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Glucocorticoids (GCs) are steroids secreted by the adrenal cortex under the hypothalamic-pituitary-adrenal axis control, one of the major neuro-endocrine systems of the organism. These hormones are involved in tissue repair, immune stability, and metabolic processes, such as the regulation of carbohydrate, lipid, and protein metabolism. Globally, GCs are presented as ‘flight and fight’ hormones and, in that purpose, they are catabolic hormones required to mobilize storage to provide energy for the organism. If acute GC secretion allows fast metabolic adaptations to respond to danger, stress, or metabolic imbalance, long-term GC exposure arising from treatment or Cushing’s syndrome, progressively leads to insulin resistance and, in fine, cardiometabolic disorders. In this review, we briefly summarize the pharmacological actions of GC and metabolic dysregulations observed in patients exposed to an excess of GCs. Next, we describe in detail the molecular mechanisms underlying GC-induced insulin resistance in adipose tissue, liver, muscle, and to a lesser extent in gut, bone, and brain, mainly identified by numerous studies performed in animal models. Finally, we present the paradoxical effects of GCs on beta cell mass and insulin secretion by the pancreas with a specific focus on the direct and indirect (through insulin-sensitive organs) effects of GCs. Overall, a better knowledge of the specific action of GCs on several organs and their molecular targets may help foster the understanding of GCs’ side effects and design new drugs that possess therapeutic benefits without metabolic adverse effects.

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.

Glucocorticoids (GCs) are steroids secreted by the adrenal cortex under the hypothalamic-pituitary-adrenal axis control, one of the major neuro-endocrine systems of the organism. These hormones are involved in tissue repair, immune stability, and metabolic processes, such as the regulation of carbohydrate, lipid, and protein metabolism. Globally, GCs are presented as ‘flight and fight’ hormones and, in that purpose, they are catabolic hormones required to mobilize storage to provide energy for the organism. If acute GC secretion allows fast metabolic adaptations to respond to danger, stress, or metabolic imbalance, long-term GC exposure arising from treatment or Cushing’s syndrome, progressively leads to insulin resistance and, in fine, cardiometabolic disorders. In this review, we briefly summarize the pharmacological actions of GC and metabolic dysregulations observed in patients exposed to an excess of GCs. Next, we describe in detail the molecular mechanisms underlying GC-induced insulin resistance in adipose tissue, liver, muscle, and to a lesser extent in gut, bone, and brain, mainly identified by numerous studies performed in animal models. Finally, we present the paradoxical effects of GCs on beta cell mass and insulin secretion by the pancreas with a specific focus on the direct and indirect (through insulin-sensitive organs) effects of GCs. Overall, a better knowledge of the specific action of GCs on several organs and their molecular targets may help foster the understanding of GCs’ side effects and design new drugs that possess therapeutic benefits without metabolic adverse effects.

Understanding the genetic causes of diseases that affect pancreatic β cells and neurons can give insights into pathways essential for both cell types. Microcephaly, epilepsy, and diabetes syndrome (MEDS) is a congenital disorder with two known etiological genes, IER3IP1 and YIPF5. Both genes encode proteins involved in endoplasmic reticulum (ER) to Golgi trafficking. We used genome sequencing to identify 6 individuals with MEDS caused by biallelic variants in the potentially novel disease gene TMEM167A. All had neonatal diabetes (diagnosed at <6 months) and severe microcephaly, and 5 also had epilepsy. TMEM167A is highly expressed in developing and adult human pancreas and brain. To gain insights into the mechanisms leading to diabetes, we silenced TMEM167A in EndoC-βH1 cells and knocked-in one patient's variant, p.Val59Glu, in induced pluripotent stem cells (iPSCs). Both TMEM167A depletion in EndoC-βH1 cells and the p.Val59Glu variant in iPSC-derived β cells sensitized β cells to ER stress. The p.Val59Glu variant impaired proinsulin trafficking to the Golgi and induced iPSC-β cell dysfunction. The discovery of TMEM167A variants as a genetic cause of MEDS highlights a critical role of TMEM167A in the ER to Golgi pathway in β cells and neurons.

Aims/hypothesis: Pancreatic beta cells secrete insulin to control glucose homeostasis. Beta cells can also adapt their function and mass when more insulin is required, especially in situations of insulin resistance (IR). Beta-cell mass adaptation can be achieved through either beta-cell proliferation or beta-cell neogenesis, a process that involves de novo beta-cell production from precursor cells. Signals and mechanisms that control adult beta-cell neogenesis and regulate the balance between beta-cell proliferation and/or beta-cell neogenesis still need to be fully deciphered. To do so, we previously developed a mouse model of pancreatic adaptation in response to a severe insulin resistance induced by a chronic glucocorticoid (GC) treatment. We observed a massive insulin production due to beta-cell adaptation by both proliferation and neogenesis. In the present study, we aimed at further characterizing beta-cell adaptation in response to mild or severe IR by studying various GC doses, along with other pharmacological or genetic models of IR. Further, we characterized the impact of aging on pancreatic adaptation in response to GC-induced IR. Finally, we precisely quantified adult beta-cell neogenesis by developing an original 3D method of beta-cell mass analysis in toto after organ clearing. Methods: Glucose metabolism, insulin secretion and pancreatic beta-cell adaptation were studied in mice rendered IR either by adipose tissue specific invalidation of SEIPIN, by chronic treatment with the insulin receptor antagonist S961 or by chronic treatment with several doses of GC both in young and aged mice. Moreover, we developed and used an unbiased- 3D analysis of beta cells on whole cleared pancreas. Results: We demonstrated that beta-cell neogenesis - reflected by an increase in islet density - is constantly observed in response to genetically- or pharmacology-induced (S961 or GC) IR. Next, we observed that pancreatic adaptation mechanisms are closely defined by the level of IR. Indeed, mild IR induced by low dose of GC resulted in functional adaptation solely, while more severe IR induced by higher doses of GC resulted in an increase in both islet density and mean islet size, reflecting beta-cell neogenesis and proliferation, respectively. Then, we showed that in older mice, beta-cell adaptation through neogenesis is preserved in response to IR. Finally, using a new and unbiased 3D analysis, we confirmed the increase in islet density and mean islet size after GCs treatment. Conclusions/interpretation: Our results present evidence that beta-cell neogenesis is a preferential mechanism of pancreatic adaptation to increase insulin secretion in response to IR in mice. Moreover, aging does not preclude beta-cell neogenesis, suggesting that it could be triggered in elderly to compensate for IR. Finally, our innovative technique of 3D analysis of whole pancreas confirms the existence of adult beta-cell neogenesis and offers a new avenue to study islet cells and pancreas adaptation.Competing Interest StatementThe authors have declared no competing interest.

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead-cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.Competing Interest StatementAuthors are employed by Scipio bioscience founded by corresponding authors Pierre Walrafen and Stuart Edelstein, except authors from St. Antoine Hospital who have no competing interests.Footnotes* Expanded version with initial Supplementary Information integrated into the main text and new Supplementary Information added.