Proinflammatory Adipokines Research Articles

Obese Adipose Tissue Secretion Induces Inflammation in Preadipocytes: Role of Toll-Like Receptor-4.

In obesity, the dysfunctional adipose tissue (AT) releases increased levels of proinflammatory adipokines such as TNFα, IL-6, and IL-1β and free fatty acids (FFAs), characterizing a chronic, low-grade inflammation. Whilst FFAs and proinflammatory adipokines are known to elicit an inflammatory response within AT, their relative influence upon preadipocytes, the precursors of mature adipocytes, is yet to be determined. Our results demonstrated that the conditioned medium (CM) derived from obese AT was rich in FFAs, which guided us to evaluate the role of TLR4 in the induction of inflammation in preadipocytes. We observed that CM derived from obese AT increased reactive oxygen species (ROS) levels and NF-ĸB nuclear translocation together with IL-6, TNFα, and IL-1β in 3T3-L1 cells in a TLR4-dependent manner. Furthermore, TLR4 signaling was involved in the increased expression of C/EBPα together with the release of leptin, adiponectin, and proinflammatory mediators, in response to the CM derived from obese AT. Our results suggest that obese AT milieu secretes lipokines, which act in a combined paracrine/autocrine manner, inducing inflammation in preadipocytes via TLR4 and ROS, thus creating a paracrine loop that facilitates the differentiation of adipocytes with a proinflammatory profile.

Emerging functions of adipokines in linking the development of obesity and cardiovascular diseases.

Increasing evidence shows that obesity is the critical factor in shaping cardio-metabolic phenotypes. However, the pathogenic mechanisms remain incompletely clarified. According to the published reports, adipose tissue communicates with several diverse organs, such as heart, lungs, and kidneys through the secretion of various cytokines named adipokines. The adipocytes isolated from obese mice or humans are dysfunctional with aberrant production of pro-inflammatory adipokines, which subsequently induce both acute and chronic inflammatory reaction and facilitate the process of cardio-metabolic disorder complications. Furthermore, the microenvironment within adipose tissue under obese status also influence the secretion of adipokines. Recently, given that several important adipokines have been completely researched and causally involved in various diseases, we could make a conclusion that adipokines play an essential role in modulating the development of cardio-metabolic disorder diseases, whereas several novel adipokines continue to be explored and elucidated. In the present review, we summarized the current knowledge of the microenvironment of adipose tissue and the published mechanisms whereby adipocytes affects obesity and cardiovascular diseases. On the other hand, we also provide the evidence to elucidate the functions of adipokines in controlling and regulating the inflammatory reactions which contribute to obesity and cardiovascular disease.

Current mechanisms in obesity and tumor progression.

Hyperadiposity, as present in obesity, is a substantial threat to cancer risk and prognosis. Studies that have investigated the link between obesity and tumor progression have proposed several mechanistic frameworks, yet, these mechanisms are not fully defined. Further, a comprehensive understanding of how these various mechanisms may interact to create a dynamic disease state is lacking in the current literature. Recent work has begun to explore not only discrete mechanisms by which obesity may promote tumor growth (for instance, metabolic and growth factor functions of insulin; inflammatory cytokines; adipokines; and others), but also how these putative tumor-promoting factors may interact. This review will highlight the present understanding of obesity, as it relates to tumor development and progression. First, we will introduce the impact of obesity in cancer within the dynamic tumor microenvironment, which will serve as a theme to frame this review. The core of this review will discuss recently proposed mechanisms that implicate obesity in tumor progression, including chronic inflammation and the role of pro-inflammatory cytokines, adipokines, hormones, and genetic approaches. Furthermore, we intend to offer current insight in targeting adipose tissue during the development of cancer prevention and treatment strategies.

Leptin and exercise-induced bronchoconstriction in obese asthmatic children

Exercise-induced bronchoconstriction (EIB) in asthmatic children has been associated with obesity. Leptin, the proinflammatory adipokine, is typically increased in obesity, whereas cysteinyl leukotrienes (cysLTs) are the main inflammatory mediators implicated in the pathogenesis of EIB. The aim of this work was to study the possible impact of obesity on the severity of EIB in asthmatic children and the possible association with the adipokine leptin. The study included eighty pre-pubertal asthmatic children divided according to their body mass index (BMI) and response to exercise into four groups; obese exercise-responders (n=20), normal-weight exercise-responders (n=20), obese exercise non-responders (n=20), and normal-weight exercise non-responders (n=20). A baseline spirometry test and a standardized exercise challenge test (ECT) were performed. The severity of EIB was assessed by the maximum percentage fall in forced expiratory volume in 1 second (MF%FEV1) after exercise. The level of fasting serum leptin and the release of cysLTs during exercise were compared between the 4 groups. The MF%FEV1 during exercise was significantly greater in obese responder compared to normal-weight responder patients (p=0.004) and MF%FEV1 was positively correlated with cysLT release during exercise, BMI z-score, waist circumference and serum leptin. CysLT release during exercise was positively correlated with the level of serum leptin (r =0.514, p=0.001). This study reports that the severity of EIB is significantly greater in obese compared to normal-weight asthmatic children and suggests an association between leptin and airway hyperresponsiveness to exercise in obese asthmatic children through a mechanism related to cysLT release

D-allulose Ameliorates Metabolic Dysfunction in C57BL/KsJ-db/db Mice.

d-allulose is an uncommon sugar that provides almost no calories when consumed. Its sweetness is 70% that of sucrose. d-allulose is a metabolic regulator of glucose and lipid metabolism. However, few reports concerning its effect on diabetes and related metabolic disturbances in db/db mice are available. In this study, we evaluated d-allulose’s effect on hyperglycemia, hyperinsulinemia, diabetes and inflammatory responses in C57BL/KsJ-db/db mice. Mice were divided into normal diet, erythritol supplemented (5% w/w), and d-allulose supplemented (5% w/w) groups. Blood glucose and plasma glucagon levels and homeostatic model assessment (HOMA-IR) were significantly lower in the d-allulose group than in the normal diet group, and plasma insulin level was significantly increased. Further, d-allulose supplement significantly increased hepatic glucokinase activity and decreased hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activity. Expression of glucose transporter 4, insulin receptor substrate 1, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha and AKT serine/threonine kinase 2 were also upregulated by d-allulose supplement in adipocyte and muscle. Finally, d-allulose effectively lowered plasma and hepatic triglyceride and free fatty acid levels, and simultaneously reduced hepatic fatty acid oxidation and carnitine palmitoyl transferase activity. These changes are likely attributable to suppression of hepatic fatty acid synthase and glucose-6-phosphate dehydrogenase activity. Notably, d-allulose also reduced pro-inflammatory adipokine and cytokine levels in plasma. Our results indicate that d-allulose is an effective sugar substitute for improving lipid and glucose metabolism.

Intramuscular adipose tissue: a cause for concern.

Intramuscular adipose tissue: a cause for concern.

Obesity and COVID-19: A Fatal Alliance.

Most people infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV2) are mildly symptomatic while few progress to critical illness and succumb to the infection. The disease severity is seen to be associated with increasing age and underlying comorbid conditions. Obesity, responsible for various metabolic disorders, appears to be a risk factor in determining the severity of infection despite any age group. Though this association is clinically relevant, the mechanisms underlying are not fully elucidated. SARS CoV2 enters host cell via Angiotensin Converting Enzyme 2 receptor, expression of which is upregulated in visceral fat tissue in obese people, underscoring the fact that adipose tissue is a potential reservoir for virus. Adipose tissue is also a source of many proinflammatory mediators and adipokines. High baseline C-Reactive Protein, interleukin 6, hyperleptinemia with Leptin resistance and hypoadiponectinemia associated with obesity explains the preexisting inflammatory state in obese individuals which predisposes them to worse outcomes and fatality.

Increased Adiposity Associated With Increased Length of Stay for Infants With Bronchiolitis.

Excess adiposity upregulates proinflammatory adipokines in infancy that have also been implicated in the pathogenesis of bronchiolitis. The association between excess adiposity and severity of disease in bronchiolitis is unclear. We sought to examine the association between adiposity and length of hospitalization and risk of PICU transfer in children with bronchiolitis. We conducted a retrospective cohort study examining infants 24 months and younger hospitalized at an academic children's hospital with bronchiolitis, grouped by weight status (BMI z score and ponderal index). Data were extracted from the medical record, including the following relevant covariates: age, sex, race and/or ethnicity, and International Classification of Diseases, 10th Revision codes. Outcomes included length of stay (LOS) and PICU transfer. We used multiple regression to examine the association between each anthropometric measure and LOS and likelihood of PICU transfer. There were 765 children in the final sample, 599 without a significant comorbidity (eg, prematurity, congenital heart disease). The median LOS was 2.8 days (interquartile range 1.7-4.9 days). LOS increased with increasing ponderal index quartile (P = .001). After accounting for age and significant comorbidities, we used multivariable regression to identify a significant association between increasing ponderal index and LOS (P = .04) and no association between BMI and LOS. Logistic regression did not reveal an association between either anthropometric measure and PICU transfer. In this study, we identified an association between a measure of excess adiposity in infants and length of hospitalization for bronchiolitis. Further work is needed to confirm this association, examine potential mechanisms, and account for other potential confounders.

Apelin/APJ signal system and glomerular filtration rate in various variants of bronchial asthma

INTRODUCTION. We have previously postulated the similarity of molecular pathogenetic mechanisms in bronchial asthma (BA) and chronic kidney disease (CKD). Understanding these mechanisms in such a comorbidity pathology is of interest to the clinicians. In recent years, the attention of BA pathogenesis researchers has attracted low-investigated adipokine – apelin. On the other hand, apelin is considered as a renoprotective adipokine that can prevent the progression of CKD. THE AIM of the study is to identify the relationship between apelin/APJ signaling system and glomerular filtration rate in different BA variants. PATIENTS AND METHODS. The 12 of practically healthy persons and 36 bronchial asthma patients were examined. Levels of apelin-12, apelin-36, and APJ receptor of apelines on peripheral blood lymphocytes were determined, as well as levels of TNF-α, IL-6 IL-4 by immunoenzyme method according to standard protocol. Glomerular filtration rate (eGFR) by CKD-EPI was calculated. RESULTS. With the help of factor analysis, it was revealed that the glomerular filtration rate in bronchial asthma is associated with the level of apelin-36. A high level of glomerular filtration rate corresponds to a high level of apelin-36. In bronchial asthma, the negative association of pro-inflammatory adipokines TNF-α and IL-6 with the glomerular filtration rate was revealed. On the other hand, the IL-4 was found to be directly related to the glomerular filtration rate according to the factorial analysis. CONCLUSION. The obtained data suggest a possible renoprotective effect of apelin-36 in bronchial asthma.

O Papel das Adipocinas na Imunologia da Obesidade

A obesidade é vista como um grande problema de saúde pública e caracteriza-se pelo excesso de tecido adiposo no corpo. Este tecido é subdividido em tecido adiposo branco (TAB) e tecido adiposo marrom (TAM). Estudos demonstram que o tecido adiposo é responsável pela liberação de substâncias sinalizadoras, denominadas adipocinas, estas possuem diversas funções sendo seu papel imunológico a atribuição de maior destaque. O presente trabalho tem como objetivo abordar as características da obesidade e sua relação com o sistema imunológico. Esta condição passou a ser vista como uma doença inflamatória, já que adipocinas pró-inflamatórias e anti-inflamatórias se encontram com níveis alterados. O estudo trata-se de uma revisão bibliográfica, com abordagem qualitativa, do tipo exploratória. Os resultados obtidos nesta pesquisa bibliográfica ratificam que a resistina e leptina que são adipocinas pró-inflamatórias estão em quantidades elevadas em pacientes com obesidade, enquanto a adiponectina, Interleucina-4 e Interleucina-10, que são adipocinas anti-inflamatórias, estão em quantidades mais baixas que o normal em pacientes com obesidade. O estudo das moléculas envolvidas na fisiopatologia da obesidade é de extrema importância, visto que esta é uma condição cada vez mais frequente no mundo, e que leva a diversos prejuízos, incluindo o desenvolvimento de doenças inflamatórias e crônicas.

Muscle, Bone, and Fat Crosstalk: the Biological Role of Myokines, Osteokines, and Adipokines.

Skeletal muscle and bone are connected anatomically and physiologically, and play a crucial role in human locomotion and metabolism. Historically, the coupling between muscle and bone has been viewed in light of mechanotransduction, which dictates that the mechanical forces applied to muscle are transmitted to the skeleton to initiate bone formation. However, these organs also communicate through the endocrine system, orchestrated by a family of cytokines namely myokines (derived from myocytes) and osteokines (derived from bone cells). A third player in this biochemical crosstalk is adipose tissue and the secretion of adipokines (derived from adipocytes). In this review, we discuss the bidirectional effects of myokines and osteokines on muscle and bone metabolism, and the impact of adipokines on both of these secretory organs. Several myokines, notably, IL6, irisin, IGF-1, BDNF, myostatin, and FGF2 exert anabolic/catabolic effects on bone, while the osteokines osteocalcin and sclerostin have shown to induce muscle anabolism and catabolism, respectively. Adipokines, such as leptin, resistin, adiponectin, and TNFα (released from adipose tissue), can also modulate muscle and bone metabolism. Contrarily, exercise-mediated release of lipolytic myokines (IL6, irisin, and LIF) stimulates thermogenesis by promoting the browning of adipocytes. Myokines, osteokines, and adipokines exert autocrine/paracrine effects locally as well as through the endocrine system, to regulate muscle, bone, and fat metabolism. Reductions in physical activity and increases in energy intake, both linked with aging, leads to adipocyte hypertrophy and the recruitment of immunological cells (macrophages). In turn, this releases pro-inflammatory adipokines which induces chronic low-grade inflammation (LGI), a key player in the pathology of several diseases. However, exercise-induced stimulation of bioactive cytokines, through muscle-bone-fat crosstalk, increases muscle anabolism, bone formation, mitochondrial biogenesis, glucose utilization, and fatty acid oxidation, and attenuates chronic LGI.

The Effect of the Ultra-Marathon Run at a Distance of 100 Kilometers on the Concentration of Selected Adipokines in Adult Men

Pro-inflammatory adipokines have a multifunctional role in adipogenesis, angiogenesis, glucose homeostasis, and inflammation. The aim of the present study is to evaluate the effect of running a 100 km ultra-marathon on serum levels of two adipokines: resistin and chemerin. Fifteen male participants complete a medical questionnaire and their body composition is assessed. Serum resistin, chemerin, high sensitivity C-reactive protein (hs-CRP), glucose, and lactate levels are measured at baseline and post-race. During-race data on fluid and food consumption and energy expenditure are calculated. There is a higher (p < 0.001) post-race concentration of resistin and hs-CRP compared with resting values, with no change in chemerin levels. There is an inverse correlation of the change in resistin levels with post-run glucose values (r = 0.742, p < 0.001) and a positive correlation between changes in hs-CRP and energy expenditure (r = 0.782, p < 0.001). The present results show the impact of running an ultra-marathon on serum levels of pro-inflammatory markers released by adipose tissue. It is difficult to establish whether these results may be due to the stress of exercise, high energy expenditure or caloric deficit. However, we suggest that an addition of resistin to traditional pro-inflammatory markers (including CRP) may improve the assessment of inflammation in conditions of high-energy expenditure.

FRI0375 VISFATIN EFFECTS ON MSCS DURING OD VIA DIFFERENTIAL REGULATION OF LNCRNA H19 AND MICRO RNA 675-3P

Background:Long non-coding (lnc-)RNA are regulatory molecules transcribed from DNA similar to mRNA and interact directly with DNA, RNA and proteins. Some lncRNAs have been shown to contain micro (mi-)RNAs in their sequence that can be released by splicing and lead to active miRNA molecules, e.g. lncRNA H19 includes two miRNAs 675-3p and -5p in its sequence.Adipose tissue derived factors (adipokines) are involved in inflammation processes and osteoarthritis (OA) development. The proinflammatory adipokine visfatin has been shown to alter osteogenic differentiation (OD) of pluripotent mesenchymal stem cells (MSCs) and reduces elastic fiber expression, increases matrix mineralization and proinflammatory cytokine and chemokine production(1).Objectives:We evaluated a novel effect of visfatin on lncRNA H19 in MSCs during OD. The goal was to explore the kinetics of the visfatin effect during OD with regard to H19 regulation and to investigate H19 downstream mechanisms leading to the observed altered MSC differentiation and osteoblast activity.Methods:MSCs isolated from OA hip or knee bone (phMSC) and commercially obtained healthy human (h-)MSCs were differentiated towards osteoblasts with/without visfatin, resistin, leptin, TNF and Wnt/TGFβ1 pathway inhibitors. Supernatants were collected at days 2, 7, 9, 14 and 21 of OD, cell lysates at day 2, 7, 9, 14 and matrix mineralization assays conducted at day 21. H19 and miRNA expression was evaluated by real-time PCR after mi-/RNA isolation. IL-6 was analyzed by ELISA.Results:H19 was continuously upregulated in unstimulated controls as expected during OD but also when stimulated with other adipokines. In contrast, stimulation with visfatin significantly decreased H19 (day 2 to 14 of OD, hip-phMSCs: p = 0.0097, knee-phMSCs: p=0.0075, h-MSC: p = 0.044). Visfatin increased matrix mineralization and IL-6 production as expected (hMSC: p = 0.03, phMSC: p = 0.013)(1). TNF stimulation during OD did not lead to a downregulation of H19 nor increased matrix mineralization, thus showing that the effects were visfatin-dependent. H19s endogenous miRNA 675-5p was changed in parallel with H19, increased during control OD and significantly down-regulated by visfatin (e.g. day 14 p = 0.015). However, H19s endogenous miRNA 675-3p was inversely regulated, downregulated during control OD while visfatin stimulation attenuated this effect (e.g. day 14 p = 0.025). Altered Wnt-signaling and involvement of the TGFβ1 pathway could not be observed.Conclusion:H19 is upregulated during OD and may therefore play a regulatory role in the process of osteogenesis. Visfatin stimulation of MSCs during OD showed pro-inflammatory effects, increased matrix mineralization while reducing elastic fiber production(1). These effects were associated with a downregulation of H19, a specific visfatin effect not triggered by other adipokines or TNF. The H19 sequence includes two endogenous micro-RNAs 675-3p and 5p. We demonstrated miRNA 675-5p to be regulated in parallel to H19, whereas miRNA 675-3p was inversely regulated and increased continuously upon visfatin stimulation. Based on these results, we hypothesize that visfatin provides a specific stimulus for the splicing of miRNA 675-3p from H19, in turn leading to H19 reduction. miRNA 675-3p thus represents an effector mechanism of visfatin that contributes to the observed functional effects in differentiating MSCs.

RESPONSES OF PLASMA ADIPOKINES TO HIGH INTENSITY INTERVAL TRAINING: SYSTEMATIC REVIEW

ABSTRACT Introduction Obesity is one of the major diseases of modern times. However, the explanation for its pathophysiology is recent and has not yet been fully elucidated. White adipose tissue synthesizes and secretes adipokines that affect several pathologies related to obesity. Excessive growth of this tissue results in increased levels of pro-inflammatory adipokines and a consequent decrease in anti-inflammatory adipokines. Nevertheless, most studies use moderate intensity training, limiting the understanding of high intensity interval training in these proteins. Objective To verify the latest information on the effects of HIIT in improving the profile of circulating adipokines. Methods A search was performed on the databases PUBMED, Lilacs, HighWire, BVS and the Cochrane Database of Systematic Reviews, with the following keywords: HIIT adipokines, HIIT leptin, HIIT adiponectin. Eleven studies were selected, published in English and Portuguese between 2013 and 2017. Results HIIT proved to be effective in increasing adiponectin in the adolescent population and in Olympic athletes, but this depended on a good prescription parameter and exercise intensity. However, maximum or supramaximal intensities were superior to low and moderate intensities. In turn, leptin presented a significant decrease in response to HIIT due to the reduction of adipose tissue, demonstrating a directly proportional relation. Other adipokines, such as omentin-1 and interleukin-10, also responded positively to HIIT, resulting in improved anti-inflammatory status. Conclusion HIIT proved to be an efficient method to reduce inflammation due to obesity, as well as inducing an improvement in sports performance. However, the effects depend on training volume, intensity and prescription method. Level of evidence I; Therapeutic study–Investigating the results of treatment.

Modulation of inter-organ signalling in obese mice by spontaneous physical activity during mammary cancer development

Accumulative evidence links breast cancer development to excess weight and obesity. During obesity, dysregulations of adipose tissue induce an increase in pro-inflammatory adipokine secretions, such as leptin and oestrogen secretions. Furthermore, a raise in oxidative stress, along with a decrease in antioxidant capacity, induces and maintains chronic inflammation, which creates a permissive environment for cancer development. Physical activity is recommended as a non-pharmacological therapy in both obese and cancer situations. Physical activity is associated with a moderation of acute inflammation, higher antioxidant defences and adipokine regulation, linked to a decrease of tumour-cell proliferation. However, the biological mechanisms underlying the relationship between oxidative stress, low-grade inflammation, carcinogenesis, obesity and physical activity are poorly understood. Our study is based on old, ovariectomised mice (C57BL/6J mice, 33 weeks old), fed with a high fat diet which increases adipose tissue favouring overweight and obesity, and housed in either an enriched environment, promoting physical activity and social interactions, or a standard environment constituting close to sedentary conditions. Our model of mammary carcinogenesis allowed for the exploration of tissue secretions and signalling pathway activation as well as the oxidative status in tumours to clarify the mechanisms involved in a multiple factorial analysis of the data set. The multiple factorial analysis demonstrated that the most important variables linked to moderate, spontaneous physical activity were the increase in growth factor (epithelial growth factor (EGF), hepatocyte growth factor (HGF)) and the activation of the signalling pathways (STAT3, c-jun n-terminal kinases (JNK), EKR1/2, nuclear factor-kappa B (NF-κB)) in the gastrocnemius (G). In inguinal adipose tissue, the NF-κB inflammation pathway was activated, increasing the IL-6 content. The adiponectin plasma (P) level increased and presented an inverse correlation with tumour oxidative status. Altogether, these results demonstrated that spontaneous physical activity in obesity conditions could slow down tumour growth through crosstalk between muscle, adipose tissue and tumour. A spontaneous moderate physical activity was able to modify the inter-organ exchange in a paracrine manner. The different tissues changed their signalling pathways and adipokine/cytokine secretions, such as adiponectin and leptin, resulting in a decrease in anti-oxidative response and inflammation in the tumour environment. This model showed that moderate, spontaneous physical activity suppresses tumour growth via a dialogue between the organs close to the tumour.

Interferon regulatory factor 7 mediates obesity-associated MCP-1 transcription.

Hypertrophy, associated with adipocyte dysfunction, causes increased pro-inflammatory adipokine, and abnormal glucose and lipid metabolism, leading to insulin resistance and obesity-related-health problems. By combining DNA microarray and genomic data analyses to predict DNA binding motifs, we identified the transcription factor Interferon Regulatory Factor 7 (IRF7) as a possible regulator of genes related to adipocyte hypertrophy. To investigate the role of IRF7 in adipocytes, we examined gene expression patterns in 3T3-L1 cells infected with a retrovirus carrying the IRF7 gene and found that enforced IRF7 expression induced the expression of monocyte chemoattractant protein-1 (MCP-1), a key initial adipokine in the chronic inflammation of obesity. CRISPR/Cas9 mediated-suppression of IRF7 significantly reduced MCP-1 mRNA. Luciferase assays, chromatin immunoprecipitation PCR analysis and gel shift assay showed that IRF7 transactivates the MCP-1 gene by binding to its proximal Interferon Stimulation Response Element (ISRE), a putative IRF7 binding motif. IRF7 knockout mice exhibited lower expression of MCP-1 in epidydimal white adipose tissue under high-fat feeding conditions, suggesting the transcription factor is physiologically important for inducing MCP-1. Taken together, our results suggest that IRF7 transactivates MCP-1 mRNA in adipocytes, and it may be involved in the adipose tissue inflammation associated with obesity.

OR20-03 Transcriptional Changes in Lipid Metabolism of Adipocytes Derived from Subcutaneous Abdominal Adipose Stem Cells of Normal-Weight Polycystic Ovary Syndrome Women

Normal-weight polycystic ovary syndrome (PCOS) women exhibit increased adipose insulin resistance in vivo (1) accompanying enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with greater lipid accumulation per cell in vitro (2). To determine whether this phenomenon is associated with abnormal adipogenic gene transcription during ASC differentiation into adipocytes, SC abdominal ASCs isolated from three non-Hispanic Caucasian normal-weight PCOS women and three age- and BMI-matched controls were cultured in adipogenic differentiation medium for 3–12 days. After RNA isolation, gene expression levels were determined by RNA sequencing at days 3, 7, and 12. Differentially expressed genes were filtered for significance (padj<0.05) and fold change (>2-fold); upstream regulator genes and gene ontology (GO) functions were determined using Ingenuity Pathway Analysis. Gene set enrichment analysis (GSEA) also was used to identify enriched cellular processes (3). Differentially expressed genes in PCOS vs. control cells were either upregulated (466, 768 and 441 genes on days 3, 7 and 12, respectively) or downregulated (742, 974 and 605 genes on days 3, 7 and 12, respectively) over time, with critical genes governing adipocyte cell differentiation in PCOS cells increased 2–6 fold at days 3, 7 and 12 (PPARγ, CEBPα, ADIPOQ, AGPAT2, FABP4, LPL, PLIN1). The predicted upstream regulator genes TGFβ1 (an adipogenic inhibitor) and TNF (a pro-inflammatory adipokine) were significantly reduced in PCOS relative to control cells at all time points. The GO functions lipid oxidation and free fatty acid (FFA) beta-oxidation were enriched amongst upregulated genes in PCOS cells across all time points, while acylglycerol synthesis was increased at days 7 and 12 alone (z>2, p<0.05, all GO functions). In parallel, GSEA showed in PCOS cells significantly increased transcripts related to oxidative phosphorylation, peroxisome activity and adipogenesis at all time points (p<0.05). Thus, adipocytes derived from SC abdominal ASCs of normal-weight PCOS women exhibit early activation of adipogenic genes, potentially underlying their exaggerated lipid accumulation in vitro, as previously described (2). These PCOS-related changes in gene expression involve an increase in both oxidative phosphorylation and FFA beta oxidation, which could disrupt the balance between energy production and lipid storage, particularly when caloric intake exceeds energy utilization.

SUN-126 Distinct Molecular Phenotypes of Non-Diseased Breast Adipose Tissue of Pre-Menopausal Obese and Non-Obese Women May Underlie Differing Breast Cancer Risks

Obesity is a major risk factor for many chronic diseases including postmenopausal breast cancer. Paradoxically, breast cancer susceptibility is inversely linked to obesity in pre-menopausal women. Adipose tissues are active endocrine organs that play major roles in tumor development and progression; however, fat depots at different anatomical sites are biologically and functionally distinct and their singular influence on breast epithelial biology remains unclear. To study the early events by which breast adiposity may provide a microenvironment predisposing normal breast epithelial cells to tumorigenesis, we collected breast tissue from pre-menopausal (n=10/group) non-obese (NO, BMI=27.6±0.8) and obese (O, BMI=44.5±2.8) women of comparable ages (NO: 36.1± 3.3; O: 40.0±2.0) with no breast cancer and undergoing elective breast reduction surgery. Breast adipose tissue and corresponding glandular cells were analyzed histologically and evaluated for expression of genes (adipokines, cytokines, steroid hormone signaling) by QPCR and proteins (proliferation, apoptosis, inflammation) by IHC. Adipocyte size distributions from NO and O breasts did not differ (P=0.9). However, adipose mRNA levels for pro-inflammatory cytokines (IL-6, IL-8, CSF-1, MCP-1) and adipokines (LEP, CFD) were higher for O than NO (P<0.05). AdipoQ, ER-α, and ER-β transcript levels were lower for O than NO (P<0.05), while those for CYP19 and PTGS2 showed reverse trends (O>NO, P<0.05). In the corresponding glandular cells, NO had higher mRNA levels for IL-6, IL-8, ER-α, and ER-β than O (P<0.05). Immunostaining with anti-Ki67 antibodies indicated that O glandular cells were 3-fold less proliferative than those for NO, consistent with their lower Cyclin D1 mRNA levels (P<0.05). Galectin-1, a pro-fibrotic protein, showed predominant myo- vs. luminal epithelial localization, with staining intensities for O tending to be higher (P=0.07) than for NO. Perilipin immunostaining was specific for adipocytes and did not differ for O and NO. A non-targeted approach using a Human Cytokine Array (R&D Systems) was employed to further evaluate the inflammation status of O vs. NO adipose. The analyses confirmed the higher expression of IL-8, Leptin and CFD (by QPCR) in O vs. NO and identified C-reactive protein, EMMPRIN, Trefoil Factor-3, Cystatin-3 and Macrophage Migration Inhibitory Factor-1 as greater in O than NO (~2-fold). Our findings demonstrate marked differences in gene and protein expression patterns of O and NO breast adipose tissue, which were accompanied by a suppression of proliferation of O relative to NO breast epithelium. We speculate that early exposure of the breast epithelium to a highly inflammatory environment fueled by breast adiposity may promote a senescent state that confers protection from pre-menopausal breast cancer.

The effect of high sugar diet on adipokine expression in 3T3-L1 cells and weight in mice

One of the major contributing factors to the prevalence of obesity in United States is the Western Diet, characterized by consumption of high fat and high simple sugars. Even though the issue of obesity has become a national interest, few studies have explored the relationship of high sugary diets (HSD) and obesity. The objective of this study was to determine the effects of HSD on weight gain and adipokine expression in adipose tissues. 3T3-L1 cells were cultured and differentiated in recommended differentiation medium supplemented with stock sugar solutions. Lipid accumulation was quantified with Oil Red O assay. Total RNA was isolated, first strand cDNA synthesized, and qRT-PCR performed. C57BL/6 male mice were fed normal rodent chow and water with the following sugar concentrations: fructose (10% solution), glucose (10% solution) and a combined fructose/glucose (55% fructose and 45% glucose for a 10% solution) for 16 weeks. Animal weights were recorded and water solutions were measured and replaced every two days. The in vitro data showed significant increase in lipogenesis for fructose/glucose as compared to the control. Pro-inflammatory adipokine expression of 3T3-L1 cells is significantly increased by two-fold when exposed to fructose/glucose solution. HSD-fed mice exhibited an addictive behavior with a significant increase in fructose/glucose water consumption, and weight gain. While all HSD-fed mice consumed more water and gained weight over time, the fructose/glucose group substantially consumed more water which resulted in major weight gain. Understanding the deleterious effects of over consumption of HSD is paramount to avoiding obesity-related diseases and disorders.

Serum Visfatin does not seem to be a Useful Marker to Guide Glucocorticoid Substitution in Adrenal Insufficiency.

Primary adrenal insufficiency (Addison's disease, AD) requires lifelong steroid substitution. Excess exogenous glucocorticoids promote abdominal obesity, insulin-glucose imbalance, and hypertension. Reliable markers of the adequate glucocorticoid replacement are lacking. Visfatin is a pro-inflammatory adipokine, with enzymatic activity of nicotinamide phosphoribosyltransferase. It enhances leukocyte function and synthesis of tumour necrosis factor α (TNFα) and interleukin-6 (IL-6). Serum visfatin is elevated in autoimmunity, but also in obesity, insulin resistance, and metabolic syndrome. This study was aimed to investigate whether serum visfatin could guide the glucocorticoid substitution in AD. Biochemical analyses were performed in 96 patients with AD (mean age 43.3±14.9 years) and 91 controls (43.5±12.5 years). Visfatin level was significantly elevated in patients with AD compared to controls (p<0.0001). Higher circulating IL-6 was also detected among subjects with AD (p=0.006). In AD, visfatin level was positively correlated with IL-6 (p=0.014), TNFα (p=0.001), body mass (p=0.015), fasting insulin (p=0.001) and HOMA-IR (p=0.001). No relationship was noticed with daily hydrocortisone (p=0.096) and urinary free cortisol excretion (p=0.499). Only the correlations with IL-6 and fasting insulin survived multiple regression analysis (p=0.049 and p=0.005, respectively). Additionally, positive correlation between visfatin and autoantibodies to 21-hydroxylase was noted (p=0.005). In the control group serum visfatin was correlated with IL-6 (p=0.009) and TNFα (p=0.0002). The current study reveals elevated serum visfatin in autoimmune AD. Visfatin does not seem a useful marker of the glucocorticoid replacement, although it correlates with fasting insulin and pro-inflammatory molecules. Further functional analyses are warranted to elucidate the role of visfatin in autoimmunity.