DGAT1 Induced Lipid Turnover Figure 4. Three weeks of high-fat feeding compared to rats on CHOW decreased whole-body insulin sensitivity as assessed by hyperinsulinemic eulgycemic clamp. DGAT1 overexpression in TA muscle increased muscle specific insulin sensitivity both in rats on CHOW as HFD. (A) Steady-state whole-body glucose infusion rates in rats on CHOW and HFD for three weeks. (B) 2DG uptake in TA muscle of CHOW fed rats after bolus injection of 2-deoxy-[3H]glucose under the same clamping conditions as in A. (C) 2DG uptake in TA muscle of HFD fed rats after bolus injection of 2-deoxy-[3H]glucose under the same clamping conditions as in A. Data are expressed as mean 6 SEM (n = 10–12). *P,0.05 CHOW-DGAT1 and HFD-control vs. CHOW-control, # P,0.05 HFD-DGAT1 vs. HFD-control. doi:10.1371/journal.pone.0014503.g004 0.9760.31 AU vs. 0.9660.31 AU, p = 0.96, Complex III: 0.8360.29 AU vs. 0.9760.34 AU, p = 0.35, Complex V: 0.9460.30 AU vs. 1.0660.34 AU, p = 0.20 in CHOW-DGAT1 vs. CHOW-control leg, figure 5B). Western blotting of UCP3 revealed a marked ,33% decrease in protein content after overexpression of DGAT1 in HFD-fed rats, which was nearly significant (p = 0.09, HFD-DGAT1 vs. HFD-control leg, figure 5C). UCP3 protein levels were not significantly different between the HFD-DGAT1 overexpressing leg and both the CHOW-DGAT1 and CHOW-control legs (p = 0.88, CHOW- DGAT1 vs. CHOW-control leg, figure 5C). Electron microscopy, OXPAT Oil Red O staining showed a marked increase in the number of intramyocellular lipid droplets upon DGAT1 overexpression, both in CHOW- and HFD-fed animals. However, electron microscopy revealed that DGAT1 overexpression on CHOW resulted in a massive increase in the number of lipid droplets of submicron size, which do not exceed the detection limit of light microscopy (figure 6A, upper panel). Upon high-fat feeding however, DGAT1 overexpression mainly increased the size of the lipid droplets, rather than the number (figure 6A lower panel). In contrast, in CHOW-fed rats, DGAT1 overexpression did not further increase the diameter of the lipid droplets (Figure 6A, upper panel). The increased size of the lipid droplets in the HFD-DGAT overexpressing leg, compared to the empty-vector control leg, corresponded with an increase in the lipid droplet coating protein OXPAT (p = 0.08, HFD-DGAT1 vs. HFD- control leg, and p = 0.14, CHOW-DGAT1 vs. CHOW-control leg, figure 6B). ATGL, CGI58, ADRP The finding that IMCL, DAG and markers of oxidative capacity were increased in DGAT1 overexpressing muscle may suggest that the turnover of IMCL is increased in the insulin- sensitive DGAT1 overexpressing legs. To test this hypothesis we determined the protein expression of ATGL and CGI58 as markers of intramyocellular triglyceride lipolysis. DGAT1 over- expression was associated with a ,35% increase in ATGL content in the HFD-DGAT1 overexpressing leg compared to the control leg of the animals (p = 0.04, HFD-DGAT1 vs. HFD-control leg, figure 7A). DGAT1 overexpression in CHOW-fed rats tended to further enhance the ATGL protein content (p = 0.06, CHOW- DGAT1 vs. CHOW-control leg, figure 7A). Western blotting of CGI58, a cofactor of ATGL showed similar results. DGAT1 overexpression led to a ,25% increase in CGI58 protein in the HFD-DGAT1 overexpressing leg compared to the control leg of the animals (p = 0.009, HFD-DGAT1 vs. HFD-control leg, figure 7B). DGAT1 overexpression in CHOW animals did not affect CGI58 protein expression (p = 0.16, CHOW-DGAT1 vs. CHOW-control leg, figure 7B). Accordingly, ADRP, a lipid droplet coating protein suggested to be involved in the regulation of lipolysis, was ,25% increased in the HFD-DGAT1 overex- pressing leg compared to the control leg of the animals (p = 0.05, HFD-DGAT1 vs. HFD-control leg, figure 7C). DGAT1 overexpression in CHOW animals had no effect on ADRP PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e14503