Browning of White Adipose Tissue Uncouples Glucose Uptake from Insulin Signaling

Presence of thermogenically active adipose tissue in adult humans has been inversely associated with obesity and type 2 diabetes. While it had been shown that insulin is crucial for the development of classical brown fat, its role in development and function of inducible brown-in-white (brite) adipose tissue is less clear. Here we show that insulin deficiency impaired differentiation of brite adipocytes. However, adrenergic stimulation almost fully induced the thermogenic program under these settings. Although brite differentiation of adipocytes as well as browning of white adipose tissue entailed substantially elevated glucose uptake by adipose tissue, the capacity of insulin to stimulate glucose uptake surprisingly was not higher in the brite state. Notably, in line with the insulin-independent stimulation of glucose uptake, our data revealed that brite recruitment results in induction of solute carrier family 2 (GLUT-1) expression in adipocytes and inguinal WAT. These results for the first time demonstrate that insulin signaling is neither essential for brite recruitment, nor is it improved in cells or tissues upon browning.     

Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor γ (PPARγ) in the Conversion of White-to-brown Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE₂. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE₂ levels. In turn, PGE₂ suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE₂ on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B₄). Taken together, these findings identify PGE₂ as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.     

miR-133a Regulates Adipocyte Browning In Vivo

Prdm16 determines the bidirectional fate switch of skeletal muscle/brown adipose tissue (BAT) and regulates the thermogenic gene program of subcutaneous white adipose tissue (SAT) in mice. Here we show that miR-133a, a microRNA that is expressed in both BAT and SATs, directly targets the 3′ UTR of Prdm16. The expression of miR-133a dramatically decreases along the commitment and differentiation of brown preadipocytes, accompanied by the upregulation of Prdm16. Overexpression of miR-133a in BAT and SAT cells significantly inhibits, and conversely inhibition of miR-133a upregulates, Prdm16 and brown adipogenesis. More importantly, double knockout of miR-133a1 and miR-133a2 in mice leads to elevations of the brown and thermogenic gene programs in SAT. Even 75% deletion of miR-133a (a1^−/−a2^+/−) genes results in browning of SAT, manifested by the appearance of numerous multilocular UCP1-expressing adipocytes within SAT. Additionally, compared to wildtype mice, miR-133a1^−/−a2^+/− mice exhibit increased insulin sensitivity and glucose tolerance, and activate the thermogenic gene program more robustly upon cold exposure. These results together elucidate a crucial role of miR-133a in the regulation of adipocyte browning in vivo.     

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.     

Intrinsic differences in BRITE adipogenesis of primary adipocytes from two different mouse strains

BRITE (brown-in-white) cells are brown adipocyte-like cells found in white adipose tissue (WAT) of rodents and/or humans. The recruitment of BRITE adipocytes, referred to as the browning of WAT, is hallmarked by the expression of UCP1 and exerts beneficial metabolic effects. Here we address whether beyond systemic cues depot- and strain-specific variation in BRITE recruitment is determined by a cellular program intrinsic to progenitors. Therefore we compared the browning capacity of serum and investigated brown and BRITE adipogenesis in primary cultures of stromal-vascular cells isolated from interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) in two inbred mouse strains C57BL/6J (B6, a strain with low browning propensity) and 129/S6SvEv (129, a strain with high browning propensity). Paradoxically, serum collected from B6 mice was more potent in the promotion of browning than serum collected from 129 mice. Nevertheless, we demonstrate that depot- and strain-specific differences observed in vivo are pheno-copied in primary cultures in vitro, as judged by UCP1 expression and by functional analysis. Notably, primary adipocytes from 129 mice had a higher capacity for isoproterenol-induced uncoupled respiration than B6. We conclude that cues intrinsic to the progenitor cells contribute to differential BRITE adipogenesis. Further analyses demonstrate that these cues are independent of autocrine/paracrine mechanisms, BRITE progenitor abundance and genetic variation in the gene regulatory region of Ucp1 but rather depend on trans-acting factors. These results provide new insights on the molecular basis of strain and depot-specific differences in BRITE adipogenesis.     

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.     

Recruited vs. nonrecruited molecular signatures of brown, "brite," and white adipose tissues

Mainly from cell culture studies, a series of genes that have been suggested to be characteristic of different types of adipocytes have been identified. Here we have examined gene expression patterns in nine defined adipose depots: interscapular BAT, cervical BAT, axillary BAT, mediastinic BAT, cardiac WAT, inguinal WAT, retroperitoneal WAT, mesenteric WAT, and epididymal WAT. We found that each depot displayed a distinct gene expression fingerprint but that three major types of depots were identifiable: the brown, the brite, and the white. Although differences in gene expression pattern were generally quantitative, some gene markers showed, even in vivo, remarkable depot specificities: Zic1 for the classical BAT depots, Hoxc9 for the brite depots, Hoxc8 for the brite and white in contrast to the brown, and Tcf21 for the white depots. The effect of physiologically induced recruitment of thermogenic function (cold acclimation) on the expression pattern of the genes was quantified; in general, the depot pattern dominated over the recruitment effects. The significance of the gene expression patterns for classifying the depots and for understanding the developmental background of the depots is discussed, as are the possible regulatory functions of the genes.     

A novel brown adipocyte-enriched long non-coding RNA that is required for brown adipocyte differentiation and sufficient to drive thermogenic gene program in white adipocytes

The thermogenic activities of brown and beige adipocytes can be exploited to reduce energy surplus and counteract obesity. Recent RNA sequencing studies have uncovered a number of long noncoding RNAs (lncRNAs) uniquely expressed in white and brown adipose tissues (WAT and BAT), but whether and how these lncRNAs function in adipogenesis remain largely unknown. Here, we report the identification of a novel brown adipocyte-enriched LncRNA (AK079912), and its nuclear localization, function and regulation. The expression of AK079912 increases during brown preadipocyte differentiation and in response to cold-stimulated browning of white adipocytes. Knockdown of AK079912 inhibits brown preadipocyte differentiation, manifested by reductions in lipid accumulation and down-regulation of adipogenic and BAT-specific genes. Conversely, ectopic expression of AK079912 in white preadipocytes up-regulates the expression of genes involved in thermogenesis. Mechanistically, inhibition of AK079912 reduces mitochondrial copy number and protein levels of mitochondria electron transport chain (ETC) complexes, whereas AK079912 overexpression increases the levels of ETC proteins. Lastly, reporter and pharmacological assays identify Pparγ as an upstream regulator of AK079912. These results provide new insights into the function of non-coding RNAs in brown adipogenesis and regulating browning of white adipocytes.     

Apple polyphenols induce browning of white adipose tissue

We and others have shown that apple polyphenols decrease adipose tissue mass. To better understand the underlying mechanisms and to expand clinical applicability, we herein examine whether apple polyphenols induce adipose thermogenic adaptations (browning) and prevent diet-induced obesity and related insulin resistance. In mice fed a standard diet, daily apple polyphenol consumption induced thermogenic adaptations in inguinal white adipose tissue (iWAT), based on increases in the expression of brown/beige adipocyte selective genes (Ucp1, Cidea, Tbx1, Cd137) and protein content of uncoupling protein 1 and mitochondrial oxidative phosphorylation enzymes. Among the upstream regulatory factors of browning, fibroblast growth factor 21 (FGF21) and peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC-1α) levels were concomitantly up-regulated by apple polyphenols. In the primary cell culture experiment, the results did not support a direct action of apple polyphenols on beige adipogenesis. Instead, apple polyphenols increased tyrosine hydroxylase (a rate-limiting enzyme of catecholamine synthesis) in iWAT, which activates the adipocyte thermogenic program possibly via intratissue cellular communications. In high-fat fed mice, apple polyphenols induced beige adipocyte development in iWAT, reduced fat accumulation, and increased glucose disposal rates in the glucose and insulin tolerance tests. Taken together, dietary administration of apple polyphenols induced beige adipocyte development in iWAT possibly via activation/induction of the peripheral catecholamine synthesis–FGF21–PGC-1α cascade. Results from diet-induced obese mice indicate that apple polyphenols have therapeutic potential for obesity and related metabolic disorders.     

Expression of Adipocyte Biomarkers in a Primary Cell Culture Models Reflects Preweaning Adipobiology

A cohort of genes was selected to characterize the adipogenic phenotype in primary cell cultures from three tissue sources. We compared the quantitative expression of biomarkers in culture relative to their expression in vivo because the mere presence or absence of expression is minimally informative. Although all biomarkers analyzed have biochemical functions in adipocytes, the expression of some of the biomarkers varied enormously in culture relative to their expression in the adult fat tissues in vivo, i.e. inguinal fat for white adipocytes and brite cells, interscapular brown adipose tissue for brown adipocytes, and ear mesenchymal stem cells for white adipocytes from adult mice. We propose that the pattern of expression in vitro does not reflect gene expression in the adult mouse; rather it is predominantly the expression pattern of adipose tissue of the developing mouse between birth and weaning. The variation in gene expression among fat depots in both human and rodent has been an extensively studied phenomenon, and as recently reviewed, it is related to subphenotypes associated with immune function, the inflammatory response, fat depot blood flow, and insulin sensitivity. We suggest that adipose tissue biology in the period from birth to weaning is not just a staging platform for the emergence of adult white fat but that it has properties to serve the unique needs of energy metabolism in the newborn. A case in point is the differentiation of brite cells that occurs during this period followed by their involution immediately following weaning.     

Browning attenuates murine white adipose tissue expansion during postnatal development

During postnatal development of mice distinct white adipose tissue depots display a transient appearance of brown-like adipocytes. These brite (brown in white) adipocytes share characteristics with classical brown adipocytes including a multilocular appearance and the expression of the thermogenic protein uncoupling protein 1. In this study, we compared two inbred mouse strains 129S6sv/ev and C57BL6/N known for their different propensity to diet-induced obesity. We observed transient browning in retroperitoneal and inguinal adipose tissue depots of these two strains. From postnatal day 10 to 20 the increase in the abundance of multilocular adipocytes and uncoupling protein 1 expression was higher in 129S6sv/ev than in C57BL6/N pups. The parallel increase in the mass of the two fat depots was attenuated during this browning period. Conversely, epididymal white and interscapular brown adipose tissue displayed a steady increase in mass during the first 30 days of life. In this period, 129S6sv/ev mice developed a significantly higher total body fat mass than C57BL6/N. Thus, while on a local depot level a high number of brite cells is associated with the attenuation of adipose tissue expansion the strain comparison reveals no support for a systemic impact on energy balance. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Long non-coding RNAs regulation in adipogenesis and lipid metabolism: Emerging insights in obesity

Obesity is a widespread health problem that brings about various adipose tissue dysfunctions. The balance of energy storage and energy expenditure is critical for normal fat accumulation and lipid metabolism. Therefore, understanding the molecular basis of adipogenesis and thermogenesis is essential to maintain adipose development and lipid homeostasis. Increasing evidence demonstrated that lncRNAs (long non-coding RNAs), a class of non-protein coding RNAs of >200 nucleotides in length, are identified as key regulators in obesity-related biological processes through diverse regulatory mechanisms. In this review, we concentrate on recent and relevant studies on the roles of lncRNAs in regulation of white adipogenesis, brown adipocyte differentiation and lipid metabolism. In addition, the diagnostic and therapeutic potential of lncRNAs is highlighted, and that will make recommendations for the future application of lncRNAs in the treatment of obesity.     

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.     

GM13133 is a negative regulator in mouse white adipocytes differentiation and drives the characteristics of brown adipocytes

Obesity is tightly associated with the disturbance of white adipose tissue storing excess energy. Thermogenic adipocytes (brown and beige) exert a critical role of oxidizing nutrients at the high rates through non‐shivering thermogenesis. The recruitment of brown characteristics in white adipocytes, termed browning, has been considered as a promising strategy for treating obesity and associated metabolic complications. Recently, long noncoding RNAs play a crucial role in regulating tissue development and participating in disease pathogenesis, yet their effects on the conversion of white into brown‐like adipocytes and thermogenic function were not totally understood. Here, we identified a mouse brown adipose specific expressed lncRNA, termed GM13133. Moreover, a considerable amount of GM13133 is expressed in adipocytes and actively modulated by cold, β₃‐adrenergic agonist and cAMP stimuli, implying a potential role in the conversion from white to brown adipocytes. Overexpression of GM13133 did not affect the proliferation of mouse white pre‐adipocytes, but inhibited white adipocyte differentiation by decreasing lipid accumulation. The forced expression of GM13133 also significantly drove the conversion of white into brown‐like adipocytes with the enhanced mitochondrial biogenesis and the induced expression of brown adipocytes specific markers. A global mRNA analysis further indicated the possible regulatory role of cAMP signaling pathway in GM13133 mediated white‐to‐brown adipocytes conversion. Our results identified a lncRNA‐mediated modulation in primary mouse white adipocyte differentiation and indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity.     

Raspberry promotes brown and beige adipocyte development in mice fed high-fat diet through activation of AMP-activated protein kinase (AMPK) α1

Development of brown and beige/brite adipocytes increases thermogenesis and helps to reduce obesity and metabolic syndrome. Our previous study suggests that dietary raspberry can ameliorate metabolic syndromes in diet-induced obese mice. Here, we further evaluated the effects of raspberry on energy expenditure and adaptive thermogenesis and determined whether these effects were mediated by AMP-activated protein kinase (AMPK). Mice deficient in the catalytic subunit of AMPKα1 and wild-type (WT) mice were fed a high-fat diet (HFD) or HFD supplemented with 5% raspberry (RAS) for 10 weeks. The thermogenic program and related regulatory factors in adipose tissue were assessed. RAS improved the insulin sensitivity and reduced fat mass in WT mice but not in AMPKα1^-/- mice. In the absence of AMPKα1, RAS failed to increase oxygen consumption and heat production. Consistent with this, the thermogenic gene expression in brown adipose tissue and brown-like adipocyte formation in subcutaneous adipose tissue were not induced by RAS in AMPKα1^-/- mice. In conclusion, AMPKα1 is indispensable for the effects of RAS on brown and beige/brite adipocyte development, and prevention of obesity and metabolic dysfunction.     

Study of lactoferrin gene expression in human and mouse adipose tissue,human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers

Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.     

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.