Studies on the expression of adipocyte-specific cell surface antigens during the differentiation of adipocyte precursor cells in vitro

Using species and cell specific antiadipocyte sera an immunoprecipitation procedure was developed which allowed the nature of adipocyte cell surface antigens to be investigated. Analysis of immunoprecipitates from mature adipocyte plasma membranes of rat, ox and chicken and similar 125I-labelled membranes revealed the presence of specific externally disposed adipocyte specific antigens which were also species specific. For mature cells the specific antigens had molecular weights of 124,000, 92,000 and 59,000 in the case of the rat, 87,000 in the case of the ox and 56,000, 47,000 and 37,000 in the case of the chicken. None of these antigens were cross immunoprecipated by antisera to non-homologous adipocytes. The presence of the antigens at the surface of differentiating rat while adipocyte precursor cells was demonstrated using a labelled-second antibody cellular immunoassay and the expression of this reactivity revealed to be an early event in the differentiation programme of the cells. The increase in cell surface immunoreactivity during the differentiation of the cells was shown to be dependent upon the expression of two of the antigens previously shown to be markers of the mature adipocyte phenotype. The functional identity and possible role of these antigens in the control of adipocyte differentiation in vitro and in vivo now becomes accessible to investigation experimentally.     

Cyclic AMP metabolism and lipolysis in cultured human adipocyte precursors

Triacylglycerol breakdown (lipolysis) results from a series of reactions culminated by activation of "hormone-stimulated" triacylglycerol lipase, an enzyme unique to adipose tissue. We have studied various components of the lipolytic process in human omental adipocyte precursors differentiating in culture. The levels of cyclic AMP, the "second messenger" of lipolytic hormones, were about sixfold higher in fat cell precursors than those in abdominal skin fibroblasts. L-Isoproterenol resulted in significant elevation of cyclic AMP levels in both cell types. Preincubation of intact adipocyte precursors with insulin resulted in significant enhancement of "low Km" cyclic AMP phosphodiesterase activity; in contrast, this hormone had no effect on fibroblast phosphodiesterase activity, a distinctive biochemical difference despite the morphological similarities between the two cell types during the early stages of adipocyte precursor maturation. Incubation of adipocyte precursors with isoproterenol resulted in the release of fatty acids into the medium, findings indicative of "hormone-stimulated" lipase activity and, hence, the operation of the entire "lipolytic cascade"; isoproterenol-stimulated lipolysis was inhibited by insulin. Release of fatty acids from fibroblasts was not observed. Thus, "hormone-stimulated" lipolysis and insulin stimulation of cyclic AMP phosphodiesterase activity are expressed during early stages of human adipocyte precursor differentiation.     

A study of adipocyte precursor cells derived from brown adipose tissue: the expression of specific cell surface antigens during their differentiation in culture

Using cell specific anti-adipocyte sera and an immuno-precipitation procedure, the nature of the cell surface antigens characterizing adipocytes from rat brown adipose tissue was investigated. Initially the ability of anti-sera, raised against adipose plasma membrane preparations of white or brown adipose tissue, to distinguish between membrane preparations derived from either tissue was confirmed. Analysis of the plasma membranes derived from brown adipose and similar preparations labelled with 125I revealed the presence of specific externally disposed mature brown adipocyte-specific antigens. The specifically immunoprecipated antigens had molecular weights of 70,000, 56,000 and 23,000. None of these antigens were cross immunoprecipated by antisera to mature white adipocyte membranes. The presence of the brown adipose specific antigens on the surface of differentiating adipocyte precursor cells derived from rat brown adipose tissue was demonstrated using a labelled-secon antibody cellular immunoassay. The expression of the immunoreactivity associated with these antigens was shown to be an early event in the differentiation programme of the cells in vitro. The functional identity and possible roles of these antigens in the control of brown adipocyte differentiation now becomes accessible to further experimental investigation.     

Cytotoxic and stimulatory effects of antilymphocyte globulin (ALG) on hematopoiesis

Four different preparations of antilymphocyte/antithymocyte globulin were tested in vitro for their toxicity to lymphocytes and to hematopoietic precursor cells, depending on concentration and time. Complete lymphocytotoxicity was observed at concentrations from 6.3 to 25 micrograms/ml, and suppression of colony formation by hematopoietic precursors was seen at concentrations from 12.5 to 250 micrograms/ml. Prolonged incubation time did not increase lymphocytotoxicity but augmented precursor cell damage. Lymphocytotoxicity was comparable among the four preparations tested whereas precursor cell toxicity varied widely. Antilymphocyte globulin is mitogenic and stimulates the release of hematopoietic growth factor activity by peripheral blood cells. Absorption of ALG with human T-cells eliminated precursor cell toxicity and mitogenicity but not the capacity to release hematopoietic growth factors. These results show that dose/time schedules for ALG administration may be relevant and ALG acts by virtue of inhibitory and stimulatory antibody effects.     

Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells.

We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.     

Identification of chicken (Gallus domesticus) adipocyte plasma membrane and differentiation specific proteins using SDS-PAGE and western blotting.

1. Affinity-purified adipocyte membrane proteins were used to raise antisera in two sheep. 2. Using one of the antisera 15 proteins were identified as being adipocyte specific by comparison on Western blots of plasma membrane proteins from various tissues. 3. Of these 15 proteins eight appeared to be present only in mature adipocytes and not in the adipocyte precursor. 4. In the presence of guinea pig complement the two antisera raised were cytotoxic to adipocytes and their precursors. 5. Characterization and further study of these adipocyte differentiation specific proteins will provide valuable information about the process of adipocyte differentiation.     

Retinol-Binding Protein 4 and Its Membrane Receptor STRA6 Control Adipogenesis by Regulating Cellular Retinoid Homeostasis and Retinoic Acid Receptor α Activity

Retinoids are vitamin A (retinol) derivatives and complex regulators of adipogenesis by activating specific nuclear receptors, including the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Circulating retinol-binding protein 4 (RBP4) and its membrane receptor STRA6 coordinate cellular retinol uptake. It is unknown whether retinol levels and the activity of RAR and RXR in adipocyte precursors are linked via RBP4/STRA6. Here, we show that STRA6 is expressed in precursor cells and, dictated by the apo- and holo-RBP4 isoforms, mediates bidirectional retinol transport that controls RARα activity and subsequent adipocyte differentiation. Mobilization of retinoid stores in mice by inducing RBP4 secretion from the liver activated RARα signaling in the precursor cell containing the stromal-vascular fraction of adipose tissue. Retinol-loaded holo-RBP4 blocked adipocyte differentiation of cultured precursors by activating RARα. Remarkably, retinol-free apo-RBP4 triggered retinol efflux that reduced cellular retinoids, RARα activity, and target gene expression and enhanced adipogenesis synergistically with ectopic STRA6. Thus, STRA6 in adipocyte precursor cells links nuclear RARα activity to the circulating RBP4 isoforms, whose ratio in obese mice was shifted toward limiting the adipogenic potential of their precursors. This novel cross talk identifies a retinol-dependent metabolic function of RBP4 that may have important implications for the treatment of obesity.     

Complete differentiation of adipocyte precursors

Summary Evidence for the complete morphological maturation of precursor cells into adipocytes in vitro is presented. Cells were isolated from the stromal fraction of adipose tissue from adult humans and from rats and were grown in culture. Abdominal skin fibroblasts were used as controls. All cell strains were initially fusiform and replicated. On reaching monolayer confluency, they were transferred to an enriched growth medium in which the human and rat adipocyte precursors differentiated into a homogeneous population of cells, morphologically indistinguishable from mature adipocytes. In contrast, skin fibroblasts from the same person or animal, and grown under identical culture conditions, did not accumulate lipid and retained their fusiform contour. The same results were obtained in the first six subcultures that were studied. Thus, there is firm evidence that fat tissue of adult humans and rats contains adipocyte precursors that differentiate into mature fat cells. The culture system that has been described will facilitate the elucidation of the factors involved in replication and differentiation of adipocyte precursors.This work was supported by The Medical Research Council of Canada Grant MA-5827, The Ontario Heart Foundation, The Atkinson Charitable Foundation, The Banting Research Foundation, The J.P. Bickell Foundation, and the Physicians' Services Incorporated Foundation     

Differentiation and function of rat adipocyte precursor cells in primary culture

Precursor cells to adipocytes were purified from the epididymal fat pads of small rats and studied in primary culture. A culture system in which substrate and cofactors were not rate-limiting for complete adipocyte conversion was used by utilizing an agarose feeding-layer. Detachment of cells from the culture dish was prevented by addition of a viscous layer of culture medium, containing methyl cellulose. This system allowed quantitation and definite characterization of formed adipocytes, defined as cells accumulating a lipid droplet >20 micro m in diameter. The cells could be subcultured but then gradually lost their adipocyte conversion ability. Age of the donor depressed the adipocyte conversion which, however, never seemed to stop completely. Prostaglandin E(1) and F(2alpha) had no definite effect in the physiological concentration range while indomethachin possibly had a weak inhibitory effect. Insulin, heparin, and isobutylmethylxanthine increased adipocyte formation. Development of characteristic adipocyte functions with time was examined. Lipoprotein lipase activity was very low in the isolated precursor cells before culture, but developed in culture at confluence and was a thousand-fold higher within a few days. At this peak lipoprotein lipase activity was 50-fold higher than in mature adipocytes from the same donor animal. Triglyceride synthesis from glucose peaked in parallel but never reached the value of mature adipocytes and very little fatty acid was synthesized. Hormone-sensitive glycerol release developed at confluence and reached the level of activity of mature adipocytes. This study and previous work have indicated a role for the cyclase system in the development of adipocytes from precursor cells. Dibutyrylcyclic AMP caused an enhancement of lipoprotein lipase activity and adipocyte conversion. In suspension media, the nucleotide caused inhibition. These results are compatible with an effect of the nucleotide, not directly on lipoprotein lipase and cell determination, but via events taking place at confluence associated with cell to cell interactions. In comparison with previously described cells from an established cell line which undergo adipose conversion (3T3 cells), the cells described in the present work, like adipocytes, showed more metabolic activity in pathways for fatty acid incorporation from exogenous lipid sources (lipoprotein lipase activity) than from de novo synthesis. Furthermore, host-factors could be followed such as in the age- and site-dependence of adipocyte formation. Physiological stimuli such as insulin, lipid substrate, and heparin had effects on adipocyte formation. It was therefore concluded that this cell preparation has a potential of yielding information of physiological significance in studies of the regulation of adipocyte multiplication.-Bj?rntorp, P., M. Karlsson, P. Pettersson, and G. Sypniewska. Differentiation and function of rat adipocyte precursor cells in primary culture.     

Generation of megakaryocytes and platelets from human subcutaneous adipose tissues

Recent advances in regenerative medicine have created a broad spectrum of stem cell research. Among them, tissue stem cell regulations are important issues to clarify the molecular mechanism of differentiation. Adipose tissues have been shown to contain abundant preadipocytes, which are multipotent to differentiate into cells including adipocytes, chondrocytes, and osteoblasts. In this study, we have first shown that megakaryocytes and platelets can be generated from adipocyte precursor cells. Human adipocyte precursor cells were cultured in conditioned media for 12 days to differentiate adipocytes, followed by 12 days of culture in media containing thrombopoietin. The ultrastructures of adipocyte precursor cell- and bone marrow CD34-positive cell-derived megakaryocytes and platelets were similar. In addition, adipocyte precursor cell-derived platelets exhibited surface expression of P-selectin and bound fibrinogen upon stimulation with platelet agonists, suggesting that these platelets were functional. This is the first demonstration that human subcutaneous adipocyte precursor cells can generate megakaryocyte and functional platelets in an in vitro culture system.     

Live imaging of mouse endogenous neural progenitors migrating in response to an induced tumor

Adult neurogenesis is restricted to specific brain regions. Although involved in the continuous supply of interneurons for the olfactory function, the role of neural precursors in brain damage-repair remains an open question. Aiming to in vivo identify endogenous neural precursor cells migrating towards a brain damage site, the monoclonal antibody Nilo2 recognizing cell surface antigens on neuroblasts, was coupled to magnetic glyconanoparticles (mGNPs). The Nilo2-mGNP complexes allowed, by magnetic resonance imaging in living animals, the in vivo identification of endogenous neural precursors at their niche, as well as their migration to a lesion site (induced brain tumor), which was fast (within hours) and orderly. Interestingly, the rapid migration of neuroblasts towards a damage site is a characteristic that might be exploited to precisely localize early damage events in neurodegenerative diseases. In addition, it might facilitate the study of regenerative mechanisms through the activation of endogenous neural cell precursors. A similar approach, combining magnetic glyconanoparticles linked to appropriate antibodies could be applied to flag other small cell subpopulations within the organism, track their migration, localize stem cell niches, cancer stem cells or even track metastatic cells.     

SORLA is required for insulin-induced expansion of the adipocyte precursor pool in visceral fat

Visceral adipose tissue shows remarkable plasticity, constantly replacing mature adipocytes from an inherent pool of adipocyte precursors. The number of precursors is set in the juvenile organism and remains constant in adult life. Which signals drive precursor pool expansion in juveniles and why they operate in visceral but not in subcutaneous white adipose tissue (WAT) are unclear. Using mouse models, we identified the insulin-sensitizing receptor SORLA as a molecular factor explaining the distinct proliferative capacity of visceral WAT. High levels of SORLA activity in precursors of juvenile visceral WAT prime these cells for nutritional stimuli provided through insulin, promoting mitotic expansion of the visceral precursor cell pool in overfed juvenile mice. SORLA activity is low in subcutaneous precursors, blunting their response to insulin and preventing diet-induced proliferation of this cell type. Our findings provide a molecular explanation for the unique proliferative properties of juvenile visceral WAT, and for the genetic association of SORLA with visceral obesity in humans.     

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.     

Netrin 1 mediates spinal cord oligodendrocyte precursor dispersal

In spinal cord, oligodendrocyte precursors that give rise to myelin-forming cells originate in a restricted domain of the ventral ventricular zone. During development, these cells migrate widely throughout the spinal cord. Netrin 1 is expressed at the ventral ventricular zone during oligodendrocyte precursors emigration, and, in vitro, netrin 1 acts as chemorepellent and antagonizes platelet-derived growth factor (PDGF) chemoattraction. Oligodendrocyte precursors express the netrin receptors DCC and UNC5 and function-blocking anti-DCC antibody inhibits chemorepulsion of ventral spinal cord explants and netrin-secreting cells. In spinal cord slice preparations, addition of function-blocking anti-DCC antibody or netrin 1 dramatically inhibits oligodendrocyte precursor migration from the ventral ventricular zone. These data indicate the initial dispersal of oligodendrocyte precursors from their localized origin is guided by a chemorepellent response to netrin 1.     

胰岛素介体产生机制的探讨

胰岛素介体──肌醇磷酸多糖,被认为是胰岛素的第二信使,存在于细胞膜上的糖肌醇磷脂是产生该介体的前体,经胰岛素或磷脂酰肌醇特异性的磷脂酶Ｃ（ＰＩＰＬＣ）水解,产生介体和二酰甘油（ＤＧ）．本实验以人红细胞为材料,用３￣Ｈ同位素标记、有机溶剂提取、薄层层析及放射性自动计数等方法,分析胰岛素或ＰＩＰＬＣ作用于红细胞后前体和ＤＧ的变化情况,以推测介体的产生机制．结果显示：胰岛素使红细胞膜上及释放至胞外上清的前体量均较对照升高,且使体系中的ＤＧ量升高;ＰＩＰＬＣ则使红细胞膜上的前体量下降,使释放至胞外上清的前体量升高,推测：胰岛素或ＰＩＰＬＣ作用于完整细胞时,激活了某种酶,使前体先从膜上释放至胞外上清,再被水解为介体和ＤＧ,同时胰岛素还可能激活完整细胞内再合成前体的机制,而ＰＬＰＬＣ却不能．     

Phenotypic and functional characterization of murine B lymphocyte precursors isolated from fetal and adult tissues

Murine fetal liver and adult bone marrow cells identified by monoclonal 14.8 antibody were enriched on antibody-coated polystyrene petri dishes. Cell surface immunoglobulin (sig)-bearing cells were depleted before this enrichment procedure, and the resulting preparations of 14.8+, slg- cells were characterized as to morphology, immunoglobulin gene expression, and functional potential in vivo and in vitro. All cells with detectable mu chains of IgM in the cytoplasm (cmu) were found to be included in the 14.8+ population. The enriched cells did not contain significant numbers of committed granulocyte-macrophage progenitor cells or putative hemopoietic stem cells. Selected cells from 16-day fetal liver were large, a majority of the cells had a lobulated rather than a spherical nuclear outline, and less than 1% had detectable cmu. Enriched cells from 19-day fetal liver were on the average smaller than those from 16-day-gestation liver and had a more typical lymphoid morphology; 30% were cmu+. Adult bone marrow 14.8+, slg- cells were similar to 19-day fetal liver cells in morphology, and approximately half were cmu+. These selected precursor cells retained the capacity to mature in vivo and in vitro. Fetal and adult 14.8+, slg- cells were efficient in generating newly formed B cells in vivo, and this maturation step appeared to be dependent on the presence of microenvironmental accessory cells. However, the ability of positively selected cells to mature in vitro was markedly decreased, and this potential was not rescued by providing known sources of accessory cells. Possible reasons for this difference are considered. This technique for positively selecting cells has allowed us to directly compare for the first time B cell precursors from fetal and adult tissues and will be invaluable for resolution of the cell compartments in the differentiation of B lymphocyte precursors, in the study of accessory cells known to facilitate this process, in the definition of humoral factors which may act on pre-B cells, in the study of immunoglobulin gene rearrangements which take place during normal differentiation, and for further comparative studies of fetal and adult lymphopoiesis.     

Phospholipase A2 is a differentiation-dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture

Phospholipase A2 enzymatic activity was measured in the teratoma-derived adipogenic cell line 1246 and in adipocyte precursors in primary cultures. It was shown that enzymatic activity was low while the cells were undifferentiated and increased by 20-24-fold after the cells had undergone adipocyte differentiation. The increase of phospholipase A2 activity follows the same time course as that observed for glycerol-3-phosphate dehydrogenase activity used as a marker of differentiation. In contrast, the differentiation-deficient, insulin-independent cell line 1246-3A always contained very low levels of phospholipase A2 activity. Phospholipase A2 activity measured in the 1246 cells was inhibited in a dose-dependent fashion by incubation with ONO-RS-082 and quinacrine which are inhibitors of phospholipase A2 activity. Measurements of arachidonate metabolites in 1246 cells showed that production of prostaglandin F2 alpha by the 1246 cells followed the same time course as the increase of phospholipase A2 activity during differentiation. Similar results were obtained with primary cultures of adipocyte precursors. These results indicate that phospholipase A2 is a differentiation-dependent enzymatic activity for the adipogenic cell line 1246 and for adipocyte precursors in primary culture. These data suggest that metabolic pathways controlled by phospholipase A2 activity could play an important physiological role in adipose tissue differentiation.     

Immunological versatility and carbon regulation of Cellulomonas fimi endo-1,4-beta-glucanases.

More than 10 protein molecules with endo-1,4-beta-glucanase activity were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram in Cellulomonas fimi culture supernatants, grown in CMC as carbon source. These molecules are shown to belong to at least four immunologically different groups, against three of which polyclonal antibodies were raised. The protein species used as antigens showed significant differences in cross reactivity, carbon regulation, and affinity to crystalline cellulose. Three intracellular precursors of the first group were detected, two of which were under carbon catabolite control with the third apparently being synthesized constitutively. In the extracellular environment this group showed the largest versatility in protein molecules. The second group appeared to originate from two intracellular precursors both synthesized constitutively and subject to minor extracellular modifications as compared to the first group. The main extracellular protein of this group showed high affinity toward crystalline cellulose. One intracellular precursor was identified for the third group, which was subject to carbon catabolite control. Only one extracellular molecule without binding ability to crystalline cellulose corresponded to this precursor, indicating that the latter was resistant to proteolytic modifications after excretion. It appears that the C. fimi cellulases are more complex than expected and reconstitution of the whole system will be difficult.     

Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus

Experiments involving sequential transplantations of the chick embryonic thymus at E9 to E12 into a first 3-day host quail embryo and then into a second chick host allowed demonstration of the cyclic periodicity of hemopoietic cell seeding of the embryonic thymus. After a first wave of colonization occurring between E6.5 and E8, the thymus becomes refractory to hemopoietic cell entry for about 4 days. It resumes its capacity to be seeded by a second wave of blood-borne stem cells at E12. After a second period of non receptivity starting at E14, a third wave of incoming cells reaches the thymus around E18. Therefore, with a slightly different periodicity, the same cyclic mechanism regulates the renewal of lymphocytes in chick and quail embryos. Quail hemopoietic cells were immunostained in the chimeric thymuses, with a species specific monoclonal antibody (anti-MB1) which recognizes a common surface antigenic determinant on all endothelial and blood cells of the quail (except erythrocytes). Two steps could thus be distinguished in the seeding process. When the thymus becomes receptive for hemopoietic cells, the latter first accumulate in the intrathymic blood vessels before penetrating massively in the thymic parenchyma. The quail chick-chimera system combined with the use of a species- and cell-type-specific antibody provides a unique tool for studying thymic colonization by lymphocyte precursors.