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, 117 (8), 2325-36

AMPK Is Essential for Energy Homeostasis Regulation and Glucose Sensing by POMC and AgRP Neurons

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AMPK Is Essential for Energy Homeostasis Regulation and Glucose Sensing by POMC and AgRP Neurons

Marc Claret et al. J Clin Invest.

Abstract

Hypothalamic AMP-activated protein kinase (AMPK) has been suggested to act as a key sensing mechanism, responding to hormones and nutrients in the regulation of energy homeostasis. However, the precise neuronal populations and cellular mechanisms involved are unclear. The effects of long-term manipulation of hypothalamic AMPK on energy balance are also unknown. To directly address such issues, we generated POMC alpha 2KO and AgRP alpha 2KO mice lacking AMPK alpha2 in proopiomelanocortin- (POMC-) and agouti-related protein-expressing (AgRP-expressing) neurons, key regulators of energy homeostasis. POMC alpha 2KO mice developed obesity due to reduced energy expenditure and dysregulated food intake but remained sensitive to leptin. In contrast, AgRP alpha 2KO mice developed an age-dependent lean phenotype with increased sensitivity to a melanocortin agonist. Electrophysiological studies in AMPK alpha2-deficient POMC or AgRP neurons revealed normal leptin or insulin action but absent responses to alterations in extracellular glucose levels, showing that glucose-sensing signaling mechanisms in these neurons are distinct from those pathways utilized by leptin or insulin. Taken together with the divergent phenotypes of POMC alpha 2KO and AgRP alpha 2KO mice, our findings suggest that while AMPK plays a key role in hypothalamic function, it does not act as a general sensor and integrator of energy homeostasis in the mediobasal hypothalamus.

Figures

Figure 1
Figure 1. Reduction in hypothalamic AMPKα2 activity in POMCα2KO and AgRPα2KO mice.
Detection of deletion of AMPKα2 allele in POMCα2KO (A) and AgRPα2KO mice (B). DNA was extracted from different tissues (T, tail; Hy, hypothalamus; C, cerebral cortex; L, liver; F, fat; M, skeletal muscle; H, heart; K, kidney) and recombination of the floxed AMPKα2 allele detected by PCR. Recombination was only detected in the hypothalamus of POMCα2KO (A) and AgRPα2KO mice (B). A PCR reaction with IL-2 as internal control is also shown. AMPKα2 activity in hypothalamic lysates from POMCα2KO (C; n = 11–13) and AgRPα2KO mice (D; n = 7). (E) AMPKα1 kinase activity in hypothalamic lysates from α1HetPOMCα2KO mice (n = 11–12). All values are mean ± SEM. *P < 0.05; ***P < 0.001.
Figure 2
Figure 2. Mice lacking AMPKα2 in POMC neurons are obese and have increased food intake and reduced energy expenditure.
(A) Weight curves of male control and POMCα2KO mice on a chow diet; n = 8. (B) Percentage body fat determined by DEXA scanning in 19-week-old male control and POMCα2KO mice; n = 6. (C) Twenty-four-hour food intake under ad libitum feeding conditions in 12-week-old male control and POMCα2KO mice; n = 8. (D) Cumulative 24-hour food intake in 12-week-old male control and POMCα2KO mice in response to an overnight fast; n = 8. (E) RMR determined by open-flow respirometry in 18-week-old control and POMCα2KO mice; n = 11 and n = 8, respectively. (F) PPARγ coactivator–1 (Pgc1) and uncoupling-protein 1 (Ucp1) mRNA levels in brown adipose tissue (BAT) assessed by quantitative RT-PCR; n = 5–7. Probes for GAPDH were used to adjust for total RNA content. (G) Weight curves of male control and POMCα2KO mice on exposure to HFD; n = 11–15. P < 0.05 at all time points, except weeks 7 and 9, where P < 0.01. (H) Percentage body fat determined by DEXA scanning in male control and POMCα2KO mice after 18 weeks on a HFD; n = 5. All values are mean ± SEM. *P < 0.05.
Figure 3
Figure 3. Obesity phenotype in POMCα2KO mice is not due to anatomical or functional disruption of POMC neurons or compensatory upregulation of AMPKα1.
Immunoreactivity for α-MSH in ARC of control (A), POMCα2KO (B), and α1KOPOMCα2KO (C) mice. Representative sections from 4 mice for each genotype are presented. Population size and distribution (D and E) for POMC neurons within the ARC in control and POMCα2KO mice (n = 4–6). POMC somatic area (F) and diameter (G) in control and POMCα2KO mice (n = 4–6). A minimum of 500 neurons were analyzed per group. 3V, third ventricle. Scale bars: 50 μm. (H) Weight curves of male control and α1HetPOMCα2KO mice on a chow diet; n = 8. (I) Weight curves of male control and α1HetPOMCα2KO mice on exposure to HFD; n = 10. P < 0.05 at all time points, except weeks 0, 2, 7, 8, 11, 12, 13, 14, and 15, where P < 0.01. All values are mean ± SEM. *P < 0.05.
Figure 4
Figure 4. POMCα2KO mice are sensitive to leptin and a melanocortin agonist.
(A) Fasting leptin levels in 4- and 12-week-old male control and POMCα2KO mice; n = 5–15. (B) Body weight change in male control and POMCα2KO mice on a chow diet after 3 consecutive days treatment with either vehicle or leptin (2 doses of 1.5 mg/kg body weight/d); n = 8. (C) Cumulative food intake in male control and POMCα2KO mice on a chow diet after 3 consecutive days treatment with either vehicle or leptin (2 doses of 1.5 mg/kg body weight/d); n = 8. Cumulative food intake at the times indicated after injection of vehicle or MT-II following an overnight fast in 16-week-old male control (D) and POMCα2KO mice (E); n = 8. (F) Agrp/Pomc and (G) Npy/Pomc mRNA expression ratio in control and POMCα2KO mice assessed by quantitative RT-PCR; n = 9–10. Probes for hypoxanthine guanine phosphoribosyl transferase (HPRT) were used to adjust for total RNA content. All values are mean ± SEM. *P < 0.05; ***P < 0.001.
Figure 5
Figure 5. Mice lacking AMPKα2 in AgRP neurons display an age-dependent lean phenotype and have increased sensitivity to a melanocortin agonist.
(A) Weight curves of male control and AgRPα2KO mice on a chow diet; n = 40. (B and C) Twenty-four-hour food intake under ad libitum feeding conditions in 12- and 26-week-old male control and AgRPα2KO mice on a chow diet; n = 8. (D and E) Cumulative 24-hour food intake in 12- and 26-week-old male control and AgRPα2KO mice in response to an overnight fast; n = 8. (F) Weight curves of male control and AgRPα2KO mice on exposure to HFD; n = 8. Cumulative food intake at the times indicated after injection of vehicle or MT-II following an overnight fast in 16-week-old male control (G) and AgRPα2KO mice (H); n = 6. (I) Percentage reduction in 24-hour food intake after injection of MT-II following an overnight fast in 16-week-old male control and AgRPα2KO mice; n = 6. All values are mean ± SEM. *P < 0.05; **P < 0.01.
Figure 6
Figure 6. POMC neurons lacking AMPKα2 respond to anorexigenic hormones but are glucose insensitive.
Current-clamp recordings were made using the perforated patch technique from POMCα2KO (A, B, and D) and control (C) POMC ARC neurons. Ten nanomolar leptin (A) and 20 nM insulin (B) were locally applied for 1–2 minutes (where indicated), inducing depolarization and hyperpolarization, respectively. The leptin-induced depolarization and insulin-induced hyperpolarization were associated with increased and decreased action potential frequency, respectively, as shown in the expanded section and in subsequent figures (lower panels). Note that spike amplitudes are truncated in the expanded sections to demonstrate changes in Vm. Reducing glucose from 2 to 0.1 mM reversibly hyperpolarizes and reduces firing frequency in control POMC neurons (C) but has no effect in POMC neurons lacking the AMPKα2 subunit (D). The broken white line in the traces represents 0 mV.
Figure 7
Figure 7. A minority of AgRP neurons are glucose responsive, a property absent in AgRPα2KO mice.
Perforated patch, current-clamp recordings were made from control (A and C) and AMPKα2KO (B and E) AgRP ARC neurons. Control (A) and AMPKα2-deleted (B) AgRP neurons were depolarized by locally applied insulin (20 nM, where indicated). (C) A minority (n = 4 of 14) of AgRP neurons respond in a concentration dependent and reversible manner to reduction (2 to 0.1 mM) in external glucose by membrane hyperpolarization. (D) Representative glucose dose response curve for the recording shown in C. (E) AgRPα2KO neurons do not respond to reduced external glucose. The broken white line in the traces represents 0 mV.

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