Incretins modulate progesterone biosynthesis by regulating bone morphogenetic protein activity in rat granulosa cells

Abstract

The effects of incretins on ovarian steroidogenesis have not been clarified. In this study, we investigated the effects of incretins, including GIP and GLP-1, on ovarian steroidogenesis using rat primary granulosa cells. Treatment with incretins significantly suppressed progesterone synthesis in the presence of FSH, and the effect of GIP was more potent than that of GLP-1. In contrast, incretins had no significant effect on estrogen synthesis by rat granulosa cells. In accordance with the effects of incretins on steroidogenesis, GIP and GLP-1 suppressed the expression of progesterogenic factors and enzymes, including StAR, P450scc, 3βHSD, but not P450arom, and cellular cAMP synthesis induced by FSH. In addition, incretins moderately increased FSHR mRNA expression in granulosa cells. Of note, treatment with GIP, but not treatment with GLP-1, augmented Smad1/5/8 phosphorylation and transcription of the BMP target gene Id-1 induced by BMP-6 stimulation, suggesting that GIP upregulates BMP receptor signaling that can inhibit FSH-induced progesterone synthesis in rat granulosa cells. On the other hand, BMP-6 treatment suppressed the expression of GIP receptor but not that of GLP-1 receptor. Expression of the BMP type-I receptor ALK-3 was upregulated by treatment with GIP and GLP-1 and that of ALK-6 was also increased by GIP, while inhibitory Smad6 expression was impaired by GIP and GLP-1 in rat granulosa cells. Collectively, the results indicate that incretins, particularly GIP, impair FSH-induced progesterone production, at least in part, by upregulating BMP signaling in rat granulosa cells. The modulatory effects of incretins on endogenous BMP activity may be applicable to treatment of dysregulated steroidogenesis such as polycystic ovary syndrome.

A network pharmacological approach to evaluate Jia-Wei-Xiao-Yao-san formula’s mechanistic pathways and its implication in the symptomatology of polycystic ovarian syndrome

“…66 Thecal cells of normal patients showed an increased level of LHCGR and CYP17A1 to facilitate folliculogenesis, 67 indicating that JWXYS formula compounds could have a constitutive role in regulating thecal and granulosa cells against the pathophysiology of PCOS. There have been other studies that point out the role of GIP that moderately induced FSHR gene transcript in granulosa cells, 68 as this node belongs to the third cluster ID and is shown in Figure 4C interacting with INS, LHCGR, and GLP1R, the latter receptor involved with the gut hormone GLP-1 which is impaired in obese women with PCOS and when stimulated by GLP-1 it can have a positive effect on the body weight, serum testosterone levels and even the polycystic ovarian morphology. [69][70][71] The interaction between GIP and GLP1R is currently unknown in PCOS subjects, but GLP1R agonists have been clinically reported in the treatment of type 2 diabetes and obesity, 72 and since JWXYS formula compounds interact with GLP1R and support why the PCOS subjects express higher insulin and total GIP levels and lower GLP-1 levels in a late phase of oral glucose tolerance test when compared with healthy women control.…”

Interaction of ovarian steroidogenesis and clock gene expression modulated by bone morphogenetic protein-7 in human granulosa cells

“…In the present experiments, it was found that BMP-7 affected the levels of 3βHSD expression rather than StAR, though this discrepancy could be due to the characteristic differences between rat primary granulosa cells and human KGN cells. As other humoral modulators related to ovarian steroidogenesis, androgens [15], incretins [16], orexins [17] and melatonin [35], which can affect endogenous BMP-Smad signaling activity in granulosa cells [36,37], may also be involved in the modulation of Clock gene expression in the ovary. Further studies are necessary to determine the functional interaction between the activities of Clock-related molecules and ovarian steroidogenesis.…”

“…KGN cells (1 × 10 5 cells/mL) were treated with forskolin (1 μM) or BMPs (100 ng/mL) in 12-well plates containing serum-free DMEM/F12 for the indicated periods. Concentrations of forskolin and BMP ligands used in the current experiments were selected based on our earlier data obtained from the same in vitro experiments [14][15][16][17]. Total cellular RNA was extracted using TRI Reagent ® (Cosmo Bio Co., Ltd., Tokyo, Japan) and the concentration of extracted RNA were determined by NanoDrop TM One spectrophotometer (Thermo Fisher Scientific, Waltham, MA).…”

“…Total cellular RNA was extracted using TRI Reagent ® (Cosmo Bio Co., Ltd., Tokyo, Japan) and the concentration of extracted RNA were determined by NanoDrop TM One spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Primer pairs for detecting the following genes: steroidogenic acute regulatory protein (StAR), steroid side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), aromatase (P450arom), and a housekeeping gene, ribosomal protein L19 (RPL19), were utilized as we earlier reported [13,[15][16][17][18][19]. Other primers were also selected from different exons to eliminate PCR products originated from chromosomal DNA as follows: 966-985 and 1,099-1,118 for Bmal1 (from GenBank accession #AB000812); 2,072-2,091 and 2,262-2,281 for Clock (NM_001267843); 1,614-1,633 and 1,870-1,889 for Per2 (NM_0022817); and 2,203-2,222 and 2,461-2,480 for Cry1 (NM_ 004075).…”

Modulation of bone morphogenetic protein activity by melatonin in ovarian steroidogenesis

“…Among the examined factors, androgens, growth hormone, and insulin‐like growth factor‐I were found to be key molecules that induce smad6/7expression. In contrast, prolactin (PRL), somatostatins, and incretins were found to be suppressors of inhibitory smad6/7 expression in the granulosa cells (Figure ). Thus, the modulatory effects on BMP activity in granulosa cells via the expression of smad6/7 molecules could be critical for integrating steroidogenesis through controlling endogenous BMP signaling.…”