Histochem Cell Biol (2012) 137:205–216 12-well plate) using 10 L/ml Lipofectamine 2,000 as transfection reagent (Invitrogen, Breda, The Netherlands). Cells were harvested 24 h posttransfection. Human study Eleven healthy, lean, male volunteers without a family his- tory of diabetes or any other endocrine disorder participated in this study. The mean age of the subjects was 23.6 § 1.0 years; mean BMI was 22.6 § 0.5 kg/m2. Sub- jects were fed in energy balance for 60 h (50–35–15% of energy as carbohydrates, fat and protein, respectively). After 60 h, a muscle biopsy of the vastus lateralis was taken using the Bergstrom technique (Bergstrom 1975). Muscle samples were immediately frozen in melting iso- pentane and stored at 80°C until assayed. The study proto- col was reviewed and approved by the Medical Ethical Committee of Maastricht University Medical Centre and all subjects gave their written informed consent before partici- pating in the study. ZDF rat study design Twenty-six male, ZDF rats (ZDF/Gmi, fa/fa) were pur- chased from Charles River (Chatillon-sur-Chalaronne, France). The rats arrived at the age of 5 or 11 weeks and were housed in pairs with ad libitum access to tap water and chow. At 6, 12, and 19 weeks of age (n = 8, n = 8, and n = 10, respectively), rats were killed by cervical disloca- tion, and hind leg muscles were rapidly dissected. The tib- ialis anterior muscle was processed for mitochondrial isolation (Lenaers et al. 2010) and soleus and EDL mus- cles were processed for electron microscopy or rapidly frozen in melting isopentane (Fluka, Zwijndrecht, The Netherlands), and stored at ¡80°C. All experiments were approved by the The Animal Care and Use Committee of Maastricht University (approval number 2005-058) and the study complied with the principles of laboratory ani- mal care. Electroporation study design A total of eighteen 8-week-old male Wistar rats were pur- chased from Charles River (Wilmington, Massachusetts, USA). Rats were housed individually on a 12:12 h light– dark cycle (light from 7:00 am to 7:00 pm), at room tem- perature (21–22°C) with ad libitum access to tap water. Rats were fed a high fat diet (45% energy from fat, D01060502, Research Diets, New Brunswick, NJ, USA) for the duration of the 3-week intervention. The Animal Care and Use Committee of Maastricht University approved the experiments (approval number 2010-036) and the study complied with the principles of laboratory animal 207 care. During the experiments, all eVorts were made to mini- mise suVering of the animals. Electroporation Two weeks after the start of the diet, overexpression of Plin5 in either the right or left tibialis anterior (TA) muscle of the rat was accomplished by an in vivo DNA electro- transfer technique to obtain overexpression of mouse perilipin 5 in one leg; the contralateral TA served as a sham-electro- porated internal control. Plin5 was electroporated randomly in the left or the right TA. DNA electroporation was per- formed under isoXurane anaesthesia. TA muscles were transcutaneously injected with either 150 g (2 g/ l) pcDNA3.1-CMV-Plin5 or pcDNA3.1-empty vector in 0.9% sterile NaCl. Within 15 s after the last injection 5 electric pulses were applied by two stainless steel plate electrodes placed at the ventral and dorsal side of the leg. 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, CA, USA) as described previously (Bruce et al. 2007, 2009). Rats were sacriWced 8 days postelectroporation. TA muscles were excised and processed for mitochondrial isolations and electron micros- copy. The remainder of the TA was rapidly frozen in melt- ing isopentane for histology and protein isolations. Mitochondrial isolations After excision, the right and left TA muscles were placed into ice-cold medium containing 100 mM sucrose, 100 Mm KCl, 50 mM Tris–HCl, 1 mM KH2PO4, 0.1 mM EGTA, and 0.2% (wt/vol) FA-free bovine serum albumin (pH 7.4). The TA was immediately processed for mitochondrial iso- lation, essentially according to Tonkonogi and Sahlin (1997). The protein concentration in the mitochondrial pel- let was measured using Xuorescamine (Fluram; Fluka, Zwijndrecht, the Netherlands) with bovine serum albumin as a standard (Udenfriend et al. 1972). Freshly isolated mitochondria were used immediately for mitochondrial res- pirometry. Remaining mitochondria were stored at ¡80°C for further analysis. High-resolution respirometry in isolated mitochondria Mitochondrial oxygen consumption was measured essen- tially according to Hoeks et al. (2008) at 37°C using a two- chamber oxygraph (OROBOROS Instruments, Innsbruck, Austria) and expressed as pmol O2/mg mitochondrial pro- tein per second. Mitochondria were characterised using a carbohydrate-derived substrate (5 mM pyruvate) and a FA-derived substrate (50 M palmitoyl-CoA plus 2 mM carnitine). For this purpose, mitochondria (0.1 mg for pyruvate 123