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IRE1α-XBP1 is a novel branch in the transcriptional regulation of Ucp1 in brown adipocytes.

Asada R, Kanemoto S, Matsuhisa K, Hino K, Cui M, Cui X, Kaneko M, Imaizumi K - Sci Rep (2015)

Bottom Line: We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1.Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression.These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Biomedical &Health Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

ABSTRACT
The unfolded protein response (UPR) not only resolves endoplasmic reticulum (ER) stress, but also regulates cellular physiological functions. In this study, we first linked the UPR to the physiological roles of brown adipose tissue (BAT). BAT is one of the tissues that control energy homeostasis in the body. Brown adipocytes are able to dissipate energy in the form of heat owing to their mitochondrial protein, uncoupling protein 1 (UCP1). We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1. Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression. However, the activation of the IRE1α-XBP1 pathway by ER stress never upregulated Ucp1. On the other hand, the activation of protein kinase A (PKA) induced Ucp1 transcription through the activation of IRE1α-XBP1. The inhibition of PKA abrogated the activation of IRE1α-XBP1 pathway, while the inhibition of a p38 mitogen activated protein kinase (p38 MAPK), which is one of the downstream molecules of PKA, never suppressed the activation of IRE1α-XBP1 pathway. These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

No MeSH data available.


Related in: MedlinePlus

IRE1α was predominantly activated by treatment with β3-AR agonist or forskolin.(a–c) WB analysis of PERK, eIF2α (a), ATF6 (b), and IRE1α (c) in brown adipocytes treated with either 1 μM CL 316,243 or 2 μM Tg for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Note that phosphorylation of IRE1α was significantly increased by treatment with CL 316,243. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), ***P < 0.001, ns: not significant. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, ***P < 0.001. cont.: control. (e–g) WB analysis of PERK, eIF2α (e), ATF6 (f), and IRE1α (g) in brown adipocytes treated with 20 μM FSK for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, ns: not significant. (h) RT-PCR analysis of Xbp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), ***P < 0.001. cont.: control.
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f2: IRE1α was predominantly activated by treatment with β3-AR agonist or forskolin.(a–c) WB analysis of PERK, eIF2α (a), ATF6 (b), and IRE1α (c) in brown adipocytes treated with either 1 μM CL 316,243 or 2 μM Tg for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Note that phosphorylation of IRE1α was significantly increased by treatment with CL 316,243. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), ***P < 0.001, ns: not significant. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, ***P < 0.001. cont.: control. (e–g) WB analysis of PERK, eIF2α (e), ATF6 (f), and IRE1α (g) in brown adipocytes treated with 20 μM FSK for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, ns: not significant. (h) RT-PCR analysis of Xbp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), ***P < 0.001. cont.: control.

Mentions: Next, we examined which branch in the UPR is important for the transcriptional induction of Ucp1 in brown adipocytes. Because the largest increase in Ucp1 mRNA was observed 3 h after the treatment of primary brown adipocytes with CL 316,243, we conducted WB analysis to measure a change in the amount of phosphorylated PERK, eIF2α, and IRE1α, or N-terminus of ATF6 until 3 h after treatment. As shown in Fig. 2a, treatment with 1 μM CL 316,243 never induced the phosphorylation of PERK or its substrate eIF2α, whereas treatment with the ER stressor thapsigargin (Tg) substantially increased the phosphorylation of both proteins in primary brown adipocytes. The cleaved fragment of ATF6 (ATF6 N-terminus) did not increase after treatment with CL 316,243 (Fig. 2b). In contrast, the phosphorylation of IRE1α significantly increased 30 min after treatment with CL 316,243 (Fig. 2c). These data indicated that IRE1α is predominantly activated downstream of the β3-AR pathway. It is not known the reason why the phosphorylation of IRE1α swiftly went back to the basal level, while sXbp1 mRNA was detected until 6 h after CL 316,243 treatment. We speculated two possibilities: 1) Because our antibody detects only phosphorylation of Ser 724 of IRE1α, the other phosphorylation sites in multiple phosphorylation sites of IRE1α could be phosphorylated with CL 316,243 treatment time. 2) Because the stability of sXbp1 mRNA is high, sXbp1 mRNA could escape from rapid degradation.


IRE1α-XBP1 is a novel branch in the transcriptional regulation of Ucp1 in brown adipocytes.

Asada R, Kanemoto S, Matsuhisa K, Hino K, Cui M, Cui X, Kaneko M, Imaizumi K - Sci Rep (2015)

IRE1α was predominantly activated by treatment with β3-AR agonist or forskolin.(a–c) WB analysis of PERK, eIF2α (a), ATF6 (b), and IRE1α (c) in brown adipocytes treated with either 1 μM CL 316,243 or 2 μM Tg for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Note that phosphorylation of IRE1α was significantly increased by treatment with CL 316,243. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), ***P < 0.001, ns: not significant. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, ***P < 0.001. cont.: control. (e–g) WB analysis of PERK, eIF2α (e), ATF6 (f), and IRE1α (g) in brown adipocytes treated with 20 μM FSK for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, ns: not significant. (h) RT-PCR analysis of Xbp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), ***P < 0.001. cont.: control.
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Related In: Results  -  Collection

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f2: IRE1α was predominantly activated by treatment with β3-AR agonist or forskolin.(a–c) WB analysis of PERK, eIF2α (a), ATF6 (b), and IRE1α (c) in brown adipocytes treated with either 1 μM CL 316,243 or 2 μM Tg for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Note that phosphorylation of IRE1α was significantly increased by treatment with CL 316,243. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), ***P < 0.001, ns: not significant. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, ***P < 0.001. cont.: control. (e–g) WB analysis of PERK, eIF2α (e), ATF6 (f), and IRE1α (g) in brown adipocytes treated with 20 μM FSK for indicated time periods. Black arrow and Mark (#) indicate ATF6 N-terminus and non-specific detections, respectively. β-actin was used as a loading control. Graphs show the ratio of phosphorylated to total PERK, eIF2α, or IRE1α, and the amount of ATF6 N-terminus. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, ns: not significant. (h) RT-PCR analysis of Xbp1 in brown adipocytes treated with either 1 μM CL 316,243 (CL) or 20 μM forskolin (FSK) for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), ***P < 0.001. cont.: control.
Mentions: Next, we examined which branch in the UPR is important for the transcriptional induction of Ucp1 in brown adipocytes. Because the largest increase in Ucp1 mRNA was observed 3 h after the treatment of primary brown adipocytes with CL 316,243, we conducted WB analysis to measure a change in the amount of phosphorylated PERK, eIF2α, and IRE1α, or N-terminus of ATF6 until 3 h after treatment. As shown in Fig. 2a, treatment with 1 μM CL 316,243 never induced the phosphorylation of PERK or its substrate eIF2α, whereas treatment with the ER stressor thapsigargin (Tg) substantially increased the phosphorylation of both proteins in primary brown adipocytes. The cleaved fragment of ATF6 (ATF6 N-terminus) did not increase after treatment with CL 316,243 (Fig. 2b). In contrast, the phosphorylation of IRE1α significantly increased 30 min after treatment with CL 316,243 (Fig. 2c). These data indicated that IRE1α is predominantly activated downstream of the β3-AR pathway. It is not known the reason why the phosphorylation of IRE1α swiftly went back to the basal level, while sXbp1 mRNA was detected until 6 h after CL 316,243 treatment. We speculated two possibilities: 1) Because our antibody detects only phosphorylation of Ser 724 of IRE1α, the other phosphorylation sites in multiple phosphorylation sites of IRE1α could be phosphorylated with CL 316,243 treatment time. 2) Because the stability of sXbp1 mRNA is high, sXbp1 mRNA could escape from rapid degradation.

Bottom Line: We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1.Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression.These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Biomedical &Health Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

ABSTRACT
The unfolded protein response (UPR) not only resolves endoplasmic reticulum (ER) stress, but also regulates cellular physiological functions. In this study, we first linked the UPR to the physiological roles of brown adipose tissue (BAT). BAT is one of the tissues that control energy homeostasis in the body. Brown adipocytes are able to dissipate energy in the form of heat owing to their mitochondrial protein, uncoupling protein 1 (UCP1). We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1. Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression. However, the activation of the IRE1α-XBP1 pathway by ER stress never upregulated Ucp1. On the other hand, the activation of protein kinase A (PKA) induced Ucp1 transcription through the activation of IRE1α-XBP1. The inhibition of PKA abrogated the activation of IRE1α-XBP1 pathway, while the inhibition of a p38 mitogen activated protein kinase (p38 MAPK), which is one of the downstream molecules of PKA, never suppressed the activation of IRE1α-XBP1 pathway. These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

No MeSH data available.


Related in: MedlinePlus