High fat diet induced obesity alters ovarian phosphatidylinositol-3 kinase signaling gene expression

Abstract

Insulin regulates ovarian phosphatidylinositol-3-kinase (PI3 K) signaling, important for primordial follicle viability and growth activation. This study investigated diet-induced obesity impacts on: (1) insulin receptor (Insr) and insulin receptor substrate 1 (Irs1); (2) PI3K components (Kit ligand (Kitlg), kit (c-Kit), protein kinase B alpha (Akt1) and forkhead transcription factor subfamily 3 (Foxo3a)); (3) xenobiotic biotransformation (microsomal epoxide hydrolase (Ephx1), Cytochrome P450 isoform 2E1 (Cyp2e1), Glutathione S-transferase (Gst) isoforms mu (Gstm) and pi (Gstp)) and (4) microRNA's 184, 205, 103 and 21 gene expression. INSR, GSTM and GSTP protein levels were also measured. Obese mouse ovaries had decreased Irs1, Foxo3a, Cyp2e1, MiR-103, and MiR-21 but increased Kitlg, Akt1, and miR-184 levels relative to lean littermates. These results support that diet-induced obesity potentially impairs ovarian function through aberrant gene expression.

Keywords: Obesity; Ovary; Phosphatidylinositol-3 kinase.

Figure 1. Obesity does not alter ovarian weight in mice

Six weeks old C57Bl/6J female mice (n = 6 per treatment group) were fed either a standard chow mice diet or a high-fat diet for approximately 7 months and were euthanized by CO₂ asphyxiation. Ovaries were removed, trimmed of excess fat and weighed. (A) Ovarian weight; (B) lean and (C) obese ovarian sections stained with hematoxylin and eosin (H&E).

Figure 2. Obesity decreases ovarian Irs1 mRNA levels in mice

Ovaries were removed from lean and obese mice (n = 6 per treatment group). (A) RNA was isolated and Insr and Irs1 mRNA levels were evaluated by quantitative RT-PCR. Values represent fold-change relative to a control value of 1 ± SE, normalized to Actb. (B) Total protein was isolated and Western blotting performed to measure INSR level. Values represent fold-change relative to a control value of 1 ± SE, normalized to ACTB. Different from control, *P < 0.05. (C) Representative Western blot for INSR and ACTB; Lean = L, Obese = O.

Figure 3. Obesity enhances ovarian KITLG/KIT-PI3K/AKT1 signaling pathway in mice

Ovaries were removed from lean and obese mice (n = 6 per treatment group). RNA was isolated and Kitlg, cKit, Akt1 and Foxo3a mRNA levels were evaluated by quantitative RT-PCR. Values represent fold-change relative to a control value of 1 ± SE, normalized to Actb. Different from control, *P < 0.05.

Figure 4. Altered pAKT^Ser473 levels are observed in the ovaries of obese mice

Ovaries were removed from (A) lean and (B) obese mice, serially sectioned and mounted onto slides (n = 6 per treatment group). Immunoflourescence staining to detect pAKT^Ser473 was performed, with counterstaining to detect the nucleus. Image J software was used to quantify levels of pAKT^Ser473 in (C) small (pre-antral; indicated by arrowhead) and (D) large oocytes (antral; indicated by broken arrow) and (E) theca cells (indicated by unbroken arrow).

Figure 5. Obesity down-regulates ovarian Cyp2e1 mRNA levels

Ovaries were removed from lean and obese mice and RNA isolated (n = 6 per treatment group). (A) Cyp2e1, Ephx1, Gstp and Gstm mRNA levels were measured by quantitative RT-PCR. Values represent fold-change relative to a control value of 1 ± SE, normalized to Actb. Different from control, *P < 0.05. (B) Western blotting was performed to determine any impact of obesity on GSTP. (C) Representative Western blot for GSTP and ACTB; Lean = L, Obese = O.

Figure 6. Obesity alters levels of ovarian miR

Ovaries were removed from lean and obese mice and RNA isolated (n = 3 per treatment group). (A) Enriched miR fractions were analyzed using a miR array. (B) qRT-PCR was performed on miR-21 to confirm the array data. Values represent fold-change relative to a control value of 1 ± SE, normalized to RAU43. Different from control, *P < 0.05.

Figure 7. Proposed model of obesity effects on AKT1 signalinga

Under basal conditions, AKT1 is positively regulated by both KITLG and IRS1. In addition, AKT1 is positively regulated by miR21 and negatively regulated by miR184. Our data indicates that obesity decreases IRS1 levels but increases KITLG leading to increased AKT1 levels. The increased levels of miR184 may counteract the decreased miR21 in AKT1 activation. AKT regulates xenobiotic metabolism, primordial follicle activation and viability as well as ovarian steroidogenesis. Thin arrows indicate positive regulation, negative regulation is indicated by the broken arrow. Block arrows indicate the impact of obesity.