Since 17βEstradiol was shown to fail to protect ovariectomized Munich Ins2C95S
mutants from ER-stress and β-cell loss, the influence of other hormones such as progesterone or testosterone or of other substances produced in the ovaries, e.g. growth factors such as IGF1 and IGF2, on β-cell survival should be examined in female Munich Ins2C95S mutant mice.
In order to get further insights into the role of ER-stress in the pathogenesis of diabetes mellitus and the protective effects of ovarian hormones/substances in female Munich Ins2C95S mutant mice, treatment of ovariectomized mutant mice with chemical chaperones, like 4-phenyl butyric acid or tauroursodeoxycholic acid, which increase ER folding capacity, would be useful.
To examine the role of estrogen and the estrogen receptors in ER-stress and β-cell loss it would be helpful to cross Munich Ins2C95S mutant mice with estrogen-receptor knock out mice (αERKO, βERKO or αβERKO mice). Moreover it would be interesting to use selective estrogen receptor modulators (SERMs) such as propyl-pyrazole-triol (PPT) for ERα and diarylpropionitrile (DPN) for ERβ to examine the estrogenic effects on the appropriate receptors.
7 Summary
Impact of 17βEstradiol on β-cell survival of female Munich Ins2C95S mutant mice
Munich Ins2C95S mutant mice were generated within the Munich ENU mouse mutagenesis project and exhibit a point mutation in the insulin 2 gene. In recent years, several mutations in the INS gene were also observed in humans, leading to early onset of diabetes with severe hyperglycaemia.
It could be shown that male Munich Ins2C95S mutant mice develop a severe progressive diabetic phenotype with profound loss of functional β-cell mass, whereas female mutants only showed mild glucose intolerance throughout life and no β-cell loss. The aim of this study was to investigate the impact of 17βEstradiol (E2) on the development of diabetes mellitus and β-cell survival in female Munich Ins2C95S
mutant mice. For this purpose, female mutant mice were ovariectomized and received either 17βEstradiol replacement therapy via subcutaneous E2 long-term pellets or non-treatment/placebo pellet. Non-/placebo-treated sham-operated mutant and wild-type mice served as controls. Clinical parameters, such as fasted and randomly fed blood glucose concentration, oral glucose tolerance, insulin sensitivity, glucose induced insulin secretion as well as fasted serum insulin concentration were determined. Moreover, serum was analysed with regard to oxidative-stress and isolated pancreatic islets were used to determine ER-stress markers. Qualitative histological and quantitative stereological analyses of the pancreas were performed, and β-cell apoptosis as well as β-cell replication were determined.
Ovariectomy without E2 supplementation led to the development of a severe and progressive diabetic phenotype. The supplementation of 17βEstradiol after ovariectomy normalised blood glucose concentrations of female mutant mice to the level of wild-type mice.
Both, E2-treated ovariectomized and non-/placebo-treated sham-operated mutant mice, demonstrated an improved oral glucose tolerance, preserved insulin sensitivity and higher glucose induced insulin secretion as compared to non-/placebo-treated ovariectomized mutant mice. However, mice of all three mutant groups showed a severely disturbed glucose-stimulated insulin secretion as compared to wild-type
controls, indicating that the mutation in Ins2 and the resulting misfolding of proinsulin 2 leads to disturbed insulin secretion.
In addition, it could be shown that the normalised or only mildly increased blood glucose concentrations of E2-treated ovariectomized and non-/placebo-treated sham-operated mutant mice led to reduced oxidative stress, as evidenced by significantly reduced malondialdehyde serum levels.
Non-/placebo- and E2-treated ovariectomized mutant mice exhibited a lower total volume and volume density of β-cells in the islets as well as a lower β-cell/non-β-cell ratio as compared to sham-operated non-/placebo-treated mutants, indicating that ovarian hormones/substances, other than 17βEstradiol, are responsible for the preserved β-cell mass of female Munich Ins2C95S mutants. Since the abundance of the ER-stress marker BIP was only increased in non-/placebo- and E2-treated mutant mice but not in sham-operated non-/placebo-treated mutant mice, ER-stress is likely to be responsible for reduced β-cell mass, and a substance produced in the ovaries other than E2 seems to protect sham-operated mutant mice from ER-stress. Since the volume density of isolated β-cells in the pancreas, which indicate islet neogenesis, was decreased in an almost identical manner in all three mutant groups as compared to wild-type mice, decreased islet neogenesis may explain the reduced β-cell mass apparent in all mutant mice.
Ultrastructural changes in the β-cells were most prominent in placebo-treated ovariectomized mutant mice. Nevertheless, all mutant mice demonstrated accumulation of electron dense material in the dilated endoplasmic reticulum, suggesting that ER-stress is existent not only in ovariectomized, but also to a lower degree in sham-operated mutant mice.
In conclusion, 17βEstradiol could ameliorate the diabetic phenotype by improving insulin secretion and insulin sensitivity but did not prevent ER-stress or loss of functional β-cell mass in ovariectomized Munich Ins2C95S mutant mice, suggesting that ovarian hormones/substances other than E2, or other substances produced in the whole female body, prevent female Munich Ins2C95S mutant mice from ER-stress and β-cell loss.