Selective Activation of Estrogen Receptor α Activation Function-1 Is Sufficient to Prevent Obesity, Steatosis, and Insulin Resistance in Mouse.

Estrogen receptor α (ERα) regulates gene transcription through two activation functions (ERα-AF1 and ERα-AF2). We recently found that the protection conferred by 17ß-estradiol against obesity and insulin resistance requires ERα-AF2 but not ERα-AF1. However, the interplay between the two ERα-AFs is poorly understood in vivo and the metabolic influence of a specific ERα-AF1 action remains to be explored. To this end, wild-type, ERα-deficient, or ERα-AF1-deficient ovariectomized female mice were fed a high-fat diet and concomitantly administered with vehicle or tamoxifen, a selective ER modulator that acts as a ERα-AF1 agonist/ERα-AF2 antagonist. In ovariectomized wild-type mice, tamoxifen significantly reduced food intake and totally prevented adiposity, insulin resistance, and steatosis. These effects were abolished in ERα-deficient and ERα-AF1-deficient mice, revealing the specific role of ERα-AF1 activation. Finally, hepatic gene expression changes elicited by tamoxifen in wild-type mice were abrogated in ERα-AF1-deficient mice. The combination of pharmacologic and transgenic approaches thus indicates that selective ERα-AF1 activation by tamoxifen is sufficient to elicit metabolic protection, contrasting with the specific requirement of ERα-AF2 in the metabolic actions of 17ß-estradiol. This redundancy in the ability of the two ERα-AFs to separately mediate metabolic prevention strikingly contrasts with the contribution of both ERα-AFs in breast cancer proliferation, shedding new light on the therapeutic potential of selective ER modulation.

Associations between hepatic miRNA expression, liver triacylglycerols and gut microbiota during metabolic adaptation to high-fat diet in mice.

AIMS/HYPOTHESIS: Despite the current pandemic of metabolic diseases, our understanding of the diverse nature of the development of metabolic alterations in people who eat a high-fat diet (HFD) is still poor. We recently demonstrated a cardio-metabolic adaptation in mice fed an HFD, which was characterised by a specific gut and periodontal microbiota profile. Since the severity of hepatic disease is characterised by specific microRNA (miRNA) signatures and the gut microbiota is a key driver of both hepatic disease and miRNA expression, we analysed the expression of three hepatic miRNA and studied their correlation with hepatic triacylglycerol content and gut microbiota. METHODS: Two cohorts of C57BL/6 4-week-old wild-type (WT) male mice (n = 62 and n = 96) were fed an HFD for 3 months to provide a model of metabolic adaptation. Additionally 8-week-old C57BL/6 mice, either WT or of different genotypes, with diverse gut microbiota (ob/ob, Nod1, Cd14 knockout [Cd14KO] and Nod2) or without gut microbiota (axenic mice) were fed a normal chow diet. Following which, glycaemic index, body weight, blood glucose levels and hepatic triacylglycerol levels were measured. Gut (caecum) microbiota taxa were analysed by pyrosequencing. To analyse hepatic miRNA expression, real-time PCR was performed on total extracted miRNA samples. Data were analysed using two-way ANOVA followed by the Dunnett's post hoc test, or by the unpaired Student's t test. A cluster analysis and multivariate analyses were also performed. RESULTS: Our results demonstrated that the expression of miR-181a, miR-666 and miR-21 in primary murine hepatocytes is controlled by lipopolysaccharide in a dose-dependent manner. Of the gut microbiota, Firmicutes were positively correlated and Proteobacteria and Bacteroides acidifaciens were negatively correlated with liver triacylglycerol levels. Furthermore, the relative abundance of Firmicutes was negatively correlated with hepatic expression of miR-666 and miR-21. In contrast, the relative abundance of B. acidifaciens was positively correlated with miR-21. CONCLUSIONS/INTERPRETATION: We propose the involvement of hepatic miRNA, liver triacylglycerols and gut microbiota as a new triad that underlies the molecular mechanisms by which gut microbiota governs hepatic pathophysiology during metabolic adaptation to HFD.

Cardiovascular therapy use, modification, and in-hospital death in patients with COVID-19: A cohort study.

AIMS: To assess the associations of exposure and modifications in exposure (i.e., discontinuation on admission, initiation during hospitalization) to eight common cardiovascular therapies with the risk of in-hospital death among inpatients with coronavirus disease 2019 (COVID-19). METHODS: In this observational study including 838 hospitalized unvaccinated adult patients with confirmed COVID-19, the use of cardiovascular therapies was assessed using logistic regression models adjusted for potential confounders. RESULTS: No cardiovascular therapy used before hospitalization was associated with an increased risk of in-hospital death. During hospitalization, the use of diuretics (aOR 2.59 [1.68-3.98]) was associated with an increase, and the use of agents acting on the renin-angiotensin system (aOR 0.39 [0.23-0.64]) and lipid-lowering agents (aOR 0.41 [0.24-0.68]) was associated with a reduction in the odds of in-hospital death. Exposure modifications associated with decreased survival were the discontinuation of an agent acting on the renin-angiotensin system (aOR 4.42 [2.08-9.37]), a ß-blocker (aOR 5.44 [1.16-25.46]), a lipid-modifying agent (aOR 3.26 [1.42-7.50]) or an anticoagulant (aOR 5.85 [1.25-27.27]), as well as the initiation of a diuretic (aOR 5.19 [2.98-9.03]) or an antiarrhythmic (aOR 6.62 [2.07-21.15]). Exposure modification associated with improved survival was the initiation of an agent acting on the renin-angiotensin system (aOR 0.17 [0.03-0.82]). CONCLUSION: In hospitalized and unvaccinated patients with COVID-19, there was no detrimental association of the prehospital use of any regular cardiovascular medication with in-hospital death, and these therapies should be continued as recommended.

Symptoms and quality of life at 1-year follow up of patients discharged after an acute COVID-19 episode.

AIM OF THE STUDY: Patients surviving COVID-19 have been described as being at risk of developing sequelae. We aimed to investigate and elicit persistent symptoms, emotional status and quality-of-life in patients discharged after an acute COVID-19 episode. METHODS: Patient-reported outcome measures were collected during a telephone interview 30 days and 1 year after discharge. Patients' general health status was evaluated using questions based on their symptoms, emotional status was assessed using the items 9 to 12 of the HeartQoL questionnaire and quality of life was assessed at 1 year through the EQ-5D-5L. In patients with a history of cardiovascular disease, all 14 items of the HeartQoL questionnaire were completed to derive the HeartQoL global score. RESULTS: Among 687 patients who survived after being hospitalised for COVID-19 at the University Hospitals of Geneva between 26 February and 26 April 2020, 184 (27%) and 165 (24%), respectively, participated in the follow-up at 30 days and 1 year. Of these 184 participants, 62% were male, median age was 58 years and 21% had a past medical history of cardiovascular disease. At one month after discharge, 61% (113/184) of patients presented fatigue and 28% (52/184) dyspnoea. One year after discharge, the main complaints were persistent fatigue in 27% (45/165) of patients, neurological problems in 17% (28/165) and dyspnoea in 14% (23/165). Eight percent (14/184) of patients declared being significantly worried 1 month after discharge and 5% (9/184) feeling depressed. The number of patients reporting being significantly worried or depressed at 1 year was lower. Regarding the quality of life at 1 year, the median EQ-5D-5L visual analogue scale score was 80 (interquartile range 70-90). CONCLUSIONS: Approximately half of patients reported some symptoms 1 year after discharge following an acute episode of COVID-19. The predominant symptom was persistent fatigue both at 1-month and at 1-year follow-up. Emotional status and quality of life appeared satisfactory.

Let-7e-5p Regulates GLP-1 Content and Basal Release From Enteroendocrine L Cells From DIO Male Mice.

Characterization of enteroendocrine L cells in diabetes is critical for better understanding of the role of glucagon-like peptide-1 (GLP-1) in physiology and diabetes. We studied L-cell transcriptome changes including microRNA (miRNA) dysregulation in obesity and diabetes. We evaluated the regulation of miRNAs through microarray analyses on sorted enteroendocrine L cells from control and obese glucose-intolerant (I-HFD) and hyperglycemic (H-HFD) mice after 16 weeks of respectively low-fat diet (LFD) or high-fat diet (HFD) feeding. The identified altered miRNAs were studied in vitro using the mouse GLUTag cell line to investigate their regulation and potential biological functions. We identified that let-7e-5p, miR-126a-3p, and miR-125a-5p were differentially regulated in L cells of obese HFD mice compared with control LFD mice. While downregulation of let-7e-5p expression was observed in both I-HFD and H-HFD mice, levels of miR-126a-3p increased and of miR-125a-5p decreased significantly only in I-HFD mice compared with controls. Using miRNA inhibitors and mimics we observed that modulation of let-7e-5p expression affected specifically GLP-1 cellular content and basal release, whereas Gcg gene expression and acute GLP-1 secretion and cell proliferation were not affected. In addition, palmitate treatment resulted in a decrease of let-7e-5p expression along with an increase in GLP-1 content and release, suggesting that palmitate acts on GLP-1 through let-7e-5p. By contrast, modulation of miR-125a-5p and miR-126a-3p in the same conditions did not affect content or secretion of GLP-1. We conclude that decrease of let-7e-5p expression in response to palmitate may constitute a compensatory mechanism contributing to maintaining constant glycemia in obese mice.

The Role of Sexual Hormones on the Enteroinsular Axis.

Sex steroid estrogens, androgens, and progesterone, produced by the gonads, which have long been considered as endocrine glands, are implicated in sexual differentiation, puberty, and reproduction. However, the impact of sex hormones goes beyond these effects through their role on energy metabolism. Indeed, sex hormones are important physiological regulators of glucose homeostasis and, in particular, of the enteroinsular axis. In this review, we describe the roles of estrogens, androgens, and progesterone on glucose homeostasis through their effects on pancreatic α- and ß-cells, as well as on enteroendocrine L-cells, and their implications in hormonal biosynthesis and secretion. The analysis of their mechanisms of action with the dissection of the receptors implicated in the several protective effects could provide some new aspects of the fine-tuning of hormonal secretion under the influence of the sex. This knowledge paves the way to the understanding of transgender physiology and new potential therapeutics in the field of type 2 diabetes.

miR-132-3p is a positive regulator of alpha-cell mass and is downregulated in obese hyperglycemic mice.

OBJECTIVE: Diabetes is a complex disease implicating several organs and cell types. Within the islets, dysregulation occurs in both alpha- and beta-cells, leading to defects of insulin secretion and increased glucagon secretion. Dysregulation of alpha-cells is associated with transcriptome changes. We hypothesized that microRNAs (miRNAs) which are negative regulators of mRNA stability and translation could be involved in alpha-cell alterations or adaptations during type 2 diabetes. METHODS: miRNA microarray analyses were performed on pure alpha- and beta-cells from high-fat diet fed obese hyperglycemic mice and low-fat diet fed controls. Then, the most regulated miRNA was overexpressed or inhibited in primary culture of mouse and human alpha-cells to determine its molecular and functional impact. RESULTS: 16 miRNAs were significantly regulated in alpha-cells of obese hyperglycemic mice and 28 in beta-cells. miR-132-3p had the strongest regulation level in alpha-cells, where it was downregulated, while we observed an opposite upregulation in beta-cells. In vitro experiments showed that miR-132-3p, which is inversely regulated by somatostatin and cAMP, is a positive modulator of alpha-cell proliferation and implicated in their resistance to apoptosis. These effects are associated with the regulation of a series of genes, including proliferation and stress markers Mki67 and Bbc3 in mouse and human alpha-cells, potentially involved in miR-132-3p functions. CONCLUSIONS: Downregulation of miR-132-3p in alpha-cells of obese diabetic mice may constitute a compensatory mechanism contributing to keep glucagon-producing cell number constant in diabetes.

High-fat diet impacts more changes in beta-cell compared to alpha-cell transcriptome.

Characterization of endocrine-cell functions and associated molecular signatures in diabetes is crucial to better understand why and by which mechanisms alpha and beta cells cause and perpetuate metabolic abnormalities. The now recognized role of glucagon in diabetes control is a major incentive to have a better understanding of dysfunctional alpha cells. To characterize molecular alterations of alpha cells in diabetes, we analyzed alpha-cell transcriptome from control and diabetic mice using diet-induced obesity model. To this aim, we quantified the expression levels of total mRNAs from sorted alpha and beta cells of low-fat and high-fat diet-treated mice through RNAseq experiments, using a transgenic mouse strain allowing collections of pancreatic alpha- and beta-cells after 16 weeks of diet. We now report that pancreatic alpha cells from obese hyperglycemic mice displayed minor variations of their transcriptome compared to controls. Depending on analyses, we identified 11 to 39 differentially expressed genes including non-alpha cell markers mainly due to minor cell contamination during purification process. From these analyses, we identified three new target genes altered in diabetic alpha cells and potently involved in cellular stress and exocytosis (Upk3a, Adcy1 and Dpp6). By contrast, analysis of the beta-cell transcriptome from control and diabetic mice revealed major alterations of specific genes coding for proteins involved in proliferation and secretion. We conclude that alpha cell transcriptome is less reactive to HFD diet compared to beta cells and display adaptations to cellular stress and exocytosis.

17-ß Estradiol regulates proglucagon-derived peptide secretion in mouse and human α- and L cells.

Clinical and experimental data indicate a beneficial effect of estrogens on energy and glucose homeostasis associated with improved insulin sensitivity and positive effects on insulin secretion. The aim of the study was to investigate the impact of estrogens on proglucagon-producing cells, pancreatic α cells, and enteroendocrine L cells. The consequences of sexual hormone deprivation were evaluated in ovariectomized mice (ovx). Ovx mice exhibited impaired glucose tolerance during oral glucose tolerance tests (OGTT), which was associated with decreased GLP-1 intestinal and pancreatic secretion and content, an effect that was reversed by estradiol (E2) treatment. Indeed, E2 increased oral glucose-induced GLP-1 secretion in vivo and GLP-1 secretion from primary culture of mouse and human α cells through the activation of all 3 estrogen receptors (ERs), whereas E2-induced GLP-1 secretion from mouse and human intestinal explants occurred only by ERß activation. Underlying the implication of ERß, its selective agonist WAY20070 was able to restore glucose tolerance in ovx mice at least partly through plasma GLP-1 increase. We conclude that E2 directly controls both α- and L cells to increase GLP-1 secretion, in addition to its effects on insulin and glucagon secretion, highlighting the potential beneficial role of the estrogenic pathway and, more particularly, of ERß agonists to prevent type 2 diabetes.

α-Cell Dysfunctions and Molecular Alterations in Male Insulinopenic Diabetic Mice Are Not Completely Corrected by Insulin.

Glucagon and α-cell dysfunction are critical in the development of hyperglycemia during diabetes both in humans and rodents. We hypothesized that α-cell dysfunction leading to dysregulated glucagon secretion in diabetes is due to both a lack of insulin and intrinsic defects. To characterize α-cell dysfunction in diabetes, we used glucagon-Venus transgenic male mice and induced insulinopenic hyperglycemia by streptozotocin administration leading to alterations of glucagon secretion. We investigated the in vivo impact of insulinopenic hyperglycemia on glucagon-producing cells using FACS-sorted α-cells from control and diabetic mice. We demonstrate that increased glucagonemia in diabetic mice is mainly due to increases of glucagon release and biosynthesis per cell compared with controls without changes in α-cell mass. We identified genes coding for proteins involved in glucagon biosynthesis and secretion, α-cell differentiation, and potential stress markers such as the glucagon, Arx, MafB, cMaf, Brain4, Foxa1, Foxa3, HNF4α, TCF7L2, Glut1, Sglt2, Cav2.1, Cav2.2, Nav1.7, Kir6.2/Sur1, Pten, IR, NeuroD1, GPR40, and Sumo1 genes, which were abnormally regulated in diabetic mice. Importantly, insulin treatment partially corrected α-cell function and expression of genes coding for proglucagon, or involved in glucagon secretion, glucose transport and insulin signaling but not those coding for cMAF, FOXA1, and α-cell differentiation markers as well as GPR40, NEUROD1, CAV2.1, and SUMO1. Our results indicate that insulinopenic diabetes induce marked α-cell dysfunction and molecular alteration, which are only partially corrected by in vivo insulin treatment.

Functional and Molecular Adaptations of Enteroendocrine L-Cells in Male Obese Mice Are Associated With Preservation of Pancreatic α-Cell Function and Prevention of Hyperglycemia.

Glucose homeostasis depends on the coordinated secretion of glucagon, insulin, and Glucagon-like peptide (GLP)-1 by pancreas and intestine. Obesity, which is associated with an increased risk of developing insulin resistance and type 2 diabetes, affects the function of these organs. Here, we investigate the functional and molecular adaptations of proglucagon-producing cells in obese mice to better define their involvement in type 2 diabetes development. We used GLU-Venus transgenic male mice specifically expressing Venus fluorochrome in proglucagon-producing cells. Mice were subjected to 16 weeks of low-fat diet or high-fat diet (HFD) and then subdivided by measuring glycated hemoglobin (HbA1c) in 3 groups: low-fat diet mice and I-HFD (glucose-intolerant) mice with similar HbA1c and H-HFD (hyperglycemic) mice, which exhibited higher HbA1c. At 16 weeks, both HFD groups exhibited similar weight gain, hyperinsulinemia, and insulin resistance. However, I-HFD mice exhibited better glucose tolerance compared with H-HFD mice. I-HFD mice displayed functional and molecular adaptations of enteroendocrine L-cells resulting in increased intestinal GLP-1 biosynthesis and release as well as maintained pancreatic α- and ß-cell functions. By contrast, H-HFD mice exhibited dysfunctional L, α- and ß-cells with increased ß- and L-cell numbers. Administration of the GLP-1R antagonist Exendin9-39 in I-HFD mice led to hyperglycemia and alterations of glucagon secretion without changes in insulin secretion. Our results highlight the cross-talk between islet and intestine endocrine cells and indicate that a compensatory adaptation of L-cell function in obesity plays an important role in preserving glucose homeostasis through the control of pancreatic α-cell functions.

Prevention of obesity and insulin resistance by estrogens requires ERα activation function-2 (ERαAF-2), whereas ERαAF-1 is dispensable.

The beneficial metabolic actions of estrogen-based therapies are mainly mediated by estrogen receptor α (ERα), a nuclear receptor that regulates gene transcription through two activation functions (AFs): AF-1 and AF-2. Using mouse models deleted electively for ERαAF-1 (ERαAF-1°) or ERαAF-2 (ERαAF-2°), we determined their respective roles in the actions of estrogens on body composition and glucose homeostasis in response to either a normal diet or a high-fat diet (HFD). ERαAF-2° males and females developed accelerated weight gain, massive adiposity, severe insulin resistance, and glucose intolerance--quite reminiscent of the phenotype observed in mice deleted for the entire ERα protein (ERα(-/-)). In striking contrast, ERαAF-1° and wild-type (wt) mice shared a similar metabolic phenotype. Accordingly, 17ß-estradiol administration regulated key metabolic genes in insulin-sensitive tissues and conferred a strong protection against HFD-induced metabolic disturbances in wt and ERαAF-1° ovariectomized mice, whereas these actions were totally abrogated in ERαAF-2° and ERα(-/-) mice. Thus, whereas both AFs have been previously shown to contribute to endometrial and breast cancer cell proliferation, the protective effect of estrogens against obesity and insulin resistance depends on ERαAF-2 but not ERαAF-1, thereby delineating new options for selective modulation of ERα.