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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

The UPR-related genes were upregulated during the transcriptional induction of Ucp1 in brown adipocytes.(a,b) Real-time PCR analysis of Ucp1 (a), and UPR-related genes (b) in BAT exposed to cold (4 °C) for 24 h, or in control (28 °C) BAT. (c) RT-PCR analysis of Xbp1 in BAT exposed to cold (left panel). uXbp1 and sXbp1 indicate unspliced and spliced forms of Xbp1, respectively. Graph on right shows the quantification of Xbp1 splicing levels. (d) Real-time analysis of a target gene of sXBP1, Erdj4 in BAT exposed to cold. Differences between control and cold exposure were analyzed by Student’s t-test. Data are mean ± S.D. (control : n = 3, cold: n = 4) *P < 0.05, **P < 0.01, ***P < 0.001. (e,f) Real-time PCR analysis of Ucp1 (e), and UPR-related genes (f) in brown adipocytes treated with 1 μM CL 316,243 for indicated time periods. (g) RT-PCR analysis of Xbp1 in brown adipocytes treated with CL 316,243 (left panel). Graph on right shows the quantification of Xbp1 splicing levels. Note that splicing levels were increased in accordance with the expression pattern of Ucp1. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.
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f1: The UPR-related genes were upregulated during the transcriptional induction of Ucp1 in brown adipocytes.(a,b) Real-time PCR analysis of Ucp1 (a), and UPR-related genes (b) in BAT exposed to cold (4 °C) for 24 h, or in control (28 °C) BAT. (c) RT-PCR analysis of Xbp1 in BAT exposed to cold (left panel). uXbp1 and sXbp1 indicate unspliced and spliced forms of Xbp1, respectively. Graph on right shows the quantification of Xbp1 splicing levels. (d) Real-time analysis of a target gene of sXBP1, Erdj4 in BAT exposed to cold. Differences between control and cold exposure were analyzed by Student’s t-test. Data are mean ± S.D. (control : n = 3, cold: n = 4) *P < 0.05, **P < 0.01, ***P < 0.001. (e,f) Real-time PCR analysis of Ucp1 (e), and UPR-related genes (f) in brown adipocytes treated with 1 μM CL 316,243 for indicated time periods. (g) RT-PCR analysis of Xbp1 in brown adipocytes treated with CL 316,243 (left panel). Graph on right shows the quantification of Xbp1 splicing levels. Note that splicing levels were increased in accordance with the expression pattern of Ucp1. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

Mentions: To examine whether UPR is activated in response to stimuli leading to thermogenesis in brown adipocytes, we exposed C57BL/6 mice to cold (4 °C) for 24 h, isolated mRNA from BAT, and then measured the expression levels of UPR-related genes by real-time PCR analysis. It is well known that cold exposure promotes the transcription of Ucp1 for thermogenesis2728. In our study, a significant elevation of Ucp1 mRNA levels was observed in BAT of mice exposed to cold (4 °C) for 24 h compared with control mice that were kept at 28 °C (Fig. 1a). The expression of the ER-resident chaperones Bip, Grp94, and Calreticulin was significantly upregulated by cold exposure (Fig. 1b). According to western -blotting (WB) analysis, the levels of Bip proteins significantly increased, and GRP94 and a component of the UPR signal, ATF4, tended to increase after cold exposure (Supplementary Fig. 1). RT-PCR analysis showed an increase in the level of sXbp1 mRNA in BAT exposed to cold (Fig. 1c). Additionally, the mRNA level of ERdj4, which is a target gene of sXBP1, also increased (Fig. 1d). Cold exposure promotes not only thermogenesis, but also various physiological responses in BAT, such as angiogenesis34. Therefore, we next asked whether the increase in the expression of UPR-related genes was due to thermogenesis in brown adipocytes in response to cold. Norepinephrine is a major activator of brown adipocytes that promotes Ucp1 expression by binding to the β3 adrenergic receptor (β3-AR), which is expressed mainly in adipose tissue. Treatment with a β3-AR agonist such as CL 316,243 can mimic cold exposure in brown adipocytes27. Indeed, subcutaneous injection of CL 316,243 (1 mg/kg) induced a significant increase in Ucp1 mRNA levels in BAT (Supplementary Fig. 2a). The expression of the ER-resident chaperones, Bip, Grp94, and Calreticulin, as well as sXbp1 mRNA, was increased by CL 316,243 (Supplementary Fig. 2b,c). Treatment of primary brown adipocytes with 1 μM CL 316,243 transiently elevated Ucp1 mRNA levels, which peaked 3 h after treatment (Fig. 1e). The expression of Bip and Grp94 was also upregulated by treatment with CL 316,243, and this increase in expression remained until 9 h after treatment (Fig. 1f). On the other hand, splicing of Xbp1 mRNA increased in accordance with the expression pattern of Ucp1 (Fig. 1g), implying that sXBP1 acts during an early phase of the physiological response of brown adipocytes to norepinephrine, although ER-resident chaperones may not be needed until a later phase.


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)

The UPR-related genes were upregulated during the transcriptional induction of Ucp1 in brown adipocytes.(a,b) Real-time PCR analysis of Ucp1 (a), and UPR-related genes (b) in BAT exposed to cold (4 °C) for 24 h, or in control (28 °C) BAT. (c) RT-PCR analysis of Xbp1 in BAT exposed to cold (left panel). uXbp1 and sXbp1 indicate unspliced and spliced forms of Xbp1, respectively. Graph on right shows the quantification of Xbp1 splicing levels. (d) Real-time analysis of a target gene of sXBP1, Erdj4 in BAT exposed to cold. Differences between control and cold exposure were analyzed by Student’s t-test. Data are mean ± S.D. (control : n = 3, cold: n = 4) *P < 0.05, **P < 0.01, ***P < 0.001. (e,f) Real-time PCR analysis of Ucp1 (e), and UPR-related genes (f) in brown adipocytes treated with 1 μM CL 316,243 for indicated time periods. (g) RT-PCR analysis of Xbp1 in brown adipocytes treated with CL 316,243 (left panel). Graph on right shows the quantification of Xbp1 splicing levels. Note that splicing levels were increased in accordance with the expression pattern of Ucp1. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.
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Related In: Results  -  Collection

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f1: The UPR-related genes were upregulated during the transcriptional induction of Ucp1 in brown adipocytes.(a,b) Real-time PCR analysis of Ucp1 (a), and UPR-related genes (b) in BAT exposed to cold (4 °C) for 24 h, or in control (28 °C) BAT. (c) RT-PCR analysis of Xbp1 in BAT exposed to cold (left panel). uXbp1 and sXbp1 indicate unspliced and spliced forms of Xbp1, respectively. Graph on right shows the quantification of Xbp1 splicing levels. (d) Real-time analysis of a target gene of sXBP1, Erdj4 in BAT exposed to cold. Differences between control and cold exposure were analyzed by Student’s t-test. Data are mean ± S.D. (control : n = 3, cold: n = 4) *P < 0.05, **P < 0.01, ***P < 0.001. (e,f) Real-time PCR analysis of Ucp1 (e), and UPR-related genes (f) in brown adipocytes treated with 1 μM CL 316,243 for indicated time periods. (g) RT-PCR analysis of Xbp1 in brown adipocytes treated with CL 316,243 (left panel). Graph on right shows the quantification of Xbp1 splicing levels. Note that splicing levels were increased in accordance with the expression pattern of Ucp1. Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 4), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.
Mentions: To examine whether UPR is activated in response to stimuli leading to thermogenesis in brown adipocytes, we exposed C57BL/6 mice to cold (4 °C) for 24 h, isolated mRNA from BAT, and then measured the expression levels of UPR-related genes by real-time PCR analysis. It is well known that cold exposure promotes the transcription of Ucp1 for thermogenesis2728. In our study, a significant elevation of Ucp1 mRNA levels was observed in BAT of mice exposed to cold (4 °C) for 24 h compared with control mice that were kept at 28 °C (Fig. 1a). The expression of the ER-resident chaperones Bip, Grp94, and Calreticulin was significantly upregulated by cold exposure (Fig. 1b). According to western -blotting (WB) analysis, the levels of Bip proteins significantly increased, and GRP94 and a component of the UPR signal, ATF4, tended to increase after cold exposure (Supplementary Fig. 1). RT-PCR analysis showed an increase in the level of sXbp1 mRNA in BAT exposed to cold (Fig. 1c). Additionally, the mRNA level of ERdj4, which is a target gene of sXBP1, also increased (Fig. 1d). Cold exposure promotes not only thermogenesis, but also various physiological responses in BAT, such as angiogenesis34. Therefore, we next asked whether the increase in the expression of UPR-related genes was due to thermogenesis in brown adipocytes in response to cold. Norepinephrine is a major activator of brown adipocytes that promotes Ucp1 expression by binding to the β3 adrenergic receptor (β3-AR), which is expressed mainly in adipose tissue. Treatment with a β3-AR agonist such as CL 316,243 can mimic cold exposure in brown adipocytes27. Indeed, subcutaneous injection of CL 316,243 (1 mg/kg) induced a significant increase in Ucp1 mRNA levels in BAT (Supplementary Fig. 2a). The expression of the ER-resident chaperones, Bip, Grp94, and Calreticulin, as well as sXbp1 mRNA, was increased by CL 316,243 (Supplementary Fig. 2b,c). Treatment of primary brown adipocytes with 1 μM CL 316,243 transiently elevated Ucp1 mRNA levels, which peaked 3 h after treatment (Fig. 1e). The expression of Bip and Grp94 was also upregulated by treatment with CL 316,243, and this increase in expression remained until 9 h after treatment (Fig. 1f). On the other hand, splicing of Xbp1 mRNA increased in accordance with the expression pattern of Ucp1 (Fig. 1g), implying that sXBP1 acts during an early phase of the physiological response of brown adipocytes to norepinephrine, although ER-resident chaperones may not be needed until a later phase.

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