HDAC3 ensures stepwise epidermal stratification via NCoR/SMRT-reliant mechanisms independent of its histone deacetylase activity.

Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.

Balanced control of thermogenesis by nuclear receptor corepressors in brown adipose tissue.

Brown adipose tissue (BAT) is a key thermogenic organ whose expression of uncoupling protein 1 (UCP1) and ability to maintain body temperature in response to acute cold exposure require histone deacetylase 3 (HDAC3). HDAC3 exists in tight association with nuclear receptor corepressors (NCoRs) NCoR1 and NCoR2 (also known as silencing mediator of retinoid and thyroid receptors [SMRT]), but the functions of NCoR1/2 in BAT have not been established. Here we report that as expected, genetic loss of NCoR1/2 in BAT (NCoR1/2 BAT-dKO) leads to loss of HDAC3 activity. In addition, HDAC3 is no longer bound at its physiological genomic sites in the absence of NCoR1/2, leading to a shared deregulation of BAT lipid metabolism between NCoR1/2 BAT-dKO and HDAC3 BAT-KO mice. Despite these commonalities, loss of NCoR1/2 in BAT does not phenocopy the cold sensitivity observed in HDAC3 BAT-KO, nor does loss of either corepressor alone. Instead, BAT lacking NCoR1/2 is inflamed, particularly with respect to the interleukin-17 axis that increases thermogenic capacity by enhancing innervation. Integration of BAT RNA sequencing and chromatin immunoprecipitation sequencing data revealed that NCoR1/2 directly regulate Mmp9, which integrates extracellular matrix remodeling and inflammation. These findings reveal pleiotropic functions of the NCoR/HDAC3 corepressor complex in BAT, such that HDAC3-independent suppression of BAT inflammation counterbalances stimulation of HDAC3 activity in the control of thermogenesis.

Establishment and characterization of a primary murine adipose tissue-chip.

Better experimental models are needed to enhance our understanding of metabolic regulation which is seen in obesity and metabolic disorders, such as type 2 diabetes. In vitro models based on microfluidics enable physiological representations of tissues with several advantages over conventional culture systems, such as perfused flow to better mimic the physiological environment. Although cell lines such as 3T3-L1 have been incorporated in microfluidic devices, murine primary preadipocytes have not been differentiated and maintained for long-term monitoring in these culture systems. We describe the differentiation of these cells into white adipose depots on a perfused microfluidic chip. We compare the effects of shear flow on these cells, and show with a direct comparison of high/low shear conditions that direct shear is detrimental to the viability of preadipocytes. We further develop a dual-chamber microfluidic chip that enables perfusion while at the same time protects the cells from direct fluidic shear. We show that the dual-layer microfluidic device enables long-term culture of cells and allows stimulation of cells through perfusion-we can culture, differentiate, and maintain the differentiated adipose tissue for over multiple weeks in the device. Both triglycerides and lipolytic glycerol production increased significantly by several folds during differentiation. After successful differentiation, the adipocytes had upregulated expression of leptin and adiponectin, which are important makers of the final stage of adipogenic differentiation. In conclusion, the dual-layer microfluidic device incorporated with primary adipocytes improves the understanding of adipose differentiation under dynamic conditions and is positioned to serve as a disease model for studying obesity and other metabolic disorders.

Protein kinase A induces UCP1 expression in specific adipose depots to increase energy expenditure and improve metabolic health.

Adipose tissue PKA has roles in adipogenesis, lipolysis, and mitochondrial function. PKA transduces the cAMP signal downstream of G protein-coupled receptors, which are being explored for therapeutic manipulation to reduce obesity and improve metabolic health. This study aimed to determine the overall physiological consequences of PKA activation in adipose tissue. Mice expressing an activated PKA catalytic subunit in adipose tissue (Adipoq-caPKA mice) showed increased PKA activity in subcutaneous, epididymal, and mesenteric white adipose tissue (WAT) depots and brown adipose tissue (BAT) compared with controls. Adipoq-caPKA mice weaned onto a high-fat diet (HFD) or switched to the HFD at 26 wk of age were protected from diet-induced weight gain. Metabolic health was improved, with enhanced insulin sensitivity, glucose tolerance, and ß-cell function. Adipose tissue health was improved, with smaller adipocyte size and reduced macrophage engulfment of adipocytes. Using metabolic cages, we found that Adipoq-caPKA mice were shown to have increased energy expenditure, but no difference to littermate controls in physical activity or food consumption. Immunoblotting of adipose tissue showed increased expression of uncoupling protein-1 (UCP1) in BAT and dramatic UCP1 induction in subcutaneous WAT, but no induction in the visceral depots. Feeding a HFD increased PKA activity in epididymal WAT of wild-type mice compared with chow, but did not change PKA activity in subcutaneous WAT or BAT. This was associated with changes in PKA regulatory subunit expression. This study shows that adipose tissue PKA activity is sufficient to increase energy expenditure and indicates that PKA is a beneficial target in metabolic health.

Systemic regulation of adipose metabolism.

White adipose tissue serves as a critical energy storage depot and endocrine organ. Adipocytes are subject to numerous levels of regulation, including neuronal, endocrine and metabolic. While insulin is the classical endocrine regulator of lipid metabolism in adipose tissue, other important endocrine hormones also control adipose tissue physiology. In this review, we will focus on the contribution of the pituitary in the modulation of adipocyte function, through the direct release of growth hormone as well as via the regulation of the thyroid gland and release of thyroid hormone. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Atomic force microscopy characterization of white and beige adipocyte differentiation.

Adipose tissue plays an essential role in systemic metabolism with white adipose tissue (WAT) making up most of the tissue and being involved in the regulation of energy homeostasis, and brown and beige adipose tissue (BAT) exhibiting thermogenic activity. There is promise in the conversion of white adipocytes into beige ones as a therapeutic potential to control and enhance systemic metabolism, but it is difficult to maintain this transformation in vivo because we do not fully understand the mechanism of conversion. In this study, we applied atomic force microscopy (AFM) to characterize beige or white adipocytes during the process of differentiation for morphology, roughness, adhesion, and elasticity at different time points. As cells differentiated to white and beige adipocytes, they exhibited morphological changes as they lipid loaded, transitioning from flattened elongated cells to a rounded shape indicating adipogenesis. While there was an initial decrease in elasticity for both beige and white adipocytes, white adipocytes exhibited a higher elasticity than beige adipocytes at all time points. Beige and white adipogenesis exhibited a decrease in adhesion energy compared to preadipocytes, yet at day 12, white adipocytes had a significant increase in adhesion energy compared to beige adipocytes. This work shows significant differences in the mechanical properties of white vs. beige adipocytes during differentiation. Results from this study contribute to a better understanding of the differentiation of adipocytes which are vital to the therapeutic induction, engineered models, and maintenance of beige adipocytes as a potential approach for enhancing systemic metabolism.

Investigation of insulin resistance through a multiorgan microfluidic organ-on-chip.

The development of hepatic insulin resistance (IR) is a critical factor in developing type 2 diabetes (T2D), where insulin fails to inhibit hepatic glucose production but retains its capacity to promote hepatic de novo lipogenesis leading to hyperglycemia and hypertriglyceridemia. Improving insulin sensitivity can be effective in preventing and treating T2D. However, selective control of glucose and lipid synthesis has been difficult. It is known that excess white adipose tissue is detrimental to insulin sensitivity, whereas brown adipose tissue transplantation can restore it in diabetic mice. However, challenges remain in our understanding of liver-adipose communication because the confounding effects of hypothalamic regulation of metabolic function cannot be ruled out in previous studies. There is a lack ofin vitromodels that use primary cells to study cellular-crosstalk under insulin resistant conditions. Building upon our previous work on the microfluidic primary liver and adipose organ-on-chips, we report for the first time, the development of an integrated insulin resistant liver-adipose (white and brown) organ-on-chip. The design of the microfluidic device was carried out using computational fluid dynamics; the experimental studies were conducted by carrying out detailed biochemical analysis RNA-seq analysis on both cell types. Further, we tested the hypothesis that brown adipocytes (BAC) regulated both hepatic insulin sensitivity and de novo lipogenesis. Our results show that BAC effectively restored insulin sensitivity and supressed hepatic glucose production and de novo lipogenesis suggesting that the experimental platform could be useful for identifying potential therapeutics to treat IR and diabetes.

Nuclear corepressor SMRT acts as a strong regulator of both ß-oxidation and suppressor of fibrosis in the differentiation process of mouse skeletal muscle cells.

BACKGROUND: Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor (CoR) which has been recognized as an important player in the regulation of hepatic lipogenesis and in somatic development in mouse embryo. SMRT protein is also widely expressed in mouse connective tissues, for example adipocytes and muscle. We recently reported that mice with global deletion of SMRT develop significant obesity and muscle wasting which are independent from thyroid hormone (TH) signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still not clear. METHODS: To clarify role of SMRT in muscle differentiation, we made myogenic C2C12 clones which lack SMRT protein (C2C12-SKO) by using CRISPR-Cas9. Wild-type C2C12 (C2C12-WT) and C2C12-SKO cells were cultured in differentiation medium, and the resulting gene and protein profiles were compared between the two cell lines both before and after differentiation. We also analyzed muscle tissues which were dissected from whole body SMRT knockout (KO) mice and their controls. RESULTS: We found significant up-regulation of muscle specific ß-oxidation markers; Peroxisome proliferator-activated receptor δ (PPARδ) and PPARγ coactivator-1α (PGC-1α) in the C2C12-SKO cells, suggesting that the cells had a similar gene profile to what is found in exercised rodent skeletal muscle. On the other hand, confocal microscopic analysis showed the significant loss of myotubes in C2C12-SKO cells similar to the morphology found in immature myoblasts. Proteomics analysis also confirmed that the C2C12-SKO cells had higher expression of markers of fibrosis (ex. Collagen1A1; COL1A1 and Fibroblast growth factor-2; FGF-2), indicating the up-regulation of Transforming growth factor-ß (TGF-ß) receptor signaling. Consistent with this, treatment with a specific TGF-ß receptor inhibitor ameliorated both the defects in myotube differentiation and fibrosis. CONCLUSION: Taken together, we demonstrate that SMRT functions as a pivotal transcriptional mediator for both ß-oxidation and the prevention for the fibrosis via TGF-ß receptor signaling in the differentiation of C2C12 myoblasts. In contrast to the results from C2C12 cells, SMRT does not appear to play a role in adult skeletal muscle of whole body SMRT KO mice. Thus, SMRT plays a significant role in the differentiation of myoblasts.

Laminin-α4 Negatively Regulates Adipocyte Beiging Through the Suppression of AMPKα in Male Mice.

Laminin-α4 (LAMA4) is an extracellular matrix protein implicated in the regulation of adipocyte differentiation and function. Prior research describes a role for LAMA4 in modulating adipocyte thermogenesis and uncoupling protein-1 (UCP1) expression in white adipose; however, the mechanisms involved are poorly understood. Here, we describe that Lama4 knockout mice (Lama4-/-) exhibit heightened mitochondrial biogenesis and peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) expression in subcutaneous white adipose tissue (sWAT). Furthermore, the acute silencing of LAMA4 with small interfering RNA in primary murine adipocytes was sufficient to upregulate the expression of thermogenic markers UCP1 and PR domain containing 16 (PRDM16). Silencing also resulted in an upregulation of PGC1-α and adenosine 5'-monophosphate-activated protein kinase (AMPK)-α expression. Subsequently, we show that integrin-linked kinase (ILK) is downregulated in the sWAT of Lama4-/- mice, and its silencing in adipocytes similarly resulted in elevated expression of UCP1 and AMPKα. Last, we demonstrate that treatment of human induced pluripotent stem cell-derived thermogenic adipocytes with LAMA4 (LN411) inhibited the expression of thermogenic markers and AMPKα. Overall, our results indicate that LAMA4 negatively regulates a thermogenic phenotype and pathways involving mitochondrial biogenesis in adipocytes through the suppression of AMPKα.

Natural angiogenesis inhibitor signals through Erk5 activation of peroxisome proliferator-activated receptor gamma (PPARgamma).

Erk-5, a member of the MAPK superfamily, has a catalytic domain similar to Erk1/2 and a unique C-terminal domain enabling binding with transcription factors. Aberrant vascularization in the Erk5-null mice suggested a link to angiogenesis. Ectopic expression of constitutively active Erk5 blocks endothelial cell morphogenesis and causes HIF1-alpha destabilization/degradation. However the mechanisms by which endogenous Erk5 regulates angiogenesis remain unknown. We show that Erk5 and its activating kinase MEK5 are the upstream mediators of the anti-angiogenic signal by the natural angiogenesis inhibitor, pigment epithelial-derived factor (PEDF). We demonstrate that Erk5 phosphorylation allows activation of PPARgamma transcription factor by displacement of SMRT co-repressor. PPARgamma, in turn is critical for NFkappaB activation, PEDF-dependent apoptosis, and anti-angiogenesis. The dominant negative MEK5 mutant and Erk5 shRNA diminished PEDF-dependent apoptosis, inhibition of the endothelial cell chemotaxis, and angiogenesis. This is the first evidence of Erk5-dependent transduction of signals by endogenous angiogenesis inhibitors.

The silencing mediator of retinoid and thyroid hormone receptors (SMRT) regulates adipose tissue accumulation and adipocyte insulin sensitivity in vivo.

The silencing mediator of retinoid and thyroid hormone receptors (SMRT) serves as a corepressor for nuclear receptors and other factors. Recent evidence suggests that SMRT is an important regulator of metabolism, but its role in adipocyte function in vivo remains unclear. We generated heterozygous SMRT knock-out (SMRT(+/-)) mice to investigate the function of SMRT in the adipocyte and the regulation of adipocyte insulin sensitivity. We show that SMRT(+/-) mice are normal weight on a regular diet, but develop increased adiposity on a high-fat diet (HFD). The mechanisms underlying this phenotype are complex, but appear to be due to a combination of an increased number of smaller subcutaneous adipocytes as well as decreased leptin expression, resulting in greater caloric intake. In addition, adipogenesis of mouse embryonic fibroblasts (MEFs) derived from these mice was increased. However, adipocyte insulin sensitivity, measured by insulin-induced Akt phosphorylation and insulin-mediated suppression of lipolysis, was enhanced in SMRT(+/-) adipocytes. These finding suggest that SMRT regulates leptin expression and limits the ability of fat mass to expand with increased caloric intake, but that SMRT also negatively regulates adipocyte insulin sensitivity.

Laminins in metabolic tissues.

Laminins are extracellular matrix proteins that reside in the basement membrane and provide structural support in addition to promoting cellular adhesion and migration. Through interactions with cell surface receptors, laminins stimulate intracellular signaling cascades which direct specific survival and differentiation outcomes. In metabolic tissues such as the pancreas, adipose, muscle, and liver, laminin isoforms are expressed in discrete temporal and spatial patterns suggesting that certain isoforms may support the development and function of particular metabolic cell types. This review focuses on the research to date detailing the expression of laminin isoforms, their potential function, as well as known pathways involved in laminin signaling in metabolic tissues. We will also discuss the current biomedical therapies involving laminins in these tissues in addition to prospective applications, with the goal being to encourage future investigation of laminins in the context of metabolic disease.

Towards an Insulin Resistant Adipose Model on a Chip.

INTRODUCTION: Adipose tissue and adipocytes are primary regulators of insulin sensitivity and energy homeostasis. Defects in insulin sensitivity of the adipocytes predispose the body to insulin resistance (IR) that could lead to diabetes. However, the mechanisms mediating adipocyte IR remain elusive, which emphasizes the need to develop experimental models that can validate the insulin signaling pathways and discover new mechanisms in the search for novel therapeutics. Currently in vitro adipose organ-chip devices show superior cell function over conventional cell culture. However, none of these models represent disease states. Only when these in vitro models can represent both healthy and disease states, they can be useful for developing therapeutics. Here, we establish an organ-on-chip model of insulin-resistant adipocytes, as well as characterization in terms of insulin signaling pathway and lipid metabolism. METHODS: We differentiated, maintained, and induced insulin resistance into primary adipocytes in a microfluidic organ-on-chip. We then characterized IR by looking at the insulin signaling pathway and lipid metabolism, and validated by studying a diabetic drug, rosiglitazone. RESULTS: We confirmed the presence of insulin resistance through reduction of Akt phosphorylation, Glut4 expression, Glut4 translocation and glucose uptake. We also confirmed defects of disrupted insulin signaling through reduction of lipid accumulation from fatty acid uptake and elevation of glycerol secretion. Testing with rosiglitazone showed a significant improvement in insulin sensitivity and fatty acid metabolism as suggested by previous reports. CONCLUSIONS: The adipose-chip exhibited key characteristics of IR and can serve as model to study diabetes and facilitate discovery of novel therapeutics.

A 3D human adipose tissue model within a microfluidic device.

An accurate in vitro model of human adipose tissue could assist in the study of adipocyte function and allow for better tools for screening new therapeutic compounds. Cell culture models on two-dimensional surfaces fall short of mimicking the three-dimensional in vivo adipose environment, while three-dimensional culture models are often unable to support long-term cell culture due, in part, to insufficient mass transport. Microfluidic systems have been explored for adipose tissue models. However, current systems have primarily focused on 2D cultured adipocytes. In this work, a 3D human adipose microtissue was engineered within a microfluidic system. Human adipose-derived stem cells (ADSCs) were used as the cell source for generating differentiated adipocytes. The ADSCs differentiated within the microfluidic system formed a dense lipid-loaded mass with the expression of adipose tissue genetic markers. Engineered adipose tissue showed a decreased adiponectin secretion and increased free fatty acid secretion with increasing shear stress. Adipogenesis markers were downregulated with increasing shear stress. Overall, this microfluidic system enables the on-chip differentiation and development of a functional 3D human adipose microtissue supported by the interstitial flow. This system could potentially serve as a platform for in vitro drug testing for adipose tissue-related diseases.

Laminin-α4 Is Upregulated in Both Human and Murine Models of Obesity.

Obesity affects nearly one billion globally and can lead to life-threatening sequelae. Consequently, there is an urgent need for novel therapeutics. We have previously shown that laminin, alpha 4 (Lama4) knockout in mice leads to resistance to adipose tissue accumulation; however, the relationship between LAMA4 and obesity in humans has not been established. In this study we measured laminin-α chain and collagen mRNA expression in the subcutaneous white adipose tissue (sWAT) of mice placed on chow (RCD) or 45% high fat diet (HFD) for 8 weeks, and also in HFD mice then placed on a "weight loss" regimen (8 weeks HFD followed by 6 weeks RCD). To assess extracellular matrix (ECM) components in humans with obesity, laminin subunit alpha mRNA and protein expression was measured in sWAT biopsies of female control subjects (BMI<30) or subjects with obesity undergoing bariatric surgery at the University of Chicago Medical Center (BMI>35) both before and three months after surgery. Lama4 was significantly higher in sWAT of HFD compared to RCD mice at both the RNA and protein level (p<0.001, p<0.05 respectively). sWAT from human subjects with obesity also showed significantly higher LAMA4 mRNA (p<0.01) and LAMA4 protein expression (p<0.05) than controls. Interestingly, even though LAMA4 expression was increased in both humans and murine models of obesity, no significant difference in Lama4 or LAMA4 expression was detected following short-term weight loss in either mouse or human samples, respectively. From these results we propose a significant association between obesity and elevated LAMA4 expression in humans, as well as in mouse models of obesity. Further studies should clarify the mechanisms underlying this association to target LAMA4 effectively as a potential therapy for obesity.

Integrins and extracellular matrix proteins modulate adipocyte thermogenic capacity.

Obesity and the metabolic disease epidemic has led to an increase in morbidity and mortality. A rise in adipose thermogenic capacity via activation of brown or beige fat is a potential treatment for metabolic diseases. However, an understanding of how local factors control adipocyte fate is limited. Mice with a null mutation in the laminin α4 (LAMA4) gene (KO) exhibit resistance to obesity and enhanced expression of thermogenic fat markers in white adipose tissue (WAT). In this study, changes in WAT extracellular matrix composition in the absence of LAMA4 were evaluated using liquid chromatography/tandem mass spectrometry. KO-mice showed lower levels of collagen 1A1 and 3A1, and integrins α7 (ITA7) and ß1 (ITB1). ITA7-ITB1 and collagen 1A1-3A1 protein levels were lower in brown adipose tissue compared to WAT in wild-type mice. Immunohistochemical staining confirmed lower levels and different spatial distribution of ITA7 in KO-WAT. In culture studies, ITA7 and LAMA4 levels decreased following a 12-day differentiation of adipose-derived stem cells into beige fat, and knock-down of ITA7 during differentiation increased beiging. These results demonstrate that extracellular matrix interactions regulate adipocyte thermogenic capacity and that ITA7 plays a role in beige adipose formation. A better understanding of the mechanisms underlying these interactions can be used to improve systemic energy metabolism and glucose homeostasis.

Enhanced glycogen metabolism in adipose tissue decreases triglyceride mobilization.

Adipose tissue is a primary site for lipid storage containing trace amounts of glycogen. However, refeeding after a prolonged partial fast produces a marked transient spike in adipose glycogen, which dissipates in coordination with the initiation of lipid resynthesis. To further study the potential interplay between glycogen and lipid metabolism in adipose tissue, the aP2-PTG transgenic mouse line was utilized since it contains a 100- to 400-fold elevation of adipocyte glycogen levels that are mobilized upon fasting. To determine the fate of the released glucose 1-phosphate, a series of metabolic measurements were made. Basal and isoproterenol-stimulated lactate production in vitro was significantly increased in adipose tissue from transgenic animals. In parallel, basal and isoproterenol-induced release of nonesterified fatty acids (NEFAs) was significantly reduced in transgenic adipose tissue vs. control. Interestingly, glycerol release was unchanged between the genotypes, suggesting that enhanced triglyceride resynthesis was occurring in the transgenic tissue. Qualitatively similar results for NEFA and glycerol levels between wild-type and transgenic animals were obtained in vivo during fasting. Additionally, the physiological upregulation of the phosphoenolpyruvate carboxykinase cytosolic isoform (PEPCK-C) expression in adipose upon fasting was significantly blunted in transgenic mice. No changes in whole body metabolism were detected through indirect calorimetry. Yet weight loss following a weight gain/loss protocol was significantly impeded in the transgenic animals, indicating a further impairment in triglyceride mobilization. Cumulatively, these results support the notion that the adipocyte possesses a set point for glycogen, which is altered in response to nutritional cues, enabling the coordination of adipose glycogen turnover with lipid metabolism.

Optimization of Co-Culture Conditions for a Human Vascularized Adipose Tissue Model.

In vitro adipose tissue models can be used to provide insight into fundamental aspects of adipose physiology. These systems may serve as replacements for animal models, which are often poor predictors of obesity and metabolic diseases in humans. Adipose tissue consists of a rich vasculature that is essential to its function. However, the study of endothelial cell-adipocyte interactions has been challenging due to differences in culture conditions required for the survival and function of each cell type. To address this issue, we performed an extensive evaluation of the cell culture media composition to identify the conditions optimal for the co-culture of endothelial cells and adipocytes. The effects of individual media factors on cell survival, proliferation, and differentiation were systematically explored. Several media factors were determined to disrupt the co-culture system. Optimized culture conditions were identified and used to generate a vascularized human adipose microtissue. An interconnected vascular network was established within an adipose micro-tissue, and the networks were anastomosed with perfused channels to form a functional network. In conclusion, media conditions were identified that enabled endothelial cell-adipocyte co-culture and were used to support the formation of a vascularized adipose tissue within a microfluidic device.