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Obesity-associated type 2 diabetes (DM) leads to adipose tissue dysfunction. Lumican is a proteoglycan implicated in obesity, insulin resistance (IR), and adipocyte dysfunction. Using human visceral adipose tissue (VAT) from subjects with and without DM, we studied lumican effects on adipocyte function. Lumican was increased in VAT and adipocytes in DM. Lumican knockdown in adipocytes decreased lipolysis and improved adipogenesis and insulin sensitivity in VAT adipocytes in DM, while treatment with human recombinant lumican increased lipolysis and impaired insulin-sensitivity in an ERK-dependent manner. We demonstrate that lumican impairs adipocyte metabolism, partially via ERK signalling, and is a potential target for developing adipose tissue-targeted therapeutics in DM.

Objective Spigelian hernia is an uncommon congenital or acquired defect in the transversus abdominis aponeurosis with non-specific symptoms posing a diagnostic challenge. There is a paucity of radiology literature on imaging findings of Spigelian hernia. The objective of this study is to explore the role of MDCT in evaluating Spigelian hernia along with clinical and surgical implications. Materials and methods In this IRB approved, HIPAA compliant retrospective observational analysis MDCT imaging findings of 43 Spigelian hernias were evaluated by two fellowship-trained radiologists. Imaging features evaluated were: presence of Spigelian hernia, laterality, relation to “hernia belt” (between 0 and 6 cm cranial to an imaginary axial line between both anterior superior iliac spines), the hernia neck and sac sizes, hernia content, and other coexistent hernias (umbilical, incisional, inguinal). Patient's demographics (age, gender, BMI, conditions with increased intra-abdominal pressure) were also recorded for any correlation. Results 60% (26/43) of Spigelian hernias were located below the hernia belt while 33% (14/43) within the hernia belt and 7% (3/43) above the hernia belt. The most common subtype of Spigelian hernia encountered was interparietal (84%). The mean hernia neck diameter was 3.4 cm, mean hernia sac volume was 329 cc. Hernia content included: fat (43/43) bowel (23/43), fluid (3/43). 3 patients had no clinical history provided, the remaining 37 patients' clinical presentation was asymptomatic in 73% (27/37), acute abdominal pain in 5% (2/37) and chronic abdominal pain in 22% (8/37). None of the hernia were incarcerated and none of the patients underwent emergent surgery. No significant correlation was noted between Spigelian hernia and causes of increased intra-abdominal pressure. 90% of our patients had other abdominal hernias. 30.9 was the mean BMI (20.8–69.1). Conclusion Most of the Spigelian hernia occurred below the traditionally described hernia belt and the majority are of interparietal subtype that can be best diagnosed with MDCT in contrast to physical examination.

Single-cell and single-nuclei RNA sequencing data (scRNAseq and snRNAseq, respectively) have revealed substantial heterogeneity in the AT (AT) cellular landscape in rodents and humans depending on depot and disease status. We used snRNAseq to characterize the cellular landscape of human visceral (VAT) and subcutaneous AT (SAT) samples from lean subjects and subjects with obesity. We identified multiple cell types in the AT cellular repertoire, including three major AT stromal cell (ASC) subpopulations, multiple types of adipocyte (ADIPO) populations that retain properties similar to ASC, endothelial cell (EC), T-cell, and macrophage (MAC) populations that are in concordance and expand upon other published datasets. ADIP and EC are more prominent in SAT compared to VAT which has a higher proportion of ASC. Of two dominant ASC subpopulations, one (inflammatory-mesothelial, IM- ASC) is present in VAT, but absent in SAT, while the other (fibroadipogenic, FA-ASC) is present in both VAT and SAT. Both populations retain their properties in in vitro culture and have adipogenic capacity with different metabolic features. Informatic and in situ studies support ADIP derived from IM- and FA- ASC are found in human VAT. We also identified a wide range of EC subtypes in human AT with features of lymphatic, venous, and arterial EC, with identification of a PRDM16 expression EC population with features of an EC progenitor. Immune cell populations match recent experimental validation of lipid activated macrophage (LAM) phenotypes, TIM4 macrophages, and a prominent population of MRC1/CD206+ resident AT macrophages with gene expression signatures related to glucocorticoid activation. Overall, our study demonstrates depot-specific cellular diversity in human VAT and SAT in which distinct ASC subpopulations may differently contribute to AT dysfunction in obesity. Also, our results highlight an unprecedented EC heterogeneity suggesting AT EC as highly specialized cells and potentially important regulators of depot-specific functions.METHODS STATEMENTHuman subjects provided informed consent and were enrolled with approval from Institutional Review Boards at the University of Michigan and Veterans Affairs Ann Arbor Healthcare System. Enrollment, consent, and all aspects of human subject research were carried out in accordance with the Belmont Report from the National Research Act of 1974, and the Declaration of Helsinki set forth by the World Medical Association. This manuscript contains no human participants’ names or other HIPAA identifiers.

BACKGROUND: Dysfunctional adipose tissue plays a central role in the pathogenesis of obesity-related metabolic disease, including type 2 diabetes. Targeting adipose tissue using biopolymer implants is a novel therapeutic approach for metabolic disease. METHODS: We transplanted porous poly(lactide-co-glycolide) (PLG) implants coated with human interleukin 4 (hIL-4)-expressing lentivirus into epididymal white adipose tissue (eWAT) of mice fed high-fat diet. Tissue and systemic inflammation and metabolism were studied with flow cytometry, immunohistochemistry, qRTPCR, adipose tissue histology and in vivo glucose tolerance testing at 2 weeks and 10 weeks of high-fat diet. RESULTS: PLG implants carrying hIL-4-expressing lentivirus implanted into epididymal white adipose tissue of mice regulated adipose tissue inflammation, including increased CD3(+)CD4(+) T-cell frequency, increased eWAT adipocyte hypertrophy, and decreased FASN and ATGL expression, along with reduced fasting blood glucose levels. These effects were observed in early obesity, but were not maintained in established obesity. CONCLUSIONS: Local delivery of bioimplants loaded with cytokine-expressing lentivirus vectors to adipose tissue influences tissue inflammation and systemic metabolism in early obesity. Further study will be required to show more durable metabolic effects. These data demonstrate that polymer biomaterials implanted into adipose tissue have the potential to modulate local tissue and systemic inflammation and metabolism.

Subcutaneous (SAT) and visceral (VAT) adipose tissues have distinct metabolic phenotypes. We hypothesized that the extracellular matrix (ECM) regulates depot-specific differences in adipocyte metabolic function in murine obesity. VAT and SAT preadipocytes from lean or obese mice were subject to adipogenic differentiation in standard 2D culture on plastic tissue culture plates or in 3D culture in ECM, followed by metabolic profiling. Adipocytes from VAT relative to SAT manifested impaired insulin-stimulated glucose uptake and decreased adipogenic capacity. In 3D-ECM-adipocyte culture, ECM regulated adipocyte metabolism in a depot-specific manner, with SAT ECM rescuing defects in glucose uptake and adipogenic gene expression in VAT adipocytes, while VAT ECM impaired adipogenic gene expression in SAT adipocytes. These findings demonstrate that ECM-adipocyte crosstalk regulates depot-specific differences in adipocyte metabolic dysfunction in murine obesity.

Dysfunctional visceral adipose tissue (VAT) in obesity is associated with type 2 diabetes (DM) but underlying mechanisms remain unclear. Our objective in this discovery analysis was to identify genes and proteins regulated by DM to elucidate aberrant cellular metabolic and signaling mediators. We performed label-free proteomics and RNA-sequencing analysis of VAT from female bariatric surgery subjects with DM and without DM (NDM). We quantified 1965 protein groups, 23 proteins, and 372 genes that were differently abundant in DM vs. NDM VAT. Proteins downregulated in DM were related to fatty acid synthesis and mitochondrial function (fatty acid synthase, FASN; dihydrolipoyl dehydrogenase, mitochondrial, E3 component, DLD; succinate dehydrogenase-α, SDHA) while proteins upregulated in DM were associated with innate immunity and transcriptional regulation (vitronectin, VTN; endothelial protein C receptor, EPCR; signal transducer and activator of transcription 5B, STAT5B). Transcriptome indicated defects in innate inflammation, lipid metabolism, and extracellular matrix (ECM) function, and components of complement classical and alternative cascades. The VAT proteome and transcriptome shared 13 biological processes impacted by DM, related to complement activation, cell proliferation and migration, ECM organization, lipid metabolism, and gluconeogenesis. Our data revealed a marked effect of DM in downregulating FASN. We also demonstrate enrichment of complement factor B (CFB), coagulation factor XIII A chain (F13A1), thrombospondin 1 (THBS1), and integrins at mRNA and protein levels, albeit with lower q-values and lack of Western blot or PCR confirmation. Our findings suggest putative mechanisms of VAT dysfunction in DM.

Despite studies implicating adipose tissue T cells (ATT) in the initiation and persistence of adipose tissue inflammation, fundamental gaps in knowledge regarding ATT function impedes progress toward understanding how obesity influences adaptive immunity. We hypothesized that ATT activation and function would have tissue-resident–specific properties and that obesity would potentiate their inflammatory properties. We assessed ATT activation and inflammatory potential within mouse and human stromal vascular fraction (SVF). Surprisingly, murine and human ATTs from obese visceral white adipose tissue exhibited impaired inflammatory characteristics upon stimulation. Both environmental and cell-intrinsic factors are implicated in ATT dysfunction. Soluble factors from obese SVF inhibit ATT activation. Additionally, chronic signaling from macrophage major histocompatibility complex II (MHCII) is necessary for ATT impairment in obese adipose tissue but is independent of increased PD1 expression. To assess intracellular signaling mechanisms responsible for ATT inflammation impairments, single-cell RNA sequencing of ATTs was performed. ATTs in obese adipose tissue exhibit enrichment of genes characteristic of T cell exhaustion and increased expression of coinhibitory receptor Btla. In sum, this work suggests that obesity-induced ATTs have functional characteristics and gene expression resembling T cell exhaustion induced by local soluble factors and cell-to-cell interactions in adipose tissue.

Increased morbidity and mortality from SARS‐CoV‐2 coronavirus disease (COVID‐19) in people with obesity has illuminated the intersection of obesity with impaired responses to infections. While data is being rapidly generated on mechanisms by which COVID‐19 impacts health, there is a critical need to better understand the pulmonary, vascular, metabolic, and immunologic aspects that drive the increased risk for complications from COVID‐19 in people with obesity. In this review, we provide a broad overview of the intersection between COVID‐19 and physiology of obesity to highlight potential mechanisms by which COVID‐19 disease severity is increased by obesity, and to identify areas for future investigation towards developing tailored therapy for people with obesity who develop COVID‐19.

Increased adipose tissue macrophages (ATMs) correlate with metabolic dysfunction in humans and are causal in development of insulin resistance in mice. Recent bulk and single-cell transcriptomics studies reveal a wide spectrum of gene expression signatures possible for macrophages that depends on context, but the signatures of human ATM subtypes are not well defined in obesity and diabetes. We profiled 3 prominent ATM subtypes from human adipose tissue in obesity and determined their relationship to type 2 diabetes. Visceral adipose tissue (VAT) and s.c. adipose tissue (SAT) samples were collected from diabetic and nondiabetic obese participants to evaluate cellular content and gene expression. VAT CD206+CD11c- ATMs were increased in diabetic participants, were scavenger receptor-rich with low intracellular lipids, secreted proinflammatory cytokines, and diverged significantly from 2 CD11c+ ATM subtypes, which were lipid-laden, were lipid antigen presenting, and overlapped with monocyte signatures. Furthermore, diabetic VAT was enriched for CD206+CD11c- ATM and inflammatory signatures, scavenger receptors, and MHC II antigen presentation genes. VAT immunostaining found CD206+CD11c- ATMs concentrated in vascularized lymphoid clusters adjacent to CD206-CD11c+ ATMs, while CD206+CD11c+ were distributed between adipocytes. Our results show ATM subtype-specific profiles that uniquely contribute to the phenotypic variation in obesity.

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.

Altered adipocyte function underlies type 2 diabetes (DM) pathogenesis. We hypothesized that adipocyte dysfunction in visceral adipose tissue (VAT) in DM originates from distinct adipocyte progenitor cell (APC) subpopulations. VAT was collected from bariatric surgery patients with (HbA1c>6.5) and without DM (NDM, HbA1c Disclosure C. Strieder-Barboza: None. C.G. Flesher: None. L.M. Geletka: None. R.W. ORourke: None. C.N. Lumeng: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK115190, DK090262)

Background Obesity-induced chronic inflammation and fibrosis in adipose tissue contributes to the progression of type 2 diabetes mellitus (DM). While fibrosis is known to induce mechanical stiffening of numerous tissue types, it is unknown whether DM is associated with alterations in adipose tissue mechanical properties. Objective The purpose of this study was to investigate whether DM is associated with differences in bulk viscoelastic properties of adipose tissue from diabetic (DM) and non-diabetic (NDM) obese subjects. Methods Bulk shear rheology was performed on visceral (VAT) and subcutaneous (SAT) adipose tissue, collected from obese subjects undergoing elective bariatric surgery. Rheology was also performed on the remaining extracellular matrix (ECM) from decellularized VAT (VAT ECM). Linear mixed models were used to assess whether correlations existed between adipose tissue mechanical properties and DM status, sex, age, and body mass index (BMI). Results DM was not associated with significant differences in adipose tissue viscoelastic properties for any of the tissue types investigated. Tissue type dependent differences were however detected, with VAT having significantly lower shear storage and loss moduli than SAT and VAT ECM independent of DM status. Conclusion Although DM is typically associated with adipose tissue fibrosis, it is not associated with differences in macroscopic adipose tissue mechanical properties.

The adipose tissue extracellular matrix (ECM) regulates adipocyte cellular metabolism and is altered in obesity and type 2 diabetes, but mechanisms underlying ECM-adipocyte metabolic crosstalk are poorly defined. Advanced glycation end-product (AGE) formation is increased in diabetes. AGE alter tissue function via direct effects on ECM and by binding scavenger receptors on multiple cell types and signaling through Rho GTPases. Our goal was to determine the role and underlying mechanisms of AGE in regulating human ECM-adipocyte metabolic crosstalk. Visceral adipocytes from diabetic and non-diabetic humans with obesity were studied in 2D and 3D-ECM culture systems. AGE is increased in adipose tissue from diabetic compared to non-diabetic subjects. Glycated collagen 1 and AGE-modified ECM regulate adipocyte glucose uptake and expression of AGE scavenger receptors and Rho signaling mediators, including the DIAPH1 gene, which encodes the human Diaphanous 1 protein (hDia1). Notably, inhibition of hDia1, but not scavenger receptors RAGE or CD36, attenuated AGE-ECM inhibition of adipocyte glucose uptake. These data demonstrate that AGE-modification of ECM contributes to adipocyte insulin resistance in human diabetes, and implicate hDia1 as a potential mediator of AGE-ECM-adipocyte metabolic crosstalk.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

Adipose tissue induced chronic low-grade inflammation is a critical link between obesity and insulin resistance. Despite studies demonstrating the importance of adipose tissue T cells (ATT) in insulin resistance, fundamental gaps in knowledge regarding CD4+ ATT function impedes progress towards understanding their role in diabetes. Little is known about the mechanisms of ATT activation and chemokine profiles. We hypothesized that high fat diet induced obesity would potentiate ATT inflammatory properties. To maximize the viability and number of ATTs in-vitro, T cell activation assays were performed by adding α-CD3/CD28 beads to the entire stromal vascular fraction (SVF). Three days after T cell receptor (TCR) stimulation, supernatants were collected and cells were analyzed using flow cytometry. Surprisingly, ATTs from obese epididymal white adipose tissue had senescent characteristics. Stimulation failed to induce CD25 upregulation, ATT proliferation, or heightened IFNγ and IL-2 secretion, which is seen in lean samples. Human samples collected from diabetic bariatric surgery patients also have an exhausted phenotype unlike nondiabetic controls. Both cell intrinsic and extrinsic factors are implicated in ATT dysfunction. Diabetic humans and mice both have significantly increased expression of the co-inhibitory receptor, PD1, on the cell surface. Despite experiments showing that T cell intrinsic factors, such as major-histocompatibility complex II signals are necessary for ATT dysfunction in obese mice, blocking PD1 fails to rescue this phenotype. Most likely this is due to additional extrinsic factors, since co-culturing lean ATTs in obese SVF results in reduced pro-inflammatory cytokine secretion post-TCR stimulation. Further studies are required to elucidate whether ATT dysfunction is a protective mechanism that reduces inflammation, and whether relieving intrinsic and extrinsic blocks on ATTs results in a more robust inflammatory response and worsened glucose tolerance. Disclosure C.E. Porsche: None. J. DelProposto: None. L.M. Geletka: None. R.W. O’Rourke: None. C. Lumeng: None. Funding National Institutes of Health (F31DK118811, T32HL125242 to C.E.P.), R01DK090262-06A1 (to C.L.)

Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitro PanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation., Highlights • Adipocytes promote pancreatic cancer cell proliferation in nutrient-poor conditions. • Adipocytes secrete glutamine. • Adipocytes rescue pancreatic cancer cell proliferation in the absence of glutamine. • Glutamine transfer may underlie adipocyte-induced pancreatic cancer cell proliferation.

Bariatric surgery is currently the most effective treatment for obesity. Not only do these types of surgeries produce significant weight loss but also they improve insulin sensitivity and whole body metabolic function. The aim of this review is to explore how altered physiology of adipose tissue may contribute to the potent metabolic effects of some of these procedures. This includes specific effects on various fat depots, the function of individual adipocytes and the interaction between adipose tissue and other key metabolic tissues. Besides a dramatic loss of fat mass, bariatric surgery shifts the distribution of fat from visceral to the subcutaneous compartment favoring metabolic improvement. The sensitivity towards lipolysis controlled by insulin and catecholamines is improved, adipokine secretion is altered and local adipose inflammation as well as systemic inflammatory markers decreases. Some of these changes have been shown to be weight loss independent, and novel hypothesis for these effects includes include changes in bile acid metabolism, gut microbiota and central regulation of metabolism. In conclusion bariatric surgery is capable of improving aspects of adipose tissue function and do so in some cases in ways that are not entirely explained by the potent effect of surgery. © 2016 World Obesity.

Objective The relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in the context of obesity and the correlation of these tissue-based phenomena with systemic metabolic disease are poorly defined. The goal of this study was to clarify the relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in human obesity and determine the correlation of these adipose-tissue based phenomena with diabetes. Methods Visceral and subcutaneous adipose tissues from humans with obesity collected during bariatric surgery were studied with QRTPCR, immunohistochemistry, and flow cytometry for expression of collagens and fibrosis-related proteins, adipocyte size, and preadipocyte frequency. Results were correlated with clinical characteristics including diabetes status. Results Fibrosis was decreased, hypertrophy was increased, and preadipocyte frequency and fibrotic gene expression were decreased in adipose tissues from diabetic subjects compared to non-diabetic subjects. These differences were greater in visceral compared to subcutaneous adipose tissue. Conclusions These data are consistent with the hypothesis that adipose tissue fibrosis in the context of human obesity limits adipocyte hypertrophy and is associated with a reciprocal increase in adipocyte hyperplasia, with beneficial effects on systemic metabolism. These findings suggest adipose tissue fibrosis as a potential target for manipulation of adipocyte metabolism.

Objective Expansion of visceral adipose tissue (VAT) and metabolic inflammation are consequences of obesity and associated with type 2 diabetes (T2DM). Metabolically activated adipose tissue macrophages (ATMs) undergo qualitative and quantitative changes that influence their inflammatory responses. How these cells contribute to insulin resistance (IR) in humans is not well understood. Cholesterol 25-Hydroxylase (CH25H) converts cholesterol into 25-Hydroxycholesterol (25-HC), an oxysterol that modulates immune responses. Using human and murine models, we investigated the role of CH25H in metabolic inflammation. Methods We performed transcriptomic (RNASeq) analysis on the human whole AT biopsies and sorted ATMs from obese non-diabetic (NDM) and obese diabetic (DM) subjects to inquire if CH25H was increased in DM. We challenged mice lacking Ch25h with a high-fat diet (HFD) to characterize their metabolic and immunologic profiling. Ch25h KO mice and human adipose tissue biopsies from NDM and DM subjects were analyzed. LC-MS was conducted to measure 25-HC level in AT. In vitro analysis permitted us to investigate the effect of 25-HC on cytokine expression. Results In our RNASeq analysis of human visceral and subcutaneous biopsies, gene pathways related to inflammation were increased in obese DM vs. non-DM subjects that included CH25H. CH25H was enriched in the stromal vascular fraction of human adipose tissue and highly expressed in CD206+ human ATMs by flow cytometry analysis. We measured the levels of the oxysterols, 25-HC and 7α25diHC, in human visceral adipose tissue samples and showed a correlation between BMI and 25-HC. Using mouse models of diet-induced obesity (DIO), we found that HFD-induced Ch25h expression in eWAT and increased levels of 25-HC in AT. On HFD, Ch25h KO mice became obese but exhibited reduced plasma insulin levels, improved insulin action, and decreased ectopic lipid deposit. Improved insulin sensitivity in Ch25h KO mice was due to attenuation of CD11c+ adipose tissue macrophage infiltration in eWAT. Finally, by testing AT explants, bone marrow-derived macrophages (BMDMs) and SVF cells from Ch25h deficient mice, we observed that 25-HC is required for the expression of pro-inflammatory genes. 25-HC was also able to induce inflammatory genes in preadipocytes. Conclusions Our data suggest a critical role for CH25H/25-HC in the progression of meta-inflammation and insulin resistance in obese humans and mouse models of obesity. In response to obesogenic stimuli, CH25H/25-HC could exert a pro-inflammatory role., HIGHLIGHTS • CH25H upregulation in visceral adipose tissue is associated with diabetes in humans. • ATMs are the primary site of CH25H expression in humans and mice. • DIO in mice activates Ch25h expression and 25-HC production in visceral adipose tissue. • Obese Ch25h KO mice have improved insulin sensitivity due to attenuated adipose tissue inflammation. • In response to inflammatory stimuli, Ch25h/25-HC potentiates myeloid activation.

Objective: Type 2 diabetes mellitus (DM) is a known risk factor for pancreatic ductal adenocarcinoma (PDAC), but the mechanisms underlying this association remain poorly understood. Multiple reports implicate adipocytes and preadipocytes as putative promoters of PDAC in humans and mice. To test whether preadipocytes regulate PDAC tumorigenic functions in the context of DM, we explored the effects of conditioned media (CM) prepared from preadipocytes from nondiabetic (NDM) and DM patients on PDAC cells. Methods: Preadipocytes were isolated from omental adipose tissue obtained from NDM and DM patients undergoing bariatric surgery. Serum-free conditioned media was prepared from these preadipocytes cultured in vitro for 72 hours. Sphere assays using T3M4 and Capan1 PDAC lines were performed in the presence or absence of conditioned media, and sphere surface area was measured over 2-3 weeks. Modifications of the sphere assays, including boiling of conditioned media as well as addition of neutralizing antibodies or recombinant proteins, were implemented. A proteome profiler array was used to identify differences in cytokine composition between CM from DM and NDM. A phospho-kinome array was used to identify differentially activated signaling pathways when PDAC cells were treated with NDM and DM conditioned media. Results: Conditioned media from human DM preadipocytes, compared to media from NDM preadipocytes, increased PDAC cell sphere surface area in both T3M4 and Capan1 cell lines. Sphere surface area correlated directly with hemoglobin A1c (HbA1c) but not sex or body mass index of the human subjects from which preadipocytes were derived. Boiling of preadipocyte supernatants abrogated their pro-growth effects. Proteome profiler array analysis identified different proteins that were differentially expressed by DM and NDM preadipocytes, among which chitinase-3-like protein 1 (CHI3L1) and interleukin-6 (IL-6) were increased over 2-fold in DM relative to NDM preadipocyte supernatants. Neutralizing antibody to human CHI3L1 and IL-6 attenuated the increase in PDAC sphere surface area in response to DM preadipocyte conditioned media in a dose-dependent manner. Recombinant human CHI3L1 and IL-6 enhanced the growth of PDAC cells when they were exposed to NDM conditioned media. Phospho-kinome analysis demonstrated that treatment of PDAC cells with DM-derived conditioned media increased activation of inflammation-associated signal transduction pathways. Conclusions: Preadipocyte-PDAC cell crosstalk regulates PDAC cell growth in a DM-specific and CHI3L1 and IL-6-dependent manner. These results suggest that the positive clinical correlation between DM and PDAC may be mediated by preadipocytes expressing higher levels of CHI3L1 and IL-6. Preadipocytes CHI3L1 and IL-6 represent targets for further research directed towards developing novel therapies for PDAC based on manipulation of the tumor microenvironment. Citation Format: Lawrence Delrosario, Carmen Flesher, Nicki Baker, Jenny Lazarus, Mirna Perusina-Lanfranca, Robert O'Rourke, Timothy Frankel. Human preadipocytes promote pancreatic cancer cellular growth in a diabetes-specific manner [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A08.

Sepsis is the leading cause of death in the intensive care unit with an overall mortality rate of 20%. Individuals who are obese and have type 2 diabetes have increased recurrent, chronic, nosocomial infections that worsen the long-term morbidity and mortality from sepsis. Additionally, animal models of sepsis have shown that obese, diabetic mice have lower survival rates compared with nondiabetic mice. Neutrophils are essential for eradication of bacteria, prevention of infectious complications, and sepsis survival. In diabetic states, there is a reduction in neutrophil chemotaxis, phagocytosis, and reactive oxygen species (ROS) generation; however, few studies have investigated the extent to which these deficits compromise infection eradication and mortality. Using a cecal ligation and puncture model of sepsis in lean and in diet-induced obese mice, we demonstrate that obese diabetic mice have decreased “emergency hematopoiesis” after an acute infection. Additionally, both neutrophils and monocytes in obese, diabetic mice have functional defects, with decreased phagocytic ability and a decreased capacity to generate ROS. Neutrophils isolated from obese diabetic mice have decreased transcripts of Axl and Mertk, which partially explains the phagocytic dysfunction. Furthermore, we found that exogenous GM-CSF administration improves sepsis survival through enhanced neutrophil and monocytes phagocytosis and ROS generation abilities in obese, diabetic mice with sepsis.

Background The utility of serum biomarkers related to inflammation and adiposity as predictors of metabolic disease prevalence and outcomes after bariatric surgery are not well-defined. Methods Associations between pre- and post-operative serum levels of four biomarkers (C-reactive protein (CRP), cystatin C (CC), leptin, ghrelin) with baseline measures of adiposity and metabolic disease prevalence (asthma, diabetes, sleep apnea), and weight loss and metabolic disease remission after bariatric surgery were studied in the Longitudinal Assessment of Bariatric Surgery (LABS) cohort. Results Baseline CRP levels were positively associated with the odds of asthma but not diabetes or sleep apnea; baseline CC levels were positively associated with asthma, diabetes, and sleep apnea; baseline leptin levels were positively associated with asthma and negatively associated with diabetes and sleep apnea; baseline ghrelin levels were negatively associated with diabetes and sleep apnea. Increased weight loss was associated with increased baseline levels of leptin and CRP and decreased baseline levels of CC. Remission of diabetes and asthma was not associated with baseline levels of any biomarker. A higher likelihood of asthma remission was associated with a greater decrease in leptin levels, and a higher likelihood of diabetes remission was predicted by a lesser decrease in CC. Bariatric surgery was associated with decreased post-operative CC, CRP, and leptin levels, and increased post-operative ghrelin levels. Conclusion This is the largest study to date of serum biomarkers of inflammation and adiposity in a bariatric surgery cohort. Biomarker levels correlate with metabolic disease prevalence prior to bariatric surgery, and with weight loss but not metabolic disease remission after surgery. Bariatric surgery regulates serum biomarker levels in a manner consistent with anti-inflammatory and compensatory orexigenic effects. These data contribute to our understanding of the mechanisms underlying the biologic effects of bariatric surgery.

Objective In addition to adipocytes, adipose tissue contains large numbers of immune cells. A wide range of evidence links the activity of these cells to regulation of adipocyte and systemic metabolic function. Bariatric surgery improves several aspects of metabolic derangements and at least some of these effects occur in a weight-loss independent manner. We sought to investigate the impact of vertical sleeve gastrectomy (VSG) on adipose immune cell frequencies. Methods We analyzed the frequencies of immune cells within distinct adipose tissue depots in obese mice that had VSG or sham surgery with a portion of the latter group pair-fed such that their body mass was matched to the VSG animals. Results We demonstrate that VSG induced a shift in the epididymal adipose tissue leukocyte profile including increased frequencies of CD11c− macrophages, increased frequencies of T cells (CD4+, CD8+, and CD4−/CD8− T cells all increased), but a significantly decreased frequency of adipose tissue dendritic cells (ATDC) that, despite the continued high fat feeding of the VSG group, dropped below control diet levels. Conclusions These results indicate that VSG induces substantial changes in the immune populations residing in the adipose depots independent of weight loss., Highlights • Sleeve gastrectomy in mice altered the adipose leukocyte populations. • CD11c− macrophages were selectively upregulated independent from weight loss. • All identified T cell populations increased manifold independent from weight loss. • The dendritic cell fraction was decreased below lean control levels with surgery.

Predictors of weight loss responses are not well-defined. We hypothesized that adipose tissue phenotypic features related to remodeling would be associated with bariatric surgery weight loss responses. Visceral and subcutaneous adipose tissues collected from patients during bariatric surgery were studied with flow cytometry, immunohistochemistry, and QRTPCR, and results correlated with weight loss outcomes. Age, male sex, and a diagnosis of type 2 diabetes were associated with less weight loss. Adipocyte size was increased and preadipocyte frequency was decreased in visceral adipose tissue from diabetic subjects. Decreased adipose tissue preadipocyte frequency was associated with less weight loss in women but not men. These data suggest that phenotypic features of adipose tissue remodeling may predict responses to weight loss interventions.

Adipocytes differentiated from preadipocytes provide a valuable model for the study of human adipocyte metabolism. We describe methods for isolation of human stromal vascular cells, expansion of preadipocytes, differentiation into mature adipocytes, and in vitro metabolic interrogation of adipocytes.

Summary An adaptive immune response triggered by obesity is characterized by the activation of adipose tissue CD4 + T cells by unclear mechanisms. We have examined whether interactions between adipose tissue macrophages (ATMs) and CD4 + T cells contribute to adipose tissue metainflammation. Intravital microscopy identifies dynamic antigen-dependent interactions between ATMs and T cells in visceral fat. Mice deficient in major histocompatibility complex class II (MHC II) showed protection from diet-induced obesity. Deletion of MHC II expression in macrophages led to an adipose tissue-specific decrease in the effector/memory CD4 + T cells, attenuation of CD11c + ATM accumulation, and improvement in glucose intolerance by increasing adipose tissue insulin sensitivity. Ablation experiments demonstrated that the maintenance of proliferating conventional T cells is dependent on signals from CD11c + ATMs in obese mice. These studies demonstrate the importance of MHCII-restricted signals from ATMs that regulate adipose tissue T cell maturation and metainflammation.

Adipose tissue dysfunction underlies the pathogenesis of metabolic disease. The metrics used to quantify adiposity and its association with metabolic disease, including body mass index, have limitations with important clinical implications. An understanding of the molecular and cellular mechanisms by which adipose tissue regulates systemic metabolism and contributes to metabolic disease will lead to next-generation adipose tissue-based therapy.

Adipose tissue inflammation is a cause of obesity-related metabolic disease. Natural killer (NK) cells are an understudied cell type in the context of obesity. The goal of this study was to determine the phenotype of human adipose tissue NK cells.We used flow cytometry phenotyping to study adipose tissue and peripheral blood NK cells from obese and lean humans.Human adipose tissue NK cells, relative to peripheral blood NK cells, express increased levels of activation markers. Adipose tissue NK cells also demonstrate an activated phenotype in obese relative to lean subjects, with increased expression of the activating receptor NKG2D.These data are the first detailed phenotypic characterization of human adipose tissue NK cells, and suggest a role for NK cells in adipose tissue inflammation in obesity.

Why do obese patients get sick? What underlies the pathogenesis of the many diseases associated with obesity? As recently as 50 years ago, the answer was mechanical stress on a variety of organ systems from the increased body weight. However, although this might explain osteoarthritis and venous stasis disease, it is not a satisfying explanation for diabetes, liver disease, allergic disease, cancer, and the many other diseases that are increased in incidence in the presence of obesity. What is it about excess adipose tissue that causes metabolic disease? The answer lies in one of the most important realizations in the field of obesity in the past 20 years: that obesity is associated with a state of chronic systemic inflammation and this inflammatory state, this hyperactivation of the immune system, underlies the pathogenesis of virtually every metabolic disease that afflicts the obese. An exciting recent study came from investigators at the University of Pennsylvania, who treated patients with endstage leukemia with their own immune T cells, genetically modified in the test tube to target leukemic cells. This “high-tech” immunotherapy achieved durable and complete remission in most of a small group of subjects, who were otherwise expected to live only months, and has generated much excitement and hope [1]. Manipulating physiologic processes as fundamental as the immune system also entails risk. In 2006, TeGenero, Incorporated, a German Biotech company, administered a monoclonal antibody designed to treat autoimmune disease to human volunteers in a Phase I safety trial. Despite previous safety testing in rodents and

Background Although it is recognized that a standardized approach to reporting weight change is essential to meaningful comparisons among cohorts and across studies, consensus is lacking. This study aimed to propose a method of reporting weight change that would allow meaningful comparisons among studies of patients who underwent bariatric surgery and to demonstrate its utility using an example from the Longitudinal Assessment of Bariatric Surgery (LABS). Methods Relationships among several measures of weight change are described. Results from an observational, longitudinal cohort study of adults undergoing bariatric surgery and from simulation studies are used to illustrate the proposed method. Results Baseline weight is a critical parameter when assessing weight change. Men undergoing a bariatric procedure other than gastric bypass or adjustable band tended to have greater weight loss 12 months after surgery than men undergoing gastric bypass when not accounting for baseline weight, but the opposite was found when results were adjusted for baseline weight. Simulation results show that with relatively modest sample sizes, the adjusted weight loss was significantly different between the 2 groups of men. Conclusion A consistent metric for reporting weight loss after bariatric surgery is essential to interpret outcomes across studies and among subgroups. The baseline weight adjusted percent of weight loss (A%WL) uses a standard population (e.g., the LABS cohort) to account for differences between cohorts with respect to baseline weight, and its use can change the interpretation of results compared with an unadjusted measure.

Obesity represents a public health crisis. While the Food and Drug Administration approved two weight loss drugs in 2012, bariatric surgery will likely remain the most efficacious treatment for the morbidly obese. Bariatric surgery is prescribed for class III obesity [body mass index (BMI) ≥40 kg/m2] or class II obesity (BMI 35–39.9kg/m2) associated with life-threatening co-morbid disease, such as type 2 diabetes, cardiovascular disease or cardiopulmonary problems. Rouxen-Y gastric bypass (RYGB), the most commonly utilized bariatric operation, divides the stomach into a small upper pouch and rearranges the small intestines producing dramatic weight loss primarily by reducing food intake1. RYGB is being used with increasing frequency in the United States. The American Society for Metabolic and Bariatric Surgery estimated that 220,000 of these procedures were performed in 2005.

Dynamic changes of adipose tissue leukocytes, including adipose tissue macrophage (ATM) and adipose tissue dendritic cells (ATDCs), contribute to obesity-induced inflammation and metabolic disease. However, clear discrimination between ATDC and ATM in adipose tissue has limited progress in the field of immunometabolism. In this study, we use CD64 to distinguish ATM and ATDC, and investigated the temporal and functional changes in these myeloid populations during obesity. Flow cytometry and immunostaining demonstrated that the definition of ATM as F4/80+CD11b+ cells overlaps with other leukocytes and that CD45+CD64+ is specific for ATM. The expression of core dendritic cell genes was enriched in CD11c+CD64− cells (ATDC), whereas core macrophage genes were enriched in CD45+CD64+ cells (ATM). CD11c+CD64− ATDCs expressed MHC class II and costimulatory receptors, and had similar capacity to stimulate CD4+ T cell proliferation as ATMs. ATDCs were predominantly CD11b+ conventional dendritic cells and made up the bulk of CD11c+ cells in adipose tissue with moderate high-fat diet exposure. Mixed chimeric experiments with Ccr2−/− mice demonstrated that high-fat diet–induced ATM accumulation from monocytes was dependent on CCR2, whereas ATDC accumulation was less CCR2 dependent. ATDC accumulation during obesity was attenuated in Ccr7−/− mice and was associated with decreased adipose tissue inflammation and insulin resistance. CD45+CD64+ ATM and CD45+CD64−CD11c+ ATDCs were identified in human obese adipose tissue and ATDCs were increased in s.c. adipose tissue compared with omental adipose tissue. These results support a revised strategy for unambiguous delineation of ATM and ATDC, and suggest that ATDCs are independent contributors to adipose tissue inflammation during obesity.

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

Obesity is a strong risk factor for multiple types of cancer. The mechanisms underlying this association are multiple and include increased systemic inflammation, increased alterations in the systemic hormonal milieu, and direct paracrine tumor-promoting effects of adipose tissue, adipose tissue stem cells, and mature adipocytes. These complex mechanisms generate a carcinogenic milieu in multiple tissues in obesity.

Adipose tissue macrophages are important mediators of inflammation and insulin resistance in obesity. IFN-γ is a central regulator of macrophage function. The role of IFN-γ in regulating systemic inflammation and insulin resistance in obesity is unknown. We studied obese IFN-γ knockout mice to identify the role of IFN-γ in regulating inflammation and insulin sensitivity in obesity. IFN-γ-knockout C57Bl/6 mice and wild-type control litter mates were maintained on normal chow or a high fat diet for 13 weeks and then underwent insulin sensitivity testing then sacrifice and tissue collection. Flow cytometry, intracellular cytokine staining, and QRTPCR were used to define tissue lymphocyte phenotype and cytokine expression profiles. Adipocyte size was determined from whole adipose tissue explants examined under immunofluorescence microscopy. Diet-induced obesity induced systemic inflammation and insulin resistance, along with a pan-leukocyte adipose tissue infiltrate that includes macrophages, T-cells, and NK cells. Obese IFN-γ-knockout animals, compared with obese wild-type control animals, demonstrate modest improvements in insulin sensitivity, decreased adipocyte size, and an M2-shift in ATM phenotype and cytokine expression. These data suggest a role for IFN-γ in the regulation of inflammation and glucose homeostasis in obesity though multiple potential mechanisms, including effects on adipogenesis, cytokine expression, and macrophage phenotype.

Obesity activates both innate and adaptive immune responses in adipose tissue, but the mechanisms critical for regulating these responses remain unknown. CD40/CD40L signaling provides bidirectional costimulatory signals between antigen-presenting cells and CD4+ T cells, and CD40L expression is increased in obese humans. Therefore, we examined the contribution of CD40 to the progression of obesity-induced inflammation in mice. CD40 was highly expressed on adipose tissue macrophages in mice, and CD40/CD40L signaling promoted the expression of antigen-presenting cell markers in adipose tissue macrophages. When fed a high fat diet, Cd40-deficient mice had reduced accumulation of conventional CD4+ T cells (Tconv: CD3+CD4+Foxp3−) in visceral fat compared with wild-type mice. By contrast, the number of regulatory CD4+ T cells (Treg: CD3+CD4+Foxp3+) in lean and obese fat was similar between wild-type and knockout mice. Adipose tissue macrophage content and inflammatory gene expression in fat did not differ between obese wild-type and knockout mice; however, major histocompatibility complex class II and CD86 expression on adipose tissue macrophages was reduced in visceral fat from knockout mice. Similar results were observed in chimeric mice with hematopoietic Cd40-deficiency. Nonetheless, neither whole body nor hematopoietic disruption of CD40 ameliorated obesity-induced insulin resistance in mice. In human adipose tissue, CD40 expression was positively correlated with CD80 and CD86 expression in obese patients with type 2 diabetes. These findings indicate that CD40 signaling in adipose tissue macrophages regulates major histocompatibility complex class II and CD86 expression to control the expansion of CD4+ T cells; however, this is largely dispensable for the development of obesity-induced inflammation and insulin resistance in mice.

Proper anastomotic healing is dependent upon many factors including adequate blood flow to healing tissue. The aim of this study was to investigate the impact of vascular endothelial growth factor (VEGF(165)) transfection on anastomotic healing in an ischemic gastrointestinal anastomosis model. Utilizing an established opossum model of esophagogastrectomy followed by esophageal-gastric anastomosis, the gastric fundus was transfected with recombinant human vascular endothelial growth factor via direct injection of a plasmid-based nonviral delivery system. Twenty-nine animals were divided into three groups: two concentrations of VEGF and a control group. Outcomes included VEGF mRNA transcript levels, neovascularization, tissue blood flow, and anastomotic bursting pressure. To determine whether local injection resulted in a systemic effect, distant tissues were evaluated for VEGF transcript levels. Successful gene transfection was demonstrated by quantitative polymerase chain reaction analysis of anastomotic tissue, with significantly higher VEGF mRNA expression in treated animals compared to controls. At the gastric side of the anastomosis, there was significantly increased neovascularization, blood flow, and bursting pressure in experimental animals compared to controls. There were no differences in outcome measures between low- and high-dose VEGF groups; however, the high-dose group demonstrated increased VEGF mRNA expression across the anastomosis. VEGF production was not increased at distant sites in treated animals. In this animal model, VEGF gene therapy increased VEGF transcription at a healing gastrointestinal anastomosis without systemic VEGF upregulation. This treatment led to improved healing and strength of the acutely ischemic anastomosis. These findings suggest that VEGF gene therapy has the potential to reduce anastomotic morbidity and improve surgical outcomes in a wide array of patients.

BACKGROUND: The functional lumen imaging probe (FLIP) uses impedance planimetry to measure the geometry of a distensible organ. The purpose of this study was to evaluate FLIP as a method to determine structural changes at the gastroesophageal junction (GEJ) following transoral incisionless fundoplication (TIF) and compare these findings with the accepted methods of esophageal testing. METHODS: Two different approaches (TIF1.0 and 2.0) using the EsophyX device were performed in six and five animals, respectively. Three dogs underwent a sham procedure. FLIP measurements were performed pre- and post-procedure and at 2-week follow-up. Upper endoscopy, manometry, and 48-h pH testing were also performed at each time point. FLIP was performed in ten patients before and 3 months after TIF. RESULTS: Following TIF procedures, there was a significant decrease in cross-sectional area (CSA) of GEJ compared to baseline; however, the CSA of both groups returned to baseline at 2-week follow-up. The FLIP results were supported with pH testing and correlated highly with both measures of GEJ structural integrity (LES and cardia circumference). Following TIF in humans, there was a decrease in GEJ distensibility compared to baseline that persisted to the 3-month evaluation. CONCLUSION: FLIP is able to measure and display changes in tissue distensibility at the GEJ, and results correlate with established methods of testing. FLIP may represent a single testing modality by which to diagnose GERD and evaluate the outcome after antireflux surgery.