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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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Related in: MedlinePlus

Lack of effects of GABABR ligands on adipocytic differentiation.(A) 3T3-L1 cells were transfected with the luciferase vector containing 4 tandem copies of CRE site, followed by exposure to the GABABR agonist baclofen, the GABABR antagonist CPG46381 or forskolin for 24 h and subsequent determination of luciferase activity. 3T3-L1 cells were cultured for 8 to 16 days, in either the presence or absence of baclofen and the antagonist saclofen, followed by Oil red O staining. Typical micrographic pictures are shown in the panel (B), while in the panel (C) quantitative data are shown as the mean ± S.E in 5 independent determinations. (D) EF was isolated from WT and GABABR1- mice, followed by culture for 5 to 16 days and subsequent staining with Oil red O. Values are the mean ± S.E. from 6 to 11 different experiments. Neither agonist nor antagonist for GABABR exhibited significant effects on adipogenesis. Ba, baclofen; Gb1, GABABR1; Sa, saclofen.
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pone-0020167-g002: Lack of effects of GABABR ligands on adipocytic differentiation.(A) 3T3-L1 cells were transfected with the luciferase vector containing 4 tandem copies of CRE site, followed by exposure to the GABABR agonist baclofen, the GABABR antagonist CPG46381 or forskolin for 24 h and subsequent determination of luciferase activity. 3T3-L1 cells were cultured for 8 to 16 days, in either the presence or absence of baclofen and the antagonist saclofen, followed by Oil red O staining. Typical micrographic pictures are shown in the panel (B), while in the panel (C) quantitative data are shown as the mean ± S.E in 5 independent determinations. (D) EF was isolated from WT and GABABR1- mice, followed by culture for 5 to 16 days and subsequent staining with Oil red O. Values are the mean ± S.E. from 6 to 11 different experiments. Neither agonist nor antagonist for GABABR exhibited significant effects on adipogenesis. Ba, baclofen; Gb1, GABABR1; Sa, saclofen.

Mentions: In order to evaluate the possible function of GABABR1 subunit expressed by adipocytes as metabotropic GABABR, an attempt was made to determine whether GABABR ligands affect luciferase activity in 3T3-L1 cells transfected with a cAMP responsive element (CRE) reporter plasmid. Exposure to forskolin resulted in a drastic increase in the luciferase activity in 3T3-L1 cells transfected with a CRE reporter plasmid, whereas the further addition of the GABABR agonist baclofen or the GABABR antagonist CGP46381 did not significantly affect the increase by forskolin (Figure 2A). Furthermore, 3T3-L1 cells were cultured for 8 to 16 days in either the presence or absence of the GABABR agonist baclofen or the GABABR antagonist saclofen, followed by determination of the lipid droplet accumulation with Oil red O staining. As shown in Figure 2B, lipid droplet accumulation was detected in 3T3-L1 cells cultured for 8 days with a marked increase at 16 days, while sustained exposure to either baclofen or saclofen failed to significantly affect the lipid droplet accumulation throughout the culture periods (Figure 2C). For further confirmation of the absence of functional GABABR from adipocytic cells, EF was isolated from mice defective of GABABR1 subunit, followed by culture in standard adipogenic induction medium to promote differentiate into adipocytes for subsequent Oil red O staining. As seen in 3T3-L1 cells, lipid droplet was drastically accumulated in EF cultured for 5 to 16 days irrespective of the knockout of GABABR1 subunit, whereas no significant difference was detected in lipid droplet accumulation throughout the cultured periods up to 16 days between EF isolated from WT and GABABR1- mice (Figure 2D). Thus, GABABR1 subunit alone would be constitutively expressed by adipocytic cells devoid of the partner GABABR2 subunit required for functional GABABR assembly.


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)

Lack of effects of GABABR ligands on adipocytic differentiation.(A) 3T3-L1 cells were transfected with the luciferase vector containing 4 tandem copies of CRE site, followed by exposure to the GABABR agonist baclofen, the GABABR antagonist CPG46381 or forskolin for 24 h and subsequent determination of luciferase activity. 3T3-L1 cells were cultured for 8 to 16 days, in either the presence or absence of baclofen and the antagonist saclofen, followed by Oil red O staining. Typical micrographic pictures are shown in the panel (B), while in the panel (C) quantitative data are shown as the mean ± S.E in 5 independent determinations. (D) EF was isolated from WT and GABABR1- mice, followed by culture for 5 to 16 days and subsequent staining with Oil red O. Values are the mean ± S.E. from 6 to 11 different experiments. Neither agonist nor antagonist for GABABR exhibited significant effects on adipogenesis. Ba, baclofen; Gb1, GABABR1; Sa, saclofen.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105007&req=5

pone-0020167-g002: Lack of effects of GABABR ligands on adipocytic differentiation.(A) 3T3-L1 cells were transfected with the luciferase vector containing 4 tandem copies of CRE site, followed by exposure to the GABABR agonist baclofen, the GABABR antagonist CPG46381 or forskolin for 24 h and subsequent determination of luciferase activity. 3T3-L1 cells were cultured for 8 to 16 days, in either the presence or absence of baclofen and the antagonist saclofen, followed by Oil red O staining. Typical micrographic pictures are shown in the panel (B), while in the panel (C) quantitative data are shown as the mean ± S.E in 5 independent determinations. (D) EF was isolated from WT and GABABR1- mice, followed by culture for 5 to 16 days and subsequent staining with Oil red O. Values are the mean ± S.E. from 6 to 11 different experiments. Neither agonist nor antagonist for GABABR exhibited significant effects on adipogenesis. Ba, baclofen; Gb1, GABABR1; Sa, saclofen.
Mentions: In order to evaluate the possible function of GABABR1 subunit expressed by adipocytes as metabotropic GABABR, an attempt was made to determine whether GABABR ligands affect luciferase activity in 3T3-L1 cells transfected with a cAMP responsive element (CRE) reporter plasmid. Exposure to forskolin resulted in a drastic increase in the luciferase activity in 3T3-L1 cells transfected with a CRE reporter plasmid, whereas the further addition of the GABABR agonist baclofen or the GABABR antagonist CGP46381 did not significantly affect the increase by forskolin (Figure 2A). Furthermore, 3T3-L1 cells were cultured for 8 to 16 days in either the presence or absence of the GABABR agonist baclofen or the GABABR antagonist saclofen, followed by determination of the lipid droplet accumulation with Oil red O staining. As shown in Figure 2B, lipid droplet accumulation was detected in 3T3-L1 cells cultured for 8 days with a marked increase at 16 days, while sustained exposure to either baclofen or saclofen failed to significantly affect the lipid droplet accumulation throughout the culture periods (Figure 2C). For further confirmation of the absence of functional GABABR from adipocytic cells, EF was isolated from mice defective of GABABR1 subunit, followed by culture in standard adipogenic induction medium to promote differentiate into adipocytes for subsequent Oil red O staining. As seen in 3T3-L1 cells, lipid droplet was drastically accumulated in EF cultured for 5 to 16 days irrespective of the knockout of GABABR1 subunit, whereas no significant difference was detected in lipid droplet accumulation throughout the cultured periods up to 16 days between EF isolated from WT and GABABR1- mice (Figure 2D). Thus, GABABR1 subunit alone would be constitutively expressed by adipocytic cells devoid of the partner GABABR2 subunit required for functional GABABR assembly.

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