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.

Canadian survey on pandemic flu preparations.

BACKGROUND: The management of pandemic influenza creates public health challenges.An ethical framework, 'Stand on Guard for Thee: ethical considerations in pandemic influenza preparedness' that served as a template for the World Health Organization's global consultation on pandemic planning, was transformed into a survey administered to a random sample of 500 Canadians to obtain opinions on key ethical issues in pandemic preparedness planning. METHODS: All framework authors and additional investigators created items that were pilot-tested with volunteers of both sexes and all socioeconomic strata. Surveys were telephone administered with random sampling achieved via random digit dialing (RDD). Eligible participants were adults, 18 years or older, with per province stratification equaling provincial percent of national population. Descriptive results were tabulated and logistic regression analyses were used to assess whether demographic factors were significantly associated with outcomes. RESULTS: 5464 calls identified 559 eligible participants of whom 88.5% completed surveys. Over 90% of subjects agreed the most important goal of pandemic influenza preparations was saving lives, with 41% endorsing saving lives solely in Canada and 50% endorsing saving lives globally as the highest priority. Older age (OR = 8.51, p < 0.05) and current employment (OR = 9.48, p < 0.05) were associated with an endorsement of saving lives globally as highest priority. About 90% of respondents supported the obligation of health care workers to report to work and face influenza pandemic risks excepting those with a serious health condition that increased risks. Over 84% supported the government's provision of disability insurance and death benefits for health care workers facing elevated risk. Strong majorities favored stocking adequate protective antiviral dosages for all Canadians (92%) and, if effective, influenza vaccinations (95%). Over 70% agreed Canada should provide international assistance to poorer countries for pandemic preparation, even if resources for Canadians were reduced. While 92% of this group, believed provision should be 7 to 10% of all resources generated, 43% believed the provision should be greater than 10%. CONCLUSIONS: Results suggest trust in public health officials to make difficult decisions, providing emphasis on reciprocity and respect for individual rights.

Project examining effectiveness in clinical ethics (PEECE): phase 1-- descriptive analysis of nine clinical ethics services.

OBJECTIVE: The field of clinical ethics is relatively new and expanding. Best practices in clinical ethics against which one can benchmark performance have not been clearly articulated. The first step in developing benchmarks of clinical ethics services is to identify and understand current practices. DESIGN AND SETTING: Using a retrospective case study approach, the structure, activities, and resources of nine clinical ethics services in a large metropolitan centre are described, compared, and contrasted. RESULTS: The data yielded a unique and detailed account of the nature and scope of clinical ethics services across a spectrum of facilities. General themes emerged in four areas-variability, visibility, accountability, and complexity. There was a high degree of variability in the structures, activities, and resources across the clinical ethics services. Increasing visibility was identified as a significant challenge within organisations and externally. Although each service had a formal system for maintaining accountability and measuring performance, differences in the type, frequency, and content of reporting impacted service delivery. One of the most salient findings was the complexity inherent in the provision of clinical ethics services, which requires of clinical ethicists a broad and varied skill set and knowledge base. Benchmarks including the average number of consults/ethicist per year and the hospital beds/ethicist ratio are presented. CONCLUSION: The findings will be of interest to clinical ethicists locally, nationally, and internationally as they provide a preliminary framework from which further benchmarking measures and best practices in clinical ethics can be identified, developed, and evaluated.

Sugar-cellulose composites VII. A comparative assessment of corn syrup as a fiber substitute in paper.

The ability of the mixture of alpha-linked glucose oligomers in corn syrup to function as intrafiber components of paper is statistically assessed at the 95% confidence level for two degrees of refining (257 and 402 mL CSF) of never-dried commercial softwood and hardwood/softwood pulps. Independent of the extent of refining, the replacement of the beaten fibers by corn syrup at about the 6% level does not result in a statistically significant deterioration (two-sided t-test, p=0.05) of the ISO brightness or of the tensile, tear, burst or surface strength values of the resultant papers. The economic potential of such fiber replacement is discussed. As paper additives, the mixed alpha-oligomers (corn syrup), glucose and lactose perform indistinguishably.

Health and productivity of older workers.

The purpose of this paper is to present a summary of a critical review of the literature on age, health, and work. The health of older workers is a concern to the extent that the productivity of older workers is considered to be a function of age-related health, the general conclusion being that age-related declines in health inevitably lead to decreased productivity. This paper investigates the implications of age-related physiological and psychological changes on the productivity of older workers, by focusing on a critical examination of the ways in which productivity has been conceptualized and operationalized. This paper concludes that the emerging concept of "work capacity" has the greatest potential for addressing the productivity issues of workforce aging. Implications for public and corporate policy and for further research are also examined.