Human Adipose Tissue Research Articles

Novel Potential Markers of Myofibroblast Differentiation Revealed by Single-Cell RNA Sequencing Analysis of Mesenchymal Stromal Cells in Profibrotic and Adipogenic Conditions

Mesenchymal stromal cells (MSCs) are the key regulators of tissue homeostasis and repair after damage. Accumulating evidence indicates the dual contribution of MSCs into the development of fibrosis induced by chronic injury: these cells can suppress the fibrotic process due to paracrine activity, but their promoting role in fibrosis by differentiating into myofibroblasts has also been demonstrated. Many model systems reproducing fibrosis have shown the ability of peroxisome proliferator-activated receptor (PPAR) agonists to reverse myofibroblast differentiation. Thus, the differentiation of multipotent cells into myofibroblasts and adipocytes can be considered as processes that require the activation of opposite patterns of gene expression. To test this hypothesis, we analyzed single cell RNA-Seq transcriptome of human adipose tissue MSCs after stimulation of the myofibroblast or adipogenic differentiation and revealed several genes that changed their expression in a reciprocal manner upon these conditions. We validated the expression of selected genes by RT-PCR, and evaluated the upregulation of several relevant proteins using immunocytochemistry, refining the results obtained by RNA-Seq analysis. We have shown, for the first time, the expression of neurotrimin (NTM), previously studied mainly in the nervous tissue, in human adipose tissue MSCs, and demonstrated its increased gene expression and clustering of membrane receptors upon the stimulation of myofibroblast differentiation. We also showed an increased level of CHD3 (Chromodomain-Helicase-DNA-binding protein 3) in MSCs under profibrotic conditions, while retinol dehydrogenase-10 (RDH10) was detected only in MSCs after adipogenic induction, which contradicted the data of transcriptomic analysis and again highlights the need to validate the data obtained by omics methods. Our findings suggest the further analysis of the potential contribution of neurotrimin and CHD3 in the regulation of myofibroblast differentiation and the development of fibrosis.

Sustained Release of Human Adipose Tissue Stem Cell Secretome from Star-Shaped Poly(ethylene glycol) Glycosaminoglycan Hydrogels Promotes Motor Improvements after Complete Transection in Spinal Cord Injury Rat Model.

Adipose tissue-derived stem cells (ASCs) have been shown to assist regenerative processes after spinal cord injury (SCI) through their secretome, which promotes several regenerative mechanisms, such as inducing axonal growth, reducing inflammation, promoting cell survival, and vascular remodeling, thus ultimately leading to functional recovery. However, while systemic delivery (e.g., i.v. [intravenous]) may cause off-target effects in different organs, the local administration has low efficiency due to fast clearance by body fluids. Herein, a delivery system for human ASCs secretome based on a hydrogel formed of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan heparin (Hep) that is suitable to continuously release pro-regenerative signaling mediators such as interleukin (IL)-4, IL-6, brain-derived neurotrophic factor, glial-cell neurotrophic factor, and beta-nerve growth factor over 10 days, is reported. The released secretome is shown to induce differentiation of human neural progenitor cells and neurite outgrowth in organotypic spinal cord slices. In a complete transection SCI rat model, the secretome-loaded hydrogel significantly improves motor function by reducing the percentage of ameboid microglia and systemically elevates levels of anti-inflammatory cytokines. Delivery of ASC-derived secretome from starPEG-Hep hydrogels may therefore offer unprecedented options for regenerative therapy of SCI.

TNFα-induced NLRP3 inflammasome mediates adipocyte dysfunction and activates macrophages through adipocyte-derived lipocalin 2

Background and aimsObesity is a state of chronic low-grade systemic inflammation. Recent studies showed that NLRP3 inflammasome initiates metabolic dysregulation in adipose tissues, primarily through activation of adipose tissue infiltrated macrophages. However, the mechanism of NLRP3 activation and its role in adipocytes remains elusive. Therefore, we aimed to examine the activation of TNFα-induced NLRP3 inflammasome in adipocytes and its role on adipocyte metabolism and crosstalk with macrophages. MethodsThe effect of TNFα on adipocyte NLRP3 inflammasome activation was measured. Caspase-1 inhibitor (Ac-YVAD-cmk) and primary adipocytes from NLRP3 and caspase-1 knockout mice were utilized to block NLRP3 inflammasome activation. Biomarkers were measured by using real-time PCR, western blotting, immunofluorescence staining, and enzyme assay kits. Conditioned media from TNFα-stimulated adipocytes was used to establish the adipocyte-macrophage crosstalk. Chromatin immunoprecipitation assay was used to identify the role of NLRP3 as a transcription factor. Mouse and human adipose tissues were collected for correlation analysis. ResultsTNFα treatment induced NLRP3 expression and caspase-1 activity in adipocytes, partly through autophagy dysregulation. The activated adipocyte NLRP3 inflammasome participated in mitochondrial dysfunction and insulin resistance, as evidenced by the amelioration of these effects in Ac-YVAD-cmk treated 3T3-L1 cells or primary adipocytes isolated from NLRP3 and caspase-1 knockout mice. Particularly, the adipocyte NLRP3 inflammasome was involved in glucose uptake regulation. Also, TNFα induced expression and secretion of lipocalin 2 (Lcn2) in a NLRP3-dependent manner. NLRP3 could bind to the promoter and transcriptionally regulate Lcn2 in adipocytes. Treatment with adipocyte conditioned media revealed that adipocyte-derived Lcn2 was responsible for macrophage NLRP3 inflammasome activation, working as a second signal. Adipocytes isolated from high-fat diet mice and adipose tissue from obese individuals showed a positive correlation between NLRP3 and Lcn2 gene expression. ConclusionsThis study highlights the importance of adipocyte NLRP3 inflammasome activation and novel role of TNFα-NLRP3-Lcn2 axis in adipose tissue. It adds rational for the current development of NLRP3 inhibitors for treating obesity-induced metabolic diseases.

Exploring Adipose Tissue Functions.

Cho, D. S., Doles, J. D. Preparation of adipose progenitor cells from mouse epididymal adipose tissues. Journal of Visualized Experiments. (162), doi: 10.3791/61694 (2020). Peics, J. et al. Isolation of adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal adipose depots. Journal of Visualized Experiments. (162), doi: 10.3791/61610 (2020). Estrada-Gutierrez, G. et al. Isolation of viable adipocytes and stromal vascular fraction from human visceral adipose tissue suitable for RNA analysis and macrophage phenotyping. Journal of Visualized Experiments. (164), doi: 10.3791/61884 (2020). Gilleron, J. et al. Exploring adipose tissue structure by methylsalicylate clearing and 3D imaging. Journal of Visualized Experiments. (162), doi: 10.3791/61640 (2020). Czepielewski, R. S. et al. Lymphatic and blood network analysis during obesity. Journal of Visualized Experiments. (165), doi: 10.3791/61814 (2020). Jager, J., Gaudfrin, M., Gilleron, J., Cormont, M., Tanti, J. F. An adipocyte cell culture model to study the impact of protein and micro-RNA modulation on adipocyte function. Journal of Visualized Experiments. (171), doi: 10.3791/61925 (2021). Poret, J. M., Molina, P. E., Simon, L. Isolation, proliferation and differentiation of rhesus macaque adipose-derived stem cells. Journal of Visualized Experiments. (171), doi: 10.3791/61732 (2021). Batista Jr., M. L., Meshulam, T., Desevin, K., Rabhi, N., Farmer, S. R. Three-dimensional adipocyte culture as a model to study cachexia-induced white adipose tissue remodeling. Journal of Visualized Experiments. (167), doi: 10.3791/61853 (2021). Akbar, N., Pinnick, K. E., Paget, D., Choudhury, R. P. Isolation and characterization of human adipocyte-derived extracellular vesicles using filtration and ultracentrifugation. Journal of Visualized Experiments. (170), doi: 10.3791/61979 (2021).

Gender differences in the risk of developing obesity related disease can be explained by sex‐specific molecular signatures that regulates adipose endothelial cell growth

Obesity is the excessive growth of adipose tissue resulting from the chronic consumption of excess calories and is a recognised risk factor for developing cardio-metabolic diseases. There is however a significant variation in the size and expandibility of different adipose tissue in humans. The inability of adipose tissue to expand “safely” leads to ectopic fat deposition, causing insulin resistance, type 2 diabetes and cardiovascular conditions – which are more prevalent in men than women. Thus, the mechanisms that control the expandability of adipose tissue are key factors in determining diabetes risk in obesity. An interesting study from York University have provided new insights on the biological underpinning in gender differences in the risk of developing obesity-related diseases. Previous study from this group has observed that when mice become obese, females grow more new blood vessels to supply the expanding fat tissue with oxygen and nutrients, whereas males grow a lot less. To understand the mechanism regulating this, the team used software to help sift through thousands of genes to zero in on the ones that would be associated with blood vessel growth. They discovered that processes associated with the proliferation of new blood vessels were high in the female mice, whereas the males had a high level of processes associated with inflammation. Thereafter, the investigators examined the behaviour of the endothelial cells ex-vivo. Interestingly, they observed that even when these cells were studied ex-vivo, male and female endothelial cells still behave very differently from each other - female endothelial cells replicated faster, while male endothelial cells displayed greater sensitivity to an inflammatory stimulus. Further studies to investigate the effects of specific genes in humans that regulate endothelial cells growth will provide potential therapeutic target to manage and treat obesity related cardio-metabolic diseases. The study was funded by a grant through the Canadian Institutes of Health Research, as well as the Natural Science and Engineering Research Council of Canada and York's Faculty of Health.

MESENCHYMAL STEM-LIKE STROMAL CELLS FROM HUMAN SUBCUTANEOUS FAT AND POLYVINYLPYRROLIDONE-BASED POLYMERIC HYDROGELS: TOXICITY AND ADHESION

Relevance. Mesenchymal stem-like stromal cells (MSCs) represent a promising material for the therapy of restoration or regeneration of bone and cartilage tissues in the treatment of patients with injuries and diseases of the musculoskeletal system. The use of hydrogels for cultivating MSCs will make it possible to protect them after being introduced into the body from to the potentially “hostile” environment of damaged or diseased tissues. To date, the properties of MSCs in their cultivation on matrices based on natural and synthetic materials have not been sufficiently studied. The aim of this work is to evaluate the toxicity and adhesive properties of hydrogels based on polyvinylpyrrolidone (PVP), obtained by two technolo-gies, for MSCs from human adipose tissue (AT) in vitro. Material and methods. Four sterile PVP hydrogels were used in the experiment, differing in the technology of preparation, the presence or absence of antibiotics. Toxicity was assessed by the number of vital cells (stained with 0.1% trypan blue solution) after adding the MSC suspension to the gels after 24 h. The behavior of MSCs (AT) was studied in dynamics (on days 2 and 5 of cultivation) in terms of the rate and quality of the formed cell monolayer. Morphological analysis of cells, the state of chromatin in the nucleus, and the presence of cytoplasmic inclusions were performed in sam-ples stained with Giemsa dye. Results. As a result, it was found that human MSCs (AT) are able to attach to the surface of all 4 matrices, represented by a physicochemically modi-fied artificial material based on PVP. Non-toxicity of all 4 hydrogels and their suitability for short-term cell culture were revealed. MSCs (AT) on PVP hy-drogels with the addition of antibiotics showed slower growth, while morphological changes were observed in the form of vacuoles in the protoplasm and pycnosis in the chromatin of the cell nucleus. Conclusion. Thus, the results obtained can be used for further research and improvement of methods for analyzing the cytotoxicity of the drugs un-der development.

Study on promoting the regeneration of grafted fat by cell-assisted lipotransfer

Cell-assisted lipotransfer (CAL), a modified adipose-derived stromal/stem cells (ADSCs)-based approach for autologous fat grafting that is an ideal option for soft tissue augmentation, has many shortcomings in terms of retention and adverse effects. The objective of our study was to improve the treatment efficacy of CAL by adding fibroblasts. ADSCs and fibroblasts were isolated from human adipose and dermal tissues, with fibroblasts identified by immunofluorescence and ADSCs identified by the multilineage differentiation method. We performed cell proliferation, apoptosis, migration, adipogenic, and hemangioendothelial differentiation experiments, qPCR and Western blotting analysis in co-cultures of fibroblasts and ADSCs. Subsequently, we conducted animal experiments with BALB/c nude mice. Masson's staining, immunofluorescence staining and ultrasound were used to analyze the occurrence of adverse reactions of the grafted fat, and CT and three-dimensional reconstruction were used to accurately evaluate the volume of the grafted fat. We found that the co-culture of fibroblasts and ADSCs promoted their mutual proliferation, adipogenic differentiation, hemangioendothelial differentiation and proliferation and migration of HUVECs. Fibroblasts inhibit the apoptosis of ADSCs. Moreover, in animal experiments, the autografted adipose group combined with ADSCs and fibroblasts had the least occurrence of oily cysts, and fat had the best form of survival. We enhanced adipocyte regeneration and angiogenesis in ADSCs and fibroblast cells after adding fibroblasts to conventional CAL autologous fat grafts. In turn, the volume retention rate of the grafted fat is improved, and the adverse reactions are reduced.

IDF2022-0574 Oxidative stress in human epicardial versus subcutaneous adipose tissue in heart failure patients

IDF2022-0574 Oxidative stress in human epicardial versus subcutaneous adipose tissue in heart failure patients

Abstract P2-15-06: Light-based bioprinting of vascularized fatty tissue for the development of biological breast implants

Abstract Although multi-modal and ablative treatment strategies for breast cancer are becoming more popular, partial or full mastectomy is still a most common intervention. While proving to be effective in curing the cancer, it leaves the patient disfigured, which can pose a considerable psychological burden. Hence, reconstructive breast surgery is continuing to increase in importance as it offers the possibility to at least partially restore the original look of the breast. Autologous adipose tissue transfer techniques, amongst them the surgical gold standard flap surgery and liposuction-based fat grafting, present natural solutions with the patient’s own tissue, but either produce a considerable secondary wound or are insufficient to address larger defects. Synthetic solutions like silicone implants on the other hand can also be used to fill large defects and only require minimal surgical intervention, but suffer from foreign body-associated complications like fibrosis. Tissue engineering biological breast implants of arbitrary size with no or minor insult to the patient’s body and excellent biocompatibility, exclusively using biomaterials and patient-specific adipose cells, could open a new chapter for breast reconstruction. Such an approach has become an intriguing possibility through the rise of powerful biofabrication technologies in the last two decades, namely three-dimensional bioprinting. Bioprinting marries additive manufacturing with biomaterials and living cells with the aim to create life-mimicking tissues and organs, and while it is mainly focused on research today, current endeavors push for application in regenerative medicine. In our work we sought to fabricate adipose tissue constructs using light-based bioprinting to investigate the potential application of such technology for future reconstructive breast surgery. The engineered tissue blocks were designed to mimic native human adipose tissue by comprising fat, connective tissue and endothelial cells. Being specifically placed by the bioprinter within extracellular matrix-like hydrogels, the cells were cultured in vitro and differentiated within the cultivation time of up to 4 weeks to form an adipose-like tissue. Live imaging showed high viability of bioprinted tissue blocks over the whole cultivation period and pronounced cellular reorganization. Confocal and histological analyses revealed a dynamic tissue maturation process with increasing proportions of differentiated adipocytes, ECM remodeling and formation of microvascular structures. In conclusion, the obtained results gave proof to the concept of engineering adipose tissue through bioprinting and could lay the groundwork for the development of all-natural biological breast implants. Citation Format: Nina Hedemann, Alexander Thomas, Nils Tribian, Lutz Kloke, Dirk Bauerschlag, Marion van Mackelenbergh. Light-based bioprinting of vascularized fatty tissue for the development of biological breast implants [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-15-06.

HASCs-derived exosomal miR-155-5p targeting TGFβR2 promotes autophagy and reduces pyroptosis to alleviate intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a complex chronic disease involving nucleus pulposus cells (NPCs) senescence, apoptosis, autophagy and extracellular matrix (ECM) degradation. In this study, we aimed to investigate the role of human adipose tissue stem cells (hASCs)-derived exosomal miR-155-5p targeting TGFβR2 in IDD and the mechanisms involved. Then miRNA sequencing was performed, and hASCs-derived Exo (hASCs-Exo) was extracted and characterized. First, NPCs were treated with different concentrations of LPS. Then miRNA sequencing was performed, and hASCs-Exo was extracted and characterized. NPCs were treated with PBS or autophagy inhibitor 3-MA. NPCs were transfected with miR-155-5p mimic, si-TGFβR2 and negative control. Cell viability, apoptosis, ROS, caspase-1+PI, pyroptosis markers, inflammatory cytokines, autophagy markers, Aggrecan, MMP13, and Akt/mTOR pathway-related factors were measured. Bioinformatics prediction and dual-luciferase were performed to verify the binding sites of miR-155-5p to TGFβR2. Finally, we validated the role of hASCs-derived exosomal miR-155-5p on IDD in vivo. LPS promoted pyroptosis of NPCs, and inhibited autophagy and ECM synthesis. MiR-155-5p was characterized as an inflammation-related miRNA in NPCs. HASCs-derived exosomal miR-155-5p inhibited pyroptosis of NPCs and promoted autophagy and ECM synthesis. After bioinformatics prediction and verification, it was found that miR-155-5p targeted TGFβR2. Moreover, miR-155-5p targeted TGFβR2 to promote autophagy and inhibit pyroptosis in NPCs. In vivo experiments revealed that hASCs-derived exosomal miR-155-5p alleviated IDD in rats. HASCs-derived exosomal miR-155-5p alleviated IDD by targeting TGFβR2 to promote autophagy and reduce pyroptosis. Our study may provide a new therapeutic target for IDD. HASCs-derived exosomal miR-155-5p is expected to be a biomarker for clinical treatment of IDD. Our study may provide a new therapeutic target for IDD.

Imaging-based assessment of fatty acid composition in human bone marrow adipose tissue at 7T: Method comparison and in vivo feasibility.

To demonstrate the feasibility and accuracy of chemical shift-encoded imaging of the fatty acid composition (FAC) of human bone marrow adipose tissue at 7T, and to determine suitable image-acquisition parameters using simulations. The noise performance of FAC estimation was investigated using simulations with a range of inter-echo time, and accuracy was assessed using a phantom experiment. Furthermore, one knee of 8 knee-healthy subjects (ages 35-54 years) was imaged, and the fractions of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) were mapped. Values were compared between reconstruction methods, and between anatomical regions. Based on simulations, ΔTE=0.6ms was chosen. The phantom experiment demonstrated high accuracy of especially SFA using a constrained reconstruction model (slope=1.1, average bias=-0.2%). The lowest accuracy was seen for PUFA using a free model (slope=2.0, average bias=9.0%). For in vivo images, the constrained model resulted in lower intersubject variation compared with the free model (e.g., in the femoral shaft, the SFA percent-point range was within 1.0% [vs. 3.0%]). Furthermore, significant regional FAC differences were detected. For example, using the constrained approach, the femoral SFA in the medial condyle was lower compared with the shaft (median [range]: 27.9% [27.1%, 28.4%] vs. 32.5% [31.8%, 32.8%]). Bone marrow adipose tissue FAC quantification using chemical-shift encoding is feasible at 7T. Both the noise performance and accuracy of the technique are superior using a constrained signal model.

Machine Learning Model Based on Insulin Resistance Metagenes Underpins Genetic Basis of Type 2 Diabetes.

Insulin resistance (IR) is considered the precursor and the key pathophysiological mechanism of type 2 diabetes (T2D) and metabolic syndrome (MetS). However, the pathways that IR shares with T2D are not clearly understood. Meta-analysis of multiple DNA microarray datasets could provide a robust set of metagenes identified across multiple studies. These metagenes would likely include a subset of genes (key metagenes) shared by both IR and T2D, and possibly responsible for the transition between them. In this study, we attempted to find these key metagenes using a feature selection method, LASSO, and then used the expression profiles of these genes to train five machine learning models: LASSO, SVM, XGBoost, Random Forest, and ANN. Among them, ANN performed well, with an area under the curve (AUC) > 95%. It also demonstrated fairly good performance in differentiating diabetics from normal glucose tolerant (NGT) persons in the test dataset, with 73% accuracy across 64 human adipose tissue samples. Furthermore, these core metagenes were also enriched in diabetes-associated terms and were found in previous genome-wide association studies of T2D and its associated glycemic traits HOMA-IR and HOMA-B. Therefore, this metagenome deserves further investigation with regard to the cardinal molecular pathological defects/pathways underlying both IR and T2D.

Methanol promotes the biodegradation of 2,2′,3,4,4′,5,5′-heptachlorobiphenyl (PCB 180) by the microbial consortium QY2: Metabolic pathways, toxicity evaluation and community response

As one of the most frequently detected PCB congeners in human adipose tissue, 2,2′,3,4,4′,5,5′-heptachlorobiphenyl (PCB 180) has attracted much attention. However, PCB 180 is difficult to be directly utilized by microorganisms due to its hydrophobicity and obstinacy. Herein, methanol (5 mM) as a co-metabolic carbon source significantly stimulated the degradation performance of microbial consortium QY2 for PCB 180 (51.9% higher than that without methanol addition). Six metabolic products including low-chlorinated PCBs and chlorobenzoic acid were identified during co-metabolic degradation, denoting that PCB 180 was metabolized via dechlorination, hydroxylation and ring-opening pathways. The oxidative stress and apoptosis induced by PCB 180 were dose-dependent, but the addition of methanol effectively promoted the tolerance of consortium QY2 to resist unfavorable environmental stress. Additionally, the significant reduction of intracellular reactive oxygen species (ROS) and enhancement of cell viability during methanol co-metabolic degradation proved that the degradation was a detoxification process. The microbial community and network analyses suggested that the potential PCB 180 degrading bacteria in the community (e.g., Achromobacter, Cupriavidus, Methylobacterium and Sphingomonas) and functional abundance of metabolic pathways were selectively enriched by methanol, and the synergies among species whose richness increased after methanol addition might dominate the degradation process. These findings provide new insights into the biodegradation of PCB 180 by microbial consortium.

Phenotypic diversity of human adipose tissue-resident NK cells in obesity.

Natural killer (NK) cells have emerged as key mediators of obesity-related adipose tissue inflammation. However, the phenotype of NK cell subsets residing in human adipose tissue are poorly defined, preventing a detailed understanding of their role in metabolic disorders. In this study, we applied multicolor flow cytometry to characterize CD56bright and CD56dim NK cells in blood and adipose tissue depots in individuals with obesity and identified surface proteins enriched on adipose tissue-resident CD56bright NK cells. Particularly, we found that adipose tissue harbored clusters of tissue-resident CD56bright NK cells signatured by the expression of CD26, CCR5 and CD63, possibly reflecting an adaptation to the microenvironment. Together, our findings provide broad insights into the identity of NK cells in blood and adipose tissue in relation to obesity.

Role of epicardial adipose tissue in human atrial fibrillation.

A recent meta-analysis among which four reports were conducted in Japan demonstrated that epicardial adipose tissue (EAT) is closely associated with an increased risk of atrial fibrillation (AF) recurrence after catheter ablation. We previously investigated the role of EAT in AF in humans. Left atrial (LA) appendage samples were obtained from AF patients during cardiovascular surgery. Histologically, the severity of fibrotic EAT remodeling was associated with LA myocardial fibrosis. Total collagen in the LA myocardium (i.e., LA myocardial fibrosis) was positively correlated with proinflammatory and profibrotic cytokines/chemokines, including interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α, in EAT. Human peri-LA EAT and abdominal subcutaneous adipose tissue (SAT) were obtained by autopsy. EAT- or SAT-derived conditioned medium was applied to the rat LA epicardial surface using an organo-culture system. EAT-conditioned medium induced atrial fibrosis in organo-cultured rat atrium. The profibrotic effect of EAT was greater than that of SAT. The fibrotic area of the organo-cultured rat atrium treated with EAT from patients with AF was greater than in patients without AF. Treatment with human recombinant angiopoietin-like protein 2 (Angptl2) induced fibrosis in organo-cultured rat atrium, which was suppressed by concomitant treatment with anti-Angptl2 antibody. Finally, we attempted to detect fibrotic EAT remodeling on computed tomography (CT) images, which demonstrated that the percent change in EAT fat attenuation was positively correlated with EAT fibrosis. Based on these findings, we conclude that the percent change in EAT fat attenuation determined using CT non-invasively detects EAT remodeling.

Human epicardial adipose tissue expresses glucose-dependent insulinotropic polypeptide, glucagon, and glucagon-like peptide-1 receptors as potential targets of pleiotropic therapies.

Human epicardial adipose tissue (EAT) plays a crucial role in the development and progression of coronary artery disease, atrial fibrillation, and heart failure. Microscopically, EAT is composed of adipocytes, nerve tissues, inflammatory, stromovascular, and immune cells. Epicardial adipose tissue is a white adipose tissue, albeit it also has brown fat-like or beige fat-like features. No muscle fascia divides EAT and myocardium; this allows a direct interaction and crosstalk between the epicardial fat and the myocardium. Thus, it might be a therapeutic target for pharmaceutical compounds acting on G-protein-coupled receptors, such as those for glucose-dependent insulinotropic polypeptide (GIP), glucagon (GCG), and glucagon-like peptide-1 (GLP-1), whose selective stimulation with innovative drugs has demonstrated beneficial cardiovascular effects. The precise mechanism of these novel drugs and their tissue and cellular target(s) need to be better understood. We evaluate whether human EAT expresses GIP, GCG, and GLP-1 receptors and whether their presence is related to EAT transcriptome. We also investigated protein expression and cell-type localization specifically for GIP receptor (GIPR) and glucagon receptor (GCGR). Epicardial adipose tissue samples were collected from 33 patients affected by cardiovascular diseases undergoing open heart surgery (90.9% males, age 67.2 ± 10.5 years mean ± SD). Microarray and immunohistochemistry analyses were performed. Microarray analysis showed that GIPR and GCGR messenger ribonucleic acids (mRNAs) are expressed in EAT, beyond confirming the previously found GLP-1 [3776 ± 1377 arbitrary unit (A.U.), 17.77 ± 14.91 A.U., and 3.41 ± 2.27 A.U., respectively]. The immunohistochemical analysis consistently indicates that GIPR and GCGR are expressed in EAT, mainly in macrophages, isolated, and in crown-like structures. In contrast, only some mature adipocytes of different sizes showed cytoplasmic immunostaining, similar to endothelial cells and pericytes in the capillaries and pre-capillary vascular structures. Notably, EAT GIPR is statistically associated with the low expression of genes involved in free fatty acid (FFA) oxidation and transport and those promoting FFA biosynthesis and adipogenesis (P < 0.01). Epicardial adipose tissue GCGR, in turn, is related to genes involved in FFA transport, mitochondrial fatty acid oxidation, and white-to-brown adipocyte differentiation, in addition to genes involved in the reduction of fatty acid biosynthesis and adipogenesis (P < 0.01). Having reported the expression of the GLP-1 receptor previously, here, we showed that GIPR and GCGR similarly present at mRNA and protein levels in human EAT, particularly in macrophages and partially adipocytes, suggesting these G-protein-coupled receptors as pharmacological targets on the ongoing innovative drugs, which seem cardiometabolically healthy well beyond their effects on glucose and body weight.

Administration of Secretome Derived from Human Mesenchymal Stem Cells Induces Hepatoprotective Effects in Models of Idiosyncratic Drug-Induced Liver Injury Caused by Amiodarone or Tamoxifen.

Drug-induced liver injury (DILI) is one of the leading causes of acute liver injury. While many factors may contribute to the susceptibility to DILI, obese patients with hepatic steatosis are particularly prone to suffer DILI. The secretome derived from mesenchymal stem cell has been shown to have hepatoprotective effects in diverse in vitro and in vivo models. In this study, we evaluate whether MSC secretome could improve DILI mediated by amiodarone (AMI) or tamoxifen (TMX). Hepatic HepG2 and HepaRG cells were incubated with AMI or TMX, alone or with the secretome of MSCs obtained from human adipose tissue. These studies demonstrate that coincubation of AMI or TMX with MSC secretome increases cell viability, prevents the activation of apoptosis pathways, and stimulates the expression of priming phase genes, leading to higher proliferation rates. As proof of concept, in a C57BL/6 mouse model of hepatic steatosis and chronic exposure to AMI, the MSC secretome was administered endovenously. In this study, liver injury was significantly attenuated, with a decrease in cell infiltration and stimulation of the regenerative response. The present results indicate that MSC secretome administration has the potential to be an adjunctive cell-free therapy to prevent liver failure derived from DILI caused by TMX or AMI.

MSR1 is not required for obesity-associated inflammation and insulin resistance in mice

Obesity induces a chronic inflammatory state associated with changes in adipose tissue macrophages (ATMs). Macrophage scavenger receptor 1 (MSR1) has been implicated in the regulation of adipose tissue inflammation and diabetes pathogenesis; however, reports have been mixed on the contribution of MSR1 in obesity and glucose intolerance. We observed increased MSR1 expression in VAT of obese diabetic individuals compared to non-diabetic and single nuclear RNA sequencing identified macrophage-specific expression of MSR1 in human adipose tissue. We examined male Msr1−/− (Msr1KO) and WT controls and observed protection from obesity and AT inflammation in non-littermate Msr1KO mice. We then evaluated obese littermate Msr1+/− (Msr1HET) and Msr1KO mice. Both Msr1KO mice and Msr1HET mice became obese and insulin resistant when compared to their normal chow diet counterparts, but there was no Msr1-dependent difference in body weight, glucose metabolism, or insulin resistance. Flow cytometry revealed no significant differences between genotypes in ATM subtypes or proliferation in male and female mice. We observed increased frequency of proliferating ATMs in obese female compared to male mice. Overall, we conclude that while MSR1 is a biomarker of diabetes status in human adipose tissue, in mice Msr1 is not required for obesity-associated insulin resistance or ATM accumulation.

Liver saturated fat content associates with hepatic DNA methylation in obese individuals

BackgroundAccumulation of saturated fatty acids (SFAs) in the liver is known to induce hepatic steatosis and inflammation causing non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although SFAs have been shown to affect the epigenome in whole blood, pancreatic islets, and adipose tissue in humans, and genome-wide DNA methylation studies have linked epigenetic changes to NAFLD and NASH, studies focusing on the association of SFAs and DNA methylation in human liver are missing. We, therefore, investigated whether human liver SFA content associates with DNA methylation and tested if SFA-linked alterations in DNA methylation associate with NAFLD-related clinical phenotypes in obese individuals.ResultsWe identified DNA methylation (Infinium HumanMethylation450 BeadChip) of 3169 CpGs to be associated with liver total SFA content (q-value < 0.05) measured using proton NMR spectroscopy in participants of the Kuopio Obesity Surgery Study (n = 51; mean ± SD:49.3 ± 8.5 years old; BMI:43.7 ± 6.2 kg/m2). Of these 3169 sites, 797 overlapped with previously published NASH-associated CpGs (NASH-SFA), while 2372 CpGs were exclusively associated with SFA (Only-SFA). The corresponding annotated genes of these only-SFA CpGs were found to be enriched in pathways linked to satiety and hunger. Among the 54 genes mapping to these enriched pathways, DNA methylation of CpGs mapping to PRKCA and TSPO correlated with their own mRNA expression (HumanHT-12 Expression BeadChip). In addition, DNA methylation of another ten of these CpGs correlated with the mRNA expression of their neighboring genes (p value < 0.05). The proportion of CpGs demonstrating a correlation of DNA methylation with plasma glucose was higher in NASH-SFA and only-SFA groups, while the proportion of significant correlations with plasma insulin was higher in only-NASH and NASH-SFA groups as compared to all CpGs on the Illumina 450 K array (Illumina, San Diego, CA, USA).ConclusionsOur results suggest that one of the mechanisms how SFA could contribute to metabolic dysregulation in NAFLD is at the level of DNA methylation. We further propose that liver SFA-related DNA methylation profile may contribute more to hyperglycemia, while insulin-related methylation profile is more linked to NAFLD or NASH. Further research is needed to elucidate the molecular mechanisms behind these observations.

Acute and long-term exercise adaptation of adipose tissue and skeletal muscle in humans: a matched transcriptomics approach after 8-week training-intervention

BackgroundExercise exerts many health benefits by directly inducing molecular alterations in physically utilized skeletal muscle. Molecular adaptations of subcutaneous adipose tissue (SCAT) might also contribute to the prevention of metabolic diseases.AimTo characterize the response of human SCAT based on changes in transcripts and mitochondrial respiration to acute and repeated bouts of exercise in comparison to skeletal muscle.MethodsSedentary participants (27 ± 4 yrs) with overweight or obesity underwent 8-week supervised endurance exercise 3×1h/week at 80% VO2peak. Before, 60 min after the first and last exercise bout and 5 days post intervention, biopsies were taken for transcriptomic analyses and high-resolution respirometry (n = 14, 8 female/6 male).ResultsIn SCAT, we found 37 acutely regulated transcripts (FC > 1.2, FDR < 10%) after the first exercise bout compared to 394, respectively, in skeletal muscle. Regulation of only 5 transcripts overlapped between tissues highlighting their differential response. Upstream and enrichment analyses revealed reduced transcripts of lipid uptake, storage and lipogenesis directly after exercise in SCAT and point to β-adrenergic regulation as potential major driver. The data also suggest an exercise-induced modulation of the circadian clock in SCAT. Neither term was associated with transcriptomic changes in skeletal muscle. No evidence for beigeing/browning was found in SCAT along with unchanged respiration.ConclusionsAdipose tissue responds completely distinct from adaptations of skeletal muscle to exercise. The acute and repeated reduction in transcripts of lipid storage and lipogenesis, interconnected with a modulated circadian rhythm, can counteract metabolic syndrome progression toward diabetes.