Modulation of Estrogen Receptor Signaling Sensitizes Female Mice to the Development of Diabetes

Abstract

Type 1 (T1) and type 2 (T2) diabetes mellitus (DM), along with their accompanying hyperglycemia, are associated with a multitude of comorbidities, including the development of diabetic kidney disease and nonalcoholic steatohepatitis. Although the hallmarks of these metabolic disorders have been well‐characterized in population and animal studies, it is becoming increasingly apparent that DM manifests itself differently in men and women. Epidemiological studies indicate that men are at higher risk of developing T2DM than pre‐menopausal women, suggesting that estrogen may serve a protective role in the development of the disorder. Sex differences have also been observed in animal models of DM where females tend to resist diabetic induction and have trouble attaining the hyperglycemic threshold, resulting in a heavy reliance on male preclinical models.Previously, we used streptozotocin (STZ; 55mg/kg BW) to induce T1DM in both male and female C57BL/6 mice to characterize their development of diabetic comorbidities. While male mice developed robust hyperglycemia (threshold was 2‐hour fasting glucose > 230 mg/dL) compared to their vehicle controls, the female mice were unaffected by STZ treatment (Schiazza et al. 2021). It has been found that estrogen receptor (ER) signaling has anti‐apoptotic activity; thus, we hypothesized that this signaling pathway may be offering protection from STZ challenge. To test this, male and female C57BL/6 mice were implanted with slow release pellets of either tamoxifen (1.25mg over 60 days) or its active metabolite, 4‐hydroxytamoxifen (4OH; 0.6mg over 21 days), to modulate ER signaling. Following implantation, mice were challenged with STZ or vehicle (citrate buffer; 55 mg/kg BW) for 5 consecutive days. Two weeks following the last injection, two hour fasting blood glucose measurements were collected. While tamoxifen failed to induce persistent hyperglycemia or impaired glucose tolerance (likely due to the low circulating levels of 4OH as measured by mass spectrometry), implantation of 4OH promoted appreciable hyperglycemia in both males and females indicating that ER signaling does have an anti‐diabetic role in both sexes (Males: 4OH Vehicle ‐ 185 ± 4.95 mg/dL, Placebo STZ ‐ 450 ± 11.6 mg/dL, 4OH STZ ‐ 575 ± 4.95 mg/dL; Females: 4OH vehicle ‐ 178 ± 3.50 mg/dL, Placebo STZ ‐ 330 ± 8.22 mg/dL, 4OH STZ ‐ 436 ± 4.65 mg/dL). Given the effectiveness of ER modulation, we next aimed to understand the downstream effects that 4OH has on both the kidney and liver. TaqMan qPCR for > 90 ER‐regulated genes was performed on cDNA isolated from all treatment groups. Initial analysis revealed that 4OH treatment modulates membrane dynamics with differential expression observed in genes associated with caveolin and lipid rafts. We also observed changes in genes regulating cellular iron handling. Efforts are currently underway to confirm these changes at both the gene and protein level, and to understand the global impact that ER modulation may have on DM. Collectively, these data have implications both for producing an effective murine model of female DM, and for illustrating potential cellular changes that occur in breast cancer patients who are treated with tamoxifen.