Gut microbiota and its derived SCFAs regulate the HPGA to reverse obesity-induced precocious puberty in female rats

Abstract

The intestinal microbiota and its derived short-chain fatty acids (SCFAs) can reverse obesity and obesity-related metabolic diseases, but whether it has an effect on obesity complicated by precocious puberty and its potential mechanism need to be further understood. The purpose of this study was to investigate the effect of the gut microbiota and its derived short-chain fatty acids (SCFAs) on obesity-induced precocious puberty rats and their regulatory mechanisms. We constructed obesity-induced precocious puberty rats using a high-fat diet (HFD) had notable similarity to precocious puberty caused by obesity due to overeating in children. We then added acetate, propionate, butyrate or their mixture to the HFD, and investigated the effect of intestinal microbiota and its derived SCFAs on the hypothalamic-pituitary-gonadal axis (HPGA) in rats with obesity-induced precocious puberty. We found that obesity-induced precocious puberty rats had an early first estrous cycle, increased hypothalamic mRNA expression of Kiss1, GPR54 and GnRH, and early gonadal maturation. Meanwhile, the intestinal microbiota imbalance and the main SCFAs production decreased in the colon. The addition of acetate, propionate, butyrate or their mixture to the HFD could significantly reverse the precocious puberty of rats, reduce GnRH release from the hypothalamus and delay the development of the gonadal axis through the Kiss1-GPR54-PKC-ERK1/2 pathway. Our findings suggest that gut microbiota-derived SCFAs are promising therapeutic means for the prevention of obesity-induced precocious puberty and provide new therapeutic strategies with clinical value.

Keywords: high-fat diet; microbiota; obesity; puberty; short chain fatty acids (SCFAs).

Figure 1

HFD induces obesity and precocious puberty in female rats. (A) Evolution of body weight. (B) Serum total cholesterol. (C) Serum triglyceride. (D) Serum low density lipoprotein cholesterol (LDL-C). (E) Serum high density lipoprotein cholesterol (HDL-C). (F) Cumulative percentage of vaginal opening. (G) Cumulative percentage of first estrus. (H) Uterine wall thickness. (I) Relative uterus weight. (J) Histological score of follicular development and ovulation. (K) Relative ovary weight. (L) Serum LH levels. (M) Serum FSH levels. (N) GnRH secretion in the hypothalamus. n = 6 per group for rat samples. *P<0.05; **P<0.01.

Figure 2

HFD promotes the gene expression of the HPGA in rats with obesity-induced precocious puberty. (A) The gene expression of Kiss1 in the hypothalamus. (B) The gene expression of GPR54 in the hypothalamus. (C) The gene expression of GnRH in the hypothalamus. (D) The gene expression of ERα in the hypothalamus. (E) The gene expression of GnRHR in the pituitary. (F) The gene expression of LHR in the ovary. (G) The gene expression of FSR in the ovary. (H) The gene expression of ERα in the ovary. n = 6 per group for rat samples. *P<0.05; **P<0.01.

Figure 3

HFD alters gut microbiota in obesity-induced precocious puberty female rats. During postnatal day (PND) 21, female rats were fed a HFD and a normal diet until the rats were sacrificed during diestrus after completing the estrous cycle. Microbial 16S rDNA genes were subsequently sequenced. (A) PCoA plot of Bray-Curtis distance of the gut microbiota. (B) PCoA plot Jaccard distance of gut microbiota. (C) PCoA plot of unweighted Uni-Frac distance of gut microbiota. (D–F) Community composition distribution at the phylum, family and genus level. (G–I) The most differentially abundant proportions between the HF and CTR group at the phylum, family and genus levels. n=6 per group for gut microbiota analysis. Significance was tested with the Wilcoxon rank-sum test.

Figure 4

HFD alters the levels of fecal SCFAs and their related receptors in the colon in the rats with obesity-induced precocious puberty. (A) The content of acetic acid in fecal. (B) The content of propanoic acid in fecal. (C) The content of butyric acid in fecal. (D) The content of valeric acid in fecal. (E) The content of hexanoic acid in fecal. (F) The content of isobutyric acid in fecal. (G) The content of isovaleric acid in fecal. (H) The gene expression of GPR43 in the colon. (I) The gene expression of GPR41 in the colon. (J) The gene expression of GPR109a in the colon. Fecal SCFAs levels were detected by GC-MS. n = 6 per group for rat samples. *P<0.05; **P<0.01; ***P<0.001. ns, not significant.

Figure 5

SCFAs reverse obesity and precocious puberty in obesity-induced precocious puberty rats. (A) Evolution of body weight. (B) Serum total cholesterol. (C) Serum triglyceride. (D) Serum low density lipoprotein cholesterol (LDL-C). (E) Serum high density lipoprotein cholesterol (HDL-C). (F) Cumulative percentage of vaginal opening. (G) Cumulative percentage of first estrus. (H) Serum LH levels. (I) Serum FSH levels. (J) Morphological and histopathological changes in uterine and uterine wall thickness after addition of SCFAs. (K) Relative uterus weight. (L) Histological score of follicular development and ovulation after addition of SCFAs diet. (M) Relative ovary weight. n = 6 per group for rat samples. *P<0.05; **P<0.01.

Figure 6

SCFAs reverse the expression of genes related to the HPGA in rats with obesity-induced precocious puberty. (A)The gene expression of Kiss1 in the hypothalamus. (B) The gene expression of GPR54 in the hypothalamus. (C) The gene expression of GnRH in the hypothalamus. (D) The gene expression of ERα in the hypothalamus. (E) Gene expression of GnRHR in the pituitary. (F) The gene expression of FSR in the ovary. (G) The gene expression of LHR in the ovary. (H) The gene expression of ERα in the ovary. n = 6 per group for rat samples. *P<0.05; **P<0.01.

Figure 7

SCFAs regulate microbiota and its receptors change in obesity-induced precocious puberty rats. During PND21, female rats were fed HFD, and 5% acetate, propionate, butyrate diet and their mixtures were added until the rats were sacrificed during diestrus after completing in estrous cycle. Microbial 16S rDNA genes were subsequently sequenced. (A) α-diversity of the chao1, shannon and simpon index in different groups. (B) PCoA plot of Bray-Curtis distance of the gut microbiota. (C) PCoA plot Jaccard distance of gut microbiota. (D) PCoA plot of unweighted Uni-Frac distance of gut microbiota. (E, F) Community composition distribution at the phylum and genus levels. (G, H) The most differentially abundant proportions between the HF, CTR, HF-A, HF-P, HF-B and HF-SCFA groups at the phylum and genus levels. n=6-8 per group for gut microbiota analysis. Significances was tested with the Wilcoxon rank-sum test. (I) The gene expression of GPR43 in the colon. (J) The gene expression of GPR41 in the colon. (K) The gene expression of GPR109a in the colon. n = 6 per group for rat samples. *P<0.05; **P<0.01.

Figure 8

SCFAs regulate hypothalamic GnRH release through GPR54-PKC-ERK1/2 pathway. (A) Representative Western blot images showing GPR54, PKC, p-ERK1/2, t-ERK1/2 and β-actin expression in rats added with SCFAs. (B–D) Statistical analysis of GPR54, PKC and p-ERk1/2 expression in each group. (E) GnRH secretion in the hypothalamus. (F) HFD led to a decrease in SCFAs in the gut and promoted the expression of the GPR54-PKC-ERK1/2 pathway. Supplementation with SCFAs with a HFD reversed the high expression of the GPR54-PKC-ERK1/2 pathway and further decreased the secretion of GnRH, weakening the activation of the pituitary gland and inhibiting gonadal development. n = 6 per group for rat samples. *P<0.05; **P<0.01; ***P<0.001.