Redundant roles of the phosphatidate phosphatase family in triacylglycerol synthesis in human adipocytes.

AIMS/HYPOTHESIS: In mammals, the evolutionary conserved family of Mg(2+)-dependent phosphatidate phosphatases (PAP1), involved in phospholipid and triacylglycerol synthesis, consists of lipin-1, lipin-2 and lipin-3. While mutations in the murine Lpin1 gene cause lipodystrophy and its knockdown in mouse 3T3-L1 cells impairs adipogenesis, deleterious mutations of human LPIN1 do not affect adipose tissue distribution. However, reduced LPIN1 and PAP1 activity has been described in participants with type 2 diabetes. We aimed to characterise the roles of all lipin family members in human adipose tissue and adipogenesis. METHODS: The expression of the lipin family was analysed in adipose tissue in a cross-sectional study. Moreover, the effects of lipin small interfering RNA (siRNA)-mediated depletion on in vitro human adipogenesis were assessed. RESULTS: Adipose tissue gene expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell line, alters expression levels of adipogenic transcription factors and lipid biosynthesis genes in early stages of differentiation. Lipin-1 knockdown alone causes a 95% depletion of PAP1 activity. Despite the reduced PAP1 activity and alterations in early adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids. Even combinatorial knockdown of lipins shows mild effects on triacylglycerol accumulation in mature adipocytes. CONCLUSIONS/INTERPRETATION: Overall, our data support the hypothesis of alternative pathways for triacylglycerol synthesis in human adipocytes under conditions of repressed lipin expression. We propose that induction of alternative lipid phosphate phosphatases, along with the inhibition of lipid hydrolysis, contributes to the maintenance of triacylglycerol content to near normal levels.This study was supported by research grants from the ‘Instituto de Salud Carlos III’ (ISCIII, Spanish Ministry of Economy and Competitiveness) (PI10/00967 and CP11/0 0021 to MM); the R. Barri Private Foundation (PV12142S to MM); the Medical Research Council (G0701446 to SS); and National Institutes of Health Grant (GM028140 to GMC). CIBER de Diabetes y Enfermedades Metabólicas asociadas (CB07708/0012) is an initiative of the ISCIII. MM acknowledges support from the ‘Miguel Servet’ tenure track programme (CP11/00021), from the Fondo de Investigación Sanitaria (FIS) co-financed by the European Regional Development Fund (ERDF), and supported by a Salvador de Madariaga Mobility fellowship from the Spanish Ministry of Education (PR2011-0584). AT is the recipient of a FI-DGR fellowship (9015-97318/2012) from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR)This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Springer

Transcriptional regulation of the heavy subunit chain of γ-glutamylcysteine synthetase by ionizing radiation

Since glutathione (GSH) protects against oxidative stress, we determined the regulation of cellular GSH by ionizing radiation in human hepatoblastoma cells, HepG2. The levels of GSH increased in irradiated HepG2 due to a greater γ-glutamylcysteine synthetase (γ-GCS) activity, which was paralleled by γ-GCS heavy subunit chain (γ-GCS-HS) mRNA levels. Transcription of deletion constructs of the γ-GCS-HS promoter cloned in a reporter vector was associated with activator protein-1 (AP-1), consistent with the DNA binding of AP-1 in nuclear extracts of irradiated HepG2. Hence, the transcriptional regulation of γ-GCS by ionizing radiation emerges as an adaptive mechanism, which may be of significance to control the consequences of the oxidative stress induced by radiation.The present work was supported by National Institute of Alcohol Abuse and Alcoholism AA09526, Dirección General Polı́tica Cientı́fica y Técnica, PM 95-0185, Fondo Investigaciones Sanitarias, FISS 94-0046/01, Plan Nacional de I+D grant SAF 97-0087-C01 and Europharma. A.M. is a Fellow from the FISS and A.C. from EuropharmaPeer Reviewe

Proposed DRLs for Mammography in Switzerland.


The aim of this study is to propose Diagnostic Reference Levels (DRLs) values for mammography in Switzerland.
Methods:
For the data collection, a survey was conducted among a sufficient number of centres, including 5 University hospitals, several cantonal hospitals, and large private clinics, covering all linguistic regions of Switzerland to be representative of the clinical practice. The data gathered contained the mean glandular dose (MGD), the compressed breast thickness (CBT), the mammography model and the examination parameters for each acquisition. The data collected was sorted into the following categories: 2D or digital breast tomosynthesis (DBT) examination, craniocaudal (CC) or mediolateral oblique (MLO) projection, and 8 categories of compressed breast thickness (CBT) ranging from 20mm to 100mm in 10mm intervals.
Results:
24762 acquisitions acquired in 31 centres on 36 mammography units from 6 different manufacturers were collected. The analysis showed that the data reflects the practice in Switzerland.The results revealed that the MGD is larger for DBT than for 2D acquisitions for the same CBT. From 20-30mm to 90-100mm of CBT, the 75th percentile of the MGD values obtained increased from 0.81mGy to 2.55mGy for 2D CC acquisitions, from 0.83mGy to 2.96mGy for 2D MLO acquisitions, from 1.22mGy to 3.66mGy for DBT CC acquisitions and from 1.33mGy to 4.04mGy for DBT MLO acquisitions.
Conclusion:
The results of the survey allow us to propose Swiss DRLs for mammography according to the examination type (2D/DBT), projection (CC/MLO) and CBT. The proposed values are very satisfactory in comparison with other studies.&#xD

Tumor necrosis factor increases hepatocellular glutathione by transcriptional regulation of the heavy subunit chain of γ-glutamylcysteine synthetase

Tumor necrosis factor (TNF) is an inflammatory cytokine that causes cell injury by generation of oxidative stress. Since glutathione (GSH) is a key cellular antioxidant that detoxifies reactive oxygen species, the purpose of our work was to examine the regulation of cellular GSH, the expression of heavy subunit chain of γ-glutamylcysteine synthetase (γ-GCS-HS), and control of intracellular generation of reactive oxygen species in cultured rat hepatocytes treated with TNF. Exposure of cells to TNF (10,000 units/ml) resulted in depletion of cellular GSH levels (50-70%) and overproduction of hydrogen peroxide (2-3-fold) and lipid peroxidation. However, cells treated with lower doses of TNF (250-500 units/ml) exhibited increased levels of GSH (60-80% over control). TNF treatment increased (70-100%) the levels of γ- GCS-HS mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthesis. Furthermore, intact nuclei isolated from hepatocytes treated with TNF transcribed the γ-GCS-HS gene to a greater extent than control cells, indicating that TNF regulates γ-GCS-HS at the transcriptional level. The capacity to synthesize GSH de novo determined in cell-free extracts incubated with GSH precursors was greater (50-70%) in hepatocytes that were treated with TNF; however, the activity of GSH synthetase remained unaltered by TNF treatment indicating that TNF selectively increased the activity of γGCS. Despite activation of nuclear factor-≃B (NF-≃B) by TNF, this transcription factor was not required for TNF-induced transcription of γ- GCS-HS as revealed by deletion constructs of the γ-GCS-HS promoter subcloned in a chloramphenicol acetyltransferase reporter vector and transfected into HepG2 cells. In contrast, a construct containing AP-1 like/metal response regulatory elements increased chloramphenicol acetyltransferase activity upon exposure to TNF. Thus, TNF increases hepatocellular GSH levels by transcriptional regulation of γ-GCS-HS gene, probably through AP-1/metal response element-like binding site(s) in its promoter, which may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines.This work was supported in part by the National Institute on Alcohol Abuse and Alcoholism Grant AA09526, Dirección General Polı́tica Cientı́fica y Técnica Grant PM 95-0185, Fondo Investigaciones Sanitarias Grant FISS 94-0046/01, Plan Nacional de I+D Grant SAF 97-0087-C01, and EuropharmaPeer Reviewe

FABP4 dynamics in obesity: discrepancies in adipose tissue and liver expression regarding circulating plasma levels.

BACKGROUND: FABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile. OBJECTIVE: In patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed. METHODS: The expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot. RESULTS: In obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group. CONCLUSION: The inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity

<i>FABP4</i> expression in adipose and liver tissues from mice.

<p>Fasting mRNA expression of <i>FABP4</i> visceral adipose tissue in ob/ob (n = 16) (grey bar) and WT (n = 16) (black bar) mouse. Adipose tissue expression level of the gene was normalized using <i>β-actin</i>. The results are given as the mean ± SD. ^*indicates significant differences between the means of the two groups (<i>P</i><0.05). (B) Fasting mRNA expression levels of hepatic <i>FABP1</i> and <i>FABP4</i> in ob/ob (n = 16) (grey bar) and WT (n = 16) (black bar) mouse. Hepatic tissue expression levels for each gene were normalized using <i>GADPH</i>. The results are given as the mean±SD. ^*indicates significant differences between the means of the two groups (<i>P</i><0.05).</p

Anthropometric and biochemical characteristics and mRNA expression of <i>FABP1</i>, <i>FABP4</i> and <i>FABP5</i> in a subgroup of morbidly obese and morbidly obese IR patients with liver biopsies.

<p>Data are mean ± SD;</p>*<p>indicates significant differences between the means of the two groups.</p><p>(<i>P</i><0.05). BMI, body mass index; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; HOMA-IR, Homeostasis model assessment of insulin resistance; SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure.</p

<i>FABP4</i> expression is higher in isolated adipocytes (ADI) than in the stromovascular fraction (SVF).

<p>(A) <i>FABP4</i> mRNA expression levels in ADI and SVF from n = 15 subjects with paired adipose biopsies (ADI <i>vs.</i> SVF, *<i>P</i> = 0.001). Data are expressed as median and IQR. (B) FABP4 protein levels in total VAT, and in ADI and SVF fractions, from n = 4 subjects. Data are expressed as mean and SD. (ADI <i>vs.</i> SVF, ^¶<i>P</i><0.05) (C) Representative blot of FABP4 protein in total VAT and SAT, and in ADI and SVF fractions. (D) Immunofluorescence detection of FABP4 (red) and CD68 (green) in VAT. The counterstaining of nuclei (DAPI) is shown in blue. Images are representative of VAT collected from five subjects. (E) Immnunohistochemical detection of FABP4 (brown, right panel) and CD68 (macrophage-specific antigen, middle panel) and negative control (left panel) in VAT. Images are representative of VAT sections collected from five subjects. A: Adipocyte; M: Macrophage. Arrow-heads indicate the specific signal.</p