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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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Expression profiles of GABAergic signaling molecules.(A) Total RNA was extracted from 3T3-L1 cells cultured for 2 to 8 days and epididymal WAT of male mice, followed by RT-PCR using specific primers for each molecule. (B) Both 3T3-L1 cells and EF were cultured until confluence, followed by detection of GABABR1 subunit protein on immunoblotting analysis. Mouse whole brain was used as a positive control. Expression of mRNA and corresponding protein for GABABR1 subunit was constitutively seen in adipocytic cells.
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pone-0020167-g001: Expression profiles of GABAergic signaling molecules.(A) Total RNA was extracted from 3T3-L1 cells cultured for 2 to 8 days and epididymal WAT of male mice, followed by RT-PCR using specific primers for each molecule. (B) Both 3T3-L1 cells and EF were cultured until confluence, followed by detection of GABABR1 subunit protein on immunoblotting analysis. Mouse whole brain was used as a positive control. Expression of mRNA and corresponding protein for GABABR1 subunit was constitutively seen in adipocytic cells.

Mentions: Under these conditions, mRNA expression for GABAergic signaling machineries was analyzed in 3T3-L1 cells. As shown in Figure 1A, mRNA expression was not seen for GABAAα1-6, β1-3, γ1-3, and δ subunits of GABAAR, and ρ3 subunits of GABACR in 3T3-L1 cells cultured for different periods up to 8 days, with marked expression of mRNA for all GABAAR subunits and GABACR subunits in mouse whole brain. Although constitutive expression was also seen with mRNA for GABABR-1a and -1b isoforms, in addition to GABABR2 subunit, in mouse whole brain, mRNA was constitutively detected for both GABABR-1a and -1b subunits, but not for GABABR2 subunit, in 3T3-L1 cells. No mRNA expression was seen for GAD, GAT and VGAT in 3T3-L1 cells cultured for different days examined. Sequencing analysis on amplified PCR products clearly confirmed the expression of mRNA for the corresponding GABABR-1a and -1b subunits in 3T3-L1 cells (data not shown). In addition, mRNA expression for GABAergic signaling machineries was analyzed in WAT isolated from ddY strain mice. In accordance with expression profiles in 3T3-L1 cells, mRNA expression was seen for both GABABR-1a and -1b subunits, but not for GABABR2 subunit, in WAT (Figure 1A). Mouse whole brain, 3T3-L1 cells and EF were homogenized in buffer containing protease inhibitors, followed by immunoblotting analysis. Immunoreactivities were found to GABABR1a subunit at approximately 130 kDa and GABABR1b subunit at approximately 100 kDa, respectively, in both differentiated 3T3-L1 cells and EF, with much less amounts than in whole brain (Figure 1B).


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)

Expression profiles of GABAergic signaling molecules.(A) Total RNA was extracted from 3T3-L1 cells cultured for 2 to 8 days and epididymal WAT of male mice, followed by RT-PCR using specific primers for each molecule. (B) Both 3T3-L1 cells and EF were cultured until confluence, followed by detection of GABABR1 subunit protein on immunoblotting analysis. Mouse whole brain was used as a positive control. Expression of mRNA and corresponding protein for GABABR1 subunit was constitutively seen in adipocytic cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105007&req=5

pone-0020167-g001: Expression profiles of GABAergic signaling molecules.(A) Total RNA was extracted from 3T3-L1 cells cultured for 2 to 8 days and epididymal WAT of male mice, followed by RT-PCR using specific primers for each molecule. (B) Both 3T3-L1 cells and EF were cultured until confluence, followed by detection of GABABR1 subunit protein on immunoblotting analysis. Mouse whole brain was used as a positive control. Expression of mRNA and corresponding protein for GABABR1 subunit was constitutively seen in adipocytic cells.
Mentions: Under these conditions, mRNA expression for GABAergic signaling machineries was analyzed in 3T3-L1 cells. As shown in Figure 1A, mRNA expression was not seen for GABAAα1-6, β1-3, γ1-3, and δ subunits of GABAAR, and ρ3 subunits of GABACR in 3T3-L1 cells cultured for different periods up to 8 days, with marked expression of mRNA for all GABAAR subunits and GABACR subunits in mouse whole brain. Although constitutive expression was also seen with mRNA for GABABR-1a and -1b isoforms, in addition to GABABR2 subunit, in mouse whole brain, mRNA was constitutively detected for both GABABR-1a and -1b subunits, but not for GABABR2 subunit, in 3T3-L1 cells. No mRNA expression was seen for GAD, GAT and VGAT in 3T3-L1 cells cultured for different days examined. Sequencing analysis on amplified PCR products clearly confirmed the expression of mRNA for the corresponding GABABR-1a and -1b subunits in 3T3-L1 cells (data not shown). In addition, mRNA expression for GABAergic signaling machineries was analyzed in WAT isolated from ddY strain mice. In accordance with expression profiles in 3T3-L1 cells, mRNA expression was seen for both GABABR-1a and -1b subunits, but not for GABABR2 subunit, in WAT (Figure 1A). Mouse whole brain, 3T3-L1 cells and EF were homogenized in buffer containing protease inhibitors, followed by immunoblotting analysis. Immunoreactivities were found to GABABR1a subunit at approximately 130 kDa and GABABR1b subunit at approximately 100 kDa, respectively, in both differentiated 3T3-L1 cells and EF, with much less amounts than in whole brain (Figure 1B).

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