Rnf20 deficiency in adipocyte impairs adipose tissue development and thermogenesis

RNF20,an E3 ligase critical for monoubiquitination of histone H2B at lysine 120 (H2Bub),has been implicated in the regulation of various cellar processes;however,its physiological roles in adipocytes remain poorly characterized.Here,we report that the adipocyte-speci-fic knockout of Rnf20 (ASKO) in mice led to progressive fat loss,organomegaly and hyperinsulinemia.Despite signs of hyperinsulinemia,normal insulin sensitivity and improved glucose tolerance were observed in the young and aged CD-fed ASKO mice.In addition,high-fat diet-fed ASKO mice developed severe liver steatosis.More-over,we observed that the ASKO mice were extremely sensitive to a cold environment due to decreased expression levels of brown adipose tissue (BAT) selec-tive genes,including uncoupling protein 1 (Ucp1),and impaired mitochondrial functions.Significantly decreased levels of peroxisome proliferator-activated receptor gamma (Ppary) were observed in the gonadal white adipose tissues (gWAT) from the ASKO mice,suggesting that Rnf20 regulates adipogenesis,at least in part,through Ppary.Rosiglitazone-treated ASKO mice exhibited increased fat mass compared to that of the non-treated ASKO mice.Collectively,our results illus-trate the critical role of RNF20 in control of white and brown adipose tissue development and physiological function.     

Mitigation of isolation-associated adipocyte interleukin-6 secretion following rapid dissociation of adipose tissue

Primary adipocyte isolation by collagenase digestion is a widely used technique to study metabolic regulation and insulin action in adipocytes. However, induction of a proinflammatory response characterized by enhanced secretion of interleukin (IL)-6 has been tightly linked to the isolation process itself. To test the hypothesis that the shaking mechanical force exerted on adipocytes stimulates inflammation during isolation, rat primary adipocytes were prepared by collagenase digestion in orbital shaking incubators maintained at varying speeds. Contrary to expectation, the isolation-induced release of IL-6 was attenuated by increasing the rotational speed of digestion and the concentration of collagenase, both of which resulted in rapid dissociation of adipocytes from the vasculature. In addition, the attenuation of IL-6 secretion was associated with decreased phosphorylation of the stress-related p38 mitogen-activated protein kinase (p38 MAPK) and preserved insulin action. The data suggest that optimization of parameters including, but not limited to, mincing technique, time of digestion, and collagenase concentration will make it possible to isolate primary adipocytes without activation of a proinflammatory response leading to elevated secretion of IL-6.     

In vivo NMR detection of diet-induced changes in adipose tissue composition

We introduce an in vivo spectroscopic method to assess the effects of diet on fatty acid composition of the predominant chemical constituent of adipocytes in mice. To do this, we make use of a nonlinear NMR signal that, unlike a standard NMR signal, is intrinsically insensitive to local magnetic field inhomogeneities and which naturally suppresses the large water signal from nonfatty tissues. Our method yields fat composition information from fat depots distributed over large sample volumes in a single experiment, without requiring the use of tedious shimming procedures, voxel selection, or water suppression. Our results suggest that this method can reveal clear differences in adipose tissue composition of mice fed a standard chow diet compared with mice fed a diet rich in polyunsaturated fatty acids. With further developments this method could be used to obtain information on human lipid composition noninvasively and to track changes in lipid composition induced by diet intervention, pharmaceutical drugs, and exercise.     

In vivo lipolysis in adipose tissue from two anatomical locations measured by microdialysis

In this study, the difference in lipolytic response in inguinal subcutaneous and epididymal adipose tissues of male Sprague-Dawley rats was assessed in vivo by microdialysis. Probes were perfused with Ringer solution in which increasing concentrations of isoproterenol (10(-7) - 10(-4) mol/L) were added. Glycerol release, expressed as extracellular glycerol concentration, was used as lipolytic index. The effect of isoproterenol on local blood flow was investigated using the ethanol technique. No differences were found in the interstitial glycerol concentration between both adipose tissues under basal conditions. When isoproterenol was perfused, a dose-response increase in glycerol production was induced in both tissues. Interstitial glycerol concentration from epididymal adipose tissue was higher than that of inguinal subcutaneous depot at all isoproterenol concentrations. No vasodilatory effect of isoproterenol was found. These results suggest that epididymal adipose tissue is more responsive in vivo to beta-adrenergic lipolysis stimulation than is subcutaneous fat pad from the inguinal region.     

In vivo human lipolytic activity in preperitoneal and subdivisions of subcutaneous abdominal adipose tissue

We studied eight normal-weight male subjects to examine whether the lipolytic rate of deep subcutaneous and preperitoneal adipose tissues differs from that of superficial abdominal subcutaneous adipose tissue. The lipolytic rates in the superficial anterior and deep posterior subcutaneous abdominal adipose tissues and in the preperitoneal adipose tissue in the round ligament were measured by microdialysis and (133)Xe washout under basal, postabsorptive conditions and during intravenous epinephrine infusion (0.15 nmol. kg(-1). min(-1)). Both in the basal state and during epinephrine stimulation, the superficial subcutaneous adipose tissue had higher interstitial glycerol concentrations than the two other depots. Similarly, the calculated glycerol outputs from the superficial depot were significantly higher than those from the deep subcutaneous and the preperitoneal depots. Thus, it is concluded that the lipolytic rate of the superficial subcutaneous adipose tissue on the anterior abdominal wall is higher than that of the deep subcutaneous adipose tissue on the posterior abdominal wall and that of the preperitoneal adipose tissue in the round ligament.     

Glycerolipid biosynthesis in rat adipose tissue. Influence of adipocyte size.

A simple one-step filtration method is described to separate larger adipocytes from the smaller ones by using nylon screen (52 microM pore size). Adipocytes retained on the screen were larger (60-90 micrometers) compared with those that passed through the screen. By using this separation technique, activities of various enzymes involved in triacylglycerol formation from sn-glycerol 3-phosphate were measured in the larger and smaller adipocytes isolated from gonadal fat-depots. The homogenates from larger adipocytes were more active in lipid formation compared with those derived from small adipocytes. This was evident from the increased activities of sn-glycerol 3-phosphate acyltransferase. Mg2+-dependent phosphatidate phosphohydrolase and diacylglycerol acyltransferase in the larger adipocytes. The activities of these enzymes were also measured in the adipocytes isolated from gonadal, perirenal and subcutaneous fat-depots. Subcutaneous adipocytes were smaller and were less active in lipid formation than gonadal and perirenal adipocytes. These measurements in the activities of individual enzymes provide evidence that the entire pathway of esterification via sn-glycerol 3-phosphate is accelerated in the larger adipocytes.     

The spatiotemporal development of adipose tissue

Adipose tissue is a structure highly specialized in energy storage. The adipocyte is the parenchymal component of adipose tissue and is known to be mesoderm or neuroectoderm in origin; however, adipocyte development remains poorly understood. Here, we investigated the development of adipose tissue by analyzing postnatal epididymal adipose tissue (EAT) in mouse. EAT was found to be generated from non-adipose structure during the first 14 postnatal days. From postnatal day 1 (P1) to P4, EAT is composed of multipotent progenitor cells that lack adipogenic differentiation capacity in vitro, and can be regarded as being in the 'undetermined' state. However, the progenitor cells isolated from P4 EAT obtain their adipogenic differentiation capacity by physical interaction generated by cell-to-matrix and cell-to-cell contact both in vitro and in vivo. In addition, we show that impaired angiogenesis caused by either VEGFA blockade or macrophage depletion in postnatal mice interferes with adipose tissue development. We conclude that appropriate interaction between the cellular and matrix components along with proper angiogenesis are mandatory for the development of adipose tissue.     

Distribution of adenosine metabolising enzymes between adipocyte and stromal-vascular cells of adipose tissue

Rat adipose tissue was digested with collagenase and separated into adipocytes and stromal-vascular cells. The adipocytes accounted for 40% of the total adipose tissue adenosine deaminase activity, 32% of 5'-nucleotidase activity and 87% of adenosine kinase activity. This distribution suggests that adipocyte are the major cell type involved in adenosine utilization in adipose tissue. Furthermore, it suggests that the high sensitivity of isolated adipocytes to adenosine is representative of their sensitivity of isolated adipocytes to adenosine is representative of their sensitivity in vivo.     

Thyroid hormones and adipose tissue development

The effect of thyroid hormones on the cellularity of the retroperitoneal adipose tissue (R.P.A.T.) was investigated in rats that were 3, 6 and 12 weeks old. Two groups of rats were respectively made hypothyroid by the antithyroid compound propylthiouracil, or hyperthyroid by thyroxine. The number of adipocytes was less in the hypothyroid rats than in the controls; it was higher in the hyperthyroid rats without any concomitant increase in the weight of their R.P.A.T. Moreover, there was no significant correlation between adipose cell number and adipose tissue weight within any group of T4 or control rats. In all groups of rats, the number of adipose cells in the R.P.A.T. was larger in males than in females; the difference was highly significant in 12 week old control rats.     

Complete differentiation in vivo of implanted cultured adipocyte precursors from adult rats

Summary The formation of fully differentiated fat cells from adipocyte precursors, implanted into the same adult rats from which they were derived, is described. Precursor strains of rat epididymal adipocyte strains were grown through five subcultures, some in the presence of radioactive thymidine. While still at a relatively undifferentiated stage, the precursors were re-implanted into a superficial intramuscular location. At the time of resection six months later, fat pads were observed at the sites of implantation. These pads contained sheets of cells morphologically identical to mature epididymal adipocytes. The fat cells in pads developing from precursors grown in the presence of [³H]thymidine, were radiolabelled. Therefore, they represent fat cells that have differentiated in vivo from the implanted cultured precursors. Implanted skin fibroblasts did not lead to the formation of adipocytes. The finding that cultured adipocyte precursors from adult rats can differentiate fully not only in vitro, but also in adult animals, supports the probable physiological significance of these cells. The precursors probably participate in fat cell turnover, which likely persists throughout adulthood.This work was supported by Medical Research Council Grant MT-5827 and Ontario Heart Foundation Grant T1-46     

Enhancement of adipose tissue formation by implantation of adipogenic-differentiated preadipocytes

Engineered adipose tissue could be used for the reconstruction or augmentation of soft tissues lost due to mastectomy or lumpectomy in plastic and reconstructive surgery. Preadipocytes are a feasible cell source for adipose tissue regeneration. However, the enhancement of the in vivo adipogenic conversion of preadipocytes remains a major task. In vitro, the adipogenic differentiation of preadipocytes prior to implantation might enhance the adipose tissue regeneration. In the present study, we investigated whether implantation of adipogenic-differentiated preadipocytes enhances the adipose tissue formation compared with implantation of undifferentiated preadipocytes. We also investigated whether basic fibroblast growth factor (bFGF) further enhances the adipose tissue formation mediated by the implantation of adipogenic-differentiated preadipocytes. A fibrin matrix containing human preadipocytes cultured in adipogenic differentiation-inducing conditions with (group 1) or without (group 2) bFGF was injected into the subcutaneous spaces of athymic mice. Fibrin matrices containing undifferentiated human preadipocytes with (group 3) or without (group 4) bFGF were also implanted. Six weeks after implantation, the implanted cells formed new tissues in all groups. Importantly, the implantation of adipogenic-differentiated preadipocytes resulted in more extensive adipogenesis than the implantation of undifferentiated preadipocytes, as evaluated by adipose tissue area and human adipocyte-specific gene expression in the newly formed tissues. In addition, bFGF enhanced neovascularization in the newly formed tissues and further enhanced the adipogenesis mediated by the adipogenic-differentiated preadipocytes. The present study demonstrates that the implantation of adipogenic-differentiated preadipocytes enhances adipose tissue regeneration, as compared with the implantation of undifferentiated preadipocytes, and that cell transplantation-mediated adipogenesis can be further enhanced by the delivery of bFGF.     

Invited review: Pre- and postnatal adipose tissue development in farm animals: from stem cells to adipocyte physiology

Both white and brown adipose tissues are recognized to be differently involved in energy metabolism and are also able to secrete a variety of factors called adipokines that are involved in a wide range of physiological and metabolic functions. Brown adipose tissue is predominant around birth, except in pigs. Irrespective of species, white adipose tissue has a large capacity to expand postnatally and is able to adapt to a variety of factors. The aim of this review is to update the cellular and molecular mechanisms associated with pre- and postnatal adipose tissue development with a special focus on pigs and ruminants. In contrast to other tissues, the embryonic origin of adipose cells remains the subject of debate. Adipose cells arise from the recruitment of specific multipotent stem cells/progenitors named adipose tissue-derived stromal cells. Recent studies have highlighted the existence of a variety of those cells being able to differentiate into white, brown or brown-like/beige adipocytes. After commitment to the adipocyte lineage, progenitors undergo large changes in the expression of many genes involved in cell cycle arrest, lipid accumulation and secretory functions. Early nutrition can affect these processes during fetal and perinatal periods and can also influence or pre-determinate later growth of adipose tissue. How these changes may be related to adipose tissue functional maturity around birth and can influence newborn survival is discussed. Altogether, a better knowledge of fetal and postnatal adipose tissue development is important for various aspects of animal production, including neonatal survival, postnatal growth efficiency and health.     

Xenotransplantation of human fetal adipose tissue: a model of in vivo adipose tissue expansion and adipogenesis

Obesity during childhood and beyond may have its origins during fetal or early postnatal life. At present, there are no suitable in vivo experimental models to study factors that modulate or perturb human fetal white adipose tissue (WAT) expansion, remodeling, development, adipogenesis, angiogenesis, or epigenetics. We have developed such a model. It involves the xenotransplantation of midgestation human WAT into the renal subcapsular space of immunocompromised SCID-beige mice. After an initial latency period of approximately 2 weeks, the tissue begins expanding. The xenografts are healthy and show robust expansion and angiogenesis for at least 2 months following transplantation. Data and cell size and gene expression are consistent with active angiogenesis. The xenografts maintain the expression of genes associated with differentiated adipocyte function. In contrast to the fetal tissue, adult human WAT does not engraft. The long-term viability and phenotypic maintenance of fetal adipose tissue following xenotransplantation may be a function of its autonomous high rates of adipogenesis and angiogenesis. Through the manipulation of the host mice, this model system offers the opportunity to study the mechanisms by which nutrients and other environmental factors affect human adipose tissue development and biology.     

Activin receptor ALK4 promotes adipose tissue hyperplasia by suppressing differentiation of adipocyte precursors

Adipocyte hyperplasia and hypertrophy are the two main processes contributing to adipose tissue expansion, yet the mechanisms that regulate and balance their involvement in obesity are incompletely understood. Activin B/GDF-3 receptor ALK7 is expressed in mature adipocytes and promotes adipocyte hypertrophy upon nutrient overload by suppressing adrenergic signaling and lipolysis. In contrast, the role of ALK4, the canonical pan-activin receptor, in adipose tissue is unknown. Here, we report that, unlike ALK7, ALK4 is preferentially expressed in adipocyte precursors, where it suppresses differentiation, allowing proliferation and adipose tissue expansion. ALK4 expression in adipose tissue increases upon nutrient overload and positively correlates with fat depot mass and body weight, suggesting a role in adipose tissue hyperplasia during obesity. Mechanistically, ALK4 signaling suppresses expression of CEBPα and PPARγ, two master regulators of adipocyte differentiation. Conversely, ALK4 deletion enhances CEBPα/PPARγ expression and induces premature adipocyte differentiation, which can be rescued by CEBPα knockdown. These results clarify the function of ALK4 in adipose tissue and highlight the contrasting roles of the two activin receptors in the regulation of adipocyte hyperplasia and hypertrophy during obesity.     

In vivo development of tissue engineered vascular grafts: a fluid-solid-growth model

Biomechanics and Modeling in Mechanobiology - Methods of tissue engineering continue to advance, and multiple clinical trials are underway evaluating tissue engineered vascular grafts (TEVGs)....