Proteomic Signatures of Human Visceral and Subcutaneous Adipocytes.

CONTEXT: Adipose tissue distribution is a key factor influencing metabolic health and risk in obesity-associated comorbidities. OBJECTIVE: Here we aim to compare the proteomic profiles of mature adipocytes from different depots. METHODS: Abdominal subcutaneous (SA) and omental visceral adipocytes (VA) were isolated from paired adipose tissue biopsies obtained during bariatric surgery on 19 severely obese women (body mass index > 30 kg/m2) and analyzed using state-of-the-art mass spectrometry. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to investigate proteome signature properties and to examine a possible association of the protein expression with the clinical data. RESULTS: We identified 3686 protein groups and found 1140 differentially expressed proteins (adj. P value < 0.05), of which 576 proteins were upregulated in SA and 564 in VA samples. We provide a global protein profile of abdominal SA and omental VA, present the most differentially expressed pathways and processes distinguishing SA from VA, and correlate them with clinical and body composition data. We show that SA are significantly more active in processes linked to vesicular transport and secretion, and to increased lipid metabolism activity. Conversely, the expression of proteins involved in the mitochondrial energy metabolism and translational or biosynthetic activity is higher in VA. CONCLUSION: Our analysis represents a valuable resource of protein expression profiles in abdominal SA and omental VA, highlighting key differences in their role in obesity.

miR-146a-5p modulates cellular senescence and apoptosis in visceral adipose tissue of long-lived Ames dwarf mice and in cultured pre-adipocytes.

MicroRNAs (miRNAs) are potent regulators of multiple biological processes. Previous studies have demonstrated that miR-146a-5p increases in normal mice during aging, while long-living Ames dwarf (df/df) mice maintain youthful levels of this miRNA. The aim of this study was to elucidate the involvement of miR-146a-5p in modulating cellular senescence and apoptosis in visceral adipose tissue of df/df mice and cultured pre-adipocytes. To test the effects of miR-146a-5p overexpression on visceral adipose tissue, wild-type, and df/df mice, were treated with miRNA-negative control-base and df/df were transfected with 4 or 8 µg/g of a miR-146a-5p mimetic, respectively. Effects of miR-146a-5p overexpression were also evaluated in 3T3-L1 cells cultured under high and normal glucose conditions. Treatment with miR-146a-5p mimetic increased cellular senescence and inflammation and decreased pro-apoptotic factors in visceral adipose tissue of df/df mice. The miR-146a-5p mimetic induced similar effects in 3T3-L1 cells cultivated at normal but not high glucose levels. Importantly, 3T3-L1 HG cells in high glucose conditions showed significantly higher expression of miR-146a-5p than 3T3-L1 grown in normal glucose conditions. These results indicate that miR-146a-5p can be a marker for cellular senescence. This miRNA represents one of the significant SASP factors that if not precisely regulated, can accentuate inflammatory responses and stimulate senescence in surrounding non-senescent cells. The role of miR-146a-5p is different in healthy versus stressed cells, suggesting potential effects of this miRNA depend on overall organismal health, aging, and metabolic state.

TRIB3 Is Highly Expressed in the Adipose Tissue of Obese Patients and Is Associated With Insulin Resistance.

CONTEXT: The upregulation of TRIB3 (Tribbles homolog 3), a stress-inducible gene encoding a pseudokinase, has been implicated in the development of insulin resistance in the skeletal muscle and liver of patients with obesity and type 2 diabetes. However, there is little information regarding TRIB3 expression in human adipose tissue. OBJECTIVE: To investigate whether TRIB3 expression is dysregulated in human adipose tissue in the context of obesity and type 2 diabetes and whether TRIB3 expression in adipose tissues is associated with insulin resistance. METHODS: We measured metabolic parameters and TRIB3 expression in abdominal subcutaneous and visceral adipose tissue in obese (with or without type 2 diabetes) and normal-weight women. Regulation of TRIB3 expression was studied in human adipocytes. RESULTS: TRIB3 expression in both fat depots was higher in patients with obesity and/or type 2 diabetes; in addition, the expression level was significantly associated with insulin resistance. Incubating adipocytes under conditions mimicking the microenvironment of obese adipose tissue, including increased endoplasmic reticulum (ER) stress, induced TRIB3 expression. In human adipocytes, the overexpression of TRIB3 impaired insulin-stimulated protein kinase B (AKT) phosphorylation and caused dysregulation of the transcription of genes encoding bioactive molecules released from adipocytes, such as proinflammatory cytokines, adiponectin, and leptin. Pioglitazone, an insulin-sensitizing agent, reduced both these effects of TRIB3 and the ER stressor-induced expression of TRB3. CONCLUSION: Our data indicate that TRIB3 expression in adipose tissue is enhanced in patients with obesity and suggest that increased TRIB3 dysregulates adipocyte function, which may contribute to the development of insulin resistance.

Exploring the Cell Stemness and the Complexity of the Adipose Tissue Niche.

Adipose tissue is a complex organ composed of different cellular populations, including mesenchymal stem and progenitor cells, adipocytes, and immune cells such as macrophages and lymphocytes. These cellular populations alter dynamically during aging or as a response to pathophysiology such as obesity. Changes in the various inflammatory cells are associated with metabolic complications and the development of insulin resistance, indicating that immune cells crosstalk with the adipocytes. Therefore, a study of the cell populations in the adipose tissue and the extracellular matrix maintaining the tissue niche is important for the knowledge on the regulatory state of the organ. We used a combination of methods to study various parameters to identify the composition of the resident cells in the adipose tissue and evaluate their profile. We analyzed the tissue structure and cells based on histology, immune fluorescence staining, and flow cytometry of cells present in the tissue in vivo and these markers' expression in vitro. Any shift in cells' composition influences self-renewal of the mesenchymal progenitors, and other cells affect the functionality of adipogenesis.

SMAD2 and SMAD3 differentially regulate adiposity and the growth of subcutaneous white adipose tissue.

Adipose tissue is the primary site of energy storage, playing important roles in health. While adipose research largely focuses on obesity, fat also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety and total energy homeostasis. SMAD2/3 proteins are downstream mediators of activin signaling, which regulate critical preadipocyte and mature adipocyte functions. Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity. The direct contributions of Smad2 and Smad3 in adipose tissues, however, are unknown. Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 on diet-induced adiposity using Smad2 or Smad3 "floxed" mice intercrossed with Adiponectin-Cre mice. Additionally, we examined visceral and subcutaneous preadipocyte differentiation efficiency in vitro. Almost all wild type subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A suppressed differentiation of preadipocytes from both depots. Smad2 conditional knockout (Smad2cKO) mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3 conditional knockout (Smad3cKO) male mice displayed a trend of reduced body weight on high-fat diet. On both diets, Smad3cKO mice displayed an adipose depot-selective phenotype, with a significant reduction in subcutaneous fat mass but not visceral fat mass. Our data suggest that Smad3 is an important contributor to the maintenance of subcutaneous white adipose tissue in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation.

Identification of markers that distinguish adipose tissue and glucose and insulin metabolism using a multi-modal machine learning approach.

The study of metabolomics has improved our knowledge of the biology behind type 2 diabetes and its related metabolic physiology. We aimed to investigate markers of adipose tissue morphology, as well as insulin and glucose metabolism in 53 non-obese male individuals. The participants underwent extensive clinical, biochemical and magnetic resonance imaging phenotyping, and we also investigated non-targeted serum metabolites. We used a multi-modal machine learning approach to evaluate which serum metabolomic compounds predicted markers of glucose and insulin metabolism, adipose tissue morphology and distribution. Fasting glucose was associated with metabolites of intracellular insulin action and beta-cell dysfunction, namely cysteine-s-sulphate and n-acetylgarginine, whereas fasting insulin was predicted by myristoleoylcarnitine, propionylcarnitine and other metabolites of beta-oxidation of fatty acids. OGTT-glucose levels at 30 min were predicted by 7-Hoca, a microbiota derived metabolite, as well as eugenol, a fatty acid. Both insulin clamp and HOMA-IR were predicted by metabolites involved in beta-oxidation of fatty acids and biodegradation of triacylglycerol, namely tartrate and 3-phosphoglycerate, as well as pyruvate, xanthine and liver fat. OGTT glucose area under curve (AUC) and OGTT insulin AUC, was associated with bile acid metabolites, subcutaneous adipocyte cell size, liver fat and fatty chain acids and derivates, such as isovalerylcarnitine. Finally, subcutaneous adipocyte size was associated with long chain fatty acids, markers of sphingolipid metabolism, increasing liver fat and dopamine-sulfate 1. Ectopic liver fat was predicted by methylmalonate, adipocyte cell size, glutathione derived metabolites and fatty chain acids. Ectopic heart fat was predicted visceral fat, gamma-glutamyl tyrosine and 2-acetamidophenol sulfate. Adipocyte cell size, age, alpha-tocopherol and blood pressure were associated with visceral fat. We identified several biomarkers associated with adipose tissue pathophysiology and insulin and glucose metabolism using a multi-modal machine learning approach. Our approach demonstrated the relative importance of serum metabolites and they outperformed traditional clinical and biochemical variables for most endpoints.

Viability, yield and expansion capability of feline MSCs obtained from subcutaneous and reproductive organ adipose depots.

BACKGROUND: The source of multipotent stromal cells (MSC) can have a significant influence on the health and expansion capacity of the cells. As the applications for allogeneic MSCs in the treatment of feline diseases increase, the location of the initial donor tissue must be analyzed. To date, comparisons have only been made between feline MSCs collected from bone marrow or abdominal fat. This is the first report to compare cells obtained from different adipose depots in the cat with a focus on clinically relevant donor tissues. The tissue was collected from 34 healthy cats undergoing spaying (fat around the ovaries and uterine horn) or subcutaneous fat collected during surgical procedures. RESULTS: The amount of starting material is essential to isolate sufficient MSCs. The total tissue yield from the subcutaneous fat was significantly greater than could be obtained from around the reproductive organs, leading to 3 times more MSCs per donor. However, the concentration of MSCs obtained from reproductive fat was higher than from subcutaneous fat. In addition, the viability of the MSCs from the reproductive fat was significantly higher than the subcutaneous fat. Since most spaying occurs in young cats (under 18 months) reproductive fat was collected from adult cats during spaying, illustrating that age did not alter the yield or viability of the MSCs. When sufficient tissue was collected, it was digested either mechanically or enzymatically. Mechanical digestion further decreased the viability and yield of MSCs from subcutaneous fat compared to enzymatic digestion. Biomarkers of stem cell characterization, expansion capacity and function were detected using qPCR. CD70, CD90 and CD105 were all expressed in high levels in the 3 groups. However, the reproductive fat had higher levels of CD73 with the mechanically digested subcutaneous fat having the least. Gata6 was detected in all samples while Sox2 and Sox17 were also detected with higher quantities found in the enzymatically digested subcutaneous fat. Negative control genes of Gata4 and Pdx1 showed no detection prior to 50 cycles. During the first three passages, age of the donor, location of the donor tissue, or digestion protocol had no effect on cell culture doubling times or cell viability. CONCLUSIONS: While MSCs from reproductive fat had superior cells/tissue weight and initial viability, there were still dramatically fewer cells obtained compared to subcutaneous fat due to the limited amount of tissue surrounding the reproductive organs. Further, in P1-P3 cultures there were no differences noted in doubling time or cell viability between tissue obtained from reproductive or subcutaneous fat depots.

Mesothelial cells are not a source of adipocytes in mice.

Visceral adipose tissue (VAT) depots are associated with the adverse metabolic consequences of obesity, such as insulin resistance. The developmental origin of VAT depots and the identity and regulation of adipocyte progenitor cells have been active areas of investigation. In recent years, a paradigm of mesothelial cells as a source of VAT adipocyte progenitor cells has emerged based on lineage tracing studies using the Wilms' tumor gene, Wt1, as a marker for cells of mesothelial origin. Here, we show that Wt1 expression in adipose tissue is not limited to the mesothelium but is also expressed by a distinct preadipocyte population in mice and humans. We identify keratin 19 (Krt19) as a highly specific marker for the adult mouse mesothelium and demonstrate that Krt19-expressing mesothelial cells do not differentiate into visceral adipocytes. These results contradict the assertion that the VAT mesothelium can serve as a source of adipocytes.

Chronic and Intermittent Hyperglycemia Modulates Expression of Key Molecules of PI3K/AKT Pathway in Differentiating Human Visceral Adipocytes.

BACKGROUND: Due to its prominence in the regulation of metabolism and inflammation, adipose tissue is a major target to investigate alterations in insulin action. This hormone activates PI3K/AKT pathway which is essential for glucose homeostasis, cell differentiation, and proliferation in insulin-sensitive tissues, like adipose tissue. The aim of this work was to evaluate the impact of chronic and intermittent high glucose on the expression of biomolecules of insulin signaling pathway during the differentiation and maturation of human visceral preadipocytes. METHODS: Human visceral preadipocytes (HPA-V) cells were treated with high glucose (30 mM)during the proliferation and/or differentiation and/or maturation stage. The level of mRNA (by Real-Time PCR) and protein (by Elisa tests) expression of IRS1, PI3K, PTEN, AKT2, and GLUT4 was examined after each culture stage. Furthermore, we investigated whether miR-29a-3p, miR-143-3p, miR-152-3p, miR-186-5p, miR-370-3p, and miR-374b-5p may affect the expression of biomolecules of the insulin signaling pathway. RESULTS: Both chronic and intermittent hyperglycemia affects insulin signaling in visceral pre/adipocytes by upregulation of analyzed PI3K/AKT pathway molecules. Both mRNA and protein expression level is more dependent on stage-specific events than the length of the period of high glucose exposure. What is more, miRs expression changes seem to be involved in PI3K/AKT expression regulation in response to hyperglycemic stimulation.

LMO3 reprograms visceral adipocyte metabolism during obesity.

Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. We recently identified LIM domain only 3 (LMO3) in human mature visceral adipocytes; however, its function in these cells is currently unknown. The aim of this study was to determine the potential involvement of LMO3-dependent pathways in the modulation of key functions of mature adipocytes during obesity. Based on a recently engineered hybrid rAAV serotype Rec2 shown to efficiently transduce both brown adipose tissue (BAT) and white adipose tissue (WAT), we delivered YFP or Lmo3 to epididymal WAT (eWAT) of C57Bl6/J mice on a high-fat diet (HFD). The effects of eWAT transduction on metabolic parameters were evaluated 10 weeks later. To further define the role of LMO3 in insulin-stimulated glucose uptake, insulin signaling, adipocyte bioenergetics, as well as endocrine function, experiments were conducted in 3T3-L1 adipocytes and newly differentiated human primary mature adipocytes, engineered for transient gain or loss of LMO3 expression, respectively. AAV transduction of eWAT results in strong and stable Lmo3 expression specifically in the adipocyte fraction over a course of 10 weeks with HFD feeding. LMO3 expression in eWAT significantly improved insulin sensitivity and healthy visceral adipose tissue expansion in diet-induced obesity, paralleled by increased serum adiponectin. In vitro, LMO3 expression in 3T3-L1 adipocytes increased PPARγ transcriptional activity, insulin-stimulated GLUT4 translocation and glucose uptake, as well as mitochondrial oxidative capacity in addition to fatty acid oxidation. Mechanistically, LMO3 induced the PPARγ coregulator Ncoa1, which was required for LMO3 to enhance glucose uptake and mitochondrial oxidative gene expression. In human mature adipocytes, LMO3 overexpression promoted, while silencing of LMO3 suppressed mitochondrial oxidative capacity. LMO3 expression in visceral adipose tissue regulates multiple genes that preserve adipose tissue functionality during obesity, such as glucose metabolism, insulin sensitivity, mitochondrial function, and adiponectin secretion. Together with increased PPARγ activity and Ncoa1 expression, these gene expression changes promote insulin-induced GLUT4 translocation, glucose uptake in addition to increased mitochondrial oxidative capacity, limiting HFD-induced adipose dysfunction. These data add LMO3 as a novel regulator improving visceral adipose tissue function during obesity. KEY MESSAGES: LMO3 increases beneficial visceral adipose tissue expansion and insulin sensitivity in vivo. LMO3 increases glucose uptake and oxidative mitochondrial activity in adipocytes. LMO3 increases nuclear coactivator 1 (Ncoa1). LMO3-enhanced glucose uptake and mitochondrial gene expression requires Ncoa1.

IFNγ-producing NK cells in adipose tissue are associated with hyperglycemia and insulin resistance in obese women.

BACKGROUND/OBJECTIVES: Innate lymphoid cells (ILCs) play an important role in the maintenance of immune and metabolic homeostasis in adipose tissue (AT). The crosstalk between AT ILCs and adipocytes and other immune cells coordinates adipocyte differentiation, beiging, glucose metabolism and inflammation. Although the metabolic and homeostatic functions of mouse ILCs have been extensively investigated, little is known about human adipose ILCs and their roles in obesity and insulin resistance (IR). SUBJECTS/METHODS: Here we characterized T and NK cell populations in omental AT (OAT) from women (n = 18) with morbid obesity and varying levels of IR and performed an integrated analysis of metabolic parameters and adipose tissue transcriptomics. RESULTS: In OAT, we found a distinct population of CD56-NKp46+EOMES+ NK cells characterized by expression of cytotoxic molecules, pro-inflammatory cytokines, and markers of cell activation. AT IFNγ+ NK cells, but not CD4, CD8 or γδ T cells, were positively associated with glucose levels, glycated hemoglobin (HbA1c) and IR. AT NK cells were linked to a pro-inflammatory gene expression profile in AT and developed an effector phenotype in response to IL-12 and IL-15. Moreover, integrated transcriptomic analysis revealed a potential implication of AT IFNγ+ NK cells in controlling adipose tissue inflammation, remodeling, and lipid metabolism. CONCLUSIONS: Our results suggest that a distinct IFNγ-producing NK cell subset is involved in metabolic homeostasis in visceral AT in humans with obesity and may be a potential target for therapy of IR.

Adipocyte calcium sensing receptor is not involved in visceral adipose tissue inflammation or atherosclerosis development in hyperlipidemic Apoe-/- mice.

The calcium sensing receptor (CaSR) is a G-protein coupled receptor that especially plays an important role in the sensing of extracellular calcium to maintain its homeostasis. Several in-vitro studies demonstrated that CaSR plays a role in adipose tissue metabolism and inflammation, resulting in systemic inflammation and contributing to atherosclerosis development. The aim of this study was to investigate whether adipocyte CaSR plays a role in adipose tissue inflammation in-vivo and atherosclerosis development. By using a newly established conditional mature adipocyte specific CaSR deficient mouse on a hyperlipidemic and atherosclerosis prone Apoe-/- background it could be shown that CaSR deficiency in adipocytes does neither contribute to initiation nor to progression of atherosclerotic plaques as judged by the unchanged lesion size or composition. Additionally, CaSR deficiency did not influence gonadal visceral adipose tissue (vAT) inflammation in-vivo, although a small decrease in gonadal visceral adipose cholesterol content could be observed. In conclusion, adipocyte CaSR seems not to be involved in vAT inflammation in-vivo and does not influence atherosclerosis development in hyperlipidemic Apoe-/- mice.

Src family kinases involved in the differentiation of human preadipocytes.

BACKGROUND: Obesity is characterized by the excess accumulation of white adipose tissue (WAT). Src family kinases (SFKs) are non-receptor tyrosine kinases consisting of eight members (SRC, FYN, YES1, HCK, LCK, LYN, FGR and BLK) that have been studied extensively in mammalian cells. Although individual members in murine cells provide some clues that are associated with the regulation of adipogenesis, the specific role of this family in adipocyte differentiation has rarely been assessed, especially in human adipocytes. METHODS: Herein, we first explored the expression profiles of SFKs during human preadipocyte differentiation. Then, we used the pyrazolo-pyrimidinyl-amine compound PP1, a potent SFK inhibitor, to evaluate the function of SFKs during adipocyte differentiation. Furthermore, we adopted a loss-of-function strategy with siRNAs to determine the role of FGR in adipocyte differentiation. RESULTS: Here, we found that SRC, FYN, YES1, LYN and FGR were expressed in human preadipocytes and induced after the initiation of differentiation. Furthermore, the SFK inhibitor PP1 suppressed adipocyte differentiation. We also found that PP1 significantly suppressed the SFK activity in preadipocytes and decreased the expression of adipogenic genes in early and late differentiation. Given that FGR exhibited the most expression enhancement in mature adipocytes, we focused on FGR and found that its knockdown reduced lipid accumulation and adipogenic gene expression. CONCLUSIONS: Collectively, these findings suggest that SFKs, especially FGR, are involved in the differentiation of human preadipocytes. Our results lay a foundation for further understanding the role of SFKs in adipocyte differentiation and provide new clues for anti-obesity therapies.

High expression of CD52 in adipocytes: a potential therapeutic target for obesity with type 2 diabetes.

The aim of the present study was to evaluate the involvement of CD52 in adipocytes as well as to explore its effect on type 2 diabetes mellitus (T2DM), and to improve our understanding of the potential molecular events of obesity with type 2 diabetes. Global changes in the CD52 expression patterns were detected in adipocytes and preadipocytes derived from obese and lean individuals. In particular, CD52 was identified as significantly differentially upregulated and was analyzed, both in vitro and in vivo, using various approaches. In vitro experiments, CD52 was a significantly up-regulated mRNA in mature adipocytes and preadipocytes. In addition, CD52 gradually increased with the differentiation of preadipocytes. In vivo experiments, the expression of CD52 in high-fat diet (HFD) -fed mice tended to be higher than that in regular diet (RD) -fed mice. Further analysis showed that CD52 expression was positively correlated with Smad3 and TGF-ß in mice, and the downregulation of CD52 was accompanied by increased glucose tolerance and insulin sensitivity. Moreover, a comparison of CD4+CD52high T cells and CD4+CD52low T cells showed that many T2DM-related genes were aberrantly expressed. Overall, CD52 may functioned as an important potential target for obesity with T2DM via TGF-ß/Smad3 axis.

Tissue regulatory T cells: regulatory chameleons.

The FOXP3+CD4+ regulatory T (Treg) cells located in non-lymphoid tissues differ in phenotype and function from their lymphoid organ counterparts. Tissue Treg cells have distinct transcriptomes, T cell receptor repertoires and growth and survival factor dependencies that arm them to survive and operate in their home tissue. Their functions extend beyond immune surveillance to tissue homeostasis, including regulation of local and systemic metabolism, promotion of tissue repair and regeneration, and control of the proliferation, differentiation and fate of non-lymphoid cell progenitors. Treg cells in diverse tissues share a common FOXP3+CD4+ precursor located within lymphoid organs. This precursor undergoes definitive specialization once in the home tissue, following a multilayered array of common and tissue-distinct transcriptional programmes. Our deepening knowledge of tissue Treg cell biology will inform ongoing attempts to harness Treg cells for precision immunotherapeutics.

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy.

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species.

Visceral adipose tissue mesenchymal stromal cells in the intersection of immunology and metabolism.

Visceral adipose tissue (VAT) is now recognized as an endocrine organ that plays a key role in organismal homeostasis by integrating metabolic and immunological aspects. In healthy individuals, this fat depot participates in the storage and release of lipids as per physiological demand, while maintaining a local anti-inflammatory environment. In this regard, recent findings highlight the pivotal role of distinct subtypes of mesenchymal stromal cells (mSCs) as orchestrators of metabolic homeostasis by engendering adipocytes to sustain adequate lipid storage as well as immune regulators via cross-talk with specialized tissue-resident immunocytes, especially regulatory T cells (Tregs) and group 2 innate lymphoid cells (ILC2s) to prevent the development of local inflammation. In addition, these stromal-immunocyte interactions are influenced by a number of physiological conditions such as aging and sex hormones. Perturbation of VAT equilibrium occurring during obesity appreciably alters the distribution and phenotype of mSCs, immunocytes, and other cell types, thereby promoting the development of chronic, low-grade inflammation locally and systemically. These alterations impair metabolic signaling and substantially contribute to the onset of disease, including type 2 diabetes. The present mini-review discusses the latest advances in this area, with an emphasis on the newly uncovered heterogeneity of mSCs, how they communicate with Tregs and ILC2s under different physio-pathological circumstances and future challenges to face.

Cardiopulmonary fitness but not muscular fitness associated with visceral adipose tissue mass.

PURPOSE: The purpose of this study is to identify the association between physical fitness (cardiopulmonary and musculoskeletal) and the distribution of abdominal fat in overweight and obese adults. METHODS: Of the total 102 overweight and obese participants, 99 participants completed all measurements. Cardiopulmonary fitness was assessed by maximal oxygen consumption test and muscular fitness was assessed using 10 repetition max. Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were assessed by computed tomography (CT) at the level of lumbar spine 4-5. RESULTS: Cardiopulmonary fitness was inversely associated with both VAT and SAT, while muscular fitness only inversely associated with SAT. Multiple linear regression analyses indicated that gender, age, and cardiopulmonary fitness, but not muscular fitness, were associated with VAT, and age, cardiopulmonary fitness, and muscular fitness were significantly associated with SAT. CONCLUSIONS: Cardiopulmonary fitness is more closely related to both VAT and SAT while muscular fitness is related with SAT.

Anti-adipogenic signals at the onset of obesity-related inflammation in white adipose tissue.

Chronic inflammation that affects primarily metabolic organs, such as white adipose tissue (WAT), is considered as a major cause of human obesity-associated co-morbidities. However, the molecular mechanisms initiating this inflammation in WAT are poorly understood. By combining transcriptomics, ChIP-seq and modeling approaches, we studied the global early and late responses to a high-fat diet (HFD) in visceral (vWAT) and subcutaneous (scWAT) AT, the first being more prone to obesity-induced inflammation. HFD rapidly triggers proliferation of adipocyte precursors within vWAT. However, concomitant antiadipogenic signals limit vWAT hyperplastic expansion by interfering with the differentiation of proliferating adipocyte precursors. Conversely, in scWAT, residing beige adipocytes lose their oxidizing properties and allow storage of excessive fatty acids. This phase is followed by tissue hyperplastic growth and increased angiogenic signals, which further enable scWAT expansion without generating inflammation. Our data indicate that scWAT and vWAT differential ability to modulate adipocyte number and differentiation in response to obesogenic stimuli has a crucial impact on the different susceptibility to obesity-related inflammation of these adipose tissue depots.

Obesity induced by high-fat diet is associated with critical changes in biological and molecular functions of mesenchymal stromal cells present in visceral adipose tissue.

The mesenchymal stromal cells (MSCs) residing within the stromal component of visceral adipose tissue appear to be greatly affected by obesity, with impairment of their functions and presence of senescence. To gain further insight into these phenomena, we analyzed the changes in total proteome content and secretome of mouse MSCs after a high-fat diet (HFD) treatment compared to a normal diet (ND). In healthy conditions, MSCs are endowed with functions mainly devoted to vesicle trafficking. These cells have an immunoregulatory role, affecting leukocyte activation and migration, acute inflammation phase response, chemokine signaling, and platelet activities. They also present a robust response to stress. We identified four signaling pathways (TGF-ß, VEGFR2, HMGB1, and Leptin) that appear to govern the cells' functions. In the obese mice, MSCs showed a change in their functions. The immunoregulation shifted toward pro-inflammatory tasks with the activation of interleukin-1 pathway and of Granzyme A signaling. Moreover, the methionine degradation pathway and the processing of capped intronless pre-mRNAs may be related to the inflammation process. The signaling pathways we identified in ND MSCs were replaced by MET, WNT, and FGFR2 signal transduction, which may play a role in promoting inflammation, cancer, and aging.