Effects of hypercaloric diet-induced hyperinsulinemia and hyperlipidemia on the ovarian follicular development in mice

Abstract

Long-term hypercaloric diets may adversely affect the development of ovarian follicles. We investigated the effects of high sugar (HS), high fat low sugar (HFLS), and high fat normal sugar (HFNS) diets on the ovarian follicle development in mice fed with these diets as compared to those fed with normal diet (control) for 180 days. Body weight, gonadal fat, glucose, lipid, insulin, estrous cycle, sex hormones and ovarian tissues were examined, and metabolism-related protein expression in the ovaries was evaluated by immunoblotting. The mice fed with hypercaloric diets showed hyperinsulinemia and hyperlipidemia, and exhibited heavier body and gonadal fat weights, longer estrous cycles, and fewer preantral and antral follicles than mice fed with normal diet. The sex hormone levels in the blood were similar to those in controls, except for significantly elevated estradiol levels in the HS diet group. The AMPKα phosphorylation was reduced, while AKT phosphorylation and caspase-3 levels were increased in the ovarian tissues of mice in all three hypercaloric diet groups than those in control. Taken together, the results suggest hyperinsulinemia and hyperlipidemia as possible mechanisms that impair the development of ovarian follicles in response to long-term exposure to unhealthy hypercaloric diets.

Keywords: Follicular development; Hypercaloric diets; Hyperinsulinemia; Hyperlipidemia.

Fig. 1.

Body weight change curves and the weight of gonadal fat pads in mice fed with normal (control) or hypercaloric diets. (A) Body weight change curves in mice fed with normal (control), HS, HFLS, or HFNS diets; (B) The weight of gonadal fat pads; (C) The ratio of gonadal fat pad weight to body weight. Data are represented as mean ± SE from 30 different mice per experimental group. C57BL/6J female mice were 28-day-old, weighing 13–16 g at the start of the diet treatments. * P < 0.05 vs. Control. Summary of lipid and glucose metabolic parameters in mice fed with normal (Control) or hypercaloric diets for 180 days. (D) The levels of serum TG, TC, HDL-C, and LDL-C; (E) The levels of FBG, FBI, and HOMA-IR. Data are presented as mean ± SE from 20 different mice per experimental group. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. Control. HS, high sugar; HFLS, high fat low sugar; HFNS, high fat normal sugar; SE, standard errors; TG, triglycerides; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; FBG, fasting blood glucose; FBI, fasting blood insulin; HOMA-IR, homeostasis model assessment index of insulin resistance.

Fig. 2.

The concentrations of serum sex hormones in mice fed with normal (Control) or hypercaloric diets. (A) The average serum concentrations of estradiol (E₂) and testosterone (T) from 20 different mice per experimental group; (B) The proestrus, estrus, metestrus and diestrus phases, and estrous cycle in 10 different mice per experimental group. Data are presented as mean ± SE. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. Control. HS, high sugar; HFLS, high fat low sugar; HFNS, high fat normal sugar.

Fig. 3.

Hematoxylin and eosin histopathological assessment of morphological changes in the ovaries of mice fed with normal (Control), HS, HFLS, or HFNS diet for 180 days. The representative ovary from the normal mouse shows follicles at different stages, including primordial follicles, primary follicles, preantral follicles and antral follicles, and corpora lutea in the cortical ovarian zone. The morphological abnormalities and decreased follicle number in mice fed with the HS, HFLS, or HFNS diets are visible. Arrows indicate the same follicle in each group. The number of follicles at different stages in mice fed with normal (Control) or hypercaloric diets. (A) The number of primordial follicles per ovary; (B) The number of primary follicles per ovary; (C) The number of preantral follicles per ovary; (D) The number of antral follicles per ovary. Data are presented as mean ± SE from six different mice per experimental group. * P < 0.05 vs. Control. HS, high sugar; HFLS, high fat low sugar; HFNS, high fat normal sugar.

Fig. 4.

Effects of hypercaloric diets on levels of AMPK/mTOR signaling pathway in the ovarian tissues in mice fed with normal (Control) or hypercaloric diets. (A) Western blotting analyses of p-LKB1, LKB1, p-AMPKα, AMPKα, p-mTOR, mTOR, and β-actin; Quantitative analyses of: (B) p-LKB1/LKB1; (C) p-AMPKα/AMPKα; and (D) p-mTOR/mTOR. β-actin is used as an internal loading control. Data are presented as mean ± SE from six different mice per each experimental group. * P < 0.05 vs. Control.

Fig. 5.

Effects of hypercaloric diets on levels of PI3K/AKT signaling pathway in the ovarian tissues in mice fed with normal (Control) or hypercaloric diets. (A) Western blotting analyses of p85, p110α, p110γ, p-AKT, AKT, caspase-3, and β-actin; Quantitative analyses of: (B) p85/β-actin; (C) p110α/β-actin; (D) p110γ/β-actin; (E) p-AKT/AKT; and (F) Caspase-3/β-actin. β-actin is used as an internal loading control. Data are presented as mean ± SE from six different mice for each experimental group. * P < 0.05 vs. Control.