Muscles, Membranes and Exercise- 10th International Conference on the Biochemistry of Exercise 
David S. Rowlands, School of Physical Education, Dunedin, New Zealand

Sportscience News Sept-Oct 1997
Muscle Membranes, Diet and Exercise: Muscle Membranes, Diet and Exercise. Striking research on how dietary fats influence exercise metabolism via muscle membranes.
Carbohydrate-Lipid Interactions During Exercise: Fat gets used only in the absence of carbohydrate. 
Fatty Acid Binding Proteins and Muscle Lipid Metabolism: Free fatty acids are transported into muscle. 
Genetics and Exercise Science: Molecular biology is revolutionizing exercise science. 
The 10th International Conference on the Biochemistry of Exercise was held at the University of Sydney, Australia, July 15-19, 1997. The conference was dedicated to the memory of John Sutton, the prominent Australian exercise scientist who died recently. Dr Sutton was instrumental in bringing this conference to Australia for the first time.

The title of the conference was Membranes, Muscle and Exercise, but a broader range of topics was presented, integrating exercise biochemistry, physiology and metabolism. I will focus this report on the topics that are most relevant to my present research and interests. I found the conference to be very well organized and informative, a credit to Dr Mark Hargreaves and the committee.  I was disappointed, however, that some of the presenters failed to discuss their findings in a broader context. The conference proceedings will be published in review form in "Biochemistry of Exercise X", sometime in the next two years.

Muscle Membranes, Diet and Exercise

A highlight was the presentation by Professor Storlien from the University of Wollongong on the effect of dietary fat on membrane composition and function, and the implications for exercise. Skeletal muscle is a major player in whole body energy balance, lipid-based energy flux and insulin-stimulated glucose uptake. Dr Storlien's research on metabolic syndromes such as diabetes has shown that the specific fatty acid composition of the major structural lipids in the sarcolemmal membrane significantly influences membrane morphology and behavior and, in turn, certain aspects of cellular and whole body metabolism and physiology.

It appears that both the degree of unsaturation, and the omega-6:omega-3 ratio influences membrane, cellular, and ultimately whole body function. For example, the higher the level of membrane unsaturation, the higher the insulin sensitivity. A diet with a low polyunsaturated:saturated fat ratio is believed to be a causal factor in hyperinsulinemia. A higher unsaturation ratio increases GLUT 4 activity, which is important for glucose transport into the muscle during exercise. Omega-3 (n-3) polyunsaturated fatty acids, found in foods from marine origin and in a few seed oils (e.g. canola), may also play a particularly important role in insulin action. A high n-6:n-3 ratio, typical of a modern western diet containing high levels of vegetable oil, appears deleterious and is related to an increased incidence of some cancers, behavioral problems, a dysfunctional immune system and cardiovascular disease (Okuyama et al., Prog. Lipid Res. 35, 409-457, 1997). Insulin action also appears to be influenced by the activity of the delta-5-desaturase enzyme, which is involved in the competitive conversion of essential n-6 and n-3 fatty acids to their membrane storage forms. The activity of delta-5-desaturase enzyme has been shown to be increased by high protein diets, glucose, and insulin, and depressed by glucagon, epinephrine, cAMP, and fasting.

With relevance to obesity research, Dr Storlien explained that the lower the n-6:n-3 ratio, the greater the membrane fluidity, the greater the proton leakage from the cell, and consequently the greater the cellular metabolic rate. On the other hand, high rates of proton leakage may be detrimental to exercise performance by increasing the amount of energy required to fuel active ion transporters responsible for ionic and acid-base homeostasis. Helge et al. (J. Physiol. 491, 63P, 1996) recently reported that endurance time tended to be greater in trained rats consuming a high saturated fat diet, than in rats on a high unsaturated fat and high carbohydrate diets.

In relation to fiber type, highly oxidative and insulin-sensitive type I and type IIa fibers appear to possess relatively unsaturated sarcolemmal membranes, with a greater percentage of n-3 fatty acid integration, compared to type IIb fibers. Endurance training has only a small effect on increasing the degree of unsaturation of the membrane. In contrast, dietary fatty acid content exerts a powerful influence. Training alone does appear to increase membrane fluidity by decreasing the level of cholesterol. Studies of Caucasian and Pima Indian populations suggest there is a genetic factor determining the processing and incorporation of fatty acids into the sarcolemmal membrane. Pima Indians have high rates of heart disease, diabetes, obesity, low levels of n-3 membrane fatty acids and delta-5-desaturase enzyme activity, and a relatively high proportion of type IIb fibers, all compared to Caucasian populations.

Heavy exercise has been shown to release non-esterified fatty acids directly from the sarcolemma into plasma, where they become available for uptake and eventual oxidation. Unsaturated fatty acids were recently suggested to be more easily oxidized as fuels during exercise. This is based on the observation of a lower respiratory exchange ratio in a group of rats fed a high unsaturated fat diet compared to high saturated fat and high carbohydrate diets (Helge et al, 1996). Others have argued, on biochemical grounds, that saturated fats, especially short and medium chain, should be the best fuel for exercise because they do not require biotransformation before they can be oxidized as fuel. Athletes and physically active people may benefit from habitually increasing their intake of dietary unsaturated fatty acids, particularly mono-unsaturates and polyunsaturated n-3, by increasing membrane fluidity and insulin sensitivity. Athletes may also benefit from ingesting saturated fatty acids as a pre-exercise meal and an exercise supplement.

The striking message I took home from Dr Storlien's talk was that diet, exercise and genetics are likely to interact in complex ways to influence cellular physiology and whole body responses. And although we still have much to learn, it seems that science is moving steadily towards optimizing diets for health and athletic performance.

Carbohydrate-Lipid Interactions During Exercise

Dr Larry Spriet presented the first of two lectures on the regulation of carbohydrate and lipid metabolism for energy provision during exercise. There seems to be little direct evidence to support classical glucose-fatty acid cycle regulation of carbohydrate (CHO) oxidation in human skeletal muscle. Rather, CHO availability appears to be a more powerful regulator of fat oxidation. Nevertheless, there are some indicators that fat oxidation has a regulatory effect on carbohydrate oxidation in skeletal muscle. For example, elevated plasma free fatty acid (FFA) availability through intralipid infusion and heparin has been shown to spare glycogen. This effect is related to decreased accumulation of intracellular phosphate (Pi), free AMP and free ADP during sustained moderate exercise. In the intralipid studies, citrate was consistently elevated and pyruvate dehydrogenase activity significantly lower (in line with glucose-fatty acid cycle regulation), but there was no associated elevation in acetyl-CoA. Dr Spriet concluded that 80-85% of the reduction in glycolytic flux in the presence of elevated FFA was due to a reduction in glycogen breakdown by the enzyme glycogen phosphorylase.

Dr Spriet discussed the findings of recent studies suggesting that CHO oxidation may regulate fat oxidation. The theory is that malonyl CoA, elevated during exercise situations associated with high rates of CHO oxidation (e.g. heavy exercise and exercise after a high CHO diet), inhibits carnitine (palmityl) transferase 1 (CPT1) activity, thereby suppressing the entry of fatty acids into mitochondria. The theory is supported indirectly by the following evidence: reduced CPT1 activity associated with high rates of CHO oxidation; in vitro studies showing that malonyl CoA is a potent inhibitor of CPT1 activity; and recent tracer studies showing suppressed oxidation of long chain, but not medium-chain fatty acid when muscles are primed to oxidize glucose (medium-chain fatty acids do not require CPT1 for entry into the mitochondria). There is little direct evidence to support this malonyl CoA theory. For example, no increase in malonyl CoA was detected during exercise in humans at 65 and 95% of VO2max. There was, however, a significant increase in acetyl CoA after 10 min. Since carnitine is a buffer of acetyl CoA, acetyl CoA binding may reduce CPT1 activity. In any event, it seems that simple regulation of CPT1 by malonyl CoA is unlikely and that other factors are involved.

The second lecture on the regulation of carbohydrate and lipid metabolism was given by Dr Edward Coyle from the University of Texas. Dr Coyle focused on the regulatory roles of lipolysis, FFA availability and glycolytic flux. Insulin is a potent regulator of substrate oxidation. Elevated insulin after carbohydrate ingestion inhibits adipose and intramuscular hormone-sensitive lipase, increases glucose uptake and glycolytic flux, and may suppress FFA entry into the mitochondria by reducing the activity of CPT1. Plasma insulin concentration is related to the amount of glucose entering the blood. The rate of lipolysis is inversely related to plasma insulin concentration, while the rate of fat oxidation is directly related to the availability of plasma FFA. As little as 20 grams of ingested glucose can raise insulin and decrease fat availability and oxidation during exercise. If the aim of the exercise is to maximize fat oxidation rate (e.g. a body fat reduction program), then the pre-exercise consumption of even a very small quantity CHO will have a detrimental effect. Any commercially available product or diet that claims to increase FFA mobilization and oxidation would have to almost totally eliminate the insulin response to CHO in food. The bottom line: don't ingest carbohydrates if you want to maximize fat burning.

Lactate, a metabolic by product of glycolysis, reduces FFA availability by suppressing mobilization from the adipose tissue. Reduced FFA availability leads to a reduction in fat oxidation and an increase in rates of glycogen breakdown and CHO oxidation.

Carbohydrate ingestion has been shown to suppress fat oxidation for up to 8 hours, despite the return of plasma insulin to baseline after a few hours (Montain et al., J. Appl. Physiol., 70, 882-888, 1991). The mechanism responsible for this sustained suppression of fat oxidation is not known. Future research will use molecular techniques to study the effects of insulin and other metabolic hormones on the synthesis of regulatory enzymes.

To summarize, I understood the data presented by Coyle and Spriet to show that carbohydrate was a more powerful regulator of fat metabolism than fat was of carbohydrate metabolism in exercise. Fat ingestion or infusion is effective in enhancing fat oxidation only in the absence of CHO.

Fatty Acid Binding Proteins and Muscle Lipid Metabolism

Dr Lorraine Turcotte presented evidence that fatty acids are transported across the plasma membrane and around the cell interior by fatty-acid binding proteins. Transport across the muscle plasma membrane shows saturation properties. However a gradient-dependent diffusion component exists in addition to carrier mediated transport (a dual diffusion-transport scenario which has also been observed for lactate). Transport of FFA into the muscle cell is dependent on plasma FFA concentration, blood flow, fiber type, and rate of fat oxidation. Training, a high fat diet, or fasting increase the concentration of fatty-acid binding proteins in the membrane and the rate of FFA transport. Intramuscular triglyceride synthesis is related to the rate of FFA uptake, and is greater in rat red than in rat white fibers. Uptake rates can exceed oxidation rates, but the rate of fat oxidation is limited by the rate of uptake.

The intramuscular triglyceride pool may be in equilibrium with the plasma pool. During light to moderate exercise there appears to be little depletion of intramuscular stores; that is, any fat taken from the intramuscular pool and oxidized during exercise is replaced by FFAs from the plasma pool. During heavier exercise eliciting oxidation rates in excess of maximal uptake rates, net depletion of intramuscular triglyceride may occur. The intramuscular pool is difficult to measure, so it may be some time before we understand the role of this pool of fat.

Genetics and Exercise Science

One of the most important themes emerging from the conference was the increasing role of molecular biology in exercise science. Dr Darrell Neufer lectured on contractile activity and skeletal muscle gene expression. The muscle remodels itself in response to training. Mitochondria density and oxidative enzymes levels increase. Muscles hypertrophy and changes in myosin isoform expression occur. We can measure changes in membrane protein concentrations, structure and functionality. Capillary density increases. Underlying all of these adaptive changes are changes in gene expression, alterations in protein synthesis, and changes in the activity of regulatory factors. We know that training responses are specific to recruitment patterns; that is, the magnitude of changes are related to the level of fiber recruitment during the exercise. The oxidative capacity of type IIb fibers, for example, appears to only increase when near maximal exercise is employed during training. The big question remains: what signaling events induce the synthesis of proteins important in the adaptive response to exercise? If we know what proteins are of most benefit to exercise performance and can determine what signals induce their synthesis, and we know what training causes the strongest signals, then we could pinpoint what training strategies should be of most benefit.

Little is currently known about the signaling events in response to training. Candidate signals are: sympathetic stimulation, hormones, stretch-activation and deformation of membranes, intracellular calcium, and changes in ATP/ADP. Recent experiments have also focused on "energy charge" (free phosphate). It is known that many proteins are regulated by phosphorylation, and several phosphorylated proteins regulate gene expression. The greater the levels of free Pi present in the cell, the greater the potential for gene expression. Post-transcriptional and post-translational regulation may be important in determining exactly how much of a given protein actually ends up being synthesized. Some genes are activated and the coding proteins synthesized during the exercise (e.g. c-fos and HSP 70), while the concentrations of other proteins do not rise until hours or even days after the exercise (e.g. GLUT4, hexokinase, lipoprotein lipase and several mitochondrial enzymes). The adaptive response to training over a period of weeks can be seen as a cumulative effect of gene expression. During the first stage of skeletal muscle hypertrophy (e.g. the day after starting a weights program), evidence suggests that increased protein synthesis can be attributed to increased translational efficiency and/or capacity as inferred from an increase in RNA levels and activity. With continued training, protein synthesis is associated with an increased abundance of myofibrillar mRNA, which suggests increased transcription rate and mRNA stability may be a response to continued exercise stress. The training response appears to involve a multistage alteration of expression and processing of genes and gene products to enable specific adaptations to the training stress to occur (Carson, Exerc. Sport Sci. Rev. 25, 301-320, 1997).

In his closing address, Dr Frank Booth gave us his perspective on "exercise biochemistry beyond 2000." We are likely to see rapid advances in exercise biochemistry with the completion of the Human Genome Project in 2003 and further advances in biotechnology. With new technology, such as DNA chips, we will be able to identify the genes that are expressed in response to a particular treatment or stimulus. Current methods limit investigation to one or two genes or gene products at a time. The use of transgenic animals and "knockout" rats to examine the role of genes and gene products is likely to increase. With the introduction of new technology and the increasing molecular focus, a number of present metabolic paradigms may become outdated.

A number of health-related issues: obesity, aging, cardiovascular disease, and immune system dysfunction, are driving exercise research.  But funding is always a worry for exercise scientists.  Drug companies, a major source of funding, are keen to fund research to find pharmacological treatments but don't like to hear that up to 95% of chronic diseases of western society can be treated with appropriate exercise and dietary advice. If we adapt our research techniques to keep up with molecular biologists, we may have more opportunities for funding.  Exercise scientists, by using the tools of both molecular biology and physiology, are in a powerful position to unravel the cellular complexity underlying seemingly simple questions like "what should I eat?" and "how much exercise do I need?"

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