Rapid, nongenomic estrogen actions protect pancreatic islet survival

Abstract

The gonadal steroid, 17β-estradiol (E₂), acts as a protective hormone preventing β-cell apoptosis in vivo in mice of both sexes and in cultured mouse and human islets. E₂ signals via the classical estrogen receptor (ER)α and ERβ, an extranuclear form of ERα and the G protein-coupled estrogen receptor (GPER). In a recent study, we determined the contribution of these receptors to β-cell survival, using a combination of genetic and pharmacological tools in mice and cultured mouse and human islets. We showed that E₂ favors islet survival by preventing apoptosis via ERα and ERβ through ERE-independent, extra-nuclear mechanisms and with a predominant ERα effect. We also revealed that E₂ prevents apoptosis via GPER-dependent mechanisms. Here, we show that E₂ prevents apoptosis independently of gene transcription or de novo protein synthesis suggesting that E₂ cytoprotection happens independently of nuclear events. Furthermore, we report that E₂ islet cytoprotection can be mimicked by the nonfeminizing E₂ stereoisomer, 17α-estradiol, suggesting that it is partially non-estrogen receptor mediated. These studies identify novel estrogen pathways and targets to protect islet survival.

The gonadal steroid, 17β-estradiol (E₂), is primarily known to be a female sex hormone, but the actions of E₂ are not limited to reproductive tissues. E₂ influences the development, differentiation, growth, and function of various tissues. E₂ is also an important survival factor. For example, E₂ is a neuroprotective hormone against multiple oxidative and pro-apoptotic injuries. In addition, E₂ protects islet survival from pro-apoptotic insults (1). Using a mouse model of estrogen deficiency by deletion of the aromatase gene, we initially showed that circulating E₂ acts as a protective hormone preventing pancreatic β-cell apoptosis in vivo in both sexes (2). Male and female mice develop a vulnerability to streptozotocin (STZ)-induced β-cell damage and insulin deficiency when estradiol production is genetically suppressed (2). We showed that E₂ prevents STZ-induced β-cell apoptosis at least partially via the estrogen receptor (ER)α (2). E₂ signals via classical ER-α, ERβ, and an extranuclear form of ERα. In a recent study, we determined the contribution of these receptors to β-cell survival (3). In the classical ER signaling pathway, E₂-activated ERα binds as a homodimer to either an ERE or a non-ERE tethered promoter to initiate gene transcription (4). To investigate whether an ERα-ERE or non-ERE signaling mechanism protects β-cell survival in vivo, we used an ERα knock-in mouse with a mutation of the DNA-binding domain of ERα that eliminates ERα binding to the ERE. We observed that ERα cytoprotection of islets in vivo is ERE independent. We also observed that both ERα and ERβ showed predominant cytosolic localization in mouse and human β-cells. We showed that E₂ prevents islet apoptosis via extra-nuclear and membrane ERs with a predominant ERα effect in mouse and human islets. The existence of both ERα and ERβ in β-cells did not demonstrate evidence of ERβ antagonism of ERα action, since pharmacological inhibition or genetic elimination of ERβ in islets did not enhance E₂ anti-apoptotic action via ERα. In addition, the combined elimination of these receptors did not synergize to abolish E₂ cytoprotection following exposure of islets to oxidative stress. We conclude that ERα and ERβ favor islet survival using non-redundant and distinct cellular pathways. These pathways are under investigation.

The G protein-coupled estrogen receptor (GPER), also known as GPR30, is a membrane receptor for estrogens that mediates rapid non-genomic signals (5, 6). Ten years ago, Angel Nadal reported the existence of a membrane GPCR in β-cells, unrelated to ERα and ERβ, which may be GPER (7, 8). A more recent study showed that GPER-deficient mice have impaired E₂-stimulated insulin release from isolated islets and impaired glucose-stimulated insulin secretion in vivo, suggesting that GPER is involved in islet biology (9). We found that GPER protein is expressed in mouse and human islets and that elimination of GPER predisposes to STZ-induced islet apoptosis in female mice (2). In addition, we showed that pharmacological activation of GPER using the agonist G1, which selectively activates GPER in a cellular environment containing ERα and ERβ (10), is efficient in protecting H₂O₂-induced apoptosis in cultured mouse and human islets. However, E₂ cytoprotection from apoptosis was retained in cultured GPER-deficient islets demonstrating that ERα can compensate for GPER deficiency. Thus, E₂ protects islet survival by preventing apoptosis via ERα and ERβ and GPER pathways with a predominant ERα effect.

It is interesting to observe that during the initial five most critical hours of the induction of apoptosis (length of islet exposure to H₂O₂), the E₂-mediated pro-survival effect does not necessitate the presence of an ER in the nucleus. Furthermore, we provide additional evidence that E₂ signals favor survival independently of nuclear events since E₂ anti-apoptotic action is not impaired by inhibition of gene transcription or de novo protein synthesis (Fig. 1). This suggests that ER- and GPER-mediated antiapoptotic actions involve rapid events such as the alteration in the phosphorylation of proteins or the function of ion channels. These pathways are under investigation.

Figure 1. E₂ prevents β-cell apoptosis independently of gene transcription and de novo protein synthesis.

Percentage of apoptotic MIN6 cells treated with Cycloheximide or Actinomycin D. Apoptosis was measured by nuclear morphology as described (3). V: vehicle. # P<0.001, * P<0.05, ** P<0.01

Evidence indicates that estrogens provide cytoprotection via mechanisms independent of ERs (11). For example, the neuroprotective effect of E₂ can be reproduced by the nonfeminizing nonestrogenic E₂ stereoisomer 17α-estradiol (12). It is not clear whether 17α-estradiol selectively binds a specific extranuclear pool of ERα and ERβ providing selective non-genomic effects with minimal transcription, or binds to a membrane receptor independent from ERα and ERβ (13). It is clear, however, that 17α-estradiol is not capable of activating GPER (10). 17α-Estradiol has also been reported to intercalate into cell membranes, where it terminates lipid peroxidation, thereby preserving membrane integrity in an ER-independent manner (14). In MIN6 β-cells, we observe that 17α-estradiol shows weak transcriptional activity compared to E₂ on a reporter construct containing an ERE (Fig. 2A). However, we find that exposure of human islets to E₂ or 17 α-estradiol produced a similar and robust protection against H₂O₂-induced apoptosis (Fig. 2B). Thus, estrogens protect islet survival via extranuclear, membrane, and perhaps non-receptor mediated events. Importantly, 17α-estradiol may be a candidate for gender-neutral antiapoptotic therapy in diabetes, because it has few of the biological effects associated with the female hormone activity.

Figure 2. 17α-estradiol protects islet survival.

(A) Relative luciferase activity in MIN6 cells transfected with an ERE reporter construct and treated with E₂ (β-E₂) or 17α-estradiol (α-E₂) (10⁻⁸M). (B) Percentage of apoptotic cells in cultured human islets. Islets were treated with E₂ (β-E₂) or 17α-estradiol (α-E₂) (10⁻⁸M) for 48 h, followed by exposure to H₂O₂ (100μM) for the last 5 hours. Apoptosis was assessed by nuclear morphology as described (3). Values represent the mean ± SE of five independent experiments. * P<0.05, ** P<0.01.