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Positive regulation by GABA(B)R1 subunit of leptin expression through gene transactivation in adipocytes.

Nakamura Y, Hinoi E, Takarada T, Takahata Y, Yamamoto T, Fujita H, Takada S, Hashizume S, Yoneda Y - PLoS ONE (2011)

Bottom Line: However, no prominent expression was seen with mRNA for GABA(B)R2 subunit required for heteromeric orchestration of the functional GABA(B)R by any adipocytic cells and tissues.Knockdown by siRNA of GABA(B)R1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells.Our results indicate that GABA(B)R1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner of heterodimeric assembly to the functional GABA(B)R.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan.

ABSTRACT

Background: The view that γ-aminobutyric acid (GABA) plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes.

Methodology/principal findings: GABA(B) receptor 1 (GABA(B)R1) subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABA(B)R ligands. However, no prominent expression was seen with mRNA for GABA(B)R2 subunit required for heteromeric orchestration of the functional GABA(B)R by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABA(B)R1- mice than in wild-type mice. Knockdown by siRNA of GABA(B)R1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells.

Conclusions/significance: Our results indicate that GABA(B)R1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner of heterodimeric assembly to the functional GABA(B)R.

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Transfection with GABABR1 siRNA in 3T3-L1 cells.(A) 3T3-L1 cells transfected with si-control and si-GABABR1 were cultured for 3 days, followed by immunoblotting for GABABR1 subunit. (B) 3T3-L1 cells transfected with siRNA were cultured for 2 days in the presence of inducers of adipocytic differentiation, followed by real time-PCR for GABABR1 subunit, leptin and PGC1α. (C) 3T3-L1 cells were transfected with the luciferase vector containing leptin promoter together with siRNA, followed by culture in either the presence or absence of inducers such as insulin, DEX and IBMX for 2 days and subsequent determination of luciferase activity. (D) 3T3-L1 cells were transfected with the luciferase vector containing PGC1α promoter along with siRNA, followed by culture for 3 days and subsequent determination of luciferase activity. Values are the mean ± S.E. from 3 different experiments. *P<0.05; **P<0.01, significantly different from each control value. ††P<0.01, significantly different from the value obtained in control cells cultured in the presence of inducers. Knockdown by siRNA of GABABR1 subunit led to decreased leptin and increased PGC1α mRNA expression levels through modulation of transactivation of corresponding genes. Gb1, GABABR1.
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pone-0020167-g005: Transfection with GABABR1 siRNA in 3T3-L1 cells.(A) 3T3-L1 cells transfected with si-control and si-GABABR1 were cultured for 3 days, followed by immunoblotting for GABABR1 subunit. (B) 3T3-L1 cells transfected with siRNA were cultured for 2 days in the presence of inducers of adipocytic differentiation, followed by real time-PCR for GABABR1 subunit, leptin and PGC1α. (C) 3T3-L1 cells were transfected with the luciferase vector containing leptin promoter together with siRNA, followed by culture in either the presence or absence of inducers such as insulin, DEX and IBMX for 2 days and subsequent determination of luciferase activity. (D) 3T3-L1 cells were transfected with the luciferase vector containing PGC1α promoter along with siRNA, followed by culture for 3 days and subsequent determination of luciferase activity. Values are the mean ± S.E. from 3 different experiments. *P<0.05; **P<0.01, significantly different from each control value. ††P<0.01, significantly different from the value obtained in control cells cultured in the presence of inducers. Knockdown by siRNA of GABABR1 subunit led to decreased leptin and increased PGC1α mRNA expression levels through modulation of transactivation of corresponding genes. Gb1, GABABR1.

Mentions: To evaluate a role of GABABR1 subunit in leptin expression in adipocytes, 3T3-L1 cells were transiently transfected with siRNA for GABABR1 subunit by RNA-mediated interference. GABABR1 subunit protein levels were markedly decreased in 3T3-L1 cells transfected with the three different siRNA for GABABR1 subunit for 72 h compared to cells with scrambled control siRNA (Figure 5A). In 3T3-L1 cells transfected with GABABR1 siRNA, no marked alterations were found in fibroblastic morphology, viability and proliferation rate (data not shown). In cells with GABABR1 siRNA#3, a significant decrease was seen in leptin mRNA expression with increased PGC1α mRNA expression (Figure 5B). To further explore the mechanisms underlying the regulation of leptin and PGC1α gene expression, luciferase reporter plasmids were constructed with leptin and PGC1α promoters, respectively, for subsequent transfection to 3T3-L1 cells. The addition of insulin, DEX and IBMX significantly increased luciferase activity with leptin promoter plasmid, while introduction of GABABR1 siRNA#3 led to significant prevention of the increase in luciferase activity without significantly affecting basal leptin promoter activity (Figure 5C). By contrast, PGC1α promoter activity was significantly increased in 3T3-L1 cells transfected with GABABR1 siRNA#3 for 72 h (Figure 5D). Therefore, GABABR1 subunit protein would directly promote transactivation of leptin gene in adipocytic cells.


Positive regulation by GABA(B)R1 subunit of leptin expression through gene transactivation in adipocytes.

Nakamura Y, Hinoi E, Takarada T, Takahata Y, Yamamoto T, Fujita H, Takada S, Hashizume S, Yoneda Y - PLoS ONE (2011)

Transfection with GABABR1 siRNA in 3T3-L1 cells.(A) 3T3-L1 cells transfected with si-control and si-GABABR1 were cultured for 3 days, followed by immunoblotting for GABABR1 subunit. (B) 3T3-L1 cells transfected with siRNA were cultured for 2 days in the presence of inducers of adipocytic differentiation, followed by real time-PCR for GABABR1 subunit, leptin and PGC1α. (C) 3T3-L1 cells were transfected with the luciferase vector containing leptin promoter together with siRNA, followed by culture in either the presence or absence of inducers such as insulin, DEX and IBMX for 2 days and subsequent determination of luciferase activity. (D) 3T3-L1 cells were transfected with the luciferase vector containing PGC1α promoter along with siRNA, followed by culture for 3 days and subsequent determination of luciferase activity. Values are the mean ± S.E. from 3 different experiments. *P<0.05; **P<0.01, significantly different from each control value. ††P<0.01, significantly different from the value obtained in control cells cultured in the presence of inducers. Knockdown by siRNA of GABABR1 subunit led to decreased leptin and increased PGC1α mRNA expression levels through modulation of transactivation of corresponding genes. Gb1, GABABR1.
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pone-0020167-g005: Transfection with GABABR1 siRNA in 3T3-L1 cells.(A) 3T3-L1 cells transfected with si-control and si-GABABR1 were cultured for 3 days, followed by immunoblotting for GABABR1 subunit. (B) 3T3-L1 cells transfected with siRNA were cultured for 2 days in the presence of inducers of adipocytic differentiation, followed by real time-PCR for GABABR1 subunit, leptin and PGC1α. (C) 3T3-L1 cells were transfected with the luciferase vector containing leptin promoter together with siRNA, followed by culture in either the presence or absence of inducers such as insulin, DEX and IBMX for 2 days and subsequent determination of luciferase activity. (D) 3T3-L1 cells were transfected with the luciferase vector containing PGC1α promoter along with siRNA, followed by culture for 3 days and subsequent determination of luciferase activity. Values are the mean ± S.E. from 3 different experiments. *P<0.05; **P<0.01, significantly different from each control value. ††P<0.01, significantly different from the value obtained in control cells cultured in the presence of inducers. Knockdown by siRNA of GABABR1 subunit led to decreased leptin and increased PGC1α mRNA expression levels through modulation of transactivation of corresponding genes. Gb1, GABABR1.
Mentions: To evaluate a role of GABABR1 subunit in leptin expression in adipocytes, 3T3-L1 cells were transiently transfected with siRNA for GABABR1 subunit by RNA-mediated interference. GABABR1 subunit protein levels were markedly decreased in 3T3-L1 cells transfected with the three different siRNA for GABABR1 subunit for 72 h compared to cells with scrambled control siRNA (Figure 5A). In 3T3-L1 cells transfected with GABABR1 siRNA, no marked alterations were found in fibroblastic morphology, viability and proliferation rate (data not shown). In cells with GABABR1 siRNA#3, a significant decrease was seen in leptin mRNA expression with increased PGC1α mRNA expression (Figure 5B). To further explore the mechanisms underlying the regulation of leptin and PGC1α gene expression, luciferase reporter plasmids were constructed with leptin and PGC1α promoters, respectively, for subsequent transfection to 3T3-L1 cells. The addition of insulin, DEX and IBMX significantly increased luciferase activity with leptin promoter plasmid, while introduction of GABABR1 siRNA#3 led to significant prevention of the increase in luciferase activity without significantly affecting basal leptin promoter activity (Figure 5C). By contrast, PGC1α promoter activity was significantly increased in 3T3-L1 cells transfected with GABABR1 siRNA#3 for 72 h (Figure 5D). Therefore, GABABR1 subunit protein would directly promote transactivation of leptin gene in adipocytic cells.

Bottom Line: However, no prominent expression was seen with mRNA for GABA(B)R2 subunit required for heteromeric orchestration of the functional GABA(B)R by any adipocytic cells and tissues.Knockdown by siRNA of GABA(B)R1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells.Our results indicate that GABA(B)R1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner of heterodimeric assembly to the functional GABA(B)R.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan.

ABSTRACT

Background: The view that γ-aminobutyric acid (GABA) plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes.

Methodology/principal findings: GABA(B) receptor 1 (GABA(B)R1) subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABA(B)R ligands. However, no prominent expression was seen with mRNA for GABA(B)R2 subunit required for heteromeric orchestration of the functional GABA(B)R by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABA(B)R1- mice than in wild-type mice. Knockdown by siRNA of GABA(B)R1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells.

Conclusions/significance: Our results indicate that GABA(B)R1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner of heterodimeric assembly to the functional GABA(B)R.

Show MeSH
Related in: MedlinePlus