Insulin and Leptin Signaling Interact in the Mouse Kiss1 Neuron during the Peripubertal Period - PubMed (original) (raw)

Insulin and Leptin Signaling Interact in the Mouse Kiss1 Neuron during the Peripubertal Period

Xiaoliang Qiu et al. PLoS One. 2015.

Abstract

Reproduction requires adequate energy stores for parents and offspring to survive. Kiss1 neurons, which are essential for fertility, have the potential to serve as the central sensors of metabolic factors that signal to the reproductive axis the presence of stored calories. Paradoxically, obesity is often accompanied by infertility. Despite excess circulating levels of insulin and leptin, obese individuals exhibit resistance to both metabolic factors in many neuron types. Thus, resistance to insulin or leptin in Kiss1 neurons could lead to infertility. Single deletion of the receptors for either insulin or the adipokine leptin from Kiss1 neurons does not impair adult reproductive dysfunction. However, insulin and leptin signaling pathways may interact in such a way as to obscure their individual functions. We hypothesized that in the presence of genetic or obesity-induced concurrent insulin and leptin resistance, Kiss1 neurons would be unable to maintain reproductive function. We therefore induced a chronic hyperinsulinemic and hyperleptinemic state in mice lacking insulin receptors in Kiss1 neurons through high fat feeding and examined the impact on fertility. In an additional, genetic model, we ablated both leptin and insulin signaling in Kiss1 neurons (IR/LepRKiss mice). Counter to our hypothesis, we found that the addition of leptin insensitivity did not alter the reproductive phenotype of IRKiss mice. We also found that weight gain, body composition, glucose and insulin tolerance were normal in mice of both genders. Nonetheless, leptin and insulin receptor deletion altered pubertal timing as well as LH and FSH levels in mid-puberty in a reciprocal manner. Our results confirm that Kiss1 neurons do not directly mediate the critical role that insulin and leptin play in reproduction. However, during puberty kisspeptin neurons may experience a critical window of susceptibility to the influence of metabolic factors that can modify the onset of fertility.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Electrophysiological response of Kiss1 neurons to insulin.

A: Averaged membrane potential (Vm) recordings from an insulin-responsive Kiss1 neuron elicited with (solid line) and without (dashed line) a 4 x 20 ms pressure application of 200 nM insulin in ACSF. Gray shading represents the mean ± SEM from traces averaged across 11 repetitions obtained every 10 sec. B: same as A with recordings averaged across 11 repetitions from a Kiss1 neuron that did not respond to pressure pulses of 200 nM insulin. Time course of pressure pulses is shown below Vm traces in A—B. C: recordings from same neuron in A in which APs were elicited by 0.2 ms depolarizing current pulses during (solid line) and without (dashed line) 4 x 20 ms pressure application of 200 nM insulin in ACSF. D: APs from C displayed on magnified time scale and aligned at peak to identify whether any changes occurred in AP time course. E: Locations of recordings from EGFP positive Kiss1 neurons. Red Xs represent insulin-responsive neurons, and the blue Xs represent insulin nonresponsive neurons. Modified from Mouse Brain in Stereotaxic Coordinates, 3 rd Edition by Franklin and Paxinos (used with permission).

Fig 2

Fig 2. Metabolic parameters in IRKiss mice after HFD treatment.

(A) Female body weight growth curve after HFD (n = 8, 8). Body weights of a low-fat diet cohort included for comparison, + indicates control low and high fat diet groups differ significantly, # indicates IRKiss low and high fat diet groups differ significantly, * indicates two HFD groups differ significantly. (B) Male body weight growth curve after HFD (Control, n = 7; IRKiss, n = 5). Body weights of a low-fat diet cohort included for comparison. + indicates control low and high fat diet groups differ significantly, # indicates IRKiss low and high fat diet groups differ significantly. (C) Female body composition measured by NMR 3 months after HFD (n = 8, 8). (D) Male body composition measured by NMR 3 months after HFD (n = 7, 5). (E) Serum leptin levels in lean males (n = 16, 8) and males after 3 months of exposure to HFD (n = 7, 4) (F) Serum leptin levels in lean females (n = 7, 7) and females after 3 months of exposure to HFD (n = 4, 9) (G) Serum insulin levels in lean males (n = 11, 10) and males after 3 months of exposure to HFD (n = 6, 5) (H) Serum insulin levels in lean females (n = 9, 10) and females after 3 months of exposure to HFD (n = 3, 9).

Fig 3

Fig 3. Reproductive axis function in IRKiss mice after HFD treatment.

(A) Serum estradiol in females (n = 3, 6) after 3 months on HFD in adulthood. (B) Serum testosterone levels in males (n = 4, 5) and females (n = 4, 7) after 3 months on HFD in adulthood. (C) GnRH gene expression after 4 months on HFD in adulthood in males (n = 6, 4) and females (n = 5, 4). (D) Serum LH levels in females (n = 13, 6) after 3 months on HFD in adulthood. (E) Serum testosterone levels in females (n = 13, 6) after 3 months on HFD in adulthood. (F) Body weight growth curve in females placed on HFD at weaning (n = 4, 4). + indicates control low and high fat diet groups differ significantly, # indicates IRKiss low and high fat diet groups differ significantly. (G) Age of vaginal opening and first estrus in females placed on HFD at weaning (n = 11, 10). (H) Age of vaginal opening and first estrus in females placed on HFD at weaning (n = 11, 10).

Fig 4

Fig 4. Generation of IR/LepRΔKiss mice.

(A) Construct in making IR/LepRΔKiss mice. Adapted from previous publications [48, 50, 81]. (B) PCR of DNA from different tissues. The exercised LepR and IR both appear as a 500bp band, whereas unexercised LepR shown as 1kb band and unexercised IR as a 2.2kb band. (C) Representative IRβ expression in different tissues and densitometry.

Fig 5

Fig 5. Kisspeptin cell number and process density in IR/LepRKiss females.

(A) Female representative Kiss1 immunostaining: Upper left square = AVPV/PeN of control mouse, lower left square = ARC of control mouse, upper right square = AVPV/PeN of IR/LepRKiss mouse, lower right square = ARC of IR/LepRKiss mouse. Left square = ARC of control mouse, right square = ARC of IR/LepRKiss mouse. (B) Male representative Kiss1 immunostaining (C) Quantification of Kiss1 cell number in the AVPV/PeN (n = 2, n = 4 mice) and Kiss1 immunoreactive area in the ARC (n = 4, 4) of adult females. (D) Quantification of Kiss1 immunoreactive area in the ARC in adult males (n = 3, 3). AVPV/PeN, anteroventral periventricular and anterior periventricular nuclei; ARC, arcuate nucleus.

Fig 6

Fig 6. Puberty in IR/LepRKiss mice.

(A) Balanopreputial separation age in male control (n = 12), IRKiss (n = 10) and IR/LepRKiss (n = 3) mice. Estimated age of sexual maturation calculated by subtraction of 20 days from the time required for delivery of a litter sired by male control (n = 8), IRKiss (n = 10) and IR/LepRKiss (n = 4) mice. (B) Vaginal opening and first estrus was evaluated in female control (n = 9–12), IRKiss (n = 10) and IR/LepRKiss (n = 7–8) mice. (C) LH serum levels on postnatal day 31 in male control (n = 7) and IR/LepRKiss (n = 7) mice and female control (n = 10) and IR/LepRKiss (n = 6) mice. (D) FSH serum levels on postnatal day 31 in male control (n = 7) and IR/LepRKiss (n = 7) mice and in female control (n = 10) and IR/LepRKiss (n = 6) mice.

Fig 7

Fig 7. Female reproduction in IR/LepRKiss mice.

(A) Estrous cycle analysis (Control, n = 6; IR/LepRKiss, n = 5) and (B) estrous cycle length (Control, n = 6; IR/LepRKiss, n = 5) in 3 months old females. (C) Estradiol in adult females in control (n = 9) and IR/LepRKiss (n = 6) mice. (D-E) LH and FSH levels in adult female control (n = 10) and IR/LepRKiss (n = 16) mice. (F) Ovarian mass in adult females in control (n = 9) and IR/LepRKiss (n = 6) mice. (G) Representative light photomicrographs of an adult IR/LepRKiss mouse ovary (n = 4). CL, corpora lutea. Scale bar, 100μm. (H) Fertility data from 5–6 months old females paired with established male breeders. Interval from mating to birth of a litter and litter size were compared between control (n = 12) and IR/LepRKiss (n = 6) mice.* P<0.05.

Fig 8

Fig 8. Male reproduction in IR/LepRKiss mice.

(A) Testosterone in adult males in control (n = 9) and IR/LepRKiss (n = 8) mice. (B-C) LH and FSH levels in adult male control (n = 10–12) and IR/LepRKiss (n = 10) mice. (D) Testis mass in control (n = 9) and IR/LepRKiss (n = 8) mice. (E) Representative sections of adult testis in IR/LepRKiss mice (n = 4). Scale bar, 100μm. (F-G) Fertility data from 5–6 months old males paired with established female breeders. Interval from mating to birth of a litter and litter size were compared between control (n = 12) and IR/LepRKiss (n = 11) mice.

Fig 9

Fig 9. Metabolic phenotype in IR/LepRKiss mice.

(A) Weekly body weight of female mice (Control n = 15 and IR/LepRKiss n = 8) mice (B) Weekly body weight of male mice (n = 16 each group). (C) Female average daily food intake calculated from weekly measurement. (D) Male average daily food intake calculated from weekly measurement. (E) Female body composition at the age of 4 months in control (n = 13) and IR/LepRKiss (n = 7) mice. (F) Male body composition at the age of 4 months in control (n = 15) and IR/LepRKiss (n = 12) mice.

Fig 10

Fig 10. Glucose regulation in IR/LepRKiss mice (A) 4–5 months old female GTT and area under curve (AUC) (inset) in control (n = 11) and IR/LepRKiss (n = 5) mice.

(B) 4–5 months old male GTT and AUC (inset) in control (n = 10) and IR/LepRKiss (n = 8) mice. (C) 5–6 months old female ITT and AUC (inset) in control and IR/LepRKiss mice (n = 7). (D) 5–6 months old male ITT and AUC (inset) in control (n = 11) and IR/LepRKiss (n = 7) mice.

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