Search

Background: Adipose tissue-derived stromal vascular fraction (SVF) harbors multipotent cells with potential therapeutic relevance. We developed a method to form adipose spheroids (AS) from the SVF with complex organoid structure and enhanced leptin secretion upon insulin stimulation., Methods: SVF was generated from the interscapular brown adipose tissue of newborn mice. Immunophenotype and stemness of cultured SVF were determined by flow cytometry and in vitro differentiation, respectively. Spheroids were generated in hanging drops and non-adherent plates and compared by morphometric methods. The adipogenic potential was compared between preadipocyte monolayers and spheroids. Extracellular leptin was quantified by immunoassay. Lipolysis was stimulated with isoprenaline and quantified by colorimetric methods. AS viability and ultrastructure were determined by confocal and transmission electron microscopy analyses., Results: Cultured SVF contained Sca1 + CD29 + CD44 + CD11b- CD45- CD90- cells with adipogenic and chondrogenic but no osteogenic potential. Culture on non-adherent plates yielded the highest quantity and biggest size of spheroids. Differentiation of AS for 15 days in a culture medium supplemented with insulin and rosiglitazone resulted in greater Pparg, Plin1, and Lep expression compared to differentiated adipocytes monolayers. AS were viable and maintained leptin secretion even in the absence of adipogenic stimulation. Glycerol release after isoprenaline stimulation was higher in AS compared to adipocytes in monolayers. AS were composed of outer layers of unilocular mature adipocytes and an inner structure composed of preadipocytes, immature adipocytes and an abundant loose extracellular matrix., Conclusion: Newborn mice adipose SVF can be efficiently differentiated into leptin-secreting AS. Prolonged stimulation with insulin and rosiglitazone allows the formation of structurally complex adipose organoids able to respond to adrenergic lipolytic stimulation., (© 2023. The Author(s).)

Objective: Beclin1 is a core molecule of the macroautophagy machinery. Although dysregulation of macroautophagy is known to be involved in metabolic disorders, the function of Beclin1 in adipocyte metabolism has not been investigated. In the present study, we aimed to study the role of Beclin1 in lipolysis and mitochondrial homeostasis of adipocytes., Methods: Autophagic flux during lipolysis was examined in adipocytes cultured in vitro and in the adipose tissue of mice. Adipocyte-specific Beclin1 knockout (KO) mice were used to investigate the activities of Beclin1 in adipose tissues., Results: cAMP/PKA signaling increased the autophagic flux in adipocytes differentiated from C3H10T1/2 cells. In vivo autophagic flux was higher in the brown adipose tissue (BAT) than that in the white adipose tissue and was further increased by the β3 adrenergic receptor agonist CL316243. In addition, surgical denervation of BAT greatly reduced autophagic flux, indicating that sympathetic nerve activity is a major regulator of tissue autophagy. Adipocyte-specific KO of Beclin1 led to a hypertrophic enlargement of lipid droplets in BAT and impaired CL316243-induced lipolysis/lipid mobilization and energy expenditure. While short-term effects of Beclin1 deletion were characterized by an increase in mitochondrial proteins, long-term Beclin1 deletion led to severe disruption of autophagy, resulting in mitochondrial loss, and dramatically reduced the expression of genes involved in lipid metabolism. Consequently, adipose tissue underwent increased activation of cell death signaling pathways, macrophage recruitment, and inflammation, particularly in BAT., Conclusions: The present study demonstrates the critical roles of Beclin1 in the maintenance of lipid metabolism and mitochondrial homeostasis in adipose tissues., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Dysregulation of macroautophagy/autophagy is implicated in obesity and insulin resistance. However, it remains poorly defined how autophagy regulates adipocyte development. Using adipose-specific rptor/raptor knockout (KO), atg7 KO and atg7 rptor double-KO mice, we show that inhibiting MTORC1 by RPTOR deficiency led to autophagic sequestration of lipid droplets, formation of LD-containing lysosomes, and elevation of basal and isoproterenol-induced lipolysis in vivo and in primary adipocytes. Despite normal differentiation at an early phase, progressive degradation and shrinkage of cellular LDs and downregulation of adipogenic markers PPARG and PLIN1 occurred in terminal differentiation of rptor KO adipocytes, which was rescued by inhibiting lipolysis or lysosome. In contrast, inactivating autophagy by depletion of ATG7 protected adipocytes against RPTOR deficiency-induced formation of LD-containing lysosomes, LD degradation, and downregulation of adipogenic markers in vitro . Ultimately, atg7 rptor double-KO mice displayed decreased lipolysis, restored adipose tissue development, and upregulated thermogenic gene expression in brown and inguinal adipose tissue compared to RPTOR-deficient mice in vivo . Collectively, our study demonstrates that autophagy plays an important role in regulating adipocyte maturation via a lipophagy and lipolysis-dependent mechanism., Abbreviations: ATG7: autophagy related 7; BAT: brown adipose tissue; CEBPB/C/EBPβ: CCAAT enhancer binding protein beta; DGAT1: diacylglycerol O-acyltransferase 1; eWAT: epididymal white adipose tissue; iWAT: inguinal white adipose tissue; KO: knockout; LD: lipid droplet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; PLIN1: perepilin 1; PNPLA2/ATGL: patatin-like phospholipase domain containing 2; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; RPTOR: regulatory associated protein of MTOR complex1; TG: triglyceride; ULK1: unc-51 like kinase 1; UCP1: uncoupling protein 1; WAT: white adipose tissue.

Reduced neo-adipogenesis and dysfunctional lipid-overloaded adipocytes are hallmarks of hypertrophic obesity linked to insulin resistance. Identifying molecular features of hypertrophic adipocytes requires appropriate in vitro models. We describe the generation of a model of human hypertrophic-like adipocytes directly comparable to normal adipose cells and the pathologic evolution toward hypertrophic state. We generate in vitro hypertrophic cells from mature adipocytes, differentiated from human mesenchymal stem cells. Combining optical, confocal, and transmission electron microscopy with mRNA/protein quantification, we characterize this cellular model, confirming specific alterations also in subcutaneous adipose tissue. Specifically, we report the generation and morphological/molecular characterization of human normal and hypertrophic-like adipocytes. The latter displays altered morphology and unbalance between canonical and dominant negative (PPARGΔ5) transcripts of PPARG , paralleled by reduced expression of PPARγ targets, including GLUT4 . Furthermore, the unbalance of PPARγ isoforms associates with GLUT4 down-regulation in subcutaneous adipose tissue of individuals with overweight/obesity or impaired glucose tolerance/type 2 diabetes, but not with normal weight or glucose tolerance. In conclusion, the hypertrophic-like cells described herein are an innovative tool for studying molecular dysfunctions in hypertrophic obesity and the unbalance between PPARγ isoforms associates with down-regulation of GLUT4 and other PPARγ targets, representing a new hallmark of hypertrophic adipocytes.

Under caloric restriction, bone marrow adipocytes (BM-Ads) do not decrease in size compared to white adipocytes, suggesting they harbor unique metabolic properties. We compare human primary BM-Ads with paired subcutaneous adipocytes (SC-Ads) using proteomic and lipidomic approaches. We find that, although SC-Ads and BM-Ads share similar morphological features, they possess distinct lipid metabolism. Although BM-Ad shows enrichment in proteins involved in cholesterol metabolism, correlating with increased free cholesterol content, proteins involved in lipolysis were downregulated. In particular, monoacylglycerol lipase expression is strongly reduced in BM-Ads, leading to monoacylglycerol accumulation. Consequently, basal and induced lipolytic responses are absent in BM-Ads, affirming their differences in metabolic fitness upon caloric restriction. These specific metabolic features are not recapitulated in vitro using common protocols to differentiate bone marrow mesenchymal stem cells. Thus, contrary to classical SC-Ads, BM-Ads display a specific lipid metabolism, as they are devoid of lipolytic activity and exhibit a cholesterol-orientated metabolism., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Lipid droplet (LD) binding proteins in mammary glands and in adipocytes were previously compared and striking similar sets of these specific proteins demonstrated. Xanthine oxidoreductase (XOR) together with perilipins and the lactating mammary gland protein butyrophilin play an important role in the secretion process of LDs into milk ducts. In contrast, in adipose tissue and in adipocytes, mainly perilipins have been described. Moreover, XOR was reported in mouse adipose tissue and adipocyte culture cells as "novel regulator of adipogenesis". This obvious coincidence of protein sets prompted us to revisit the formation of LDs in human-cultured adipocytes in more detail with special emphasis on the possibility of a LD association of XOR. We demonstrate by electron and immunoelectron microscopy new structural details on LD formation in adipocytes. Surprisingly, by immunological and proteomic analysis, we identify in contrast to previous data showing the enzyme XOR, predominantly the expression of aldehyde oxidase (AOX). AOX could be detected tightly linked to LDs when adipocytes were treated with starvation medium. In addition, the majority of cells show an enormous interconnected, tubulated mitochondria network. Here, we discuss that (1) XOR is involved-together with perilipins-in the secretion of LDs in alveolar epithelial cells of the lactating mammary gland and is important in the transcytosis pathway of capillary endothelial cells. (2) In cells, where LDs are not secreted, XOR cannot be detected at the protein level, whereas in contrast in these cases, AOX is often present. We detect AOX in adipocytes together with perilipins and find evidence that these proteins might direct LDs to mitochondria. Finally, we here report for the first time the exclusive and complementary localization of XOR and AOX in diverse cell types.

Stem cells undergo drastic morphological alterations during differentiation. While extensive studies have been performed to examine the cytoskeletal remodeling, there is a growing interest to determine the morphological, structural and functional changes of the nucleus. The current study is therefore aimed at quantifying the extent of remodeling of the nuclear morphology of human mesenchymal stem cells during biochemically-induced adipogenic differentiation. Results show the size of nuclei decreased exponentially over time as the lipid accumulation is up-regulated. Increases in the lipid accumulation appear to lag the nuclear reorganization, suggesting the nuclear deformation is a prerequisite to adipocyte maturation. Furthermore, the lamin A/C expression was increased and redistributed to the nuclear periphery along with a subsequent increase in the nuclear aspect ratio. To further assess the role of the nucleus, a nuclear morphology with a high aspect ratio was achieved using microcontact-printed substrate. The cells with an elongated nuclear shape did not efficiently undergo adipogenesis, suggesting the cellular and nuclear processes associated with stem cell differentiation at the early stage of adipogenesis cause a change in the nuclear morphology and cannot be abrogated by the morphological cues. In addition, a novel computational biomechanical model was generated to simulate the nuclear shape change during differentiation and predict the forces acting upon the nucleus. This effort led to the development of computational scaling approach to simulate the experimentally observed adipogenic differentiation processes over 15 days in less than 1.5 hours.

Unlike white and brown adipose tissues, the bone marrow adipocyte (BMA) exists in a microenvironment containing unique populations of hematopoietic and skeletal cells. To study this microenvironment at the sub-cellular level, we performed a three-dimensional analysis of the ultrastructure of the BMA niche with focused ion beam scanning electron microscopy (FIB-SEM). This revealed that BMAs display hallmarks of metabolically active cells including polarized lipid deposits, a dense mitochondrial network, and areas of endoplasmic reticulum. The distinct orientations of the triacylglycerol droplets suggest that fatty acids are taken up and/or released in three key areas - at the endothelial interface, into the hematopoietic milieu, and at the bone surface. Near the sinusoidal vasculature, endothelial cells send finger-like projections into the surface of the BMA which terminate near regions of lipid within the BMA cytoplasm. In some regions, perivascular cells encase the BMA with their flattened cellular projections, limiting contacts with other cells in the niche. In the hematopoietic milieu, BMAT adipocytes of the proximal tibia interact extensively with maturing cells of the myeloid/granulocyte lineage. Associations with erythroblast islands are also prominent. At the bone surface, the BMA extends organelle and lipid-rich cytoplasmic regions toward areas of active osteoblasts. This suggests that the BMA may serve to partition nutrient utilization between diverse cellular compartments, serving as an energy-rich hub of the stromal-reticular network. Lastly, though immuno-EM, we've identified a subset of bone marrow adipocytes that are innervated by the sympathetic nervous system, providing an additional mechanism for regulation of the BMA. In summary, this work reveals that the bone marrow adipocyte is a dynamic cell with substantial capacity for interactions with the diverse components of its surrounding microenvironment. These local interactions likely contribute to its unique regulation relative to peripheral adipose tissues., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Firstly identified by anatomists, the fat tissue is nowadays an area of intense research due to increased global prevalence of obesity and its associated diseases. Histologically, there are four types of fat tissue cells which are currently recognized (white, brown, beige, and perivascular adipocytes). Therefore, in this study we are reviewing the most recent data regarding the origin, structure, and molecular mechanisms involved in the development of adipocytes. White adipocytes can store triglycerides as a consequence of lipogenesis, under the regulation of growth hormone or leptin and adiponectin, and release fatty acids resulted from lipolysis, under the regulation of the sympathetic nervous system, glucocorticoids, TNF-α, insulin, and natriuretic peptides. Brown adipocytes possess a mitochondrial transmembrane protein thermogenin or UCP1 which allows heat generation. Recently, thermogenic, UCP positive adipocytes have been identified in the subcutaneous white adipose tissue and have been named beige adipocytes. The nature of these cells is still controversial, as current theories are suggesting their origin either by transdifferentiation of white adipocytes, or by differentiation from an own precursor cell. Perivascular adipocytes surround most of the arteries, exhibiting a supportive role and being involved in the maintenance of intravascular temperature. Thoracic perivascular adipocytes resemble brown adipocytes, while abdominal ones are more similar to white adipocytes and, consequently, are involved in obesity-induced inflammatory reactions. The factors involved in the regulation of adipose stem cells differentiation may represent potential pathways to inhibit or to divert adipogenesis. Several molecules, such as pro-adipogenic factors (FGF21, BMP7, BMP8b, and Cox-2), cell surface proteins or receptors (Asc-1, PAT2, P2RX5), and hypothalamic receptors (MC4R) have been identified as the most promising targets for the development of future therapies. Further investigations are necessary to complete the knowledge about adipose tissue and the development of a new generation of therapeutic tools based on molecular targets., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Obesity is characterized by an excessive triacylglycerol accumulation in white adipocytes. Various mechanisms allowing the tight regulation of triacylglycerol storage and mobilization by lipid droplet-associated proteins as well as lipolytic enzymes have been identified. Increasing energy expenditure by inducing a mild uncoupling of mitochondria in adipocytes might represent a putative interesting anti-obesity strategy as it reduces the adipose tissue triacylglycerol content (limiting alterations caused by cell hypertrophy) by stimulating lipolysis through yet unknown mechanisms, limiting the adverse effects of adipocyte hypertrophy. Herein, the molecular mechanisms involved in lipolysis induced by a mild uncoupling of mitochondria in white 3T3-L1 adipocytes were characterized. Mitochondrial uncoupling-induced lipolysis was found to be independent from canonical pathways that involve lipolytic enzymes such as HSL and ATGL. Finally, enhanced lipolysis in response to mitochondrial uncoupling relies on a form of autophagy as lipid droplets are captured by endolysosomal vesicles. This new mechanism of triacylglycerol breakdown in adipocytes exposed to mild uncoupling provides new insights on the biology of adipocytes dealing with mitochondria forced to dissipate energy., (© 2017 Wiley Periodicals, Inc.)

An inherent compromise must be made between transverse resolution and depth of focus (DOF) in spectral domain optical coherence tomography (SD-OCT). Thus far, OCT has not been capable of providing a sufficient DOF to stably acquire cellular-resolution images. We previously reported a novel technique named multiple aperture synthesis (MAS) to extend the DOF in high-resolution OCT [Optica4, 701 (2017)]. In this technique, the illumination beam is scanned across the objective lens pupil plane by being steered at the pinhole using a custom-made microcylindrical lens. Images captured via multiple distinctive apertures were digitally refocused, which is similar to synthetic aperture radar. In this study, we applied this technique for the first time to image both a homemade microparticle sample and biological tissue. The results demonstrated the feasibility and efficacy of high-resolution biological tissue imaging with a dramatic DOF extension.

We compared histological structure of the orbital and subcutaneous adipose tissue. Quantitative morphometry showed that the mean diameter of adipocytes and their volume density in the orbital adipose tissue were significantly lower than in the subcutaneous fat, while volume density of vessels and elements of the connective tissue in the orbital adipose tissue was higher. Previously known and revealed here differences in the structure of orbital and subcutaneous adipose tissue do not answer the question, whether subcutaneous adipose tissue can be used for replacement of the orbital tissue deficit.

Background: Morbid obesity is related to chronic inflammation and many metabolic complications. Interleukin (IL)-6 plays a pivotal pathophysiological role in obesity, and IL-6 trans-signaling through the soluble IL-6 receptor (sIL-6R) has a major proinflammatory effect. The aim of this study was to investigate the association between sIL-6R, adipocyte size, and insulin resistance in morbidly obese individuals., Methods: We measured concentrations of sIL-6R, high-sensitivity C-reactive protein, and lipid parameters and estimated homeostasis model assessment of insulin resistance (HOMA-IR) before the patients underwent bariatric surgery. Mesenteric adipose tissue was collected during surgery, and adipocyte size and concentrations of membrane-bound IL-6 receptor (mIL-6R) were evaluated. In total, 35 adults (20 men and 15 women) were recruited., Results: The subjects with high HOMA-IR (≥2.4) had higher fasting glucose/insulin, triglycerides, sIL-6R, and adipocyte size and lower high-density lipoprotein cholesterol and mIL-6R than those with low HOMA-IR (<2.4). Adipocyte size positively correlated with sIL-6R (r = 0.559, P = 0.001) and HOMA-IR (r = 0.773, P ≤ 0.001) independent of age, gender, body mass index (BMI), waist, and use of diabetic drugs. In addition, every 1 ng/mL increase in sIL-6R concentration corresponded to a 10.2% decrease in the likelihood of maintaining lower insulin resistance. Furthermore, an sIL-6R level of 77.45 ng/mL was a reasonable cutoff level to propose lower insulin resistance in morbidly obese subjects., Conclusions: Circulating sIL-6R is more closely associated with insulin resistance status than waist-to-hip ratio or BMI in morbidly obese Taiwanese adults. sIL-6R may be a useful biomarker to assess insulin resistance among morbidly obese subjects.

Context: The role of the extracellular matrix (ECM) in regulating adipocyte metabolism in the context of metabolic disease is poorly defined., Objective: The objective of this study was to define the metabolic phenotype of adipocytes associated with human diabetes (DM) and the role of the ECM in regulating adipocyte metabolism., Design: Adipose tissues from obese patients were studied in standard 2-dimensional (2D) cell culture and an in vitro model of decellularized adipose tissue ECM repopulated with human adipocytes, and results were correlated with DM status., Setting: This study was conducted at the Academic University Medical Center and Veteran's Administration Hospital., Patients: Seventy patients with morbid obesity undergoing bariatric surgery were included in the study., Interventions: Visceral and subcutaneous adipose tissues were collected at the time of bariatric surgery., Outcome Measures: This study used metabolic assays for glucose uptake, lipolysis, and lipogenesis in adipocytes in 2D cell culture and 3-dimensional ECM culture., Results: Adipocytes from subjects with DM manifest decreased glucose uptake and decreased lipolysis in 2D culture. ECM supports differentiation of mature adipocytes and recapitulates DM-specific differences in adipocyte metabolism observed in 2D culture. ECM from subjects without DM partially rescues glucose uptake and lipolytic defects in adipocytes from subjects with DM, whereas ECM from subjects with DM impairs glucose uptake in adipocytes from subjects without DM., Conclusions: DM is associated with adipocyte metabolic dysfunction. The ECM regulates adipocyte metabolism. Nondiabetic ECM rescues metabolic dysfunction in DM adipocytes, whereas DM ECM imparts features of metabolic dysfunction to nondiabetic adipocytes. These findings suggest the ECM as a target for manipulating adipose tissue metabolism.

Nanoscale chemical mapping of newly-synthesised phospholipid molecules inside a mammalian cell is demonstrated using tip-enhanced Raman spectroscopy (TERS) for the first time using mouse pre-adipocyte cells as a model system. Newly-synthesised membrane phospholipid distribution within a pre-adipocyte cell is mapped with <20 nm spatial resolution, overcoming the diffraction limit of confocal Raman spectroscopy via plasmonic enhancement of Raman signals at a TERS tip-apex.

Aims: Enlarged adipocytes are a prime feature of adipose tissue dysfunction, and may be an appropriate target to decrease disease risk in obesity. We aimed to assess the change in adipocyte size in response to lifestyle and surgical weight loss interventions for overweight or obesity; and to explore whether certain participant and intervention characteristics influence this response., Methods: We systematically searched MEDLINE, EMBASE, CINAHL and Cochrane electronic databases to identify weight loss studies that quantified adipocyte size before and after the intervention. Using meta-regression analysis, we assessed the independent effects of weight loss, age, sex, adipocyte region, and intervention type (surgical vs. lifestyle) on adipocyte size reduction. We repeated the model as a sensitivity analysis including only the lifestyle interventions., Results: Thirty-five studies met our eligibility criteria. In our main model, every 1.0% weight loss was associated with a 0.64% reduction in adipocyte size (p=0.003); and adipocytes from the upper body decreased 5% more in size than those in the lower body (p=0.009). These relationships were no longer significant when focusing only on lifestyle interventions. Moreover, age, sex and intervention type did not independently affect adipocyte size reduction in either model., Conclusions: Weight loss in obese individuals is consistently associated with a decrease in adipocyte size that is more pronounced in upper-body adipocytes. It remains to be clarified how biological differences and intervention characteristics influence this relationship, and whether it corresponds with reductions in other aspects of adipose tissue dysfunction and disease risk., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The pro-inflammatory profile of M1 macrophage accumulation in adipose tissue is a central event leading to the metabolic complications of obesity. However, the mechanisms by which M1 macrophages are enriched in adipose tissue during weight gain remain incompletely understood. Here, we investigated the effects of adipocyte-derived microvesicles (ADM) on modulating macrophage phenotype in mice and explored the involved molecular signalling pathways. We found that, compared with ADM from lean mice (SD ADM), ADM from obese mice (HFD ADM) significantly enhanced M1 marker expression. The quantitative RT-PCR assay demonstrated that miR-155 was upregulated in both HFD ADM and HFD ADM-treated macrophages. By depleting miR-155 expression in HFD ADM and increasing miR-155 level in SD ADM, we further illustrated that miR-155 in ADM-induced M1 macrophage polarization. Functionally, in contrast to SD ADM, HFD ADM significantly decreased the protein level of SOCS1, a proven miR-155 target, leading to activation of STAT1, and suppression of STAT6 signalling; these effects were reversed by silencing miR-155 in HFD ADM. Furthermore, the supernatant of bone marrow-derived macrophages pre-stimulated with miR-155-bearing ADM interfered with insulin signalling and insulin-induced glucose uptake in adipocytes. Collectively, these results provide the first evidence that M1 macrophage polarization can be mediated by miR-155-bearing ADM, which reciprocally regulates insulin signalling and glucose uptake in adipocytes. Our study reveals a novel mechanism through which obesity induces an imbalance in the M1-to-M2 macrophage ratio in adipose tissue, thus causing chronic inflammation and local insulin resistance., (© The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

Adipose-derived stem cells (ASCs) with multilineage potential can be induced into osteoblasts, adipocytes and chondrocytes. ASCs as seed cell are widely used in the field of tissue engineering, but most studies either use autologous cells as the source or an immunodeficient animal as the host. In our present study, we explored the feasibility of applying allogeneic ASCs and demineralized bone matrix (DBM) scaffolds for repairing tubular bone defects without using immunosuppressive therapy. Allogeneic ASCs were expanded and seeded on DBM scaffolds and induced to differentiate along the osteogenic lineage. Eight Sprague-Dawley (SD) rats were used in this study and bilateral critical-sized defects (8 mm) of the ulna were created and divided into two groups: with ASC-DBM constructs or DBM alone. The systemic immune response and the extent of bone healing were evaluated post-operatively. Twenty-four weeks after implantation, digital radiography (DR) testing showed that new bones had formed in the experimental group. By contrast, no bone tissue formation was observed in the control group. This study demonstrated that allogeneic ASCs could promote bone regeneration and repair tubular bone defects combined with DBM by histologically typical bone without systemic immune response., (© 2015 by the Society for Experimental Biology and Medicine.)

Adipose tissue, an active metabolic and endocrine organ mainly composed of unilocular adipocytes, is implicated in various obesity related diseases. Developing morphologically and functionally accurate in vitro models of the adipose tissue is therefore critically important for basic biological studies, drug screening/testing, and clinical implants to advance the understanding and treatment of these diseases. However, current adipose tissue engineering technologies either cannot replicate the unilocular morphologies of mature adipocytes, or lack the ease of monitoring, handling, and scaling up required in the above mentioned applications. This paper presents the differentiation of adipose derived stem cells (ADSCs) to mature adipocytes in highly observable and highly handleable 3D fiber shaped constructs exhibiting morphologies and functions of native adipose tissues. Using the cell fiber technology, ADSCs were encapsulated in hydrogel microfibers, allowed to form into fiber shaped constructs, and differentiated to mature unilocular adipocytes. These adipocyte fibers are observed and maintained for up to 91 d, and secretion of adipose tissue-specific factor, adiponectin, is further confirmed. The handleability of the adipocyte fibers is demonstrated by assembling the adipocyte fibers into doll shaped constructs. Such highly observable, highly handleable, and scalable characteristics of the adipocyte fibers make them suitable for biological studies, high-throughput drug screening/testing, and clinical applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The insulin-sensitive pool of glucose transporter isoform 4 (GLUT4) can be isolated from total cell membranes using the 16K fractionation protocol, described here. This method produces a light membrane-containing supernatant that includes the insulin-sensitive pool of GLUT4 in GLUT4 storage vesicles. The 16K pellet fraction contains the heavy membranes (including the plasma membrane, mitochondria, nuclei, Golgi apparatus, and endoplasmic reticulum). The distribution of proteins between the two fractions is determined via immunoblotting. By subjecting insulin-stimulated versus unstimulated cells to this protocol, the mobilization of proteins out of the insulin-sensitive GLUT4 pool can be assessed., (© 2016 Cold Spring Harbor Laboratory Press.)

The dynamic nature of insulin-sensitive glucose transporter isoform 4 (GLUT4) storage vesicles (GSVs) makes their characterization challenging. Fractionation techniques can facilitate isolation of GSVs from insulin-sensitive cells. In this protocol, we describe preparation of a total membrane fraction from 3T3-L1 adipocytes. The resulting pellet contains all membranes and allows for easier identification of membrane proteins, including the insulin-sensitive pool of GLUT4. A method for concentration of the soluble fraction is also included., (© 2016 Cold Spring Harbor Laboratory Press.)

The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals., (© 2016 Society for Endocrinology.)

Background: Fat tissue graft has been commonly used for soft tissue augmentation. However, the mechanisms underlying the maintenance of graft volume and weight are still unclear. As morphological features provide direct evidences for cell death and survival, we aimed to investigate the fate of grafted adipocytes and the dynamic changes in the remodeling of adipose tissues by transmission electron microscopy technique., Methods: The unilateral inguinal fat pad of C57BL/6J mice was autografted to the dorsa of the mice. Perilipin expression and morphological changes were investigated by immunohistochemistry staining and transmission electron microscopy, respectively, in grafted tissues collected at posttransplantation days 0, 1, 3, 5, 7, 14, and 30., Results: Transmission electron microscopy analysis revealed that most adipocytes in grafts showed traits of cell death on postgrafting day 3. Multilocular adipocytes with naive nuclei were observed as early as day 5 and a larger number of multilocular adipocytes were found on day 14. Perilipin immunostaining revealed that only some adipocytes located in the margin of grafts survived through the ischemic injury. New adipocytes were visualized at the periphery of the grafts, although the scope of viable adipocyte zonal areas increased from day 5 to day 30., Conclusions: The results provide ultrastructural evidences associated with the remodeling dynamics of adipose tissue grafts. It is suggested that maximized volume of graft should be obtained through promoting regeneration other than improving survival of grafted adipose tissues.

Obesity-induced endoplasmic reticulum (ER) stress and inflammation lead to adipocytes dysfunction. Autophagy helps to adapt to cellular stress and involves in regulating innate inflammatory response. In present study, we examined the activity of rapamycin, a mTOR kinase inhibitor, against endoplasmic reticulum stress and inflammation in adipocytes. An in vitro model was used in which 3T3-L1 adipocytes were preloaded with palmitate (PA) to generate artificial hypertrophy mature adipocytes. Elevated autophagy flux and increased number of autophagosomes were observed in response to PA and rapamycin treatment. Rapamycin attenuated PA-induced PERK and IRE1-associated UPR pathways, evidenced by decreased protein levels of eIF2α phosphorylation, ATF4, CHOP, and JNK phosphorylation. Inhibiting autophagy with chloroquine (CQ) exacerbated these ER stress markers, indicating the role of autophagy in ameliorating ER stress. In addition, cotreatment of CQ abolished the anti-ER stress effects of rapamycin, which confirms the effect of rapamycin on ERs is autophagy-dependent. Furthermore, rapamycin decreased PA-induced nuclear translocation of NFκB P65 subunit, thereby NFκB-dependent inflammatory cytokines MCP-1 and IL-6 expression and secretion. In conclusion, rapamycin attenuated PA-induced ER stress/NFκB pathways to counterbalance adipocytes stress and inflammation. The beneficial of rapamycin in this context partly depends on autophagy. Stimulating autophagy may become a way to attenuate adipocytes dysfunction.

Subcutaneous white adipose tissue harvested by liposuction has been studied with the aim to understand how the adipocytes modify their morphology when subjected to the passage in a needle for liposuction and to cryopreservation. The work try to clarify the ultrastructural aspects of adipose tissue, in the conditions described before, examining samples of body fat employed in fat graft procedures, and samples after cryopreservation. Scanning and transmission electron microscopy show that the first event that occur in the adipocytes is a lesion of mild degree detectable early in the samples fixed immediately after liposuction. The sequence of events following the adipocyte stress appeared composed by different phases: plasmatic membrane interruption, loss of lipid charge, formation of cup-like adipocytes and formation of post-adipocytes (i.e. cells that survive to traumatic events and restart to internalize lipid droplets). In conclusion, the study suggests that the loss of lipid charge in adipose cell is an active process that can be due to a small hole in the cytoplasmic membrane with the preservation of a large part of the cytoplasmatic content and that at the end of the process of lipid extrusion the cell can maintain viability.

The lysosomal degradation pathway, autophagy, is essential for the maintenance of cellular homeostasis. Recently, autophagy has been demonstrated to be required in the process of adipocyte conversion. However, its role in mature adipocytes under physiological and pathological conditions remains unclear. Here, we report a major function of BECN1 in the regulation of basal autophagy in mature adipocytes. We also show that berberine, a natural plant alkaloid, inhibits basal autophagy in adipocytes and adipose tissue of mice fed a high-fat diet via downregulation of BECN1 expression. We further demonstrate that berberine has a pronounced effect on the stability of Becn 1 mRNA through the Mir30 family. These findings explore the potential of BECN1 as a key molecule and a drug target for regulating autophagy in mature adipocytes.

Objective: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined., Methods: EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes., Results: Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR., Conclusions: A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated., (Copyright © 2013 The Obesity Society.)

Objective: To determine the cellular architecture of the inflammatory infiltrate in adipose tissue from obese mice, and identify the source of inflammatory cytokines in adipose tissue at a single cell level., Methods: Adipose tissue from diet-induced obese mice was digested by collagenase treatment and fractionated by density centrifugation to obtain an adipocyte floating layer and a pellet of stromal vascular cells. The cellular architecture of the adipocyte-macrophage interaction in both intact white adipose tissue (WAT) and the separated density gradient floating layer fraction was analyzed by confocal immunohistochemistry. Cytokine expression was detected by semi-quantitative real time PCR and immunohistochemical analysis., Results: Three dimensional image analysis of WAT and the separated "adipocyte" floating layer revealed lipid-engorged macrophages, macrophages in contact with lipid droplets and sheath-like assemblies of macrophages surrounding adipocytes. The macrophages immunostained for TNFα and to a lesser extent for the immunoregulatory cytokine IL-10. TNFα staining was associated only with macrophages indicating that macrophages and not adipocytes are the source of TNFα expression in the adipocyte floating layer., Conclusion: Macrophages form assemblies that tightly adhere to and cover adipocytes and lipid droplets. TNFα found in low density adipocyte preparations is due to contamination with macrophages., (Copyright © 2013 The Obesity Society.)

We report on the heterogeneity and diversity of lipid droplets (LDs) in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN) family, we could distinguish between endogenously derived LDs (endogenous LDs) positive for perilipin from exogenously induced LDs (exogenous LDs) positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF) vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.

Adipose tissue is composed of a variety of cell types that include mature adipocytes, endothelial cells, fibroblasts, adipocyte progenitors, and a range of inflammatory leukocytes. These cells work in concert to promote nutrient storage in adipose tissue depots and vary widely based on location. In addition, overnutrition and obesity impart significant changes in the architecture of adipose tissue that are strongly associated with metabolic dysfunction. Recent studies have called attention to the importance of adipose tissue microenvironments in regulating adipocyte function and therefore require techniques that preserve cellular interactions and permit detailed analysis of three-dimensional structures in fat. This chapter summarizes our experience with the use of laser scanning confocal microscopy for imaging adipose tissue in rodents., (© 2014 Elsevier Inc. All rights reserved.)

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.

Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol's role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Adipocytes are a cell population largely located in the human bone marrow cavity. In this specific microenvironment where adipocytes can interact with a variety of different cells, the role of fat is mainly unknown. To our knowledge, this report is the first to characterize mature adipocytes isolated from human bone marrow (BM-A) molecularly and functionally to better understand their roles into the hematopoietic microenvironment. Healthy BM-A were isolated after collagenase digestion and filtration. We studied the morphology of BM-A, their gene expression and immunophenotypic profile and their functional ability in the hematopoietic microenvironment, comparing them with adipocytes derived from adipose tissue (AT-A). BM-A showed a unilocular lipid morphology similar to AT-A and did not lose their morphology in culture; they showed a comparable pattern of stem cell-surface antigens to AT-A. In line with these observations, molecular data showed that BM-A expressed some embryonic stem cells genes, such as Oct4, KLf4, c-myc, Gata4, Tbx1, and Sox17, whereas they did not express the stem cell markers Sox2 and Nanog. Moreover, BM-A had long telomeres that were similar to bone marrow mesenchymal stem cells. Notably, BM-A supported the survival and differentiation of hematopoietic stem cells in long-term cultures. These results showed that BM-A are stromal cells with a gene expression pattern that distinguished them from AT-A. BM-A showed stem cell properties through their hematopoietic supporting function, which was certainly linked to their role in the maintenance of the bone marrow microenvironment. Depending on specific demands, BM-A may acquire different functions based on their local environment., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. Here, we studied the gene expression and functional properties of mature adipocytes isolated from human omental and subcutaneous fat tissues. We also focused on dedifferentiated adipocytes in culture and their morphologies and functional changes with respect to mature adipocytes, stromal-vascular fraction (SVF)-derived mesenchymal stem cells (MSCs) and bone marrow (BM)-derived MSCs. Isolated mature adipocytes expressed stem cell and reprogramming genes. They replicated in culture after assuming a fibroblast-like shape and expanded similarly to SVF- and BM-derived MSCs. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the typical mesenchymal morphology and immunophenotype, expressed stem cell genes and differentiated into multilineage cells. Moreover, during the dedifferentiation process, we showed changes in the epigenetic status of mature adipocytes, which led dedifferentiated adipocytes to display a similar DNA methylation condition to BM-derived MSCs. Like SVF- and BM-derived MSCs, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature adipocytes stimulated their growth. Furthermore, dedifferentiated adipocytes maintained the survival and complete differentiation characteristic of hematopoietic stem cells. This is the first study that in addition to characterizing isolated and dedifferentiated adipocytes also reports on the immunoregulatory and hematopoietic supporting functions of these cells. This structural and functional characterization might have clinical applications of both mature and dedifferentiated adipocytes in such fields, as regenerative medicine., (Copyright © 2012 AlphaMed Press.)

Fish final product can be affected by excessive lipid accumulation. Therefore, it is important to develop strategies to control obesity in cultivated fish to strengthen the sustainability of the aquaculture industry. As in mammals, the development of adiposity in fish depends on hormonal, cytokine and dietary factors. In this study, we investigated the proliferation and differentiation of preadipocytes isolated from the large yellow croaker and examined the effects of critical factors such as insulin, TNFα and DHA on the proliferation, differentiation and lipolysis of adipocytes. Preadipocytes were isolated by collagenase digestion, after which their proliferation was evaluated. The differentiation process was optimized by assaying glycerol-3-phosphate dehydrogenase (GPDH) activity. Oil red O staining and electron microscopy were performed to visualize the accumulated triacylglycerol. Gene transcript levels were measured using SYBR green quantitative real-time PCR. Insulin promoted preadipocytes proliferation, stimulated cell differentiation and decreased lipolysis of mature adipocytes. TNFα and DHA inhibited cell proliferation and differentiation. While TNFα stimulated mature adipocyte lipolysis, DHA showed no lipolytic effect on adipocytes. The expressions of adipose triglyceride lipase (ATGL), fatty acid synthase (FAS), lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor α, γ (PPARα, PPARγ) were quantified during preadipocytes differentiation and adipocytes lipolysis to partly explain the regulation mechanisms. In summary, the results of this study indicated that although preadipocytes proliferation and the differentiation process in large yellow croaker are similar to these processes in mammals, the effects of critical factors such as insulin, TNFα and DHA on fish adipocytes development are not exactly the same. Our findings fill in the gaps in the basic data regarding the effects of critical factors on adiposity development in fish and will facilitate the further study of molecular mechanism by which these factors act in fish and the application of this knowledge to eventually control obesity in cultured species.

Background: Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species., Methods: Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro-computed tomography and histology., Results: Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells., Conclusions: Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal cells.

Decreased serum creatinine concentration has been recently described to constitute a new risk factor of type 2 diabetes. Increased free radicals have been consistently associated with decreased serum creatinine and with cellular senescence. Telomere length is considered as a biological marker for senescence. We aimed to study the association of telomere length with serum creatinine. Telomere length of subcutaneous adipose tissue cells was measured in a sample of obese and nonobese subjects (n = 49). Telomere length of subcutaneous adipose tissue cells was positively associated with serum creatinine (r = 0.40, P = 0.004), i.e., the lower the telomere length, the lower the serum creatinine, but not with glomerular filtration rate (GFR). In addition, telomere length was negatively associated with BMI (r = -0.45, P = 0.001) and systolic blood pressure (r = -0.41, P = 0.003). In a multiple linear regression analysis, BMI (P = 0.005), systolic blood pressure (P = 0.01) and telomere length (P = 0.03) independently contributed to 37% of serum creatinine variance after controlling for sex and age. In conclusion, the association of serum creatinine with a marker of cellular senescence suggests an underlying mechanism influencing both decreased serum creatinine and increased risk of type 2 diabetes.

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.

In the present work the effects of a new low frequency, high intensity ultrasound technology on human adipose tissue ex vivo were studied. In particular, we investigated the effects of both external and surgical ultrasound-irradiation (10 min) by evaluating, other than sample weight loss and fat release, also histological architecture alteration as well apoptosis induction. The influence of saline buffer tissue-infiltration on the effects of ultrasound irradiation was also examined. The results suggest that, in our experimental conditions, both transcutaneous and surgical ultrasound exposure caused a significant weight loss and fat release. This effect was more relevant when the ultrasound intensity was set at 100 % (~2.5 W/cm², for external device; ~19-21 W/cm2, for surgical device) compared to 70 % (~1.8 W/cm² for external device; ~13-14 W/cm2 for surgical device). Of note, the effectiveness of ultrasound was much higher when the tissue samples were previously infiltrated with saline buffer, in accordance with the knowledge that ultrasonic waves in aqueous solution better propagate with a consequently more efficient cavitation process. Moreover, the overall effects of ultrasound irradiation did not appear immediately after treatment but persisted over time, being significantly more relevant at 18 h from the end of ultrasound irradiation. Evaluation of histological characteristics of ultrasound-irradiated samples showed a clear alteration of adipose tissue architecture as well a prominent destruction of collagen fibers which were dependent on ultrasound intensity and most relevant in saline buffer-infiltrated samples. The structural changes of collagen bundles present between the lobules of fat cells were confirmed through scanning electron microscopy (SEM) which clearly demonstrated how ultrasound exposure induced a drastic reduction in the compactness of the adipose connective tissue and an irregular arrangement of the fibers with a consequent alteration in the spatial architecture. The analysis of the composition of lipids in the fat released from adipose tissue after ultrasound treatment with surgical device showed, in agreement with the level of adipocyte damage, a significant increase mainly of triglycerides and cholesterol. Finally, ultrasound exposure had been shown to induce apoptosis as shown by the appearance DNA fragmentation. Accordingly, ultrasound treatment led to down-modulation of procaspase-9 expression and an increased level of caspase-3 active form.

Background: Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT., Methods and Results: Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-β highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%., Conclusion: Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes.

A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the 'linker of nucleus and cytoskeleton' (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.

This communication extends the recently reported cell-specific finite element (FE) method in Slomka and Gefen (2010) in which geometrically realistic FE cell models are created from confocal microscopy scans for large deformation analyses. The cell-specific FE method is extended here in the following aspects: (i) we demonstrate that cell-specific FE is versatile enough to deal with cells of substantially different geometrical shapes. The examples of an "elongated" pre-adipocyte and a "round" mature adipocyte are used to demonstrate this feature. (ii) We demonstrate that cell-specific FE can be used to analyze the mechanical behavior of cells that incorporate complex intracellular structures and are subjected to large deformations--again through the example of an adipocyte which contains a multitude of lipid droplets, each having a different size and shape. By demonstrating feasibility of inclusion of such inhomogeneities in the cytoplasm, the present work paves the way for modeling cellular organelles such as Golgi bodies, lysosomes and mitochondria in mechanically loaded cells using cell-specific FE., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Abnormal changes during fat formation are closely related to the prevalence of many diseases. In order to understand the formation mechanism of fat, we used atomic force microscopy (AFM) to characterize the morphology and mechanical properties of porcine preadipocytes during the differentiation. Preadipocytes and adipocytes were different morphologically. The surface roughness of adipocytes was less than preadipocytes by detection of the ultrastructure. The mechanical properties of preadipocytes were changed during differentiation with AFM-based force spectroscopy. Preadipocytes were 20% higher than adipocytes in the adhesion force, stiffness and Young's modulus. Therefore, AFM analysis of membrane changes related to adipocytes formation provided quantitative data in the nanometer level for further studying the formation mechanism of the adipocytes.

Adipose tissue is currently being recognized as an important endocrine organ, carrying defects in a number of metabolic diseases. Mitochondria play a key role in normal adipose tissue function and mitochondrial alterations can result in pathology, like lipodystrophy or type 2 diabetes. Although Pgc1α is regarded as the main regulator of mitochondrial function, downstream Nrf1 is the key regulator of mitochondrial biogenesis. Nrf1 is also involved in a wide range of other processes, including proliferation, innate immune response, and apoptosis. To determine transcriptional targets of Nrf1, 3T3-L1 preadipocytes were transfected with either pNrf1 or a control vector. Two days post-confluence, 3T3-L1 preadipocytes were allowed to differentiate. At day 8 of differentiation, Nrf1 overexpressing cells had an increased mtDNA copy number and reduced lipid content. This was not associated with an increased ATP production rate per cell. Using global gene expression analysis, we observed that Nrf1 overexpression stimulated cell proliferation, apoptosis, and cytokine expression. In addition, prolonged Nrf1 induced an adipokine expression profile of insulin resistant adipocytes. Nrf1 has a wide range of transcriptional targets, stimulators as well as inhibitors of adipose tissue functioning. Therefore, post-transcriptional regulation of Nrf1, or stimulating specific Nrf1 targets may be a more suitable approach for stimulating mitochondrial biogenesis and treating adipose tissue defects, instead of directly stimulating Nrf1 expression. In addition, our results show that short-term effects can drastically differ from long-term effects., (© 2010 Wiley-Liss, Inc.)

Ultrastructural and histochemical studies of bioptic and postmortem tissue samples from ten Fabry hemizygotes showed lysosomal storage in adipocytes as a constant feature of the classic phenotype of α-galactosidase (GLA) deficiency. The storage was represented by a crescent-shaped line of storage lysosomes of varying thicknesses restricted to the perinuclear subplasmalemmal area. The ultrastructure of the storage lysosomes was dominated by concentric lipid membranes modified by simultaneous deposition of autofluorescent ceroid. Storage was widely expressed in adipose tissue. The number of storage lysosomes was increased, and the lysosomes were more clustered in adipocytes with less voluminous lipid content. The findings should attract interest to studies of adipose tissue biology in Fabry disease, a topic that has not been studied so far. In terms of cell biology, the observations represent indirect evidence of significant lysosomal turnover of α-galactose lipid conjugates in adipocytes demasked by GLA deficiency. The results extend the thus far limited information on the adipocyte lysosomal system and its participation in lysosomal storage disorders.

TNF-alpha was the first proinflammatory cytokine identified linking obesity, insulin resistance and chronic inflammation. However, the mechanism of TNF-alpha in the etiology of insulin resistance is still far from clear. Because the mitochondria play an important role in energy metabolism, we investigated whether mitochondrial dysfunction is involved in pathogenesis of TNF-alpha-mediated insulin resistance. First, a fully differentiated insulin-resistant 3T3-L1 adipocyte model was established by incubating with 4 ng/ml TNF-alpha for 4 d, and then the mitochondrial morphology and functions were observed. TNF-alpha treatment induced pronounced morphological changes in the mitochondria, which became smaller and condensed, and some appeared hollow and absent of cristae. Mitochondrial dynamics changes were observed as increased mitofusion protein mfn1 and mitofission protein Drp1 levels compared with controls. No obvious effects on mitochondrial biogenesis were found. PGC-1alpha levels decreased, but no significant changes were found in mtTFA mRNA expression, NRF1mRNA expression and mitochondrial DNA (mtDNA). TNFalpha treatment also led to decreased mitochondrial membrane potential and reduced production of intracellular ATP, as well as accumulation of significant amounts of reactive oxygen species (ROS). Further research is required to determine if mitochondrial dysfunction is involved in the inflammatory mechanism of insulin resistance and may be a potential target for the treatment of insulin resistance., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

NYGGF4 is a recently discovered gene that is involved in obesity-associated insulin resistance. It has been suggested that mitochondrial dysfunction might be responsible for the development of insulin resistance induced by NYGGF4 overexpression. In the present study, we aimed to define the impact of down-regulating NYGGF4 expression by RNA interference (RNAi) on the insulin sensitivity and mitochondrial function of 3T3-L1 adipocytes. The results revealed that NYGGF4 knockdown enhanced the glucose uptake of adipocytes, which reconfirmed the regulatory function of NYGGF4 in adipocyte insulin sensitivity. However, an unexpected observation was that knockdown of NYGGF4 reduced intracellular ATP concentration and promoted an increase in mitochondrial transmembrane potential (ΔΨm) and reactive oxygen species (ROS) level without affecting mitochondrial morphology or mtDNA. Therefore, the role of NYGGF4 in mitochondrial function remains unclear, and further animal studies are needed to explore the biological function of this gene.

Adipose tissue lipoatrophy caused by caveolin gene deletion in mice is not linked to defective adipocyte differentiation. We show that adipose tissue development cannot be rescued by endothelial specific caveolin-1 re-expression, indicating primordial role of caveolin in mature adipocytes. Partial or total caveolin deficiency in adipocytes induced broad protein expression defects, including but not limited to previously described downregulation of insulin receptor. Global alterations in protein turnover, and accelerated degradation of long-lived proteins were found in caveolin-deficient adipocytes. Lipidation of endogenous LC3 autophagy marker and distribution of GFP-LC3 into aggregates demonstrated activated autophagy in the absence of caveolin-1 in adipocytes. Furthermore, electron microscopy revealed autophagic vacuoles in caveolin-1 deficient but not control adipocytes. Surprisingly, significant levels of lipidated LC3-II were found around lipid droplets of normal adipocytes, maintained in nutrient-rich conditions or isolated from fed mice, which do not display autophagy. Altogether, these data indicate that caveolin deficiency induce autophagy in adipocytes, a feature that is not a physiological response to fasting in normal fat cells. This likely resulted from defective insulin and lipolytic responses that converge in chronic nutrient shortage in adipocytes lacking caveolin-1. This is the first report of a pathological situation with autophagy as an adaptative response to adipocyte failure.

Long-term clinical use of glucocorticoids often causes the serious side effect of non-traumatic avascular osteonecrosis. The aim of this study was to examine the effects and mechanisms of a glucocorticoid, dexamethasone (Dex), on differentiation of primary cultured rat bone marrow mesenchymal cells (BMCs). We also tried to block the inhibitory effects of Dex on osteoblast differentiation. Adipocyte markers (peroxisome proliferator-activated receptorgamma-2 and aP2) were increased in response to Dex treatment in a dose- and time-dependent manner, while osteoblastic markers [Runx2, COL 1, osterix, alkaline phosphatase (ALP) and OC] were down-regulated, consistent with ALP and osteocalcin promoter activity. To validate the effects of Runx2 on the expression of osteogenesis and adipocyte genes, pCMV/Flag-Runx2 was transfected into BMCs, and relevant markers were detected after 10(-7) M Dex treatment for 48 h. The results indicated that Dex treatment induced adipogenic differentiation and suppressed proliferation. No significant difference was detected in expressions of these genes between Runx2-transfected cells and Dex-treated BMCs. These data suggest that Dex primarily induced adipocyte differentiation of BMCs. Exogenous Runx2 can antagonize the effect of Dex on osteoblast differentiation.