Effects of glucocorticoids on adipose tissue plasticity.

Glucocorticoids (GCs) play an important role in metabolic adaptation, regulating carbohydrate-lipid homeostasis and the immune system. Because they also have anti-inflammatory and immunosuppressive properties, synthetic analogues of GCs have been developed and are widely used in the treatment of chronic inflammatory conditions and in organ transplantation. GCs are among the most commonly prescribed drugs in the world. However, long term and high GC doses can cause side effects such as GC-induced diabetes and lipodystrophy. In recent years, a large number of independent studies have reported the effects of constitutive and adipocyte-specific deletion of the GC receptor (GR) in mice under different diets and treatments, resulting in contrasting phenotypes. To avoid potential compensatory mechanisms associated with the constitutive adipocyte GR silencing during adipose tissue development, our team has generated an inducible mouse model of GR deletion specifically in the adipocyte (AdipoGR-KO). Using this mouse model, we were able to demonstrate the critical role of the adipocyte GR in GC-induced metabolic changes. Indeed, under conditions of hypercorticism, AdipoGR-KO mice showed an improvement in glucose tolerance and insulin sensitivity, as well as in lipid profile, despite a massive increase in adiposity. This result is explained by a densification of adipose tissue vascularization, highlighting the repressive role of adipocyte GR in the healthy expansion of this tissue. Our work has largely contributed to the demonstration of the important role of the adipocyte GR in the physiology and pathophysiology of the adipose tissue and its impact on energy homeostasis.

Pink adipose tissue: A paradigm of adipose tissue plasticity.

Adipose tissue is highly plastic, as illustrated mainly by the transdifferentiation of white adipocytes into beige adipocytes, depending on environmental conditions. However, during gestation and lactation in rodent, there is an amazing phenomenon of transformation of subcutaneous adipose tissue into mammary glandular tissue, known as pink adipose tissue, capable of synthesizing and secreting milk. Recent work using transgenic lineage-tracing experiments, mainly carried out in Saverio Cinti's team, has demonstrated very convincingly that this process does indeed correspond to a transdifferentiation of white adipocytes into mammary alveolar cells (pink adipocytes) during gestation and lactation. This phenomenon is reversible, since during the post-lactation phase, pink adipocytes revert to the white adipocyte phenotype. The molecular mechanisms underlying this reversible transdifferentiation remain poorly understood.

Adipocyte Glucocorticoid Receptor Activation With High Glucocorticoid Doses Impairs Healthy Adipose Tissue Expansion by Repressing Angiogenesis.

In humans, glucocorticoids (GCs) are commonly prescribed because of their anti-inflammatory and immunosuppressive properties. However, high doses of GCs often lead to side effects, including diabetes and lipodystrophy. We recently reported that adipocyte glucocorticoid receptor (GR)-deficient (AdipoGR-KO) mice under corticosterone (CORT) treatment exhibited a massive adipose tissue (AT) expansion associated with a paradoxical improvement of metabolic health compared with control mice. However, whether GR may control adipose development remains unclear. Here, we show a specific induction of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic vascular endothelial growth factor A (VEGFA) expression in GR-deficient adipocytes of AdipoGR-KO mice compared with control mice, together with an increased adipose vascular network, as assessed by three-dimensional imaging. GR activation reduced HIF-1α recruitment to the Vegfa promoter resulting from Hif-1α downregulation at the transcriptional and posttranslational levels. Importantly, in CORT-treated AdipoGR-KO mice, the blockade of VEGFA by a soluble decoy receptor prevented AT expansion and the healthy metabolic phenotype. Finally, in subcutaneous AT from patients with Cushing syndrome, higher VEGFA expression was associated with a better metabolic profile. Collectively, these results highlight that adipocyte GR negatively controls AT expansion and metabolic health through the downregulation of the major angiogenic effector VEGFA and inhibition of vascular network development.

RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding.

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.

The necroptosis-inducing pseudokinase mixed lineage kinase domain-like regulates the adipogenic differentiation of pre-adipocytes.

Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/ß-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.

Deficiency of the RNA-binding protein Cth2 extends yeast replicative lifespan by alleviating its repressive effects on mitochondrial function.

Iron dyshomeostasis contributes to aging, but little information is available about the molecular mechanisms. Here, we provide evidence that in Saccharomyces cerevisiae, aging is associated with altered expression of genes involved in iron homeostasis. We further demonstrate that defects in the conserved mRNA-binding protein Cth2, which controls stability and translation of mRNAs encoding iron-containing proteins, increase lifespan by alleviating its repressive effects on mitochondrial function. Mutation of the conserved cysteine residue in Cth2 that inhibits its RNA-binding activity is sufficient to confer longevity, whereas Cth2 gain of function shortens replicative lifespan. Consistent with its function in RNA degradation, Cth2 deficiency relieves Cth2-mediated post-transcriptional repression of nuclear-encoded components of the electron transport chain. Our findings uncover a major role of the RNA-binding protein Cth2 in the regulation of lifespan and suggest that modulation of iron starvation signaling can serve as a target for potential aging interventions.

Inhibition of Adipose Tissue Beiging by HIV Integrase Inhibitors, Dolutegravir and Bictegravir, Is Associated with Adipocyte Hypertrophy, Hypoxia, Elevated Fibrosis, and Insulin Resistance in Simian Adipose Tissue and Human Adipocytes.

For people living with HIV, treatment with integrase-strand-transfer-inhibitors (INSTIs) can promote adipose tissue (AT) gain. We previously demonstrated that INSTIs can induce hypertrophy and fibrosis in AT of macaques and humans. By promoting energy expenditure, the emergence of beige adipocytes in white AT (beiging) could play an important role by limiting excess lipid storage and associated adipocyte dysfunction. We hypothesized that INSTIs could alter AT via beiging inhibition. Fibrosis and gene expression were measured in subcutaneous (SCAT) and visceral AT (VAT) from SIV-infected, dolutegravir-treated (SIVART) macaques. Beiging capacity was assessed in human adipose stromal cells (ASCs) undergoing differentiation and being exposed to dolutegravir, bictegravir, or raltegravir. Expression of beige markers, such as positive-regulatory-domain-containing-16 (PRDM16), were lower in AT of SIVART as compared to control macaques, whereas fibrosis-related genes were higher. Dolutegravir and bictegravir inhibited beige differentiation in ASCs, as shown by lower expression of beige markers and lower cell respiration. INSTIs also induced a hypertrophic insulin-resistant state associated with a pro-fibrotic phenotype. Our results indicate that adipocyte hypertrophy induced by INSTIs is involved via hypoxia (revealed by a greater hypoxia-inducible-factor-1-alpha gene expression) in fat fibrosis, beiging inhibition, and thus (via positive feedback), probably, further hypertrophy and associated insulin resistance.

Genome-Wide Analysis of Translation in Replicatively Aged Yeast.

Protein synthesis is an essential process that affects major cellular functions including growth, energy production, cell signaling, and enzymatic reactions. However, how it is impacted by aging and how the translation of specific proteins is changed during the aging process remain understudied. Although yeast is a widely used model for studying eukaryotic aging, analysis of age-related translational changes using ribosome profiling in this organism has been challenging due to the need for isolating large quantities of old cells. Here, we provide a detailed protocol for genome-wide analysis of protein synthesis using ribosome profiling in replicatively aged yeast. By combining genetic enrichment of old cells with the biotin affinity purification step, this method allows large-scale isolation of aged cells sufficient for generating ribosome profiling libraries. We also describe a strategy for normalization of samples using a spike-in with worm lysates that permits quantitative comparison of absolute translation levels between young and old cells.

Molecular Mechanisms of Glucocorticoid-Induced Insulin Resistance.

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.

(Un)folding mechanisms of adaptation to ER stress: lessons from aneuploidy.

During stress, accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers activation of the adaptive mechanisms that restore protein homeostasis. One mechanism that eukaryotic cells use to respond to ER stress is through activation of the unfolded protein response (UPR) signaling pathway, which initiates degradation of misfolded proteins and leads to inhibition of translation and increased expression of chaperones and oxidative folding components that enhance ER protein folding capacity. However, the mechanisms of adaptation to ER stress are not limited to the UPR. Using yeast Saccharomyces cerevisiae, we recently discovered that the protein folding burden in the ER can be alleviated in a UPR-independent manner through duplication of whole chromosomes containing ER stress-protective genes. Here we discuss our findings and their implication to our understanding of the mechanisms by which cells respond to protein misfolding in the ER.

Genetic screen identifies adaptive aneuploidy as a key mediator of ER stress resistance in yeast.

The yeast genome becomes unstable during stress, which often results in adaptive aneuploidy, allowing rapid activation of protective mechanisms that restore cellular homeostasis. In this study, we performed a genetic screen in Saccharomyces cerevisiae to identify genome adaptations that confer resistance to tunicamycin-induced endoplasmic reticulum (ER) stress. Whole-genome sequencing of tunicamycin-resistant mutants revealed that ER stress resistance correlated significantly with gains of chromosomes II and XIII. We found that chromosome duplications allow adaptation of yeast cells to ER stress independently of the unfolded protein response, and that the gain of an extra copy of chromosome II alone is sufficient to induce protection from tunicamycin. Moreover, the protective effect of disomic chromosomes can be recapitulated by overexpression of several genes located on chromosome II. Among these genes, overexpression of UDP-N-acetylglucosamine-1-P transferase (ALG7), a subunit of the 20S proteasome (PRE7), and YBR085C-A induced tunicamycin resistance in wild-type cells, whereas deletion of all three genes completely reversed the tunicamycin-resistance phenotype. Together, our data demonstrate that aneuploidy plays a critical role in adaptation to ER stress by increasing the copy number of ER stress protective genes. While aneuploidy itself leads to proteotoxic stress, the gene-specific effects of chromosome II aneuploidy counteract the negative effect resulting in improved protein folding.

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

CAN1 Arginine Permease Deficiency Extends Yeast Replicative Lifespan via Translational Activation of Stress Response Genes.

Transcriptional regulation plays an important role in the control of gene expression during aging. However, translation efficiency likely plays an equally important role in determining protein abundance, but it has been relatively understudied in this context. Here, we used RNA sequencing (RNA-seq) and ribosome profiling to investigate the role of translational regulation in lifespan extension by CAN1 gene deletion in yeast. Through comparison of the transcriptional and translational changes in cells lacking CAN1 with other long-lived mutants, we were able to identify critical regulatory factors, including transcription factors and mRNA-binding proteins, that coordinate transcriptional and translational responses. Together, our data support a model in which deletion of CAN1 extends replicative lifespan through increased translation of proteins that facilitate cellular response to stress. This study extends our understanding of the importance of translational control in regulating stress resistance and longevity.

The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation.

To maintain bone mass turnover and bone mineral density (BMD), bone marrow (BM) mesenchymal stem cells (MSCs) are constantly recruited and subsequently differentiated into osteoblasts. HIV-infected patients present lower BMD than non-HIV infected individuals and a higher prevalence of osteopenia/osteoporosis. In antiretroviral treatment (ART)-naive patients, encoded HIV proteins represent pathogenic candidates. They are released by infected cells within BM and can impact on neighbouring cells. In this study, we tested whether HIV proteins Tat and/or Nef could induce senescence of human BM-MSCs and reduce their capacity to differentiate into osteoblasts. When compared to nontreated cells, MSCs chronically treated with Tat and/or Nef up to 30 days reduced their proliferative activity and underwent early senescence, associated with increased oxidative stress and mitochondrial dysfunction. The antioxidant molecule N-acetyl- cysteine had no or minimal effects on Tat- or Nef-induced senescence. Tat but not Nef induced an early increase in NF-κB activity and cytokine/chemokine secretion. Tat-induced effects were prevented by the NF-κB inhibitor parthenolide, indicating that Tat triggered senescence via NF-κB activation leading to oxidative stress. Otherwise, Nef- but not Tat-treated cells displayed early inhibition of autophagy. Rapamycin, an autophagy inducer, reversed Nef-induced senescence and oxidative stress. Moreover, Tat+Nef had cumulative effects. Finally, Tat and/or Nef decreased the MSC potential of osteoblastic differentiation. In conclusion, our in vitro data show that Tat and Nef could reduce the number of available precursors by inducing MSC senescence, through either enhanced inflammation or reduced autophagy. These results offer new insights into the pathophysiological mechanisms of decreased BMD in HIV-infected patients.

HIV protease inhibitors induce senescence and alter osteoblastic potential of human bone marrow mesenchymal stem cells: beneficial effect of pravastatin.

HIV-infected patients receiving antiretroviral therapy present an increased prevalence of age-related comorbidities, including osteoporosis. HIV protease inhibitors (PIs) have been suspected to participate to bone loss, but the mechanisms involved are unknown. In endothelial cells, some PIs have been shown to induce the accumulation of farnesylated prelamin-A, a biomarker of cell aging leading to cell senescence. Herein, we hypothesized that these PIs could induce premature aging of osteoblast precursors, human bone marrow mesenchymal stem cells (MSCs), and affect their capacity to differentiate into osteoblasts. Senescence was studied in proliferating human MSCs after a 30-day exposure to atazanavir and lopinavir with or without ritonavir. When compared to untreated cells, PI-treated MSCs had a reduced proliferative capacity that worsened with increasing passages. PI treatment led to increased oxidative stress and expression of senescence markers, including prelamin-A. Pravastatin, which blocks prelamin-A farnesylation, prevented PI-induced senescence and oxidative stress, while treatment with antioxidants partly reversed these effects. Moreover, senescent MSCs presented a decreased osteoblastic potential, which was restored by pravastatin treatment. Because age-related bone loss is associated with increased bone marrow fat, we also evaluated the capacity of PI-treated MSCs to differentiate into adipocyte. We observed an altered adipocyte differentiation in PI-treated MSCs that was reverted by pravastatin. We have shown that some PIs alter osteoblast formation by affecting their differentiation potential in association with altered senescence in MSCs, with a beneficial effect of statin. These data corroborate the clinical observations and allow new insight into pathophysiological mechanisms of PI-induced bone loss in HIV-infected patients.