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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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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.
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Figure 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.

Mentions: To investigate the potential link between ER stress and resistin downregulation, mouse 3T3-L1 adipocytes were incubated with thapsigargin, which causes ER stress by inhibiting the sarco/ER Ca2+ pump, and led to dramatically reduced resistin mRNA levels (Fig. 1A). To confirm ER stress as the mechanism underlying resistin downregulation, adipocytes were treated with tunicamycin, which induces ER stress by inhibiting N-linked glycosylation of newly synthesized proteins. Tunicamycin treatment markedly downregulated resistin mRNA levels in a time-dependent fashion (Fig. 1B). By contrast, the same treatment induced expression of ATF3, which is activated by ER stress (26). The decrease of resistin mRNA by tunicamycin was also dose-dependent (Fig. 1C). To examine the effects of tunicamycin on resistin protein levels, cells were treated for 24 h to lower resistin mRNA as in Fig. 1C, then treated with fresh tunicamycin- or vehicle-containing media, after which the accumulation of secreted resistin as well as the intracellular resistin levels were measured by ELISA. Resistin protein was substantially decreased in the media and cell lysate of tunicamycin-treated cells (Fig. 1D), similar to that observed with resistin knockdown, which reduced the mRNA to similar extent but did not induce upregulation of BiP mRNA that would have signified ER stress (Fig. 1E). Finally, the effects of tunicamycin on resistin secretion also appeared to be dose dependent (data not shown). Collectively, these results indicate that ER stress is a potent regulator of resistin mRNA and protein levels in 3T3-L1 adipocytes.


Endoplasmic reticulum stress regulates adipocyte resistin expression.

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

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 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.
Mentions: To investigate the potential link between ER stress and resistin downregulation, mouse 3T3-L1 adipocytes were incubated with thapsigargin, which causes ER stress by inhibiting the sarco/ER Ca2+ pump, and led to dramatically reduced resistin mRNA levels (Fig. 1A). To confirm ER stress as the mechanism underlying resistin downregulation, adipocytes were treated with tunicamycin, which induces ER stress by inhibiting N-linked glycosylation of newly synthesized proteins. Tunicamycin treatment markedly downregulated resistin mRNA levels in a time-dependent fashion (Fig. 1B). By contrast, the same treatment induced expression of ATF3, which is activated by ER stress (26). The decrease of resistin mRNA by tunicamycin was also dose-dependent (Fig. 1C). To examine the effects of tunicamycin on resistin protein levels, cells were treated for 24 h to lower resistin mRNA as in Fig. 1C, then treated with fresh tunicamycin- or vehicle-containing media, after which the accumulation of secreted resistin as well as the intracellular resistin levels were measured by ELISA. Resistin protein was substantially decreased in the media and cell lysate of tunicamycin-treated cells (Fig. 1D), similar to that observed with resistin knockdown, which reduced the mRNA to similar extent but did not induce upregulation of BiP mRNA that would have signified ER stress (Fig. 1E). Finally, the effects of tunicamycin on resistin secretion also appeared to be dose dependent (data not shown). Collectively, these results indicate that ER stress is a potent regulator of resistin mRNA and protein levels in 3T3-L1 adipocytes.

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