Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor ß (TGFß) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

CD36 Drives Metastasis and Relapse in Acute Myeloid Leukemia.

Identifying mechanisms underlying relapse is a major clinical issue for effective cancer treatment. The emerging understanding of the importance of metastasis in hematologic malignancies suggests that it could also play a role in drug resistance and relapse in acute myeloid leukemia (AML). In a cohort of 1,273 AML patients, we uncovered that the multifunctional scavenger receptor CD36 was positively associated with extramedullary dissemination of leukemic blasts, increased risk of relapse after intensive chemotherapy, and reduced event-free and overall survival. CD36 was dispensable for lipid uptake but fostered blast migration through its binding with thrombospondin-1. CD36-expressing blasts, which were largely enriched after chemotherapy, exhibited a senescent-like phenotype while maintaining their migratory ability. In xenograft mouse models, CD36 inhibition reduced metastasis of blasts and prolonged survival of chemotherapy-treated mice. These results pave the way for the development of CD36 as an independent marker of poor prognosis in AML patients and a promising actionable target to improve the outcome of patients. SIGNIFICANCE: CD36 promotes blast migration and extramedullary disease in acute myeloid leukemia and represents a critical target that can be exploited for clinical prognosis and patient treatment.

Lactate fluxes mediated by the monocarboxylate transporter-1 are key determinants of the metabolic activity of beige adipocytes.

Activation of energy-dissipating brown/beige adipocytes represents an attractive therapeutic strategy against metabolic disorders. While lactate is known to induce beiging through the regulation of Ucp1 gene expression, the role of lactate transporters on beige adipocytes' ongoing metabolic activity remains poorly understood. To explore the function of the lactate-transporting monocarboxylate transporters (MCTs), we used a combination of primary cell culture studies, 13C isotopic tracing, laser microdissection experiments, and in situ immunofluorescence of murine adipose fat pads. Dissecting white adipose tissue heterogeneity revealed that the MCT1 is expressed in inducible beige adipocytes as the emergence of uncoupling protein 1 after cold exposure was restricted to a subpopulation of MCT1-expressing adipocytes suggesting MCT1 as a marker of inducible beige adipocytes. We also observed that MCT1 mediates bidirectional and simultaneous inward and outward lactate fluxes, which were required for efficient utilization of glucose by beige adipocytes activated by the canonical ß3-adrenergic signaling pathway. Finally, we demonstrated that significant lactate import through MCT1 occurs even when glucose is not limiting, which feeds the oxidative metabolism of beige adipocytes. These data highlight the key role of lactate fluxes in finely tuning the metabolic activity of beige adipocytes according to extracellular metabolic conditions and reinforce the emerging role of lactate metabolism in the control of energy homeostasis.

Short exposure to cold atmospheric plasma induces senescence in human skin fibroblasts and adipose mesenchymal stromal cells.

Cold Atmospheric Plasma (CAP) is a novel promising tool developed in several biomedical applications such as cutaneous wound healing or skin cancer. Nevertheless, in vitro studies are lacking regarding to CAP effects on cellular actors involved in healthy skin healing and regarding to the mechanism of action. In this study, we investigated the effect of a 3 minutes exposure to CAP-Helium on human dermal fibroblasts and Adipose-derived Stromal Cells (ASC) obtained from the same tissue sample. We observed that CAP treatment did not induce cell death but lead to proliferation arrest with an increase in p53/p21 and DNA damages. Interestingly we showed that CAP treated dermal fibroblasts and ASC developed a senescence phenotype with p16 expression, characteristic morphological changes, Senescence-Associated ß-galactosidase expression and the secretion of pro-inflammatory cytokines defined as the Senescence-Associated Secretory Phenotype (SASP). Moreover this senescence phenotype is associated with a glycolytic switch and an increase in mitochondria content. Despite this senescence phenotype, cells kept in vitro functional properties like differentiation potential and immunomodulatory effects. To conclude, we demonstrated that two main skin cellular actors are resistant to cell death but develop a senescence phenotype while maintaining some functional characteristics after 3 minutes of CAP-Helium treatment in vitro.

A New Role for Browning as a Redox and Stress Adaptive Mechanism?

The worldwide epidemic of obesity and metabolic disorders is focusing the attention of the scientific community on white adipose tissue (WAT) and its biology. This tissue is characterized not only by its capability to change in size and shape but also by its heterogeneity and versatility. WAT can be converted into brown fat-like tissue according to different physiological and pathophysiological situations. The expression of uncoupling protein-1 in brown-like adipocytes changes their function from energy storage to energy dissipation. This plasticity, named browning, was recently rediscovered and convergent recent accounts, including in humans, have revived the idea of using these oxidative cells to fight against metabolic diseases. Furthermore, recent reports suggest that, beside the increased energy dissipation and thermogenesis that may have adverse effects in situations such as cancer-associated cachexia and massive burns, browning could be also considered as an adaptive stress response to high redox pressure and to major stress that could help to maintain tissue homeostasis and integrity. The aim of this review is to summarize the current knowledge concerning brown adipocytes and the browning process and also to explore unexpected putative role(s) for these cells. While it is important to find new browning inducers to limit energy stores and metabolic diseases, it also appears crucial to develop new browning inhibitors to limit adverse energy dissipation in wasting-associated syndromes.

An enhanced immune response of Mclk1⁺/⁻ mutant mice is associated with partial protection from fibrosis, cancer and the development of biomarkers of aging.

The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1⁺/⁻ mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1⁺/⁻ mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1⁺/⁻ mutants, as well as during spontaneous tumorigenesis in Mclk1⁺/⁻Trp53⁺/⁻ double mutants. Tumour latency was increased by the Mclk1⁺/⁻ genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8⁺ T lymphocytes from Mclk1⁺/⁻ graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1⁺/⁻ mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.

ERK1/2 phosphorylate Raptor to promote Ras-dependent activation of mTOR complex 1 (mTORC1).

The Ras/mitogen-activated protein kinase (MAPK) pathway regulates a variety of cellular processes by activating specific transcriptional and translational programs. Ras/MAPK signaling promotes mRNA translation and protein synthesis, but the exact molecular mechanisms underlying this regulation remain poorly understood. Increasing evidence suggests that the mammalian target of rapamycin (mTOR) plays an essential role in this process. Here, we show that Raptor, an essential scaffolding protein of the mTOR complex 1 (mTORC1), becomes phosphorylated on proline-directed sites following activation of the Ras/MAPK pathway. We found that ERK1 and ERK2 interact with Raptor in cells and mediate its phosphorylation in vivo and in vitro. Using mass spectrometry and phosphospecific antibodies, we found three proline-directed residues within Raptor, Ser(8), Ser(696), and Ser(863), which are directly phosphorylated by ERK1/2. Expression of phosphorylation-deficient alleles of Raptor revealed that phosphorylation of these sites by ERK1/2 normally promotes mTORC1 activity and signaling to downstream substrates, such as 4E-BP1. Our data provide a novel regulatory mechanism by which mitogenic and oncogenic activation of the Ras/MAPK pathway promotes mTOR signaling.

mTORC1-activated S6K1 phosphorylates Rictor on threonine 1135 and regulates mTORC2 signaling.

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3 alpha/beta (GSK3 alpha/beta) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.

Regulation of mTOR complex 1 (mTORC1) by raptor Ser863 and multisite phosphorylation.

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser(696)/Thr(706) and cluster 2, Ser(855)/Ser(859)/Ser(863)/Ser(877)) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser(863) phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser(863) via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser(863) phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser(863) as well as on the five other identified sites (e.g. Ser(859), Ser(855), Ser(877), Ser(696), and Thr(706)). Strikingly, raptor Ser(863) phosphorylation is absolutely required for raptor Ser(859) and Ser(855) phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser(863) phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser(859) and Ser(855)). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.

Oncogenic MAPK signaling stimulates mTORC1 activity by promoting RSK-mediated raptor phosphorylation.

BACKGROUND: The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that controls cell growth in response to mitogens, as well as amino acid and energy sufficiency. The scaffolding protein Raptor binds to mTOR and recruits substrates to the rapamycin-sensitive mTOR complex 1 (mTORC1). Although Raptor has been shown to be essential for mTORC1 activity, the mechanisms regulating Raptor function remain unknown. RESULTS: Here, we demonstrate that Raptor becomes highly phosphorylated on RXRXXpS/T consensus motifs after activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. Using pharmacological inhibitors and RNA interference, we show that the p90 ribosomal S6 kinases (RSKs) 1 and 2 are required for Raptor phosphorylation in vivo and directly phosphorylate Raptor in vitro. Quantitative mass spectrometry and site-directed mutagenesis revealed that RSK specifically phosphorylates Raptor within an evolutionarily conserved region with no previously known function. Interestingly, expression of oncogenic forms of Ras and MEK that elevate mTORC1 activity induced strong and constitutive phosphorylation of Raptor on these residues. Importantly, we demonstrate that expression of Raptor mutants lacking RSK-dependent phosphorylation sites markedly reduced mTOR phosphotransferase activity, indicating that RSK-mediated phosphorylation of Raptor is important for mTORC1 activation by the Ras/MAPK pathway. CONCLUSIONS: We propose a unique mode of mTOR regulation in which RSK-mediated phosphorylation of Raptor regulates mTORC1 activity and thus suggest a means by which the Ras/MAPK pathway might promote rapamycin-sensitive signaling independently of the PI3K/Akt pathway.

The RSK factors of activating the Ras/MAPK signaling cascade.

The p90 ribosomal S6 kinase (RSK) constitute a family of serine/threonine kinases activated downstream of the Ras/mitogen-activated protein kinase (MAPK) pathway. In mammals, four RSK genes have been identified (RSK1, RSK2, RSK3 and RSK4), and RSK orthologues have also been described in D. melanogaster and C. elegans, but not in yeast or plants. The RSK isoforms are composed of two distinct and functional kinase domains that are activated in a sequential manner by a series of phosphorylation events. These enzymes were among the first substrates of extracellular signal-regulated kinase (ERK) to be discovered and have proven to be ubiquitous and multifunctional mediators of ERK signal transduction. While the RSK isoforms promote cell survival though the inactivation of several apoptotic effectors, they also appear to mediate cell growth and proliferation by simultaneously regulating substrates involved in gene transcription and mRNA translation. RSK1-4 are ubiquitously expressed in cell lines and tissues, and at present, little is known about specific and overlapping functions of individual RSK isoforms. The upregulation of RSK1 and RSK2 expression in different types of cancer suggest that they may be involved in oncogenesis and could potentially be targeted in anti-cancer therapies. The recent identification of specific RSK inhibitors will likely help addressing the biological functions of the RSK isoforms and their contributions in pathological conditions.

Site specific changes of redox metabolism in adipose tissue of obese Zucker rats.

Adipose tissues are differently involved in lipid metabolism and obesity according to their type and location. Increasing reports stress on the impact of redox metabolism on obesity and metabolic syndrome. The aim of this work is to investigate the site-specific redox metabolism in three different adipose tissues and its changes occurring in obesity. We analysed enzymatic and non-enzymatic parameters, and focused on the reduced/oxidized glutathione and coenzyme Q couples. In lean compared with obese non-diabetic Zucker rats, interscapular brown fat seems well protected against oxidative stress and epididymal adipose tissue shows a more reduced glutathione redox state, associated with a higher susceptibility to lipophilic oxidative stress than inguinal adipose tissue. Epididymal adipose tissue redox metabolism significantly differs from inguinal one by its limited redox metabolism adaptation. Our results demonstrate site-specific managements of reactive oxygen species metabolism in obese Zucker rats. These results are not consistent with the classic deciphering of inflammatory situation and produce a new conception of the redox parameters implication in the development of the metabolic syndrome.

Adipose tissue proadipogenic redox changes in obesity.

The role of inflammation and oxidative stress in the development of obesity and associated metabolic disorders is under debate. We investigated the redox metabolism in a non-diabetic obesity model, i.e. 11-week-old obese Zucker rats. Antioxidant enzyme activities, lipophilic antioxidant (alpha-tocopherol, coenzymes Q) and hydrophilic antioxidant (glutathione, vitamin C) contents and their redox state (% oxidized form), were studied in inguinal white fat and compared with blood and liver. The adipose tissues of obese animals showed a specific higher content of hydrophilic molecules in a lower redox state than those of lean animals, which were associated with lower lipophilic molecule content and lipid peroxidation. Conversely and as expected, glutathione content decreased and its redox state increased in adipose tissues of rats subjected to lipopolysaccharide-induced systemic oxidative stress. In these in vivo models, oxidative stress and obesity thus had opposite effects on adipose tissue redox state. Moreover, the increase in glutathione content and the decrease of its redox state by antioxidant treatment promoted in vitro the accumulation of triglycerides in preadipocytes. Taken together and contrary to the emergent view, our results suggest that obesity is associated with an intracellular reduced redox state that promotes on its own the development of a deleterious proadipogenic process.