The isoforms of sterol regulatory element-binding proteins (SREBP) (1a, 1c, and 2) are key transcriptional regulators of lipid biosynthesis. We examined their regulation by gonadotropin and insulin in human granulosa cells. After removal of leukocytes, granulosa cells were exposed to hormonal additions for 16 h starting on d 2 of culture. Progesterone, lactate, and IGF binding protein-1 were measured in culture medium and cellular mRNA measured by competitive RT-PCR. Addition of human chorionic gonadotropin (hCG) (100 ng/ml) stimulated progesterone production (7.0-fold, P < 0.001 vs. control), whereas lactate was increased by hCG (1.6-fold, P < 0.001) and insulin (1.4-fold, P < 0.001; 1000 ng/ml). Insulin decreased IGF binding protein-1 production by 85% (P < 0.001). There were no significant effects on the expression of SREBP-1a but significant increases in mRNA for SREBP-1c with insulin (6.3-fold), hCG (10.4-fold) and in combination (15.2-fold; P < 0.01 for all comparisons). No consistent effects on SREBP-2 were observed. The expression of mRNA for fatty acid synthase, a target gene for SREBP-1c, was increased by hCG (24-fold, P = 0.006) and insulin (19-fold, P = 0.024), which also increased the level of cellular, total fatty acid (1.34-fold; P = 0.03). Thus, hCG and insulin cause a switch toward expression of the SREBP-1c isoform with consequent effects on fatty acid synthesis. We suggest that high circulating insulin, associated with clinically defined insulin resistance, may up-regulate SREBP-1c expression in the ovary.
Context: Production of 3-carbon units (as lactate) by granulosa cells (GCs) is important in follicular and oocyte development and may be modulated by metformin. Objective: The aim of the study was to examine the action of metformin on GC lactate production and potential mediation via AMP-activated protein kinase (AMPK). Design: GCs were prepared from follicular aspirates. After exposure to metformin and other potential modulators of AMPK in culture, aspects of cellular function were examined. Setting: The study was conducted in a private fertility clinic/university academic center. Patients: Women undergoing routine in vitro fertilization participated in the study. Interventions: All agents were added in culture. Main Outcome Measures: Lactate output of GCs was measured. Cell extracts were prepared after culture, and phosphorylated forms of AMPK and acetyl CoA carboxylase (ACC) were assayed using Western analysis. Results: Metformin led to a rapid increase in lactate production by GCs [minimum effective dose, 250 μm; maximum dose studied, 1 mm (1.22-fold; P < 0.01)]. This dose range of metformin was similar to that required for stimulation of phospho-AMPK in GCs [minimum effective dose, 250 μm; maximum effect, 500 μm (2.01-fold; P < 0.001)]. Increasing phospho-ACC, as a representative downstream target regulated by AMPK, was apparent over a lower range (minimum effective dose, 31 μm; maximum effect, 250 μm; P < 0.001). A level of metformin (125 μm) insufficient for the stimulation of lactate output when used alone potentiated the effects of suboptimal doses of insulin on lactate production. Adiponectin (2.5 μg/ml) had a small but significant effect on lactate output. Conclusions: Metformin activates AMPK in GCs, stimulating lactate production and increasing phospho-ACC. Metformin also enhances the action of suboptimal insulin concentrations to stimulate lactate production.
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