sensitivity is related to the upregulation of ADRP levels [30]. These data suggest that hydrolysis of TAG in lipid droplets might indeed be under control of ADRP. Also, OXPAT seems to promote long-chain fatty acid oxidation besides being involved in fatty acid-induced triacylglycerol accumulation [31]. By converting fatty acyl-CoA and DAG into TAG, DGAT1 is considered to modulate the levels of the lipotoxic fatty acid derivate DAG, which is marked as a possible causative fatty acid intermediate in the onset of insulin resistance [17]. Paradoxically, to our knowledge, this study is the first to show that myocellular DAG levels were increased upon muscle specific DGAT1 overexpression. The present study showed an increase in total DAG content upon DGAT1 overexpression in TA muscle compared to the empty-vector control leg, which was most pronounced upon high-fat feeding. In line with our observation, it has recently been shown by others that muscle-specific overex- pression of DGAT1 resulted in a profound increase in IMCL [17]. In their model though, the increase in IMCL was associated with a reduction in muscle DAG content. Recently, the same group showed that muscle-specific transgenic overexpression of DGAT1 in mice enhanced muscle triglyceride synthesis as reflected by the 70% increase in muscle IMCL content; however, no data were reported on muscle DAG content [14]. In interpreting our results it is important to keep in mind that we used a specific muscle- orientated model to unilaterally overexpress DGAT1. Therefore, the effect of DGAT1 overexpression in muscle can be compared to control muscle in the same animal, preventing the influence of systemic differences, which are likely to interfere the comparison between a transgenic animal and a wild-type control. Indeed, the transgenic overexpression of DGAT1 by use of a muscle-specific MCK promotor also resulted in differences in body weight gain [14,17]. The advantage of our model is that such whole-body phenotypic changes are prevented, allowing the conclusion that the results obtained in our study are truly muscle-specific. In agreement with our hypothesis that increasing the lipogenic capacity of skeletal muscle can improve insulin sensitivity, the results of the present study indicate that DGAT1 overexpression did improve skeletal muscle insulin resistance. DGAT1 overex- pression in left TA muscle of high-fat-fed rats was associated with a ,16% higher uptake of bolus-injected 2DG in skeletal muscle compared to the sham-electroporated right leg of the animals. Previous studies confirm the maintenance of insulin sensitivity in skeletal muscle overexpressing DGAT1 by increasing the storage of fatty acids inside the muscle as TAG [14,16,17]. Moreover, these insulin-sensitizing effects observed in skeletal muscle overexpressing DGAT1 recapitulates the findings induced by exercise training, a phenomenon referred to as the athlete’s paradox. Indeed, Liu et al. [17] compared the effects of upregulating myocellular DGAT1 expression with exercise and found that the mechanism underlying insulin sensitivity was similar in both conditions, namely the channelling of reactive fatty acid substrates into storage in the form of TAG. These results suggest that exercise-induced DGAT1 activation may explain the athlete’s paradox. Here we confirm that DGAT1 overexpression may improve insulin sensitivity, but not via a decrease in total DAG content, but rather an elevated DAG content, although we can not exclude the possibility that a subfraction of ‘‘active’’ DAG was decreased in our study. Very interestingly, it was recently shown that endurance trained athletes are also not characterized by low, but rather by increased DAG levels in skeletal muscle [32], consistent with our observation of elevated DAG levels and improved insulin sensitivity. From these and our results, we suggested that increased turnover of DAG and TAG, especially upon high-fat feeding, may be beneficial for muscle insulin DGAT1 Induced Lipid Turnover sensitivity. Accordingly, ATLG protein expression - the main triglyceride lipase in myotubes and in rat skeletal muscle [33] - was significantly elevated upon DGAT1 overexpression in left TA muscle of both HFD- and CHOW-fed rats compared to their control legs respectively. Moreover, the expression of CGI-58, an activating cofactor of ATGL [34], was significantly upregulated upon DGAT1 overexpression in rats receiving the HFD. Also, the lipid droplet coating protein ADRP, which is known to interact with CGI58 to regulate the access of this co-activator to the TAG stores, was significantly upregulated in the leg of HFD-fed rats subjected to DGAT1 overexpression [35,36]. Hence, these data suggest that skeletal muscle triglyceride lipolysis may have been increased upon DGAT1 overexpression upon high-fat feeding. Again, this may resemble the endurance-trained state, as some [37] but not all endurance training studies [38,39] observe an increase in ATGL content, indicating that the utilization of IMCL is increased. In endurance training, increased muscle lipid content is balanced by elevated mitochondrial oxidative capacity [40,41], allowing the oxidation of liberated fatty acids. Interestingly, in our overexpression model we show an increase in total OXPHOS protein content in the DGAT1 overexpressing TA muscle in rats receiving a HFD compared to their empty vector control legs. This pattern of protein expression was also shown for the individual complexes I, II and V of the OXPHOS complexes. In CHOW-fed animals, DGAT1 overexpression did not further enhance the expression level of the OXPHOS complexes. Also, PGC1a protein content, a marker of mitochondrial biogenesis, was significantly upregulated in the DGAT1 overexpressing leg of animals receiving a HFD, whereas no effect of DGAT1 overexpression on PGC1a protein levels were noticed upon CHOW-feeding. We have previously shown – as reviewed in [42] – that UCP3 protein content is elevated under conditions when fatty acid supply exceeds oxidative capacity, such as high-fat feeding and fasting, and UCP3 is reduced when oxidative capacity increases as is the case with endurance training. In line with our hypothesis, UCP3 was upregulated by HF feeding in the sham-electroporated leg, but DGAT1 overexpression reversed the increase in UCP3 protein content to levels observed in CHOW. This matches with the observed increase in the capacity of oxidative substrate degrada- tion upon DGAT1 overexpression. These findings implicate that especially in case of increased delivery of free fatty acids (i.e. high fat feeding) to skeletal muscle, enhanced intramuscular lipid turnover together with the improvement in markers of mitochon- drial function and biogenesis might be responsible for the improved skeletal muscle insulin sensitivity, even despite elevated muscle DAG levels. Next to DAG, other fatty acid intermediates have been suggested to interfere with muscle insulin signalling. Thus, several studies have implicated a role for ceramides in the development of insulin resistance in conditions of increased fatty acid supply to skeletal muscle [43,44,45,46]. Moreover, conditions of increased DGAT1 expression, such as exercise training [40,47] have been linked to reductions in ceramide levels. Therefore, theoretically the insulin-sensitizing effect of DGAT1 overexpression could be due to reductions in ceramide levels. However, in our study, muscle ceramide content was not affected by increasing the intramyocel- lular lipid storage through DGAT1 overexpression, neither in animals on CHOW or on HFD. Thus, it seems that by specifically stimulating triglyceride biosynthesis in skeletal muscle, fatty acids are not directed towards de novo ceramide synthesis. More important, it shows that the positive effects of DGAT1 overexpression in muscle insulin sensitivity in our study could not be attributed to changes in ceramides levels. Alternatively, the PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e14503