Paradoxical Increase in TAG and DAG Content Parallel the Insulin Sensitizing Effect of Unilateral DGAT1 Overexpression in Rat Skeletal Muscle Silvie Timmers1,2., Johan de Vogel-van den Bosch1,2., Matthijs K. C. Hesselink3, Denis van Beurden 2 , Gert Schaart3, Maria Joao Ferraz4, Mario Losen 5 , Pilar Martinez-Martinez5, Marc H. De Baets5, Johannes M. F. G. Aerts4, Patrick Schrauwen1,2* 1 Top Institute Food and Nutrition (TIFN), Wageningen, The Netherlands, 2 Department of Human Biology, Maastricht University, Maastricht, The Netherlands, 3 Human Movement Sciences, School for Nutrition, Toxicology and Metabolism (NUTRIM), Maastricht University, Maastricht, The Netherlands, 4 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 5 Department of Neuroscience, School of Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands Abstract Background: The involvement of muscle triacylglycerol (TAG) storage in the onset of insulin resistance is questioned and the attention has shifted towards inhibition of insulin signalling by the lipid intermediate diacylglycerol (DAG). The enzyme 1,2-acylCoA:diacylglyceroltransferase-1 (DGAT1) esterifies a fatty acyl-CoA on DAG to form TAG. Therefore, the aim of the present study was to investigate if unilateral overexpression of DGAT1 in adult rat Tibialis anterior (TA) muscle will increase conversion of the lipid intermediate DAG into TAG, thereby improving muscle insulin sensitivity. Methodology/Principal Findings: The DGAT1 gene construct was injected in the left TA muscle of male rats on chow or high-fat (45% kcal) diet for three weeks, followed by application of one 800 V/cm and four 80 V/cm pulses, using the contralateral leg as sham-electroporated control. Seven days after electroporation, muscle specific insulin sensitivity was assessed with a hyperinsulinemic euglycemic clamp using 2-deoxy-[3H]glucose. Here, we provide evidence that unilateral overexpression of DGAT1 in TA muscle of male rats is associated with an increased rather than decreased DAG content. Strikingly, this increase in DAG content was accompanied by improved muscle insulin sensitivity. Interestingly, markers of muscle lipolysis and mitochondrial function were also increased in DGAT1 overexpressing muscle. Conclusions/Significance: We conclude that unilateral DGAT1 overexpression can rescue insulin sensitivity, possibly by increasing DAG and TAG turnover in skeletal muscle. In case of a proper balance between the supply and oxidation of fatty acids in skeletal muscle, the lipid intermediate DAG may not exert harmful effects on insulin signalling. Citation: Timmers S, de Vogel-van den Bosch J, Hesselink MKC, van Beurden D, Schaart G, et al. (2011) Paradoxical Increase in TAG and DAG Content Parallel the Insulin Sensitizing Effect of Unilateral DGAT1 Overexpression in Rat Skeletal Muscle. PLoS ONE 6(1): e14503. doi:10.1371/journal.pone.0014503 Editor: Gian Paolo Fadini, University of Padova, Italy Received July 23, 2010; Accepted December 15, 2010; Published January 14, 2011 Copyright: 2011 Timmers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by Top Institute Food and Nutrition. TI Food and Nutrition was formerly known as WCFS. Partners are major Dutch food companies and research organizations. A VICI (grant 918.96.618) and a VIDI (grant 917.66.359) for innovative research from the Netherlands Organization for Scientific Research (NWO) supports the work of P. Schrauwen and M. Hesselink, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This study was funded by Top Institute Food and Nutrition. TI Food and Nutrition was formerly known as WCFS. Partners are major Dutch food companies and research organizations. A VICI (grant 918.96.618) and a VIDI (grant 917.66.359) for innovative research from the Netherlands Organization for Scientific Research (NWO) supports the work of P. Schrauwen and M. Hesselink, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Therefore, this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: p.schrauwen@hb.unimaas.nl . These authors contributed equally to this work. Introduction Obesity is characterized by excessive fatty acid mobilization, giving rise to many obesity-related metabolic complications including insulin resistance [1,2]. Indeed, a high rate of fatty acid availability and subsequent uptake by skeletal muscle can augment intramuscular lipid deposition [1,2], and there is a strong correlation between the intramyocellular lipid (IMCL) concentra- tion and the severity of insulin resistance, both in humans and rodents [3,4,5,6]. However, evidence from the last decade supports the hypothesis that IMCL accumulation does not directly affect insulin action [7,8,9,10], rather other fatty acid intermedi- ates, such as DAG, are proposed to impede insulin signalling [11,12,13]. In fact, endurance trained athletes are considered among the most insulin-sensitive people, despite having high concentrations of IMCL; a finding that is referred to as ‘‘the athlete’s paradox’’ [7,8,9,10]. Therefore, IMCL is considered a reservoir for fatty acids that can be used for substrate delivery, such as during exercise, and if IMCL accumulation is accompa- nied by high fat oxidative capacity, detrimental effects of IMCL PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e14503