1. Metformin alters insulin signaling and viability of human granulosa cells 
Barbara Sonntag, M.D., Martin Götte, Ph.D., Pia Wülfing, M.D., Andreas N. Schüring, M.D., Ludwig Kiesel, M.D., Robert R. Greb, M.D. 
Fertility and Sterility 
Volume 84, Pages (October 2005)
DOI: /j.fertnstert
Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

2. FIGURE 1
Human basal granulosa cell viability is significantly increased by metformin with no additional stimulatory effect of insulin and IGF. Primary granulosa luteal cells (GC) from IVF patients were cultured for 48 hours in serum-free medium with or without metformin (1 mmol/L) in the absence of any other ligands, or with the addition of insulin (Ins), IGF-1, or IGF-2 after a 24-hour preincubation period. Cell viability was determined by a colorimetric proliferation assay (WST-1). Addition of metformin (Met) significantly increases basal granulosa cell viability (P=.041) and significantly changes the stimulatory effect of ligands on granulosa cell viability (−Met vs. +Met, P=.018). Insulin, IGF-1, and IGF-2 significantly increase granulosa cell viability relative to control only when not coincubated with metformin (P<.001). The results are presented as the mean ± SEM relative to control arbitrarily set as 1. Bars with no common superscripts are significantly different (P<.05).
Sonntag. Metformin and granulosa insulin signaling. Fertil Steril 2005.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

3. FIGURE 2
Metformin preincubation significantly increases basal and insulin-induced phosporylated AKT protein expression in HGL5 cells. Following preincubation with or without metformin for 24 hours, total cell lysates were prepared from the human granulosa cell line HGL5 after stimulation with insulin for up to 60 minutes. Differences between total and phosphorylated protein were detected by immunoblotting using phosphospecific antibodies. Metformin significantly increases basal and stimulated phosphorylated AKT protein expression (P=.028). A significant increase following insulin stimulation was present after 5 and 15 minutes only when cells were not coincubated with metformin (P<.05). (Upper panel) Typical immunoblotting of phosporylated AKT and total AKT protein. (Lower panel) Data (mean ± SEM) for the ratios of protein levels relative to unstimulated control arbitrarily set as 1. Bars with no common superscripts are significantly different (P<.05).
Sonntag. Metformin and granulosa insulin signaling. Fertil Steril 2005.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

4. FIGURE 3
Metformin preincubation significantly decreases basal and insulin-induced phosphorylated p44/42 mitogen-activated protein kinase (MAPK) protein expression in HGL5 cells. Following preincubation with or without metformin for 24 hours, total cell lysates were prepared from the human granulosa cell line HGL5 after stimulation with insulin for up to 60 minutes. Differences between total and phosphorylated protein were detected by immunoblotting using phosphospecific antibodies. Metformin significantly decreases basal and insulin-induced phosphorylated MAPK protein expression (P=.008). Insulin stimulation did not significantly increase phosphorylated MAPK expression with or without metformin preincubation. (Upper panel) Typical immunoblotting of phosporylated MAPK and total MAPK protein. (Lower panel) Data (mean ± SEM) for the ratios of protein levels relative to unstimulated control arbitrarily set as 1. Bars with no common superscripts are significantly different (P<.05).
Sonntag. Metformin and granulosa insulin signaling. Fertil Steril 2005.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions