The role of somatosensory innervation of adipose tissues.

Adipose tissues communicate with the central nervous system to maintain whole-body energy homeostasis. The mainstream view is that circulating hormones secreted by the fat convey the metabolic state to the brain, which integrates peripheral information and regulates adipocyte function through noradrenergic sympathetic output1. Moreover, somatosensory neurons of the dorsal root ganglia innervate adipose tissue2. However, the lack of genetic tools to selectively target these neurons has limited understanding of their physiological importance. Here we developed viral, genetic and imaging strategies to manipulate sensory nerves in an organ-specific manner in mice. This enabled us to visualize the entire axonal projection of dorsal root ganglia from the soma to subcutaneous adipocytes, establishing the anatomical underpinnings of adipose sensory innervation. Functionally, selective sensory ablation in adipose tissue enhanced the lipogenic and thermogenetic transcriptional programs, resulting in an enlarged fat pad, enrichment of beige adipocytes and elevated body temperature under thermoneutral conditions. The sensory-ablation-induced phenotypes required intact sympathetic function. We postulate that beige-fat-innervating sensory neurons modulate adipocyte function by acting as a brake on the sympathetic system. These results reveal an important role of the innervation by dorsal root ganglia of adipose tissues, and could enable future studies to examine the role of sensory innervation of disparate interoceptive systems.

Early postnatal interactions between beige adipocytes and sympathetic neurites regulate innervation of subcutaneous fat.

While beige adipocytes have been found to associate with dense sympathetic neurites in mouse inguinal subcutaneous white fat (iWAT), little is known about when and how this patterning is established. Here, we applied whole-tissue imaging to examine the development of sympathetic innervation in iWAT. We found that parenchymal neurites actively grow between postnatal day 6 (P6) and P28, overlapping with early postnatal beige adipogenesis. Constitutive deletion of Prdm16 in adipocytes led to a significant reduction in early postnatal beige adipocytes and sympathetic density within this window. Using an inducible, adipocyte-specific Prdm16 knockout model, we found that Prdm16 is required for guiding sympathetic growth during early development. Deleting Prdm16 in adult animals, however, did not affect sympathetic structure in iWAT. Together, these findings highlight that beige adipocyte-sympathetic neurite communication is crucial to establish sympathetic structure during the early postnatal period but may be dispensable for its maintenance in mature animals.

Mammals have two types of fatty tissue: white fat that mainly stores energy, and brown and beige fat, also known as thermogenic fat, which burns energy to generate heat. In humans, brown fat is associated with potent anti-obesity and anti-diabetes effects. A better understanding of how this type of fat develops and functions could lead to therapeutic strategies to treat these conditions. Adult human brown fat is similar to rodent inducible brown fat, also known as beige fat. In adult mice, beige fat cells need stimulation from the environment to form. Cold can lead to the generation of beige fat cells by activating a part of the nervous system known as the sympathetic nervous system. In order for this cold-induced formation of beige fat cells to take place, nerves from the sympathetic nervous system must first innervate the fatty tissue. Beige fat cells themselves are important for establishing this innervation, but it was not well understood when and how this occurs. To study the role of beige fat cells in the establishment of nerve innervation, Chi et al. used genetically modified mice whose beige fat cells are removed when they are treated with an antibiotic called doxycycline. If mice that had not been genetically modified were treated with doxycycline, they developed beige fat cells soon after birth, and these cells shortly became densely innervated by the sympathetic nervous system. However, if the mutant mice were treated with doxycycline around birth, these mice could not make beige fat cells during the treatment and failed to develop dense innervation even when they grew older. These results showed that beige fat cells that form soon after birth are necessary to establish sympathetic nervous system innervation. But are beige fat cells required to maintain this innervation as the mice grow older? To test this, Chi et al. removed them after the innervation was fully established. These mice maintained their innervation, showing that beige fat cells appear to only be required during the establishment of innervation. Understanding how the sympathetic nervous system establishes its connection to fat so cold can stimulate beige fat formation is a first step to finding new treatments for conditions such as diabetes or obesity. Exploring the timing that underlies the interactions between the sympathetic nervous system and beige fat cells may provide therapeutic targets in this direction.

A leptin-BDNF pathway regulating sympathetic innervation of adipose tissue.

Mutations in the leptin gene (ob) result in a metabolic disorder that includes severe obesity1, and defects in thermogenesis2 and lipolysis3, both of which are adipose tissue functions regulated by the sympathetic nervous system. However, the basis of these sympathetic-associated abnormalities remains unclear. Furthermore, chronic leptin administration reverses these abnormalities in adipose tissue, but the underlying mechanism remains to be discovered. Here we report that ob/ob mice, as well as leptin-resistant diet-induced obese mice, show significant reductions of sympathetic innervation of subcutaneous white and brown adipose tissue. Chronic leptin treatment of ob/ob mice restores adipose tissue sympathetic innervation, which in turn is necessary to correct the associated functional defects. The effects of leptin on innervation are mediated via agouti-related peptide and pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus. Deletion of the gene encoding the leptin receptor in either population leads to reduced innervation in fat. These agouti-related peptide and pro-opiomelanocortin neurons act via brain-derived neurotropic factor-expressing neurons in the paraventricular nucleus of the hypothalamus (BDNFPVH). Deletion of BDNFPVH blunts the effects of leptin on innervation. These data show that leptin signalling regulates the plasticity of sympathetic architecture of adipose tissue via a top-down neural pathway that is crucial for energy homeostasis.

Neuropathy and neural plasticity in the subcutaneous white adipose depot.

The difficulty in obtaining as well as maintaining weight loss, together with the impairment of metabolic control in conditions like diabetes and cardiovascular disease, may represent pathological situations of inadequate neural communication between the brain and peripheral organs and tissues. Innervation of adipose tissues by peripheral nerves provides a means of communication between the master metabolic regulator in the brain (chiefly the hypothalamus), and energy-expending and energy-storing cells in the body (primarily adipocytes). Although chemical and surgical denervation studies have clearly demonstrated how crucial adipose tissue neural innervation is for maintaining proper metabolic health, we have uncovered that adipose tissue becomes neuropathic (ie: reduction in neurites) in various conditions of metabolic dysregulation. Here, utilizing both human and mouse adipose tissues, we present evidence of adipose tissue neuropathy, or loss of proper innervation, under pathophysiological conditions such as obesity, diabetes, and aging, all of which are concomitant with insult to the adipose organ as well as metabolic dysfunction. Neuropathy is indicated by loss of nerve fiber protein expression, reduction in synaptic markers, and lower neurotrophic factor expression in adipose tissue. Aging-related adipose neuropathy particularly results in loss of innervation around the tissue vasculature, which cannot be reversed by exercise. Together with indications of neuropathy in muscle and bone, these findings underscore that peripheral neuropathy is not restricted to classic tissues like the skin of distal extremities, and that loss of innervation to adipose may trigger or exacerbate metabolic diseases. In addition, we have demonstrated stimulation of adipose tissue neural plasticity with cold exposure, which may ameliorate adipose neuropathy and be a potential therapeutic option to re-innervate adipose and restore metabolic health.

Zic1 mRNA is transiently upregulated in subcutaneous fat of acutely cold-exposed mice.

In the mammalian adipose organ cold exposure not only activates typical brown adipose tissue, but also induces browning, that is the formation of thermogenic multilocular adipocytes in white, or predominantly white, adipose depots such as subcutaneous fat. Unlike typical brown adipocytes, newly formed thermogenic adipocytes have been reported not to express the gene zinc finger of the cerebellum 1 (Zic1). Here, a time course approach enabled us to document a significant increase in Zic1 messenger RNA in inguinal subcutaneous fat from acutely (24 hr) cold-exposed mice, which was paralleled by an increase in multilocular and paucilocular uncoupling protein 1-positive adipocytes and in parenchymal noradrenergic innervation. This transient, depot-specific molecular signature was associated not to Zic1 promoter demethylation, but to chromatin remodeling through an H3K9me3 histone modification. These findings challenge the notion that Zic1 is exclusively expressed by typical brown adipocytes and suggest its involvement in brown adipocyte precursor differentiation and/or white-to-brown adipocyte transdifferentiation.

Nerve detection during surgery: optical spectroscopy for peripheral nerve localization.

Precise nerve localization is of major importance in both surgery and regional anesthesia. Optically based techniques can identify tissue through differences in optical properties, like absorption and scattering. The aim of this study was to evaluate the potential of optical spectroscopy (diffuse reflectance spectroscopy) for clinical nerve identification in vivo. Eighteen patients (8 male, 10 female, age 53 ± 13 years) undergoing inguinal lymph node resection or resection or a soft tissue tumor in the groin were included to measure the femoral or sciatic nerve and the surrounding tissues. In vivo optical measurements were performed using Diffuse Reflectance Spectroscopy (400-1600 nm) on nerve, near nerve adipose tissue, muscle, and subcutaneous fat using a needle-shaped probe. Model-based analyses were used to derive verified quantitative parameters as concentrations of optical absorbers and several parameters describing scattering. A total of 628 optical spectra were recorded. Measured spectra reveal noticeable tissue specific characteristics. Optical absorption of water, fat, and oxy- and deoxyhemoglobin was manifested in the measured spectra. The parameters water and fat content showed significant differences (P < 0.005) between nerve and all surrounding tissues. Classification using k-Nearest Neighbor based on the derived parameters revealed a sensitivity of 85% and a specificity of 79%, for identifying nerve from surrounding tissues. Diffuse Reflectance Spectroscopy identifies peripheral nerve bundles. The differences found between tissue groups are assignable to the tissue composition and structure.

Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density.

While the cell-intrinsic pathways governing beige adipocyte development and phenotype have been increasingly delineated, comparatively little is known about how beige adipocytes interact with other cell types in fat. Here, we introduce a whole-tissue clearing method for adipose that permits immunolabeling and three-dimensional profiling of structures including thermogenic adipocytes and sympathetic innervation. We found that tissue architecture and sympathetic innervation differ significantly between subcutaneous and visceral depots. Subcutaneous fat demonstrates prominent regional variation in beige fat biogenesis with localization of UCP1+ beige adipocytes to areas with dense sympathetic neurites. We present evidence that the density of sympathetic projections is dependent on PRDM16 in adipocytes, providing another potential mechanism underlying the metabolic benefits mediated by PRDM16. This powerful imaging tool highlights the interaction of tissue components during beige fat biogenesis and reveals a previously undescribed mode of regulation of the sympathetic nervous system by adipocytes.

Differential sympathetic outflow to adipose depots is required for visceral fat loss in response to calorie restriction.

The sympathetic nervous system (SNS) regulates energy homeostasis in part by governing fatty acid liberation from adipose tissue. We first examined whether SNS activity toward discrete adipose depots changes in response to a weight loss diet in mice. We found that SNS activity toward each adipose depot is unique in timing, pattern of activation, and habituation with the most dramatic contrast between visceral and subcutaneous adipose depots. Sympathetic drive toward visceral epididymal adipose is more than doubled early in weight loss and then suppressed later in the diet when weight loss plateaued. Coincident with the decline in SNS activity toward visceral adipose is an increase in activity toward subcutaneous depots indicating a switch in lipolytic sources. In response to calorie restriction, SNS activity toward retroperitoneal and brown adipose depots is unaffected. Finally, pharmacological blockage of sympathetic activity on adipose tissue using the ß3-adrenergic receptor antagonist, SR59230a, suppressed loss of visceral adipose mass in response to diet. These findings indicate that SNS activity toward discrete adipose depots is dynamic and potentially hierarchical. This pattern of sympathetic activation is required for energy liberation and loss of adipose tissue in response to calorie-restricted diet.

Sympathetic nervous system activity and anti-lipolytic response to iv-glucose load in subcutaneous adipose tissue of obese and obese type 2 diabetic subjects.

The study aim was to investigate the effect of endogenous insulin release on lipolysis in subcutaneous adipose tissue after adrenergic stimulation in obese subjects diagnosed with type 2 diabetes (T2D). In 14 obese female T2D subjects, or 14 obese non-T2D controls, glycerol concentration was measured in response to the α1,2,ß-agonist norepinephrine, the α1-agonist norfenefrine and the ß2-agonist terbutaline (each 10-4 M), using the microdialysis technique. After 60 minutes of stimulation, an intravenous glucose load (0.5 g/kg lean body mass) was given. Local blood flow was monitored by means of the ethanol technique. Norepinephrine and norfenefrine induced a four and three fold rise in glycerol dialysate concentration (p<0.001, each), with a similar pattern in adipose tissue. Following agonist stimulation and glucose infusion, endogenous insulin release inhibited lipolysis in the presence of norepinephrine, which was more rapid and pronounced in healthy obese controls than in T2D subjects (p = 0.024 obese vs T2D subjects). Insulin-induced inhibition of lipolysis in the presence of norfenefrine was similar in all study participants. In the presence of terbutaline the lipolysis rate increased two fold until the effect of endogenous insulin (p<0.001). A similar insulin-induced decrease in lipolysis was observed for each of the norfenefrine groups and the terbutaline groups, respectively. Adipose tissue blood flow remained unchanged after the iv-glucose load. Both norepinephrine and norfenefrine diminished blood flow slightly, but insulin reversed this response (p<0.001 over the entire time). Terbutaline alone and terbutaline plus increased endogenous insulin augmented local blood flow (p<0.001 over the entire time). In conclusion, a difference in insulin-induced inhibition of lipolysis was observed in obese T2D subjects compared to obese healthy controls following modulation of sympathetic nervous system activity and is assumed to be due to ß1-adrenoceptor mediated stimulation by norepinephrine.

SMAS Fusion Zones Determine the Subfascial and Subcutaneous Anatomy of the Human Face: Fascial Spaces, Fat Compartments, and Models of Facial Aging.

BACKGROUND: Fusion zones between superficial fascia and deep fascia have been recognized by surgical anatomists since 1938. Anatomical dissection performed by the author suggested that additional superficial fascia fusion zones exist. OBJECTIVES: A study was performed to evaluate and define fusion zones between the superficial and the deep fascia. METHODS: Dissection of fresh and minimally preserved cadavers was performed using the accepted technique for defining anatomic spaces: dye injection combined with cross-sectional anatomical dissection. RESULTS: This study identified bilaminar membranes traveling from deep to superficial fascia at consistent locations in all specimens. These membranes exist as fusion zones between superficial and deep fascia, and are referred to as SMAS fusion zones. CONCLUSIONS: Nerves, blood vessels and lymphatics transition between the deep and superficial fascia of the face by traveling along and within these membranes, a construct that provides stability and minimizes shear. Bilaminar subfascial membranes continue into the subcutaneous tissues as unilaminar septa on their way to skin. This three-dimensional lattice of interlocking horizontal, vertical, and oblique membranes defines the anatomic boundaries of the fascial spaces as well as the deep and superficial fat compartments of the face. This information facilitates accurate volume augmentation; helps to avoid facial nerve injury; and provides the conceptual basis for understanding jowls as a manifestation of enlargement of the buccal space that occurs with age.

Central sympathetic innervations to visceral and subcutaneous white adipose tissue.

There is a link between visceral white adipose tissue (WAT) and the metabolic syndrome in humans, with health improvements produced with small visceral WAT reduction. By contrast, subcutaneous WAT provides a site for lipid storage that is rather innocuous relative to ectopic lipid storage in muscle or liver. The sympathetic nervous system (SNS) is the principal initiator for lipolysis in WAT by mammals. Nothing is known, however, about the central origins of the SNS circuitry innervating the only true visceral WAT in rodents, mesenteric WAT (MWAT), which drains into the hepatic portal vein. We tested whether the central sympathetic circuits to subcutaneous [inguinal WAT (IWAT)] and visceral WAT (MWAT) are separate or shared and whether they possess differential sympathetic drives with food deprivation in Siberian hamsters. Using two isogenic strains of pseudorabies virus, a retrograde transneuronal viral tract tracer within the same hamsters, we found some overlap (∼20-55% doubly infected neurons) between the two circuitries across the neural axis with lesser overlap proximal to the depots (spinal cord and sympathetic chain) and with more neurons involved in the innervation of IWAT than MWAT in some brain regions. Food deprivation triggered a greater sympathetic drive to subcutaneous (IWAT) than visceral (MWAT) depots. Collectively, we demonstrated both shared and separate populations of brain, spinal cord, and sympathetic chain neurons ultimately project to a subcutaneous WAT depot (IWAT) and the only visceral WAT depot in rodents (MWAT). In addition, the lipolytic stimulus of food deprivation only increased SNS drive to subcutaneous fat (IWAT).

Neurochemical characterization and sexual dimorphism of projections from the brain to abdominal and subcutaneous white adipose tissue in the rat.

Retroperitoneal white adipose tissue (rWAT) and subcutaneous (inguinal) white adipose tissue (iWAT) are both innervated and regulated by sympathetic efferents, but the distribution and identity of the cells in the brain that regulate sympathetic outflow are poorly characterized. Our aim was to use two isogenic strains of a neurotropic virus (pseudorabies, Bartha) tagged with either green or red fluorescent reporters to identify cells in the brain that project to rWAT and/or iWAT. These viruses were injected into separate WAT depots in male and female Sprague Dawley rats. Retrogradely labeled neurons in the CNS were characterized by immunohistochemistry and PCR. For the latter, laser capture of individual virally labeled neurons was used. All virally labeled brain regions contained neurons projecting to either and both WAT depots. Neurons to abdominal fat were the most abundant in males, whereas females contained a greater proportion of neurons to subcutaneous via private lines and collateral branches. Retrogradely labeled neurons directed to WAT expressed estrogen receptor-α (ERα), and fewer neurons to subcutaneous WAT expressed ERα in males. Regardless of sex, projections from the arcuate nucleus were predominantly from pro-opiomelanocortin cells, with a notable lack of projections from agouti-related protein-expressing neurons. Within the lateral hypothalamus, neurons directed to rWAT and iWAT expressed orexin and melanin-concentrating hormone (MCH), but male rats had a predominance of MCH directed to iWAT. In conclusion, the neurochemical substrates that project through polysynaptic pathways to iWAT and rWAT are different in male and female rats, suggesting that metabolic regulation of rWAT and iWAT is sexually dimorphic.

Vagus nerve preservation results in visceral fat maintenance after distal gastrectomy.

BACKGROUND/AIMS: Although preservation of the vaguas nerve is recommended in surgery for earlystage gastric cancer, the physiological effect of vagotomy on the postoperative course has not been well documented. We assessed the effect of vagotomy on the change in fat volume after gastrectomy. METHODOLOGY: Subcutaneous fat area (SFA) and visceral fat area (VFA) were separately measured in computed tomographic images taken before and more than 6 months after surgery, using Fat Scan software. The ratios of postoperative/ preoperative values of these two fat areas as well as body weight were calculated in 45 patients who underwent DG with (n=24) or without (n=21) vagotomy. RESULTS: Vagotomy did not affect the change in body weight (91.3±1.7% vs. 92.1±1.7%). In patients with vagotomy, VFA was reduced to 59.0±5.1%, which was significantly greater than the reduction in SFA (74.3±8.7%, p=0.042). In contrast, the reduction ratios of VFA and SFA were equal in vagus nerve-preserved patients (78.4±6.7% vs. 78.2±6.9%, p=0.97). CONCLUSIONS: The vagus nerve may have a function to locally regulate the intra-abdominal fat volume and preservation of the vagus nerve results in the maintenance of visceral fat after DG.

A pilot study on lipolytic effect of subcutaneous botulinum toxin injection in rabbits.

OBJECTIVE: To determine whether botulinum toxin type A (BTX-A) exerts a lipolytic effect by interfering with acetylcholine transmission at the cholinergic parasympathetic nerve endings. STUDY DESIGN: Fifteen male rabbits were divided into 3 equal groups: 1 control group (A) and 2 case groups (B and C). The abdomens of all rabbits were divided into a 3 x 3-square grid. The groups received 9 subcutaneous injections of 0.9% normal saline, 1 U BTX-A (group B) and 2 U BTX-A (group C), respectively. Four weeks later the entire grid was excised from the abdominal area. Hematoxylin-eosin-stained tissue was used for stereologic analysis to estimate cell surface and volume in 100 randomly selected cells. RESULTS: Gross thinning of subcutaneous fat and shattering and disappearance of fat globules were seen in both case groups. Fat cell volume was reduced by 65% in group B (p = 0.009) and 77% in group C (p = 0.009) compared to control animals. Fat cell surface also decreased by 51% in group B (p = 0.009) and 63% in group C rabbits (p = 0.009) compared to control animals. CONCLUSION: Our pilot animal study revealed a dose-dependent lipolytic effect of subcutaneous BTX-A injection.

Comparison of skin incision vs. skin plus deep tissue incision on ongoing pain and spontaneous activity in dorsal horn neurons.

Surgery injures both skin and deep tissue causing pain at rest and evoked pain with activities. In this study, we examined the extent of injury by incision and dorsal horn neuron (DHN) spontaneous activity (SA) in rats that underwent a sham operation, skin incision or skin plus deep tissue incision. Pain behaviors were measured 1 day later followed by DHN recordings in the same rats. On postoperative day (POD) 1, guarding pain, assessed with an abbreviated pain score, was increased in the skin plus deep tissue incision group (7.0+/-0.7 vs. 0.1+/-0.6 in control, P<0.001), but not in the skin incision group (1.8+/-1.0); yet, mechanical and heat hyperalgesia were similar in both incised groups. In the rats that underwent skin plus deep tissue incision, more DHNs expressed SA (78.1% vs. 35.7% in control, P<0.01) and SA rate also tended to be greater (13.8+/-2.9 vs. 5.6+/-2.0 imp/s). Bupivacaine infiltration into the incision decreased SA in both skin incision and skin plus deep tissue incision (POD1) groups to the same level as in the sham-operated rats. In a separate group of rats that underwent skin plus deep tissue incision, guarding pain was not present (0.1+/-0.6) on POD7 and the percentage and rate of DHN SA were the same as in the sham control. These data demonstrate that incised deep tissue rather than skin is critical for the development of guarding pain and increased SA of DHNs. Skin incision alone is sufficient for primary mechanical and heat hyperalgesia.

Increased lipolysis of subcutaneous abdominal adipose tissue and altered noradrenergic activity in patients with Cushing's syndrome: an in-vivo microdialysis study.

Cushing's syndrome is associated with typical central redistribution of adipose tissue. The aim of the study was to assess lipolysis and catecholamines and their metabolites in subcutaneous abdominal adipose tissue using an in-vivo microdialysis technique. Nine patients with Cushing's syndrome and nine age-, gender- and body mass index (BMI)-matched control subjects were included in the study. Local glycerol concentrations were significantly increased in subcutaneous adipose tissue of patients with Cushing's syndrome (p<0.001). Plasma noradrenaline, dihydroxyphenylglycol and dihydroxyphenylalanine were decreased in patients with Cushing's syndrome (p<0.02, p<0.05, and p<0.02, respectively). Adrenaline, noradrenaline, dihydroxyphenylglycol and dihydroxyphenylalanine concentrations in subcutaneous abdominal adipose were non-significantly higher in patients with Cushing's syndrome. In conclusion, we showed that lipolysis in subcutaneous adipose tissue of patients with Cushing's syndrome is significantly increased as compared to healthy subjects. This finding together with non-significantly increased local catecholamine concentrations in these patients suggests a possible link between increased lipolysis and catecholaminergic activity in subcutaneous adipose tissue.