein are reduced in tunicamycin-treated adipocytes (Fig. 4A and B). This treatment also abolished C/EBPα occupancy at the resistin gene as measured by ChIP both at the previously characterized C/EBPα binding site (31) as well as an additional upstream site identified in a recent genome-wide ChIP-chip study (33) (Fig. 4C). Similar decreases in occupancy were noted on the known C/EBPα binding sites on the promoters of PPARγ and adiponectin (data not shown).

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.

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.

Show MeSH
Related in: MedlinePlus