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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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Klf4 overexpression increased Agrp in GT1-7 cells, whereas overnight food deprivation augmented hypothalamic Agrp and Klf4. A: The minimal promoter of AGRP contains two CACCC-boxes (underlined) that are putative bindings sites for KLF4 (proximal [A] and distal [B]). B: Transient transfection of hypothalamic GT1-7 cells with a KLF4 expression construct increased AGRP mRNA and (D) Agrp protein levels. C and E: KLF4 overexpression also increased endogenous Agrp mRNA and protein levels in mouse whole-brain primary cultures from two male mice, respectively. F: Immunocytochemistry of dividing GT1-7 hypothalamic cells displaying elevated Klf4 and Agrp protein levels. G: Transiently transfected GT1-7 cells with a KLF4 expression construct displayed accumulation of KLF4 in the nucleus (arrows) and overexpression of endogenous Agrp. Hypothalamic Agrp mRNA (H) and Klf4 mRNA (I) were significantly increased in mice that were food deprived overnight (n = 6 fed mice, n = 7 food-deprived mice). AU, arbitrary units representing Klf4 or Agrp mRNA levels adjusted by cyclophilin mRNA as endogenous control. Data are shown as means ± SE (*P < 0.05; **P < 0.01). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 1: Klf4 overexpression increased Agrp in GT1-7 cells, whereas overnight food deprivation augmented hypothalamic Agrp and Klf4. A: The minimal promoter of AGRP contains two CACCC-boxes (underlined) that are putative bindings sites for KLF4 (proximal [A] and distal [B]). B: Transient transfection of hypothalamic GT1-7 cells with a KLF4 expression construct increased AGRP mRNA and (D) Agrp protein levels. C and E: KLF4 overexpression also increased endogenous Agrp mRNA and protein levels in mouse whole-brain primary cultures from two male mice, respectively. F: Immunocytochemistry of dividing GT1-7 hypothalamic cells displaying elevated Klf4 and Agrp protein levels. G: Transiently transfected GT1-7 cells with a KLF4 expression construct displayed accumulation of KLF4 in the nucleus (arrows) and overexpression of endogenous Agrp. Hypothalamic Agrp mRNA (H) and Klf4 mRNA (I) were significantly increased in mice that were food deprived overnight (n = 6 fed mice, n = 7 food-deprived mice). AU, arbitrary units representing Klf4 or Agrp mRNA levels adjusted by cyclophilin mRNA as endogenous control. Data are shown as means ± SE (*P < 0.05; **P < 0.01). (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Algorithmic analysis of the human AGRP promoter (Tess/Transfac and Alibaba2) revealed the presence of two conserved CACCC-boxes at position +163/+169 (position A or proximal position), and position −277/−283 that is a reverse CACCC-box/G-rich stretch (position B or distal position) (Fig. 1A). CACCC-boxes are typical binding motifs for the transcription factor KLF4 (12–15), which became a candidate effector of AGRP. The direct effect of KLF4 on Agrp was tested by transiently transfecting GT1-7 cells with a KLF4 expression construct, which resulted in an increase of endogenous Agrp mRNA and protein levels (Fig. 1B and D, respectively). This effect was recapitulated in mouse whole-brain primary cultures from two male mice (Fig. 1C and E).


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)

Klf4 overexpression increased Agrp in GT1-7 cells, whereas overnight food deprivation augmented hypothalamic Agrp and Klf4. A: The minimal promoter of AGRP contains two CACCC-boxes (underlined) that are putative bindings sites for KLF4 (proximal [A] and distal [B]). B: Transient transfection of hypothalamic GT1-7 cells with a KLF4 expression construct increased AGRP mRNA and (D) Agrp protein levels. C and E: KLF4 overexpression also increased endogenous Agrp mRNA and protein levels in mouse whole-brain primary cultures from two male mice, respectively. F: Immunocytochemistry of dividing GT1-7 hypothalamic cells displaying elevated Klf4 and Agrp protein levels. G: Transiently transfected GT1-7 cells with a KLF4 expression construct displayed accumulation of KLF4 in the nucleus (arrows) and overexpression of endogenous Agrp. Hypothalamic Agrp mRNA (H) and Klf4 mRNA (I) were significantly increased in mice that were food deprived overnight (n = 6 fed mice, n = 7 food-deprived mice). AU, arbitrary units representing Klf4 or Agrp mRNA levels adjusted by cyclophilin mRNA as endogenous control. Data are shown as means ± SE (*P < 0.05; **P < 0.01). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 1: Klf4 overexpression increased Agrp in GT1-7 cells, whereas overnight food deprivation augmented hypothalamic Agrp and Klf4. A: The minimal promoter of AGRP contains two CACCC-boxes (underlined) that are putative bindings sites for KLF4 (proximal [A] and distal [B]). B: Transient transfection of hypothalamic GT1-7 cells with a KLF4 expression construct increased AGRP mRNA and (D) Agrp protein levels. C and E: KLF4 overexpression also increased endogenous Agrp mRNA and protein levels in mouse whole-brain primary cultures from two male mice, respectively. F: Immunocytochemistry of dividing GT1-7 hypothalamic cells displaying elevated Klf4 and Agrp protein levels. G: Transiently transfected GT1-7 cells with a KLF4 expression construct displayed accumulation of KLF4 in the nucleus (arrows) and overexpression of endogenous Agrp. Hypothalamic Agrp mRNA (H) and Klf4 mRNA (I) were significantly increased in mice that were food deprived overnight (n = 6 fed mice, n = 7 food-deprived mice). AU, arbitrary units representing Klf4 or Agrp mRNA levels adjusted by cyclophilin mRNA as endogenous control. Data are shown as means ± SE (*P < 0.05; **P < 0.01). (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Algorithmic analysis of the human AGRP promoter (Tess/Transfac and Alibaba2) revealed the presence of two conserved CACCC-boxes at position +163/+169 (position A or proximal position), and position −277/−283 that is a reverse CACCC-box/G-rich stretch (position B or distal position) (Fig. 1A). CACCC-boxes are typical binding motifs for the transcription factor KLF4 (12–15), which became a candidate effector of AGRP. The direct effect of KLF4 on Agrp was tested by transiently transfecting GT1-7 cells with a KLF4 expression construct, which resulted in an increase of endogenous Agrp mRNA and protein levels (Fig. 1B and D, respectively). This effect was recapitulated in mouse whole-brain primary cultures from two male mice (Fig. 1C and E).

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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