Hypothalamic AMP-activated protein kinase regulates glucose production

Abstract

Objective: The fuel sensor AMP-activated protein kinase (AMPK) in the hypothalamus regulates energy homeostasis by sensing nutritional and hormonal signals. However, the role of hypothalamic AMPK in glucose production regulation remains to be elucidated. We hypothesize that bidirectional changes in hypothalamic AMPK activity alter glucose production.

Research design and methods: To introduce bidirectional changes in hypothalamic AMPK activity in vivo, we first knocked down hypothalamic AMPK activity in male Sprague-Dawley rats by either injecting an adenovirus expressing the dominant-negative form of AMPK (Ad-DN AMPKα2 [D(157)A]) or infusing AMPK inhibitor compound C directly into the mediobasal hypothalamus. Next, we independently activated hypothalamic AMPK by delivering either an adenovirus expressing the constitutive active form of AMPK (Ad-CA AMPKα1(312) [T172D]) or the AMPK activator AICAR. The pancreatic (basal insulin)-euglycemic clamp technique in combination with the tracer-dilution methodology was used to assess the impact of alternations in hypothalamic AMPK activity on changes in glucose kinetics in vivo.

Results: Injection of Ad-DN AMPK into the hypothalamus knocked down hypothalamic AMPK activity and led to a significant suppression of glucose production with no changes in peripheral glucose uptake during the clamps. In parallel, hypothalamic infusion of AMPK inhibitor compound C lowered glucose production as well. Conversely, molecular and pharmacological activation of hypothalamic AMPK negated the ability of hypothalamic nutrients to lower glucose production.

Conclusions: These data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo.

FIG. 1.

Molecular knockdown of hypothalamic AMPK by the dominant-negative form of AMPK (DN AMPK) is sufficient to lower glucose production. A: Schematic representation of the working hypothesis: Inhibition of hypothalamic AMPK activity by DN AMPK or compound C leads to the lowering of hepatic glucose production. B: Experimental procedure and clamp protocol. A bilateral MBH catheter was implanted on day 0. Adenovirus tagged with GFP (Ad-GFP) or adenovirus-expressing DN AMPK (Ad-DN AMPK) was injected into the MBH of a group of rats immediately after MBH catheter implantation. Venous and arterial cannulations were done on day 5, and the pancreatic clamp protocol was performed on day 8. In the Ad-GFP and Ad-DN AMPK–injected rats, no MBH infusions were given during the clamp experiments. In rats with no adenovirus injection, 5% DMSO control or compound C was infused into the MBH during the clamps. C: Hypothalamic AMPK activity was significantly diminished in animals injected with Ad-DN AMPK, compared with control animals with injection of Ad-GFP (*P < 0.001). Hypothalamic injection of Ad-DN AMPK led to an increase in glucose infusion rate (D) (*P < 0.01) and a decrease in glucose production (E) (*P < 0.001) compared with the GFP control. F: Suppression of glucose production during the clamp period (180–210 min) expressed as percentage reduction from basal steady state (60–90 min) (*P < 0.01 vs. GFP control). G: Glucose uptake was not significantly different from that of GFP control. Values are shown as means ± SEM. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

Hypothalamic administration of compound C, the pharmacological inhibitor of AMPK, lowers glucose production. Direct infusion of compound C (Cmpd C), the pharmacological inhibitor of AMPK, into the MBH significantly increased the glucose infusion rate (A) (*P < 0.001) and decreased the glucose production (B) (*P < 0.05) during the clamps compared with the 5% DMSO control group. C: Suppression of glucose production during the clamp period (180–210 min) expressed as the percentage reduction from the basal steady state (60–90 min) (*P < 0.001). D: Glucose uptake in the compound C–treated group did not differ significantly from that of the 5% DMSO treated–control group. Values are shown as means ± SE.

FIG. 3.

Hypothalamic administration of AICAR, the pharmacological activator of AMPK, negates the ability of hypothalamic glucose/lactate-sensing mechanisms to decrease glucose production. A: Schematic representation of the working hypothesis: Activation of hypothalamic AMPK by AICAR or the constitutively active form of AMPK (CA AMPK) prevents the ability of hypothalamic glucose/lactate to decrease glucose production. B: Experimental procedure and clamp protocol. A bilateral MBH catheter was implanted on day 0. Adenovirus tagged with GFP (Ad-GFP) or adenovirus expressing CA AMPK (Ad-CA AMPK) was injected into the MBH of a group of rats immediately after MBH catheter implantation. Venous and arterial cannulations were done on day 5, and the pancreatic clamp protocol was performed on day 8. In rats with no adenovirus injection, AICAR, saline, glucose, lactate, AICAR+glucose, or AICAR+lactate was infused into the MBH during the clamp experiments. In rats injected with Ad-GFP or Ad-CA AMPK, saline, glucose, or lactate was infused into the MBH during the clamp studies. C: Direct MBH infusion of glucose or lactate during the clamps increased glucose infusion rate (*P < 0.001) and lowered glucose production (D) (*P < 0.001) compared with those of MBH vehicle (AICAR/saline) treatments. MBH glucose or lactate coinfused with AICAR failed to increase glucose infusion rate (C) and lower glucose production (D) compared with those of vehicle treatments. E: Suppression of glucose production during the clamp period (180–210 min) expressed as the percentage reduction from the basal steady state (60–90 min) (*P < 0.05 vs. other groups). F: Glucose uptake was comparable in all groups. Values are shown as means ± SE. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

Hypothalamic administration of the constitutively active form of AMPK (CA AMPK) negates the ability of hypothalamic glucose/lactate-sensing mechanisms to decrease glucose production. Direct MBH administration of glucose or lactate to the GFP treatment groups increased glucose infusion rate (A) (*P < 0.01) and lowered glucose production (B) (*P < 0.001) compared to those of GFP/saline and CA AMPK/saline groups during the clamps. Direct MBH administration of glucose or lactate during the clamps to the CA AMPK treatment groups failed to increase glucose infusion rate (A) and lower glucose production (B). C: Suppression of glucose production during the clamp period (180–210 min) expressed as the percentage reduction from the basal steady state (60–90 min) (*P < 0.01 vs. other groups). D: Glucose uptake was comparable in all groups. Values are shown as means ± SE.