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Molecular mechanisms for activation of the agouti-related protein and stimulation of appetite.

Ilnytska O, Stütz AM, Park-York M, York DA, Ribnicky DM, Zuberi A, Cefalu WT, Argyropoulos G - Diabetes (2010)

Bottom Line: We found that the Krüppel-like factor 4 (Klf4) is a potent activator of Agrp by binding to a specific CACCC-box in its minimal promoter.We also found that an extract of tarragon, termed PMI-5011, activated hypothalamic Klf4 and Agrp.In addition, the tarragon extract PMI-5011 activated Klf4 and orexigenic neuropeptides and reduced peripheral insulin and leptin levels leading to positive energy balance.

View Article: PubMed Central - PubMed

Affiliation: Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, USA.

ABSTRACT

Objective: The agouti-related protein (Agrp) is a powerful orexigenic peptide, but little is known about its transcriptional regulation. The objective of this study was to determine molecular mechanisms for the activation of hypothalamic Agrp and identify compounds that stimulate appetite.

Research design and methods: We used promoter analyses methods, hypothalamic cell culture and transfection, immunohistochemistry, luciferase-expressing transgenic mice, in vivo bioluminescence, anitisense RNA, mouse feeding studies, indirect calorimetry, real-time PCR, and Western blots.

Results: We found that the Krüppel-like factor 4 (Klf4) is a potent activator of Agrp by binding to a specific CACCC-box in its minimal promoter. We also found that an extract of tarragon, termed PMI-5011, activated hypothalamic Klf4 and Agrp. In vivo, PMI-5011 increased Agrp promoter activity in luciferase-expressing transgenic mice, increased hypothalamic Klf4 and Agrp expression, increased hypothalamic Orexin and melanin-concentrating hormone, increased food intake, reduced circulating insulin and leptin levels, attenuated energy expenditure, and enhanced body weight but only when using a high-fat diet.

Conclusions: These data show that Klf4 augmented hypothalamic Agrp by binding to a specific CACCC-box onto its minimal promoter. In addition, the tarragon extract PMI-5011 activated Klf4 and orexigenic neuropeptides and reduced peripheral insulin and leptin levels leading to positive energy balance.

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AGRP promoter analysis showed KLF4 specificity for the proximal CACCC-box. A: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box significantly diminished the effect of KLF4 on AGRP promoter activity in transiently transfected clonal hypothalamic N38 cells. B: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box completely abrogated the effect of KLF4 on AGRP promoter activity in transiently transfected human adrenocarcinoma NCI-h295R cells. C: Sp1 did not increase AGRP promoter activity on its own or when cotransfected with KLF4, whereas mutagenesis of the proximal CACCC-box (mutA) abrogated the effect of KLF4 on its own or in combination with Sp1. D: EMSA experiments using wild-type (Wt) and mutant (mutA) competition (Compet) primers. E: Antibody-supershift EMSA confirmed that Klf4 directly bound the promoter of AGRP at the proximal CACCC-box (mutA). Nuclear extracts (NE) were obtained from clonal hypothalamic N38 cells transfected either with the empty-vector pcDNA3.1 (P) or the KLF4 expression vector (K). The position of the KLF4-containing protein complex is marked by an arrow. Basic transcription element (BTE) corresponds to a known KLF4 binding site as the BTE of the CYP1A1 gene (30), as a positive control. F: Chromatin immunoprecipitation (ChIP) assays, using lysates from KLF4-transfected cells (second bar) or cells cotransfected with a combination of KLF4− and AGRP−796/+373 promoter. Increased immunoprecipitates were found in the cotransfected cells because of enrichment for the CACCC-box (RLA, relative luciferase activity adjusted by renilla). Data are shown as means ± SE (*P < 0.05; **P < 0.01; ***P < 0.001).
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Figure 2: AGRP promoter analysis showed KLF4 specificity for the proximal CACCC-box. A: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box significantly diminished the effect of KLF4 on AGRP promoter activity in transiently transfected clonal hypothalamic N38 cells. B: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box completely abrogated the effect of KLF4 on AGRP promoter activity in transiently transfected human adrenocarcinoma NCI-h295R cells. C: Sp1 did not increase AGRP promoter activity on its own or when cotransfected with KLF4, whereas mutagenesis of the proximal CACCC-box (mutA) abrogated the effect of KLF4 on its own or in combination with Sp1. D: EMSA experiments using wild-type (Wt) and mutant (mutA) competition (Compet) primers. E: Antibody-supershift EMSA confirmed that Klf4 directly bound the promoter of AGRP at the proximal CACCC-box (mutA). Nuclear extracts (NE) were obtained from clonal hypothalamic N38 cells transfected either with the empty-vector pcDNA3.1 (P) or the KLF4 expression vector (K). The position of the KLF4-containing protein complex is marked by an arrow. Basic transcription element (BTE) corresponds to a known KLF4 binding site as the BTE of the CYP1A1 gene (30), as a positive control. F: Chromatin immunoprecipitation (ChIP) assays, using lysates from KLF4-transfected cells (second bar) or cells cotransfected with a combination of KLF4− and AGRP−796/+373 promoter. Increased immunoprecipitates were found in the cotransfected cells because of enrichment for the CACCC-box (RLA, relative luciferase activity adjusted by renilla). Data are shown as means ± SE (*P < 0.05; **P < 0.01; ***P < 0.001).

Mentions: The effect of KLF4 on the AGRP promoter was evaluated by site directed mutagenesis of the two candidate CACCC-boxes at positions A and B. The mouse clonal hypothalamic cell line (N38) and the human adrenocortical carcinoma cell line NCI-h295R were used for these experiments because they both express endogenous Agrp whereas the NCI-h295R cell line also represents a “human” environment because we are using the human AGRP promoter. Mutagenesis of the proximal (position A or mutA) but not the distal CACCC-box (mutB) diminished significantly the effect of KLF4 on AGRP promoter in the mouse N38 (Fig. 2A) and completely abolished promoter activity in the human environment of the NCI-h295R cells (Fig. 2B). Because KLF4 had been suggested to interact with the Sp1 family of transcription factors (30), we used a Sp1 expression construct but found that it had no effect on AGRP promoter activity, and neither did it enhance the effect of KLF4 (Fig. 2C).


Molecular mechanisms for activation of the agouti-related protein and stimulation of appetite.

Ilnytska O, Stütz AM, Park-York M, York DA, Ribnicky DM, Zuberi A, Cefalu WT, Argyropoulos G - Diabetes (2010)

AGRP promoter analysis showed KLF4 specificity for the proximal CACCC-box. A: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box significantly diminished the effect of KLF4 on AGRP promoter activity in transiently transfected clonal hypothalamic N38 cells. B: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box completely abrogated the effect of KLF4 on AGRP promoter activity in transiently transfected human adrenocarcinoma NCI-h295R cells. C: Sp1 did not increase AGRP promoter activity on its own or when cotransfected with KLF4, whereas mutagenesis of the proximal CACCC-box (mutA) abrogated the effect of KLF4 on its own or in combination with Sp1. D: EMSA experiments using wild-type (Wt) and mutant (mutA) competition (Compet) primers. E: Antibody-supershift EMSA confirmed that Klf4 directly bound the promoter of AGRP at the proximal CACCC-box (mutA). Nuclear extracts (NE) were obtained from clonal hypothalamic N38 cells transfected either with the empty-vector pcDNA3.1 (P) or the KLF4 expression vector (K). The position of the KLF4-containing protein complex is marked by an arrow. Basic transcription element (BTE) corresponds to a known KLF4 binding site as the BTE of the CYP1A1 gene (30), as a positive control. F: Chromatin immunoprecipitation (ChIP) assays, using lysates from KLF4-transfected cells (second bar) or cells cotransfected with a combination of KLF4− and AGRP−796/+373 promoter. Increased immunoprecipitates were found in the cotransfected cells because of enrichment for the CACCC-box (RLA, relative luciferase activity adjusted by renilla). Data are shown as means ± SE (*P < 0.05; **P < 0.01; ***P < 0.001).
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Related In: Results  -  Collection

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Figure 2: AGRP promoter analysis showed KLF4 specificity for the proximal CACCC-box. A: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box significantly diminished the effect of KLF4 on AGRP promoter activity in transiently transfected clonal hypothalamic N38 cells. B: Mutagenesis of the proximal (mutA) but not the distal (mutB) CACCC-box completely abrogated the effect of KLF4 on AGRP promoter activity in transiently transfected human adrenocarcinoma NCI-h295R cells. C: Sp1 did not increase AGRP promoter activity on its own or when cotransfected with KLF4, whereas mutagenesis of the proximal CACCC-box (mutA) abrogated the effect of KLF4 on its own or in combination with Sp1. D: EMSA experiments using wild-type (Wt) and mutant (mutA) competition (Compet) primers. E: Antibody-supershift EMSA confirmed that Klf4 directly bound the promoter of AGRP at the proximal CACCC-box (mutA). Nuclear extracts (NE) were obtained from clonal hypothalamic N38 cells transfected either with the empty-vector pcDNA3.1 (P) or the KLF4 expression vector (K). The position of the KLF4-containing protein complex is marked by an arrow. Basic transcription element (BTE) corresponds to a known KLF4 binding site as the BTE of the CYP1A1 gene (30), as a positive control. F: Chromatin immunoprecipitation (ChIP) assays, using lysates from KLF4-transfected cells (second bar) or cells cotransfected with a combination of KLF4− and AGRP−796/+373 promoter. Increased immunoprecipitates were found in the cotransfected cells because of enrichment for the CACCC-box (RLA, relative luciferase activity adjusted by renilla). Data are shown as means ± SE (*P < 0.05; **P < 0.01; ***P < 0.001).
Mentions: The effect of KLF4 on the AGRP promoter was evaluated by site directed mutagenesis of the two candidate CACCC-boxes at positions A and B. The mouse clonal hypothalamic cell line (N38) and the human adrenocortical carcinoma cell line NCI-h295R were used for these experiments because they both express endogenous Agrp whereas the NCI-h295R cell line also represents a “human” environment because we are using the human AGRP promoter. Mutagenesis of the proximal (position A or mutA) but not the distal CACCC-box (mutB) diminished significantly the effect of KLF4 on AGRP promoter in the mouse N38 (Fig. 2A) and completely abolished promoter activity in the human environment of the NCI-h295R cells (Fig. 2B). Because KLF4 had been suggested to interact with the Sp1 family of transcription factors (30), we used a Sp1 expression construct but found that it had no effect on AGRP promoter activity, and neither did it enhance the effect of KLF4 (Fig. 2C).

Bottom Line: We found that the Krüppel-like factor 4 (Klf4) is a potent activator of Agrp by binding to a specific CACCC-box in its minimal promoter.We also found that an extract of tarragon, termed PMI-5011, activated hypothalamic Klf4 and Agrp.In addition, the tarragon extract PMI-5011 activated Klf4 and orexigenic neuropeptides and reduced peripheral insulin and leptin levels leading to positive energy balance.

View Article: PubMed Central - PubMed

Affiliation: Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, USA.

ABSTRACT

Objective: The agouti-related protein (Agrp) is a powerful orexigenic peptide, but little is known about its transcriptional regulation. The objective of this study was to determine molecular mechanisms for the activation of hypothalamic Agrp and identify compounds that stimulate appetite.

Research design and methods: We used promoter analyses methods, hypothalamic cell culture and transfection, immunohistochemistry, luciferase-expressing transgenic mice, in vivo bioluminescence, anitisense RNA, mouse feeding studies, indirect calorimetry, real-time PCR, and Western blots.

Results: We found that the Krüppel-like factor 4 (Klf4) is a potent activator of Agrp by binding to a specific CACCC-box in its minimal promoter. We also found that an extract of tarragon, termed PMI-5011, activated hypothalamic Klf4 and Agrp. In vivo, PMI-5011 increased Agrp promoter activity in luciferase-expressing transgenic mice, increased hypothalamic Klf4 and Agrp expression, increased hypothalamic Orexin and melanin-concentrating hormone, increased food intake, reduced circulating insulin and leptin levels, attenuated energy expenditure, and enhanced body weight but only when using a high-fat diet.

Conclusions: These data show that Klf4 augmented hypothalamic Agrp by binding to a specific CACCC-box onto its minimal promoter. In addition, the tarragon extract PMI-5011 activated Klf4 and orexigenic neuropeptides and reduced peripheral insulin and leptin levels leading to positive energy balance.

Show MeSH