The nervous system and adipose tissues: a tale of dialogues.

White, beige, and brown adipose tissues play a crucial role in maintaining energy homeostasis. Due to the heterogeneous and diffuse nature of fat pads, this balance requires a fine and coordinated control of many actors and therefore permanent dialogues between these tissues and the central nervous system. For about two decades, many studies have been devoted to describe the neuro-anatomical and functional complexity involved to ensure this dialogue. Thus, if it is now clearly demonstrated that there is an efferent sympathetic innervation of different fat depots controlling plasticity as well as metabolic functions of the fat pad, the crucial role of sensory innervation capable of detecting local signals informing the central nervous system of the metabolic state of the relevant pads is much more recent. The purpose of this review is to provide the current state of knowledge on this subject.

From whole-organ imaging to in-silico blood flow modeling: A new multi-scale network analysis for revisiting tissue functional anatomy.

We present a multi-disciplinary image-based blood flow perfusion modeling of a whole organ vascular network for analyzing both its structural and functional properties. We show how the use of Light-Sheet Fluorescence Microscopy (LSFM) permits whole-organ micro-vascular imaging, analysis and modelling. By using adapted image post-treatment workflow, we could segment, vectorize and reconstruct the entire micro-vascular network composed of 1.7 million vessels, from the tissue-scale, inside a ∼ 25 × 5 × 1 = 125mm3 volume of the mouse fat pad, hundreds of times larger than previous studies, down to the cellular scale at micron resolution, with the entire blood perfusion modeled. Adapted network analysis revealed the structural and functional organization of meso-scale tissue as strongly connected communities of vessels. These communities share a distinct heterogeneous core region and a more homogeneous peripheral region, consistently with known biological functions of fat tissue. Graph clustering analysis also revealed two distinct robust meso-scale typical sizes (from 10 to several hundred times the cellular size), revealing, for the first time, strongly connected functional vascular communities. These community networks support heterogeneous micro-environments. This work provides the proof of concept that in-silico all-tissue perfusion modeling can reveal new structural and functional exchanges between micro-regions in tissues, found from community clusters in the vascular graph.

Tissue Regeneration: The Dark Side of Opioids.

Opioids are regarded as among the most effective analgesic drugs and their use for the management of pain is considered standard of care. Despite their systematic administration in the peri-operative period, their impact on tissue repair has been studied mainly in the context of scar healing and is only beginning to be documented in the context of true tissue regeneration. Indeed, in mammals, growing evidence shows that opioids direct tissue repair towards scar healing, with a loss of tissue function, instead of the regenerative process that allows for recovery of both the morphology and function of tissue. Here, we review recent studies that highlight how opioids may prevent a regenerative process by silencing nociceptive nerve activity and a powerful anti-inflammatory effect. These data open up new perspectives for inducing tissue regeneration and argue for opioid-restricted strategies for managing pain associated with tissue injury.

Rapid and Efficient Production of Human Functional Mast Cells through a Three-Dimensional Culture of Adipose Tissue-Derived Stromal Vascular Cells.

Mast cells (MC) are innate immune cells involved in many physiological and pathological processes. However, studies of MC function and biology are hampered by the difficulties to obtain human primary MC. To solve this problem, we established a new method to produce easily and rapidly high numbers of MC for in vitro studies using human adipose tissue, which is an abundant and easy access tissue. Stromal vascular fraction of adipose tissue, obtained from human abdominal dermolipectomy, was cultured as spheroids in serum free medium supplemented in stem cell factor. Using this method, we generated, within 3 wk, a highly pure population of connective tissue-type MC expressing MC typical peptidases (tryptase, chymase, and carboxypeptidase-A3) with a yield increasing over time. Stem cell factor was required for this culture, but unlike MC derived from CD34+ cells, this culture did not depend on IL-3 and -6. MC obtained with this method degranulated following FcεRI cross-linking or stimulation by C5a, compound 48/80, and substance P. Interestingly, activation by anti-IgE of both white adipose tissue-MC and MC obtained from peripheral blood-derived CD34+ pluripotent progenitor cells induced the production of PGs as well as proinflammatory cytokines (TNF-α, Il-6, and GM-CSF). In conclusion, we developed a new time saving and reproducible method to produce highly pure and functional human MC in 3 wk from human adipose tissue.

Collective and experimental research project for master's students on the pathophysiology of obesity.

We describe here a collective and experimental research project-based learning (ERPBL) for master's students that can be used to illustrate some basic concepts on glucose/lipid homeostasis and renal function around a topical issue. The primary objective of this ERPBL was to strengthen students' knowledge and understanding of physiology and pathophysiology. The secondary objectives were to help students to develop technical/practical abilities and acquire transversal skills with real-world connections. Obesity is a worldwide public health problem that increases the risk for developing type 2 diabetes and nephropathies. To study the impact of western dietary habits, students evaluated the effects of a diet enriched with fat and cola [high-fat and cola diet (HFCD)] on metabolism and renal function in mice. Students mainly worked in tandem to prepare and perform experiments, but also collectively to compile, analyze, and discuss data. Students showed that HFCD-fed mice 1) developed obesity; 2) exhibited glucose homeostasis impairments associated to ectopic fat storage; and 3) displayed reduced glomerular filtration. The educational benefit of the program was estimated using three evaluation metrics: a conventional multicriteria assessment by teachers, a pre-/posttest, and a self-evaluation questionnaire. They showed that the current approach successfully strengthened scientific student knowledge and understanding of physiology/pathophysiology. In addition, it helped students develop new skills, such as technical and transversal skills. We concluded that this ERPBL dealing with the pathophysiology of obesity was strongly beneficial for master's students, thereby appearing as an efficient and performing educational tool.

Fibre crosslinking drives the emergence of order in a three-dimensional dynamical network model.

The extracellular-matrix (ECM) is a complex interconnected three-dimensional network that provides structural support for the cells and tissues and defines organ architecture as key for their healthy functioning. However, the intimate mechanisms by which ECM acquire their three-dimensional architecture are still largely unknown. In this paper, we study this question by means of a simple three-dimensional individual based model of interacting fibres able to spontaneously crosslink or unlink to each other and align at the crosslinks. We show that such systems are able to spontaneously generate different types of architectures. We provide a thorough analysis of the emerging structures by an exhaustive parametric analysis and the use of appropriate visualization tools and quantifiers in three dimensions. The most striking result is that the emergence of ordered structures can be fully explained by a single emerging variable: the number of links per fibre in the network. If validated on real tissues, this simple variable could become an important putative target to control and predict the structuring of biological tissues, to suggest possible new therapeutic strategies to restore tissue functions after disruption, and to help in the development of collagen-based scaffolds for tissue engineering. Moreover, the model reveals that the emergence of architecture is a spatially homogeneous process following a unique evolutionary path, and highlights the essential role of dynamical crosslinking in tissue structuring.

Food intake adaptation to dietary fat involves PSA-dependent rewiring of the arcuate melanocortin system in mice.

Hormones such as leptin and ghrelin can rapidly rewire hypothalamic feeding circuits when injected into rodent brains. These experimental manipulations suggest that the hypothalamus might reorganize continually in adulthood to integrate the metabolic status of the whole body. In this study, we examined whether hypothalamic plasticity occurs in naive animals according to their nutritional conditions. For this purpose, we fed mice with a short-term high-fat diet (HFD) and assessed brain remodeling through its molecular and functional signature. We found that HFD for 3 d rewired the hypothalamic arcuate nucleus, increasing the anorexigenic tone due to activated pro-opiomelanocortin (POMC) neurons. We identified the polysialic acid molecule (PSA) as a mediator of the diet-induced rewiring of arcuate POMC. Moreover, local pharmacological inhibition and genetic disruption of the PSA signaling limits the behavioral and metabolic adaptation to HFD, as treated mice failed to normalize energy intake and showed increased body weight gain after the HFD challenge. Altogether, these findings reveal the existence of physiological hypothalamic rewiring involved in the homeostatic response to dietary fat. Furthermore, defects in the hypothalamic plasticity-driven adaptive response to HFD are obesogenic and could be involved in the development of metabolic diseases.

Pain sensing neurons promote tissue regeneration in adult mice.

Tissue repair after injury in adult mammals, usually results in scarring and loss of function in contrast to lower vertebrates such as the newt and zebrafish that regenerate. Understanding the regulatory processes that guide the outcome of tissue repair is therefore a concerning challenge for regenerative medicine. In multiple regenerative animal species, the nerve dependence of regeneration is well established, but the nature of the innervation required for tissue regeneration remains largely undefined. Using our model of induced adipose tissue regeneration in adult mice, we demonstrate here that nociceptive nerves promote regeneration and their removal impairs tissue regeneration. We also show that blocking the receptor for the nociceptive neuropeptide calcitonin gene-related peptide (CGRP) inhibits regeneration, whereas CGRP administration induces regeneration. These findings reveal that peptidergic nociceptive neurons are required for adult mice tissue regeneration.

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.

Detection of extracellular glucose by GLUT2 contributes to hypothalamic control of food intake.

The sugar transporter GLUT2, present in several tissues of the gut-brain axis, has been reported to be involved in the control of food intake. GLUT2 is a sugar transporter sustaining energy production in the cell, but it can also function as a receptor for extracellular glucose. A glucose-signaling pathway is indeed triggered, independently of glucose metabolism, through its large cytoplasmic loop domain. However, the contribution of the receptor function over the transporter function of GLUT2 in the control of food intake remains to be determined. Thus, we generated transgenic mice that express a GLUT2-loop domain, blocking the detection of glucose but leaving GLUT2-dependent glucose transport unaffected. Inhibiting GLUT2-mediated glucose detection augmented daily food intake by a mechanism that increased the meal size but not the number of meals. Peripheral hormones (ghrelin, insulin, leptin) were unaffected, leading to a focus on central aspects of feeding behavior. We found defects in c-Fos activation by glucose in the arcuate nucleus and changes in the amounts of TRH and orexin neuropeptide mRNA, which are relevant to poorly controlled meal size. Our data provide evidence that glucose detection by GLUT2 contributes to the control of food intake by the hypothalamus. The sugar transporter receptor, i.e., "transceptor" GLUT2, may constitute a drug target to treat eating disorders and associated metabolic diseases, particularly by modulating its receptor function without affecting vital sugar provision by its transporter function.

Involvement of hippocampal CA3 K(ATP) channels in contextual memory.

This paper evaluates the involvement of hippocampal ATP-sensitive potassium channels (K(ATP)) in learning and memory. After confirming expression of the Kir6.2 subunit in the CA3 region of C57BL/6J mice, we performed intra-hippocampal pharmacological injections of specific openers and blockers of K(ATP) channels. The opener diazoxide, the blocker tolbutamide, or a mixture of both, were bilaterally injected in the CA3 region before we subjected the animals to a fear conditioning paradigm. Diazoxide strongly impaired contextual memory of mice at both doses tested. This impairment was specifically reversed by co-injecting the blocker tolbutamide. Moreover, we studied the mnemonic abilities of mice deleted for the Kir6.2 subunit. These mice were backcrossed to C57BL/6J mice and tested in two learning paradigms. We found a significant impairment of contextual and tone memories in the Kir6.2 knock-out mice when compared with heterozygous or wild-type animals. Furthermore, these animals were also slightly impaired in a spatial version of the Morris water maze task. Our data suggest a specific involvement of hippocampal K(ATP) Kir6.2/SUR1 channels in memory processes.

3D analysis of the whole subcutaneous adipose tissue reveals a complex spatial network of interconnected lobules with heterogeneous browning ability.

Adipose tissue, as the main energy storage organ and through its endocrine activity, is interconnected with all physiological functions. It plays a fundamental role in energy homeostasis and in the development of metabolic disorders. Up to now, this tissue has been analysed as a pool of different cell types with very little attention paid to the organization and putative partitioning of cells. Considering the absence of a complete picture of the intimate architecture of this large soft tissue, we developed a method that combines tissue clearing, acquisition of autofluorescence or lectin signals by confocal microscopy, segmentation procedures based on contrast enhancement, and a new semi-automatic image analysis process, allowing accurate and quantitative characterization of the whole 3D fat pad organization. This approach revealed the unexpected anatomic complexity of the murine subcutaneous fat pad. Although the classical picture of adipose tissue corresponds to a superposition of simple and small ellipsoidal lobules of adipose cells separated by mesenchymal spans, our results show that segmented lobules display complex 3D poly-lobular shapes. Despite differences in shape and size, the number of these poly-lobular subunits is similar from one fat pad to another. Finally, investigation of the relationships of these subunits between each other revealed a never-described organization in two clusters with distinct molecular signatures and specific vascular and sympathetic nerve densities correlating with different browning abilities. This innovative procedure reveals that subcutaneous adipose tissue exhibits a subtle functional heterogeneity with partitioned areas, and opens new perspectives towards understanding its functioning and plasticity.

Role for mitochondrial reactive oxygen species in brain lipid sensing: redox regulation of food intake.

The ability for the brain to sense peripheral fuel availability is mainly accomplished within the hypothalamus, which detects ongoing systemic nutrients and adjusts food intake and peripheral metabolism as needed. Here, we hypothesized that mitochondrial reactive oxygen species (ROS) could trigger sensing of nutrients within the hypothalamus. For this purpose, we induced acute hypertriglyceridemia in rats and examined the function of mitochondria in the hypothalamus. Hypertriglyceridemia led to a rapid increase in the mitochondrial respiration in the ventral hypothalamus together with a transient production of ROS. Cerebral inhibition of fatty acids-CoA mitochondrial uptake prevented the hypertriglyceridemia-stimulated ROS production, indicating that ROS derived from mitochondrial metabolism. The hypertriglyceridemia-stimulated ROS production was associated with change in the intracellular redox state without any noxious cytotoxic effects, suggesting that ROS function acutely as signaling molecules. Moreover, cerebral inhibition of hypertriglyceridemia-stimulated ROS production fully abolished the satiety related to the hypertriglyceridemia, suggesting that hypothalamic ROS production was required to restrain food intake during hypertriglyceridemia. Finally, we found that fasting disrupted the hypertriglyceridemia-stimulated ROS production, indicating that the redox mechanism of brain nutrient sensing could be modulated under physiological conditions. Altogether, these findings support the role of mitochondrial ROS as molecular actors implied in brain nutrient sensing.

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.

[Brain glucose sensing]. / La sensibilité cérébrale au glucose.

Brain nutrient sensing permits fine regulation of physiological functions such as food intake and blood glucose regulation related to energy homeostasis. The mechanism of glucose sensing is the most extensively studied, and parallels have been drawn between pancreatic beta cells and neurons. Two types of glucose-sensing neuron have been identified, namely those whose activity is directly proportional to the glucose concentration, and those whose activity is inversely proportional to the glucose concentration. It was recently demonstrated that the mechanism depends on the amplitude of change in the glucose concentration. In some cases detection is probably not ensured by neurons themselves but by astrocytes, indicating that the two cell types are coupled in some way. Glucose sensing can be modulated by other nutrients (particularly fatty acids) and also by hormones (insulin, leptin and ghrelin) and peptides (NPY). The subtle cellular and molecular mechanisms involved in glucose sensing probably explain reported discrepancies in the expression of glucose transporters, hexokinases and channels. Astrocytes might also be involved in one type of response, thus adding a new level of complexity.

Regionalization of browning revealed by whole subcutaneous adipose tissue imaging.

OBJECTIVE: White and brown adipose tissues play a major role in the regulation of metabolic functions. With the explosion of obesity and metabolic disorders, the interest in adipocyte biology is growing constantly. While several studies have demonstrated functional differences between adipose fat pads, especially in their involvement in metabolic diseases, there are no data available on possible heterogeneity within an adipose depot. METHODS: This study investigated the three-dimensional (3-D) organization of the inguinal fat pad in adult mice by combining adipose tissue clearing and autofluorescence signal acquisition by confocal microscopy. In addition, the study analyzed the expression of genes involved in adipocyte biology and browning at the mARN and protein levels in distinct areas of the inguinal adipose tissue, in control conditions and after cold exposure. RESULTS: Semiautomated 3-D image analysis revealed an organization of the fat depot showing two regions: the core was structured into segmented lobules, whereas the periphery appeared unsegmented. Perilipin immunostaining showed that most of the adipocytes located in the core region had smaller lipid droplets, suggesting a brown-like phenotype. qPCR analysis showed a higher expression of the browning markers Ucp1, Prdm16, Ppargc1a, and Cidea in the core region than at the periphery. Finally, cold exposure induced upregulation of thermogenic gene expression associated with an increase of UCP1 protein, specifically in the core region of the inguinal fat depot. CONCLUSIONS: Altogether, these data demonstrate a structural and functional heterogeneity of the inguinal fat pad, with an anatomically restricted browning process in the core area.

A new ATP-sensitive K+ channel-independent mechanism is involved in glucose-excited neurons of mouse arcuate nucleus.

Glucose is known to modify electrical activity of neurons in different hypothalamic areas such as the arcuate nucleus (ARC) or the ventromedian nucleus. In these structures, it has been demonstrated that glucose-induced excitation of neurons involves ATP-sensitive K(+) (K(ATP)) channel closure. The aim of the present study was to determine whether ARC neurons were able to detect high extracellular glucose concentrations and which mechanisms were involved in this detection by using whole-cell and cell-attached patch-clamp techniques in acute mouse brain slices. An increase from 5 to 20 mmol/l glucose stimulated 19% and inhibited 9% of ARC neurons. Because of the high-glucose concentrations used, we called these neurons high-glucose-excited (HGE) and high-glucose-inhibited (HGI) neurons, respectively. Glucose-induced depolarization of HGE neurons was not abolished by tetrodotoxin treatment and was correlated with an increase of membrane conductance that reversed at approximately 20 mV. Experiments with diazoxide, pinacidil, or tolbutamide showed that K(ATP) channels were present and functional in most of the ARC neurons but were mostly closed at 5 mmol/l glucose. Moreover, HGE neurons were also present in ARC of Kir6.2 null mice. These results suggested that ARC neurons have the ability to sense higher glucose concentrations than 5 mmol/l through a new K(ATP) channel-independent mechanism.

Hypothalamic eIF2α signaling regulates food intake.

The reversible phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) is a highly conserved signal implicated in the cellular adaptation to numerous stresses such as the one caused by amino acid limitation. In response to dietary amino acid deficiency, the brain-specific activation of the eIF2α kinase GCN2 leads to food intake inhibition. We report here that GCN2 is rapidly activated in the mediobasal hypothalamus (MBH) after consumption of a leucine-deficient diet. Furthermore, knockdown of GCN2 in this particular area shows that MBH GCN2 activity controls the onset of the aversive response. Importantly, pharmacological experiments demonstrate that the sole phosphorylation of eIF2α in the MBH is sufficient to regulate food intake. eIF2α signaling being at the crossroad of stress pathways activated in several pathological states, our study indicates that hypothalamic eIF2α phosphorylation could play a critical role in the onset of anorexia associated with certain diseases.

Fatty acid transporter CD36 mediates hypothalamic effect of fatty acids on food intake in rats.

Variations in plasma fatty acid (FA) concentrations are detected by FA sensing neurons in specific brain areas such as the hypothalamus. These neurons play a physiological role in the control of food intake and the regulation of hepatic glucose production. Le Foll et al. previously showed in vitro that at least 50% of the FA sensing in ventromedial hypothalamic (VMH) neurons is attributable to the interaction of long chain FA with FA translocase/CD36 (CD36). The present work assessed whether in vivo effects of hypothalamic FA sensing might be partly mediated by CD36 or intracellular events such as acylCoA synthesis or ß-oxidation. To that end, a catheter was implanted in the carotid artery toward the brain in male Wistar rats. After 1 wk recovery, animals were food-deprived for 5 h, then 10 min infusions of triglyceride emulsion, Intralipid +/- heparin (IL, IL(H), respectively) or saline/heparin (SH) were carried out and food intake was assessed over the next 5 h. Experimental groups included: 1) Rats previously injected in ventromedian nucleus (VMN) with shRNA against CD36 or scrambled RNA; 2) Etomoxir (CPT1 inhibitor) or saline co-infused with IL(H)/S(H); and 3) Triacsin C (acylCoA synthase inhibitor) or saline co-infused with IL(H)/S(H). IL(H) significantly lowered food intake during refeeding compared to S(H) (p<0.001). Five hours after refeeding, etomoxir did not affect this inhibitory effect of IL(H) on food intake while VMN CD36 depletion totally prevented it. Triacsin C also prevented IL(H) effects on food intake. In conclusion, the effect of FA to inhibit food intake is dependent on VMN CD36 and acylCoA synthesis but does not required FA oxidation.

A physiological increase of insulin in the olfactory bulb decreases detection of a learned aversive odor and abolishes food odor-induced sniffing behavior in rats.

Insulin is involved in multiple regulatory mechanisms, including body weight and food intake, and plays a critical role in metabolic disorders such as obesity and diabetes. An increasing body of evidence indicates that insulin is also involved in the modulation of olfactory function. The olfactory bulb (OB) contains the highest level of insulin and insulin receptors (IRs) in the brain. However, a role for insulin in odor detection and sniffing behavior remains to be elucidated. Using a behavioral paradigm based on conditioned olfactory aversion (COA) to isoamyl-acetate odor, we demonstrated that an intracerebroventricular (ICV) injection of 14 mU insulin acutely decreased olfactory detection of fasted rats to the level observed in satiated animals. In addition, whereas fasted animals demonstrated an increase in respiratory frequency upon food odor detection, this effect was absent in fasted animals receiving a 14 mU insulin ICV injection as well as in satiated animals. In parallel, we showed that the OB and plasma insulin levels were increased in satiated rats compared to fasted rats, and that a 14 mU insulin ICV injection elevated the OB insulin level of fasted rats to that of satiated rats. We further quantified insulin receptors (IRs) distribution and showed that IRs are preferentially expressed in the caudal and lateral parts of the main OB, with the highest labeling found in the mitral cells, the main OB projection neurons. Together, these data suggest that insulin acts on the OB network to modulate olfactory processing and demonstrate that olfactory function is under the control of signals involved in energy homeostasis regulation and feeding behaviors.