Intramyocellular lipid storage and insulin resistance
Insulin resistance is associated with high levels of intramyocellular lipid (IMCL), which is predominantly dispersed into small lipid droplets throughout the muscle cells (Figure 1). In insulin resistant white adipose tissue, insulin mediated suppression of lipolysis is compromised; hence circulating levels of fatty acids are high. Sequestering of fatty acids from the circulation in non-adipose tissue (like skeletal muscle) is referred to as ectopic lipid storage. The capacity to oxidize fatty acids is compromised in most insulin resistant subjects. Thus, spillover of fatty acids from the white adipose tissue along with a reduced fat oxidative capacity may promote fat storage in muscle. Multiple studies have reported a negative correlation between IMCL content and insulin sensitivity, suggesting that a high IMCL content in skeletal muscle is detrimental for insulin sensitivity. Most of the IMCL is stored as inert triacylglycerol. However, in synthesis and degradation of IMCL transient elevations in bio-active lipid intermediates, like diacylglycerol and ceramide, may occur, which potentially impede insulin signaling and hence compromise myocellular glucose uptake. This section shortly describes how lipids are stored in skeletal muscle and how these lipids are degraded for oxidation. Subsequently possible mechanisms how increased IMCL content impedes insulin signaling will be described.
Storage and degradation of lipids in skeletal muscle
Figure 1. Intramyocellular lipids in skeletal muscle of a type 2 diabetes patient. IMCL is dispersed in small lipid droplets (stained in green). Cell membranes are shown in blue. (Click to enlarge)Circulating fatty acids are taken up by the skeletal muscle cells in various ways. Once in the muscle, fatty acids can be incorporated into triacylglycerol (TAG), which is the main component of IMCL, or can be oxidized by the mitochondria. In TAG synthesis, three fatty acids bind to glycerol. The first step in the TAG formation is an ester bond between one fatty acid and glycerol, resulting in monoacylglycerol (MAG). Subsequently another fatty acid is bound to MAG facilitated by MGAT forming diacylglycerol (DAG). The final step for TAG formation is the addition of a third fatty acid to DAG by DGAT.
Lipolysis of TAG occurs when fatty acids are required for oxidation. In this process fatty acids are subsequently released from glycerol. Upon complete lipolysis this results in the release of three fatty acids. The first step in lipolysis is catalyzed by adipose triglyceride lipase (ATGL) resulting in DAG and one fatty acid.
Subsequently another fatty acid is hydrolyzed from glycerol catalyzed by hormone sensitive lipase (HSL). Finally, MAG is hydrolyzed by monoglyceride lipase . Lipid droplet lipolysis is a complex and strictly orchestrated process with many more players like (co-)activators and suppressors involved.
Intramuscular lipid storage and insulin resistance
The negative association between IMCL content and insulin sensitivity has been observed in a population consisting of sedentary lean, obese and type 2 diabetic subjects. This association, however, is not present in endurance trained athletes. Although athletes have elevated levels of IMCL they usually are very insulin sensitive . So, the relationship between lipid storage in skeletal muscle with insulin sensitivity is unlikely to be causal. This has led to the suggestion that lipid intermediates, like DAG and ceramides, are able to interfere with myocellular insulin signaling, and that increased IMCL is merely a surrogate measure of the presence of elevated DAG or ceramide levels.
The higher levels of lipid intermediates in insulin resistant subjects could be a consequence of a lower fat oxidative capacity, which is low in insulin resistance. This blunted oxidative capacity is due to fewer mitochondria and/or compromised mitochondrial function. In combination with an elevated IMCL content, mitochondrial dysfunction could result in increased availability of lipid intermediates.
In contrast, endurance trained athletes have a high capacity to oxidize fat and have elevated IMCL storage, serving as a readily available energy source during exercise. Thus, if lipid turnover is high and fatty acids can be readily oxidized, the toxic effect of lipid intermediates is likely limited. An imbalance between ATGL and HSL activity, which in principle could raise DAG levels, has been suggested for the insulin resistant state .
In cellular systems, it has been shown that these lipid intermediates interfere with insulin signaling and reduce insulin stimulated glucose uptake. Diacylglycerol inhibits the insulin signaling via PKC, which inhibits the insulin signaling cascade downstream of the insulin receptor. Induction of insulin resistance in healthy lean subjects by lipid infusion resulted in an increase in DAG and PKC levels . In line with this, weight loss or exercise reduces DAG content in skeletal muscle and improves insulin sensitivity in obese subjects . However, the reductions in DAG content due to exercise interventions were not associated with improvements in insulin sensitivity.
Paradoxically, athletes can have higher total DAG levels compared to obese insulin resistant subjects . This suggests that total DAG in itself cannot deteriorate insulin sensitivity. However, DAG presents as 3 different isoforms, or optical isomers, with a wide range in chain length and level of saturation of the fatty acids making up the DAG pool. Particular DAG species, like saturated DAG, and DAG at specific locations in the cell are particularly potent to interfere with insulin signaling. For saturated DAG content evidence is not consistent. One study shows that athletes have relatively more saturated DAG compared to obese insulin resistant subjects and more or less similar DAG levels as sedentary lean people .
In contrast another study showed that sedentary lean people have relatively higher levels of saturated DAG . Hydrolysis of TAG in the LD will result in LD bound DAG. It should be noted though that DAG can also be of phospholipid origin and be bound to phospholipid membranes of skeletal muscle. A study showed that this membrane bound fraction of DAG makes up about 80% of the total DAG pool in muscle and is higher in insulin resistant subjects compared to endurance trained athletes. This higher membrane fraction was associated with a higher PKC activation and insulin resistance . Especially membrane bound DAG, which is unlikely to be of lipid droplet origin, has the potential to interfere with the insulin signaling as it resides at the site where insulin signaling takes place.
Another lipid intermediate that has been associated with insulin resistance is ceramide. Ceramide is a sphingolipid and consists of one fatty acid and a sphingosine molecule. Also the fatty acids bound to ceramide can differ, but it requires palmitoyl-CoA to synthesize ceramide. Ceramide inhibits the insulin signaling pathway resulting in inhibition of glucose uptake. In insulin resistant obese subjects, higher ceramide levels have been observed compared to insulin sensitive subjects. In addition, ceramide levels are negatively associated with insulin sensitivity. It should be noted, that difference in ceramide levels between groups with distinct levels of insulin sensitivity is not always observed . In obese insulin resistant subjects total ceramide levels drop upon 16 weeks exercise training as an insulin-sensitizing intervention . However, reduced ceramide content induced by exercise training does not always associate with improvements of insulin sensitivity.
Also for ceramides a subtype specific effect may underlie the variation in insulin sensitivity upon interventions altering ceramide content. Interestingly, exercise-mediated reductions in specific ceramides species (C16:0 and C24:1) were associated with improvements in insulin sensitivity . These two ceramide species were higher in obese insulin resistant subjects compared to athletes suggesting that these two ceramide species may interfere with insulin sensitivity.
Subcellular location of myocellular lipid droplets
Figure 2. Subcellular location of myocellular lipid droplets. Two compartments can be distinguished (A) subsarcolemmal lipid droplets indicated by red arrows and (A and B) intermyofibrillar lipid droplets indicated by black arrows. (Click to enlarge)Insulin signaling occurs at the cell membrane and lipid intermediates from lipid droplets close to the cell membrane might impact locally on insulin signaling. Thus, subcellular location of lipid droplets may well be of importance when it comes to insulin sensitivity. Lipid droplets can be stored subsarcolemmal, in the near vicinity of the cell membrane, or between the contractile filaments of the skeletal muscle, the intermyofibrillar lipid droplets (Figure 2). Electron microscopic images showed that type 2 diabetic subjects have more lipid droplets in the subsarcolemmal region compared to obese subjects. The number of lipid droplets in the intermyofibrillar region, however, is not different. In addition, in type 2 diabetic subjects with a high amount of lipid droplets in the subsarcolemmal region, the subsarcolemmal lipid droplet content correlates with insulin resistance . Improving insulin sensitivity by training reduces the number of lipid droplets in the subsarcolemmal region, while the amount of lipid droplets in the intermyofibrillar region is unaffected . Not only the number of lipid droplets in the subsarcolemmal region is affected by training, but also lipid droplet size in the subsarcolemmal region is reduced .
To summarize, the association between lipid storage in skeletal muscle and insulin sensitivity is not straightforward. Although bioactive lipids like DAG indeed can impede insulin signaling in cell systems, it is not yet clear if this also holds true in the human in vivo situation. Moreover, the debate on the putative role of different DAG isoforms and subtypes and subcellular location with respect to their insulin inhibiting potential has only recently been initiated. It seems safe to state that in sedentary people excess IMCL directly or indirectly impedes insulin sensitivity. On the other hand, improvements in myocellular insulin sensitivity can be achieved without noticeable or consistent alterations in IMCL or bioactive lipids.
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