DGAT1 Induced Lipid Turnover Figure 1. Electroporation of DGAT1 in TA muscle of rats on CHOW and HFD is associated with an increased partitioning of fatty acids towards IMCL. IMCL levels after three weeks of dietary intervention, area fraction reflects the percentage of the total measured cell surface covered by lipid droplets. Data are expressed as mean 6 SEM (n = 10–12). *P,0.05 CHOW-DGAT1 vs. CHOW-control, # P,0.05 HFD-DGAT1 vs. HFD-control. doi:10.1371/journal.pone.0014503.g001 CHOW-fed animals, three weeks of high-fat feeding did not increase ceramide content in TA muscle. Furthermore, also DGAT1 overpression did not affect ceramide levels both in CHOW-DGAT1 overexpressing legs and in HFD-DGAT1 overexpressing legs compared to their controls, respectively. Insulin sensitivity One week after electroporation of DGAT1 whole-body and tissue insulin sensitivity were assessed in vivo by hyperinsulinemic- euglycemic clamps in combination with radioisotope-labelled Figure 2. Three weeks of high-fat feeding is associated with an increased TA DAG content compared to rats on CHOW. DGAT1 overexpression leads to an increase in TA DAG content in rats on CHOW and tended to increase DAG content in rats on HFD. Data are expressed as mean 6 SEM (n = 6). *P,0.05 CHOW-DGAT1 and HFD-control vs. CHOW-control. doi:10.1371/journal.pone.0014503.g002 Figure 3. Three weeks of high-fat feeding is not associated with an increased TA ceramide content compared to rats on CHOW. DGAT1 overexpression does not result in an increase in TA ceramide content in rats on CHOW or HFD. Data are expressed as mean 6 SEM (n = 10–12). doi:10.1371/journal.pone.0014503.g003 infusion of 2-deoxy-[3H]glucose (2DG). Compared to CHOW- fed animals, three weeks of high-fat feeding induced whole body insulin resistance as reflected by a significantly lower glucose infusion rate (30.461.2 mg/min/kg vs. 35.261.3 mg/kg/min in HFD- vs. CHOW-fed animals, p = 0.02, figure 4A). In CHOW fed rats, 2DG uptake was significantly elevated in DGAT1 overexpressing TA muscle compared to their empty vector control legs (p = 0.036, figure 4B). Also upon high-fat feeding, DGAT1 overexpression significantly increased 2DG uptake in TA muscle compared to the empty vector control legs of these rats (p = 0.05, figure 4C). These results were not confounded by altered basal GLUT4 expression, as this was not affected by DGAT1 overexpression (data not shown). Markers of mitochondrial function and biogenesis (OXPHOS, PGC1a, UCP3) To test if the increase in lipid content in DGAT1 overexpressing muscle was accompanied by a compensatory increase in mitochondrial capacity, we determined protein levels of major mitochondrial markers. DGAT1 overexpression was associated with a ,32% increase in PGC1a in the HFD-DGAT1 overexpressing leg compared to the control leg of the animals (p = 0.04, figure 5A), suggesting an increased mitochondrial biogenesis. High fat feeding did not affect the PGC1a content in TA muscle. DGAT1 overexpression in CHOW animals had no significant effect on PGC1a expression (p = 0.64, figure 5A). Western blotting of the OXPHOS complexes revealed that DGAT1 overexpression, in rats fed a HFD, led to an increase in protein levels of the individual complexes I, III and V, suggesting an increased mitochondrial density (Complex I: 1.260.4 AU vs. 0.8660.26 AU, p = 0.007, Complex II: 0.9760.31 AU vs. 0.9860.31 AU, p = 0.89 Complex III: 1.060.32 AU vs. 0.7760.23 AU, p = 0.0023, Complex V: 1.060.30 AU vs. 0.8960.26 AU, p = 0.014 in HFD-DGAT1 vs. HFD-control leg, figure 5B). DGAT1 overexpression did not enhance mitochon- drial density in rats receiving CHOW represented as differences in protein expression of the individual complexes (Complex I: 0.9560.39 AU vs. 0.9260.37 AU, p = 0.72, Complex II: PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e14503