Time-of-flight and black-blood MRI to study intracranial arteries in rats.

Intracranial aneurysms (IAs) are usually incidentally discovered by magnetic resonance imaging (MRI). Once discovered, the risk associated with their treatment must be balanced with the risk of an unexpected rupture. Although clinical observations suggest that the detection of contrast agent in the aneurysm wall using a double-inversion recovery black-blood (BB) sequence may point to IA wall instability, the exact meaning of this observation is not understood. Validation of reliable diagnostic markers of IA (in)stability is of utmost importance to deciding whether to treat or not an IA. To longitudinally investigate IA progression and enhance our understanding of this devastating disease, animal models are of great help. The aim of our study was to improve a three-dimensional (3D)-time-of-flight (TOF) sequence and to develop a BB sequence on a standard preclinical 3-T MRI unit to investigate intracranial arterial diseases in rats. We showed that our 3D-TOF sequence allows reliable measurements of intracranial artery diameters, inter-artery distances, and angles between arteries and that our BB sequence enables us to visualize intracranial arteries. We report the first BB-MRI sequence to visualize intracranial arteries in rats using a preclinical 3-T MRI unit. This sequence could be useful for a large community of researchers working on intracranial arterial diseases.Relevance statement We developed a black-blood MRI sequence to study vessel wall enhancement in rats with possible application to understanding IAs instability and finding reliable markers for clinical decision-making.Key points• Reliable markers of aneurysm stability are needed for clinical decision.• Detection of contrast enhancement in the aneurysm wall may be associated with instability.• We developed a black-blood MRI sequence in rats to be used to study vessel wall enhancement of IAs.

Revealing the complexity of ultra-soft hydrogel re-swelling inside the brain.

The brain is an ultra-soft viscoelastic matrix. Sub-kPa hydrogels match the brain's mechanical properties but are challenging to manipulate in an implantable format. We propose a simple fabrication and processing sequence, consisting of de-hydration, patterning, implantation, and re-hydration steps, to deliver brain-like hydrogel implants into the nervous tissue. We monitored in real-time the ultra-soft hydrogel re-swelling kinetics in vivo using microcomputed tomography, achieved by embedding gold nanoparticles inside the hydrogel for contrast enhancement. We found that re-swelling in vivo strongly depends on the implant geometry and water availability at the hydrogel-tissue interface. Buckling of the implant inside the brain occurs when the soft implant is tethered to the cranium. Finite-element and analytical models reveal how the shank geometry, modulus and anchoring govern in vivo buckling. Taken together, these considerations on re-swelling kinetics of hydrogel constructs, implant geometry and soft implant-tissue mechanical interplay can guide the engineering of biomimetic brain implants.

Cold exposure protects from neuroinflammation through immunologic reprogramming.

Autoimmunity is energetically costly, but the impact of a metabolically active state on immunity and immune-mediated diseases is unclear. Ly6Chi monocytes are key effectors in CNS autoimmunity with an elusive role in priming naive autoreactive T cells. Here, we provide unbiased analysis of the immune changes in various compartments during cold exposure and show that this energetically costly stimulus markedly ameliorates active experimental autoimmune encephalomyelitis (EAE). Cold exposure decreases MHCII on monocytes at steady state and in various inflammatory mouse models and suppresses T cell priming and pathogenicity through the modulation of monocytes. Genetic or antibody-mediated monocyte depletion or adoptive transfer of Th1- or Th17-polarized cells for EAE abolishes the cold-induced effects on T cells or EAE, respectively. These findings provide a mechanistic link between environmental temperature and neuroinflammation and suggest competition between cold-induced metabolic adaptations and autoimmunity as energetic trade-off beneficial for the immune-mediated diseases.

Bi-component HlgC/HlgB and HlgA/HlgB γ-hemolysins from S. aureus: Modulation of Ca2+ channels activity through a differential mechanism.

Staphylococcal bi-component leukotoxins known as *pore-forming toxins* induce upon a specific binding to membrane receptors, two independent cellular events in human neutrophils. First, they provoke the opening of pre-existing specific ionic channels including Ca2+ channels. Then, they form membrane pores specific to monovalent cations leading to immune cells death. Among these leukotoxins, HlgC/HlgB and HlgA/HlgB γ-hemolysins do act in synergy to induce the opening of different types of Ca2+ channels in the absence as in the presence of extracellular Ca2+. Here, we investigate the mechanism underlying the modulation of Ca2+-independent Ca2+ channels in response to both active leukotoxins in human neutrophils. In the absence of extracellular Ca2+, the Mn2+ has been used as a Ca2+ surrogate to determine the activity of Ca2+-independent Ca2+ channels. Our findings provide new insights about different mechanisms involved in the staphylococcal γ-hemolysins activity to regulate three different types of Ca2+-independent Ca2+ channels. We conclude that (i) HlgC/HlgB stimulates the opening of La3+-sensitive Ca2+ channels, through a cholera toxin-sensitive G protein, (ii) HlgA/HlgB stimulates the opening of Ca2+ channels not sensitive to La3+, through a G protein-independent process, and (iii) unlike HlgA/HlgB, HlgC/HlgB toxins prevent the opening of a new type of Ca2+ channels by phosphorylation/de-phosphorylation-dependent mechanisms.

Tristetraprolin Promotes Hepatic Inflammation and Tumor Initiation but Restrains Cancer Progression to Malignancy.

BACKGROUND & AIMS: Tristetraprolin (TTP) is a key post-transcriptional regulator of inflammatory and oncogenic transcripts. Accordingly, TTP was reported to act as a tumor suppressor in specific cancers. Herein, we investigated how TTP contributes to the development of liver inflammation and fibrosis, which are key drivers of hepatocarcinogenesis, as well as to the onset and progression of hepatocellular carcinoma (HCC). METHODS: TTP expression was investigated in mouse/human models of hepatic metabolic diseases and cancer. The role of TTP in nonalcoholic steatohepatitis and HCC development was further examined through in vivo/vitro approaches using liver-specific TTP knockout mice and a panel of hepatic cancer cells. RESULTS: Our data demonstrate that TTP loss in vivo strongly restrains development of hepatic steatosis and inflammation/fibrosis in mice fed a methionine/choline-deficient diet, as well as HCC development induced by the carcinogen diethylnitrosamine. In contrast, low TTP expression fostered migration and invasion capacities of in vitro transformed hepatic cancer cells likely by unleashing expression of key oncogenes previously associated with these cancerous features. Consistent with these data, TTP was significantly down-regulated in high-grade human HCC, a feature further correlating with poor clinical prognosis. Finally, we uncover hepatocyte nuclear factor 4 alpha and early growth response 1, two key transcription factors lost with hepatocyte dedifferentiation, as key regulators of TTP expression. CONCLUSIONS: Although TTP importantly contributes to hepatic inflammation and cancer initiation, its loss with hepatocyte dedifferentiation fosters cancer cells migration and invasion. Loss of TTP may represent a clinically relevant biomarker of high-grade HCC associated with poor prognosis.

The impact of short-term machine perfusion on the risk of cancer recurrence after rat liver transplantation with donors after circulatory death.

Hypothermic and normothermic ex vivo liver perfusions promote organ recovery after donation after circulatory death (DCD). We tested whether these perfusions can reduce the risk of hepatocellular carcinoma (HCC) recurrence in a 1h-DCD syngeneic transplantation model, using Fischer F344 rats. DCD grafts were machine perfused for 2h with hypothermic perfusion (HOPE) or normothermic perfusion (NORMO), and transplanted. After reperfusion, we injected HCC cells into the vena porta. On day 28 after transplantation, we assessed tumour volumes by MRI. Control rats included transplantations with Fresh and non-perfused DCD livers. We observed apoptotic-necrotic hepatocyte foci in all DCD grafts, which were more visible than in the Fresh liver grafts. Normothermic perfusion allowed a faster post-transplant recovery, with lower day 1 levels of transaminases compared with the other DCD. Overall, survival was similar in all four groups and all animals developed HCCs. Total tumor volume was lower in the Fresh liver recipients compared to the DCD and DCD+HOPE recipients. Volumes in DCD+NORMO recipients were not significantly different from those in the Fresh group. This experiment confirms that ischemia/reperfusion injury promotes HCC cell engraftment/growth after DCD liver transplantation. Using the present extreme 1h ischemia model, both hypothermic and normothermic perfusions were not effective in reducing this risk.

P-Glycoprotein 1 Affects Chemoactivities of Resveratrol against Human Colorectal Cancer Cells.

Resveratrol has been proposed to prevent tumor growth and the different steps of carcinogenesis; nevertheless, these biological effects are sometimes discordant between different cell types. Several hypotheses and works have suggested that the metabolism of resveratrol could be at the origin of a different cellular response. We show here, using colorectal tumor cell lines, that the biological effects of RSV result mainly from its carriage by carriers of the superfamily of ABC transporter, i.e., P-gP, MRP, or BCRP. Using cell lines overexpressing these different transporters, we have been able to highlight the importance of P-gP in the response of cells to RSV. These results were confirmed by invalidating the gene coding for P-gP, which restored the sensitivity of colorectal cells resistant to the polyphenol. Subsequently, the status of P-glycoprotein expression is an important element to be taken into consideration in the cytotoxic activity of resveratrol in colorectal cancer cells.

Short-term 3D culture systems of various complexity for treatment optimization of colorectal carcinoma.

Three-dimensional (3D) cultures have the potential to increase the predictive value of pre-clinical drug research and bridge the gap towards anticipating clinical outcome of proposed treatments. However, their implementation in more advanced drug-discovery programs is still in its infancy due to the lack of reproducibility and low time- and cost effectiveness. HCT116, SW620 and DLD1 cells, cell lines with distinct mutations, grade and origin, were co-cultured with fibroblasts and endothelial cells (EC) in 3D spheroids. Clinically relevant drugs, i.e. 5-fluorouracil (5-FU), regorafenib and erlotinib, were administered individually to in CRC cell cultures. In this study, we established a robust, low-cost and reproducible short-term 3D culture system addressing the various complexities of the colorectal carcinoma (CRC) microenvironment. We observed a dose-dependent increase of erlotinib sensitivity in 3D (co-)cultures compared to 2D cultures. Furthermore, we compared the drug combination efficacy and drug-drug interactions administered in 2D, 3D and 3D co-cultures. We observed that synergistic/additive drug-drug interactions for drug combinations administered at low doses shifted towards additive and antagonistic when applied at higher doses in metastatic CRC cells. The addition of fibroblasts at various ratios and EC increased the resistance to some drug combinations in SW620 and DLD1 cells, but not in HCT116. Retreatment of SW620 3D co-cultures with a low-dose 3-drug combination was as active (88% inhibition, relative to control) as 5-FU treatment at high dose (100 µM). Moreover, 3D and 3D co-cultures responded variably to the drug combination treatments, and also signalling pathways were differently regulated, probably due to the influence of fibroblasts and ECs on cancer cells. The short-term 3D co-culture system developed here is a powerful platform for screening (combination) therapies. Understanding of signalling in 3D co-cultures versus 3D cultures and the responses in the 3D models upon drug treatment might be beneficial for designing anti-cancer therapies.

Does non-activated platelet-rich plasma (PRP) enhance fat graft outcome? An assessment with 3D CT-scan in mice.

BACKGROUND: The adjunction of autologous platelet-rich plasma (PRP) is emerging as a promising approach to enhance the long-term survival of fat grafting, but there are still insufficient data on its efficacy. The aim of this in vivo study was to assess the effect of the addition of non-activated PRP on fat graft outcome. METHODS: Human adipose tissue mixed with 20% of non-activated PRP was injected under the scalp skin of nude Balb/cAnNRj mice and compared to grafted fat mixed with 20% of saline. The fat graft volume was analyzed by a computed tomography scan until day 90 and immunohistochemistry was then performed to assess adipocyte viability and graft revascularization. RESULTS: At day 90, the volume of fat graft was not enhanced by PRP compared to the saline control group. However, immunohistochemistry showed that PRP significantly increased the fat graft area occupied by intact adipocytes compared to the saline group (72.66% vs. 60.78%, respectively; p < 0.05). Vascularity was also significantly higher in the PRP group compared to the control group (6695 vs. 4244 CD31+ cells/µm2, respectively; p < 0.05). CONCLUSION: The adjunction of non-activated-PRP to fat grafts significantly increased adipocyte viability and tissue vascularity. However, in contrast to other studies adding activated-PRP, non-activated-PRP did not increase residual fat graft volume until day 90. Further studies are therefore needed to understand whether PRP has a positive effect on fat graft volume. As 3D computed tomography scan is a reproducible and precise technique, it should be used to analyze fat graft volume changes over time.

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9-18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced. This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

Experimental Model of Human Malignant Mesothelioma in Athymic Mice.

Malignant pleural mesothelioma (MPM) is a thoracic aggressive cancer caused by asbestos exposure, which is difficult to diagnose and treat. Here, we characterized an in vivo orthotopic xenograft model consisting of human mesothelioma cells (designed as H2052/484) derived from a pleural NCI-H2052 tumor injected in partially immunodeficient athymic mice. We assessed tumor formation and tumor-dependent patterns of inflammation. H2052/484 cells conserved their mesothelioma phenotype and most characteristics from the parental NCI-H2052 cells. After intra-thoracic injection of H2052/484 cells, thoracic tumors developed in nearly all mice (86%) within 14 days, faster than from parental NCI-H2052 cells. When the mice were euthanized, the pleural lavage fluid was examined for immune cell profiles. The pleural immune cell population increased with tumor development. Interestingly, the proportion of myeloid-derived suppressor cell and macrophage (especially CD206⁺ M2 macrophages) populations increased in the pleural fluid of mice with large mesothelioma development, as previously observed in immunocompetent mice. This reliable orthotopic model recapitulates human mesothelioma and may be used for the study of new treatment strategies.

* Microcomputed Tomography Technique for In Vivo Three-Dimensional Fat Tissue Volume Evaluation After Polymer Injection.

Tissue engineering technologies are new and promising techniques in fat tissue reconstruction. However, to assess their efficacy before any clinical application, in vivo experiments are mandatory. This study assesses whether microcomputed tomography (CT) scan imaging is suitable to analyze in vivo the behavior of injected engineered polymer and changes in fat tissue. The volume of mice inguinal fat pads and the resorption rate of different polymers were analyzed by CT scan for up to 3 months. Different biomaterials were used, including our innovative microspheres loaded with oleic acid. We were able to follow in vivo the polymer and the fat volume of the same animals during a long-term follow-up of 90 days. Semiautomatic three-dimensional quantification allowed to determine the fat volume enhancement after injection, as well as the resorption rate of our product compared to other biomaterials (i.e., polylactic and hyaluronic acid) until 90 days. Our results demonstrate the encouraging proof-of-principle evidence for the application of micro-CT scan technology to follow in vivo biodegradable polymers in a fat tissue engineering approach. This noninvasive technique offers the advantages of the long-term follow-up of fat tissue and synthetic materials in the same animals, which allows both a scientific evaluation of the measurements and the reduction of the number of animals used in in vivo protocols in accordance with the 3 "R" principles governing the use of animals in science.

Preclinical validations of [18F]FPyPEGCBT-c(RGDfK): a 18F-labelled RGD peptide prepared by ligation of 2-cyanobenzothiazole and 1,2-aminothiol to image angiogenesis.

BACKGROUND: αVß3, αVß5 and α5ß1 integrins are known to be involved in carcinogenesis and are overexpressed in many types of tumours compared to healthy tissues; thereby they have been selected as promising therapeutic targets. Positron emission tomography (PET) is providing a unique non-invasive screening assay to discriminate which patient is more prone to benefit from antiangiogenic therapies, and extensive research has been carried out to develop a clinical radiopharmaceutical that binds specifically to integrin receptors. We recently reported the synthesis of a new 18F-labelled RGD peptide prepared by 2-cyanobenzothiazole (CBT)/1,2-aminothiol conjugation. This study aims at characterising the preclinical biologic properties of this new tumour-targeting ligand, named [18F]FPyPEGCBT-c(RGDfK).The in vitro binding properties of [18F]FPyPEGCBT-c(RGDfK) were analysed by standard binding assay in U-87 MG and SKOV-3 cancer models and its selectivity towards integrins by siRNA depletions. Its preclinical potential was studied in mice bearing subcutaneous tumours by ex vivo biodistribution studies and in vivo microPET/CT imaging. RESULTS: In vitro, FPyPEGCBT-c(RGDfK) efficiently bound RGD-recognising integrins as compared to a control c(RGDfV) peptide (IC50 = 30.8 × 10-7 M vs. 6.0 × 10-7 M). [18F]FPyPEGCBT-c(RGDfK) cell uptake was mediated by an active transport through binding to αV, ß3 and ß5 but not to ß1 subunits. In vivo, this new tracer demonstrated specific tumour uptake with %ID/g of 2.9 and 2.4 in U-87 MG and SKOV-3 tumours 1 h post injection. Tumour-to-muscle ratios of 4 were obtained 1 h after intravenous administration of the tracer allowing good visualisation of the tumours. However, unfavourable background accumulation and high hepatobiliary excretion were observed. CONCLUSION: [18F]FPyPEGCBT-c(RGDfK) specifically detects tumours expressing RGD-recognising integrin receptors in preclinical studies. Further optimisation of this radioligand may yield candidates with improved imaging properties and would warrant the further use of this efficient labelling technique for alternative targeting vectors.

Caloric Restriction Leads to Browning of White Adipose Tissue through Type 2 Immune Signaling.

Caloric restriction (CR) extends lifespan from yeast to mammals, delays onset of age-associated diseases, and improves metabolic health. We show that CR stimulates development of functional beige fat within the subcutaneous and visceral adipose tissue, contributing to decreased white fat and adipocyte size in lean C57BL/6 and BALB/c mice kept at room temperature or at thermoneutrality and in obese leptin-deficient mice. These metabolic changes are mediated by increased eosinophil infiltration, type 2 cytokine signaling, and M2 macrophage polarization in fat of CR animals. Suppression of the type 2 signaling, using Il4ra(-/-), Stat6(-/-), or mice transplanted with Stat6(-/-) bone marrow-derived hematopoietic cells, prevents the CR-induced browning and abrogates the subcutaneous fat loss and the metabolic improvements induced by CR. These results provide insights into the overall energy homeostasis during CR, and they suggest beige fat development as a common feature in conditions of negative energy balance.

Prolonged mitotic arrest induces a caspase-dependent DNA damage response at telomeres that determines cell survival.

A delay in the completion of metaphase induces a stress response that inhibits further cell proliferation or induces apoptosis. This response is thought to protect against genomic instability and is important for the effects of anti-mitotic cancer drugs. Here, we show that mitotic arrest induces a caspase-dependent DNA damage response (DDR) at telomeres in non-apoptotic cells. This pathway is under the control of Mcl-1 and other Bcl-2 family proteins and requires caspase-9, caspase-3/7 and the endonuclease CAD/DFF40. The gradual caspase-dependent loss of the shelterin complex protein TRF2 from telomeres promotes a DDR that involves DNA-dependent protein kinase (DNA-PK). Suppression of mitotic telomere damage by enhanced expression of TRF2, or the inhibition of either caspase-3/7 or DNA-PK during mitotic arrest, promotes subsequent cell survival. Thus, we demonstrate that mitotic stress is characterised by the sub-apoptotic activation of a classical caspase pathway, which promotes telomere deprotection, activates DNA damage signalling, and determines cell fate in response to a prolonged delay in mitosis.

Role of MIF/CD74 signaling pathway in the development of pleural mesothelioma.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine implicated in acute and chronic inflammatory diseases. MIF is overexpressed in various tumors. It displays a number of functions that provide a direct link between the process of inflammation and tumor growth. Our group recently identified the MIF-receptor CD74 as an independent prognostic factor for overall survival in patients with malignant pleural mesothelioma.In the present study, we compared the levels of expression of MIF and CD74 in different human mesothelioma cell lines and investigated their physiopathological functions in vitro and in vivo.Human mesothelioma cells expressed more CD74 and secreted less MIF than non tumoral MeT5A cells, suggesting a higher sensitivity to MIF. In mesothelioma cells, high MIF levels were associated with a high multiplication rate of cells. In vitro, reduction of MIF or CD74 levels in both mesothelioma cell lines showed that the MIF/CD74 signaling pathway promoted tumor cell proliferation and protected MPM cells from apoptosis. Finally, mesothelioma cell lines expressing high CD74 levels had a low tumorigenic potential after xenogeneic implantation in athymic nude mice.All these data highlight the complexity of the MIF/CD74 signaling pathway in the development of mesothelioma.

[¹¹C]acetate and PET/CT assessment of muscle activation in rat studies.

PURPOSE: The purpose of the present study is to apply kinetic analysis to investigate exercise-related changes in the metabolism of the skeletal muscle of the rat hindlimb by [[Formula: see text]]acetate positron emission tomography and computed tomography (PET/CT). METHODS: Contractions were induced in Wistar rats' left hindlimb by electrostimulation of the Vastus Lateralis muscle motor point. After 15 min of muscle contractions, [[Formula: see text]]acetate was injected and PET/CT of both hindlimbs was acquired. The resting hindlimb was used as a control reference. The kinetic parameters [Formula: see text] and [Formula: see text] were calculated for the target muscles (exercised and control) and correlated with the corresponding standardized uptake values (SUVs). The ratio between each kinetic parameter values and the SUV extracted for the exercised muscle and the muscle at rest was computed ([Formula: see text] and [Formula: see text], respectively). RESULTS: Kinetic analysis quantitatively confirmed that net tracer uptake ([Formula: see text]) and washout ([Formula: see text]) were significantly higher in exercised muscles ([Formula: see text] for exercised muscles vs. [Formula: see text] for resting muscles, [Formula: see text]; [Formula: see text] for exercised muscle vs. [Formula: see text] for resting muscle, [Formula: see text]). On the other hand, SUV was not significantly different between active and inactive muscles ([Formula: see text] for exercised muscles vs. [Formula: see text] for resting muscles). Linear regression analysis revealed a good correlation ([Formula: see text]) between net tracer uptake ratio ([Formula: see text]) and the SUV ratio [Formula: see text]). A lower correlation was found between the net tracer washout ratio ([Formula: see text]) and the SUV ratio ([Formula: see text]). CONCLUSION: The present study showed that kinetic modelling can detect changes between active and inactive skeletal muscles with a higher sensitivity with respect to the SUV, when performed with [[Formula: see text]]acetate PET/CT.

Gut Microbiota Orchestrates Energy Homeostasis during Cold.

Microbial functions in the host physiology are a result of the microbiota-host co-evolution. We show that cold exposure leads to marked shift of the microbiota composition, referred to as cold microbiota. Transplantation of the cold microbiota to germ-free mice is sufficient to increase insulin sensitivity of the host and enable tolerance to cold partly by promoting the white fat browning, leading to increased energy expenditure and fat loss. During prolonged cold, however, the body weight loss is attenuated, caused by adaptive mechanisms maximizing caloric uptake and increasing intestinal, villi, and microvilli lengths. This increased absorptive surface is transferable with the cold microbiota, leading to altered intestinal gene expression promoting tissue remodeling and suppression of apoptosis-the effect diminished by co-transplanting the most cold-downregulated strain Akkermansia muciniphila during the cold microbiota transfer. Our results demonstrate the microbiota as a key factor orchestrating the overall energy homeostasis during increased demand.

Microbiota depletion promotes browning of white adipose tissue and reduces obesity.

Brown adipose tissue (BAT) promotes a lean and healthy phenotype and improves insulin sensitivity. In response to cold or exercise, brown fat cells also emerge in the white adipose tissue (WAT; also known as beige cells), a process known as browning. Here we show that the development of functional beige fat in the inguinal subcutaneous adipose tissue (ingSAT) and perigonadal visceral adipose tissue (pgVAT) is promoted by the depletion of microbiota either by means of antibiotic treatment or in germ-free mice. This leads to improved glucose tolerance and insulin sensitivity and decreased white fat and adipocyte size in lean mice, obese leptin-deficient (ob/ob) mice and high-fat diet (HFD)-fed mice. Such metabolic improvements are mediated by eosinophil infiltration, enhanced type 2 cytokine signaling and M2 macrophage polarization in the subcutaneous white fat depots of microbiota-depleted animals. The metabolic phenotype and the browning of the subcutaneous fat are impaired by the suppression of type 2 cytokine signaling, and they are reversed by recolonization of the antibiotic-treated or germ-free mice with microbes. These results provide insight into the microbiota-fat signaling axis and beige-fat development in health and metabolic disease.

Ectopic UCP1 Overexpression in White Adipose Tissue Improves Insulin Sensitivity in Lou/C Rats, a Model of Obesity Resistance.

Brown adipose tissue (BAT), characterized by the presence of uncoupling protein 1 (UCP1), has been described as metabolically active in humans. Lou/C rats, originating from the Wistar strain, are resistant to obesity. We previously demonstrated that Lou/C animals express UCP1 in beige adipocytes in inguinal white adipose tissue (iWAT), suggesting a role of this protein in processes such as the control of body weight and the observed improved insulin sensitivity. A ß3 adrenergic agonist was administered for 2 weeks in Wistar and Lou/C rats to activate UCP1 and delineate its metabolic impact. The treatment brought about decreases in fat mass and improvements in insulin sensitivity in both groups. In BAT, UCP1 expression increased similarly in response to the treatment in the two groups. However, the intervention induced the appearance of beige cells in iWAT, associated with a marked increase in UCP1 expression, in Lou/C rats only. This increase was correlated with a markedly enhanced glucose uptake measured during euglycemic-hyperinsulinemic clamps, suggesting a role of beige cells in this process. Activation of UCP1 in ectopic tissues, such as beige cells in iWAT, may be an interesting therapeutic approach to prevent body weight gain, decrease fat mass, and improve insulin sensitivity.