Adipocyte Subpopulations Research Articles

Tbx15 Defines a Glycolytic Subpopulation and White Adipocyte Heterogeneity.

Tbx15 is a member of the T-box gene family of mesodermal developmental genes. We have recently shown that Tbx15 plays a critical role in the formation and metabolic programming of glycolytic myofibers in skeletal muscle. Tbx15 is also differentially expressed among white adipose tissue (WAT) in different body depots. In the current study, using three independent methods, we show that even within a single WAT depot, high Tbx15 expression is restricted to a subset of preadipocytes and mature white adipocytes. Gene expression and metabolic profiling demonstrate that the Tbx15Hi preadipocyte and adipocyte subpopulations of cells are highly glycolytic, whereas Tbx15Low preadipocytes and adipocytes in the same depot are more oxidative and less glycolytic. Likewise, in humans, expression of TBX15 in subcutaneous and visceral WAT is positively correlated with markers of glycolytic metabolism and inversely correlated with obesity. Furthermore, overexpression of Tbx15 is sufficient to reduce oxidative and increase glycolytic metabolism in cultured adipocytes. Thus, Tbx15 differentially regulates oxidative and glycolytic metabolism within subpopulations of white adipocytes and preadipocytes. This leads to a functional heterogeneity of cellular metabolism within WAT that has potential impact in the understanding of human metabolic diseases.

Bone Marrow Small Adipocytes in Acute Myeloid Leukemia Correlate with Prognosis

Introduction: Adipocytes have a substantial effect on the outcome and progression of certain solid tumors. However, little attention has been paid to the role of bone marrow (BM) adipocytes in acute myeloid leukemia (AML) because it is difficult to observe adipocytes through clinical BM aspiration. Although it was reported that adipocytes affected the behavior of leukemia cells in vitro, there is still no direct in vivo evidence. In the present study, we investigated the influence of adipocytes by focusing on their changing size in BM from primary AML patients.Methods: We retrospectively analyzed the biopsy specimens of BM from 70 patients with newly diagnosed AML and 70 controls with lymphoma or solid tumors without infiltration of BM. The size and type of adipocytes were analyzed for average diameter (Ad.Dm) and area (Ad.Ar) by tracing each individual adipocyte with Image-Pro Plus 5.1. Then, AML patients were further divided into 32 patients with continuous complete remission and 38 patients who were refractory based on the treatment effects. Adipocyte number (Ad.N; per square millimeter) and the percentage of adipocyte volume per tissue volume (Ad.V/TV) were compared between these two groups. Furthermore, the prognostic impact of adipocytes on the overall survival (OS) and relapse-free survival (RFS) in AML patients was analyzed by Cox regression analysis and Kaplan-Meier curves. Finally, the phenotype of adipocytes was determined by immunohistochemistry of UCP-1 and Perilipin1 to further explore the possible mechanism of the effect of adipocytes on the prognosis.Results: The Ad.Dm and Ad.Ar in BM from AML patients were 38.3±14.7 µm and 559.4±271.9 µm2, respectively, and both values were significantly smaller than those for the controls (P<0.001). The Ad.Dm exhibited no relation to the number of blasts in BM, indicating that the decreased size of adipocytes in AML cannot be attributed to extensive marrow blasts. Adipocytes were classified as small, medium and large adipocytes according to the frequency distribution of Ad.Dm in BM from controls. A significant difference was detected only in the proportion of small adipocytes (Ad.Dm<42.6 µm) between AML patients and controls (43.9% vs 25%, P=0.005). Furthermore, the Ad.V/TV and Ad.N of the adipocytes in the refractory group were 6.70±3.18% and 31.56±11.72/mm2, respectively, which were significantly higher than those of the remission subjects (P<0.001). This outcome prompted us to further analyze the role of small adipocytes in AML. Patients with Ad.V/TV of small adipocytes≤ 2.3% exhibited a longer OS than patients with Ad.V/TV of small adipocytes >2.3% (P<.001). Similarly, the subjects with Ad.N of small adipocytes <10.6/mm2 (P<.001) had a longer OS. Meanwhile, for the remission AML patients, those with Ad.V/TV of small adipocytes ≤2.5% had a shorter RFS than patients with Ad.V/TV of small adipocytes >2.5% (P<.001), and similar significant differences were also found between patients with Ad.N of small adipocytes ≥9.2/mm2 and Ad.N of small adipocytes <9.2/mm2(P<.001). In the biopsy specimens of BM, the subgroup of small adipocytes exhibited expression of Perilipin1 but not mitochondrial membrane protein UCP-1. Combination with the unilocular lipid droplets in adipocytes indicated that small adipocytes did not play a role in the conversion to brown adipose tissue.Conclusions: We first defined a subpopulation of small adipocytes in BM and demonstrated that only these cells but not all adipocytes were related to a poor prognosis in AML patients. The morphological changes of marrow adipocytes could be helpful to judge the prognosis of leukemia and could lead to other therapeutic perspectives. DisclosuresNo relevant conflicts of interest to declare.

Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine.

The reprogramming of cellular memory in specific cell types, and in visceral adipocytes in particular, appears to be a fundamental aspect of obesity and its related negative health outcomes. We explored the hypothesis that adipose tissue contains epigenetically distinct subpopulations of adipocytes that are differentially potentiated to record cellular memories of their environment. Adipocytes are large, fragile, and technically difficult to efficiently isolate and fractionate. We developed fluorescence nuclear cytometry (FNC) and fluorescence activated nuclear sorting (FANS) of cellular nuclei from visceral adipose tissue (VAT) using the levels of the pan-adipocyte protein, peroxisome proliferator-activated receptor gamma-2 (PPARg2), to distinguish classes of PPARg2-Positive (PPARg2-Pos) adipocyte nuclei from PPARg2-Negative (PPARg2-Neg) leukocyte and endothelial cell nuclei. PPARg2-Pos nuclei were 10-fold enriched for most adipocyte marker transcripts relative to PPARg2-Neg nuclei. PPARg2-Pos nuclei showed 2- to 50-fold higher levels of transcripts encoding most of the chromatin-remodeling factors assayed, which regulate the methylation of histones and DNA cytosine (e.g., DNMT1, TET1, TET2, KDM4A, KMT2C, SETDB1, PAXIP1, ARID1A, JMJD6, CARM1, and PRMT5). PPARg2-Pos nuclei were large with decondensed chromatin. TAB-seq demonstrated 5-hydroxymethylcytosine (5hmC) levels were remarkably dynamic in gene bodies of various classes of VAT nuclei, dropping 3.8-fold from the highest quintile of expressed genes to the lowest. In short, VAT-derived adipocytes appear to be more actively remodeling their chromatin than non-adipocytes.

5hmC Distribution in Isolated Visceral Adipose Tissue Nuclei

The reprogramming of specific cell types, and in visceral adipocytes in particular, is likely to be a fundamental to obesity and its related comorbidities. We explored the hypothesis that adipose tissue contains epigenetically distinct subpopulations of adipocytes in that are differentially potentiated to record memories of their cellular environment. Adipocytes are large, fragile, and technically difficult to isolate. We developed fluorescence nuclear cytometry (FNC) and fluorescence activated nuclear sorting (FANS) of cellular nuclei from visceral adipose tissue (SsVAT) using the levels of the adipocyte‐specific protein, peroxisome proliferator‐activated receptor gamma‐2 (PPARg2). Using FANS we obtained highly purified PPARg2‐Positive (PPARg2‐Pos) nuclei, which were distinct from PPARg2‐Negative (PPARg2‐Neg) leukocyte, endothelial cell and progenitor cell nuclei. PPARg2‐Pos VAT nuclei showed 2‐ to 50‐fold higher levels of transcripts encoding most of the chromatin‐remodeling factors assayed, including those which regulate the methylation of histones and DNA cytosine (e.g., DNMT1, DNMT3A, TET2, TET3, KMT2C, SETDB1, PAXP1, ARID1A, KMT2C, JMJD6, CARM1, and PRMT5). PPARg2‐Pos nuclei have large decondensed chromatin structures. Immunofluorescence microscopy (IFM) revealed the co‐distribution of PPARg2 protein with 5‐hydroxymethylcytosine (5hmC), with nearly all nuclei staining most strongly for PPARg2 also staining strongly for 5hmC. TET‐assisted bisulfite sequencing (TAB‐seq) demonstrated 5hmCG levels were the highest for PPARg2‐High nuclei and lowest for PPARg2‐Neg nuclei across all gene regions, and 5hmC levels were remarkably dynamic in gene bodies of PPARg2‐Pos nuclei, dropping 3.8‐fold from the highest quintile of expressed genes to the lowest. In short, VAT‐derived adipocytes are epigenetically distinct with PPARg2 and 5hmC levels coordinately regulated and proportional to nuclear size.Support or Funding InformationThis work was supported by grants from UGA's Office of the Vice President for Research, UGA's Obesity Initiative and from NIH's National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, DK1003920 and DK096300) to RBM, NHGRI HG006827 grant to CH, the National Institutes of Health grant R00GM100000 to RJS, and a Patricia and Doyle Mote Biomedical Research Award to PY.

Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis

The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36intermediate/high during adipocyte differentiation in vitro. The gradual increase of CD36intermediate/high/NRpositive cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression.

Exercise improves adipose function and inflammation and ameliorates fatty liver disease in obese diabetic mice.

Adipose inflammation and dysfunction underlie metabolic obesity. Exercise improves glycemic control and metabolic indices, but effects on adipose function and inflammation are less clear. Accordingly, it was hypothesized that exercise improves adipose morphometry to reduce adipose inflammation in hyperphagic obese mice. Alms1 mutant foz/foz mice housed in pairs were fed an atherogenic or chow diet; half the cages were fitted with a computer-monitored wheel for voluntary exercise. Insulin-induced AKT-phosphorylation, adipocyte size distribution, and inflammatory recruitment were studied in visceral versus subcutaneous depots, and severity of fatty liver disease was determined. Exercise prevented obesity and diabetes development in chow-fed foz/foz mice and delayed their onset in atherogenic-fed counterparts. Insulin-stimulated phospho-AKT levels in muscle were improved with exercise, but not in adipose or liver. Exercise suppressed adipose inflammatory recruitment, particularly in visceral adipose, associated with an increased number of small adipocyte subpopulations, and enhanced expression of beige adipocyte factor PRDM16 in subcutaneous fat. In atherogenic-fed foz/foz mice liver, exercise suppressed development of nonalcoholic steatohepatitis and related liver fibrosis. Exercise confers metabo-protective effects in atherogenic-fed hyperphagic mice by preventing early onset of obesity and diabetes in association with enhanced muscle insulin sensitivity, improved adipose morphometry, and suppressed adipose and liver inflammation.

Browning of white fat: does irisin play a role in humans?

The discovery of irisin as an exercise-regulated myokine inducing browning of WAT has gained interest as a potential new strategy to combat obesity and its associated disorders, such as type 2 diabetes. However, there are inconsistencies regarding the relevance of irisin in humans. The regulation of FNDC5 mRNA expression by exercise and contraction could not be reproduced by a number of human studies using several exercise protocols and in vitro approaches. Furthermore, the nature of FNDC5 fragments and the presence of irisin in humans are questionable and probably contribute to conflicting data obtained with commercially available ELISA kits. Most importantly, the information regarding the concentration of circulating irisin in humans is not clear, as different studies using different kits measure irisin levels in a wide range. Data about the role of irisin in states of human obesity and metabolic diseases are conflicting and, in some cases, changes in irisin levels have been observed; they were only moderate in 10-20%. Independent of the presence and regulation of FNDC5/irisin in humans, the application of recombinant irisin could still represent a therapeutic strategy to fight obesity. However, the current data obtained from human cell models reveal that FNDC5/irisin has no effect on browning of the major WAT depots in humans and is likely to selectively target a small subpopulation of adipocytes, which are located in classical BAT regions, such as the supraclavicular adipose tissue. Thus, other candidates, such as BMP7 or CNPs, seem to be more prominent candidates as inducers of browning in humans.

General Theory of Body Contouring: 2. Modulation of Mechanical Properties of Subcutaneous Fat Tissue

Subcutaneous white adipose tissue (sWAT) can be described micromechanically as a foam structure. It is shown that according to this model, mechanical stiffness of this tissue is primarily dependent on the average cell size and is almost independent of the dispersion of cell sizes in a local adipocytes’ population. Whereas the influence of natural fat renewal process with a rate of 10% per year must be of minor importance for mechanical properties of sWAT, induced adipocytes’ death can substantially reduce local sWAT stiffness. The sWAT which contains two or more different subpopulations of adipocytes of varying sizes with a spatially clustered structure can demonstrate significant inhomogeneity of their mechanical properties when compared with those of sWAT consisting of a single population of adipocytes. It is proposed that this effect may be an important pathophysiological feature of cellulite. Transformation of the cell shape from quasispherical to wrinkled or elliptical forms makes adipocytes more susceptible to thermo-mechanical stress reducing the strain needed to achieve the local plastic deformation. These mechanical features of sWAT are essential for understanding the mechanisms of different non-invasive and minimal invasive body contouring procedures.

Distinct populations of adipogenic and myogenic Myf5-lineage progenitors in white adipose tissues

Brown adipose tissues (BAT) are derived from a myogenic factor 5 (Myf5)-expressing cell lineage and white adipose tissues (WAT) predominantly arise from non-Myf5 lineages, although a subpopulation of adipocytes in some WAT depots can be derived from the Myf5 lineage. However, the functional implication of the Myf5- and non-Myf5-lineage cells in WAT is unclear. We found that the Myf5-lineage constitution in subcutaneous WAT depots is negatively correlated to the expression of classical BAT and newly defined beige/brite adipocyte-specific genes. Consistently, fluorescent-activated cell sorting (FACS)-purified Myf5-lineage adipo-progenitors give rise to adipocytes expressing lower levels of BAT-specific Ucp1, Prdm16, Cidea, and Ppargc1a genes and beige adipocyte-specific CD137, Tmem26, and Tbx1 genes compared with the non-Myf5-lineage adipocytes from the same depots. Ablation of the Myf5-lineage progenitors in WAT stromal vascular cell (SVC) cultures leads to increased expression of BAT and beige cell signature genes. Strikingly, the Myf5-lineage cells in WAT are heterogeneous and contain distinct adipogenic [stem cell antigen 1(Sca1)-positive] and myogenic (Sca1-negative) progenitors. The latter differentiate robustly into myofibers in vitro and in vivo, and they restore dystrophin expression after transplantation into mdx mouse, a model for Duchenne muscular dystrophy. These results demonstrate the heterogeneity and functional differences of the Myf5- and non-Myf5-lineage cells in the white adipose tissue.

Molecular and morphometric description of adipose tissue during weight changes: A quantitative tool for assessment of tissue texture

The aim of this study was to evaluate the morphometric changes of adipose tissue of lean and obese rats as assessed by computerized image analysis (IA) system in experimental conditions, with different degrees of adiposity. Moreover, to validate measures obtained by image analysis by correlation with direct measures of adiposity (body weight, epididimal fat, mean fat cell size and serum leptin). Finally to correlate these changes to expression of genes involved in lipid deposition and mobilization in adipose tissue. Lean (Fa/?) and genetically obese (fa/fa) Zucker rats were studied. Obese rats were food-restricted or treated with retinoic acid (ATRA) in order to reduce body weight and fat content. Moreover, gene expression of two key enzymes involved in fat metabolism (HSL and DGAT) were assessed in adipose tissue by RT-PCR. Our results show that HSL expression in adipose tissue was lower in obese compared to lean rats (1.47+/-0.02 vs 0.35+/-0.03, p<0.005) and was upregulated during food restriction in obese rats. DGAT expression was similar in lean and obese rats and was reduced by treatment with ATRA in obese rats. Tissue texture assessed by IA was significantly higher in lean compared to obese rats (23.2+/-0.6 vs 11.6+/-2.4%; p=0.01). Tissue structure highly correlated with adiposity in obese rats with different amount of body fat (area fraction vs epididimal fat depot: p=0.001). Distribution of measures for each sample, an index of spread of adipose tissue texture, as expressed by the standard deviation, correlated with adiposity (standard deviation vs epididimal fat depot: p=0.002) thus suggesting that adipose tissue texture increases its heterogeneity when adiposity is lower. This observation is in agreement with the hypothesis that the process of lipid mobilization from adipose tissue is not uniform, but a subpopulation of slimming adipocytes undergoes a complete release of their fat content while the rest of the tissue is much less affected. Moreover, image analysis system seems a reliable quantitative tool for assessment of adipose tissue texture.

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) of rats treated with the beta3-adrenoceptor agonist, CL-316243 (CL). Objectives were to determine whether these multilocular adipocytes derived from cells that already existed in the WAT or from proliferation of precursor cells and whether new mitochondria contained in them were typical brown adipocyte mitochondria. Use of 5-bromodeoxyuridine to identify cells that had undergone mitosis during the CL treatment showed that most multilocular cells derived from cells already present in the WAT. Morphological techniques showed that at least a subpopulation of unilocular adipocytes underwent conversion to multilocular mitochondria-rich adipocytes. A small proportion of multilocular adipocytes ( approximately 8%) was positive for UCP1 by immunohistochemistry. Biochemical techniques showed that mitochondrial protein recovered from WAT increased 10-fold and protein isolated from brown adipose tissue (BAT) doubled in CL-treated rats. Stained gels showed a different protein composition of new mitochondria isolated from WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a much lower concentration than in BAT mitochondria, and UCP3, at a higher concentration than that in BAT mitochondria. We hypothesize that multilocular adipocytes present at 7 days of CL treatment have two origins. First, most come from convertible unilocular adipocytes that become multilocular and make many mitochondria that contain UCP3. Second, some come from a cell that gives rise to more typical brown adipocytes that express UCP1.

Depot-related and thiazolidinedione-responsive expression of uncoupling protein 2 (UCP2) in human adipocytes.

Uncoupling protein 2 (UCP2) is a recently described homologue of the uncoupling protein of brown adipocytes (UCP1), which is expressed at high levels in human white adipose tissue. Studies were undertaken (1) to establish whether the expression of UCP2 mRNA varies in a depot-related manner in isolated human adipocytes, (2) to determine whether thiazolidinedione exposure influences the expression of UCP2 mRNA in cultured human pre-adipocytes, and (3) to determine whether human UCP2 is targeted to mitochondria when transfected into mammalian cells. Abdominal subcutaneous and omental adipose tissue biopsies were obtained from adult patients undergoing elective intra-abdominal surgical procedures. A competitive reverse transcriptase-polymerase chain reaction (RT-PCR) was used to quantify UCP2 mRNA expression in human omental and subcutaneous adipocytes, and in cultured human preadipocytes differentiated in vitro using the thiazolidinedione, BRL49653. Chinese hamster ovary cells were transfected with a vector expressing human UCP2, and its cellular localization was determined by confocal immunofluorescence microscopy. Adipocytes isolated from human omentum consistently expressed more UCP2 mRNA than did subcutaneous adipocytes from the same subjects (mean fold difference 2.92+/-0.44 P<0.001, n=11) with no effect of gender or body mass index being seen. BRL49653 treatment of subcutaneously, but not omentally, derived preadipocytes stimulated expression of UCP2 mRNA (5.1+/-1.1 fold). Transfected human UCP2 was detected exclusively in mitochondria of CHO cells. Increased expression of UCP2 in human omental adipose tissue relative to subcutaneous adipose tissue is related to the expression levels in adipocytes per se, a finding which may relate to the particular functional attributes of this sub-population of adipocytes. Furthermore, BRL 49653 has site-specific effects of on the expression of UCP2 in human preadipocytes, a finding which may be relevant to the therapeutic effects of such compounds. Finally we present evidence for the mitochondrial localisation of human UCP2.

Thyrotropin receptor expression in Graves' orbital adipose/connective tissues: potential autoantigen in Graves' ophthalmopathy.

It is acknowledged that the TSH receptor (TSHr) on thyroid follicular cells is the autoantigen involved in the hyperthyroidism of Graves' disease. However, whether this receptor is expressed in extrathyroidal tissues, and whether it participates directly in the pathogenesis of Graves' ophthalmopathy (GO) are unclear. We sought to detect the expression of TSHr messenger ribonucleic acid (mRNA) and protein in orbital adipose/connective tissue specimens and in human orbital preadipocyte fibroblast cultures using liquid hybridization analysis and immunohistochemical methods. We demonstrated intact and variant TSHr mRNA transcripts and TSHr-like immunoreactivity in orbital adipose/connective tissue specimens from patients with GO. In addition, TSHr-like immunoreactivity was detected in early passage GO preadipocyte fibroblast cultures that were shown to include some adipose cells. In contrast, neither TSHr mRNA nor protein was detected in normal orbital adipose/connective tissue specimens or in late passage GO orbital fibroblast cultures containing no lipid-laden adipose cells. In conclusion, we showed that TSHr is expressed in the adipose/connective tissue of the diseased orbit in GO. In addition, TSHr is demonstrable in early passage GO preadipocyte orbital fibroblast cultures that contain a subpopulation of adipocytes. Subsequent passaging of these cells results in the loss of both TSHr expression and adipocyte-specific staining. These results suggest that both the expression of this receptor and the accumulation of adipose tissue in the orbit in GO may be induced in vivo by a humoral factor(s) not present in the cell culture environment.