Thermogenic Ability of Uncoupling Protein 1 in Beige Adipocytes in Mice

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24°C (control group) or 10°C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes.     

Brite/beige fat and UCP1 — is it thermogenesis?

The presence of two distinct types of adipose tissue, which have opposing functions, has been known for decades. White adipose tissue (WAT) is the main tissue of energy storage, while brown adipose tissue (BAT) dissipates energy as heat and is required for non-shivering thermoregulation. In the last few years, a third type of adipocyte was identified, termed the brite (“brown and white”) or beige adipocyte. Their physiological control and role, however, are not fully clarified. Brite/beige adipocytes have a positive impact on systemic metabolism that is generally explained by the thermogenesis of brite/beige adipocytes; although thermogenesis has not been directly measured but is mostly inferred by gene expression data of typical thermogenic genes such as uncoupling protein 1 (UCP1). Here we critically review functional evidence for the thermogenic potential of brite/beige adipocytes, leading to the conclusion that direct measurements of brite/beige adipocyte bioenergetics, beyond gene regulation, are pivotal to quantify their thermogenic potential. In particular, we exemplified that the massive induction of UCP1 mRNA during the browning of isolated subcutaneous adipocytes in vitro is not reflected in significant alterations of cellular bioenergetics. Herein, we demonstrate that increases in mitochondrial respiration in response to beta-adrenergic stimulus can be independent of UCP1. Using HEK293 cells expressing UCP1, we show how to directly assess UCP1 function by adequate activation in intact cells. Finally, we provide a guide on the interpretation of UCP1 activity and the pitfalls by solely using respiration measurements. The functional analysis of beige adipocyte bioenergetics will assist to delineate the impact of browning on thermogenesis, possibly elucidating additional physiological roles and its contribution to systemic metabolism, highlighting possible avenues for future research. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Taking control over intracellular fatty acid levels is essential for the analysis of thermogenic function in cultured primary brown and brite/beige adipocytes

Thermogenesis in brown adipocytes, conferred by mitochondrial uncoupling protein 1 (UCP1), is receiving great attention because metabolically active brown adipose tissue may protect humans from metabolic diseases. In particular, the thermogenic function of brown‐like adipocytes in white adipose tissue, known as brite (or beige) adipocytes, is currently of prime interest. A valid procedure to quantify the specific contribution of UCP1 to thermogenesis is thus of vital importance. Adrenergic stimulation of lipolysis is a common way to activate UCP1. We here report, however, that in this frequently applied setup, taking control over intracellular fatty acid levels is essential for the analysis of thermogenic function in cultured brown and brite adipocytes. By the application of these findings, we demonstrate that UCP1 is functionally thermogenic in intact brite adipocytes and adrenergic UCP1 activation is largely dependent on adipose triglyceride lipase (ATGL) rather than hormone sensitive lipase (HSL).     

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.     

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.     

UCP1 mRNA does not produce heat

Because of the possible role of brown adipose tissue and UCP1 in metabolic regulation, even in adult humans, there is presently considerable interest in quantifying, from in-vitro data, the thermogenic capacities of brown and brite/beige adipose tissues. An important issue is therefore to establish which parameters are the most adequate for this. A particularly important issue is the relevance of UCP1 mRNA levels as estimates of the degree of recruitment and of the thermogenic capacity resulting from differences in physiological conditions and from experimental manipulations. By solely following UCP1 mRNA levels in brown adipose tissue, the conclusion would be made that the tissue's highest activation occurs after only 6 h in the cold and then successively decreases to being only some 50% elevated after 1 month in the cold. However, measurement of total UCP1 protein levels per depot ("mouse") reveals that the maximal thermogenic capacity estimated in this way is reached first after 1 month but represents an approx. 10-fold increase in thermogenic capacity. Since this in-vitro measure correlates quantitatively and temporally with the acquisition of nonshivering thermogenesis, this must be considered the most physiologically relevant parameter. Similarly, observations that cold acclimation barely increases UCP1 mRNA levels in classical brown adipose tissue but leads to a 200-fold increase in UCP1 mRNA levels in brite/beige adipose tissue depots may overemphasise the physiological significance of these depots, as the high fold-increases are due to very low initial levels, and the UCP1 mRNA levels reached are at least an order of magnitude lower than in brown adipose tissue; furthermore, based on total UCP1 protein amounts, the brite/beige depots attain only about 10% of the thermogenic capacity of the classical brown adipose tissue depots. Consequently, inadequate conclusions may be reached if UCP1 mRNA levels are used as a proxy for the metabolic significance of recruited versus non-recruited brown adipose tissue and for estimating the metabolic significance of brown versus brite/beige adipose tissues. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

棕色和米色脂肪激活剂: 潜在的减肥靶标

全球性肥胖症及其代谢疾病已经严重影响人类健康。因此,对其进行治疗变得愈加重要。新近研究表明,激活棕色和米色脂肪可能成为对抗肥胖的有效途径。白色脂肪棕色化可使储存能量的白色脂肪转化为具有类似棕色脂肪产热特性的米色脂肪,来增加耗能,对抗肥胖。本文综述了棕色和米色脂肪激活剂及其作用机制的研究进展,并从纳米技术的角度展望了其在肥胖症治疗中的应用前景。     

Inducible beige adipocytes improve impaired glucose metabolism in interscapular BAT-removal mice

Inducible beige adipocytes are emerging as an interesting issue in obesity and metabolism research. There is a neglected possibility that brown adipocytes are equally activated when external stimuli induce the formation of beige adipocytes. Thus, the question is whether beige adipocytes have the same functions as brown adipocytes when brown adipose tissue (BAT) is lacking. This question has not been well studied. Therefore we determine the beneficial effects of beige adipocytes upon cold challenge or CL316243 treatments in animal models of interscapular BAT (iBAT) ablation by surgical denervation. We found that denervated iBAT were activated by cold exposure and CL316243 treatments. The data show that beige adipocytes partly contribute to the improvement of impaired glucose metabolism resulting from denervated iBAT. Thus, we further used iBAT-removal animal models to abolish iBAT functions completely. We found that beige adipocytes upon cold exposure or CL316243 treatments improved impaired glucose metabolism and enhanced glucose uptake in iBAT-removal mice. The insulin signaling was activated in iBAT-removal mice upon cold exposure. Both the activation of insulin signaling and up-regulation of glucose transporter expression were observed in iBAT-removal mice with CL316243 treatments. The data show that inducible beige adipocytes may have different mechanisms to improve impaired glucose metabolism. Inducible beige adipocytes can also enhance energy expenditure and lipolytic activity of white adipose tissues when iBAT is lacking. We provide direct evidences for the beneficial effect of inducible beige adipocytes in glucose metabolism and energy expenditure in the absence of iBAT in vivo.     

PEX13 is required for thermogenesis of white adipose tissue in cold-exposed mice

Non-shivering thermogenesis (NST) is a heat generating process controlled by the mitochondria of brown adipose tissue (BAT). In the recent decade, ‘functionally’ acting brown adipocytes in white adipose tissue (WAT) has been identified as well: the so-called process of the ‘browning’ of WAT. While the importance of uncoupling protein 1 (UCP1)-oriented mitochondrial activation has been intensely studied, the role of peroxisomes during the browning of white adipocytes is poorly understood. Here, we assess the change in peroxisomal membrane proteins, or peroxins (PEXs), during cold stimulation and importantly, the role of PEX13 in the cold-induced remodeling of white adipocytes. PEX13, a protein that originally functions as a docking factor and is involved in protein import into peroxisome matrix, was highly increased during cold-induced recruitment of beige adipocytes within the inguinal WAT of C57BL/6 mice. Moreover, beige-induced 3 T3-L1 adipocytes and stromal vascular fraction (SVF) cells by exposure to the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone showed a significant increase in mitochondrial thermogenic factors along with peroxisomal proteins including PEX13, and these were confirmed in SVF cells with the beta 3 adrenergic receptor (β3AR)-selective agonist CL316,243. To verify the relevance of PEX13, we used the RNA silencing method targeting the Pex13 gene and evaluated the subsequent beige development in SVF cells. Interestingly, siPex13 treatment suppressed expression of thermogenic proteins such as UCP1 and PPARγ coactivator 1 alpha (PGC1α). Overall, our data provide evidence supporting the role of peroxisomal proteins, in particular PEX13, during beige remodeling of white adipocytes.     

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.     

Establishing the potency of N-acyl amino acids versus conventional fatty acids as thermogenic uncouplers in cells and mitochondria from different tissues

The possibility that N-acyl amino acids could function as brown or brite/beige adipose tissue-derived lipokines that could induce UCP1-independent thermogenesis by uncoupling mitochondrial respiration in several peripheral tissues is of significant physiological interest. To quantify the potency of N-acyl amino acids versus conventional fatty acids as thermogenic inducers, we have examined the affinity and efficacy of two pairs of such compounds: oleate versus N-oleoyl-leucine and arachidonate versus N-arachidonoyl-glycine in cells and mitochondria from different tissues. We found that in cultures of the muscle-derived L6 cell line, as well as in primary cultures of murine white, brite/beige and brown adipocytes, the N-acyl amino acids were proficient uncouplers but that they did not systematically display higher affinity or potency than the conventional fatty acids, and they were not as efficient uncouplers as classical protonophores (FCCP). Higher concentrations of the N-acyl amino acids (as well as of conventional fatty acids) were associated with signs of deleterious effects on the cells. In liver mitochondria, we found that the N-acyl amino acids uncoupled similarly to conventional fatty acids, thus apparently via activation of the adenine nucleotide transporter-2. In brown adipose tissue mitochondria, the N-acyl amino acids were able to activate UCP1, again similarly to conventional fatty acids. We thus conclude that the formation of the acyl-amino acid derivatives does not confer upon the corresponding fatty acids an enhanced ability to induce thermogenesis in peripheral tissues, and it is therefore unlikely that the N-acyl amino acids are of specific physiological relevance as UCP1-independent thermogenic compounds.     

Aging and β3-adrenergic stimulation alter mitochondrial lipidome of adipose tissue

Mitochondrial abundance and thermogenic capacity are two imperative components that distinguish brown, beige and white adipose tissues. Most importantly, the lipid composition is vital for maintaining the quantity, quality and function of mitochondria. Therefore, we employed quantitative lipidomics to probe the mitochondrial lipidome of adipose tissues. The mitochondrial lipidome reveals β3-adrenergic stimulation and aging drastically altered the levels of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio and acyl chain desaturation. Precisely, PC_36:2 and PE_38:4 levels correlate with the increased brown and beige fat activity in young mice. While aging increased lysoPC species in white adipose tissue (WAT) mitochondria, CL-316,243 administration reduced lysoPC species and increased lyso-PE_{18:1 and 18:2} content during WAT browning. Also, non-thermogenic mitochondria accumulate sphingomyelin (SM), phosphatidylserine (PS), phosphatidic acid (PA) and ether-linked PC (ePC). Similarly, enrichment of phosphatidylglycerol (PG) and cardiolipin (CL) levels are associated with thermogenic mitochondria. Also, our in vitro experiment supports that blocking the de novo sphingolipid synthesis pathway by myriocin, SPT1 inhibitor increased the thermogenic capacity and oxygen consumption rate in mature adipocytes. Overall, our study suggests mitochondria of brown, beige and white adipose tissues own a unique pattern of lipid molecular species and their levels are altered by aging and CL-316,243 administration.     

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label‐free multiphoton redox and fluorescence lifetime microscopy

In this work, the metabolic characteristics of adipose tissues in live mouse model were investigated using a multiphoton redox ratio and fluorescence lifetime imaging technology. By analyzing the intrinsic fluorescence of metabolic coenzymes, we measured the optical redox ratios of adipocytes in vivo and studied their responses to thermogenesis. The fluorescence lifetime imaging further revealed changes in protein bindings of metabolic coenzymes in the adipocytes during thermogenesis. Our study uncovered significant heterogeneity in the cellular structures and metabolic characteristics of thermogenic adipocytes in brown and beige fat. Subgroups of brown and beige adipocytes were identified based on the distinct lipid size distributions, redox ratios, fluorescence lifetimes and thermogenic capacities. The results of our study show that this label‐free imaging technique can shed new light on in vivo study of metabolic dynamics and heterogeneity of adipose tissues in live organisms.