Adipose Tissue Extracellular Matrix Research Articles

Obesity in the 21st Century. Prevalence, Phenotypes, Course Variants and Consequences

The review article examines the issues of obesity regarding its prevalence, phenotypes, course and consequences. The anatomical, physiological and functional features of adipose tissue are discussed. The “Discussion” section presents the authors’ own clinical observations on therapeutic and surgical issues in the treatment of obesity. Currently, adipose tissue is divided into white, brown and beige. White and brown adipose tissue can turn into each other through the beige adipose tissue stage. The volume and activity of brown adipose tissue require greater expenditure in the metabolism of oxygen molecules and are more pronounced in women. The components of the extracellular matrix of adipose tissue are collagen types I, III, V, VI. The collagen structure of adipose tissue varies depending on the location, volume and size of fat, age, gender, functional state of the kidneys, thyroid gland, nature of food, energy expenditure and sleep patterns. With intense work and cold exposure, the hormone irisin is formed in skeletal muscles, which promotes the transformation of white adipose tissue into brown or beige, helps reduce body weight in obesity, and has a beneficial effect on the course of type 2 diabetes mellitus and associated diseases. Traditionally, obese individuals are divided into metabolically healthy obesity and metabolically unhealthy obesity based on the degree of metabolic disorders. In visceral fat, lipolysis occurs with high intensity, which supports the development of inflammation. In obesity, decreased expression of adiponectin accelerates the development of atherosclerotic cardiovascular diseases. As body weight increases, visceral adipose tissue acquires an inflammatory phenotype, manifested by increased expression of cytokines (interleukin-6, interleukin-1, interleukin-17, tumor necrosis factor-alpha), hyperactivation of the tissue renin-angiotensin-aldosterone system, as well as excessive cell infiltration immune system (leukocytes, neutrophils, T-lymphocytes, monocytes, macrophages). During inflammation in adipose tissue, inflammatory (M1) and atherogenic (M4) phenotypes of macrophages dominate. In morbid obesity, the number of macrophages in adipose tissue can reach up to 50% of all cells.

Engineering and characterization of a hydrogel mimicking subcutaneous interstitial space

We have synthesized and characterized a collagen-hyaluronic acid hybrid network. The aim was to create a hydrogel mimicking the extracellular matrix of adipose tissue, primarily for use in in vitro studies of protein drug transport in the subcutaneous interstitial space. The network was created by covalently crosslinking methacryloyl-functionalized collagen type I and thiol-functionalized hyaluronic acid by means of thiol-Michael and thiol-ene photo-click reaction. The degree of modification corresponded to 74 % of the lysine and arginine groups on collagen, and 16 to 29 % of the carboxylate groups on hyaluronic acid, as determined with 1H NMR. Circular dichroism measurements showed that the triple helix of modified collagen remained intact. Oscillatory shear rheology tests showed that the hydrated networks displayed viscoelastic properties characteristic of hydrogels. The storage modulus, measured at 1 Hz frequency in the linear viscoelastic range (<5%), varied in a controllable way between 1.5 and 4 kPa depending on the collagen concentration and collagen-to-hyaluronic acid ratio. The hydrogels had a lower collagen content (0.6––1.2 wt%) but similar hyaluronic acid content and shear modulus at low strain rates as the extracellular matrix in adipose tissue and were penetrable by albumin and lysozyme. The results show that the hydrogels are promising as model systems for investigations of drug transport.

Microphysiological system recapitulating the pathophysiology of adipose tissue in obesity.

A growing body of evidence has indicated that white adipose tissue (AT) remodeling is a major trigger for obesity-associated metabolic complications. However, the scarcity of translational models is an obstacle to the development of medicines that act on adipose restoration. Here, we describe a microphysiological system (MPS) that emulates the unique features of reprogrammed AT as a new in vitro tool for studying AT pathophysiology in obesity. The AT MPS contained mature adipocytes embedded in an extracellular matrix (ECM) hydrogel interfaced with AT microvascular endothelium, which was constantly perfused with fresh media. The unique biochemical signals due to the remodeled ECM in obesity were recapitulated using a decellularized AT ECM (AT dECM) hydrogel, which preserves the features of altered ECM composition in obesity. The mature adipocytes embedded in the AT dECM hydrogel maintained their function and morphology for a week without dedifferentiation. Using the AT MPS, we successfully modeled inflammation-induced AT microvascular dysfunction, the recruitment of immune cells due to the upregulation of cell adhesion molecules, and higher cancer cell adhesion as an indicator of metastasis, which are observed in obese individuals. The AT MPS may therefore represent a promising platform for understanding the dynamic cellular interplay in obesity-induced AT remodeling and validating the efficacy of drugs targeting AT in obesity. STATEMENT OF SIGNIFICANCE: The lack of translational in vitro white adipose tissue (AT) models is one of the main obstacles for understanding the obesity-induced reprogramming and the development of medicines. We report herein the AT microphysiological system (MPS), which recapitulates obesity and normal conditions and yields cell- and AT dECM-derived signals, thereby allowing accurate comparative in vitro analyses. Using the AT MPS, we successfully modeled reprogrammed AT in obesity conditions, including inflammation-induced AT vascular dysfunction, the recruitment of immune cells, and higher cancer cell metastasis, which are observed in obese individuals. Our proposed adipose tissue model providing physiological relevance and complexity may therefore enhance the understanding of obesity-associated disorders and be used to investigate their underlying molecular mechanisms to develop pharmacologic treatment strategies.

Technical note: Noninvasive monitoring of normal tissue radiation damage using spectral quantitative ultrasound spectroscopy.

While radiation therapy (RT) is a critical component of breast cancer therapy and is known to decrease overall local recurrence rates, recent studies have shown that normal tissue radiation damage may increase recurrence risk. Fibrosis is a well-known consequence of RT, but the specific sequence of molecular and mechanical changes induced by RT remains poorly understood. To improve cancer therapy outcomes, there is a need to understand the role of the irradiated tissue microenvironment in tumor recurrence. This study seeks to evaluate the use of spectral quantitative ultrasound (spectral QUS) for real time determination of the normal tissue characteristic radiation response and to correlate these results to molecular features in irradiated tissues. Murine mammary fat pads (MFPs) were irradiated to 20Gy, and spectral QUS was used to analyze tissue physical properties pre-irradiation as well as at 1, 5, and 10 days post-irradiation. Tissues were processed for scanning electron microscopy imaging as well as histological and immunohistochemical staining to evaluate morphology and structure. Tissue morphological and structural changes were observed non-invasively following radiation using mid-band fit (MBF), spectral slope (SS), and spectral intercept (SI) measurements obtained from spectral QUS. Statistically significant shifts in MBF and SI indicate structural tissue changes in real time, which matched histological observations. Radiation damage was indicated by increased adipose tissue density and extracellular matrix (ECM) deposition. Our findings demonstrate the potential of using spectral QUS to noninvasively evaluate normal tissue changes resulting from radiation damage. This supports further pre-clinical studies to determine how the tissue microenvironment and physical properties change in response to therapy, which may be important for improving treatment strategies.

Aberrant Mesenteric Adipose Extracellular Matrix Remodelling is Involved in Adipocyte Dysfunction in Crohn's Disease: The Role of TLR-4-mediated Macrophages.

Hypertrophic mesenteric adipose tissue [htMAT] is involved in the disease progression of Crohn's disease [CD] through expressing proinflammatory adipokines from dysfunctional adipocytes by unknown mechanism. Adipocyte function is affected by dynamic adipose tissue extracellular matrix [ECM] remodelling that is mainly mediated by macrophages, and our study aimed to reveal whether aberrant ECM remodelling was present in CD-htMAT and its effects on adipocyte dysfunction, as well as the mechanism. ECM remodelling was examined in MAT samples from CD patients and controls. Mice with dinitrobenzene sulphonic acid [DNBS]-induced colitis were used in vivo study, and lipopolysaccharide [LPS]-induced remodelling behaviour in macrophages was examined in vitro. Macrophages or TLR4 inhibition were used to analyse ECM remodelling mechanisms and their effects on adipocyte function. Aberrant ECM remodelling: was observed in CD-htMAT, which was characterised by a widened and deformed ECM structure accompanied by dysregulated matrix synthesis and degradation; served as a reservoir for inflammatory factors/cells dominated by macrophages; and was involved in adipocyte dysfunction. In addition, macrophages were the main source of ECM remodelling regulatory factors with activation of Toll-like receptor 4 [TLR4] in htMAT. In vivo, macrophage depletion or TLR4 inhibition largely attenuated mesenteric ECM remodelling while improving mesenteric adipocyte dysfunction during chronic enteritis. In vitro, antagonizing TLR4 significantly inhibited LPS-induced macrophage ECM remodelling behavior. The aberrant ECM remodelling in CD-htMAT contributed to mesenteric adipocyte dysfunction, which may be caused at least partly by TLR4-mediated macrophage remodelling behavior. Inhibiting ECM remodelling may be a potential therapeutic strategy for CD.

160-LB: Age-Related Adipose Tissue Fibrosis and Neuropathy in the Genetically Diverse HET3 Mouse

Changes in adipose tissue's extracellular matrix, as with fibrosis, inhibits the tissue's ability to shrink and expand as needed, resulting in sheer stress on adipocytes and chronic inflammation, as well as decreased insulin sensitivity. With aging, a state of metabolic dysregulation and increased incidence of insulin resistance, we have observed a reduction in adipose innervation. This ‘adipose neuropathy' likely poses a physiological challenge for tissue metabolic function and glucose homeostasis, as has been observed in adipose denervation studies. Using second-harmonic generation label-free imaging of adipose collagen and advanced image co-localization techniques, we found that obese adipose tissue exhibits higher co-localization of nerves with collagen fibers, indicating that changes in tissue fibrosis may tie mechanistically with tissue neuropathy. We compared C57BL/6J mice with the more genetically diverse HET3 mouse model that is used by the NIA's intervention testing program, to investigate age-related neuropathy and adipose fibrosis. We found that HET3 mice displayed mitigated neuropathy phenotypes in skin, muscle and adipose, compared to inbred C56BL/6J mice. Tissue fibrosis was significantly increased with age in subcutaneous and visceral white adipose depots of both strains of mice. Using birefringence imaging, we found that the relative distribution of type I / thick filament collagen increased with age while type III / thin filament collagen decreased. Gene expression by qPCR revealed significant increases in collagen genes Col1a1, Col3a1, and Col5a1 in adipose with age. Despite its success as a longevity treatment, the mTOR inhibitor rapamycin had little to no effect on reducing or preventing the onset of neuropathy in HET3 aged mice, and importantly it worsened the fibrotic phenotype with increased type 1 collagen in adipose. Together, we found that rapamycin's longevity effects are uncoupled from healthspan producing effects, with detrimental impacts on adipose fibrosis that may impact age-related insulin resistance. Disclosure J. Willows: None. G. Mishra: None. M. Blaszkiewicz: None. K. L. Townsend: Other Relationship; Neuright, Inc. Funding American Heart Association (AHA) Collaborative Sciences Award (18CSA34090028)

Adipose Tissue Extracellular Matrix Remodeling in Response to Dietary Patterns and Exercise: Molecular Landscape, Mechanistic Insights, and Therapeutic Approaches

Simple SummaryAdipose tissue is considered a metabolic organ that adjusts overall energy homeostasis and critical hormones to the body’s needs. In conditions of caloric intake surpassing energy expenditure, lipid accumulation occurs with constant extracellular matrix deposition. Excess lipids and adipocyte hypertrophy may reduce extracellular matrix flexibility in conjunction with hypoxia and inflammation. These processes induce the development of adipose tissue fibrosis and correlated metabolic dysfunctions, such as insulin resistance. With the increasing rate of chronic diseases worldwide, it is essential to generate a more precise knowledge of fibrotic processes, as well as to create optimal models to study potential therapies to combat the harmful effects of extracellular matrix deposition. In this work, we focused on the physiological processes in the remodeling of adipose tissue fibrosis, along with their relevance to clinical indications. Furthermore, we emphasize understanding how lifestyle can alleviate adipocyte dysfunction. Several studies showed that a nutritionally balanced diet combined with exercise is a remarkable potential strategy for lipolytic activity, preventing rapid extracellular matrix expansion in parallel with insulin and glucose action improvements. Thus, the emerging beneficial role of exercise training and low-calorie diet on adipose tissue ECM remodeling is a topic that deserves attention from health professionals.The extracellular matrix (ECM) is a 3-dimensional network of molecules that play a central role in differentiation, migration, and survival for maintaining normal homeostasis. It seems that ECM remodeling is required for adipose tissue expansion. Despite evidence indicating that ECM is an essential component of tissue physiology, adipose tissue ECM has received limited attention. Hence, there is great interest in approaches to neutralize the harmful effects of ECM enlargement. This review compiles and discusses the current literature on adipose tissue ECM remodeling in response to different dietary patterns and exercise training. High-calorie diets result in substantial adipose tissue ECM remodeling, which in turn could lead to fibrosis (excess deposition of collagens, elastin, and fibronectin), inflammation, and the onset of metabolic dysfunction. However, combining a nutritionally balanced diet with exercise is a remarkable potential strategy for lipolytic activity, preventing rapid ECM expansion in different adipose tissue depots. Despite the distinct exercise modalities (aerobic or resistance exercise) reversing adipose tissue fibrosis in animal models, the beneficial effect on humans remains controversial. Defining molecular pathways and specific mechanisms that mediate the positive effects on adipose tissue, ECM is essential in developing optimized interventions to improve health and clinical outcomes.

Chronic Binge Alcohol Differentially Affects Extracellular Matrix Phenotype in Omental and Subcutaneous Adipose Tissue of SIV‐infected Macaques

The incidence of at‐risk alcohol use among people living with HIV (PLWH) is two times greater than in the general population. Though antiretroviral therapy (ART) has significantly reduced HIV‐associated mortality, increased survival of PLWH on ART is associated with increased incidence of metabolic comorbidities. Adipose tissue maladaptation is central to metabolic alterations in PLWH and at‐risk alcohol use. This involves adipokine disruption, adipocyte dysfunction, inflammation, and dysregulation of the extracellular milieu. Using a relevant preclinical model of HIV‐infection, the objective of our study was to investigate the effects of chronic binge alcohol (CBA) administration on omental adipose tissue (OmAT) and subcutaneous adipose tissue (SAT) extracellular matrix (ECM) phenotype. Rhesus macaques were administered CBA or isovolumetric water (VEH) through an intragastric catheter for 30 min, 5 days a week, with blood alcohol concentrations reaching 50‐60 mM. Three months after initiation of CBA or VEH administration, macaques were inoculated with SIVMac251, and 2.5 months later animals were initiated on ART. CBA or VEH administration was continued throughout the duration of the study (14.5 mo). Animals were euthanized at 11.5 mo of SIV infection, and adipose (OmAT and SAT) samples excised. Collagen content, adipocyte diameter and adipocyte numbers were measured by Picrosirius Red Staining (PSR). A custom PCR Array was used to assess differential expression of 41 ECM‐related genes. CBA administration significantly increased collagen expression and decreased adipocyte diameter in the OmAT without altering adipocyte numbers. There was a strong negative correlation of OmAT adipocyte diameter and collagen expression. CBA administration did not result in significant differences in collagen expression, adipocyte diameter or number in SAT. CBA administration significantly upregulated expression of transforming growth factor beta‐1 (TGFB1), platelet‐derived growth factor receptor alpha (PDGFRA) and secreted protein acidic and rich in cysteine (SPARC), mediators of a pro‐fibrotic milieu in OmAT. In contrast, CBA did not significantly alter expression of ECM‐related genes in the SAT. These data suggest that the maladaptive adipose changes associated with CBA in SIV infection are depot specific. We speculate that changes in the ECM contribute to altered gene expression and impaired adipocyte metabolic capacity.

Adipose tissue stiffness in the development of metabolic diseases

Obesity, defined as an expansion of white adipose tissue, is also accompanied by interstitial fibrosis and overexpression of extracellular matrix (ECM) in adipose tissue in recent studies. We hypothesize that obesity induces adipose tissue ECM crosslinking and stiffening, leading to adipocyte dysfunction. First, we applied a collagen gel system to investigate the effect of ECM stiffness on adipogenesis and mature adipocyte functions. Our results showed that increased ECM stiffness impaired adipogenesis; and blunted the sensitivity to insulin and lipolytic cues, as well as secretion of adipokines, in differentiated adipocytes. In contrast, increased ECM stiffness exacerbated the sensitivity to inflammatory stimulation. Next, we found that ob/ob adipose tissue exhibited an increased signal in the polarized view of picrosirius red stained section, as well as increased crosslinks and aggregated collagen in second harmonics generation (SHG) imaging. These results suggest that ECM crosslinking was upregulated in the adipose tissue of obese mice. Ex vivo crosslinking inhibition in ob/ob adipose tissue explants increased insulin sensitivity, adiponectin expression and lipolytic activity. Finally, in vivo crosslinking inhibition reversed metabolic impairments and adipocyte functional signature genes in ob/ob mice. Taken together, our study underscores that adipose tissue crosslinking and stiffening may orchestrate metabolic disorders resulting from adipocyte dysfunction in obesity.

Osteogenic differentiation of human dental pulp stem cells in decellularised adipose tissue solid foams.

3D cell culture systems based on biological scaffold materials obtainable from both animal and human tissues constitute very interesting tools for cell therapy and personalised medicine applications. The white adipose tissue (AT) extracellular matrix (ECM) is a very promising biomaterial for tissue engineering due to its easy accessibility, malleability and proven biological activity. In the present study, human dental pulp stem cells (hDPSCs) were combined in vitro with ECM scaffolds from porcine and human decellularised adipose tissues (pDAT, hDAT) processed as 3D solid foams, to investigate their effects on the osteogenic differentiation capacity and bone matrix production of hDPSCs, compared to single-protein-based 3D solid foams of collagen type I and conventional 2D tissue-culture-treated polystyrene plates. pDAT solid foams supported the osteogenic differentiation of hDPSCs to similar levels to collagen type I, as assessed by alkaline phosphatase and alizarin red stainings, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and osteocalcin/bone gamma-carboxyglutamate protein (BGLAP) immunostaining. Interestingly, hDAT solid foams showed a markedly lower capacity to sustain hDPSC osteogenic differentiation and matrix calcification and a higher capacity to support adipogenesis, as assessed by RT-qPCR and oil red O staining. White ATs from both human and porcine origins are relatively abundant and available sources of raw material to obtain high quality ECM-derived biomedical products. These biomaterials could have promising applications in tissue engineering and personalised clinical therapy for the healing and regeneration of lesions involving not only a loss of calcified bone but also its associated soft non-calcified tissues.

Recent Developments in Extracellular Matrix Remodeling for Fat Grafting.

Remodeling of the extracellular matrix (ECM), which provides structural and biochemical support for surrounding cells, is vital for adipose tissue regeneration after autologous fat grafting. Rapid and high-quality ECM remodeling can improve the retention rate after fat grafting by promoting neovascularization, regulating stem cells differentiation, and suppressing chronic inflammation. The degradation and deposition of ECM are regulated by various factors, including hypoxia, blood supply, inflammation, and stem cells. By contrast, ECM remodeling alters these regulatory factors, resulting in a dynamic relationship between them. Although researchers have attempted to identify the cellular sources of factors associated with tissue regeneration and regulation of the microenvironment, the factors and mechanisms that affect adipose tissue ECM remodeling remain incompletely understood. This review describes the process of adipose ECM remodeling after grafting and summarizes the factors that affect ECM reconstruction. Also, this review provides an overview of the clinical methods to avoid poor ECM remodeling. These findings may provide new ideas for improving the retention of adipose tissue after fat transplantation.

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Adipose tissue exposed to high fat diet affects extracellular matrix genes in the mesenchymal stem cell population

Background and Aims: Adipose tissue influences the physiological and pathological processes in our body by regulating lipid storage and metabolic homeostasis. Extracellular matrix (ECM) is a dynamic and complex assemblage consisting of polysaccharides, proteogylcans and signaling proteins. Though both adipocytes and non-adipose cells of the stromal fraction contribute to ECM maintenance, role of adipose tissue ECM in the disease remains poorly characterized. High fat diet (HFD) and obesity represent major risk factors for atherosclerosis.

Cardiac glycosides cause cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis.

Cardiac glycosides inhibit Na+ /K+ -ATPase and are used to treat heart failure and arrhythmias. They can induce inflammasome activation and pyroptosis in macrophages, suggesting cytotoxicity, which remains to be elucidated in human tissues. To determine the cell-type specificity of this cytotoxicity, we used human monocyte-derived macrophages and non-adherent peripheral blood cells from healthy donors, plus omental white adipose tissue, stromal vascular fraction-derived pre-adipocytes and adipocytes from obese patients undergoing bariatric surgery. All these cells/tissues were treated with nanomolar concentrations of ouabain (50, 100, 500 nM) to investigate the level of cytotoxicity and the mechanisms leading to cell death. In white adipose tissue, we investigated ouabain-mediated cytotoxicity by measuring insulin sensitivity, adipose tissue function and extracellular matrix deposition ex vivo. Ouabain induced cell death through pyroptosis and apoptosis, and was more effective in monocyte-derived macrophages compared to non-adherent peripheral blood mononuclear cell populations. This cytotoxicity is dependent on K+ flux, as ouabain causes intracellular depletion of K+ and accumulation of Na+ and Ca2+ . Consistently, the cell death caused by these ion imbalances can be rescued by addition of potassium chloride to human monocyte-derived macrophages. Remarkably, when white adipose tissue explants from obese patients are cultured with nanomolar concentrations of ouabain, this causes depletion of macrophages, down-regulation of type VI collagen levels and amelioration of insulin sensitivity ex vivo. The use of nanomolar concentration of cardiac glycosides could be an attractive therapeutic treatment for metabolic syndrome, characterized by pathogenic infiltration and activation of macrophages. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Adipose Tissue Macrophage Polarization in Healthy and Unhealthy Obesity.

Over 650 million adults are obese (body mass index ≥ 30 kg/m2) worldwide. Obesity is commonly associated with several comorbidities, including cardiovascular disease and type II diabetes. However, compiled estimates suggest that from 5 to 40% of obese individuals do not experience metabolic or cardiovascular complications. The existence of the metabolically unhealthy obese (MUO) and the metabolically healthy obese (MHO) phenotypes suggests that underlying differences exist in both tissues and overall systemic function. Macrophage accumulation in white adipose tissue (AT) in obesity is typically associated with insulin resistance. However, as plastic cells, macrophages respond to stimuli in their microenvironments, altering their polarization between pro- and anti-inflammatory phenotypes, depending on the state of their surroundings. The dichotomous nature of MHO and MUO clinical phenotypes suggests that differences in white AT function dictate local inflammatory responses by driving changes in macrophage subtypes. As obesity requires extensive AT expansion, we posit that remodeling capacity with adipose expansion potentiates favorable macrophage profiles in MHO as compared with MUO individuals. In this review, we discuss how differences in adipogenesis, AT extracellular matrix deposition and breakdown, and AT angiogenesis perpetuate altered AT macrophage profiles in MUO compared with MHO. We discuss how non-autonomous effects of remote organ systems, including the liver, gastrointestinal tract, and cardiovascular system, interact with white adipose favorably in MHO. Preferential AT macrophage profiles in MHO stem from sustained AT function and improved overall fitness and systemic health.

14 Characterization of adipose tissue extracellular matrix component mRNA expression during porcine adipogenesis using real-time PCR

Abstract Given adipose tissue is histologically classified as connective tissue, we hypothesized expression of extracellular matrix (ECM) components are significantly altered during adipogenesis. However, little is known about the regulation of the ECM during adipose tissue development in the pig. Therefore, the objective of this study was to characterize expression of ECM components during porcine adipogenesis. Primary cultures of adipose tissue stromal-vascular cells were harvested from 3-day-old neonatal pigs (n=6) and preadipocytes induced to differentiate in vitro for 8 days in the presence of insulin, hydrocortisone, and rosiglitazone. Total RNA was extracted from these cultures on days 0 and 8 post-induction. Real-time PCR was then utilized to determine changes in mRNA expression for collagen type I alpha 1 chain (COL1A), collagen type I alpha 2 chain (COL2A), collagen type I alpha 3 chain (COL3A), collagen type I alpha 4 chain (COL4A), collagen type I alpha 6 chain (COL6A), biglycan, fibronectin, laminin, nitogen-1 (NID1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9), metallopeptidase inhibitor 3 (TIMP3). The mRNA abundances of COL1A, COL3A and MMP2 were significantly downregulated 2.86-fold (P &amp;lt; 0.05), 16.7-fold (P &amp;lt; 0.01) and 3.1-fold (P &amp;lt; 0.05) respectively in day 8 (differentiated) compared to day 0 (undifferentiated) cultures. Meanwhile, mRNA abundances were significantly upregulated during adipogenesis for the COL2A (2.82-fold; P &amp;lt; 0.05), COL4A (2.01-fold; P &amp;lt; 0.05), COL6A (2.8-fold; P &amp;lt; 0.05), biglycan (49.9- fold; P &amp;lt; 0.001), fibronectin (452-fold; P &amp;lt; 0.001), laminin (6.1-fold; P &amp;lt; 0.05), NID1(47.4-fold; P &amp;lt; 0.01), MMP9 (76.8- fold; P &amp;lt; 0.01), and TIMP3(3.04-fold; P &amp;lt; 0.05) genes. These data support the hypothesis that significant changes in ECM components occur during porcine adipogenesis. Modulating adipose tissue ECM remodeling might be a novel strategy to manipulate adiposity in the pig.

Elucidating nanoscale mechanical properties of diabetic human adipose tissue using atomic force microscopy

Obesity-related type 2 diabetes (DM) is a major public health concern. Adipose tissue metabolic dysfunction, including fibrosis, plays a central role in DM pathogenesis. Obesity is associated with changes in adipose tissue extracellular matrix (ECM), but the impact of these changes on adipose tissue mechanics and their role in metabolic disease is poorly defined. This study utilized atomic force microscopy (AFM) to quantify difference in elasticity between human DM and non-diabetic (NDM) visceral adipose tissue. The mean elastic modulus of DM adipose tissue was twice that of NDM adipose tissue (11.50 kPa vs. 4.48 kPa) to a 95% confidence level, with significant variability in elasticity of DM compared to NDM adipose tissue. Histologic and chemical measures of fibrosis revealed increased hydroxyproline content in DM adipose tissue, but no difference in Sirius Red staining between DM and NDM tissues. These findings support the hypothesis that fibrosis, evidenced by increased elastic modulus, is enhanced in DM adipose tissue, and suggest that measures of tissue mechanics may better resolve disease-specific differences in adipose tissue fibrosis compared with histologic measures. These data demonstrate the power of AFM nanoindentation to probe tissue mechanics, and delineate the impact of metabolic disease on the mechanical properties of adipose tissue.

Type VI collagen and its cleavage product, endotrophin, cooperatively regulate the adipogenic and lipolytic capacity of adipocytes

ObjectiveObesity-induced adipose tissue remodeling is closely associated with systemic insulin resistance. However, the mechanistic involvement of adipocyte-derived extracellular matrix proteins under pathophysiological conditions remains unclear. Our aim was to investigate the distinctive contributions of each chain of type VI collagens (Col6) and its cleavage protein endotrophin to adipocyte functions and insulin sensitivity. MethodsCol6 comprises three alpha chains: Col6a1, Col6a2, and Col6a3. We generated Col6a1-, Col6a2-, and Col6a3-deficient 3T3-L1 adipocytes using the CRISPR-Cas9 system as well as a novel Col6a3-deficient (Col6a3KO) mouse model for loss-of-function studies. Adenoviral-endotrophin and adipocyte-specific doxycycline-inducible endotrophin transgenic mice were utilized for the gain-of-function analysis. ResultsThe holo-Col6 fibrils were found to be required for mature adipocyte differentiation. Only Col6a3-deficient 3T3-L1 adipocytes showed decreased inflammation and basal adipocyte lipolysis and prevented ER-stress-induced insulin resistance. Consistently, Col6a3KO mice showed decreased adipocyte size and fat mass of epididymal adipose tissues due to a defect in adipogenic and lipolytic capacity of adipocytes. Beyond the structural role of Col6a3, overexpression of endotrophin in obese mice further augmented insulin resistance, which was tightly associated with a significant increase in lipolysis, inflammation, and cellular apoptosis in adipose tissues, whereas this showed a limited effect on adipogenesis. ConclusionsThese novel findings corroborate our previous observations suggesting that adipose tissue extracellular matrix regulates adipocyte function and insulin sensitivity in pathophysiological conditions. Mechanistically, holo-Col6 fibrils and their signaling derivative endotrophin govern adipocyte function independently of their role as structural supports via MAPK signaling pathways, and the latter could be an important metabolic effector in obesity-related metabolic diseases.

Mechanisms of adipose tissue extracellular matrix alterations in an in vitro model of adipocytes hypoxia and aging

Fibrosis has been considered as a hallmark of dysfunctional adipose tissue (AT), however the role and mechanisms of fibrosis in the age related AT dysfunction are not yet well characterized.The aim of the study was to investigate the mechanisms of extracellular matrix (ECM) alterations and the role of caveolins, using an in vitro model of adipocyte aging and hypoxia.Hypoxic adipocytes, but also aged adipocytes, were characterized by a significant increase in gene expression of pro-inflammatory cytokines and ECM components. Immunofluorescence analysis confirmed an increase in collagen VI-A3 in hypoxic and also in aged adipocytes. However aged adipocytes were characterized by only a slight increase in HIF1α immunofluorescence and by a more relevant increase in senescence compared to hypoxic and mature adipocytes, with an increase in p-53 protein and a decrease in SIRT 1 protein.Immunofluorescence and western blot analysis revealed a significant decrease in caveolin-1 expression in hypoxic adipocytes and even more in aged adipocytes.In conclusions, aging adipocytes are associated to alteration of ECM and fibrosis, by modulation of the caveolins through complex mechanisms where inflammation, hypoxia and cellular senescence are coexisting.

701-P: Effects of Exercise on Extracellular Matrix Modifiers in Subcutaneous Adipose Tissue of Obese Adults

Mechanisms underlying the direct effects of exercise on metabolic health (independently of weight loss) are poorly understood, and very little is known about the effects of exercise on adipose tissue. The purpose of this study was to examine the effects of exercise training on the key extracellular matrix (ECM) modifiers, matrix metalloproteinase 2 (MMP2) and MMP9, in abdominal subcutaneous adipose tissue (aSAT) of obese adults. Seven obese adults (BMI=33±4 kg/m2; age=32±6) completed 12 weeks of exercise training (either 45min of steady-state exercise at 70% HRmax or 10x1min intervals at 90% HRmax with 1min recovery between intervals), and they were required to maintain body weight throughout. aSAT biopsies were collected before training (Pre) and twice after training - 1 day post exercise (1d PEX) and again 4 days post exercise (4d PEX; to wash out the acute effects of exercise). Using standard immunoblotting methods, we found no evidence for effects of training on either MMP2 or MMP9. However, using gelatin zymography (a technique with more than 100-fold higher sensitivity than immunoblotting) we found a strong trend for an increased MMP9 abundance 1d PEX (3.7±1.0 vs. 3.0±1.4 arbitrary units (AU), for 1d PEX vs. Pre; P=0.08). Interestingly, however, MMP9 returned to Pre training levels by 4d PEX (1.9±1.3 AU; P&amp;lt;0.05 vs. 1d PEX). Gelatin zymography can also allow for quantification of the active-form of MMPs, and we found trends for exercise training to increase MMP2 in both its active- (P=0.08 and P=0.22 for 1d PEX and 4d PEX vs. Pre, respectively) and inactive-form (P=0.13 and P=0.12 for 1d PEX and 4d PEX vs. Pre, respectively). We were unable to detect the active form of MMP9. In summary, because increased abundance and activity of MMPs may alter the fibrotic content and composition of the ECM, exercise-induced increases in MMP2 and MMP9 within aSAT may contribute to favorable modifications within adipose tissue ECM of obese adults. Disclosure P. Varshney: None. B.J. Ryan: None. C. Ahn: None. M.W. Schleh: None. J.F. Horowitz: None. Funding National Institutes of Health (R01DK077966, P30DK089503, F32DK117522, UL1TR002240)