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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 profile of marker genes in adipocytes.(A) Total RNA was extracted from EF cultured for 2 days. (B) Total RNA was extracted from EF transfected with GABABR1 expression vector. (C) Total RNA was extracted from epididymal WAT of male mice at 4 weeks old. Values are the mean ± S.E. from 3 to 8 different experiments. *P<0.05; **P<0.01, significantly different from each control value obtained in WT mice. Selective downregulation was seen in leptin mRNA expression in both cultured EF and WAT prepared from GABABR1- mice, while marked upregulation was found in both PGC1α and UCP1 mRNA expression in WAT, but not cultured EF, of GABABR1- mice. Gb1, GABABR1; ND, not detectable.
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pone-0020167-g003: Expression profile of marker genes in adipocytes.(A) Total RNA was extracted from EF cultured for 2 days. (B) Total RNA was extracted from EF transfected with GABABR1 expression vector. (C) Total RNA was extracted from epididymal WAT of male mice at 4 weeks old. Values are the mean ± S.E. from 3 to 8 different experiments. *P<0.05; **P<0.01, significantly different from each control value obtained in WT mice. Selective downregulation was seen in leptin mRNA expression in both cultured EF and WAT prepared from GABABR1- mice, while marked upregulation was found in both PGC1α and UCP1 mRNA expression in WAT, but not cultured EF, of GABABR1- mice. Gb1, GABABR1; ND, not detectable.

Mentions: To next investigate the possible significance other than the partner of functional GABABR of GABABR1 subunit expressed by adipocytic cells, we examined the expression profile of a variety of adipocyte marker genes in cultured EF prepared from GABABR1- mice. Similarly marked expression was seen with mRNA for a variety of adipocytic marker genes related to differentiation and lipolysis between EF from WT and GABABR1- mice (Figure 3A). These included PPARγ, CCAAT/enhancer binding protein-α (C/EBPα), Fabp4, LPL, β3 adrenergic receptor (AR) and perilipin. However, mRNA expression was significantly lower with hormone sensitive lipase (HSL) in EF of GABABR1- mice than that of WT mice. Among different adipocytokines examined, moreover, a significant decrease was seen in mRNA expression for leptin, but not for adiponectin, resistin or tumor necrosis factor-α (TNFα), in EF from GABABR1- mice. Independent of the presence of GABABR1 subunit, by contrast, mRNA expression was not detected with the 2 marker genes related to mitochondrial biogenesis such as PGC1α and uncoupling protein 1 (UCP1) in EF cultured for 2 days. Transfection with GABABR1 expression vector drastically increased GABABR1 mRNA expression in EF cultured for 2 days (data not shown), but failed to prevent the reduction of leptin mRNA expression in EF prepared from GABABR1- mice (Figure 3B).


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 profile of marker genes in adipocytes.(A) Total RNA was extracted from EF cultured for 2 days. (B) Total RNA was extracted from EF transfected with GABABR1 expression vector. (C) Total RNA was extracted from epididymal WAT of male mice at 4 weeks old. Values are the mean ± S.E. from 3 to 8 different experiments. *P<0.05; **P<0.01, significantly different from each control value obtained in WT mice. Selective downregulation was seen in leptin mRNA expression in both cultured EF and WAT prepared from GABABR1- mice, while marked upregulation was found in both PGC1α and UCP1 mRNA expression in WAT, but not cultured EF, of GABABR1- mice. Gb1, GABABR1; ND, not detectable.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020167-g003: Expression profile of marker genes in adipocytes.(A) Total RNA was extracted from EF cultured for 2 days. (B) Total RNA was extracted from EF transfected with GABABR1 expression vector. (C) Total RNA was extracted from epididymal WAT of male mice at 4 weeks old. Values are the mean ± S.E. from 3 to 8 different experiments. *P<0.05; **P<0.01, significantly different from each control value obtained in WT mice. Selective downregulation was seen in leptin mRNA expression in both cultured EF and WAT prepared from GABABR1- mice, while marked upregulation was found in both PGC1α and UCP1 mRNA expression in WAT, but not cultured EF, of GABABR1- mice. Gb1, GABABR1; ND, not detectable.
Mentions: To next investigate the possible significance other than the partner of functional GABABR of GABABR1 subunit expressed by adipocytic cells, we examined the expression profile of a variety of adipocyte marker genes in cultured EF prepared from GABABR1- mice. Similarly marked expression was seen with mRNA for a variety of adipocytic marker genes related to differentiation and lipolysis between EF from WT and GABABR1- mice (Figure 3A). These included PPARγ, CCAAT/enhancer binding protein-α (C/EBPα), Fabp4, LPL, β3 adrenergic receptor (AR) and perilipin. However, mRNA expression was significantly lower with hormone sensitive lipase (HSL) in EF of GABABR1- mice than that of WT mice. Among different adipocytokines examined, moreover, a significant decrease was seen in mRNA expression for leptin, but not for adiponectin, resistin or tumor necrosis factor-α (TNFα), in EF from GABABR1- mice. Independent of the presence of GABABR1 subunit, by contrast, mRNA expression was not detected with the 2 marker genes related to mitochondrial biogenesis such as PGC1α and uncoupling protein 1 (UCP1) in EF cultured for 2 days. Transfection with GABABR1 expression vector drastically increased GABABR1 mRNA expression in EF cultured for 2 days (data not shown), but failed to prevent the reduction of leptin mRNA expression in EF prepared from GABABR1- mice (Figure 3B).

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