Expression Of Adipocyte Fatty Acid-binding Protein Research Articles

Conjugated linoleic acid regulates adipocyte fatty acid binding protein expression via peroxisome proliferator-activated receptor α signaling pathway and increases intramuscular fat content.

Intramuscular fat (IMF) is correlated positively with meat tenderness, juiciness and taste that affected sensory meat quality. Conjugated linoleic acid (CLA) has been extensively researched to increase IMF content in animals, however, the regulatory mechanism remains unclear. Adipocyte fatty acid binding protein (A-FABP) gene has been proposed as candidates for IMF accretion. The purpose of this study is to explore the molecular regulatory pathways of CLA on intramuscular fat deposition. Here, our results by cell lines indicated that CLA treatment promoted the expression of A-FABP through activated the transcription factor of peroxisome proliferator-activated receptor α (PPARα). Moreover, in an animal model, we discovered that dietary supplemental with CLA significantly enhanced IMF deposition by up-regulating the mRNA and protein expression of PPARα and A-FABP in the muscle tissues of mice. In addition, our current study also demonstrated that dietary CLA increased mRNA expression of genes and enzymes involved in fatty acid synthesis and lipid metabolism the muscle tissues of mice. These findings suggest that CLA mainly increases the expression of A-FABP through PPARα signaling pathway and regulates the expression of genes and enzymes related to IMF deposition, thus increasing IMF content. These results contribute to better understanding the molecular mechanism of IMF accretion in animals for the improvement of meat quality.

Adipocyte Fatty Acid-Binding Protein, Cardiovascular Diseases and Mortality.

It has been increasingly recognized that inflammation plays an important role in the pathogenesis of cardiovascular disease (CVD). In obesity, adipose tissue inflammation, especially in the visceral fat depots, contributes to systemic inflammation and promotes the development of atherosclerosis. Adipocyte fatty acid-binding protein (AFABP), a lipid chaperone abundantly secreted from the adipocytes and macrophages, is one of the key players mediating this adipose-vascular cross-talk, in part via its interaction with c-Jun NH2-terminal kinase (JNK) and activator protein-1 (AP-1) to form a positive feedback loop, and perpetuate inflammatory responses. In mice, selective JNK inactivation in the adipose tissue significantly reduced the expression of AFABP in their adipose tissue, as well as circulating AFABP levels. Importantly, fat transplant experiments showed that adipose-specific JNK inactivation in the visceral fat was sufficient to protect mice with apoE deficiency from atherosclerosis, with the beneficial effects attenuated by the continuous infusion of recombinant AFABP, supporting the role of AFABP as the link between visceral fat inflammation and atherosclerosis. In humans, raised circulating AFABP levels are associated with incident metabolic syndrome, type 2 diabetes and CVD, as well as non-alcoholic steatohepatitis, diabetic nephropathy and adverse renal outcomes, all being conditions closely related to inflammation and enhanced CV mortality. Collectively, these clinical data have provided support to AFABP as an important adipokine linking obesity, inflammation and CVD. This review will discuss recent findings on the role of AFABP in CVD and mortality, the possible underlying mechanisms, and pharmacological inhibition of AFABP as a potential strategy to combat CVD.

Abstract 4363: A-FABP and estrogens are independently involved in the development of breast cancer

Abstract Background: We previously reported that postmenopausal obese women exhibit increased levels of circulating adipocyte fatty acid binding protein (A-FABP), which is associated with breast cancer (BC) development. In postmenopause, increased estrogen levels are associated with increased BC risk. Hypothesis: Estrogens (estrone: E1, estradiol: E2 and estriol: E3) are associated with A-FABP in the setting of obesity and obesity-related BC development. Methods: Immunoassays for E1, E2, E3, and A-FABP were performed using serum samples from women with and without BC. Results: Statistical analysis was conducted using the Mann-Whitney test and multiple linear regression models. Considering all subjects, E1 and E2 but not E3 levels were significantly higher in pre- than in postmenopause among both non-obese and obese individuals (p<0001). E3 (p<0.01) and E1 (p<0.05) levels were higher in non-obese than in obese women. A-FABP levels were significantly higher in obese postmenopausal women with and without BC, but not in premenopausal women, regardless of BC status. When stratified by BC status, E2 levels were significantly higher, while E1 and E3 levels were significantly lower in postmenopausal healthy obese than non-obese women. These body mass index (BMI) based differences were not observed among women with BC. E3 levels were higher in obese pre (p=0.003) and postmenopausal (p=0.001) women with BC than those without. A-FABP levels were significantly higher in postmenopausal obese healthy women and women with BC. A-FABP was not highly associated with E1, E2 or E3. Impact: 1) A-FABP expression is increased in postmenopausal obese women with and without BC; 2) E1 and E2 expression is higher in pre- than postmenopausal women, regardless of BMI; 3) E1 and E3 levels are higher in lean than obese individuals, regardless of menopausal status; 4) the influence of obesity on E1, E2 and E3 expression in postmenopause is lost in women with BC, 5) E3 is increased in obese vs. non-obese women with BC, and 6) A-FABP and estrogens are independently involved in the development of BC. Citation Format: Bing Li, Jiaqing Hao, Xiaofang Yan, Maiying Kong, Edward Sauter. A-FABP and estrogens are independently involved in the development of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4363.

Down-regulation of A-FABP predicts non-muscle invasive bladder cancer progression: investigation with a long term clinical follow-up

BackgroundNon-muscle invasive bladder cancers (NMIBC: pTa, pT1) are characterised by a high risk of recurrence and/or progression. Identification of prognostic markers is needed to improve both diagnosis and management of the disease. The aim of this study was to analyse the expression of A-FABP (adipocyte-fatty acid binding protein) and to evaluate its prognostic value in bladder cancer with a long term clinical follow-up.MethodsA-FABP expression was investigated by immunohistochemistry in 236 tumours (114 pTa, 61 pT1, 61 pT2–4). Immunostaining was classified as negative (absent or weak immunostaining and moderate or strong staining on ≤10% of cells) or positive (moderate or strong staining on > 10% of cells). Event-free survival (EFS) and overall survival (OS) were determined with a 87.3 months median follow-up in the overall cohort. Recurrence-free survival (RFS) and progression-free survival (PFS) were established in NMIBC.ResultsLoss of A-FABP was associated with higher mean age, high stage/grade, and the presence of metastatic lymph nodes. It was correlated with shorter median EFS (17.5 vs 62.5 months; p = 0.001) and mean OS (76.7 vs 154.2 months; p = 0.009) and with higher risk of progression in the pTa/pT1 subgroup (HR, 0.36; 95% CI, 0.13–0.96; p = 0.041) and importantly in the pTa tumours (HR, 0.34; 95% CI, 0.10–0.97; p = 0.045).ConclusionThese results demonstrated that loss of A-FABP expression following a long follow-up was predictive of pTa and pTa/pT1 progression. Immunohistochemistry on diagnostic biopsy is easy to use and could be of value to help clinicians to propose appropriate treatment for these tumours.

Intermittent High Glucose Exacerbates A-FABP Activation and Inflammatory Response through TLR4-JNK Signaling in THP-1 Cells

Background Glucose fluctuation confers additional risks on diabetes-related vascular diseases, but the underlying mechanisms are unknown. Macrophage activation mediated by TLR4-JNK signaling plays an important role during the progress of diabetes. In the present study, we hypothesize that glucose fluctuation results in macrophage inflammation through TLR4-JNK signaling pathways. Methods THP-1 cells were treated with normal glucose (5 mM), constant high glucose (25 mM), and intermittent high glucose (rotation per 6 h in 5 mM or 25 mM) for 24 h. The mRNA and protein expression levels of TLR4, p-JNK, and adipocyte fatty acid-binding protein (A-FABP) were determined, and the proinflammatory cytokines TNF-α and IL-1β were quantified. Results In constant high glucose, TLR4 expression and JNK phosphorylation levels increased, and this effect was more pronounced in intermittent high glucose. Accordingly, the expression of A-FABP and the release of the proinflammatory cytokines TNF-α and IL-1β also increased in response to constant high glucose, an effect that also was more evident in intermittent high glucose. The inhibition of p-JNK by SP600125 did not attenuate TLR4 expression, but totally inhibited both A-FABP expression and the production of the proinflammatory cytokines TNF-α and IL-1β in both constant and intermittent high glucose. Conclusions Intermittent high glucose potentiates A-FABP activation and inflammatory responses via TLR4/p-JNK signaling in THP-1 cells. These findings suggest a more detrimental impact of glucose fluctuation on macrophage inflammation in diabetes-related vascular diseases than thus far generally assumed.

Abstract P1-02-04: Adipocyte fatty acid binding protein levels predict risk of obesity associated breast cancer

Abstract Background: Breast cancer risk has been linked to obesity, especially in postmenopausal women. Adipocyte fatty acid binding protein (A-FABP) is found in adipose tissue, and preliminary evidence suggests that its expression in serum is associated with obesity and breast cancer risk. Surgery for morbid obesity appears effective in decreasing morbidity and/or mortality from a variety diseases of adulthood, including cancer. We evaluated the association of A-FABP expression with 1) body mass index (BMI), 2) breast cancer and 3) change in BMI after obesity surgery. Hypothesis: Increased A-FABP expression is associated with 1) BMI level, 2) breast cancer, and 3) change in BMI after obesity surgery. Methods: Serum was collected under an institutional review board approved protocol from two cohorts of obese women: 1) those with or without breast cancer, and 2) those undergoing surgery for morbid obesity. In the first cohort, samples were collected from 101 women prior to surgery to diagnose and treat a concerning breast lesion. In the second, samples were collected from 82 healthy obese women at baseline, and whenever possible, at 3, 6, and 12 months after obesity surgery. A-FABP levels in serum were measured using human A-FABP4 ELISA kits. The student's t-test was used to compare A-FABP levels in the two groups. Linear mixed effect models were used to examine the relationship between A-FABP and BMI or change of BMI over time, controlling for age, menopause status and history of breast cancer. Results: A-FABP levels were significantly higher in women with cancer than healthy controls among obese women (p=0.039), but not among non-obese women (p > 0.05). A-FABP levels were higher in obese than non-obese women without (p=0.038) and with breast cancer (p<0.001). Among healthy obese controls, A-FABP was associated with BMI at baseline (p=0.016) and its change over time after surgery (p=0.005). The association of A-FABP with BMI was significant for women of normal breast cancer risk regardless of their menopause status (p=0.012 for pre-menopause and p<0.001 for post-menopause), but not for those with a family history of breast cancer (p>0.05). A-FABP was also more predictive of change in BMI in postmenopausal normal risk women (p<0.001) than in other groups (p>0.05). Impact: Circulating levels of A-FABP are associated with cancer in obese women, with baseline BMI in healthy women and with change in BMI in normal risk women undergoing surgery for morbid obesity. These findings all point to a clinically important mechanistic role for A-FABP in obesity influenced breast cancer. They also suggest that downregulation of A-FABP may be involved in the decreased risk of cancer, including breast cancer, that has been observed in follow-up after obesity surgery. Citation Format: Sauter E, Hao J, Yan X, Kong M, Li B. Adipocyte fatty acid binding protein levels predict risk of obesity associated breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-04.

Adipocyte Fatty Acid Binding Protein Potentiates Toxic Lipids-Induced Endoplasmic Reticulum Stress in Macrophages via Inhibition of Janus Kinase 2-dependent Autophagy

Lipotoxicity is implicated in the pathogenesis of obesity-related inflammatory complications by promoting macrophage infiltration and activation. Endoplasmic reticulum (ER) stress and adipocyte fatty acid binding protein (A-FABP) play key roles in obesity and mediate inflammatory activity through similar signaling pathways. However, little is known about their interplay in lipid-induced inflammatory responses. Here, we showed that prolonged treatment of palmitic acid (PA) increased ER stress and expression of A-FABP, which was accompanied by reduced autophagic flux in macrophages. Over-expression of A-FABP impaired PA-induced autophagy associating with enhanced ER stress and pro-inflammatory cytokine production, while genetic ablation or pharmacological inhibition of A-FABP reversed the conditions. PA-induced expression of autophagy-related protein (Atg)7 was attenuated in A-FABP over-expressed macrophages, but was elevated in A-FABP-deficient macrophages. Mechanistically, A-FABP potentiated the effects of PA by inhibition of Janus Kinase (JAK)2 activity, thus diminished PA-induced Atg7 expression contributing to impaired autophagy and further augmentation of ER stress. These findings suggest that A-FABP acts as autophagy inhibitor to instigate toxic lipids-induced ER stress through inhibition of JAK2-dependent autophagy, which in turn triggers inflammatory responses in macrophages. A-FABP-JAK2 axis may represent an important pathological pathway contributing to obesity-related inflammatory diseases.

Clinical significance of elevated serum A-FABP and free fatty acid in neonates with hypoxic ischemic brain damage.

The main function of adipocyte fatty acid-binding protein (A-FABP) is to regulate fatty acid metabolism as its molecular chaperone. The clinical significance of A-FABP in hypoxic-ischemic brain damage (HIBD) neonates is not yet clear. Free fatty acid (FFA) in cerebral cortex increases along with hypoxia ischemia degree. Thus, we aimed to investigate whether FFA can induce A-FABP expression and elevate the serum A-FABP level in HIBD neonates. In the present study, 42 HIBD neonates were selected including 11 cases as mild, 16 cases as moderate and 15 cases as severe. The serum was collected from peripheral vein at 72 h after the first visit (acute stage) and 7 days after birth (recovery stage), and the serum from 10 normal neonates was used as the control. The serum level of A-FABP and FFA in 42 neonates with acute phase and recovery phase HIBD were detected using ELISA and copper colorimetric method. The overall serum A-FABP content in HIBD neonates at the acute stage was significantly higher compared to the normal neonates (P<0.05). The serum A-FABP level in severe HIBD neonates was significantly higher than that in mild HIBD, moderate HIBD and normal neonates (P<0.05). The serum FFA level in HIBD neonates at the acute stage was 1,521.57±605.63 µmol/l, which was significantly higher than that in the normal neonates 838.24±294.22 µmol/l. The serum FFA levels in mild, moderate and severe HIBD neonates were significantly higher than those in the normal neonates. The overall A-FABP level in HIBD neonates at the recovery stage was significantly lower compared to the acute stage, which was significant in severe HIBD neonates. A-FABP levels in mild and moderate HIBD neonates at recovery stage were decreased compared with the acute stage, although there was no statistical difference. There was a positive correlation between serum A-FABP and FFA in HIBD neonates at acute stage (r=0.369, P<0.05). In conclusion, serum A-FABP and FFA levels were signifcantly increased in HIBD neonates at acute stage, and were positively correlated. The serum A-FABP level in HIBD neonates at recovery stage was significantly lower than that in the acute stage. The results suggested that serum A-FABP and FFA levels at acute stage can reflect the severity of HIBD. The detection of serum A-FABP and FFA can be applied as indicators for the early diagnosis of HIBD, but also provides a basis for the clinical evaluation of HIBD treatment.

Overexpression of the A-FABP gene facilitates intermuscular fat deposition in transgenic mice.

Adipocyte fatty acid-binding protein (A-FABP), the most abundant FABP in adipocytes, controls fatty acid uptake, transport, and metabolism in fat cells. We constructed a transgenic mice model that overexpressed the cattle A-FABP gene to investigate the relationship between A-FABP expression and intermuscular fat deposition. There was no significant difference in body weight and serum biochemical indexes between transgenic and wild-type mice. Further, there were no significant differences in intermuscular triglyceride content and A-FABP expression levels over three generations of transgenic mice. However, abdominal adipose rate, A-FABP protein content, and intermuscular triglyceride levels of transgenic mice were significantly higher than those of wild-type mice. In addition, triglycerides were remarkably higher in the skeletal muscle but lower in the myocardium of transgenic mice. Thus, overexpression of cattle A-FABP gene promoted fat deposition in the skeletal muscle of transgenic mice.

Oleate promotes differentiation of chicken primary preadipocytes invitro.

In addition to providing energy and constituting cell membrane, fatty acids also play an important role in adipocyte differentiation and lipid metabolism. As an important member of monounsaturated fatty acids, oleate, together with other components, is widely used to induce chicken preadipocyte differentiation. However, it is not clear whether oleate alone can induce chicken preadipocyte differentiation. In the present study, four different treatments were designed to test this question: basal medium, IDX [insulin, dexamethasone and IBMX (isobutylmethylxanthine)], oleate and IDX plus oleate. Cytoplasmic lipid droplet accumulation and mRNA expression for adipogenesis-related genes were monitored. After treatment of oleate on chicken preadipocytes, apparent lipid droplet formation and lipid accumulation were observed, accompanied by increasing expression of PPARγ (peroxisome proliferator-activated receptor-γ) and AFABP (adipocyte fatty acid-binding protein), but decreasing level of GATA2 (GATA-binding protein 2). In contrast, for cells cultured in the basal medium with or without IDX supplementation, lipid droplet barely occurred. These results suggest that exogenous oleate alone can act as an inducer of preadipocyte differentiation into adipocytes.

Two-Dimensional Fluorescence In-Gel Electrophoresis of Coronary Restenosis Tissues in Minipigs

We aimed to uncover the protein changes of coronary artery in-stent restenosis (ISR) tissue in minipigs with and without streptozotocin-induced diabetes mellitus by quantitative 2-dimensional fluorescence in-gel electrophoresis (2D-DIGE), and to investigate the influences of crucial proteins identified, particularly adipocyte fatty acid binding protein (AFABP), in human arterial smooth muscle cells. Sirolimus-eluting stents were implanted in the coronary arteries of 15 diabetic and 26 nondiabetic minipigs, and angiography was repeated after 6 months. The intima tissue of significant ISR and non-ISR segments in both diabetic and nondiabetic minipigs was analyzed by 2D-DIGE and MALDI-TOF/TOF mass spectrometry. AFABP level was significantly increased in ISR tissue than in non-ISR tissue in both diabetic and nondiabetic minipigs, with level being higher in diabetic ISR than in nondiabetic ISR tissue. In human arterial smooth muscle cells, overexpression of AFABP significantly altered phenotype and promoted growth and migration, with effects more prominent in high-glucose than in low-glucose medium, whereas AFABP knockdown inhibited these effects. AFABP overexpression increased reactive oxygen species production by upregulating the expression of NADPH oxidase subunits Nox1, Nox4, and P22 through multiple pathways, with elevation of downstream gene cyclin D1, matrix metalloproteinase-2, and monocyte chemoattractant protein-1. However, AFABP-induced effects were inhibited by diphenyleneiodonium, pathway inhibitors, and small interfering RNA. In addition, the supernatant from AFABP-expressing human arterial smooth muscle cells and recombinant AFABP also promoted cellular growth and migration. This study has demonstrated that AFABP is significantly increased in coronary artery ISR segments of both diabetic and nondiabetic minipigs. Increased AFABP expression and secretory AFABP of human arterial smooth muscle cells promote growth and migration via reactive oxygen species-mediated activation.

Beneficial effects of flaxseed oil and fish oil diet are through modulation of different hepatic genes involved in lipid metabolism in streptozotocin–nicotinamide induced diabetic rats

Dietary omega-3 fatty acids have been demonstrated to have positive physiological effects on lipid metabolism, cardiovascular system and insulin resistance. Type-2 diabetes (T2DM) is known for perturbations in fatty acid metabolism leading to dyslipidemia. Our objective was to investigate beneficial effects of dietary flaxseed oil and fish oil in streptozotocin-nicotinamide induced diabetic rats. Thirty-six adult, male, Wistar rats were divided into six groups: three diabetic and three non-diabetic. Diabetes was induced by an injection of nicotinamide (110mg/kg) and STZ (65mg/kg). The animals received either control, flaxseed oil or fish oil (10% w/w) enriched diets for 35days. Both diets lowered serum triglycerides and very low-density lipoprotein cholesterol levels and elevated serum high-density lipoprotein cholesterol levels in diabetic rats, while serum total cholesterol and LDL-C levels remained unaffected. Both the diets increased omega-3 levels in plasma and RBCs of diabetic rats. Flaxseed oil diet significantly up-regulated the key transcription factor peroxisome proliferator-activated receptor-α (PPAR-α ) and down-regulated sterol regulatory element-binding protein-1 (SREBP-1) in diabetic rats, which would have increased β-oxidation of fatty acids and concomitantly reduced lipogenesis respectively, thereby reducing TG levels. Fish oil diet, on the contrary lowered serum TG levels without altering PPAR-α while it showed a non-significant reduction in SREBP-1 expression in diabetic rats. Another key finding of the study is the activation of D5 and D6 desaturases in diabetic rats by flaxseed oil diet or fish oil diets, which may have resulted in an improved omega-3 status and comparable effects shown by both diets. The reduced expression of Liver-fatty acid binding protein in diabetic rats was restored by fish oil alone, while both diets showed equal effects on adipocyte fatty acid-binding protein expression. We also observed down-regulation of atherogenic cytokines tumor necrosis factor-α and interleukin-6 by both the diets. In conclusion, dietary flaxseed oil and fish oil have therapeutic potential in preventing lipid abnormalities in T2DM.

Pharmacological inhibition of adipocyte fatty acid binding protein alleviates both acute liver injury and non-alcoholic steatohepatitis in mice

Adipocyte fatty acid binding protein (A-FABP) is a key mediator of inflammatory response in macrophages. Increased hepatic expression and circulating levels of A-FABP have been observed in patients with non-alcoholic fatty liver disease (NAFLD). Here, we investigated the role of A-FABP in both lipopolysaccaride (LPS)-induced acute liver injury and high fat high cholesterol (HFHC) diet-induced NAFLD in mice. Mice with LPS-induced acute liver injury and HFHC diet-induced obesity were treated with the A-FABP inhibitor BMS309403. Liver tissues of the mice were analyzed by immunohistochemistry, Western blot or real-time PCR. A-FABP expression in Kupffer cells was significantly elevated in mice with LPS-induced acute liver injury and HFHC diet-induced obesity, as compared to their healthy controls. Pretreatment of mice with BMS309403 led to a diminished LPS-induced elevation in serum levels of alanine transaminase and hepatic production of pro-inflammatory cytokines. Likewise, chronic treatment of HFHC diet-induced obese mice with BMS309403 ameliorated hepatic steatosis, macrophage infiltration, and cellular ballooning of hepatocytes. Such improvements in liver function and morphology were accompanied by significantly decreased activation of both c-Jun and NF-κB. Pretreatment with BMS309403 suppressed both LPS- and palmitate-induced pro-inflammatory responses in isolated rat Kupffer cells. Adenovirus-mediated ectopic expression of A-FABP alone was sufficient to induce liver injury and inflammation in mice. These findings suggest that A-FABP is an important contributor to both LPS-induced acute liver injury and diet-induced NAFLD by potentiating inflammation in Kupffer cells. Pharmacological inhibition of A-FABP may represent a promising modality for obesity-related non-alcoholic steatohepatitis.

Expression pattern of adipocyte fatty acid-binding protein gene in different tissues and its regulation of genes related to adipocyte differentiation in duck

Abundance of duck adipocyte fatty acid-binding protein (A-FABP) mRNA was detected in this study by quantitative real-time PCR. The results showed that duck A-FABP gene was expressed in muscular tissues and many organs, except pancreas, lung, and kidney, and highly expressed in adipose tissues, especially in sebum. The expression of A-FABP and adipocyte differentiation-related genes was upregulated by oleic acid, and the A-FABP knockdown suppressed the oleic acid-stimulated expression of these genes in the cultured duck adipocytes, indicating A-FABP might play an important role in duck adipocyte differentiation.

1067 ADIPOCYT-FATTY ACID BINDING PROTEIN, A POTENTIAL PROGNOSTIC MARKER OF BLADDER TRANSITIONAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Few studies provided evidence that loss of adipocyte-fatty acid binding protein (A-FABP) expression is associated with progression of human bladder TCC and suggest that it could be a putative marker of progression of the disease. The re-expression of A-FABP could be a therapeutic approach in early stage bladder cancer to prevent disease progression. The aim of this study is first to determine whether A-FABP is a progression marker by analysing A-FABP mRNA expression within the tumor. Secondly, to demonstrate whether the expression of this potential prognostic marker could be up-regulated by Peroxisome Proliferator-Activated Receptor (PPAR) gamma agonists such as prostaglandins and thiazolidinediones (antidiabetic drugs) known to exhibit anti-neoplastic effects. METHODS: Total RNA was extracted from 26 samples of bladder TCC from clinical stages pTa to pT4 obtained from transurethral resection or cystectomy specimens. The relative A-FABP mRNA expression level was evaluated by RTqPCR. The regulation of AFABP expression was studied by RTqPCR and western-blotting in T24 cells derived from an undifferentiated grade III carcinoma of the bladder. RESULTS: We reported from bladder tumors that decrease of A-FABP transcript level significantly correlated to tumor stage and to histologic grade (p 0.05). Namely, in poor prognosis high grade pT1 tumors there was a loss of A-FABP expression compared to good prognosis tumors. We described for the first time the molecular mechanism by which this marker is up-regulated by PPAR gamma in T24 cells. This effect occurred through a PPARdependent transcriptional mechanism without modifying mRNA stability and interestingly required de novo protein synthesis. CONCLUSIONS: Data as a whole suggest a prognostic significance of A-FABP in bladder cancer outcome and the potential utility of overexpression of this protein by antidiabetic agents already in clinical use open up new perspectives in the treatment of bladder cancer by intravesical instillations of thiazolidinediones in order to inhibit malignant progression of bladder cancer.

Chronic administration of BMS309403 improves endothelial function in apolipoprotein E‐deficient mice and in cultured human endothelial cells

Adipocyte fatty acid-binding protein (A-FABP) is up-regulated in regenerated endothelial cells and modulates inflammatory responses in macrophages. Endothelial dysfunction accompanying regeneration is accelerated by hyperlipidaemia. Here, we investigate the contribution of A-FABP to the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE(-/-) ) mice and in cultured human endothelial cells. A-FABP was measured in aortae of ApoE(-/-) mice and human endothelial cells by RT-PCR, immunostaining and immunoblotting. Total and phosphorylated forms of endothelial nitric oxide synthase (eNOS) were measured by immunoblotting. Changes in isometric tension were measured in rings of mice aortae A-FABP was expressed in aortic endothelium of ApoE(-/-) mice aged 12 weeks and older, but not at 8 weeks or in C57 wild-type mice. Reduced endothelium-dependent relaxations to acetylcholine, UK14304 (selective α(2) -adrenoceptor agonist) and A23187 (calcium ionophore) and decreased protein presence of phosphorylated and total eNOS were observed in aortae of 18 week-old ApoE(-/-) mice compared with age-matched controls. A 6 week treatment with the A-FABP inhibitor, BMS309403, started in 12 week-old mice, improved endothelial function, phosphorylated and total eNOS and reduced plasma triglyceride levels but did not affect endothelium-independent relaxations. The beneficial effect of BMS309403 on UK14304-induced relaxations was attenuated by Pertussis toxin. In cultured human microvascular endothelial cells, lipid-induced A-FABP expression was associated with reduced phosphorylated eNOS and NO production and was reversed by BMS309403. Elevated expression of A-FABP in endothelial cells contributes to their dysfunction both in vivo and in vitro.

Unsaturated fatty acids repress the expression of adipocyte fatty acid binding protein via the modulation of histone deacetylation in RAW 264.7 macrophages

Adipocyte fatty acid binding protein (A-FABP) present in macrophages has been implicated in the integration of lipid metabolism and inflammatory response, contributing to development of insulin resistance and atherosclerosis. This study was conducted to test the hypothesis that the role of fatty acids in the inflammatory pathways is mediated through the modulation of A-FABP expression in macrophages. Murine RAW 264.7 macrophages were treated with inflammatory insults and fatty acids for quantitative real-time PCR and Western blot analysis. The cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor, for elucidating mechanisms for the regulation of A-FABP expression by fatty acids. RNA interference (RNAi) to knock down A-FABP was utilized to assess its role in inflammatory gene expression. When RAW 264.7 were incubated with lipopolysaccharides (LPS; 100 ng/ml) or 2.5 ng/ml of tumor necrosis factor α for 18 h, A-FABP mRNA and protein levels were drastically increased. Unsaturated fatty acids (100 μmol/l in complexed with BSA) such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid, significantly repressed the basal as well as LPS-induced A-FABP expression, whereas palmitic acid did not elicit the same effect. TSA increased A-FABP mRNA levels and abolished the repressive effect of linoleic acid on A-FABP expression in unstimulated and LPS-stimulated macrophages. Depletion of A-FABP expression by 70-80% using RNAi markedly decreased cyclooxygenase 2 mRNA abundance and potentiated the repression by linoleic acid. Unsaturated fatty acids inhibited the basal as well as LPS-induced A-FABP expression. The mechanism may involve histone deacetylation and anti-inflammatory effect of unsaturated fatty acids may be at least in part attributed to their repression of A-FABP expression in RAW 264.7 macrophages.

Adipocyte Fatty Acid-binding Protein Modulates Inflammatory Responses in Macrophages through a Positive Feedback Loop Involving c-Jun NH2-terminal Kinases and Activator Protein-1

Adipocyte fatty acid-binding protein (A-FABP) has emerged as an important mediator of inflammation in macrophages. Macrophage-selective ablation of A-FABP alone is sufficient to prevent the development of high cholesterol diet-induced atherosclerosis in apoE-deficient mice. However, the precise mechanisms whereby A-FABP modulates inflammation remain elusive. Here, we report that A-FABP forms a finely tuned positive loop between JNK and activator protein-1 (AP-1) to exacerbate lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Real time PCR and luciferase reporter analysis showed that LPS induced A-FABP expression through transcriptional activation. This effect was mediated by JNK, which promoted the recruitment of c-Jun to a highly conserved AP-1 consensus binding motif located within the proximal region of the A-FABP promoter. LPS-induced transactivation of the A-FABP gene was diminished by either pharmacological inhibition of JNK or knocking down c-Jun or by mutating the AP-1 recognition site within the proximal region (-122 to -116 bp) of the A-FABP promoter. Conversely, the LPS-evoked phosphorylation of JNK, activation of AP-1, and production of pro-inflammatory cytokines were markedly attenuated by pharmacological or genetic suppression of A-FABP in macrophages. Furthermore, the LPS-induced elevation in A-FABP expression could also be prevented by the selective A-FABP inhibitor BMS309403. These findings support the notion that pharmacological inhibition of A-FABP represents a valid strategy for treating inflammation-related disorders such as atherosclerosis.

Tissue expression characterization of chicken adipocyte fatty acid-binding protein and its expression difference between fat and lean birds in abdominal fat tissue

Fatty acid-binding proteins are considered to be the carriers for the transportation of intracellular fatty acids and play an important role in the development of fatness traits. Adipocyte fatty acid-binding protein (A-FABP) is one of the family members. The current study was designed to analyze the tissue expression characterization of chicken A-FABP and its expression difference between the fat and lean males in abdominal fat tissue to reveal the possible relationship between the expression of A-FABP and abdominal fat tissue development and growth in chicken. First, fusion protein glutathione S-transferase/A-FABP was induced and purified, and then the antiserum containing specific polyclonal antibodies was obtained by immunizing healthy female rabbits using the purified fusion protein. Second, tissue expression characterization of A-FABP was investigated by Western blot. Finally, A-FABP expression difference in abdominal fat tissue between the fat and lean males was investigated by real-time reverse transcription-PCR and Western blot methods. The results showed that A-FABP expressed specifically in abdominal fat tissue and the mRNA expression level of A-FABP in fat males was lower than that of lean males at 2, 3, 4, 6, 7, 9, and 10 wk of age (P<0.05), and the protein expression level of fat males was lower than that of lean males at 6 and 10 wk of age (P<0.05). These results suggested that chicken A-FABP might affect abdominal fat deposition through changing its expression level, and the possible mechanism may be that a high expression level of A-FABP induced the high lipolytic rate and led to the decreased abdominal fat mass accordingly.

Major Urinary Protein-1 Increases Energy Expenditure and Improves Glucose Intolerance through Enhancing Mitochondrial Function in Skeletal Muscle of Diabetic Mice

Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by approximately 30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.