Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks.

Cellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival and TGF-ß signaling by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes.This article has an associated First Person interview with the first author of the paper.

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers.

Long-term exposure to peroxisome proliferator-activated receptor γ (PPARγ) agonists such as rosiglitazone induces browning of rodent and human adipocytes; however, the transcriptional mechanisms governing this phenotypic switch in adipocytes are largely unknown. Here we show that rosiglitazone-induced browning of human adipocytes activates a comprehensive gene program that leads to increased mitochondrial oxidative capacity. Once induced, this gene program and oxidative capacity are maintained independently of rosiglitazone, suggesting that additional browning factors are activated. Browning triggers reprogramming of PPARγ binding, leading to the formation of PPARγ "superenhancers" that are selective for brown-in-white (brite) adipocytes. These are highly associated with key brite-selective genes. Based on such an association, we identified an evolutionarily conserved metabolic regulator, Kruppel-like factor 11 (KLF11), as a novel browning transcription factor in human adipocytes that is required for rosiglitazone-induced browning, including the increase in mitochondrial oxidative capacity. KLF11 is directly induced by PPARγ and appears to cooperate with PPARγ in a feed-forward manner to activate and maintain the brite-selective gene program.

New Strategies Protecting from Ischemia/Reperfusion.

This Special Issue aims to highlight new avenues in the management of Ischemia/Reperfusion (I/R) injury [...].

Akt Inhibition as Preconditioning Treatment to Protect Kidney Cells against Anoxia.

Lesions issued from the ischemia/reperfusion (I/R) stress are a major challenge in human pathophysiology. Of human organs, the kidney is highly sensitive to I/R because of its high oxygen demand and poor regenerative capacity. Previous studies have shown that targeting the hypusination pathway of eIF5A through GC7 greatly improves ischemic tolerance and can be applied successfully to kidney transplants. The protection process correlates with a metabolic shift from oxidative phosphorylation to glycolysis. Because the protein kinase B Akt is involved in ischemic protective mechanisms and glucose metabolism, we looked for a link between the effects of GC7 and Akt in proximal kidney cells exposed to anoxia or the mitotoxic myxothiazol. We found that GC7 treatment resulted in impaired Akt phosphorylation at the Ser473 and Thr308 sites, so the effects of direct Akt inhibition as a preconditioning protocol on ischemic tolerance were investigated. We evidenced that Akt inhibitors provide huge protection for kidney cells against ischemia and myxothiazol. The pro-survival effect of Akt inhibitors, which is reversible, implied a decrease in mitochondrial ROS production but was not related to metabolic changes or an antioxidant defense increase. Therefore, the inhibition of Akt can be considered as a preconditioning treatment against ischemia.

Modulation of the inflammatory response to LPS by the recruitment and activation of brown and brite adipocytes in mice.

Numerous studies have shown that the recruitment and activation of thermogenic adipocytes, which are brown and beige/brite, reduce the mass of adipose tissue and normalize abnormal glycemia and lipidemia. However, the impact of these adipocytes on the inflammatory state of adipose tissue is still not well understood, especially in response to endotoxemia, which is a major aspect of obesity and metabolic diseases. First, we analyzed the phenotype and metabolic function of white and brite primary adipocytes in response to lipopolysaccharide (LPS) treatment in vitro. Then, 8-wk-old male BALB/c mice were treated for 1 wk with a ß3-adrenergic receptor agonist (CL316,243, 1 mg/kg/day) to induce recruitment and activation of brown and brite adipocytes and were subsequently injected with LPS (Escherichia coli lipopolysaccharide, 100 µg/mouse ip) to generate acute endotoxemia. The metabolic and inflammatory parameters of the mice were analyzed 6 h later. Our results showed that in response to LPS, thermogenic activity promoted a local anti-inflammatory environment with high secretion of IL-1 receptor antagonist (IL-1RA) without affecting other anti- or proinflammatory cytokines. Interestingly, activation of brite adipocytes reduced the LPS-induced secretion of leptin. However, thermogenic activity and adipocyte function were not altered by LPS treatment in vitro or by acute endotoxemia in vivo. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes specifically in response to endotoxemia. This encourages potential therapy involving brown and brite adipocytes for the treatment of obesity and associated metabolic diseases.NEW & NOTEWORTHY Recruitment and activation of brown and brite adipocytes in the adipose tissue of mice lead to a local low-grade anti-inflammatory phenotype in response to acute endotoxemia without alteration of adipocyte phenotype and function.

Oxytocin Controls Chondrogenesis and Correlates with Osteoarthritis.

This study investigated the relationship of oxytocin (OT) to chondrogenesis and osteoarthritis (OA). Human bone marrow and multipotent adipose-derived stem cells were cultured in vitro in the absence or presence of OT and assayed for mRNA transcript expression along with histological and immunohistochemical analyses. To study the effects of OT in OA in vivo, a rat model and a human cohort of 63 men and 19 women with hand OA and healthy controls, respectively, were used. The baseline circulating OT, interleukin-6, leptin, and oestradiol levels were measured, and hand X-ray examinations were performed for each subject. OT induced increased aggrecan, collagen (Col) X, and cartilage oligomeric matrix protein mRNA transcript levels in vitro, and the immunolabelling experiments revealed a normalization of Sox9 and Col II protein expression levels. No histological differences in lesion severity were observed between rat OA groups. In the clinical study, a multivariate analysis adjusted for age, body mass index, and leptin levels revealed a significant association between OA and lower levels of OT (odds ratio = 0.77; p = 0.012). Serum OT levels are reduced in patients with hand OA, and OT showed a stimulatory effect on chondrogenesis. Thus, OT may contribute to the pathophysiology of OA.

Impact of dietary ω3 polyunsaturated fatty acid supplementation on brown and brite adipocyte function.

The recent characterization of functional brown adipose tissue in adult humans has opened new perspectives for regulation of energy expenditure with respect to obesity and diabetes. Furthermore, dietary recommendations have taken into account the insufficient dietary intake of ω3 PUFAs and the concomitant excessive intake of ω6 PUFA associated with the occurrence of overweight/obesity. We aimed to study whether ω3 PUFAs could play a role in the recruitment and function of energy-dissipating brown/brite adipocytes. We show that ω3 PUFA supplementation has a beneficial effect on the thermogenic function of adipocytes. In vivo, a low dietary ω6:ω3 ratio improved the thermogenic response of brown and white adipose tissues to ß3-adrenergic stimulation. This effect was recapitulated in vitro by PUFA treatment of hMADS adipocytes. We pinpointed the ω6-derived eicosanoid prostaglandin (PG)F2α as the molecular origin because the effects were mimicked with a specific PGF2α receptor agonist. PGF2α level in hMADS adipocytes was reduced in response to ω3 PUFA supplementation. The recruitment of thermogenic adipocytes is influenced by the local quantity of individual oxylipins, which is controlled by the ω6:ω3 ratio of available lipids. In human nutrition, energy homeostasis may thus benefit from the implementation of a more balanced dietary ω6:ω3 ratio.

Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome.

The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of Drosophila during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by N1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

IP-receptor and PPARs trigger the conversion of human white to brite adipocyte induced by carbaprostacyclin.

Brite adipocytes recently discovered in humans are of considerable importance in energy expenditure by converting energy excess into heat. This property could be useful in the treatment of obesity, and nutritional aspects are relevant to this important issue. Using hMADS cells as a human cell model which undergoes a white to a brite adipocyte conversion, we had shown previously that arachidonic acid, the major metabolite of the essential nutrient Ω6-linoleic acid, plays a major role in this process. Its metabolites PGE2 and PGF2 alpha inhibit this process via a calcium-dependent pathway, whereas in contrast carbaprostacyclin (cPGI2), a stable analog of prostacyclin, activates white to brite adipocyte conversion. Herein, we show that cPGI2 generates via its cognate cell-surface receptor IP-R, a cyclic AMP-signaling pathway involving PKA activity which in turn induces the expression of UCP1. In addition, cPGI2 activates the pathway of nuclear receptors of the PPAR family, i.e. PPARα and PPARγ, which act separately from IP-R to up-regulate the expression of key genes involved in the function of brite adipocytes. Thus dual pathways are playing in concert for the occurrence of a browning process of human white adipocytes. These results make prostacyclin analogs as a new class of interesting molecules to treat obesity and associated diseases.

The K+ channel TASK1 modulates ß-adrenergic response in brown adipose tissue through the mineralocorticoid receptor pathway.

Brown adipose tissue (BAT) is essential for adaptive thermogenesis and dissipation of caloric excess through the activity of uncoupling protein (UCP)-1. BAT in humans is of great interest for the treatment of obesity and related diseases. In this study, the expression of Twik-related acid-sensitive K(+) channel (TASK)-1 [a pH-sensitive potassium channel encoded by the potassium channel, 2-pore domain, subfamily K, member 3 (Kcnk3) gene] correlated highly with Ucp1 expression in obese and cold-exposed mice. In addition, Task1-null mice, compared with their controls, became overweight, mainly because of an increase in white adipose tissue mass and BAT whitening. Task1(-/-)-mouse-derived brown adipocytes, compared with wild-type mouse-derived brown adipocytes, displayed an impaired ß3-adrenergic receptor response that was characterized by a decrease in oxygen consumption, Ucp1 expression, and lipolysis. This phenotype was thought to be caused by an exacerbation of mineralocorticoid receptor (MR) signaling, given that it was mimicked by corticoids and reversed by an MR inhibitor. We concluded that the K(+) channel TASK1 controls the thermogenic activity in brown adipocytes through modulation of ß-adrenergic receptor signaling.

MicroRNA-26 family is required for human adipogenesis and drives characteristics of brown adipocytes.

Adipose tissue contains thermogenic adipocytes (i.e., brown and brite/beige) that oxidize nutrients at exceptionally high rates via nonshivering thermogenesis. Its recent discovery in adult humans has opened up new avenues to fight obesity and related disorders such as diabetes. Here, we identified miR-26a and -26b as key regulators of human white and brite adipocyte differentiation. Both microRNAs are upregulated in early adipogenesis, and their inhibition prevented lipid accumulation while their overexpression accelerated it. Intriguingly, miR-26a significantly induced pathways related to energy dissipation, shifted mitochondrial morphology toward that seen in brown adipocytes, and promoted uncoupled respiration by markedly increasing the hallmark protein of brown fat, uncoupling protein 1. By combining in silico target prediction, transcriptomics, and an RNA interference screen, we identified the sheddase ADAM metallopeptidase domain 17 (ADAM17) as a direct target of miR-26 that mediated the observed effects on white and brite adipogenesis. These results point to a novel, critical role for the miR-26 family and its downstream effector ADAM17 in human adipocyte differentiation by promoting characteristics of energy-dissipating thermogenic adipocytes.

In vitro brown and "brite"/"beige" adipogenesis: human cellular models and molecular aspects.

Brown adipose tissue (BAT) has long been thought to be absent or very scarce in human adults so that its contribution to energy expenditure was not considered as relevant. The recent discovery of thermogenic BAT in human adults opened the field for innovative strategies to combat overweight/obesity and associated diseases. This energy-dissipating function of BAT is responsible for adaptive thermogenesis in response to cold stimulation. In this context, adipocytes can be converted, within white adipose tissue (WAT), into multilocular adipocytes expressing UCP1, a mitochondrial protein that plays a key role in heat production by uncoupling the activity of the respiratory chain from ATP synthesis. These adipocytes have been named "brite" or "beige" adipocytes. Whereas BAT has been studied for a long time in murine models both in vivo and in vitro, there is now a strong demand for human cellular models to validate and/or identify critical factors involved in the induction of a thermogenic program within adipocytes. In this review we will discuss the different human cellular models described in the literature and what is known regarding the regulation of their differentiation and/or activation process. In addition, the role of microRNAs as novel regulators of brown/"brite" adipocyte differentiation and conversion will be depicted. Finally, investigation of both the conversion and the metabolism of white-to-brown converted adipocytes is required for the development of therapeutic strategies targeting overweight/obesity and associated diseases. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

Polyamine metabolism in macrophage-adipose tissue function and homeostasis.

Intracellular metabolism is a crucial regulator of macrophage function. Recent evidence revealed that the polyamine pathway and subsequent hypusination of eukaryotic initiation factor 5A (eIF5A) are master regulators of immune cell functions. In brown adipose tissue (BAT), macrophages show an impressive degree of heterogenicity, with specific subsets supporting adaptive thermogenesis during cold exposure. In this review, we discuss the impact of polyamine metabolism on macrophage diversity and function, with a particular focus on their role in adipose tissue homeostasis. Thus, we highlight the exploration of how polyamine metabolism in macrophages contributes to BAT homeostasis as an attractive and exciting new field of research.

Differences in birch tar composition are explained by adhesive function in the central European Iron Age.

Birch bark tar is the most widely documented adhesive in prehistoric Europe. More recent periods attest to a diversification in terms of the materials used as adhesives and their application. Some studies have shown that conifer resins and beeswax were added to produce compound adhesives. For the Iron Age, no comparative large-scale studies have been conducted to provide a wider perspective on adhesive technologies. To address this issue, we identify adhesive substances from the Iron Age in north-eastern France. We applied organic residue analysis to 65 samples from 16 archaeological sites. This included residues adhering to ceramics, from vessel surface coatings, repaired ceramics, vessel contents, and adhesive lumps. Our findings show that, even during the Iron Age in north-eastern France, birch bark tar is one of the best-preserved adhesive substances, used for at least 400 years. To a lesser extent, Pinaceae resin and beeswax were also identified. Through statistical analyses, we show that molecular composition differs in samples, correlating with adhesive function. This has implications for our understanding of birch bark tar production, processing and mode of use during the Iron Age in France and beyond.

Chondrogenic potential of stem cells derived from adipose tissue: a powerful pharmacological tool.

Chondrogenesis has been widely investigated in vitro using bone marrow-derived mesenchymal stromal cells (BM-MSCs) or primary chondrocytes. However, their use raises some issues partially circumvented by the availability of Adipose tissue-derived MSCs. Herein; we characterized the chondrogenic potential of human Multipotent Adipose-Derived Stem (hMADS) cells, and their potential use as pharmacological tool. hMADS cells are able to synthesize matrix proteins including COMP, Aggrecan and type II Collagen. Furthermore, hMADS cells express BMP receptors in a similar manner to BM-MSC, and BMP6 treatment of differentiated cells prevents expression of the hypertrophic marker type X Collagen. We tested whether IL-1ß and nicotine could impact chondrocyte differentiation. As expected, IL-1ß induced ADAMTS-4 gene expression and modulated negatively chondrogenesis while these effects were reverted in the presence of the IL-1 receptor antagonist. Nicotine, at concentrations similar to those observed in blood of smokers, exhibited a dose dependent increase of Aggrecan expression, suggesting an unexpected protective effect of the drug under these conditions. Therefore, hMADS cells represent a valuable tool for the analysis of in vitro chondrocyte differentiation and to screen for potentially interesting pharmacological drugs.

Renal Ischemia Tolerance Mediated by eIF5A Hypusination Inhibition Is Regulated by a Specific Modulation of the Endoplasmic Reticulum Stress.

Through kidney transplantation, ischemia/reperfusion is known to induce tissular injury due to cell energy shortage, oxidative stress, and endoplasmic reticulum (ER) stress. ER stress stems from an accumulation of unfolded or misfolded proteins in the lumen of ER, resulting in the unfolded protein response (UPR). Adaptive UPR pathways can either restore protein homeostasis or can turn into a stress pathway leading to apoptosis. We have demonstrated that N1-guanyl-1,7-diamineoheptane (GC7), a specific inhibitor of eukaryotic Initiation Factor 5A (eIF5A) hypusination, confers an ischemic protection of kidney cells by tuning their metabolism and decreasing oxidative stress, but its role on ER stress was unknown. To explore this, we used kidney cells pretreated with GC7 and submitted to either warm or cold anoxia. GC7 pretreatment promoted cell survival in an anoxic environment concomitantly to an increase in xbp1 splicing and BiP level while eiF2α phosphorylation and ATF6 nuclear level decreased. These demonstrated a specific modulation of UPR pathways. Interestingly, the pharmacological inhibition of xbp1 splicing reversed the protective effect of GC7 against anoxia. Our results demonstrated that eIF5A hypusination inhibition modulates distinctive UPR pathways, a crucial mechanism for the protection against anoxia/reoxygenation.

Sig1R protein regulates hERG channel expression through a post-translational mechanism in leukemic cells.

Sig1R (Sigma-1receptor) is a 25-kDa protein structurally unrelated to other mammalian proteins. Sig1R is present in brain, liver, and heart and is overexpressed in cancer cells. Studies using exogenous sigma ligands have shown that Sig1R interacts with a variety of ion channels, but its intrinsic function and mechanism of action remain unclear. The human ether-à-gogo related gene (hERG) encodes a cardiac channel that is also abnormally expressed in many primary human cancers, potentiating tumor progression through the modulation of extracellular matrix adhesive interactions. We show herein that sigma ligands inhibit hERG current density and cell adhesion to fibronectin in K562 myeloid leukemia cells. Heterologous expression in Xenopus oocytes demonstrates that Sig1R potentiates hERG current by stimulating channel subunit biosynthesis. Silencing Sig1R in leukemic K562 cells depresses hERG current density and cell adhesion to fibronectin by reducing hERG membrane expression. In K562 cells, Sig1R silencing does not modify hERG mRNA contents but reduces hERG mature form densities. In HEK cells expressing hERG and Sig1R, both proteins co-immunoprecipitate, demonstrating a physical association. Finally, Sig1R expression enhances both channel protein maturation and stability. Altogether, these results demonstrate for the first time that Sig1R controls ion channel expression through the regulation of subunit trafficking activity.

Intracellular pH Control by Membrane Transport in Mammalian Cells. Insights Into the Selective Advantages of Functional Redundancy.

Intracellular pH is a vital parameter that is maintained close to neutrality in all mammalian cells and tissues and acidic in most intracellular compartments. After presenting the main techniques used for intracellular an vesicular pH measurements we will briefly recall the main molecular mechanisms that affect and regulate intracellular pH. Following this we will discuss the large functional redundancy found in the transporters of H+ or acid-base equivalents. For this purpose, we will use mathematical modeling to simulate cellular response to persistent and/or transient acidification, in the presence of different transporters, single or in combination. We will also test the presence or absence of intracellular buffering. This latter section will highlight how modeling can yield fundamental insight into deep biological questions such as the utility of functional redundancy in natural selection.

Hierarchization of myogenic and adipogenic progenitors within human skeletal muscle.

Skeletal muscle cells constitute a heterogeneous population that maintains muscle integrity through a high myogenic regenerative capacity. More unexpectedly, this population is also endowed with an adipogenic potential, even in humans, and intramuscular adipocytes have been found to be present in several disorders. We tested the distribution of myogenic and adipogenic commitments in human muscle-derived cells to decipher the cellular basis of the myoadipogenic balance. Clonal analysis showed that adipogenic progenitors can be separated from myogenic progenitors and, interestingly, from myoadipogenic bipotent progenitors. These progenitors were isolated in the CD34(+) population on the basis of the expression of CD56 and CD15 cell surface markers. In vivo, these different cell types have been found in the interstitial compartment of human muscle. In vitro, we show that the proliferation of bipotent myoadipogenic CD56(+)CD15(+) progenitors gives rise to myogenic CD56(+)CD15(-) progenitors and adipogenic CD56(-)CD15(+) progenitors. A cellular hierarchy of muscle and fat progenitors thus occurs within human muscle. These results provide cellular bases for adipogenic differentiation in human skeletal muscle, which may explain the fat development encountered in different muscle pathological situations.

Isolation of a highly myogenic CD34-negative subset of human skeletal muscle cells free of adipogenic potential.

The differentiation of multipotent cells into undesirable lineages is a significant risk factor when performing cell therapy. In muscular diseases, myofiber loss can be associated with progressive fat accumulation that is one of the primary factors leading to decline of muscular strength. Therefore, to avoid any contribution of injected multipotent cells to fat deposition, we have searched for a highly myogenic but nonadipogenic muscle-derived cell population. We show that the myogenic marker CD56, which is the gold standard for myoblast-based therapy, was unable to separate muscle cells into myogenic and adipogenic fractions. Conversely, using the stem cell marker CD34, we were able to sort two distinct populations, CD34(+) and CD34(-), which have been thoroughly characterized in vitro and in vivo using an immunodeficient Rag2(-/-)gamma(c) (-/-) mouse model of muscle regeneration with or without adipose deposition. Our results demonstrate that both populations have equivalent capacities for in vitro amplification. The CD34(+) cells and CD34(-) cells exhibit equivalent myogenic potential, but only the CD34(-) population fails to differentiate into adipocytes in vitro and in vivo after transplantation into regenerative fat muscle. These data indicate that the muscle-derived cells constitute a heterogeneous population of cells with various differentiation potentials. The simple CD34 sorting allows isolation of myogenic cells with no adipogenic potential and therefore could be of high interest for cell therapy when fat is accumulated in diseased muscle.