Sensory or sympathetic white adipose tissue denervation differentially affects depot growth and cellularity

Functional and histological evidence for the sympathetic nervous system (SNS) innervation of white adipose tissue (WAT) exists for several species; however, its sensory innervation has only been shown in laboratory rats, and its function is unclear. We tested the effects of sensory and SNS innervation of Siberian hamster epididymal and inguinal WAT (EWAT and IWAT) by assessing calcitonin gene-related peptide (CGRP)- and tyrosine hydroxylase-immunoreactivity (ir), respectively. Next, we tested the role of the sensory innervation of WAT on growth and cellularity because WAT surgical denervation increases pad mass via selective increases in fat cell number, an effect ascribed to SNS denervation but that could be due to the accompanying surgical disruption of WAT sensory innervation. Sensory denervation was accomplished via multiple local microinjections of capsaicin into WAT, and its effects were compared with those of surgical denervation. Surgically denervated IWAT and EWAT showed significantly decreased tyrosine hydroxylase-ir and CGRP-ir, whereas capsaicin-treated WAT had only significantly decreased CGRP-ir. Surgically denervated pad masses were significantly increased; this was accompanied by increased total fat cell number in IWAT, with no change in fat cell size. EWAT only showed a significant increase in the number of small- to medium-sized adipocytes (75-125 mum diameter). By contrast, sensory-denervated pad masses were unchanged, but IWAT showed significantly increased average fat cell size. Collectively, these data provide immunohistochemical evidence for sensory and SNS innervation of WAT in Siberian hamsters and differential control of WAT cellularity by these innervations, as well as the ability of locally applied capsaicin to selectively reduce WAT sensory innervation.     

Ghrelin regulates adiposity in white adipose tissue and UCP1 mRNA expression in brown adipose tissue in mice

To examine the involvement of ghrelin in obesity, we investigated the effects of treatment with peripherally administered ghrelin on food intake, adiposity, and expression of uncoupling protein (UCP) mRNA in brown (BAT) and white (WAT) adipose tissue in mice. Acute bolus administration of ghrelin at a dose of 120 nmol/kg increased cumulative food intake over 4 and 24 h as compared to controls (p<0.05 for each), whereas 12 nmol/kg/day ghrelin showed no remarkable effect (p>0.1). Chronic repeated treatment with 12 nmol/kg/day ghrelin for 7 days increased body weight and adiposity assessed by the weight of adipose tissue, triglyceride content in WAT (p<0.05 for each versus control). In addition, the same treatment decreased and increased mRNA expression of BAT UCP1 and WAT UCP2, respectively (p<0.05 for each). In conclusion, ghrelin can regulate body weight, adiposity and UCPs mRNA expression in mice. The present results provide evidence for a new regulatory loop involving ghrelin and UCP, and add novel insights into the regulatory mechanisms of obesity.     

The Role of Thermogenic Fat Tissue in Energy Consumption

Mammalian adipose tissues are broadly divided into white adipose tissue (WAT) and thermogenic fat tissue (brown adipose tissue and beige adipose tissue). Uncoupling protein 1 (UCP1) is the central protein in thermogenesis, and cells that exhibit induced UCP1 expression and appear scattered throughout WAT are called beige adipocytes, and their induction in WAT is referred to as “beiging”. Beige adipocytes can differentiate from preadipocytes or convert from mature adipocytes. UCP1 was thought to contribute to non-shivering thermogenesis; however, recent studies demonstrated the presence of UCP1-independent thermogenic mechanisms. There is evidence that thermogenic fat tissue contributes to systemic energy expenditure even in human beings. This review discusses the roles that thermogenic fat tissue plays in energy consumption and offers insight into the possibility and challenges associated with its application in the treatment of obesity and type 2 diabetes.     

Brown and beige fat: From molecules to physiology

The recent re-discovery of brown adipose tissue (BAT) and even more recent discovery of the browning of white adipose tissue (WAT) in humans have generated intense scientific interest in the role of adipose tissue as potential target against obesity and its metabolic complications. The purpose of this review is to: i) critically evaluate the current evidence on the physiological significance of BAT and the browning of WAT in metabolic function in humans and ii) discuss factors that have been reported to regulate BAT and/or the browning of WAT in humans. The current literature supports that BAT and the browning of WAT constitute promising emerging targets for interventions aiming to prevent and/or treat of obesity and its metabolic complications. Further research is needed to better understand the physiological significance of BAT and browning of WAT in health and disease along with the factors modulating their metabolic function in humans.     

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep

Background  

The gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA).     

The potential autocrine/paracrine roles of ghrelin and its receptor in hormone-dependent cancer

Ghrelin is a recently identified 28 amino acid peptide capable of stimulating pituitary growth hormone release in humans. The actions of ghrelin are mediated via the naturally occurring ghrelin receptor, also known as the growth hormone secretagogue receptor (GHS-R). Ghrelin and its receptors are now being recognized as components of the growth hormone axis and are therefore potentially involved in tissue growth and development. As is the case for other members of this axis, evidence is rapidly emerging to indicate that ghrelin/GHS-R may play an important autocrine/paracrine role in some cancers. This review highlights the evidence for the expression, regulation and potential functional role of ghrelin and its receptor in hormone-dependent cancers, such as prostate and breast cancer.     

Genetic variants of ghrelin in metabolic disorders

An increasing understanding of the role of genes in the development of obesity may reveal genetic variants that, in combination with conventional risk factors, may help to predict an individual's risk for developing metabolic disorders. Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis and it is a reasonable candidate gene for obesity-related co-morbidities. In cross-sectional studies low total ghrelin concentrations and some genetic polymorphisms of ghrelin have been associated with obesity-associated diseases. The present review highlights many of the important problems in association studies of genetic variants and complex diseases. It is known that population-specific differences in reported associations exist. We therefore conclude that more studies on variants of ghrelin gene are needed to perform in different populations to get deeper understanding on the relationship of ghrelin gene and its variants to obesity.     

Higher ghrelin and lower leptin secretion are associated with lower LH secretion in young amenorrheic athletes compared with eumenorrheic athletes and controls

Amenorrhea is common in young athletes and is associated with low fat mass. However, hormonal factors that link decreased fat mass with altered gonadotropin pulsatility and amenorrhea are unclear. Low levels of leptin (an adipokine) and increased ghrelin (an orexigenic hormone that increases as fat mass decreases) impact gonadotropin pulsatility. Studies have not examined luteinizing hormone (LH) secretory dynamics in relation to leptin or ghrelin secretory dynamics in adolescent and young adult athletes. We hypothesized that 1) young amenorrheic athletes (AA) would have lower LH and leptin and higher ghrelin secretion than eumenorrheic athletes (EA) and nonathletes and 2) higher ghrelin and lower leptin would be associated with lower LH secretion. This was a cross-sectional study. We examined ghrelin and leptin secretory patterns (over 8 h, from 11 PM to 7 AM) in relation to LH secretory patterns in AA, EA, and nonathletes aged 14-21 yr. Ghrelin and leptin were assessed every 20 min and LH every 10 min. Groups did not differ for age, bone age, or BMI. However, fat mass was lower in AA than in EA and nonathletes. AA had lower LH and higher ghrelin pulsatile secretion and AUC than nonathletes and lower leptin pulsatile secretion and AUC than EA and nonathletes. Percent body fat was associated positively with LH and leptin secretion and inversely with ghrelin. In a regression model, ghrelin and leptin secretory parameters were associated independently with LH secretory parameters. We conclude that higher ghrelin and lower leptin secretion in AA related to lower fat mass may contribute to altered LH pulsatility and amenorrhea.     

Thematic review series: adipocyte biology. Sympathetic and sensory innervation of white adipose tissue

During our study of the reversal of seasonal obesity in Siberian hamsters, we found an interaction between receptors for the pineal hormone melatonin and the sympathetic nervous system (SNS) outflow from brain to white adipose tissue (WAT). This ultimately led us and others to conclude that the SNS innervation of WAT is the primary initiator of lipid mobilization in these as well as other animals, including humans. There is strong neurochemical (norepinephrine turnover), neuroanatomical (viral tract tracing), and functional (sympathetic denervation-induced blockade of lipolysis) evidence for the role of the SNS in lipid mobilization. Recent findings suggest the presence of WAT sensory innervation based on strong neuroanatomical (viral tract tracing, immunohistochemical markers of sensory nerves) and suggestive functional (capsaicin sensory denervation-induced WAT growth) evidence, the latter implying a role in conveying adiposity information to the brain. By contrast, parasympathetic nervous system innervation of WAT is characterized by largely negative neuroanatomical evidence (viral tract tracing, immunohistochemical and biochemical markers of parasympathetic nerves). Functional evidence (intraneural stimulation and in situ microdialysis) for the role of the SNS innervation in lipid mobilization in human WAT is convincing, with some controversy regarding the level of sympathetic nerve activity in human obesity.     

Partial Inhibition of Adipose Tissue Lipolysis Improves Glucose Metabolism and Insulin Sensitivity Without Alteration of Fat Mass

When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet–fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.     

Leucine deprivation stimulates fat loss via increasing CRH expression in the hypothalamus and activating the sympathetic nervous system

We previously showed that leucine deprivation decreases abdominal fat mass largely by increasing energy expenditure, as demonstrated by increased lipolysis in white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). The goal of the present study was to investigate the possible involvement of central nervous system (CNS) in this regulation and elucidate underlying molecular mechanisms. For this purpose, levels of genes and proteins related to lipolysis in WAT and UCP1 expression in BAT were analyzed in wild-type mice after intracerebroventricular administration of leucine or corticotrophin-releasing hormone antibodies, or in mice deleted for three β-adrenergic receptors, after being maintained on a leucine-deficient diet for 7 d. Here, we show that intracerebroventricular administration of leucine significantly attenuates abdominal fat loss and blocks activation of hormone sensitive lipase in WAT and induction of UCP1 in BAT in leucine-deprived mice. Furthermore, we provide evidence that leucine deprivation stimulates fat loss by increasing expression of corticotrophin-releasing hormone in the hypothalamus via activation of stimulatory G protein/cAMP/protein kinase A/cAMP response element-binding protein pathway. Finally, we show that the effect of leucine deprivation on fat loss is mediated by activation of the sympathetic nervous system. These results suggest that CNS plays an important role in regulating fat loss under leucine deprivation and thereby provide novel and important insights concerning the importance of CNS leucine in the regulation of energy homeostasis.     

Consummatory behavior and metabolic indicators after central ghrelin injections in rats

Ghrelin, an endogenous ligand for the growth-hormone-secretagogue receptor, is a 28-amino acid peptide with a post-translational acyl modification necessary for its activity. It has central nervous system actions that affect appetite, body mass and energy balance. An intracerebroventricular (ICV) injection protocol of sub-nanomolar doses of ghrelin, known to alter the morphology of ACTH and GH producing pituicytes and plasma levels of these hormones, was used to provide an overview of metabolic changes linked to energy metabolism. Variables measured were: food intake (FI), water intake (WI), fecal mass, urine volume, body weight (BW), retroperitoneal (RP) and epididymal (EPI) white adipose tissue (WAT), and changes in serum leptin, insulin, triglycerides, cholesterol, and glucose. Five injections of rat ghrelin or PBS (n = 8 per group) were given ICV every 24 h (1 μg/5 μL PBS) to adult male rats. Ghrelin had a positive and cumulative effect on FI, WI and BW (p < 0.05), but not feces mass or urine volume (p > 0.05). Centrally applied ghrelin clearly increased RP WAT (by 235%, p < 0.001), EPI WAT (by 85%, p < 0.05) and serum insulin levels (by 43%, p < 0.05), and decreased serum leptin levels (by 77%, p < 0.05) without (p > 0.05) evoking changes in blood triglyceride cholesterol, or glucose levels.

These data and the available literature clearly document that exposure of the brain of normal rats, over time, to sub-nanomolar doses of ghrelin results in metabolic dysregulation culminating in increased body mass, consummatory behavior, and lipid stores as well as changes in blood leptin/insulin levels. Thus, modulation of central ghrelin receptors may represent a pharmacological approach for controlling multiple factors involved in energy balance and obesity.     

Effects of white adipose tissue grafts on total body fat and cellularity are dependent on graft type and location

Surgical removal of body fat (lipectomy) triggers compensatory increases in nonexcised white adipose tissue (WAT), thus restoring adiposity levels in many species, including Siberian hamsters. In Siberian hamsters, when their lipectomized WAT is transplanted to another site (autologous grafts, no net change in body fat), healthy grafts result, but the lipectomy-induced compensatory increases in nonexcised WAT masses are exaggerated, an effect that apparently occurs only when the grafts contact intact WAT. When WAT is added to nonlipectomized hamsters to increase body fat, native WAT pads do not decrease. Thus WAT addition or removal-replacement does not induce compensatory WAT responses consistent with total body fat regulation as does WAT subtraction. Therefore, we tested whether the exaggerated response to lipectomy occurring with autologous WAT transplantation is dependent on graft site placement and whether the donor graft source [inguinal or epididymal WAT (IWAT, EWAT), sibling vs. nonsibling] affected body fat responses to WAT additions in nonlipectomized hamsters. Lipectomized hamsters received subcutaneous autologous EWAT grafts placed remotely from other WAT (ventrum) or in contact with intact WAT (dorsum), whereas intact hamsters received EWAT or IWAT grafts from sibling or nonsibling donors. The exaggerated response to lipectomy only occurred when grafts were in contact with intact WAT. EWAT, but not IWAT, additions to nonlipectomized siblings or nonsiblings increased native IWAT and retroperitoneal WAT mass but not EWAT mass compared with controls. Collectively, WAT transplantation to either lipectomized or nonlipectomized hamsters increased body fat contingent on graft contact with intact or native WAT.     

Altered desaturation and elongation of fatty acids in hormone-sensitive lipase null mice

Background

Hormone-sensitive lipase (HSL) is expressed predominantly in adipose tissue, where it plays an important role in catecholamine-stimulated hydrolysis of stored lipids, thus mobilizing fatty acids. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, retinyl esters and lipoidal esters. Despite its role in fatty acid mobilization, HSL null mice have been shown to be resistant to diet-induced obesity. The aim of this study was to define lipid profiles in plasma, white adipose tissue (WAT) and liver of HSL null mice, in order to better understand the role of this multifunctional enzyme.

Methodology/Principal Findings

This study used global and targeted lipidomics and expression profiling to reveal changed lipid profiles in WAT, liver and plasma as well as altered expression of desaturases and elongases in WAT and liver of HSL null mice on high fat diet. Decreased mRNA levels of stearoyl-CoA desaturase 1 and 2 in WAT were consistent with a lowered ratio of 16∶1n7/16∶0 and 18∶1n9/18∶0 in WAT and plasma. In WAT, increased ratio of 18∶0/16∶0 could be linked to elevated mRNA levels of the Elovl1 elongase.

Conclusions

This study illustrates the importance of HSL for normal lipid metabolism in response to a high fat diet. HSL deficiency greatly influences the expression of elongases and desaturases, resulting in altered lipid profiles in WAT, liver and plasma. Finally, altered proportions of palmitoleate, a recently-suggested lipokine, in tissue and plasma of HSL null mice, could be an important factor mediating and contributing to the changed lipid profile, and possibly also to the decreased insulin sensitivity seen in HSL null mice.     

Sympathetic innervation of white adipose tissue and its regulation of fat cell number

White adipose tissue (WAT) is innervated by the sympathetic nervous system (SNS), and the central origins of this innervation have been demonstrated for inguinal and epididymal WAT (iWAT and eWAT, respectively) using a viral transneuronal tract tracer, the pseudorabies virus (PRV). Although the more established role of this sympathetic innervation of WAT is as a major stimulator of lipid mobilization, this innervation also inhibits WAT fat cell number (FCN); thus, local denervation of WAT leads to marked increases in WAT mass and FCN. The purpose of this study was to extend our understanding of the SNS regulation of FCN using neuroanatomical and functional analyses. Therefore, we injected PRV into retroperitoneal WAT (rWAT) to compare the SNS outflow to this pad from what already is known for iWAT and eWAT. In addition, we tested the ability of local unilateral denervation of rWAT or iWAT to promote increases in WAT mass and FCN vs. their contralateral neurally intact counterparts. Although the overall pattern of innervation was more similar than different for rWAT vs. iWAT or eWAT, its SNS outflow appeared to involve more neurons in the suprachiasmatic and solitary tract nuclei. Denervation produced significant increases in WAT mass and FCN for both iWAT and rWAT, but FCN was increased significantly more in iWAT than in rWAT. These data suggest differences in origins of the sympathetic outflow to WAT and functional differences in the WAT SNS innervation that could contribute to the differential propensity for fat cell proliferation across WAT depots in vivo.     

Effects of triglycerides, obesity, and starvation on ghrelin transport across the blood-brain barrier

Human ghrelin is transported across the blood-brain barrier (BBB) of normal mice. Here, we studied the effects of triglycerides, obesity, and starvation in retired breeder mice maintained on a high fat diet, mice age-matched to the retired breeders but maintained on normal chow, and 8-week-old mice maintained on breeder chow. The rate of ghrelin transport across the BBB was studied by both the intravenous administration method of multiple-time regression analysis and by the brain perfusion method. We found that (1) obese, aged mice lost the ability to transport intravenously administered ghrelin across the BBB, resulting in an inverse relation between body weight and ghrelin BBB permeability; (2) serum triglycerides promoted transport of intravenously administered ghrelin across the BBB, whereas epinephrine had no effect; (3) fasting tended to promote ghrelin transport across the BBB as most readily shown in brain perfusion studies; (4) evidence suggested that a serum factor promoted ghrelin transport in 8-week-old mice. Overall, these results show that serum factors and physiological states influence the rate at which ghrelin is transported across the blood-brain barrier.     

The trochanteric fat pad

Technological developments based on the use of autologous white adipose tissue (WAT) attracted attention to minor fat depots as possible sources of adipose tissue. In plastic surgery, the trochanteric fatty pad is one of the most used WAT depots for its location and organoleptic characteristics that make it particularly suitable for reconstructive procedures. Despite its wide use in clinic, the structure of this depot has never been studied in detail and it is not known if structural differences exist among trochanteric fat and other subcutaneous WAT depots. The present study was performed on trochanteric fat pad with the aim to clarify the morphology of its adipocytes, stroma and microcirculation, with particular reference to the stem niches. Histological and ultrastructural studies showed that the main peculiar feature of the trochanteric fat concerns its stromal component, which appears less dense than in the other subcutaneous WATs studied. The intra-parenchymal collagen stroma is poor and the extracellular compartment shows large spaces, filled with electron-light material, in which isolated collagen bundles are present. The adipocytes are wrapped in weak and easily detachable collagen baskets. These connective sheaths are very thin compared to the sheaths in other subcutaneous WAT depots. The capillaries are covered by large, long and thin elements surrounded by an external lamina; these perivascular cells are poor in organelles and mainly contain poly-ribosomes. In conclusion, when compared to other WAT deposits, the trochanteric fatty pad shows structural peculiarities in its stroma and microcirculation suggesting a high regenerative potential. Resistance, dissociability, microvascular weft and high regenerative potential make the trochanteric fatty pad a privileged source for harvesting in autologous WAT-based regenerative procedures.     

Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues

Mitochondrial uncoupling protein 1 (UCP1) is usually expressed only in brown adipose tissue (BAT) and a key molecule for metabolic thermogenesis to avoid an excess of fat accumulation. However, there is little BAT in adult humans. Therefore, UCP1 expression in tissues other than BAT is expected to reduce abdominal fat. Here, we show reduction of abdominal white adipose tissue (WAT) weights in rats and mice by feeding lipids from edible seaweed, Undaria pinnatifida. Clear signals of UCP1 protein and mRNA were detected in WAT of mice fed the Undaria lipids, although there is little expression of UCP1 in WAT of mice fed control diet. The Undaria lipids mainly consisted of glycolipids and seaweed carotenoid, fucoxanthin. In the fucoxanthin-fed mice, WAT weight significantly decreased and UCP1 was clearly expressed in the WAT, while there was no difference in WAT weight and little expression of UCP1 in the glycolipids-fed mice. This result indicates that fucoxanthin upregulates the expression of UCP1 in WAT, which may contribute to reducing WAT weight.     

Scheduled feeding results in adipogenesis and increased acylated ghrelin

Ghrelin, known to stimulate adipogenesis, displays an endogenous secretory rhythmicity closely related to meal patterns. Therefore, a chronic imposed feeding schedule might induce modified ghrelin levels and consequently adiposity. Growing Wistar rats were schedule-fed by imposing a particular fixed feeding schedule of 3 meals/day without caloric restriction compared with total daily control intake. After 14 days, their body composition was measured by DEXA and compared with ad libitum-fed controls and to rats daily intraperitoneal injection with ghrelin. Feeding patterns, circadian activity, and pulsatile acylated ghrelin variations were monitored. After 14 days, rats on the imposed feeding schedule displayed, despite an equal daily calorie intake, a slower growth rate compared with ad libitum-fed controls. Moreover, schedule-fed rats exhibiting a feeding pattern with intermittent fasting periods had a higher fat/lean ratio compared with ad libitum-fed controls. Interestingly, ghrelin-treated rats also showed an increase in fat mass, but the fat/lean ratio was not significantly increased compared with controls. In the schedule-fed rats, spontaneous activity and acylated ghrelin levels were increased and associated with the scheduled meals, indicating anticipatory effects. Our results suggest that scheduled feeding, associated with intermittent fasting periods, even without nutrient/calorie restriction on a daily basis, results in adipogenesis. This repartitioning effect is associated with increased endogenous acylated ghrelin levels. This schedule-fed model points out the delicate role of meal frequency in adipogenesis and provides an investigative tool to clarify any effects of endogenous ghrelin without the need for ghrelin administration.