Dysregulation of the Pdx1/Ovol2/Zeb2 axis in dedifferentiated ß-cells triggers the induction of genes associated with epithelial-mesenchymal transition in diabetes.

OBJECTIVE: ß-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We previously showed that increased miR-7 levels trigger ß-cell dedifferentiation and diabetes. We used ß-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of ß-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes. METHODS: We performed bulk and single-cell RNA sequencing (RNA-seq) in islets obtained from ß-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-bH1 cell lines. We analysed previously published mouse and human T2D data sets. RESULTS: Bulk RNA-seq revealed that ß-cell dedifferentiation is associated with the induction of genes associated with epithelial-to-mesenchymal transition (EMT) in prediabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in ß-cells. These molecular changes are associated with a weakening of ß-cell: ß-cell contacts, increased extracellular matrix (ECM) deposition, and TGFß-dependent islet fibrosis. We found that the mesenchymal reprogramming of ß-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in ß-cells. Notable among genes transactivated by Pdx1 is Ovol2, which encodes a transcriptional repressor of the EMT transcription factor Zeb2. Following compromised ß-cell identity, the reduction in Pdx1 gene expression causes a decrease in Ovol2 protein, triggering mesenchymal reprogramming of ß-cells through the induction of Zeb2. We provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islets in T2D subjects. CONCLUSIONS: Our study indicates that miR-7-mediated ß-cell dedifferentiation induces EMT signalling and a chronic response to tissue injury, which alters the islet microenvironment and predisposes to fibrosis. This research suggests that regulators of EMT signalling may represent novel therapeutic targets for treating ß-cell dysfunction and fibrosis in T2D.

Role of Hepatic Glucocorticoid Receptor in Metabolism in Models of 5αR1 Deficiency in Male Mice.

Inhibition of 5α-reductases impairs androgen and glucocorticoid metabolism and induces insulin resistance in humans and rodents. The contribution of hepatic glucocorticoids to these adverse metabolic changes was assessed using a liver-selective glucocorticoid receptor (GR) antagonist, A-348441. Mice lacking 5α-reductase 1 (5αR1-KO) and their littermate controls were studied during consumption of a high-fat diet, with or without A-348441(120 mg/kg/d). Male C57BL/6 mice (age, 12 weeks) receiving dutasteride (1.8 mg/kg/d)) or vehicle with consumption of a high-fat diet, with or without A-348441, were also studied. In the 5αR1-KO mice, hepatic GR antagonism improved diet-induced insulin resistance but not more than that of the controls. Liver steatosis was not affected by hepatic GR antagonism in either 5αR1KO mice or littermate controls. In a second model of 5α-reductase inhibition using dutasteride and hepatic GR antagonism with A-348441 attenuated the excess weight gain resulting from dutasteride (mean ± SEM, 7.03 ± 0.5 vs 2.13 ± 0.4 g; dutasteride vs dutasteride plus A-348441; P < 0.05) and normalized the associated hyperinsulinemia after glucose challenge (area under the curve, 235.9 ± 17 vs 329.3 ± 16 vs 198.4 ± 25 ng/mL/min; high fat vs high fat plus dutasteride vs high fat plus dutasteride plus A-348441, respectively; P < 0.05). However, A-348441 again did not reverse dutasteride-induced liver steatosis. Thus, overall hepatic GR antagonism improved the insulin resistance but not the steatosis induced by a high-fat diet. Moreover, it attenuated the excessive insulin resistance caused by pharmacological inhibition of 5α-reductases but not genetic disruption of 5αR1. The use of dutasteride might increase the risk of type 2 diabetes mellitus and reduced exposure to glucocorticoids might be beneficial.

Metabolic dysfunction in female mice with disruption of 5α-reductase 1.

5α-Reductases irreversibly catalyse A-ring reduction of pregnene steroids, including glucocorticoids and androgens. Genetic disruption of 5α-reductase 1 in male mice impairs glucocorticoid clearance and predisposes to glucose intolerance and hepatic steatosis upon metabolic challenge. However, it is unclear whether this is driven by changes in androgen and/or glucocorticoid action. Female mice with transgenic disruption of 5α-reductase 1 (5αR1-KO) were studied, representing a 'low androgen' state. Glucocorticoid clearance and stress responses were studied in mice aged 6 months. Metabolism was assessed in mice on normal chow (aged 6 and 12 m) and also in a separate cohort following 1-month high-fat diet (aged 3 m). Female 5αR1-KO mice had adrenal suppression (44% lower AUC corticosterone after stress), and upon corticosterone infusion, accumulated hepatic glucocorticoids (~27% increased corticosterone). Female 5αR1-KO mice aged 6 m fed normal chow demonstrated insulin resistance (~35% increased area under curve (AUC) for insulin upon glucose tolerance testing) and hepatic steatosis (~33% increased hepatic triglycerides) compared with controls. This progressed to obesity (~12% increased body weight) and sustained insulin resistance (~38% increased AUC insulin) by age 12 m. Hepatic transcript profiles supported impaired lipid ß-oxidation and increased triglyceride storage. Female 5αR1-KO mice were also predisposed to develop high-fat diet-induced insulin resistance. Exaggerated predisposition to metabolic disorders in female mice, compared with that seen in male mice, after disruption of 5αR1 suggests phenotypic changes may be underpinned by altered metabolism of glucocorticoids rather than androgens.

Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry.

Estrogens circulate at concentrations less than 20pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the "reagent" group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought. Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify "FMP" derivatives of estrogens, following LC separation. Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2pg on-column and the method was linear from 1-400pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (<20%). The derivatives were stable over 24h at 10°C (7-9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5mL) 135-473, 193-722pmol/L; male plasma (1mL) 25-111, 60-180pmol/L and post-menopausal female plasma (2mL), 22-78, 29-50pmol/L. Thus FMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of estrogens in low abundance in plasma and serum, offering advantages in specificity over immunoassay and existing MS techniques.