Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity

Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympathetic nervous system. This knockdown increased uncoupling protein 1 expression in both inguinal fat and interscapular brown adipose tissue (BAT). Consistent with the activation of BAT, DMH NPY knockdown increased energy expenditure and enhanced the thermogenic response to a cold environment. This knockdown also increased locomotor activity, improved glucose homeostasis, and enhanced insulin sensitivity. Together, these results demonstrate critical roles of DMH NPY in body weight regulation through affecting food intake, body adiposity, thermogenesis, energy expenditure, and physical activity.     

Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats

The thermogenic activity of brown adipose tissue (BAT) is heavily dependent on high perfusion, through its dense vascular system. Angiogenesis must go hand-in-hand with BAT functions, but little is known about the factors controlling it. In the present study we demonstrate that: (a) vascular endothelial growth factor (VEGF) is synthesised and released in brown adipocytes in culture; (b) VEGF mRNA isoforms and protein appear in dispersed mature brown adipocytes and whole tissue; (c) VEGF expression is increased in BAT from cold-exposed rats, and in cultured brown adipocytes exposed to noradrenaline and the β₃-adrenoceptor agonists; (e) BAT from genetically obese (fa/fa) rats exhibits reduced expression of VEGF as well as a change in the ratio of mRNA isoforms. It is concluded that sympathetic control of VEGF expression via noradrenaline acting on β₃-adrenoceptors plays a major role in developmental and adaptive angiogenesis, and defects in this contribute to the reduced thermogenic capacity of BAT in genetic obesity.     

Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue

Brown adipose tissue (BAT), a major site for mammalian non‐shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca²⁺‐permeable non‐selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to β‐adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca²⁺ concentrations in wild‐type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to β‐adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high‐fat‐diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy.     

Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells

Adipose tissue is a major metabolic organ, and it has been traditionally classified as either white adipose tissue(WAT) or brown adipose tissue(BAT). WAT and BAT are characterized by different anatomical locations, morphological structures, functions, and regulations. WAT and BAT are both involved in energy balance. WAT is mainly involved in the storage and mobilization of energy in the form of triglycerides, whereas BAT specializes in dissipating energy as heat during cold- or diet-induced thermogenesis. Recently, brownlike adipocytes were discovered in WAT. These brownlike adipocytes that appear in WAT are called beige or brite adipocytes. Interestingly, these beige/brite cells resemble white fat cells in the basal state, but they respond to thermogenic stimuli with increased levels of thermogenic genes and increased respiration rates. In addition, beige/brite cells have a gene expressionpattern distinct from that of either white or brown fat cells. The current epidemic of obesity has increased the interest in studying adipocyte formation(adipogenesis), especially in beige/brite cells. This review summarizes the developmental process of adipose tissues that originate from the mesenchymal stem cells and the features of these three different types of adipocytes.     

Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells简

Adipose tissue is a major metabolic organ, and it has been traditionally classified as either white adipose tissue (WAT) or brown adipose tissue (BAT). WAT and BAT are characterized by different anatomical locations, morphological structures, functions, and regulations. WAT and BAT are both involved in energy balance. WAT is mainly involved in the storage and mobilization of energy in the form of triglycerides, whereas BAT specializes in dissipating energy as heat during cold- or diet-induced thermogenesis. Recently, brown-like adipocytes were discovered in WAT. These brown-like adipocytes that appear in WAT are called beige or brite adipocytes. Interestingly, these beige/brite cells resemble white fat cells in the basal state, but they respond to thermogenic stimuli with increased levels of thermogenic genes and increased respiration rates. In addition, beige/brite cells have a gene expression pattern distinct from that of either white or brown fat cells. The current epidemic of obesity has increased the interest in studying adipocyte formation (adipogenesis), especially in beige/brite cells. This review summarizes the developmental process of adipose tissues that originate from the mesenchymal stem cells and the features of these three different types of adipocytes.     

Thermogenically competent nonadrenergic recruitment in brown preadipocytes by a PPARgamma agonist

Most physiologically induced examples of recruitment of brown adipose tissue (BAT) occur as a consequence of chronic sympathetic stimulation (norepinephrine release within the tissue). However, in some physiological contexts (e.g., prenatal and prehibernation recruitment), this pathway is functionally contraindicated. Thus a nonsympathetically mediated mechanism of BAT recruitment must exist. Here we have tested whether a PPARgamma activation pathway could competently recruit BAT, independently of sympathetic stimulation. We continuously treated primary cultures of mouse brown (pre)adipocytes with the potent peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist rosiglitazone. In rosiglitazone-treated cultures, morphological signs of adipose differentiation and expression levels of the general adipogenic marker aP2 were manifested much earlier than in control cultures. Importantly, in the presence of the PPARgamma agonist the brown adipocyte phenotype was significantly enhanced: UCP1 was expressed even in the absence of norepinephrine, and PPARalpha expression and norepinephrine-induced PGC-1alpha mRNA levels were significantly increased. However, the augmented levels of PPARalpha could not explain the brown-fat promoting effect of rosiglitazone, as this effect was still evident in PPARalpha-null cells. In continuously rosiglitazone-treated brown adipocytes, mitochondriogenesis, an essential part of BAT recruitment, was significantly enhanced. Most importantly, these mitochondria were capable of thermogenesis, as rosiglitazone-treated brown adipocytes responded to the addition of norepinephrine with a large increase in oxygen consumption. This thermogenic response was not observable in rosiglitazone-treated brown adipocytes originating from UCP1-ablated mice; hence, it was UCP1 dependent. Thus the PPARgamma pathway represents an alternative, potent, and fully competent mechanism for BAT recruitment, which may be the cellular explanation for the enigmatic recruitment in prehibernation and prenatal states.     

Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-fed mice and in clonal brown adipocytes

Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.     

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.     

The role of insulin in norepinephrine turnover and thermogenesis in brown adipose tissue after acute cold-exposure

The role of insulin in norepinephrine turnover (NE) and thermogenesis in brown adipose tissue (BAT) after acute cold-exposure was studied using streptozocin (STZ)-induced diabetic rats. NE turnover was estimated by the NE synthesis inhibition technique with alpha-methyl-p-tyrosine. BAT thermogenesis was estimated by measuring mitochondrial guanosine-5'-diphosphate (GDP), cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at an ambient temperature of 22 degrees C and during a six-hour cold-exposure at 4 degrees C. In insulin-deficient diabetic rats, the NE turnover, mitochondrial GDP binding, cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at 22 degrees C were significantly reduced, compared with those of control rats. Treatment of STZ-induced diabetic rats with insulin prevented a decrease in NE turnover and BAT thermogenesis. Acute cold-exposure increased the NE turnover of BAT in insulin-deficient diabetic rats. The BAT thermogenic response to acute cold-exposure, however, did not occur in insulin-deficient diabetic rats. These results suggest that insulin is not essential in potentiating NE turnover in BAT after acute cold-exposure, but is required for cold-induced thermogenesis.     

The application of stem cell therapy and brown adipose tissue transplantation in metabolic disorders

The discovery of brown fat in adult humans has led to increased research of the thermogenic function of this tissue in various metabolic diseases. In addition, high levels of brown fat have been correlated with lower body mass index values. Therefore, increasing brown fat mass and/or activity through methods such as the browning of white fat is considered a promising strategy to prevent and treat obesity-associated diseases. Cell-based approaches using mesenchymal stromal cells and brown adipose tissue (BAT) have been utilized to directly increase BAT mass/activity through cell and tissue implantation into animals. In addition, recent studies evaluating the transplantation of human embryonic stem cells and induced pluripotent stem (iPS) cells have shown promising results in terms of positive metabolic function. In this comprehensive review, we provide a summary of the research over the past 10 years with regard to stem cell therapy and brown fat tissue transplantation for the effective treatment of metabolic syndrome. Recent advancements in stem cell methods have allowed for the production of brown adipocytes from human iPS cells, which represent an unlimited source of cellular material with which to study adipocyte development. In addition, this process is expected to be used to further explore drug- and cell-based therapies to treat obesity-related metabolic complications.     

Trans-10, cis-12, but not cis-9, trans-11 CLA isomer,inhibits brown adipocyte thermogenic capacity

Conjugated linoleic acid (CLA) is reported to have health benefits, including reduction of body fat. Previous studies have shown that brown adipose tissue (BAT) is particularly sensitive to CLA-supplemented diet feeding. Most of them use mixtures containing several CLA isomers, mainly cis-9, trans-11 and trans-10, cis-12 in equal concentration. Our aim was to characterize the separate effects of both CLA isomers on thermogenic capacity in cultured brown adipocytes. The CLA isomers showed opposite effects. Hence, on the one hand, trans-10, cis-12 inhibited uncoupling protein (UCP) 1 induction by norepinephrine (NE) and produced a decrease in leptin mRNA levels. These effects were associated with a blockage of CCAAT-enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma(2) mRNA expression. On the other hand, cis-9, trans-11 enhanced the UCP1 elicited by NE, an effect reported earlier for polyunsaturated fatty acids and also observed here for linoleic acid. These findings could explain, at least in part, the effects observed in vivo when feeding a CLA mixture supplemented diet as a result of the combined action of CLA isomers (reduction of adipogenesis and defective BAT thermogenesis that could be through trans-10, cis-12 and enhanced UCP1 thermogenic capacity through cis-9, trans-11).     

Effect of the beta(3)-adrenergic agonist Cl316,243 on functional differentiation of white and brown adipocytes in primary cell culture

We investigated the effect of the specific beta(3)-adrenergic receptor agonist CL 316,243 (CL) on proliferation and functional differentiation of the Siberian hamster (Phodopus sungorus) white and brown preadipocytes in primary cell culture. Proliferation of both white and brown preadipocytes was stimulated by a general beta-adrenergic agonist (isoproterenol) but not by CL. Lipolysis of differentiated white and brown adipocytes was stimulated similarly by CL with maximum effect at 10 nM. Thermogenic properties of cells were assessed by immunodetection of UCP-1, the brown adipocyte specific uncoupling protein, and measurement of cytochrome c oxidase (COx) activity as an index of mitochondrial capacity. UCP-1 content was largely increased by CL in BAT but not in WAT cultures. Basal UCP-2 mRNA levels were similar in WAT and BAT cultures and increased by both CL and isoproterenol. COx activity of BAT cultures was twice as high as that of WAT cultures but in neither cell culture system could it be increased by beta-adrenergic stimulation. We suggest (i) that white and brown preadipocyte proliferation is increased in vitro via beta1 or beta(2), but not beta(3)-adrenergic pathways, (ii) that white and brown preadipocytes represent different cell types, and (iii) that in vitro beta-adrenergic stimulation it is not sufficient to induce complete thermogenic adaptation of brown adipocytes.     

Differentiation-dependent expression of cathepsin D and importance of lysosomal proteolysis in the degradation of UCP1 in brown adipocytes

The lysosomal protease cathepsin D increased markedly in brown adipocytes during differentiation in primary cultures. Differentiated cells had 20 times the amount of immunoreactive cathepsin D found in preadipocytes. Cathepsin D mRNA, as estimated by relative RT-PCR, was also present in higher amounts in differentiated brown fat cells. Cathepsin D expression was not influenced by repeated exposures of brown adipocytes to norepinephrine (NE). Cathepsin D levels were also unchanged when NE was withdrawn for 48 h after cells had been exposed to NE for 7 days. In contrast, exposure of the cells to NE for 7 days increased their UCP1 content by more than twofold, which returned to basal levels within 48 h of withholding NE. The half-life of UCP1 under basal conditions and in cells chronically exposed to NE was estimated from reductions in [35S]methionine-labelled immunoprecipitable UCP1 over 72 h. UCP1 t1/2 under basal conditions was 3.7+/-0.4 days, which was similar to the half-lives of labelled mitochondrial translation products (3.6+/-0.8 days). The turnover rates of both UCP1 and mitochondrial translation products were reduced by NE. The turnover rate of UCP1 in the presence or absence of NE cannot account solely for the rapid loss of UCP1 from brown adipocytes upon withdrawal of NE. This loss was reduced when cells were incubated with inhibitors of phosphatidylinositol 3-kinases (PI 3-kinase), previously shown to block formation of autophagic vacuoles. Thus, brown adipocytes acquire a large capacity for both uncoupled metabolism and for lysosomal proteolysis during differentiation. Withdrawal of NE, as often occurs in vivo from suppression of sympathetic nervous system activity, would not only terminate thermogenesis but also favor formation of autophagic vacuoles to rapidly reduce the cell content of UCP1-containing mitochondria.     

Argonaute-2 is associated to brown adipose tissue activation

MicroRNAs (miRNAs) are important modulators of thermogenic brown adipose tissue (BAT). They have been involved in its differentiation and hence its functioning. While different regulators of the miRNA machinery have been shown to be essential for BAT differentiation, little is known about their implication in BAT activation. The aim of this work was to evaluate the role of AGO2, the chief miRNA mediator, in BAT activation.We took advantage of two non-genetic models of BAT activation to analyze the miRNA machinery and miRNA expression in BAT. We used principal component analysis (PCA) to obtain an overview of miRNA expression according to the BAT activation state. In vitro, we examined AGO2 expression during brown adipocyte differentiation and activation. Finally, we downregulated AGO2 to reveal its potential role in the thermogenic function of brown adipocytes.PCA analysis allowed to cluster animals on their miRNA signature in active BAT. Moreover, hierarchical clustering showed a positive correlation between global upregulation of miRNA expression and active BAT. Consistently, the miRNA machinery, particularly AGO2, was upregulated in vivo in active BAT and in vitro in mature brown adipocytes. Finally, the partial loss-of-function of AGO2 in mature brown adipocytes is sufficient to lead to a diminished expression of UCP1 associated to a decreased uncoupled respiration.Therefore, our study shows the potential contribution of AGO2 in BAT activation. Since BAT is a calorie-burning tissue these data have a translational potential in terms of therapeutic target in the field of altered fuel homeostasis associated to obesity and diabetes.     

Hormone‐induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure

Adrenergic stimulation of brown adipocytes (BA) induces mitochondrial uncoupling, thereby increasing energy expenditure by shifting nutrient oxidation towards thermogenesis. Here we describe that mitochondrial dynamics is a physiological regulator of adrenergically‐induced changes in energy expenditure. The sympathetic neurotransmitter Norepinephrine (NE) induced complete and rapid mitochondrial fragmentation in BA, characterized by Drp1 phosphorylation and Opa1 cleavage. Mechanistically, NE‐mediated Drp1 phosphorylation was dependent on Protein Kinase‐A (PKA) activity, whereas Opa1 cleavage required mitochondrial depolarization mediated by FFAs released as a result of lipolysis. This change in mitochondrial architecture was observed both in primary cultures and brown adipose tissue from cold‐exposed mice. Mitochondrial uncoupling induced by NE in brown adipocytes was reduced by inhibition of mitochondrial fission through transient Drp1 DN overexpression. Furthermore, forced mitochondrial fragmentation in BA through Mfn2 knock down increased the capacity of exogenous FFAs to increase energy expenditure. These results suggest that, in addition to its ability to stimulate lipolysis, NE induces energy expenditure in BA by promoting mitochondrial fragmentation. Together these data reveal that adrenergically‐induced changes to mitochondrial dynamics are required for BA thermogenic activation and for the control of energy expenditure.     

Characteristics of diet-induced brown adipose tissue growth and thermogenesis in rats

The characteristics of regional brown (BAT) and white adipose tissue (WAT) growth and of thermogenesis following experimental overfeeding were studied in groups of male Sprague-Dawley rats fed lab chow or cafeteria diets for 8 weeks postweaning. Regional BAT and WAT growth was determined by dissection and weighing, and thermogenesis was characterized by measurements of resting and norepinephrine (NE)-stimulated oxygen consumption, of serum thyroid hormone concentrations, and of 24-hour urinary NE excretion levels. Cafeteria feeding resulted in a 113% increase in total BAT, with the most prominent increases in the interscapular, thoracic, and perirenal regions. Retroperitoneal, epididymal, and omental WAT were significantly greater in cafeteria than in chow-fed rats. Resting oxygen consumption of cafeteria-fed rads increased by 10% and NE excretion by 64% compared to chow-fed controls, while serum T₃ concentrations were nearly doubled in the cafeteria-fed rats. The thermogenic response to NE injection in cafeteria-fed rats was 102% of their resting levels, compared to a 51% increase in the chow-fed controls. The results indicate that increased BAT growth occurs in all primary BAT depots following cafeteria-feeding in rats, and that the greater BAT mass is qualitatively proportional to their greater capacity for non-shivering thermogenesis. Also, the increased NE excretion and greater serum T₃ concentration are consistent with increased sympathetic and thyroidal activity and may in part explain the thermogenic response to diet in the rat.     

Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP

A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented. Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP). Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation. The novel, atypical beta-adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor. Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression. In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation.