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Mediobasal hypothalamic SIRT1 is essential for resveratrol's effects on insulin action in rats.

Knight CM, Gutierrez-Juarez R, Lam TK, Arrieta-Cruz I, Huang L, Schwartz G, Barzilai N, Rossetti L - Diabetes (2011)

Bottom Line: Selective inhibition of hypothalamic SIRT1 using a cell-permeable SIRT1 inhibitor or SIRT1-shRNA negated the effect of central and peripheral resveratrol on glucose production.Blockade of the K(ATP) channel and hepatic vagotomy significantly attenuated the effect of central resveratrol on hepatic glucose production.In addition, we found no evidence for hypothalamic AMPK activation after MBH resveratrol administration.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Diabetes Research Center, Albert EinsteinCollege of Medicine, Bronx, New York, USA. colette.knight@einstein.yu.edu

ABSTRACT

Objective: Sirtuin 1 (SIRT1) and its activator resveratrol are emerging as major regulators of metabolic processes. We investigate the site of resveratrol action on glucose metabolism and the contribution of SIRT1 to these effects. Because the arcuate nucleus in the mediobasal hypothalamus (MBH) plays a pivotal role in integrating peripheral metabolic responses to nutrients and hormones, we examined whether the actions of resveratrol are mediated at the MBH.

Research design and methods: Sprague Dawley (SD) male rats received acute central (MBH) or systemic injections of vehicle, resveratrol, or SIRT1 inhibitor during basal pancreatic insulin clamp studies. To delineate the pathway(s) by which MBH resveratrol modulates hepatic glucose production, we silenced hypothalamic SIRT1 expression using a short hairpin RNA (shRNA) inhibited the hypothalamic ATP-sensitive potassium (K(ATP)) channel with glibenclamide, or selectively transected the hepatic branch of the vagus nerve while infusing resveratrol centrally.

Results: Our studies show that marked improvement in insulin sensitivity can be elicited by acute administration of resveratrol to the MBH or during acute systemic administration. Selective inhibition of hypothalamic SIRT1 using a cell-permeable SIRT1 inhibitor or SIRT1-shRNA negated the effect of central and peripheral resveratrol on glucose production. Blockade of the K(ATP) channel and hepatic vagotomy significantly attenuated the effect of central resveratrol on hepatic glucose production. In addition, we found no evidence for hypothalamic AMPK activation after MBH resveratrol administration.

Conclusions: Taken together, these studies demonstrate that resveratrol improves glucose homeostasis mainly through a central SIRT1-dependent pathway and that the MBH is a major site of resveratrol action.

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Effect of hypothalamic SIRT1 silencing on glucose production. A: Representative Western blot analysis of SIRT1 knockdown in HEK293 cells. SIRT1-shRNA decreases SIRT1 expression by ∼50% compared with control virus. B: Immunohistochemical staining of SIRT1 in the arcuate nucleus. C: Timeline of injections for viral constructs and animal preparation for the study. D: Schematic of MBH infusions and insulin clamp protocol. E–H: Effect of the SIRT1-shRNA on GIR, glucose production, and glucose uptake in animals treated with and without resveratrol. Experiments represent n = 6–8 per group. All values are mean ± SEM. *P < 0.05, **P < 0.005 SIRT1-shRNA compared with control-shRNA after resveratrol treatment; #P < 0.05 in control-shRNA group after resveratrol vs. DMSO treatment. For these studies: control-shRNA, white bars; SIRT1-shRNA, black bars. Arrowheads indicate SIRT1 immunoreactive cells. (A high-quality color representation of this figure is available in the online issue.)
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Figure 3: Effect of hypothalamic SIRT1 silencing on glucose production. A: Representative Western blot analysis of SIRT1 knockdown in HEK293 cells. SIRT1-shRNA decreases SIRT1 expression by ∼50% compared with control virus. B: Immunohistochemical staining of SIRT1 in the arcuate nucleus. C: Timeline of injections for viral constructs and animal preparation for the study. D: Schematic of MBH infusions and insulin clamp protocol. E–H: Effect of the SIRT1-shRNA on GIR, glucose production, and glucose uptake in animals treated with and without resveratrol. Experiments represent n = 6–8 per group. All values are mean ± SEM. *P < 0.05, **P < 0.005 SIRT1-shRNA compared with control-shRNA after resveratrol treatment; #P < 0.05 in control-shRNA group after resveratrol vs. DMSO treatment. For these studies: control-shRNA, white bars; SIRT1-shRNA, black bars. Arrowheads indicate SIRT1 immunoreactive cells. (A high-quality color representation of this figure is available in the online issue.)

Mentions: To provide confirmation that central SIRT1 is essential for resveratrol action, we used an shRNA to selectively decrease SIRT1 expression. In cell culture, SIRT1 silencing was verified to be at least 50% by Western blot analysis (Fig. 3A). Immunohistochemical analysis confirmed that hypothalamic injection of the SIRT1-shRNA in rats reduced SIRT1 expression in the MBH compared with control (Fig. 3B, Supplementary Fig. 2A–C). Lower-magnification photomicrographs further confirmed that SIRT1 silencing was restricted to the medial portion of the arcuate nucleus because regions rostral and caudal did not show any significant difference in SIRT1 expression between control and SIRT1-shRNA–treated groups (Supplementary Fig. 2D–I). In preparation for the clamp studies, control-shRNA or SIRT1-shRNA was injected into the MBH immediately after implantation of the hypothalamic cannulae. This was followed by vascular catheterization and clamp studies after 2 weeks (Fig. 3C and D). There were no significant differences between the baseline metabolic characteristics of the control-shRNA and the SIRT1-shRNA group (Supplementary Table 2). When animals treated with the control virus or SIRT1-shRNA was infused with vehicle (5% DMSO) during the clamp study, there was no significant change in hepatic glucose production or glucose uptake between the groups. Resveratrol treatment reduced hepatic glucose production in animals who received the control-shRNA. However, similar to the cell-permeable SIRT1 inhibitor, treatment with the SIRT1-shRNA significantly blocked the effect of resveratrol on hepatic glucose production, resulting in an approximately twofold reduction of the GIR (Fig. 3E–H). Treatment with the SIRT1-shRNA had no effect on AgRP expression, but a slight increase in POMC expression was observed (Supplementary Fig. 2J and K). Taken together, the data indicate that resveratrol mediates its effect on glucose production through an SIRT1-dependent pathway and that inhibition of central SIRT1 by using a cell-permeable inhibitor or shRNA silencing is sufficient to blunt the effect of resveratrol on hepatic glucose metabolism.


Mediobasal hypothalamic SIRT1 is essential for resveratrol's effects on insulin action in rats.

Knight CM, Gutierrez-Juarez R, Lam TK, Arrieta-Cruz I, Huang L, Schwartz G, Barzilai N, Rossetti L - Diabetes (2011)

Effect of hypothalamic SIRT1 silencing on glucose production. A: Representative Western blot analysis of SIRT1 knockdown in HEK293 cells. SIRT1-shRNA decreases SIRT1 expression by ∼50% compared with control virus. B: Immunohistochemical staining of SIRT1 in the arcuate nucleus. C: Timeline of injections for viral constructs and animal preparation for the study. D: Schematic of MBH infusions and insulin clamp protocol. E–H: Effect of the SIRT1-shRNA on GIR, glucose production, and glucose uptake in animals treated with and without resveratrol. Experiments represent n = 6–8 per group. All values are mean ± SEM. *P < 0.05, **P < 0.005 SIRT1-shRNA compared with control-shRNA after resveratrol treatment; #P < 0.05 in control-shRNA group after resveratrol vs. DMSO treatment. For these studies: control-shRNA, white bars; SIRT1-shRNA, black bars. Arrowheads indicate SIRT1 immunoreactive cells. (A high-quality color representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 3: Effect of hypothalamic SIRT1 silencing on glucose production. A: Representative Western blot analysis of SIRT1 knockdown in HEK293 cells. SIRT1-shRNA decreases SIRT1 expression by ∼50% compared with control virus. B: Immunohistochemical staining of SIRT1 in the arcuate nucleus. C: Timeline of injections for viral constructs and animal preparation for the study. D: Schematic of MBH infusions and insulin clamp protocol. E–H: Effect of the SIRT1-shRNA on GIR, glucose production, and glucose uptake in animals treated with and without resveratrol. Experiments represent n = 6–8 per group. All values are mean ± SEM. *P < 0.05, **P < 0.005 SIRT1-shRNA compared with control-shRNA after resveratrol treatment; #P < 0.05 in control-shRNA group after resveratrol vs. DMSO treatment. For these studies: control-shRNA, white bars; SIRT1-shRNA, black bars. Arrowheads indicate SIRT1 immunoreactive cells. (A high-quality color representation of this figure is available in the online issue.)
Mentions: To provide confirmation that central SIRT1 is essential for resveratrol action, we used an shRNA to selectively decrease SIRT1 expression. In cell culture, SIRT1 silencing was verified to be at least 50% by Western blot analysis (Fig. 3A). Immunohistochemical analysis confirmed that hypothalamic injection of the SIRT1-shRNA in rats reduced SIRT1 expression in the MBH compared with control (Fig. 3B, Supplementary Fig. 2A–C). Lower-magnification photomicrographs further confirmed that SIRT1 silencing was restricted to the medial portion of the arcuate nucleus because regions rostral and caudal did not show any significant difference in SIRT1 expression between control and SIRT1-shRNA–treated groups (Supplementary Fig. 2D–I). In preparation for the clamp studies, control-shRNA or SIRT1-shRNA was injected into the MBH immediately after implantation of the hypothalamic cannulae. This was followed by vascular catheterization and clamp studies after 2 weeks (Fig. 3C and D). There were no significant differences between the baseline metabolic characteristics of the control-shRNA and the SIRT1-shRNA group (Supplementary Table 2). When animals treated with the control virus or SIRT1-shRNA was infused with vehicle (5% DMSO) during the clamp study, there was no significant change in hepatic glucose production or glucose uptake between the groups. Resveratrol treatment reduced hepatic glucose production in animals who received the control-shRNA. However, similar to the cell-permeable SIRT1 inhibitor, treatment with the SIRT1-shRNA significantly blocked the effect of resveratrol on hepatic glucose production, resulting in an approximately twofold reduction of the GIR (Fig. 3E–H). Treatment with the SIRT1-shRNA had no effect on AgRP expression, but a slight increase in POMC expression was observed (Supplementary Fig. 2J and K). Taken together, the data indicate that resveratrol mediates its effect on glucose production through an SIRT1-dependent pathway and that inhibition of central SIRT1 by using a cell-permeable inhibitor or shRNA silencing is sufficient to blunt the effect of resveratrol on hepatic glucose metabolism.

Bottom Line: Selective inhibition of hypothalamic SIRT1 using a cell-permeable SIRT1 inhibitor or SIRT1-shRNA negated the effect of central and peripheral resveratrol on glucose production.Blockade of the K(ATP) channel and hepatic vagotomy significantly attenuated the effect of central resveratrol on hepatic glucose production.In addition, we found no evidence for hypothalamic AMPK activation after MBH resveratrol administration.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Diabetes Research Center, Albert EinsteinCollege of Medicine, Bronx, New York, USA. colette.knight@einstein.yu.edu

ABSTRACT

Objective: Sirtuin 1 (SIRT1) and its activator resveratrol are emerging as major regulators of metabolic processes. We investigate the site of resveratrol action on glucose metabolism and the contribution of SIRT1 to these effects. Because the arcuate nucleus in the mediobasal hypothalamus (MBH) plays a pivotal role in integrating peripheral metabolic responses to nutrients and hormones, we examined whether the actions of resveratrol are mediated at the MBH.

Research design and methods: Sprague Dawley (SD) male rats received acute central (MBH) or systemic injections of vehicle, resveratrol, or SIRT1 inhibitor during basal pancreatic insulin clamp studies. To delineate the pathway(s) by which MBH resveratrol modulates hepatic glucose production, we silenced hypothalamic SIRT1 expression using a short hairpin RNA (shRNA) inhibited the hypothalamic ATP-sensitive potassium (K(ATP)) channel with glibenclamide, or selectively transected the hepatic branch of the vagus nerve while infusing resveratrol centrally.

Results: Our studies show that marked improvement in insulin sensitivity can be elicited by acute administration of resveratrol to the MBH or during acute systemic administration. Selective inhibition of hypothalamic SIRT1 using a cell-permeable SIRT1 inhibitor or SIRT1-shRNA negated the effect of central and peripheral resveratrol on glucose production. Blockade of the K(ATP) channel and hepatic vagotomy significantly attenuated the effect of central resveratrol on hepatic glucose production. In addition, we found no evidence for hypothalamic AMPK activation after MBH resveratrol administration.

Conclusions: Taken together, these studies demonstrate that resveratrol improves glucose homeostasis mainly through a central SIRT1-dependent pathway and that the MBH is a major site of resveratrol action.

Show MeSH
Related in: MedlinePlus