Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression in?vivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.     

Recruitment of brown fat and conversion of white into brown adipocytes: Strategies to fight the metabolic complications of obesity?

The role of white and brown adipose tissues in energy metabolism is well established. However, the existence of brown fat in adult humans was until very recently a matter of debate, and the molecular mechanisms underlying brown adipocyte development remained largely unknown. In 2009, several studies brought direct evidence for functional brown adipose tissue in adults. New factors involved in brown fat cell differentiation have been identified. Moreover, work on the origin of fat cells took an unexpected path with the recognition of different populations of brown fat cell precursors according to the anatomical location of the fat depots: a precursor common to skeletal muscle cells and brown adipocytes from brown fat depots, and a progenitor cell common to white adipocytes and brown adipocytes that appear in certain conditions in white fat depots. There is also mounting evidence that mature white adipocytes, including human fat cells, can be converted into brown fat-like adipocytes, and that the typical fatty acid storage phenotype of white adipocyte can be altered towards a fat utilization phenotype. These data open up new opportunities for the development of drugs for obesity and its metabolic and cardiovascular complications.     

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.     

Pharmacological and nutritional agents promoting browning of white adipose tissue

The role of brown adipose tissue in the regulation of energy balance and maintenance of body weight is well known in rodents. Recently, interest in this tissue has re-emerged due to the realization of active brown-like adipose tissue in adult humans and inducible brown-like adipocytes in white adipose tissue depots in response to appropriate stimuli (“browning process”). Brown-like adipocytes that appear in white fat depots have been called “brite” (from brown-in-white) or “beige” adipocytes and have characteristics similar to brown adipocytes, in particular the capacity for uncoupled respiration. There is controversy as to the origin of these brite/beige adipocytes, but regardless of this, induction of the browning of white fat represents an attractive potential strategy for the management and treatment of obesity and related complications. Here, the different physiological, pharmacological and dietary determinants that have been linked to white-to-brown fat remodeling and the molecular mechanisms involved are reviewed in detail. In the light of available data, interesting therapeutic perspectives can be expected from the use of specific drugs or food compounds able to induce a program of brown fat differentiation including uncoupling protein 1 expression and enhancing oxidative metabolism in white adipose cells. However, additional research is needed, mainly focused on the physiological relevance of browning and its dietary control, where the use of ferrets and other non-rodent animal models with a more similar adipose tissue organization and metabolism to humans could be of much help. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Exercise ameliorates high-fat diet-induced metabolic and vascular dysfunction, and increases adipocyte progenitor cell population in brown adipose tissue

A high-fat diet (HFD) is associated with adipose inflammation, which contributes to key components of metabolic syndrome, including obesity and insulin resistance. The increased visceral adipose tissue mass associated with obesity is the result of hyperplasia and hypertrophy of adipocytes. To investigate the effects of exercise on HFD-induced metabolic disorders, male C57BL/6 mice were divided into four groups: SED (sedentary)-ND (normal diet), EX (exercise)-ND, SED-HFD, and EX-HFD. Exercise was performed on a motorized treadmill at 15 m/min, 40 min/day, and 5 day/wk for 8 wk. Exercise resulted in a decrease in abdominal fat contents and inflammation, improvements in glucose tolerance and insulin resistance, and enhancement of vascular constriction and relaxation responses. Exercise with or without HFD increased putative brown adipocyte progenitor cells in brown adipose tissue compared with groups with the same diet, with an increase in brown adipocyte-specific gene expression in brown and white adipose tissue. Exercise training enhanced in vitro differentiation of the preadipocytes from brown adipose depots into brown adipocytes and enhanced the expression of uncoupling protein 1. These findings suggest that exercise ameliorates high-fat diet-induced metabolic disorders and vascular dysfunction, and increases adipose progenitor cell population in brown adipose tissue, which might thereby contribute to enhanced functional brown adipose.     

Conditionally immortalized white preadipocytes: a novel adipocyte model

This study describes a novel approach to generate conditionally immortalized preadipocyte cell lines from white adipose tissue (IMWAT) that can be induced to differentiate into white adipocytes even after expansion in culture. Such adipocytes express markers of white fat such as peroxisome proliferator-activated receptor gamma and aP2 but not brown fat markers, have an intact insulin signaling pathway, and express proinflammatory cytokines. They can be readily transduced with adenoviral vectors, allowing them to be used to investigate the consequences of the depletion of specific adipocyte factors using short hairpin RNA. This approach has been used to study the effect of reduced expression of the nuclear receptor corepressor receptor interacting protein 140 (RIP140), a regulator of adipocyte function. The depletion of RIP140 results in changes in metabolic gene expression that resemble those in adipose tissue of the RIP140 null mouse. Thus, IMWAT cells provide a novel model for adipocytes that are derived from preadipocytes rather than fibroblasts and provide an alternative system to primary preadipocytes for the investigation of adipocyte function.     

Adipokine gene expression in dog adipose tissues and dog white adipocytes differentiated in primary culture.

Obesity and its associated disorders are increasing in companion animals, particularly in dogs. We have investigated whether genes encoding key adipokines, some of which are implicated in the pathologies linked to obesity, are expressed in canine adipose tissues. Using RT-PCR, mRNAs encoding the following adipokines were detected in dog white adipose tissue: adiponectin, leptin, angiotensinogen, plasminogen activator inhibitor-1, IL-6, haptoglobin, metallothionein-1 and 2, and nerve growth factor. The adipokine mRNAs were present in all fat depots examined. Fractionation of adipose tissue by collagenase digestion showed that each gene was expressed in mature adipocytes. The mRNA for TNFalpha was not evident in adipose tissue, but was detected in isolated adipocytes. Fibroblastic preadipocytes from gonadal white fat were differentiated into adipocytes in primary culture and adipokine expression examined before and after differentiation (days 0 and 11, respectively). Each adipokine gene expressed in dog white adipocytes was also expressed in the differentiated cells. These results demonstrate that dog white adipose tissue expresses major adipokine genes, expression being in the adipocytes. Investigation of adipokine production and function will provide insight into the mechanisms involved in obesity-related pathologies in dogs and serve as a model for the related human diseases.     

Cellular changes during cold acclimatation in adipose tissues

Cold exposure is a well-known physiological stimulus that activates the sympathetic nervous system and induces brown adipose tissue (BAT) hyperplasia. The effects of cold exposure or cold acclimatation have been extensively studied in interscapular BAT (IBAT). However, it has been recently shown that brown adipocytes are present in adipose deposits considered as white fat such as periovarian (PO) fat pad. We have investigated the kinetic of brown precursor recruitment in adipose tissues using DNA measurement and specific marker expression. In IBAT, cold exposure induces proliferation of precursor cells and differentiation into preadipocytes characterized by the expression of A2COL6, a marker specific to early steps of the differentiation process. A chronic stimulation of the tissue is necessary to observe the full effect. In PO fat pad, no proliferation can be detected, whereas differentiation of brown preadipocytes and maybe phenotypic conversion of white adipocytes seems to be promoted. In conclusion, these data demonstrated that 1) the same stimulus (cold exposure) does not induce the same response in terms of preadipocyte proliferation and differentiation in periovarian and brown adipose tissues, although both contain brown adipocytes, and 2) preadipocyte recruitment in adipose tissues after cold exposure depends on the predominant type of fat cells. © 1996 Wiley-Liss, Inc.     

A plasmid region encoding the active fragment and the inhibitor protein of colicin E3--CA38

Thermogenin is the purine-nucleotide binding polypeptide in brown adipose tissue mitochondria (M_r 32 000) which confers upon these mitochondria the ability to produce heat. An enzyme-linked immunosorbent assay (ELISA) has been developed to demonstrate and quantitate the occurrence of thermogenin antigen in small amounts of tissue, and thus to characterize different depots of fat tissue as white or brown. The extreme sensitivity of the method allows determination of thermogenin in samples equivalent to <1 mg tissue. The results indicate that thermogenin seems to be exclusively localised in brown fat mitochondria (as compared to white fat, liver or heart muscle mitochondria), and thermogenin antigen could only be found in brown adipocytes (as compared to white adipocytes). Thus, brown and white adipose tissue are probably ontogenetically different     

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.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

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.     

Beta 3-adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat.

White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by beta-adrenoceptors but there is a controversy about the subtype(s) involved. In the present study, we exposed to cold beta 3-adrenoceptor knockout mice (beta 3KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein-1 (UCP1). In brown fat of beta 3KO mice, UCP1 expression levels were normal at 24 degrees C as well as after a 10-day cold exposure. Strikingly, under both conditions, in the white fat of beta 3KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that beta 3-adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat.     

Adipocyte lineages: Tracing back the origins of fat

The obesity epidemic has intensified efforts to understand the mechanisms controlling adipose tissue development. Adipose tissue is generally classified as white adipose tissue (WAT), the major energy storing tissue, or brown adipose tissue (BAT), which mediates non-shivering thermogenesis. It is hypothesized that brite adipocytes (brown in white) may represent a third adipocyte class. The recent realization that brown fat exist in adult humans suggests increasing brown fat energy expenditure could be a therapeutic strategy to combat obesity. To understand adipose tissue development, several groups are tracing the origins of mature adipocytes back to their adult precursor and embryonic ancestors. From these studies emerged a model that brown adipocytes originate from a precursor shared with skeletal muscle that expresses Myf5-Cre, while all white adipocytes originate from a Myf5-negative precursors. While this provided a rational explanation to why BAT is more metabolically favorable than WAT, recent work indicates the situation is more complex because subsets of white adipocytes also arise from Myf5-Cre expressing precursors. Lineage tracing studies further suggest that the vasculature may provide a niche supporting both brown and white adipocyte progenitors; however, the identity of the adipocyte progenitor cell is under debate. Differences in origin between adipocytes could explain metabolic heterogeneity between depots and/or influence body fat patterning particularly in lipodystrophy disorders. Here, we discuss recent insights into adipose tissue origins highlighting lineage-tracing studies in mice, how variations in metabolism or signaling between lineages could affect body fat distribution, and the questions that remain unresolved. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.     

Necdin controls proliferation of white adipocyte progenitor cells

White adipose tissues are composed mainly of white fat cells (adipocytes), which play a key role in energy storage and metabolism. White adipocytes are terminally differentiated postmitotic cells and arise from their progenitor cells (preadipocytes) or mesenchymal stem cells residing in white adipose tissues. Thus, white adipocyte number is most likely controlled by the rate of preadipocyte proliferation, which may contribute to the etiology of obesity. However, little is known about the molecular mechanisms that regulate preadipocyte proliferation during adipose tissue development. Necdin, which is expressed predominantly in postmitotic neurons, is a pleiotropic protein that possesses anti-mitotic and pro-survival activities. Here we show that necdin functions as an intrinsic regulator of white preadipocyte proliferation in developing adipose tissues. Necdin is expressed in early preadipocytes or mesenchymal stem cells residing in the stromal compartment of white adipose tissues in juvenile mice. Lentivirus-mediated knockdown of endogenous necdin expression in vivo in adipose tissues markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood. Furthermore, necdin-null mutant mice exhibit a greater expansion of adipose tissues due to adipocyte hyperplasia than wild-type mice when fed the high-fat diet during the juvenile and adult periods. Adipose stromal-vascular cells prepared from necdin-null mice differentiate in vitro into a significantly larger number of adipocytes in response to adipogenic inducers than those from wild-type mice. These results suggest that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development.     

The adipose organ of obesity-prone C57BL/6J mice is composed of mixed white and brown adipocytes

White and brown adipocytes are believed to occupy different sites in the body. We studied the anatomical features and quantitative histology of the fat depots in obesity and type 2 diabetes-prone C57BL/6J mice acclimated to warm or cold temperatures. Most of the fat tissue was contained in depots with discrete anatomical features, and most depots contained both white and brown adipocytes. Quantitative analysis showed that cold acclimation induced an increase in brown adipocytes and an almost equal reduction in white adipocytes; however, there were no significant differences in total adipocyte count or any signs of apoptosis or mitosis, in line with the hypothesis of the direct transformation of white into brown adipocytes. The brown adipocyte increase was accompanied by enhanced density of noradrenergic parenchymal nerve fibers, with a significant correlation between the density of these fibers and the number of brown adipocytes. Comparison with data from obesity-resistant Sv129 mice disclosed a significantly different brown adipocyte content in C57BL/6J mice, suggesting that this feature could underpin the propensity of the latter strain to develop obesity. However, the greater C57BL/6J browning capacity can hopefully be harnessed to curb obesity and type 2 diabetes in patients with constitutively low amounts of brown adipose tissue.     

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.     

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.