Fig. 3.

Generation of microvascular ATGL-deficient endothelial cells. (A, B) Endothelial cells isolated from AKO hearts showed increased capacity of triglyceride storage, impaired lipolytic activity, and (C) increased incorporation of glucose-derived carbon into cellular triglycerides. *P < 0.05; AKO vs WT at 20 h; §P < 0.05; WT 20 h vs 24 h; n = 3. (D) mRNA (n = 5–6) and (E) protein expression of eNOS (n = 6) as well as (F) basal and stimulated enzyme activity (n = 6–11) were not affected in ATGL-deficient cells. (G) Protein expression of NOX 2 (n = 3) and NOX4 (n = 6) as well as (H) NADPH oxidase-dependent lucigenin CL (n = 3–4) was similar in WT and AKO cells; *P < 0.05; untreated vs gp91ds-dat-treated WT; #P < 0.05; untreated vs gp91ds-dat-treated AKO. (I) Expression of ubiquitinated proteins was not affected by endothelial ATGL deficiency; n = 5. Expression of NOX2 and NOX4 protein as well as eNOS mRNA is presented relative to WT controls (= 1). eNOS protein is expressed as μg eNOS per mg total protein using purified eNOS as standard. eNOS activity is expressed as % of [³H]l-citrulline formed from incorporated [³H]l-arginine. Data represent mean values ± SEM of n experiments.