Ontogenetical changes in adipose tissue of the cat: Convertible adipose tissue

The ultrastructural characteristics of the inguinal, interscapular, and perirenal adipose tissue in kittens and cats were studied. There were no qualitative differences among adipocytes in the three anatomical areas. The only recorded difference was in the amount of lipids stored in the adipocytes in younger stages. Immediately after birth lipids occupied 25% of the volume in the inguinal area, 15% in interscapular fat tissue, and 10% in perirenal fat tissue. At this stage the adipose tissue morphologically resembled brown adipose tissue (BAT) of rodents. Two weeks after birth, lipids accumulated and adipocytes in the inguinal area became unilocular and appeared similar to white adipose tissue (WAT). A similar transition occurred approx 25 days after birth in interscapular fat and approx 6 weeks after birth in the perirenal area. No morphological signs of any cell degradation or destruction, nor any increased activity of preadipocytes, were seen during this conversion from BAT-like to WAT-like adipose tissue. The conversion of the adipose tissue was correlated with a decrease in vascularization and innervation, a loss of intercellular connections, and a changed mitochondrial population. Mitochondria in multilocular adipocytes resembled those in typical BAT which contain uncoupling protein (“UC-mitochondria”). After conversion to unilocular adipocytes the amount of mitochondria was halved, their cristae even more reduced, and their appearance was of a WAT-type (UCP-lacking mitochondria, which are coupled under physiological conditions; “C-mitochondria”). Since this category of adipose tissue differs from both typical brown and white adipose tissue, the name “convertible adipose tissue” (CAT) is proposed. Apparently adipose tissue from comparatively large mammals is of this convertible type.     

Convertible adipose tissue in mice

Summary Ability to express uncoupling protein (UCP) and establish UCP-dependent thermogenesis was analyzed in anatomical areas of mice that are generally considered to be white adipose tissue: mesenterial, perimetral, epididymal, inguinal, and superficial layer of interscapular white adipose tissue. The mice were acclimatized for 1 week to 4° C; the following week they were exposed to cold stress (1 h at-20° C, 2–3 times daily). In such conditions in inguinal adipose tissue, slot-blot analysis detected significant amount of UCP mRNA and lipoprotein lipase mRNA. Immuno-electron-microscopic localization of UCP showed that developed mitochondria of cold-stressed inguinal adipocytes contained UCP in the same amount as uncoupled (UC)-mitochondria of brown adipocytes. Morphological and morphometrical analysis showed that such inguinal adipose tissue appeared as brown adipose tissue. Since in control mice, inguinal adipose tissue was UCP-negative and tissue appeared as white adipose tissue, the duration of this white-to-brown adipose tissue conversion was analyzed. Mice, cold stressed for 1 week, were rewarmed at 28° C and their inguinal adipose tissue was analyzed in comparison with interscapular brown adipose tissue and epididymal white adipose tissue for another 37 days. During that time inguinal adipocytes ceased expressing UCP mRNA; UC-mitochondria in inguinal adipocytes were destroyed and replaced with common, C-mitochondria; and UCP was undetectable immunohistochemically. Adipocytes accumulated lipids, and the tissue morphologically once again resembled white adipose tissue. Described changes showed that besides typical brown and white adipose tissue in mice, there existed a third type of adipose tissue described as convertible adipose tissue.     

Developmental changes in uncoupling protein 1 and F1-ATPase subunit levels in the golden hamster brown adipose tissue mitochondria as determined by electron microscopy in situ immunocytochemistry

The postnatal developmental changes in mitochondrial uncoupling protein 1 (UCP 1) and F1-ATP synthase (ATPase) subunit levels in the interscapular brown adipose tissue (BAT) were studied in golden Syrian hamsters (Mesocricetus auratus) using electron microscopy in situ immunocytochemistry. The relatively low initial density of 5 nm gold conjugated anti-UCP 1 immunocomplexes gradually increased from 7- to 21-day-old animals and numerous immunocomplexes were found on the mitochondrial membranes of adult hamsters. At the age of 7-9 days, a positive reaction was also detected in the cytoplasm of BAT adipocytes. Immunolocalization of F1-ATPase subunit indicated its presence in BAT mitochondria and cytoplasm of 7- to 9-day-old animals. However, contrary to UCP 1, intensity of the immunostaining of F1-ATPase subunit rapidly decreased both in mitochondria and cytoplasm between the 10th and 21st postnatal day and it became stabilized in adult animals at a very low level restricted to mitochondria. These results confirm that profound changes in the enzymatic apparatus of BAT mitochondrial membranes, leading to formation of thermogenic mitochondria, occur not until the early postnatal period of hamster ontogenetic development.     

Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes

Multilocular,mitochondria-rich adipocytes appear in white adipose tissue (WAT) ofrats treated with the 3-adrenoceptor agonist, CL-316243 (CL).Objectives were to determine whether these multilocular adipocytesderived from cells that already existed in the WAT or fromproliferation of precursor cells and whether new mitochondria containedin them were typical brown adipocyte mitochondria. Use of5-bromodeoxyuridine to identify cells that had undergone mitosis duringthe CL treatment showed that most multilocular cells derived from cellsalready present in the WAT. Morphological techniques showed that atleast a subpopulation of unilocular adipocytes underwent conversion tomultilocular mitochondria-rich adipocytes. A small proportion ofmultilocular adipocytes (~8%) was positive for UCP1 byimmunohistochemistry. Biochemical techniques showed that mitochondrialprotein recovered from WAT increased 10-fold and protein isolated frombrown adipose tissue (BAT) doubled in CL-treated rats. Stained gelsshowed a different protein composition of new mitochondria isolatedfrom WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a muchlower concentration than in BAT mitochondria, and UCP3, at a higherconcentration than that in BAT mitochondria. We hypothesize thatmultilocular adipocytes present at 7 days of CL treatment have twoorigins. First, most come from convertible unilocular adipocytes thatbecome multilocular and make many mitochondria that contain UCP3.Second, some come from a cell that gives rise to more typical brownadipocytes that express UCP1.

     

Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture

Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, [35S]methionine incorporation and immunofluorescence microscopy. In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria. Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H(+)-ATPase. In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H(+)-ATPase. A selective synthesis of UCP was induced by activation of beta-adrenergic receptors or by elevated levels of cellular cAMP. UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration. Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.     

棕色脂肪的产热及其调控机制

棕色脂肪组织是小型哺乳动物重要的兼性产热器官,位于棕色脂肪细胞线粒体内膜的解偶联蛋白是此种产热机制的关键物质.产热刺激激活解偶联蛋白构成的质子通道,在线粒体内膜形成质子短路,从而解除ATP合成偶联对氧化呼吸速率的控制,使产热高速进行.去甲肾上腺素、甲状腺激素、胰岛素、pH值以及食物、环境温度等因素综合调控着棕色脂肪组织的结构与功能状态.     

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.     

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.     

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).     

布氏田鼠胎后发育过程中褐色脂肪组织和肝脏的产热特征

为探讨布氏田鼠胎后恒温能力的发育过程,本文测定了1、5、9、17、21、33 和41 日龄幼体的褐色脂肪组织(BAT)和肝脏的重量、线粒体蛋白含量和细胞色素c氧化酶(COX) 的活性。布氏田鼠胎后发育期间BAT 增补明显,主要表现为重量的增加和单位组织重量COX 活性的升高等,属典型的晚成型发育特征。布氏田鼠胎后发育过程中BAT 和肝脏产热特征的变化与幼体的产热特点和恒温能力的发育是相一致的。     

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.     

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.     

In vitro brown and “brite”/“beige” adipogenesis: Human cellular models and molecular aspects

Brown adipose tissue (BAT) has long been thought to be absent or very scarce in human adults so that its contribution to energy expenditure was not considered as relevant. The recent discovery of thermogenic BAT in human adults opened the field for innovative strategies to combat overweight/obesity and associated diseases. This energy-dissipating function of BAT is responsible for adaptive thermogenesis in response to cold stimulation. In this context, adipocytes can be converted, within white adipose tissue (WAT), into multilocular adipocytes expressing UCP1, a mitochondrial protein that plays a key role in heat production by uncoupling the activity of the respiratory chain from ATP synthesis. These adipocytes have been named “brite” or “beige” adipocytes. Whereas BAT has been studied for a long time in murine models both in vivo and in vitro, there is now a strong demand for human cellular models to validate and/or identify critical factors involved in the induction of a thermogenic program within adipocytes. In this review we will discuss the different human cellular models described in the literature and what is known regarding the regulation of their differentiation and/or activation process. In addition, the role of microRNAs as novel regulators of brown/“brite” adipocyte differentiation and conversion will be depicted. Finally, investigation of both the conversion and the metabolism of white-to-brown converted adipocytes is required for the development of therapeutic strategies targeting overweight/obesity and associated diseases. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

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.     

Let's burn whatever you have: mitofusin 2 metabolically re‐wires brown adipose tissue

Brown adipose tissue (BAT) has received enormous scientific and lay attention in the recent past as its thermogenic, energy‐consuming capacities represent prime candidates for therapeutic interventions toward obesity, glucose intolerance, and diabetes even in humans. The overall positive effects of BAT activation and recruitment on systemic energy homeostasis have been largely attributed to the inherent ability of brown adipocytes to combust fatty acid and glucose energy substrates through mitochondrial uncoupling, driven by the unique expression of uncoupling protein 1 (UCP1). Two recent reports by Boutant et al and Mahdaviani et al now identify the GTPase mitofusin (Mfn) 2 as a key determinant of BAT thermogenic function that is largely independent of its previously described role in mitochondrial fusion [1,2].     

Rat brown adipose tissue thermogenic features are altered during mid-pregnancy.

Brown adipose tissue (BAT) thermogenesis is inhibited during late-pregnancy and lactation in the rat. However, scarce information concerning BAT functionality during mid-pregnancy is available. The aim of this work was to investigate uncoupling proteins and leptin expression during placentation in rat BAT as well as other key parameters in the thermogenic function of the tissue. BAT mitochondrial content was found to be reduced 50% in 11 and 13 day pregnant rats as compared to nonpregnant controls, although uncoupling protein 1 (UCP1) content was not modified. Furthermore, UCP3 mRNA levels were found to be highly increased during this period. beta3-adrenergic receptor (beta3-AR) decreased expression resulted in a higher alpha2/beta3 ratio. Finally, leptin mRNA levels in BAT were found to be 3-fold up-regulated in pregnant animals. In conclusion, we show the existence of profound changes in thermogenic features in BAT during gestational days 11 and 13, pointing to the importance of this tissue during mid-pregnancy.