Metabolic regulation of female puberty via hypothalamic AMPK-kisspeptin signaling

Abstract

Conditions of metabolic distress, from malnutrition to obesity, impact, via as yet ill-defined mechanisms, the timing of puberty, whose alterations can hamper later cardiometabolic health and even life expectancy. AMP-activated protein kinase (AMPK), the master cellular energy sensor activated in conditions of energy insufficiency, has a major central role in whole-body energy homeostasis. However, whether brain AMPK metabolically modulates puberty onset remains unknown. We report here that central AMPK interplays with the puberty-activating gene, Kiss1, to control puberty onset. Pubertal subnutrition, which delayed puberty, enhanced hypothalamic pAMPK levels, while activation of brain AMPK in immature female rats substantially deferred puberty. Virogenetic overexpression of a constitutively active form of AMPK, selectively in the hypothalamic arcuate nucleus (ARC), which holds a key population of Kiss1 neurons, partially delayed puberty onset and reduced luteinizing hormone levels. ARC Kiss1 neurons were found to express pAMPK, and activation of AMPK reduced ARC Kiss1 expression. The physiological relevance of this pathway was attested by conditional ablation of the AMPKα1 subunit in Kiss1 cells, which largely prevented the delay in puberty onset caused by chronic subnutrition. Our data demonstrate that hypothalamic AMPK signaling plays a key role in the metabolic control of puberty, acting via a repressive modulation of ARC Kiss1 neurons in conditions of negative energy balance.

Keywords: AMPK; Kiss1; energy balance; puberty; undernutrition.

Fig. 1.

Metabolic and pharmacological activation of brain AMPK delays puberty onset. In the Upper panels, the impact of chronic subnutrition (UN) on BW (A), time course of VO, as phenotypic sign of puberty onset (B), and hypothalamic pAMPK levels (C) are shown. Peripubertal female rats were subjected to 30% reduction in daily food ration, from PND23 onwards. Age-paired rats fed ad libitum [normal nutrition (NN)] were used as controls. Group sizes were for BW: NN = 20, UN = 10 animals per group; for Western blot: NN = 5, UN = 5 animals per group. ***P < 0.001 vs. corresponding controls (Student t tests). In the Lower panels, the effects of chronic icv infusion of the pharmacological activator of AMPK, AICAR, on BW (D), VO (E), uterus weight (UW) (F), and ovarian weight (OW) (G) (at PND36) in pubertal female rats are presented. Females chronically treated with vehicle served as controls. Group sizes were: n = 16 animals per group for vehicle treatment; n = 10 animals per group for AICAR. *P < 0.05; **P < 0.01 vs. corresponding control groups.

Fig. 2.

Virogenetic overexpression of AMPK in the arcuate nucleus delays puberty onset. In A, a schematic representation of the bilateral stereotaxic injection of viruses inducing overexpression of AMPK-CA in the ARC, is shown. Representative images, at two different magnifications, of bilateral targeting of the ARC, as denoted by fluorescein labeling, are also presented in B and C, while efficiency of adenoviral-mediated infection of ARC neurons by stereotaxic delivery is documented by GFP labeling, as shown in D. In addition, the impact of AMPK-CA overexpression on BW (E), the percentage of VO (F), and serum LH levels (as marker of activation of the reproductive axis) (G) is documented. Animals injected with viral vectors overexpressing only the marker, GFP, were used as controls. Due to coadministration of fluorescein isothiocyanate in both GFP and AMPK-CA–treated animals, the injection site was evaluated postmortem, and animals with evidence for off-target injections were not considered for further analyses. Final group sizes (i.e., animals with proper viral injections) were: control-GFP = 8 animals per group; AMPK-CA = 9 animals per group. *P < 0.05 vs. control-GFP group (Student t tests).

Fig. 3.

Activation of AMPK suppresses Kiss1 expression in the arcuate nucleus. In the Upper panels (A), dark-field photomicrographs showing Kiss1 mRNA expression (white clusters of silver grains) in representative sections of the AVPV and the ARC in the hypothalamus of pubertal female rats (PND33) chronically treated with vehicle or AICAR are presented. In addition, we display data from the quantification of Kiss1 expression in the above experimental groups. **P < 0.01 vs. corresponding control group and nucleus (ANOVA followed by Student–Newman–Keuls multiple range test) (n = 4–5 animals per group). 3V, third ventricle. In the Lower panels (B), confocal images are shown of the expression of kisspeptin and pAMPK in the ARC of pubertal female rats (PND36). In the Top row, low magnification images are shown for Kisspeptin (Left), pAMPK (Central Left), and DAPI (nuclear staining; Central Right); the image on the Right corresponds to the final merge of the three signals. Two different examples at higher magnification are shown in the additional two rows below. Colocalization is denoted by white arrowheads.

Fig. 4.

Genetic ablation of AMPKα1 in Kiss1 neurons prevents pubertal delay induced by subnutrition. A summary is presented of different markers of pubertal onset in mice with genetic ablation of the AMPKα1 subunit in Kiss1-expressing cells (Kiss1-Cre^+/−; AMPKα1 ^loxP/loxP, named KAMKO), and their corresponding Kiss1-Cre^−/−; AMPKα1 ^loxP/loxP controls. The animals were explored in two metabolic conditions: fed ad libitum (normal nutrition; NN) and after chronic subnutrition (20% reduction in daily food ration from PND23 onwards; UN). Data on evolution of BW (A) and percentage of VO (B), as well as final BW (C), VO (D), ovarian weight (OW) (E), and circulating LH levels (F) on PND44, are also shown. Group sizes were: NN-control = 9; NN-KAMKO = 8; UN-control = 10; UN-KAMKO = 9 animals per group. *P < 0.05 vs. corresponding control group (ANOVA followed by Student–Newman–Keuls multiple range test).