Shc depletion stimulates brown fat activity in vivo and in vitro

Adipose tissue is an important metabolic organ that integrates a wide array of homeostatic processes and is crucial for whole‐body insulin sensitivity and energy metabolism. Brown adipose tissue (BAT) is a key thermogenic tissue with a well‐established role in energy expenditure. BAT dissipates energy and protects against both hypothermia and obesity. Thus, BAT stimulation therapy is a rational strategy for the looming pandemic of obesity, whose consequences and comorbidities have a huge impact on the aged. Shc‐deficient mice (ShcKO) were previously shown to be lean, insulin sensitive, and resistant to high‐fat diet and obesity. We investigated the contribution of BAT to this phenotype. Insulin‐dependent BAT glucose uptake was higher in ShcKO mice. Primary ShcKO BAT cells exhibited increased mitochondrial respiration; increased expression of several mitochondrial and lipid‐oxidative enzymes was observed in ShcKO BAT. Levels of brown fat‐specific markers of differentiation, UCP1, PRDM16, ELOVL3, and Cox8b, were higher in ShcKO BAT. In vitro, Shc knockdown in BAT cell line increased insulin sensitivity and metabolic activity. In vivo, pharmacological stimulation of ShcKO BAT resulted in higher energy expenditure. Conversely, pharmacological inhibition of BAT abolished the improved metabolic parameters, that is the increased insulin sensitivity and glucose tolerance of ShcKO mice. Similarly, in vitro Shc knockdown in BAT cell lines increased their expression of UCP1 and metabolic activity. These data suggest increased BAT activity significantly contributes to the improved metabolic phenotype of ShcKO mice.     

Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.     

Human brown adipose tissue: Underestimated target in metabolic disease?

Active brown adipose tissue (BAT) has, since it rediscovery in adult humans in 2009, received much attention for its ability to increase energy expenditure when activated. By means of mitochondrial uncoupling activity BAT's main function is to produce heat instead of storing energy such as in white adipose tissue (WAT). Therefore, BAT is considered a new potential target to treat obesity and the metabolic syndrome. However, the contribution of this thermogenic tissue is still a matter of debate among researchers.The aim of this review is to give an overview of the differences between classical brown adipocytes and inducible beige adipocytes in humans, and the potential activators of BAT in humans. Furthermore newly described genetic markers for identification of these two types of brown adipocytes are examined. Finally, the potential of the current measurement techniques, and the contribution of BAT activity to whole body energy expenditure are discussed.     

Mitochondrial fission: firing up mitochondria in brown adipose tissue

Brown adipose tissue (BAT) is an important site for energy expenditure, for instance to generate heat in times of cold exposure. BAT expansion and activation can increase energy dissipation of an organism. This involves the coordinated activation of mitochondrial metabolism and heat generation through uncoupling of oxidative phosphorylation. In this issue of The EMBO Journal, the Shirihai group uncovers a novel potentiation pathway for BAT energy expenditure. Changes in mitochondrial dynamics, in particular mitochondrial fission, act in synergy with fatty acid‐induced uncoupling to activate BAT metabolism in response to the hormone norepinephrine.     

N-butylidenephthalide ameliorates high-fat diet-induced obesity in mice and promotes browning through adrenergic response/AMPK activation in mouse beige adipocytes

Thermogenesis (non-exercise activity) in brown adipose tissue (BAT) promotes energy expenditure because of its higher number of mitochondria than white adipose tissue (WAT). The main function of thermogenesis in BAT can counteract obesity through the dissipation of calories as heat. N-butylidenephthalide (BP) is a natural derivative from Angelica sinensis, a Chinese herb that has been used for thousands of years. In this report, we demonstrated that BP improved the metabolic profiles of mice with high fat diet-induced obesity (DIO) by preventing weight gain, improving serum blood parameters, enhancing energy expenditure, stimulating white fat browning, and reversing hepatic steatosis. Further investigations demonstrated that BP administration upregulated the mRNA expression of beige (CD137, TMEM26) and brown fat selected genes (UCP1, PRDM16, PGC-1α, PPARγ) in white adipose tissues. In vitro studies, BP treatment increased multilocular lipid droplet levels, induced β-adrenergic receptor (cAMP/PKA) and AMP-activated protein kinase (AMPK) signaling (AMPK/acetyl-CoA carboxylase/SIRT1), and increased oxygen consumption in murine differentiated beige adipocytes, and the effects of BP were blocked by an AMPK inhibitor. BP promoted the interaction of AMPK with PGC-1α in beige adipocytes. Our findings provide novel insights into the application of BP in regulating energy metabolism and suggest its utility for clinical use in the treatment of obesity and related diseases.     

Trib1 deficiency causes brown adipose respiratory chain depletion and mitochondrial disorder

Tribbles homolog 1 (TRIB1) belongs to the Tribbles family of pseudokinases, which plays a key role in tumorigenesis and inflammation. Although genome-wide analysis shows that TRIB1 expression is highly correlated with blood lipid levels, the relationship between TRIB1 and adipose tissue metabolism remains unclear. Accordingly, the aim of the present study was to explore the role of TRIB1 on mitochondrial function in the brown adipose tissue (BAT). Trib1-knockout mice were established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. The metabolic function of the BAT was induced by a β3-adrenoceptor agonist and the energy metabolism function of mitochondria in the BAT of mice was evaluated. Trib1-knockout mice exhibited obesity and impaired BAT thermogenesis. In particular, Trib1 knockout reduced the ability of the BAT to maintain body temperature, inhibited β3-adrenoceptor agonist-induced thermogenesis, and accelerated lipid accumulation in the liver and adipose tissues. In addition, Trib1 knockout reduced mitochondrial respiratory chain complex III activity, produced an imbalance between mitochondrial fusion and fission, caused mitochondrial structural damage and dysfunction, and affected heat production and lipid metabolism in the BAT. Conversely, overexpression of Trib1 in 3T3-L1 adipocytes increased the number of mitochondria and improved respiratory function. These findings support the role of Trib1 in regulating the mitochondrial respiratory chain and mitochondrial dynamics by affecting mitochondrial function and thermogenesis in the BAT.Subject terms: Energy metabolism, Obesity     

Inhibition of in vitro and in vivo brown fat differentiation program by myostatin

Objective: Obesity arises mainly due to the imbalance between energy storage and its expenditure. Metabolically active brown adipose tissue (BAT) has recently been detected in humans and has been proposed as a new target for anti‐obesity therapy because of its unique capacity to regulate energy expenditure. Myostatin (Mst), a negative regulator of muscle mass, has been identified as a potential target to regulate overall body composition. Although the beneficial effects of Mst inhibition on muscle mass are well known, its role in the regulation of lipid metabolism, and energy expenditure is not very clear. Design and Methods: We tested the effects of Mst inhibition on the gene regulatory networks that control BAT differentiation using both in vivo and in vitro model systems. PRDM16 and UCP1, two key regulators of brown fat differentiation were significantly up regulated in levator‐ani (LA) and gastrocnemius (Gastroc) muscles as well as in epididymal (Epi) and subcutaneous (SC) fat pads isolated from Mst knock out (Mst KO) male mice compared with wild type (WT) mice. Results: Using mouse embryonic fibroblast (MEFs) primary cultures obtained from Mst KO group compared to the WT group undergoing adipogenic differentiation, we also demonstrate a significant increase in select genes and proteins that improve lipid metabolism and energy expenditure. Conclusion: Treatment of Mst KO MEFs with recombinant Mst protein significantly inhibited the gene expression levels of UCP1, PRDM16, PGC1‐α/β as well as BMP7. Future studies to extend these findings and explore the therapeutic potential of Mst inhibition on metabolic disorders are warranted.     

Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism

Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.     

3,5-Diiodo-L-Thyronine Activates Brown Adipose Tissue Thermogenesis in Hypothyroid Rats

3,5-diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.     

Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase

Strategies to prevent and treat obesity aim to decrease energy intake and/or increase energy expenditure. Regarding the increase of energy expenditure, two key intracellular targets may be considered (1) mitochondrial oxidative phosphorylation, the major site of ATP production, and (2) AMP-activated protein kinase (AMPK), the master regulator of cellular energy homeostasis. Experiments performed mainly in transgenic mice revealed a possibility to ameliorate obesity and associated disorders by mitochondrial uncoupling in metabolically relevant tissues, especially in white adipose tissue (WAT), skeletal muscle (SM), and liver. Thus, ectopic expression of brown fat-specific mitochondrial uncoupling protein 1 (UCP1) elicited major metabolic effects both at the cellular/tissue level and at the whole-body level. In addition to expected increases in energy expenditure, surprisingly complex phenotypic effects were detected. The consequences of mitochondrial uncoupling in WAT and SM are not identical, showing robust and stable obesity resistance accompanied by improvement of lipid metabolism in the case of ectopic UCP1 in WAT, while preservation of insulin sensitivity in the context of high-fat feeding represents the major outcome of muscle UCP1 expression. These complex responses could be largely explained by tissue-specific activation of AMPK, triggered by a depression of cellular energy charge. Experimental data support the idea that (1) while being always activated in response to mitochondrial uncoupling and compromised intracellular energy status in general, AMPK could augment energy expenditure and mediate local as well as whole-body effects; and (2) activation of AMPK alone does not lead to induction of energy expenditure and weight reduction.     

Regulatory networks of non-coding RNAs in brown/beige adipogenesis

BAT (brown adipose tissue) is specialized to burn fatty acids for heat generation and energy expenditure to defend against cold and obesity. Accumulating studies have demonstrated that manipulation of BAT activity through various strategies can regulate metabolic homoeostasis and lead to a healthy phenotype. Two classes of ncRNA (non-coding RNA), miRNA and lncRNA (long non-coding RNA), play crucial roles in gene regulation during tissue development and remodelling. In the present review, we summarize recent findings on regulatory role of distinct ncRNAs in brown/beige adipocytes, and discuss how these ncRNA regulatory networks contribute to brown/beige fat development, differentiation and function. We suggest that targeting ncRNAs could be an attractive approach to enhance BAT activity for protecting the body against obesity and its pathological consequences.     

Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling

Combined fatty acid esterification and lipolysis, termed lipid cycling, is an ATP‐consuming process that contributes to energy expenditure. Therefore, interventions that stimulate energy expenditure through lipid cycling are of great interest. Here we find that pharmacological and genetic inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes activates lipid cycling and energy expenditure, even in the absence of adrenergic stimulation. We show that the resulting increase in ATP demand elevates mitochondrial respiration coupled to ATP synthesis and fueled by lipid oxidation. We identify that glutamine consumption and the Malate‐Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). We thus demonstrate that energy expenditure through enhanced lipid cycling can be activated in brown adipocytes by decreasing mitochondrial pyruvate availability. We present a new mechanism to increase energy expenditure and fat oxidation in brown adipocytes, which does not require adrenergic stimulation of mitochondrial uncoupling.     

Tissue-specific functional interaction between apolipoproteins A1 and E in cold-induced adipose organ mitochondrial energy metabolism

White (WAT) and brown (BAT) adipose tissue, the two main types of adipose organ, are responsible for lipid storage and non-shivering thermogenesis, respectively. Thermogenesis is a process mediated by mitochondrial uncoupling protein 1 (UCP1) which uncouples oxidative phosphorylation from ATP production, leading to the conversion of free fatty acids to heat. This process can be triggered by exposure to low ambient temperatures, caloric excess, and the immune system. Recently mitochondrial thermogenesis has also been associated with plasma lipoprotein transport system. Specifically, apolipoprotein (APO) E3 is shown to have a bimodal effect on WAT thermogenesis that is highly dependent on its site of expression. Similarly, APOE2 and APOE4 differentially affect BAT and WAT mitochondrial metabolic activity in processes highly modulated by APOA1. Furthermore, the absence of classical APOA1 containing HDL (APOA1-HDL), is associated with no measurable non-shivering thermogenesis in WAT of mice fed high fat diet. Based on these previous observations which indicate important regulatory roles for both APOA1 and APOE in adipose tissue mitochondrial metabolic activity, here we sought to investigate the potential roles of these apolipoproteins in BAT and WAT metabolic activation in mice, following stimulation by cold exposure (7 °C). Our data indicate that APOA1-HDL promotes metabolic activation of BAT only in the presence of very low levels (virtually undetectable) of APOE3-containing HDL (APOE3-HDL), which acts as an inhibitor in this process. In contrast, induction of WAT thermogenesis is subjected to a more complicated regulation which requires the combined presence of both APOA1-HDL and APOE3-HDL.     

Oleoyl-oestrone inhibits lipogenic, but maintains thermogenic, gene expression of brown adipose tissue in overweight rats

In the present study we intended to determine how BAT (brown adipose tissue) maintained thermogenesis under treatment with OE (oleoyl-oestrone), a powerful slimming hormone that sheds off body lipid but maintains the metabolic rate. Overweight male rats were subjected to daily gavages of 10 nmol/g of OE or vehicle (control) for 10 days. A PF (pair-fed) vehicle-receiving group was used to discount the effects attributable to energy availability limitation. Interscapular BAT mass, lipid, DNA, mRNA and the RT-PCR (real-time PCR) expression of lipid and energy metabolism genes for enzymes and regulatory proteins were measured. BAT mass and lipid were decreased in OE and PF, with the latter showing a marked reduction in tissue mRNA. Maintenance of perilipin gene expression in PF and OE rats despite the loss of lipid suggests the preservation of the vacuolar interactive surface, a critical factor for thermogenic responsiveness. OE and, to a lesser extent, PF maintained the expression of genes controlling lipolysis and fatty acid oxidation, but markedly decreased the expression of those genes involved in lipogenic and acyl-glycerol synthesis. OE did not affect UCP1 (uncoupling protein 1) (decreased in PF), beta(3) adrenergic receptors or hormone-sensitive lipase gene mRNAs, which may translate in maintaining a full thermogenic system potential. OE rats were able to maintain a less energetically stressed BAT (probably through glucose utilization) than PF rats. These changes were not paralleled in PF rats, in which lower thermogenesis and glucose preservation resulted in a heavier toll on internal fat stores. Thus the mechanism of action of OE is more complex and tissue-specific than previously assumed.     

Neuregulin 4: A “Hotline” Between Brown Fat and Liver

The discovery that functional brown adipose tissue (BAT) in adult humans is inversely related to body fat mass and may reflect metabolic health has stimulated adipose tissue research to explore activation of BAT as a potential target for antiobesity treatments. In addition to the capacity of BAT to increase energy expenditure and glucose and lipid uptake, BAT secretes factors that may contribute to the regulation of whole‐body metabolism. Among signals released from BAT, neuregulin 4 (NRG4) has been recently identified as an endocrine factor that may link the activation of BAT to protection against diet‐induced obesity, insulin resistance, and hepatic steatosis. NRG4 was shown to directly reduce lipogenesis in hepatocytes, and it could indirectly activate BAT via sympathetic neurons or via inducing brown adipocyte–like signatures in white adipocytes in a paracrine manner. However, the potential relevance of NRG4 as a diagnostic tool or target for the treatment of obesity‐related diseases remains to be explored.     

Acute central infusion of leptin modulates fatty acid mobilization by affecting lipolysis and mRNA expression for uncoupling proteins

Chronic administration of leptin has been shown to reduce adiposity through energy intake and expenditure. The present study aims to examine how acute central infusion of leptin regulates peripheral lipid metabolism, as assessed by markers indicative of their mobilization and utilization. A bolus infusion of 1 microg/rat leptin into the third cerebroventricle increased the expression of mRNA for hormone-sensitive lipase (HSL), an indicator of lipolysis, in white adipose tissue (WAT). This was accompanied by elevation of plasma levels of glycerol, but not of free fatty acids, as compared to the saline control (P < 0.03). The same treatment with leptin decreased plasma insulin levels but did not affect the plasma glucose level (P < 0.05 for insulin). Among the major regulators of the transportation or utilization of energy substrates, leptin treatment increased expression of mRNA for uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), UCP2 in WAT, and UCP3 in quadriceps skeletal muscle, but not those for fatty acid-binding protein in WAT, carnitine phosphate transferase-1, a marker for beta oxidation of fatty acids in muscle, nor glucose transporter 4 in WAT and muscle (P < 0.01 for HSL, P < 0.05 for UCP1, and P < 0.005 for UCP2 and UCP3). These results indicate that, even in a single bolus, leptin may regulate the mobilization and/or utilization of energy substrates such as fatty acids by affecting lipolytic activity in WAT and by increasing the expression of UCPs in BAT, WAT, and muscle.