Increased expression of inflammation-related genes in cultured preadipocytes/stromal vascular cells from obese compared with non-obese Pima Indians.
ABSTRACT: The specific contributions made by the various cell types in adipose tissue to obesity, particularly obesity-related inflammation, need to be clarified. The aim of this study was to elucidate the potential role of adipocyte precursor cells (preadipocytes/stromal vascular cells [SVC]).We performed Affymetrix oligonucleotide microarray expression profiling of cultured abdominal subcutaneous preadipocytes/SVC isolated from the adipose tissue of 14 non-obese (BMI 25+/-4 kg/m2) and 14 obese (55+/-8 kg/m2) non-diabetic Pima Indian subjects. Quantitative real-time PCR (RT-PCR) was used to verify the differential expression of several genes in an independent group of subjects.We identified 218 differentially expressed genes with p values less than 0.01. Microarray expression profiling revealed that the expression of inflammation-related genes was significantly upregulated in preadipocytes/SVC of obese individuals. Quantitative RT-PCR confirmed the upregulation of IL8, CTSS, ITGB2, HLA-DRA, CD53, PLA2G7 and MMP9 in preadipocytes/SVC of obese subjects.The upregulation of inflammation-related genes in preadipocytes/SVC of obese subjects may increase the recruitment of immune cells into adipose tissue and may also result in changes in the extracellular matrix (tissue remodelling) to accommodate adipose tissue expansion in obesity.
Project description:Adipose tissue pathology in obese patients often features impaired adipogenesis, angiogenesis, and chronic low-grade inflammation, all of which are regulated in large part by adipose tissue stromal vascular cells [SVC; i.e., non-adipocyte cells within adipose tissue including preadipocytes, endothelial cells (ECs), and immune cells]. Exercise is known to increase subcutaneous adipose tissue lipolysis, but the impact of exercise on SVCs in adipose tissue has not been explored. The purpose of this study was to assess the effects of a session of exercise on preadipocyte, EC, macrophage, and T cell content in human subcutaneous adipose tissue. We collected abdominal subcutaneous adipose tissue samples from 10 obese adults (BMI 33 ± 3 kg/m2, body fat 41 ± 7%) 12 h after a 60 min acute session of endurance exercise (80 ± 3%HRpeak) vs. no acute exercise session. SVCs were isolated by collagenase digestion and stained for flow cytometry. We found that acute exercise reduced preadipocyte content (38 ± 7 vs. 30 ± 13%SVC; p = 0.04). The reduction was driven by a decrease in CD34hi preadipocytes (18 ± 5 vs. 13 ± 6%SVC; p = 0.002), a subset of preadipocytes that generates high lipolytic rate adipocytes ex vivo. Acute exercise did not alter EC content. Acute exercise also did not change total immune cell, macrophage, or T cell content, and future work should assess the effects of exercise on subpopulations of these cells. We conclude that exercise may rapidly regulate the subcutaneous adipose tissue preadipocyte pool in ways that may help attenuate the high lipolytic rates that are commonly found in obesity.
Project description:Sirt1 plays an important role in regulating glucose and lipid metabolism in obese animal models. Impaired adipose tissue angiogenesis in the obese state decreases adipogenesis and thereby contributes to glucose intolerance and lipid metabolism. However, the mechanism by which Sirt1 activation affects obesity-associated impairments in angiogenesis in the adipose tissue is not fully understood. Here, we show that SRT1720 treatment induces angiogenic genes in cultured 3T3-L1 preadipocytes and ex vivo preadipocytes. siRNA-mediated knockdown of Sirt1 in 3T3-L1 preadipocytes downregulated angiogenic genes in the preadipocytes. SRT1720 treatment upregulated metabolically favorable genes and reduced inflammatory gene expressions in the adipose tissue of diet-induced obese (DIO) mice. Collectively, these findings suggest a novel role of SRT1720-induced Sirt1 activation in the induction of angiogenic genes in preadipocytes, thereby reducing inflammation and fibrosis in white adipose tissue (WAT) and promoting insulin sensitivity.
Project description:BACKGROUND:Preadipocyte is closely related to obesity-induced inflammation. The impairment of autophagic flux by defective lysosomal function has been observed in adipose tissue from obese mice. While the fatty acid translocase CD36 is an important immuno-metabolic receptor, it remains unclear whether preadipocyte CD36 is involved in adipose tissue inflammation and whether CD36 regulates lysosomal function. METHODS:Using visceral adipose tissue from obese patients, a high-fat diet (HFD)-induced obese mice model, primary mouse preadipocytes and 3T3L1 cells we analyzed whether and how preadipocyte CD36 modulates lysosomal function and adipose tissue inflammation. FINDINGS:CD36 expression in preadipocytes is induced in obese patients and HFD-fed mice, accompanied with the disruption of lysosome function. CD36 knockout protects primary preadipocytes of HFD-fed mice from lysosomal impairment. In vitro, CD36 interacts with Fyn to phosphorylate and activate Inositol (1,4,5)-trisphosphate receptor 1 (IP3R1), causing excess calcium transport from endoplasmic reticulum (ER) to lysosome, which results in lysosomal impairment and inflammation. Moreover, IP3R inhibitor 2-aminoethoxydiphenyl borate (2APB) attenuates lysosomal impairment, inflammation and lipid accumulation in CD36-overexpressing preadipocytes. INTERPRETATION:Our data support that the abnormal upregulation of CD36 in preadipocytes may contribute to the development of adipose tissue inflammation. CD36/Fyn/IP3R1-mediated lysosomal calcium overload leads to lysosomal impairment and inflammation in preadipocyte. Thus targeting improving lysosomal calcium homeostasis may represent a novel strategy for treating obesity-induced inflammation.
Project description:Extracellular matrix (ECM) remodeling dynamically occurs to accommodate adipose tissue expansion during obesity. One non-fibrillar component of ECM, biglycan, is released from the matrix in response to tissue stress; the soluble form of biglycan binds to toll-like receptor 2/4 on macrophages, causing proinflammatory cytokine secretion. To investigate the pattern and regulatory properties of biglycan expression in human adipose tissues in the context of obesity and its related diseases, we recruited 21 non-diabetic obese women, 11 type 2 diabetic obese women, and 59 normal-weight women. Regardless of the presence of diabetes, obese patients had significantly higher biglycan mRNA in both visceral and subcutaneous adipose tissue. Biglycan mRNA was noticeably higher in non-adipocytes than adipocytes and significantly decreased during adipogenesis. Adipose tissue biglycan mRNA positively correlated with adiposity indices and insulin resistance parameters; however, this relationship disappeared after adjusting for BMI. In both fat depots, biglycan mRNA strongly correlated with the expression of genes related to inflammation and endoplasmic reticulum stress. In addition, culture of human preadipocytes and differentiated adipocytes under conditions mimicking the local microenvironments of obese adipose tissues significantly increased biglycan mRNA expression. Our data indicate that biglycan gene expression is increased in obese adipose tissues by altered local conditions.
Project description:In obesity, the dysfunctional adipose tissue (AT) releases increased levels of proinflammatory adipokines such as TNF?, IL-6, and IL-1? 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-?B nuclear translocation together with IL-6, TNF?, and IL-1? in 3T3-L1 cells in a TLR4-dependent manner. Furthermore, TLR4 signaling was involved in the increased expression of C/EBP? 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.
Project description:Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity.
Project description:OBJECTIVES:High-fat diet (HFD) feeding in mice is characterized by accumulation of ?? T cells in adipose tissue. However, the contribution of ?? T cells to obesity-induced inflammation of skeletal muscle, a major organ of glucose uptake, is unknown. This study was undertaken to evaluate the effect of ?? T cells on insulin sensitivity and inflammatory state of skeletal muscle and adipose tissue in obesity. Furthermore, we investigated whether CD4+IFN?+ (TH1) cells are involved in skeletal muscle and adipose tissue metabolic dysfunction that accompanies obesity. METHODS:Mice lacking ?? T cells (T cell receptor beta chain-deficient [TCRb-/-] mice) were fed HFD for 12 weeks. Obesity-induced skeletal muscle and adipose tissue inflammation was assessed by flow cytometry and quantitative RT-PCR. To investigate the effect of TH1 cells on skeletal muscle and adipose tissue inflammation and metabolic functions, we injected 5×10(5) TH1 cells or PBS weekly over 12 weeks into HFD-fed TCRb-/- mice. We also cultured C2C12 myofibers and 3T3-L1 adipocytes with TH1-conditioned medium. RESULTS:We showed that similar to adipose tissue, skeletal muscle of obese mice have higher ?? T cell content, including TH1 cells. TCRb-/- mice were protected against obesity-induced hyperglycemia and insulin resistance. We also demonstrated suppressed macrophage infiltration and reduced inflammatory cytokine expression in skeletal muscle and adipose tissue of TCRb-/- mice on HFD compared to wild-type obese controls. Adoptive transfer of TH1 cells into HFD-fed TCRb-/- mice resulted in increased skeletal muscle and adipose tissue inflammation and impaired glucose metabolism. TH1 cells directly impaired functions of C2C12 myotubes and 3T3-L1 adipocytes in vitro. CONCLUSIONS:We conclude that reduced adipose tissue and skeletal muscle inflammation in obese TCRb-/- mice is partially attributable to the absence of TH1 cells. Our results suggest an important role of TH1 cells in regulating inflammation and insulin resistance in obesity.
Project description:Obesity increases the risk of developing major diseases such as diabetes and cardiovascular disease. Adipose tissue, particularly adipocytes, may play a major role in the development of obesity and its comorbidities. The aim of this study was to characterise, in adipocytes from obese people, the most differentially expressed genes that might be relevant to the development of obesity.We carried out microarray gene profiling of isolated abdominal subcutaneous adipocytes from 20 non-obese (BMI 25+/-3 kg/m2) and 19 obese (BMI 55+/-8 kg/m2) non-diabetic Pima Indians using Affymetrix HG-U95 GeneChip arrays. After data analyses, we measured the transcript levels of selected genes based on their biological functions and chromosomal positions using quantitative real-time PCR.The most differentially expressed genes in adipocytes of obese individuals consisted of 433 upregulated and 244 downregulated genes. Of these, 410 genes could be classified into 20 functional Gene Ontology categories. The analyses indicated that the inflammation/immune response category was over-represented, and that most inflammation-related genes were upregulated in adipocytes of obese subjects. Quantitative real-time PCR confirmed the transcriptional upregulation of representative inflammation-related genes (CCL2 and CCL3) encoding the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein 1alpha. The differential expression levels of eight positional candidate genes, including inflammation-related THY1 and C1QTNF5, were also confirmed. These genes are located on chromosome 11q22-q24, a region with linkage to obesity in the Pima Indians.This study provides evidence supporting the active role of mature adipocytes in obesity-related inflammation. It also provides potential candidate genes for susceptibility to obesity.
Project description:Obesity is one of major health challenges in the industrial world. Although rice hull has been reported to show various bioactivities, no studies have evaluated its anti-obesity effect. We hope to demonstrate the anti-obesity effect of rice hull extract (RHE) and the underlying mechanism in high-fat diet (HFD)-induced obese mice and 3T3-L1 preadipocytes. Serum lipid profiles were determined by enzymatic methods. Histological analysis of liver and epididymis fat tissues was carried out with hematoxylin and eosin stain. The mRNA expression of adipogenic markers was analyzed with qRT-PCR and western blotting. Oral administration of RHE reduced body weight gain and fat accumulation in HFD-fed mice. RHE also reduced lipid accumulation by inhibiting the mRNA expression of adipogenic-related genes in HFD-fed obese mice and differentiated preadipocytes. The downregulation of adipogenesis by RHE was mediated through the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, RHE induced the phosphorylation of c-Jun N-terminal kinases (JNK) and extracellular-signal-regulated kinases (ERK) in liver and epididymis adipose tissues of HFD-fed obese mice. Taken together, these findings indicate that RHE could inhibit the differentiation of adipose cell and prevent HFD-induced obesity, suggesting its potential in the prevention of obesity and metabolic syndrome and related-disorders.
Project description:Epoxide hydrolase 2 (EPHX2) is an emerging therapeutic target in several immunometabolic disorders. EPHX2 metabolizes anti-inflammatory epoxyeicosatrienoic acids into pro-inflammatory diols. The contribution of EPHX2 activity to human obesity remains unexplored. We compared the expression of EPHX2 between lean and obese humans (n = 20 each) in subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs) using RT-PCR, Western Blot analysis, immunohistochemistry, and confocal microscopy before and after a 3-month physical activity regimen. We also assessed EPHX2 levels during preadipocyte differentiation in humans and mice. EPHX2 mRNA and protein expression were significantly elevated in obese subjects, with concomitant elevated endoplasmic reticulum (ER) stress components (the 78-kDa glucose-regulated protein; GRP78, and the Activating transcription factor 6; ATF6) and inflammatory markers (Tumor necrosis factor-?; TNF?, and Interleukin 6; IL6) as compared to controls (p < 0.05). EPHX2 mRNA levels strongly correlated with adiposity markers. In obese individuals, physical activity attenuated EPHX2 expression levels in both the SAT and PBMCs, with a parallel decrease in ER stress and inflammation markers. EPHX2 expression was also elevated during differentiation of both human primary and 3T3-L1 mouse preadipocytes. Mediators of cellular stress (palmitate, homocysteine, and macrophage culture medium) also increased EPHX2 expression in 3T3-L1 preadipocytes. Our findings suggest that EPHX2 upregulation is linked to ER stress in adiposity and that physical activity may attenuate metabolic stress by reducing EPHX2 expression.