B J Whitehouse

Effect of 17[beta]-estradiol on insulin secretion and cytosolic calcium in Min6 mouse insulinoma cells and human islets of Langerhans

Objective: Female gonadal steroids can exert an insulinotropic effect in vivo. The objective of this study was to investigate the effects in vitro of 17-β-estradiol (17β-E2) on changes in cytosolic calcium ([Ca2+]i) and on insulin secretion from the MIN6 mouse insulinoma cell line and human primary islets of Langerhans. Methods: Stimulus-induced changes in [Ca2+]i were measured in Fura-2-loaded cells by single cell microfluorimetry. The effects of 17β-E2 on insulin secretion were measured in static incubation experiments, and the rate and pattern of secretory responses were studied in multi-channel perifusion experiments. Results: 17β-E2 (1-100 nmol/L) enhanced basal (2 mmol/L glucose) insulin secretion but had no effect on secretory responses to 20 mmol/L glucose or to depolarizing stimuli (100 μmol/L tolbutamide, 20 mmol/L KCl). Approximately 60% of MIN6 cells responded to 17β-E2 (1-100 nmol/L) with a small but sustained increase in [Ca2+]i, whereas 98% of MIN6 cells responded to tolbutamide (100 μmol/L). Similar effects were observed in experiments using human primary β cells. In contrast, 17β-E2 had no detectable effect on the increases in [Ca2+]i evoked by tolbutamide (100 μmol/L) or glucose (20 mmol/L). Conclusions: Our observations are consistent with a rapid effect of 17β-E2 to depolarize β cells leading to an influx of extracellular Ca2+ and the initiation of insulin secretion by the consequent elevations in [Ca2+]i. We suggest that this may offer a mechanism through which circulating estradiol can influence β-cell responsiveness to other signals.

A role for the p42/44 isoforms of MAPK in the regulation of steroid secretion from Y1 mouse adrenocortical cells.

The mitogen-activated protein kinase (MAPK) superfamily of enzymes comprise three distinct signal transduction cascades incorporating the p54 stressactivated protein kinases (SAPKs or c-Jun N-terminal kinases), the p38 subfamilies and the p42 and p44 extracellular signal-regulated kinases (~42144) [I]. The p42/44 MAPK isoforms play a major role in cellular proliferation and differentiation and may also be involved in short term signalling events such as secretory responses. In this study we have investigated whether the ~42144 activities are important in the regulation of steroid secretion by Y1 mouse adrenocortical cells. Mitochondria1 protein extracts were prepared from Y 1 cells by differential centrifugation as described in Jones et al, 2000 [2]. Protein extracts (1 OOpg) were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with an antibody that recognises the 42 and 44 kDa isoforms of MAPK. Figure 1 clearly demonstrates the expression of p42/44 in mitochondria1 extracts of Y 1 cells.

Inhibition of protein tyrosine phosphatase activity blocks shape change & steroidogenesis in Y1 cells

Y 1 adrenocortical cells respond to forskolin stimulation with increases in steroid secretion and change of shape. The rapid rounding of flat, adherent cells which occurs is known to involve dephosphorylation of the focal adhesion protein, paxillin. We have investigated the effects of a tyrosine phosphatase inhibitor, calpeptin (CP) on steroidogenesis and shape change in Y1 cells. Forskolin treatment (FSK, 2 pM) caused marked rounding of Y1 cells (FSK = 76.3 rfI 1.5 % cells rounded after 30 minutes, untreated = 2.9 k 0.7 % rounded); calpeptin pretreatment (CP; 100 ugiml) had little effect on shape (9.6 f 2.4% rounded) but blocked the rounding response to FSK (32.1 k 2.1 % rounded. Calpetin also eliminated the steroidogenic response to FSK (FSK = 242 k 14 % control; FSK + CP = 1 13 +_ I 8 9′0 control) without affecting production of steroid from membrane permeant 22R-OH-cholesterol. The results support the view that dephosphorylation of paxillin is important in the rounding response and provide evidence for the involvement of tyrosine-phosphatase activity in cyclic AMP-stimulated steroidogenesis in Y 1 cells

The role of ERK2/1 in steroid production and StAR protein expression by Y1 cells: Studies using StAR over-expressing transfects

In studies using inhibitors of MEK-1, the upstream activator of ERK2/1, we have recently demonstrated that the 42 and 44 kDa extracellular signal-regulated kinases (ERK2/1, respectively) play a major role in regulating cyclic AMP-stimulated StAR gene expression in Y1 mouse adrenocortical cells. We have now extended these studies with the use of a stably-transfected Y1 cell-line over-expressing StAR mRNA under control of CMV promoter control. In these cells, increased StAR expression was correlated with enhanced basal steroidogenesis that was not inhibited by MEK-1 inhibitors (untransfected: 0.7 ± 0.3 pmol pregnenolone/106 cells, transfected: 4.8 ± 1.7 pmol pregnenolone/106 cells), in accordance with ERK2/1 acting to regulate steroid production before transcription of the StAR gene. However, StAR mRNA and protein expression and steroid production by transfected cells were further enhanced in the presence forskolin (FSK, 1 µM) and to a much greater extent than in control cells (untransfected: 4.4 ± 0.2 pmol pregnenolone/106 cells, transfected: 10.4 ± 0.3 pmol pregnenolone/106 cells). Furthermore, MEK-1 inhibitors did not reduce the FSK-induced increase in StAR mRNA and protein expression in transfected cells. These results suggest that activation of adenylate cyclase by FSK can regulate the expression of StAR by both increasing transcription of the endogenous gene and by enhancing stability of existing mRNA. They further suggest that ERK2/1 activation is required for FSK-induced StAR mRNA transcription but is not involved in the regulation of mRNA stability by FSK.