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165 Chapter 7 GENERAL DISCUSSION7.1 Overview Changes in lifestyle throughout the last 30 years has resulted in a huge rise in obesity and concomitant increases in the incidence of the obesity-associated metabolic diseases, insulin resistance and type 2 diabetes (WHO 2006). This provides a challenge to researchers, as the development of successful therapeutic interventions requires a thorough understanding of the underlying mechanisms. Due to its role in disposing the majority of circulating glucose following the ingestion of a meal (Ferrannini et al 1985), skeletal muscle is vitally important in maintaining glucose homeostasis. Lipid accumulation in insulin sensitive is associated with reductions in glucose tolerance, while acute exercise and life-style interventions involving a reduced calorie and fat intake and an increase in physical activity improve glucose tolerance (chapter 1). In both cases the underlying mechanisms are only partially understood. Therefore, the aims of this thesis were i) to compare resistance and endurance exercise as means to improve glucose tolerance, ii) to investigate whether ingestion of a single high fat meal reduces glucose tolerance and whether it can be restored with one bout of resistance exercise, iii) investigate whether near infrared spectroscopy (NIRS) can detect meal-induced 166 increases in muscle capillary blood volume and iv) to develop new immunofluorescence microscopy methods applied to human muscle fibres that can identify defects in lipid metabolism which potentially contribute to the mechanism leading to insulin resistance. 7.2 The effects of exercise and nutritional interventions on glucose tolerance Previous studies have demonstrated that acute bouts of endurance and resistance exercise lead to enhancements in insulin-mediated glucose uptake up to 24-48 h after exercise (chapter 1). Chapter 2 demonstrated that glucose tolerance measured with an oral glucose tolerance test, can be improved 6 h after resistance exercise when a carbohydrate/protein-hydrolysate drink is ingested after exercise to resynthesize at least part of the muscle glycogen oxidised during the exercise. On the other hand these beneficial effects on glucose tolerance were not seen 6 h after endurance exercise when the same glycogen repletion protocol was used. These results were explained (chapter 2) by the fact that resistance exercise not only leads to glycogen depletion but also to activation of early intermediates in the insulin signalling cascade (Hernandez et al 2000, Creer et al 2005), while endurance exercise only causes glycogen depletion and no activation of the insulin signalling cascade (Richter et al 2001; Wojtaszewski et al 2000). Low concentrations of glycogen following exercise have been shown to stimulate insulin stimulated glycogen synthase activity and muscle glucose uptake (chapter 1). The interpretation of the observations made in chapter 2 is based on the assumption that the muscle glycogen stores were largely or fully restored before the start of the OGTT. Although this assumption seems to be reasonable as the carbohydrate-protein beverage consumed in chapter 2 has previously been shown to lead to high rates of muscle glycogen synthesis (van Loon et al 2000), measurements of the muscle glycogen content and insulin signalling events are needed to confirm these conclusions. 167 At the time that we performed the study described in chapter 2 the available literature reported that an acute bout of endurance exercise did not activate early events in the insulin signalling cascade, including insulin-induced insulin receptor kinase activity, IRS-1 tyrosine phosphorylation, PI 3-kinase activity, and serine phosphorylation of Akt and of glycogen synthase kinase-3 (chapter 1). However, it has recently been reported that phosphorylation of Akt substrate of 160 kDa (AS160) occurs in rat skeletal muscle immediately and 4 h after endurance exercise (Arias et al 2007) and this is mediated by increases in AMPK (Treebak et al 2006). AS160 is also phosphorylated upon insulin stimulation involving the IRS-1/PI 3kinase/Akt signalling pathway (chapter 1). This indicates that phosphorylation of AS160 is a cross-point where insulin- and contraction-related stimuli converge to stimulate GLUT4 translocation. This newly discovered mechanism has revealed a potential route by which GLUT4 translocation to the plasma membrane could occur without increases in proximal insulin signalling steps in the post-exercise period. Further work is required to investigate how long AS160 remains phosphorylated after endurance exercise, if the activation remains when carbohydrates are ingested immediately after exercise (leading to acute inactivation of AMPK), if AS160 phosphorylation depends on muscle glycogen content, if resistance exercise also leads to phosphorylation of AS160, and finally whether exercise-induced AS160 phosphorylation also occurs in insulin resistant states. The results from chapter 2 could have potential implications to type 2 diabetics who want to reduce the period they are exposed to elevated blood glucose concentrations. Delaying feeding will lead to more prolonged enhancements in glycogen synthase activity and glucose transport and a reduction in blood glucose concentrations. On the other hand results from 168 chapter 2 suggest that the benefit of exercise remains when a carbohydrate/protein drink is ingested following resistance exercise. Resistance exercise, therefore, may be the preferential mode of exercise when the aim is to improve glucose control/homeostasis. Resistance exercise may also have additional benefits. Koopman et al have shown that ingesting carbohydrate and protein after resistance exercise improves net protein balance (Koopman et al 2005b) and enhances downstream insulin signalling events involved in protein synthesis (Koopman et al 2007). In chapter 3, the glucose response to an OGTT 6 h after ingestion of a single high fat meal was 36% higher than that observed after ingestion of a low fat meal, but the difference was not significant. However, following ingestion of the high fat meal, the glucose concentration failed to return to the pre-OGTT concentration at 2 h, potentially indicating a small reduction in the ability of insulin to stimulate glucose uptake in line with the set hypothesis. More prolonged periods of high fat feeding (3 days) in lean healthy subjects have been shown to impair insulin-mediated glucose uptake and glucose tolerance (Bachmann et al 2001; Pehleman et al 2005). The 36% increase in glucose response after ingestion of a high fat meal found in chapter 3, although not significant, may be relevant to some individuals, as six of the eight subjects demonstrated an elevated glucose response to the OGTT. This difference in subject response may be due to differences in individual ability to handle a lipid load. A recent study demonstrated that rats bred for high endurance running capacity had a higher basal skeletal muscle oxidative capacity and were resistant to high fat diet-induced obesity and insulin resistance (Noland et al 2007). In addition, the adipose tissue in obese and obese insulin resistant groups has a poor lipid buffering capacity leading to higher postprandrial plasma concentrations of triglycerides and free fatty acids (Bays et al 2004; Frayn 2000). 169 Therefore the effect of a single high fat meal may have greater negative effects on glucose control in populations at risk of lipid-induced metabolic impairments and in those already insulin resistant. Chapter 3 also confirmed previous (eg. chapter 2, Koopman et al 2005a, Fluckey et al 1994) and also more recent studies (Praet et al 2006) showing that a single bout of resistance exercise can improve insulin-mediated glucose control. The novel finding in chapter 3 was that a resistance exercise bout performed in the postprandial period following ingestion of a high fat meal significantly improves glucose tolerance. This study indicates that it is beneficial to perform resistance exercise following the ingestion of an energy dense meal to prevent short-term impairments in glucose control. The possible mechanisms for improvements in glucose tolerance may be related to activation of early insulin signalling intermediates (PI 3-kinase and Akt) seen in previous studies (Hernandez et al 2000, Creer et al 2005). A recent study also demonstrated that the protein concentration of enzymes involved in lipid synthesis such as diacylglycerol acyltransferase (DGAT) and mitochondrial glycerol-3phosphate acyltransferase (mGPAT) were increased the day after a single bout of endurance exercise and lead to increases in IMCL content and reduced concentrations of intracellular DAG and ceramides (Schenk and Horowitz 2007). Interestingly the single bout of endurance exercise also prevented the development of insulin resistance during a lipid/heparin infusion leading to high FFA concentrations (Schenk and Horowitz 2007). The effect of resistance exercise upon the protein expression and activity of such enzymes is not known but resistance exercise is known to lower IMCL content with resynthesis occuring during the first few hours 170 of recovery (Koopman et al 2006). Upregulation of DGAT and/or mGPAT and subsequent increases in IMCL synthesis may partially explain why an increased glucose tolerance was seen when resistance exercise was performed in a period of enhanced lipid availability. Fatty acids originating from chylomicron-TG hydrolysis by muscle lipoprotein lipase in the postprandrial period would be taken up by the muscle and used for synthesis of IMCL preventing any accumulation of intracellular FA metabolites. Therefore a bout of resistance exercise appears to be beneficial to glucose control after the ingestion of a high fat meal. This may be particularly advisable to those who are already insulin resistant and experience large daily elevations in plasma glucose concentrations. 7.3 New methods to investigate the mechanisms of insulin resistance 7.3.1 Increases in microvascular blood volume Over the last 5-10 years it has become clear that insulin-mediated increases in capillary perfusion is a vital step in the regulation of glucose homeostasis. These findings are mostly derived from novel techniques utilising contrast-enhanced ultrasound (CEU) applied to humans to measure the volume of blood in microvascular compartments. In chapter 4, we investigated whether it was possible to measure increases in microvascular blood volume noninvasively using near-infrared spectroscopy (NIRS), where changes in total haemoglobin (HbT) concentrations were considered to represent changes in blood volume in the measured compartment. However NIRS was only able to detect small changes in HbT following muscle contraction and no change in HbT following ingestion of a beverage known to elicit large increases in plasma insulin concentrations. It was concluded that the NIR-light is absorbed by red blood cells in a variety of vessels (arterioles, venules, capillaries) in different tissues (skin, subcutaneous adipose tissue, muscle, connective tissue) and, therefore, NIRS can not be used 171 to gather specific information on the volume of blood present in muscle fibre capillaries. Therefore, investigations measuring the expansion of muscle capillary blood volume continue to depend upon the detection of albumin-coated microbubbles with contrast enhanced ultrasound (Vincent et al 2006) or measurement of the capillary permeability surface area using invasive techniques (Gudsbjornsdottir et al 2003). 7.3.2 Fluorescence microscopy, skeletal muscle lipid metabolism and insulin resistance Accumulation of FA metabolites (LCFACoA, DAG and ceramides) in muscle leads to activation of PKC and downregulation of insulin signalling and is assumed to be the primary mechanism responsible for reductions in insulin-mediated glucose uptake in obese subjects with insulin resistance and during intralipid/heparin infusions leading to high plasma FFA concentrations. Abnormalities in the transport and oxidation of FFA and in the synthesis and mobilisation of IMCL could all contribute to the increase in intracellular FA metabolites in insulin resistant states and explain the paradox that athletes combine a high IMCL content with high insulin sensitivity. The abnormalities in IMCL synthesis and mobilisation have only partially been identified and much of our current knowledge of the enzymes involved in IMCL synthesis and mobilisation depends on extrapolation of data obtained in cultured adipocytes. For a summary of the processes believed to be involved in IMCL synthesis and metabolism and how they may relate to insulin resistance see figure 7.1. Chapters 5 and 6 in this thesis developed new immunohistochemical methods that can be used to study lipid metabolism in skeletal muscle. 172 Chapter 5 used double-(immuno)staining protocols to simultaneously identify type I muscle fibres and intramyocellular lipid droplets, a method previously developed by Koopman et al (2001) in Maastricht. Quantitative analysis demonstrated that the lipid content is approximately 2-fold greater in oxidative type 1 fibres than glycolytic type II fibres confrming the earlier data published by this group (van Loon et al 2003 and 2004). Additionally identification of the PAT protein adipophilin was combined with a lipid droplet stain to show that ~60% of lipid droplets colocalise with clusters of adipophilin. Adipophilin has been Figure 7.1. The regulation of triglyceride synthesis and lipolysis in skeletal muscle. Both processes help to maintain low concentrations of intracellular fatty acid metabolites by directing fatty acids to TG synthesis or transport across the mitochondrial membrane and oxidation. If defects occur in the synthesis, hydrolysis or transport an accumulation of fatty acid metabolites activate PKC leading to insulin resistance. ATGL, adipose triglyceride lipase. DAG, diacylglycerol. DGAT diacylglycerol acyltransferase. FA CoA, fatty acylCoA. FAT, fatty acid translocase. GPAT, glycerol-3-phosphate acyl transferase. HSL, hormone sensitive lipase. lysoPAT, lysophosphatidate acyltransferase. M, mitochondria. MG, monoacylglycerol. MGAT, monoacylglycerol acyltransferase. MGL, monoacylglycerol lipase. PA phosphatase, phosphatidic acid acyltransferase. PKC, protein kinase C. TG, triglyceride. 173 Fatty acid Plasma Skeletal muscle FAT Glycerol-3-phosphate GPAT Fatty acid 1-acyl-G-3-P Fattylipase acyl CoA synthetase suggested to potentially mediate access of hormone sensitive (HSL) to IMCL (Prats et lysoPAT al 2006). We also showed that adipophilin does not colocalise with mitochondria and Phosphatidic acid FA CoA therefore is unlikely to channel fatty acids from the lipid droplets to the mitochondria upon PA phosphatase LD hydrolysis as suggested earlier (Chanderbhan et al 1982, Nakamura and Fujimoto 2003). INSULIN MG PKC DAG As our studies were performed in the resting state this conclusion is restricted to this RESISTANCE MGAT condition, but is not necessarily true during exercise. DGAT M Fatty acid FAT/CPT The results from chapter 6 illustrate that the mitochondrial network has a higher density in type I than in type II fibres and the greatest region DAG content is present MG in the subsarcolemmal Glycerol TG ATGL HSL MGL ? and progressively declines from plasma membrane to the interior of the fibre. The relative HSL distribution of LDs is similar to the mitochondria network with the highest content between 0Adrenaline Contraction 174 5 μm from the plasma membrane. Chapter 6 showed that the LDs are situated in the spaces between neighbouring mitochondria in the network and in close contact to at least one and often more mitochondria in lean young individuals. Presumably this aids efficient oxidation of IMCL observed in trained individuals during exercise (van Loon et al 2003). Obese subjects have been reported to have low subsarcolemmal mitochondria content (Ritov et al 2005) and increased lipid droplets in the interior of muscle fibres (Malenfant et al 2001). Disturbances in the spatial distribution of LDs and mitochondria in skeletal muscle may impair the oxidation of LDs during exercise and provide an explanation as to why insulin resistant subjects are unable to oxidise IMCL during exercise. 7.4 Future Research Immunofluorescence staining techniques such as the novel methods developed in this thesis provide a powerful tool to investigate the underlying mechanisms of the relationship between skeletal muscle lipid metabolism and insulin resistance. Standard biochemical assays applied to homogenates and extracts have the disadvantage that they do not give information on fibre type specificity. The use of immunohistochemical techniques has revealed that the intramyocellular lipid pool significantly contributes to total fat oxidation in type I fibres of lean active individuals, while no significant IMCL utilisation has been observed in type 2 fibres (van Loon et al 2003). Similarly measurement of mitochondrial content and enzyme activity is commonly performed on homogenates containing mixed fibre types (Bizeau et al 1998, Holloway et al 2007). With the new immunohistochemical stainings developed in this thesis it is now possible to investigate the spatial distribution of mitochondria in both subsarcolemmal and intermyofibrillar regions in a fibre type specific manner in different populations. 175 Great advances have been made in recent years concerning the understanding of the regulation of the activity of transporters and enzymes involved in glucose and fatty acid metabolism in skeletal muscle. We know that the activity of transporters and enzymes is not only modulated by means of phosphorylation/dephosphorylation, but also by translocation from one subcellular compartment to another upon insulin-stimulation or exercise. For example, we have known for many years that one of the key steps in glucose uptake is the translocation of GLUT4 from intracellular vesicles to the plasma membrane (Cushman and Wardzala 1980). We also know that translocation processes are involved in the transport of free fatty acids (FFA) into muscle fibres and into the mitochondria via translocation of fatty acid translocase (FAT) (Campbell et al 2004, Holloway et al 2006). We also know that colocalisation of enzymes with a substrate or with coactivator proteins can be the end point of these translocation processes. For example Prats et al (2006) demonstrated that hormone sensitive lipase (HSL) translocates from intracellular stores upon muscle contraction of intact rat muscle fibres to colocalise with LDs and two of the PAT proteins in muscle. Although traditional biochemical techniques combining differential centrifugation and Western blots can be used to quantify the content of proteins in membrane and cytosolic fractions they do not give information on the spatial distribution of proteins, on final colocalisation or on fibre type specificity. Techniques that can visualise fibre-type specific translocation and/or colocalisation steps in human muscle biopsies with a high resolution have a huge advantage over many biochemical techniques and will be a new avenue to explore muscle lipid metabolism in the next few years. With the rise in obesity and obesity-related metabolic disorders, the effort to develop these new techniques should be substantially increased to fully 176 understand the mechanisms by which the current life-style leads to the massive increase in the frequency of obesity, metabolic syndrome, type 2 diabetes and cardiovascular disease. Immunofluorescence techniques similar to the ones developed in this thesis provide an opportunity to further investigate the mechanisms of lipid synthesis, hydrolysis and oxidation in skeletal muscle. The technique set up in Chapter 5 makes it possible to investigate the interaction of ADRP and LDs in lean, obese and insulin resistant groups both at rest and during exercise. We can test now whether a low adipophilin protein content in insulin resistant muscles plays a role in the mechanisms leading to elevated fatty acid metabolites. Following on from cross-sectional studies of different populations, acute exercise studies are also warranted to further investigate IMCL oxidation during exercise. Biopsies pre and postendurance exercise would make it possible to investigate whether the degree of LD and adipophilin colocalisation is related to the capacity of IMCL oxidation and insulin sensitivity. Similar methods should also be developed to investigate other determinants of IMCL synthesis and oxidation. The finding that HSL exerts its effect not only through increased activity but through direct translocation and interaction with the LD (Prats et al 2006) explains the large increases in lipolysis with only small increases in enzyme activity. So far the fluorescent techniques used to illustrate this have been performed on intact rat muscle fibres (Prats et al 2006). They also showed that HSL translocates to the PAT proteins, which appear to surround the majority of lipid droplets. It is important to reproduce these results in human muscle fibres and immunofluorescence on longitudinal muscle sections taken before and after exercise would be an ideal way to do this. This could also demonstrate if it is only the LDs that colocalise with adipophilin that can be subjected to HSL-stimulated lipolysis and 177 thus oxidation. Additionally defects in the translocation of HSL possibly associated with adipophilin content could potentially explain the inability of insulin resistant muscle to oxidise IMCL during exercise. This work should be extended to also define the role of other PAT proteins in the regulation of LD lipolysis in response to exercise and adrenaline. Although work has been done on some of these other enzymes/protein in rats (Prats et al 2006), no studies are available in humans and on differences between physically active, sedentary and insulin resistant populations. Furthermore, adipose triglyceride lipase (ATGL) has recently been discovered (Zimmerman et al 2004) as the lipase responsible for the first step in TG breakdown to diacylglycerol (DAG), while HSL primarily acts on DAG (figure 7.1). The role and importance of ATGL in skeletal muscle lipolysis is not known. The technique set up in chapter 6 is an ideal method to identify the network of mitochondria and LDs in muscle as both can be observed simultaneously on the same muscle fibres. On the basis of the regular positioning of the mitochondria in type I fibres we were able to create a matrix showing the theoretical maximum density of mitochondria and LDs (Figure 7.2A). In the filled matrix each LD is attached to two neighbouring mitochondria. In the lean subjects investigated in chapter 6 many of the mitochondrial positions were occupied in type I fibres while the density of the lipid droplets was lower, meaning that all LDs neighboured at least one mitochondrion (Figure 7.2B). Previous studies have shown that sedentary, obese and insulin resistant individuals have a much lower mitochondrial density and only a marginally lower density of LDs (Figure 7.2C). This could imply that a number of lipid droplets have no neighbouring mitochondria. This may explain why LDs are not oxidised efficiently during exercise and why there is an accumulation of fatty acid metabolites. In future studies we will investigate this theoretical model presented in figure 7.2 in different subject groups. The 178 images should preferentially be made in 3 dimensions, which is possible with the software available in our laboratory. To obtain optimal information on relative occupancy of the mitochondria and LD networks in different populations, fatty acid diffusion distances and their potential impact on concentrations of fatty acid metabolites, we will develop mathematical models in collaboration with Eindhoven University of Technology. These models also will include comparisons of the network images obtained before and after exercise, to investigate how fatty acid diffusion distances and mitochondrial density gradients between subsarcolemmal and deeper layers determine LD oxidation during exercise. Another interesting protein for future investigations is fatty acid translocase (FAT/CD36). FAT translocates during exercise and in response to meal induced increases in insulin from intracellular stores to the plasma membrane (Kiens 2006). Recently it has also been shown that during exercise FAT translocates to the inner mitochondrial membrane taking a position next to CPTI, reducing malonylCoA inhibition and thus increasing fatty acid oxidation during exercise (Bezaire 2006, Holloway et al 2006, Schenk and Horowitz 2006). Suggestions have been made that an increased FAT content in the plasma membrane leads to increased fatty acid uptake in animal models of type 2 diabetes (Bonen et al 2004, and 2006). A high FAT content in the plasma membrane in the presence of a low FAT content in the mitochondria would lead to an imbalance between fatty acid uptake and oxidation and could lead to accumulation of fatty acid metabolites and lead to insulin resistance. The development of immunofluorescence microscopy methods that allow us to simultaneously measure FAT content of plasma membrane and mitochondrial membranes in resting and exercise biopsies of various populations with and without insulin resistance therefore should have high priority. 179 Mitochondrial (mGPAT) and microsomal (GPAT3) glycerol-3-phophate acyltansferase and diacylglycerol acyltransferase (DGAT) are key enzymes in the synthesis of triglycerides and lipid droplets in muscle. Recent studies (Liu et al 2007, Schenk & Horowitz 2007) suggest that an increased protein content of DGAT and mGPAT leads to increased insulin sensitivity and protection against acute insulin resistance induced by an intralipid/heparin infusion. Furthermore one bout of endurance exercise is sufficient to increase the content of DGAT and mGPAT the next day and offer protection against lipid/heparin induced insulin resistance (Schenk & Horrowitz 2007). The proposed mechanism behind this observation is that fatty acids taken up by the muscle will be more efficiently channelled into the IMCL stores leading to lower concentrations of diacylglycerol, long-chain fatty acylCoAs and potentially ceramides, the fatty acid intermediates that lead to insulin resistance via PKC activation (chapter 1). Again immunohistochemical assays able to quantify DGAT, mGPAT and the recently discovered microsomal GPAT (Cao et al 2006) in muscle again should have a high priority. Such assays could be used to evaluate the effect of training interventions on the capacity to oxidise IMCL during exercise and synthesize IMCL during recovery in various populations with and without insulin resistance. As explained in chapter 1 and chapter 4, insulin action on the endothelium to increase muscle capillary perfusion is of great importance in determining glucose uptake in muscle and glucose tolerance (Rattigan et al 2006). Impairments in the endothelium of the terminal arterioles in muscle and in feeding and resistance arteries have been suggested to contribute to the development of insulin resistance, type 2 diabetes and cardiovascular disease (Wagenmakers et al 2006). Metabolism and mechanisms that operate in the endothelium of the microvasculature are particularly difficult to study due to the inaccessibility of terminal arterioles and capillaries in human muscle. Much of what we assume to know today is based on extrapolation from studies in larger vessels, cultured endothelial cells and animal models of insulin resistance. Immunofluorescent labelling of basement and plasma membranes with 180 anti-laminin shows that capillaries, arterioles and venules are visible on sections of human muscle biopsies (see figure 7.3, unpublished data of Shaw and Wagenmakers). Therefore immunolabelling of terminal arterioles with antibodies against insulin signalling intermediates (eg IRS-1 total, tyrosine and serine phosphorylated IRS-1), against eNOS (total protein and serine phosphorylated) as an indicator of basal and insulin stimulated NO production could potentially identify defects in the insulin resistance of terminal arterioles. Furthermore NADPH oxidase expression was recently shown to be increased in venous vascular endothelial cells harvested from obese subjects (Silver et al 2007). NADPH oxidase produces superoxide anions that can take away functional NO in a reaction leading to peroxynitrite production (Chapter 1.4.5). Future studies should, therefore, explore whether the enzyme systems and signalling pathways that control endothelial NO production in response to insulin and exercise can be visualised and quantified with newly developed immuonohistochemical staining methods of arterioles and capillaries applied to longitudinal sections of human muscle.