may be limited. This also implies that under conditions of high fatty acid flux, as with high-fat diets and obesity, limiting the accumulation of detrimental fatty acid metabolites such as DAG by improving the partitioning of fatty acids towards storage as IMCL, is likely to blunt lipid related insulin resistance [9]. DGAT1 is a key enzyme that catalyzes the final step in triglyceride synthesis. Overexpressing this enzyme results in increased triglyceride synthesis, increased fatty acid oxidation and preservation of insulin sensitivity despite the increased amount of fatty acids offered to the muscle [14,15]. It has therefore been suggested that elevated DGAT1 activity could be an explanation for the athlete’s paradox. Indeed, Schenk and Horowitz [16] showed that one acute bout of exercise leads to an increase in DGAT1 expression and improved insulin sensitivity. Also in rodents, exercise increases myocytic DGAT1 activity, and transgenic overexpression of DGAT1 in skeletal muscle in mice is sufficient to replicate the exercise paradox characterized by increased muscle lipid content coupled with increased muscle insulin sensitivity [14,17]. However, life-long muscle overexpres- sion of DGAT1 may not resemble the situation that is observed with endurance training, and may in fact lead to phenotypic compensatory changes, such as a lower body weight that was observed in mice overexpressing DGAT1 [14]. Therefore, we here aimed to examine the impact of acute unilateral, muscle-specific overexpression of DGAT1 in adult rats on skeletal muscle lipid metabolism. To this end, we transiently overexpressed DGAT1 by using gene electroporation in left TA muscle of adult rats while using the right leg of the same animal as a sham-electroporated control [18]. We hypothesized that overexpression of DGAT1 in TA muscle of adult male Wistar rats would lead to an increase in IMCL content and a decrease in DAG content, leading to improved insulin sensitivity. Strikingly, we observed that DGAT1 overexpression was paralleled by an increased rather than decreased DAG content yet improved insulin sensitivity. Interest- ingly, key proteins in myocellular lipolysis and oxidative substrate metabolism were increased upon DGAT1 overexpression. Methods Ethics statement The experimental protocol was approved by the Institutional Animal Care and Use Committee of Maastricht University and complied with the principles of laboratory animal care (approval number 2006-024). During the experiments all efforts were made to minimize suffering of the animals. Animals and diets Twenty-two, eight-week old male Wistar rats were obtained from Charles River Laboratories and were housed individually in an environmentally controlled laboratory (temperature 2261uC and relative humidity 5562%). Rats were randomly divided in two groups and were allowed ad libitum access to food and tap water. One group (n = 10) received standard CHOW (10 energy % as fat) (Ssniff, Germany) and the other group (n = 12) received a high-fat diet (HFD) that contained 45 energy % of fat derived from lard (Research Diets, D01060502, Wijk bij Duurstede, The Netherlands). Diets were provided for three weeks. Cannulation Two weeks before the end of the experiment the rats were sedated under isoflurane anaesthesia and permanent indwelling catheters were placed in the right jugular vein (for the sampling of blood) and the left carotid artery (for the infusion of insulin and glucose) under aseptic conditions. Both catheters were tunnelled DGAT1 Induced Lipid Turnover subcutaneously and exteriorized at the top of the head, where they were attached. To maintain patency, the catheters were filled with a mixture of 85% glycerol (Merck, Darmstadt, Germany) and 500 U/ml heparin (Leo Pharma BV, Breda, The Netherlands). All rats regained pre-surgery body mass within 3 days. Electroporation One-week before the hyperinsulinemic euglycemic clamp overexpression of mouse DGAT1 in the left Tibialis anterior (TA) muscle of the rat was obtained, with the right TA as sham- electroporated internal control. DNA electroporation was done under isoflurane anaesthesia. Left TA muscles were transcuta- neously injected with 150 mg (2 mg/ml) pcDNA sport DGAT1- construct (pCMV-sport6, Invitrogen, Breda, The Netherlands) in 0.9% sterile NaCl. Right TA muscles were injected with 150 mg (2 mg/ml) empty vector. Within 15 seconds after the last injection 5 electric pulses were applied by two stainless steel plate electrodes placed at each side of the leg to obtain transient permeabilization of the cell membrane, facilitating the passage of the DNA. One high voltage pulse of 800 V/cm and four low voltage pulses of 80 V/cm at 1 Hz were generated by an ECM 830 electroporator (BTX, San Diego) as described before [19,20,21]. Shaving the leg and the application of conductive gel minimized impedance. Hyperinsulinemic euglycemic clamp Hyperinsulinemic euglycemic clamping of conscious rats was performed after a 6 h fast. A primed continuous infusion of insulin (Actrapid HM; Novo Nordisk, Copenhagen, Denmark) was administered at a rate of 13 mU/kg/min for 120 min. Blood glucose values were monitored at 10-min intervals throughout the clamp. The glucose infusion rate (GIR) was adjusted to maintain blood glucose concentration within the range of 4.5–5.5 mmol/L. Insulin stimulated glucose disposal in vivo within the individual TA muscles was studied according to a previously described method [22]. Upon completion of the clamp, the rats were killed with an overdose of sodium pentobarbital and TA muscles were quickly dissected and frozen in liquid nitrogen-cooled isopentane and stored at 280uC until further analyses. Deoxy glucose measurement Tibialis anterior muscles were cut in three parts and the middle part was ground with a N2-cooled mortar and pestle. After homogenisa- tion, the samples were heated to 99uC for 30 min, cooled and centrifuged for 5 min at 13,000 rpm. 2-deoxy-[1-3H]-D-glucose- phosphate was separated from 2DG in the supernatant by binding to a Dowex column (Dowex 1*8-100, ion exchange resin, Sigma). Bound 2-deoxy-[1-3H]-D-glucose-phosphate was eluted from the column by using the eluate buffer (formic acid, ammoniumacetate in mQ, pH 4.9). 14 ml LCD scintillation fluid was added to 1 ml eluate, and this cocktail was counted in the scintillation counter for 5 min. Histological analysis of intramyocellular lipids (IMCL) and quantification of DGAT1 Cryosections (5 mm) from the midbelly region of the TA muscle were stained for neutral lipids with Oil Red O and DGAT1 overexpresssion was quantified as previously described [15]. Electron microscopy In a subset of 4 CHOW and 4 high-fat fed mice both the DGAT1 overexpressing muscle as well as the empty-vector control condition were processed for routine transmission electron microscopy. In brief, small blocks of muscle tissue (2*2*2 mm) from both legs were fixed by glutaraldehyde and post-fixed by PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e14503