Adipocyte Fatty Acid-binding Protein mRNA Research Articles

Molecular cloning, characterisation, and expression analysis of adipocyte fatty acid binding protein gene in Xupu goose (Anser cygnoides domesticus)

ABSTRACT 1. Adipocyte fatty acid binding protein (A-FABP) plays a key role in fatty acid uptake and intracellular transport. The objective of the present study was to identify and characterise the A-FABP gene in Xupu goose. 2. The full-length cDNA of goose A-FABP gene was cloned from the liver tissue using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The distribution of the goose A-FABP in different tissues was determined by quantitative real-time PCR (qRT-PCR). 3. The results showed that the full-length cDNA sequence of goose A-FABP was 657 bp, containing a 5ʹ-UTR of 52 bp, a 3ʹ-UTR of 206 bp and an open reading frame (ORF) of 399 bp, which encoded a polypeptide of 132 amino acids (AA). 4. The AA sequence of goose A-FABP showed 76.52%, 75.00%, 93.18% and 99.24% identities with previously described homologues from humans (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), and duck (Anas platyrhynchos), respectively, and phylogenetic analysis revealed a close relationship among them. The transcript of Xupu goose A-FABP was ubiquitously expressed in all tested tissues, and showed a high-level expression in abdominal fat, sebum and liver. 5. A significant positive correlation was identified between A-FABP mRNA abundance in the three adipose tissues and liver weight, ratio of liver to body weight, TG content, and VLDL concentration in the plasma of Xupu goose. A significant negative correlation was observed between the mRNA level of A-FABP and HDL concentration in the plasma of Xupu goose. 6. These findings provide a foundation for further research on the function and mechanism of A-FABP in the fat deposition process.

Cardiac Surgery Increases Serum Concentrations of Adipocyte Fatty Acid-Binding Protein and Its mRNA Expression in Circulating Monocytes but Not in Adipose Tissue

Adipocyte fatty acid binding protein (A-FABP) is a novel adipokine involved in the regulation of lipid and glucose metabolism and inflammation. To evaluate its potential role in the development of postoperative hyperglycemia and insulin resistance we assessed A-FABP serum concentrations and mRNA expression in skeletal and myocardial muscle, subcutaneous and epicardial adipose tissue and peripheral monocytes in 11 diabetic and 20 age- and sex-matched non-diabetic patients undergoing elective cardiac surgery. Baseline serum A-FABP did not differ between the groups (31.1+/-5.1 vs. 25.9+/-4.6 ng/ml, p=0.175). Cardiac surgery markedly increased serum A-FABP in both groups with a rapid peak at the end of surgery followed by a gradual decrease to baseline values during the next 48 h with no significant difference between the groups at any timepoint. These trends were analogous to postoperative excursions of plasma glucose, insulin and selected proinflammatory markers. Cardiac surgery increased A-FABP mRNA expression in peripheral monocytes, while no effect was observed in adipose tissue or muscle. Our data suggest that circulating A-FABP might be involved in the development of acute perioperative stress response, insulin resistance and hyperglycemia of critically ill irrespectively of the presence of diabetes mellitus.

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.

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.

Visfatin regulates genes related to lipid metabolism in porcine adipocytes

Visfatin is a visceral adipose tissue-specific adipocytokine that plays a positive role in attenuating insulin resistance by binding to the insulin receptor. Visfatin has been suggested to play a role in the regulation of lipid metabolism and inflammation; however, the mechanism remains unclear. We investigated the effects of visfatin on the regulation of gene expression in cultured porcine preadipocytes and differentiated adipocytes. In preadipocytes, the mRNA abundance of lipoprotein lipase and PPARgamma were significantly increased by visfatin or insulin treatment after 8 d (all P < 0.05). In the presence of insulin, the mRNA abundance of adipocyte fatty acid-binding protein was 24.7-fold greater than in the untreated group (P < 0.05), whereas visfatin alone had no effect on adipocyte fatty acid-binding protein mRNA abundance. Adipocyte differentiation was induced by insulin treatment for 8 d. In differentiated porcine adipocytes, exposure to insulin or visfatin for 24 h increased (P < 0.05) fatty acid synthase mRNA abundance but had no effect on the expression of sterol regulatory element binding-protein 1c mRNA. We also found a 5.8-fold upregulation of IL-6 expression in porcine adipocytes after 24 h of treatment with visfatin (P < 0.05). These results demonstrated that visfatin upregulated lipoprotein lipase expression in preadipocytes, potentially facilitating lipid uptake, and increased the gene expression of fatty acid synthase in differentiated adipocytes to potentially enhance lipogenic activity. Furthermore, visfatin can upregulate IL-6 expression in differentiated porcine adipocytes. The information presented in this study provides insights into the roles of visfatin in lipid metabolism in pigs.

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.

Adipose Tissue Fat Accumulation Is Reduced by a Single Intraperitoneal Injection of Peroxisome Proliferator-Activated Receptor Gamma Agonist When Given to Newly Hatched Chicks

Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates adipocyte differentiation and modulates lipid metabolism in mammals. The aim of the present study was to investigate whether the administration of PPARγ ligands, adipogenic cocktail, or both to newly hatched chicks regulates adipocyte differentiation in vivo and modulates fat deposition in growing broiler chickens. Levels of PPARγ, CCAAT/enhancer binding protein α, and adipocyte fatty acid-binding protein mRNA in the abdominal fat pad of 7-d-old broiler chicks given a single intraperitoneal dose of troglitazone, a synthetic PPARγ ligand, at 1 d old were significantly greater than those in control chickens. This suggests administration of troglitazone enhanced adipocyte differentiation in vivo. Adipose tissue weight in 28-d-old chickens similarly administered triolein emulsion containing troglitazone or adipogenic cocktail (i.e., dexamethasone, insulin, isobutyl-methylxanthine, and oleic acid) was also significantly less than that of control chickens. However, there was no significant difference in BW between treated and control chickens. Although BW and carcass composition were not different between troglitazone-treated and control chickens, at 48 d of age abdominal fat pad weight and feed intake were significantly decreased in chickens treated with troglitazone compared with controls. These results demonstrate that a single intraperitoneal injection of troglitazone to newly hatched chicks reduces fat deposition in mature broiler chickens.

Follistatin rescues the inhibitory effect of activin A on the differentiation of bovine preadipocyte

We investigated the effect of activin A and follistatin on the differentiation of bovine preadipocytes. Stromal-vascular (SV) cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with activin A during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase (GPDH) activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. Activin A suppressed the induction of all differentiation markers regardless of the duration of treatment. The treatment with activin A also reduced the expression of peroxisome proliferator-activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α mRNAs without affecting the expression of C/EBPβ mRNA. We also observed that follistatin completely rescued the inhibitory effect of activin A on bovine preadipocyte differentiation. Furthermore, the higher doses of follistatin increased GPDH activity even in the presence of activin A compared with the cells treated with neither activin A nor follistatin. Additionally, the SV cells expressed activin A and myostatin mRNAs. These results suggest that activin A inhibits bovine preadiopocyte differentiation via affecting transcriptional cascade upstream of PPARγ and C/EBPα expressions, and that follistatin suppresses the inhibitory effect of activin A on bovine preadipocyte differentiation. Endogenous activin A and/or myostatin possibly inhibit the differentiation of bovine preadipocytes.

Myostatin inhibits differentiation of bovine preadipocyte

We investigated the effect of myostatin on the differentiation of bovine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, the differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin, and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with myostatin during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. The treatment with myostatin throughout the differentiation period severely suppressed the induction of all differentiation markers. The treatment with myostatin in the early phase of differentiation also suppressed the induction of terminal differentiation markers but three-fold higher dose of myostatin was required for the suppression compared with its treatment throughout the differentiation period. Myostatin treatment reduced the expression of peroxisome proliferator-activated receptor (PPAR) γ mRNA and interfered with the induction of CCAAT/enhancer binding protein (C/EBP) α mRNA. We also observed that follistatin stimulates preadipocyte differentiation in the presence of myostatin. These results suggest that myostatin inhibits bovine preadiopocyte differentiation through suppressing PPARγ and C/EBPα mRNA expressions and that follistatin counteracts the suppressive effect of myostatin.

α2- to β3-Adrenoceptor switch in 3T3-L1 preadipocytes and adipocytes: modulation by testosterone, 17β-estradiol, and progesterone

Sex steroid hormones are important factors in the determination of fat distribution and accumulation. The aim of this study was to investigate the effect of testosterone (T), 17beta-estradiol (17betaE), and progesterone (P) on adrenergic receptor (AR) gene expression in 3T3-L1 preadipocytes and adipocytes and their relation to the proliferation and differentiation processes. Our data clearly show that alpha(2A)-AR was the highest AR subtype expressed in preadipocytes, whereas in mature adipocytes was by far beta(3)-AR. In the differentiation process to adipocytes, alpha(2A)-AR expression was decreased to 0.3-fold (P < 0.01), whereas beta(3)-AR was upregulated 578-fold (P < 0.001) compared with preadipocytes. In addition, the expression of alpha(2A)-AR in preadipocytes was increased upon incubation with T, 17betaE, and P, and a stimulation of proliferation was also observed in 17betaE- and P-treated cells. In mature adipocytes, 17betaE and P enhanced both alpha(2A)- and beta(3)-AR gene expression (although the effects on beta(3)-AR mRNA levels could be more relevant, since beta(3)-AR was the most highly expressed), whereas T only increased alpha(2A)-AR mRNA levels. Leptin and adipocyte fatty acid-binding protein mRNA levels were higher after 17betaE and P treatment, possibly indicating a proadipogenic effect of these hormones. In conclusion, this study indicates that AR gene expression is affected by these hormones in both preadipocytes and adipocytes, which could have potential importance when considering the role of ARs in the mechanisms underlying the sex-related differences in adipose tissue regional distribution.