Lactate induces FGF21 expression in adipocytes through a p38-MAPK pathway.

FGF21 (fibroblast growth factor 21), first described as a main fasting-responsive molecule in the liver, has been shown to act as a true metabolic regulator in additional tissues, including muscle and adipose tissues. In the present study, we found that the expression and secretion of FGF21 was very rapidly increased following lactate exposure in adipocytes. Using different pharmacological and knockout mice models, we demonstrated that lactate regulates Fgf21 expression through a NADH/NAD-independent pathway, but requires active p38-MAPK (mitogen activated protein kinase) signalling. We also demonstrated that this effect is not restricted to lactate as additional metabolites including pyruvate and ketone bodies also activated the FGF21 stress response. FGF21 release by adipose cells in response to an excess of intermediate metabolites may represent a physiological mechanism by which the sensing of environmental metabolic conditions results in the release of FGF21 to improve metabolic adaptations.

Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury.

Fibro-adipogenic progenitors (FAPs) play a crucial role in skeletal muscle regeneration, as they generate a favorable niche that allows satellite cells to perform efficient muscle regeneration. After muscle injury, FAP content increases rapidly within the injured muscle, the origin of which has been attributed to their proliferation within the muscle itself. However, recent single-cell RNAseq approaches have revealed phenotype and functional heterogeneity in FAPs, raising the question of how this differentiation of regenerative subtypes occurs. Here we report that FAP-like cells residing in subcutaneous adipose tissue (ScAT), the adipose stromal cells (ASCs), are rapidly released from ScAT in response to muscle injury. Additionally, we find that released ASCs infiltrate the damaged muscle, via a platelet-dependent mechanism and thus contribute to the FAP heterogeneity. Moreover, we show that either blocking ASCs infiltration or removing ASCs tissue source impair muscle regeneration. Collectively, our data reveal that ScAT is an unsuspected physiological reservoir of regenerative cells that support skeletal muscle regeneration, underlining a beneficial relationship between muscle and fat.

Driving regeneration, instead of healing, in adult mammals: the decisive role of resident macrophages through efferocytosis.

Tissue repair after lesion usually leads to scar healing and thus loss of function in adult mammals. In contrast, other adult vertebrates such as amphibians have the ability to regenerate and restore tissue homeostasis after lesion. Understanding the control of the repair outcome is thus a concerning challenge for regenerative medicine. We recently developed a model of induced tissue regeneration in adult mice allowing the comparison of the early steps of regenerative and scar healing processes. By using studies of gain and loss of function, specific cell depletion approaches, and hematopoietic chimeras we demonstrate here that tissue regeneration in adult mammals depends on an early and transient peak of granulocyte producing reactive oxygen species and an efficient efferocytosis specifically by tissue-resident macrophages. These findings highlight key and early cellular pathways able to drive tissue repair towards regeneration in adult mammals.

Adipose tissue sensitivity to radiation exposure.

Treatment of cancer using radiation can be significantly compromised by the development of severe acute and late damage to normal tissue. Treatments that either reduce the risk and severity of damage or that facilitate the healing of radiation injuries are being developed, including autologous adipose tissue grafts to repair tissue defects or involutional disorders that result from tumor resection. Adipose tissue is specialized in energy storage and contains different cell types, including preadipocytes, which could be used for autologous transplantation. It has long been considered a poorly proliferative connective tissue; however, the acute effects of ionizing radiation on adipose tissue have not been investigated. Therefore, the aim of this study was to characterize the alterations induced in adipose tissue by total body irradiation. A severe decrease in proliferating cells, as well as a significant increase in apoptotic cells, was observed in vivo in inguinal fat pads following irradiation. Additionally, irradiation altered the hematopoietic population. Decreases in the proliferation and differentiation capacities of non-hematopoietic progenitors were also observed following irradiation. Together, these data demonstrate that subcutaneous adipose tissue is very sensitive to irradiation, leading to a profound alteration of its developmental potential. This damage could also alter the reconstructive properties of adipose tissue and, therefore, calls into question its use in autologous fat transfer following radiotherapy.

Preconditioning by mitochondrial reactive oxygen species improves the proangiogenic potential of adipose-derived cells-based therapy.

OBJECTIVE: Transplantation of adipose-derived stroma cells (ADSCs) stimulates neovascularization after experimental ischemic injury. ADSC proangiogenic potential is likely mediated by their ability to differentiate into endothelial cells and produce a wide array of angiogenic and antiapoptotic factors. Mitochondrial reactive oxygen species (ROS) have been shown to control ADSC differentiation. We therefore hypothesized that mitochondrial ROS production may change the ADSC proangiogenic properties. METHODS AND RESULTS: The use of pharmacological strategies (mitochondrial inhibitors, antimycin, and rotenone, with or without antioxidants) allowed us to specifically and precisely modulate mitochondrial ROS generation in ADSCs. We showed that transient stimulation of mitochondrial ROS generation in ADSCs before their injection in ischemic hindlimb strongly improved revascularization and the number of ADSC-derived CD31-positive cells in ischemic area. Mitochondrial ROS generation increased the secretion of the proangiogenic and antiapoptotic factors, VEGF and HGF, but did not affect ADSC ability to differentiate into endothelial cells, in vitro. Moreover, mitochondrial ROS-induced ADSC preconditioning greatly protect ADSCs against oxidative stress-induced cell death. CONCLUSIONS: Our study demonstrates that in vitro preconditioning by moderate mitochondrial ROS generation strongly increases in vivo ADSC proangiogenic properties and emphasizes the crucial role of mitochondrial ROS in ADSC fate.

Opioids prevent regeneration in adult mammals through inhibition of ROS production.

Inhibition of regeneration and induction of tissue fibrosis are classic outcomes of tissue repair in adult mammals. Here, using a newly developed model of regeneration in adult mammals i.e. regeneration after massive resection of an inguinal fat pad, we demonstrate that both endogenous and exogenous opioids prevent tissue regeneration in adults, by inhibiting the early production of reactive oxygen species (ROS) that generally occurs after lesion and is required for regeneration. These effects can be overcome and regeneration induced by the use of an opioid antagonist. The results obtained in both our new model and the gold standard adult zebrafish demonstrate that this mechanism can be considered as a general paradigm in vertebrates. This work clearly demonstrates that ROS is required for tissue regeneration in adult mammals and shows the deleterious effect of opioids on tissue regeneration through the control of this ROS production. It thus raises questions about opioid-based analgesia in perioperative care.

Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives.

BACKGROUND: Adipose tissue development and remodeling are closely associated with the growth of vascular network. We hypothesized that adipose tissue may contain progenitor cells with angiogenic potential and that therapy based on adipose tissue-derived progenitor cells administration may constitute a promising cell therapy in patients with ischemic disease. METHODS AND RESULTS: In mice, cultured stromal-vascular fraction (SVF) cells from adipose tissue have a great proangiogenic potential, comparable to that of bone marrow mononuclear cells in the mouse ischemic hindlimb model. Similarly, cultured human SVF cells differentiate into endothelial cells, incorporate into vessels, and promote both postischemic neovascularization in nude mice and vessel-like structure formation in Matrigel plug. In vitro, these cells represent a homogeneous population of CD34- and CD13-positive cells, which can spontaneously express the endothelial cell markers CD31 and von Willebrand factor when cultured in semisolid medium. Interestingly, dedifferentiated mature human adipocytes have the potential to rapidly acquire the endothelial phenotype in vitro and to promote neovascularization in ischemic tissue and vessel-like structure formation in Matrigel plug, suggesting that cells of endothelial and adipocyte phenotypes may have a common precursor. CONCLUSIONS: This study demonstrates, for the first time, that adipocytes and endothelial cells have a common progenitor. Such adipose lineage cells participate in vascular-like structure formation in Matrigel plug and enhance the neovascularization reaction in ischemic tissue. These results also highlight the concept that adipose lineage cells represent a suitable new cell source for therapeutic angiogenesis in ischemic disease.

Mouse adipose tissue stromal cells give rise to skeletal and cardiomyogenic cell sub-populations.

We previously reported that adipose tissue could generate cardiomyocyte-like cells from crude stromal vascular fraction (SVF) in vitro that improved cardiac function in a myocardial infarction context. However, it is not clear whether these adipose-derived cardiomyogenic cells (AD-CMG) constitute a homogenous population and if AD-CMG progenitors could be isolated as a pure population from the SVF of adipose tissue. This study aims to characterize the different cell types that constitute myogenic clusters and identify the earliest AD-CMG progenitors in vitro for establishing a complete phenotype and use it to sort AD-CMG progenitors from crude SVF. Here, we report cell heterogeneity among adipose-derived clusters during their course of maturation and highlighted sub-populations that exhibit original mixed cardiac/skeletal muscle phenotypes with a progressive loss of cardiac phenotype with time in liquid culture conditions. Moreover, we completed the phenotype of AD-CMG progenitors but we failed to sort them from the SVF. We demonstrated that micro-environment is required for the maturation of myogenic phenotype by co-culture experiments. These findings bring complementary data on AD-CMG and suggest that their emergence results from in vitro events.

Browning of white adipose cells by intermediate metabolites: an adaptive mechanism to alleviate redox pressure.

The presence of brown adipose tissue (BAT) in human adults opens attractive perspectives to treat metabolic disorders. Indeed, BAT dissipates energy as heat via uncoupling protein (UCP)1. Brown adipocytes are located in specific deposits or can emerge among white fat through the so-called browning process. Although numerous inducers have been shown to drive this process, no study has investigated whether it could be controlled by specific metabolites. Here, we show that lactate, an important metabolic intermediate, induces browning of murine white adipose cells with expression of functional UCP1. Lactate-induced browning also occurs in human cells and in vivo. Lactate controls Ucp1 expression independently of hypoxia-inducible factor-1α and PPARα pathways but requires active PPARγ signaling. We demonstrate that the lactate effect on Ucp1 is mediated by intracellular redox modifications as a result of lactate transport through monocarboxylate transporters. Further, the ketone body ß-hydroxybutyrate, another metabolite that impacts redox state, is also a strong browning inducer. Because this redox-dependent increase in Ucp1 expression promotes an oxidative phenotype with mitochondria, browning appears as an adaptive mechanism to alleviate redox pressure. Our findings open new perspectives for the control of adipose tissue browning and its physiological relevance.

Metabolic endotoxemia directly increases the proliferation of adipocyte precursors at the onset of metabolic diseases through a CD14-dependent mechanism.

Metabolic endotoxemia triggers inflammation, targets cells from the stroma-vascular fraction of adipose depots, and metabolic disease. To identify these cells we here infused mice with lipopolysaccharides and showed by FACS analyses and BrdU staining that the number of small subcutaneous adipocytes, preadipocytes and macrophages increased in wild type but not in CD14-knockout (KO) mice. This mechanism was direct since in CD14KO mice grafted subcutaneously and simultaneously with fat pads from CD14KO and wild-type mice the concentration of cytokine mRNA was increased in the wild-type fat pad only. Conversely, the mRNA concentration of genes involved in glucose and lipid metabolism and the number of large adipocytes was reduced. Eventually, a pretreatment with LPS enhanced HFD-induced metabolic diseases. Altogether, these results show that metabolic endotoxemia increases the proliferation of preadipocytes through a CD14-dependent mechanism directly, without recruiting CD14-positive cells from non-adipose depot origin. This mechanism could precede the onset of metabolic diseases.

Endothelial and cardiac regeneration from adipose tissues.

For a long time, adipose tissue was only considered for its crucial role in energy balance and associated diseases. The discovery of the presence of immature cells highlights a putative role for these tissues as reservoirs of therapeutic cells. Indeed, since fat pads can be sampled by liposuction under local anesthesia in adult patients, adipose tissue represents a promising source of regenerative cells, particularly in cardiovascular regeneration. Indeed among other potentials, we and others have demonstrated the great angiogenic properties of adipose-derived stromal cells (ASCs) and the existence of peculiar cells, at least in mice, that are able to spontaneously give rise to functional cardiomyocytes. This review deciphers the different steps necessary to isolate, characterize, and manipulate such striking cells.

Adipose-derived cardiomyogenic cells: in vitro expansion and functional improvement in a mouse model of myocardial infarction.

AIMS: Cells derived from the stroma vascular fraction (SVF) of mouse adipose tissue can spontaneously give rise to rare, functional, cardiac-like cells in vitro. This study aimed to improve the production of adipose-derived cardiomyogenic cells (AD-CMG), to characterize them and to assess their cardiac fate and functional outcomes after their administration in a mouse model of acute myocardial infarction. METHODS AND RESULTS: The culture process optimized to improve in vitro cardiac specification consisted of a primary culture of murine SVF cells in semi-solid methylcellulose medium, a selection of AD-CMG cell clusters, and a secondary culture and expansion in BHK21 medium. AD-CMG cells were CD29(+), CD31(-), CD34(-), CD44(+), CD45(-), CD81(+), CD90(-), CD117(-), and Flk-1(-) and expressed several cardiac contractile proteins. After 1, 2, and 4 weeks of their injection in mice having acute myocardial infarction, a strong presence of green fluorescent protein-positive cells was identified by immunohistochemistry as well as quantitative polymerase chain reaction. Echocardiography showed a significant reduction of remodelling and stability of left ventricle ejection fraction in the AD-CMG cell-treated group vs. controls. Vascular density analysis revealed that AD-CMG administration was also associated with stimulation of angiogenesis in peri-infarct areas. CONCLUSION: Cardiomyogenic cells can be selected and expanded in large amounts from mouse adipose tissue. They can survive and differentiate in an acute myocardial infarction model, avoiding remodelling and impairment of cardiac function, and can promote neo-vascularization in the ischaemic heart.

Adult stromal cells derived from human adipose tissue provoke pancreatic cancer cell death both in vitro and in vivo.

BACKGROUND: Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation. PRINCIPAL FINDINGS: Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth. CONCLUSION: These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.

Vascular fate of adipose tissue-derived adult stromal cells in the ischemic murine brain: A combined imaging-histological study.

Increasing evidence indicates that fat tissue can provide a novel source of progenitor cells with therapeutic potential. Here, the fate of adipose tissue-derived stromal cells (ADSCs) transplanted into the mouse ischemic cortex was monitored in the long term using in vivo imaging, and subsequently characterized. The left middle cerebral artery (MCA) was occluded in C57BL/6J mice equipped with a closed cranial window chronically implanted over the left parietal cortex (n = 20). ADSCs expressing the green fluorescent protein (GFP) (approximately 18 x 10(3) cells in 0.5 microl) were transplanted into the ipsilateral cortex, 24 h after MCA occlusion. GFP+-ADSCs were monitored through the window using confocal fluorescence microscopy to assess their single fate in vivo. Co-localization of GFP with vascular, neuronal, glial or proliferation markers was investigated immunohistochemically. Repeated in vivo imaging revealed that GFP+-ADSCs migrated over 1 week toward the lesion, survived for at least 4 weeks, and exhibited a particular tropism for vessels. About 5% of the transplanted GFP+-ADSCs were scattered in the peri-ischemic area on histological sections. Immunohistochemistry evidenced that perivascular GFP+-ADSCs enfolded CD31-labeled endothelial cells, always outside their basal lamina, and occasionally expressed smooth muscle alpha-actin. Less than 1% GFP and BrdU co-labeling indicated a low proliferation rate of ADSCs. These results demonstrate that cerebral ischemia induces ADSCs survival, migration toward the lesion, especially toward microvessels, and occasional differentiation into smooth muscle cells.

From heterogeneity to plasticity in adipose tissues: site-specific differences.

In mammals, two types of adipose tissues are present, brown (BAT) and white (WAT). WAT itself can be divided into subcutaneous and internal fat deposits. All these tissues have been shown to present a great tissue plasticity, and recent data emphasized on the multiple differentiation potentials obtained from subcutaneous WAT. However, no study has compared the heterogeneity of stroma-vascular fraction (SVF) cells and their differentiation potentials according to the localization of the fat pad. This study clearly demonstrates that WAT and BAT present different antigenic features and differentiation potentials. WAT by contrast to BAT contains a large population of hematopoietic cells composed essentially of macrophages and hematopoietic progenitor cells. In WAT, the non-hematopoietic population is mainly composed of mesenchymal stem cell (MSC)-like but contains also a significant proportion of immature cells, whereas in BAT, the stromal cells do not present the same phenotype. Internal and subcutaneous WAT present some discrete differences in the phenotype of their cell populations. WAT derived SVF cells give rise to osteoblasts, endothelial cells, adipocytes, hematopoietic cells, and cardiomyoblasts only from inguinal cells. By contrast, BAT derived SVF cells display a reduced plasticity. Adipose tissues thus appear as complex tissues composed of different cell subsets according to the location of fat pads. Inguinal WAT appears as the most plastic adipose tissue and represents a potential and suitable source of stem cell, considering its easy sampling as a major advantage for cell therapy.

Macrophage characteristics of stem cells revealed by transcriptome profiling.

We previously showed that the phenotypes of adipocyte progenitors and macrophages were close. Using functional analyses and microarray technology, we first tested whether this intriguing relationship was specific to adipocyte progenitors or could be shared with other progenitors. Measurements of phagocytic activity and gene profiling analysis of different progenitor cells revealed that the latter hypothesis should be retained. These results encouraged us to pursue and to confirm our analysis with a gold-standard stem cell population, embryonic stem cells or ESC. The transcriptomic profiles of ESC and macrophages were clustered together, unlike differentiated ESC. In addition, undifferentiated ESC displayed higher phagocytic activity than other progenitors, and they could phagocytoze apoptotic bodies. These data suggest that progenitors and stem cells share some characteristics of macrophages. This opens new perspectives on understanding stem cell phenotype and functionalities such as a putative role of stem cells in tissue remodeling by discarding dead cells but also their immunomodulation or fusion properties.

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.

Reconstitution of lethally irradiated mice by cells isolated from adipose tissue.

It is suggested that hematopoietic stem cells (HSC) could be found in several tissues of mesodermic origin. Among these, adipose tissue can expand throughout adult life and its expansion is not only due to mature adipocyte hypertrophy but also to the presence of precursor cells in stroma-vascular fraction (SVF). Here we report that transplantation of cells isolated from mice adipose tissue can efficiently rescue lethally irradiated mice and results in a reconstitution of major hematopoietic lineages. Donor cells can be detected in blood and in hematopoietic tissues of recipient mice. Adipose tissue contains a significant percentage of CD34, CD45 positive cells, and SVF cells were able to give rise to hematopoietic colonies in methylcellulose. We demonstrate the presence of hematopoietic progenitors in adipose tissue by phenotypic and functional characteristics. Thus adipose tissue could be considered as an important and convenient source of cells able to support hematopoiesis.

Preadipocyte conversion to macrophage. Evidence of plasticity.

Preadipocytes are present throughout adult life in adipose tissues and can proliferate and differentiate into mature adipocytes according to the energy balance. An increasing number of reports demonstrate that cells from adipose lineages (preadipocytes and adipocytes) and macrophages share numerous functional or antigenic properties. No large scale comparison reflecting the phenotype complexity has been performed between these different cell types until now. We used profiling analysis to define the common features shared by preadipocyte, adipocyte, and macrophage populations. Our analysis showed that the preadipocyte profile is surprisingly closer to the macrophage than to the adipocyte profile. From these data, we hypothesized that in a macrophage environment preadipocytes could effectively be converted into macrophages. We injected labeled stroma-vascular cells isolated from mouse white adipose tissue or 3T3-L1 preadipocyte cell line into the peritoneal cavity of nude mice and investigated changes in their phenotype. Preadipocytes rapidly and massively acquired high phagocytic activity and index. 60-70% of preadipocytes also expressed five macrophage-specific antigens: F4/80, Mac-1, CD80, CD86, and CD45. These values were similar to those observed for peritoneal macrophages. In vitro experiments showed that cell-to-cell contact between preadipocytes and peritoneal macrophages partially induced this preadipocyte phenotype conversion. Overall, these results suggest that preadipocyte and macrophage phenotypes are very similar and that preadipocytes have the potential to be very efficiently and rapidly converted into macrophages. This work emphasizes the great cellular plasticity of adipose precursors and reinforces the link between adipose tissue and innate immunity processes.