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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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PMI-5011 increased Agrp/Klf4 and food intake, but body weight was increased only under an HFD. A: Agrp and Klf4 protein levels were significantly higher in the group receiving PMI-5011 in a chow diet for 24 days. n = 6 in the chow diet group and n = 7 in the chow diet plus PMI-5011 group. B: Total food intake was significantly higher in the PMI-5011–fed group. C: Agrp and Klf4 protein levels were significantly higher in the mice receiving PMI-5011 in HFD (data shown for HFD-1). D: Meta-analysis of the combined HFD studies showed significantly higher body weights for the PMI-5011–fed mice. Data are shown as means ± SE (*P < 0.05).
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Figure 6: PMI-5011 increased Agrp/Klf4 and food intake, but body weight was increased only under an HFD. A: Agrp and Klf4 protein levels were significantly higher in the group receiving PMI-5011 in a chow diet for 24 days. n = 6 in the chow diet group and n = 7 in the chow diet plus PMI-5011 group. B: Total food intake was significantly higher in the PMI-5011–fed group. C: Agrp and Klf4 protein levels were significantly higher in the mice receiving PMI-5011 in HFD (data shown for HFD-1). D: Meta-analysis of the combined HFD studies showed significantly higher body weights for the PMI-5011–fed mice. Data are shown as means ± SE (*P < 0.05).

Mentions: Because body weight was not increased as it would be expected given the increased food intake and reduced energy expenditure, another study was performed to examine the long-term (24-day) effects of PMI-5011 on food intake still under a chow diet. Hypothalamic Klf4 and Agrp protein levels, as well as total food intake, were significantly higher in the PMI-5011–fed group of mice (Fig. 6A and B), but body weight was again unaffected (data not shown). We then examined the hypothesis that a higher fat content may be required to be present in the diet for effects of PMI-5011 to become evident on body weight. We examined the data from three previously performed studies that had used PMI-5011 in a HFD. We found that body weight of the mice receiving the PMI-5011 was enhanced in all three studies and both Agrp and Klf4 were significantly elevated (Fig. 6C). A meta-analysis was then performed using the data from all three studies that showed a statistically significant increase for the overall body weight of mice consuming HFD supplemented with PMI-5011 (Fig. 6D).


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)

PMI-5011 increased Agrp/Klf4 and food intake, but body weight was increased only under an HFD. A: Agrp and Klf4 protein levels were significantly higher in the group receiving PMI-5011 in a chow diet for 24 days. n = 6 in the chow diet group and n = 7 in the chow diet plus PMI-5011 group. B: Total food intake was significantly higher in the PMI-5011–fed group. C: Agrp and Klf4 protein levels were significantly higher in the mice receiving PMI-5011 in HFD (data shown for HFD-1). D: Meta-analysis of the combined HFD studies showed significantly higher body weights for the PMI-5011–fed mice. Data are shown as means ± SE (*P < 0.05).
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Related In: Results  -  Collection

License
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Figure 6: PMI-5011 increased Agrp/Klf4 and food intake, but body weight was increased only under an HFD. A: Agrp and Klf4 protein levels were significantly higher in the group receiving PMI-5011 in a chow diet for 24 days. n = 6 in the chow diet group and n = 7 in the chow diet plus PMI-5011 group. B: Total food intake was significantly higher in the PMI-5011–fed group. C: Agrp and Klf4 protein levels were significantly higher in the mice receiving PMI-5011 in HFD (data shown for HFD-1). D: Meta-analysis of the combined HFD studies showed significantly higher body weights for the PMI-5011–fed mice. Data are shown as means ± SE (*P < 0.05).
Mentions: Because body weight was not increased as it would be expected given the increased food intake and reduced energy expenditure, another study was performed to examine the long-term (24-day) effects of PMI-5011 on food intake still under a chow diet. Hypothalamic Klf4 and Agrp protein levels, as well as total food intake, were significantly higher in the PMI-5011–fed group of mice (Fig. 6A and B), but body weight was again unaffected (data not shown). We then examined the hypothesis that a higher fat content may be required to be present in the diet for effects of PMI-5011 to become evident on body weight. We examined the data from three previously performed studies that had used PMI-5011 in a HFD. We found that body weight of the mice receiving the PMI-5011 was enhanced in all three studies and both Agrp and Klf4 were significantly elevated (Fig. 6C). A meta-analysis was then performed using the data from all three studies that showed a statistically significant increase for the overall body weight of mice consuming HFD supplemented with PMI-5011 (Fig. 6D).

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