IGF-1 Acts through Kiss1-expressing Cells to Influence Metabolism and Reproduction

Abstract

Objective: Kisspeptin, encoded by the Kiss1 gene, ties puberty and fertility to energy status; however, the metabolic factors that control Kiss1-expressing cells need to be clarified.

Methods: To evaluate the impact of IGF-1 on the metabolic and reproductive functions of kisspeptin producing cells, we created mice with IGF-1 receptor deletion driven by the Kiss1 promoter (IGF1R^Kiss1 mice). Previous studies have shown IGF-1 and insulin can bind to each other's receptor, permitting IGF-1 signaling in the absence of IGF1R. Therefore, we also generated mice with simultaneous deletion of the IGF1R and insulin receptor (IR) in Kiss1-expressing cells (IGF1R/IR^Kiss1 mice).

Results: Loss of IGF1R in Kiss1 cells caused stunted body length. In addition, female IGF1R^Kiss1 mice displayed lower body weight and food intake plus higher energy expenditure and physical activity. This phenotype was linked to higher proopiomelanocortin (POMC) expression and heightened brown adipose tissue (BAT) thermogenesis. Male IGF1R^Kiss1 mice had mild changes in metabolic functions. Moreover, IGF1R^Kiss1 mice of both sexes experienced delayed puberty. Notably, male IGF1R^Kiss1 mice had impaired adulthood fertility accompanied by lower gonadotropin and testosterone levels. Thus, IGF1R in Kiss1-expressing cells impacts metabolism and reproduction in a sex-specific manner. IGF1R/IR^Kiss1 mice had higher fat mass and glucose intolerance, suggesting IGF1R and IR in Kiss1-expressing cells together regulate body composition and glucose homeostasis.

Conclusions: Overall, our study shows that IGF1R and IR in Kiss1 have cooperative roles in body length, metabolism, and reproduction.

Keywords: Body weight; IGF-1 receptor; Insulin receptor; Kiss1-expressing cells; Reproduction.

Figure 1.. Loss of hypothalamic IGF1R and IR protein expression in Kiss1 neurons of IGF1R^Kiss1 mice.

(A) The IGF1R gene with exon 4 flanked by loxP sites (large arrows) before (top panel) and after (bottom panel) Cre recombination. Primers used for detection of the truncated IGF1R gene are labeled p1, p2, and p3. (B) PCR analysis of DNA from hypothalamus, liver, ovary, and testis of a IGF1R^Kiss1 mouse. The excised IGF1R gene appears as a 390-bp band and the unexercised IGF1R gene sequence as a 750-bp band. (C) Colocation of Kiss1 neuron and IGF1Rs from control and IGF1R^Kiss1 mice. (D) Quantification of colocalization. (E) Kiss1 mRNA expression in hypothalamus. Values throughout figure are means ±SEM. For entire figure, *p < 0.05, **p < 0.01, determined by Tukey’s post hoc test following one-way ANOVA.

Figure 2.. Body length and IGF-1 levels in IGF1R^Kiss1 and IGF1R/IR^Kiss1 Mice.

(A) Body length curves and (B) serum IGF-1 levels in female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (C) Body length curves and (D) serum IGF-1 levels in male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. All data are shown as means ±SEM with individual values. For the entire figure, *p < 0.05 (control vs IGF1R^Kiss1 or IGF1R/IR^Kiss1 mice), ^#p < 0.05 (control vs. IGF1R/IR^Kiss1 mice), as determined by Bonferroni’s Multiple Comparison Test following two-way ANOVA for each time point in A and C, or Tukey’s post hoc test following one-way ANOVA for B and D.

Figure 3.. Altered energy balance in female IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice.

(A) Body weight curves, (B-C) fat and lean mass percentage and weight, and (D) food intake of female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (E-F) Energy expenditure and (G-H) physical activity in 4-month-old female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (I) Relative expression of POMC and AgRP mRNA in hypothalamus as measured by quantitative PCR in female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (J) Relative expression of thermogenesis markers in BAT as measured by quantitative PCR in female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (K) Representative sliced and HE-stained paraffin-embedded white and brown adipose tissue in 5-month-old male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. N=4–12 per genotype. All data are shown as means ±SEM with individual values. For entire figure, *p < 0.05, **p < 0.01, ***p < 0.0001, ****p< 0.00001, determined by Bonferroni’s Multiple Comparison Test following two-way ANOVA for each time point in A, E, G; or Tukey’s post hoc test following one-way ANOVA.

Figure 4.. Altered energy balance in male IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice.

(A) Body weight curves, (B-C) fat and lean body mass percentage and weight, and (D) food intake of male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (E-F) Energy expenditure and (G-H) physical activity in 4-month-old male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (I) BAT weight in 5-month-old male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. N=4–12 per genotype. All data are shown as means ±SEM with individual values. For entire figure, *p < 0.05, **p < 0.01, ***p < 0.0001, ****p< 0.00001, determined by Bonferroni’s Multiple Comparison Test following two-way ANOVA for each time point in A, E, G; or Tukey’s post hoc test following one-way ANOVA.

Figure 5.. Insulin insensitivity in female IGF1R/IR^Kiss1 mice and glucose intolerance in male IGF1R/IR^Kiss1 mice.

(A) Glucose tolerance test (GTT), (B) area under curve (AUC) of GTT (GTT-AUC), (C) insulin tolerance test (ITT) (D) and AUC of ITT (ITT-AUC) in 3-month-old female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (E) Fasting glucose, (F) insulin, (G) C-peptide, and (H) insulin: C-peptide ratio in 3-month-old female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (I) GTT, (J) GTT-AUC, (K) ITT, and (L) ITT-AUC in 3-month-old male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (M) Fasting glucose, (N) insulin, (O) C-peptide, and (P) insulin: C-peptide ratio in 3-month-old male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. N=6–12 per genotype. All data are shown as means ±SEM with individual values. For entire figure, *p < 0.05, ^#p < 0.05 (control vs IGF1R/IR^Kiss1 mice), determined by Bonferroni’s Multiple Comparison Test following two-way ANOVA for each time point in A, C, I and K; or Tukey’s post hoc test following one-way ANOVA.

Figure 6.. Delayed pubertal development in IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice.

(A) Vaginal opening age and (B) first estrus age in female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (C-D) Serum levels of LH and FSH in 4 weeks-old female control, IGF1R^Kiss1, and IGF1R/IR^Kiss1 mice. (E) Balanopreputial separation age and serum levels of (F) LH and (G) FSH in male control, IGF1R^Kiss1, and IGF1R/IR^Kiss1 mice. N=5–12 per genotype. LH luteinizing hormone, FSH follicle-stimulating hormone. All data are shown as means ±SEM with individual values. For entire figure, *p < 0.05, **p < 0.01, determined by Tukey’s post hoc test following one-way ANOVA.

Figure 7.. Estrus cyclicity in IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice.

(A) estrus cycle length and (B) days spent in each estrus cycle stage in female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. N=6–12 per genotype. P proestrus, E estrus, M metestrus, D diestrus. All data are shown as means ±SEM with individual values.

Figure 8.. Adult fertility in IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice.

(A) Pregnancy rate, (B) numbers of pups/litter, (C) serum LH, (D) FSH and (E) estradiol levels on diestrus in 3- to 4-month-old female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (F) Uterine and ovary weight, and (G) ovarian follicle numbers in 5-month-old female control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (H) Pregnancy rate, (I) numbers of pups/litter, (J) serum LH (K), LH/FSH ratio and (L) testosterone levels in 3- to 4-month-old male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (M) Testis weight and (N) analysis of cross-sectional testes seminiferous tubule in 5-month-old male, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. (O) Representative sliced and HE-stained paraffin-embedded testes in 5-month-old male control, IGF1R^Kiss1 and IGF1R/IR^Kiss1 mice. N=4–12 per genotype. All data are shown as means ±SEM with individual values. For the entire figure, *p < 0.05 and **p < 0.01, determined by Tukey’s post hoc test following one-way ANOVA, except G and N determined by Bonferroni’s Multiple Comparison Test following two-way ANOVA.