Adapted from methods described previously [28]. Livers (aprox 200 mg) were homogenized for 2 min in ice-cold chloroform-methanol (2:1, vol/ vol). 
TG were extracted during 5-h shaking at room temperature. For phase separation, H2SO4 was added, samples were centrifuged, and the organic bottom layer was collected. The organic solvent was 
dried using a Speed Vac and redissolved in chloroform. TG (Randox Laboratories LTD, UK) content of each sample was measured in duplicate after evaporation of the organic solvent using an enzymatic method.Quantitative reverse transcriptase PCR (qRT-PCR) analysisRNA was extracted using TrizolH reagent (Invitrogen) according to the manufacturer’s instructions and two Tunicamycin micrograms of total RNA were used for each RT reaction and cDNA synthesis was performed using SuperScriptTM First-Strand Synthesis System (Invitrogen) and random primers as previously described [29]. Negative control reactions, containing all reagents except the sample were used to ensure specificity of the PCR amplification. For the analysis of gene expression we used real-time reversetranscription polymerase chain reaction (RT-PCR) analyses performed in a fluorescent temperature cycler (TaqManH; Applied Biosystems; Foster City, CA, USA) following the manufacturer’s instructions [29,30]. Five hundred ng of total RNA were used for each RT reaction. The PCR cycling conditions included an initial denaturation at 50uC for 10 min followed by 40 cycles at 95uC for 15 sec; 60uC for 1 min. The oligonucleotide specific primers and probes were: G6Pase Fw 59-CCA GGT CGT GGC TGG AGT CT-39, Rv 59-TGT AGA TGC CCC GGA TGT G-39, 59-FAMCAG GCA TTG CTG TGG CTG AAA CTT TCA G-TAM-39; and PEPCK1 Fw 59-CCA CAG CTG CTG CAG AAC AC-39, Rv 59-GAA GGG TCG CAT GGC AAA-39, 59-FAM-AGG GCA AGA TCA TCA TGC ACG ACC C-TAM-39. For the analysis of the data, the input value of the gene expression was standardized to the 18S value for each sample of each group and was expressed compared with the average value for the control group.Levels of plasma metabolites and hormonesPlasma glucose was measured by the glucose oxidase method (Glucose and Triglyceride Spinreact, Spain). Plasma nonesterified fatty acids (NEFA) concentrations were determined using a kit from Wako (US); triacylglycerol (TG) and cholesterol were determined using a kit from Randox Laboratories (LTD, UK). Plasma MedChemExpress 79831-76-8 insulin levels were measured by a previously described RIA [27].Data Analysis and StatisticsValues are plotted as the mean 6 SEM for each genotype. Statistical significance was determined by Student’s t -test. A P value less than 0.05 was considered statistically significant.Results Eng+/2 mice fed a standard diet do not show metabolic alterationsAge-matched male WT and Eng+/2 mice of 4 weeks of age were maintained on standard diet for 8 weeks to assess their metabolicEndoglin and Diet-Induced Insulin ResistanceFigure 2. Glucose homeostasis and insulin sensitivity in mice fed a standard diet. Basal glucose levels (A), glucose tolerance test (B), respective area under the curve (C), insulin tolerance test ( of glucose levels represented against t0) (D), and respective area under the curve (E) in 8week male wild type (WT) and endoglin heterozygous (HZ) mice fed a standard diet. n = 6?. *p,0.05. doi:10.1371/journal.pone.0054591.gphenotypes. No body weight differences were found between both genotypes (Figure 1A). 23115181 Consistently, body composition (fat mass and non fat mass) (Figure 1B and 1C) and food intake (Figure.Adapted from methods described previously [28]. Livers (aprox 200 mg) were homogenized for 2 min in ice-cold chloroform-methanol (2:1, vol/ vol). TG were extracted during 5-h shaking at room temperature. For phase separation, H2SO4 was added, samples were centrifuged, and the organic bottom layer was collected. The organic solvent was dried using a Speed Vac and redissolved in chloroform. TG (Randox Laboratories LTD, UK) content of each sample was measured in duplicate after evaporation of the organic solvent using an enzymatic method.Quantitative reverse transcriptase PCR (qRT-PCR) analysisRNA was extracted using TrizolH reagent (Invitrogen) according to the manufacturer’s instructions and two micrograms of total RNA were used for each RT reaction and cDNA synthesis was performed using SuperScriptTM First-Strand Synthesis System (Invitrogen) and random primers as previously described [29]. Negative control reactions, containing all reagents except the sample were used to ensure specificity of the PCR amplification. For the analysis of gene expression we used real-time reversetranscription polymerase chain reaction (RT-PCR) analyses performed in a fluorescent temperature cycler (TaqManH; Applied Biosystems; Foster City, CA, USA) following the manufacturer’s instructions [29,30]. Five hundred ng of total RNA were used for each RT reaction. The PCR cycling conditions included an initial denaturation at 50uC for 10 min followed by 40 cycles at 95uC for 15 sec; 60uC for 1 min. The oligonucleotide specific primers and probes were: G6Pase Fw 59-CCA GGT CGT GGC TGG AGT CT-39, Rv 59-TGT AGA TGC CCC GGA TGT G-39, 59-FAMCAG GCA TTG CTG TGG CTG AAA CTT TCA G-TAM-39; and PEPCK1 Fw 59-CCA CAG CTG CTG CAG AAC AC-39, Rv 59-GAA GGG TCG CAT GGC AAA-39, 59-FAM-AGG GCA AGA TCA TCA TGC ACG ACC C-TAM-39. For the analysis of the data, the input value of the gene expression was standardized to the 18S value for each sample of each group and was expressed compared with the average value for the control group.Levels of plasma metabolites and hormonesPlasma glucose was measured by the glucose oxidase method (Glucose and Triglyceride Spinreact, Spain). Plasma nonesterified fatty acids (NEFA) concentrations were determined using a kit from Wako (US); triacylglycerol (TG) and cholesterol were determined using a kit from Randox Laboratories (LTD, UK). Plasma insulin levels were measured by a previously described RIA [27].Data Analysis and StatisticsValues are plotted as the mean 6 SEM for each genotype. Statistical significance was determined by Student’s t -test. A P value less than 0.05 was considered statistically significant.Results Eng+/2 mice fed a standard diet do not show metabolic alterationsAge-matched male WT and Eng+/2 mice of 4 weeks of age were maintained on standard diet for 8 weeks to assess their metabolicEndoglin and Diet-Induced Insulin ResistanceFigure 2. Glucose homeostasis and insulin sensitivity in mice fed a standard diet. Basal glucose levels (A), glucose tolerance test (B), respective area under the curve (C), insulin tolerance test ( of glucose levels represented against t0) (D), and respective area under the curve (E) in 8week male wild type (WT) and endoglin heterozygous (HZ) mice fed a standard diet. n = 6?. *p,0.05. doi:10.1371/journal.pone.0054591.gphenotypes. No body weight differences were found between both genotypes (Figure 1A). 23115181 Consistently, body composition (fat mass and non fat mass) (Figure 1B and 1C) and food intake (Figure.
