In the current study, we examined the subcellular distribution of lipids in red and white muscles of lean and insulin-resistant animals. Our data demonstrate that: (1) lipid droplets are more prevalent in the intermyofibrillar region in lean and obese animals; (2) insulin-resistant white muscles are devoid of subsarcolemmal lipid droplets and therefore (3) subsarcolemmal lipids are not required for insulin resistance; and (4) within the intermyofibrillar region, lipids are trafficked away from mitochondria in obese animals, a process that (5) cannot be explained by increased abundance of triacylglycerol esterification enzymes. This diversion of lipids away from intermyofibrillar mitochondria probably contributes to lipid accumulation in this subcellular compartment in insulin-resistant muscle.
Total cellular lipids and insulin resistance
Skeletal muscle is key for glucose disposal and is known to take up ~80% of a glucose load . An increase in the intramuscular content of selected lipids has been associated with skeletal muscle insulin resistance [28–30]. Our findings here support this notion, as total intramuscular triacylglycerol and diacylglycerol contents were increased in the red and white muscles examined in the obese animals. In addition, red, but not white, skeletal muscle from obese animals displayed increased ceramide content. It is currently believed that the accumulation of bioactive lipids, such as diacylglycerol  and ceramides , contributes to the development of insulin resistance [31, 32]. In the current study, the white muscle of obese animals was insulin-resistant, which coincided with increased diacylglycerol, but unaltered ceramide content. Combined, these data suggest that increased ceramide content is not necessarily a pre-requisite for insulin resistance, supporting instead the belief that diacylglycerol content may have a direct role in this condition.
While it was originally thought that the accumulation of triacylglycerol was a direct cause of insulin resistance , the view now is that triacylglycerol is a neutral and beneficial lipid storage depot, and that the accumulation of triacylglycerol in insulin resistance skeletal muscle is likely to be a marker of altered lipid homeostasis, rather than a cause of insulin resistance. This view is supported by a number of observations, including the observation that overabundance of DGAT1 increases intramuscular triacylglycerol content, while protecting against diet-induced insulin resistance , as well as the finding that exercise-trained athletes display high intramuscular triacylglycerol content, but are highly insulin-sensitive [34, 35]. However, a recent report by Nielsen and colleagues  suggests that triacylglycerol lipid droplets can negatively affect insulin signalling if accumulated in the subsarcolemmal subcellular region, thus renewing interest in triacylglycerol accumulation as a potential cause of insulin resistance.
Subcellular distribution of lipids
Regardless of fibre composition or genotype, we found that lipid droplets were larger and more prevalent in the intermyofibrillar region. Moreover, our calculation of the relative abundance of subsarcolemmal lipids, based on assumptions of average fibre diameter and shape in red muscle, suggested that subsarcolemmal lipids only represented approximately 6% to 10% of the overall lipids in the muscle. Lipid droplet content in the intermyofibrillar region increased to a greater absolute magnitude in the muscle fibre of obese Zucker rats. However, given that subsarcolemmal lipids are virtually absent in lean animals, subsarcolemmal lipids increased to a greater relative amount with obesity. Combined, these data imply that lipid droplet accumulation in various subcellular regions in obese animals reflects alterations in total cellular lipid homeostasis. While it is possible that estimates based on fibre diameter and shape underestimated the relative content of subsarcolemmal lipids in the cell, lipid droplets were not detectable in the subsarcolemmal region of white muscle even when this was insulin-resistant, suggesting that lipid droplets in the subsarcolemmal region are not required for the development of insulin resistance. This is in contrast to a recent study by Nielsen et al. , who found that increased lipid droplet volume was exclusively limited to the subsarcolemmal compartment in patients with type 2 diabetes. The discrepancy between our study and that of Nielsen and colleagues may be related to species and/or condition. However, it should be noted that subsarcolemmal lipids also did not correlate with insulin sensitivity following aerobic training in the same report by Nielsen et al. , with five participants who displayed pronounced reductions in subsarcolemmal lipids having only modest changes in insulin sensitivity and one participant who had increased subsarcolemmal lipids with modestly improved insulin sensitivity . Therefore, based on our observations, it appears that lipid accumulation in the subsarcolemmal region is not a direct cause and/or requirement for the development of insulin resistance in muscle of obese rodents. Presumably, and similarly to biochemical determinations of intramuscular lipids, lipid droplet accumulation in the subsarcolemmal region probably reflects alterations in cellular lipid homeostasis, rather than being a direct cause of insulin resistance.
Mitochondrial subcellular alterations and fatty acid metabolism
A decrease in mitochondrial fatty acid oxidation has been proposed to contribute to lipid accumulation [8, 36]. Therefore regional differences in mitochondrial content and/or function may help to explain subcellular differences in lipid droplets. In the current study, muscle mDNA was increased in muscles of obese animals, which probably accounted for the increases in subsarcolemmal and intermyofibrillar mitochondrial density. Previously, moreover, we had shown that fatty acid oxidation in isolated subsarcolemmal and intermyofibrillar mitochondria remained unaltered (intermyofibrillar) or was increased (subsarcolemmal)  in the same animal model. The increase in subsarcolemmal mitochondrial density and fatty acid oxidation may represent compensatory mechanisms designed to prevent lipid droplet accumulation in the subsarcolemmal region. Therefore, reduced mitochondrial fatty acid oxidation apparently cannot account for the observed intramuscular lipid accumulation, a notion that is consistent with contemporary mitochondrial literature [37–41].
However, this literature is based on in vitro measurements that are optimised to determine the capacity of mitochondrial function and therefore may not represent the in vivo oxidative flux. In support of this, in the obese Zucker rat, despite increased mitochondrial density as shown here and elsewhere [9, 38], and despite increased in vitro mitochondrial function , we (present study) and others  found that rates of total muscle fatty acid oxidation were decreased, while, as found here, rates of triacylglycerol esterification were increased. It should be noted that the notion that fatty acid oxidation is reduced in obese Zucker rats [15, 42, 43] is controversial. Nevertheless, the discrepancy between mitochondrial oxidative capacity and in vivo oxidative flux is echoed in the Zucker diabetic fatty rat, a model of type 2 diabetes where mitochondrial content and isolated mitochondrial function are increased , yet in vivo oxidative capacity, as assessed by 31P magnetic resonance spectroscopy, is unaltered . Several mechanisms may explain these data, including the observation that levels of malonyl-CoA, a biological inhibitor of carnitine palmitoyl-transferase I and mitochondrial fatty acid oxidation, are increased in obesity and in individuals with type 2 diabetes . Combined, these data suggest the possibility that fewer fatty acids are transported into mitochondria in obese insulin-resistant muscle, a phenomenon that could contribute to lipid accumulation.
Intramyocellular fatty acid trafficking
To ascertain whether lipids are trafficked away from mitochondria in the obese animal, we incubated muscle in the presence of [3H]palmitate and subsequently isolated subsarcolemmal and intermyofibrillar mitochondria. The tibialis anterior muscle is not appropriate for this in vitro analysis, and so we used the soleus muscle, another oxidative muscle that also displays insulin resistance and lipid accumulation in these animals , for these studies. The results with this approach suggest that fewer lipids are transported into intermyofibrillar mitochondria in the in vivo situation. This finding is somewhat surprising, given the now well-recognised observation that rates of fatty acid transport in the obese Zucker rat are substantially increased [9, 15, 41]. Nevertheless, it appears that less [3H]palmitate was transported into intermyofibrillar mitochondria, despite a greater delivery of fatty acids into the muscle’s interior, suggesting that lipids were trafficked away from the mitochondria. The observed phenomenon was unlikely to have been caused by decreased transport capacity into mitochondria, as we have previously shown that carnitine palmitoyl-transferase I activity and rates of mitochondrial fatty acid oxidation are not reduced in these mitochondria . Combined, these data suggest that lipids were trafficked away from the intermyofibrillar mitochondria in an unknown manner.
Given the proximity of intermyofibrillar mitochondria to the majority of lipid droplets in the obese animals, we hypothesised that triacylglycerol esterification enzymes would be increased in the intermyofibrillar region, providing a mechanism for the apparent stealing of lipids away from mitochondrial oxidation. However, similarly to previous reports [47, 48], increases in whole-muscle content of several esterification enzymes were not observed. In addition, lipin-1 and DGAT1 were not detectable on intermyofibrillar mitochondria, while GPAT1 content was unaltered with obesity. We cannot rule out the possibility that levels of these enzymes were increased specifically on the endoplasmic reticulum in the intermyofibrillar region. In addition, adipose tissue triacylglycerol lipase protein was unaltered (lean 100 ± 4, obese 89 ± 4 arbitrary units), while hormone-sensitive lipase cellular protein was increased (lean 100 ± 6, obese 138 ± 16 arbitrary units), suggesting that decreased lipolysis cannot account for the observed trafficking of lipids into lipid droplets. Alternatively, the observation that fewer lipids are transferred into intermyofibrillar mitochondria may simply reflect a situation where mitochondria are already oversupplied with lipids . Nevertheless, as far as we can determine, ours is the first attempt to directly show that fatty acid transport into mitochondria is decreased in insulin-resistant muscle, possibly providing a potential mechanism for lipid accumulation in the intermyofibrillar region.
In the current study, we determined the subcellular distribution of lipids in insulin-resistant skeletal muscle. While others have suggested that subsarcolemmal lipids contribute to attenuations in insulin signalling , the current data suggest that subsarcolemmal lipids are not required for the development of insulin resistance, as insulin-resistant white muscles were devoid of lipids in the subsarcolemmal region. Therefore, similarly to increases in total triacylglycerol content, lipid droplet accumulation in the subsarcolemmal region is likely to simply reflect alterations in total cellular lipid homeostasis. In addition, we provide evidence that in insulin-resistant muscle fatty acids are trafficked away from intermyofibrillar mitochondria and probably into lipid droplets in the intermyofibrillar region. While the underlying mechanism for this diversion of fatty acids remains unknown, we have provided evidence that this is not a direct effect of increased triacylglycerol esterification enzyme content on intermyofibrillar mitochondria.