Metabolic aspects of low carbohydrate diets and exercise
© Peters and LeBlanc; licensee BioMed Central Ltd. 2004
Received: 22 September 2004
Accepted: 30 September 2004
Published: 30 September 2004
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© Peters and LeBlanc; licensee BioMed Central Ltd. 2004
Received: 22 September 2004
Accepted: 30 September 2004
Published: 30 September 2004
Following a low carbohydrate diet, there is a shift towards more fat and less carbohydrate oxidation to provide energy to skeletal muscle, both at rest and during exercise. This review summarizes recent work on human skeletal muscle carbohydrate and fat metabolic adaptations to a low carbohydrate diet, focusing mainly on pyruvate dehydrogenase and pyruvate dehydrogenase kinase, and how these changes relate to the capacity for carbohydrate oxidation during exercise.
Exercise, an acute bout of muscular activity, requires an expenditure of energy above resting levels. This required mechanical energy is provided through the conversion of metabolic fuels into ATP, the base currency of chemical energy. Once produced, ATP is the only direct form of energy that is transferred and utilized by the contractile apparatus within the muscle. Fats are the predominant fuel source of resting skeletal muscle and during exercise, there is a complex interaction between skeletal muscle fat and carbohydrate (CHO) metabolism (see  for review). When evaluating the effects of exercise on skeletal muscle fuel utilization, there are many facets that must be taken into consideration. These include intensity and duration of the bout of exercise and the training status of the subjects. During low intensity physical activity (25% maximal oxygen uptake (VO2max)), fat supplies the majority of metabolic fuel to exercising skeletal muscle . As physical activity increases to moderate levels (65–70% VO2max), there is a shift to more reliance on CHO, specifically muscle glycogen . However, at this level of physical activity, fat oxidation becomes increasingly important as the duration of exercise increases  or as training status improves . The studies presented in this review utilize moderately active subjects (maximal oxygen uptake, 50–60 ml·kg-1·min-1) exercising at a workload of 65–75% VO2max for 30–48 min.
The sources of chemical energy that fuel exercising skeletal muscle are available through endogenous depots (intramuscular glycogen and triglycerides) or exogenous sources (plasma glucose and free fatty acids). In turn, these exogenous and endogenous fuel sources are replenished through dietary intake. As a result, there is an important relationship between diet and fuel metabolism in skeletal muscle.
Diets low in carbohydrate content have become increasingly popular as a method of weight loss. These diets that limit daily dietary carbohydrate intake are termed low-carbohydrate diets (LCD). When evaluating the effects of LCD, there are a couple of factors that must be considered, as they may influence the measured outcome. These include the composition of the diet (since a LCD may replace the missing CHOs with either protein or fat), and the duration of the dietary period. For the purpose of this review, LCD will refer primarily to high-fat low-carbohydrate isocaloric diets with <50 g of CHO per day, with a composition of 3–8% CHO, 22–46% protein, and 51–75% fat, and consumed for 3–6 days.
The present paper will briefly review human skeletal muscle metabolism during exercise and the importance of dietary CHO for metabolic energy production. It has been well documented that diets low in carbohydrates result in several metabolic and hormonal adaptations that improve fat oxidation and promote glycogen sparing in exercising skeletal muscle (see  for review). However, the mechanism(s) responsible for these changes in exercising skeletal muscle are still debatable, but could be the result of up-regulated fat and/or down-regulated carbohydrate metabolism. The emphasis of the present paper is on adaptive skeletal muscle CHO and fat metabolism in humans, and will compare recent studies that examine the effects of altered diets on key enzymes and how fatty acid composition and re-feeding of carbohydrates following these altered diets affect these enzymes. Data from other mammals are cited where necessary.
Each of the covalent regulatory enzymes of PDH is subject to allosteric regulation. Phosphorylation of the complex is catalyzed by a family of four PDK isoforms (PDK1-4) which differ in their responsiveness to allosteric inhibition by pyruvate or activation by energy charge (ATP/ADP ratio), redox (NADH/NAD+ ratio), and acetyl-CoA-to-free CoA ratio (see  for review). In addition, the kinases differ in their specificity for the different phosphorylation sites . Thus, the relative activities of the PDK isoform population will determine the response of the PDH complex in acute situations. An intrinsic pair of phosphatases (PDP1 and 2) catalyze the dephosphorylation and activation of PDH . PDP1 is the isoform which is activated in the presence of increasing concentrations of Ca2+ ions (as would be expected during exercise), while PDP2 is activated when insulin levels are increased during dietary manipulations .
At rest, PDH is mainly phosphorylated and inactive due to high energy charge, redox, and acetyl-CoA-to-free CoA ratio and low pyruvate concentration, which maintain a high PDK activity. Phosphatase activity is low at rest, due to low intramuscular Ca2+ levels. During exercise, Ca2+ release from the sarcoplasmic reticulum is the primary stimulus that coarsely activates PDH whereas changes to pyruvate concentration, energy charge, and possibly redox fine-tune this activation (see  for review), in order to match PDH activation to the demand for CHO oxidation .
In addition to the importance of intramitochondrial effectors to the acute regulation of PDH activation in the first few seconds or minutes, long-term or chronic alterations to the activation state of PDH can be accomplished through stable changes in the absolute levels of PDK and/or PDP. The rate of activation of PDH is dependent on the activity ratio of PDK and PDP, and changes in the expression of either covalent modifier would alter the rate of activation or inactivation of PDH. These chronic alterations occur over hours or days and are independent of acute changes in intramitochondrial effector concentrations.
These increased levels of PDK4 protein and PDK activity would be expected to render the complex resistant to activation during exercise, as observed by Putman et al.  for two reasons: 1) increased multi-site phosphorylation of the PDH complex, and/or 2) decreased sensitivity of the complex to regulation by pyruvate. Increased PDK activity would be expected to enhance multi-site phosphorylation of the E1 subunit and make the complex more resistant to dephosphorylation and activation by the phosphatase . Under normal dietary conditions the predominant isoform in human skeletal muscle is PDK2, which has a greater affinity for phosphorylation of site 1 (the inactivating site) of the E1 subunit [15–17]. However, as the population of PDK4 isoform increased, there would be enhanced phosphorylation of the 2nd (barrier) site, since this isoform has a greater affinity for both site 1 and site 2 [15–17]. As well, PDK2 has a greater sensitivity to inactivation by pyruvate than PDK4 . Thus at the onset of exercise with increased glycolytic flux, the increased levels of PDK4 protein would render the complex more resistant to activation due to increased PDK4 kinase activity even in the face of elevated muscle pyruvate concentrations .
A confounding factor in the Putman study was that subjects had undergone intense glycogen depleting exercise protocols prior to both dietary interventions, so the initial levels of skeletal muscle glycogen and glycogen utilization was considerably lower following the LCD . In a subsequent study, subjects were asked to refrain from intense exercise throughout the study, and a LCD (~3% carbohydrate) was compared to a mixed diet (~55–60% carbohydrate) instead of a high-carbohydrate diet . Subjects followed each 6 d dietary intervention with 30 min exercise at a slightly lower workload (65% VO2max). The object of the study was to match as closely as possible the glycogen utilization during exercise between the two trials. Although the initial skeletal muscle glycogen concentration was still ~50% lower in the LCD compared to the mixed diet, skeletal muscle glycogen utilization and pyruvate accumulation were similar during the 30 min of exercise in both trials. Unlike the attenuated activation of PDHa at the onset of exercise which was observed in the Putman study , these authors observed that the activation during exercise was identical between the two conditions. Thus, in spite of the fact that PDK activity and PDK4 isoform would be expected to increase to a similar extent as previous studies , these effects were overridden when initial muscle glycogen levels were higher and glycolytic flux to pyruvate was maintained . It is clear from these studies that the intensity and duration of the exercise play a role in the regulatory changes observed during exercise following a LCD. As exercise intensity increases, the demand for muscle and liver glycogenolysis and muscle carbohydrate oxidation increases. These stores are not fully replenished following a very low carbohydrate diet, and therefore during intense exercise glycogenolytic flux and PDH activation are decreased following a LCD.
In humans, there is little information on how rapidly the LCD-adapted increase in PDK activity and PDK4 protein may be reversed with carbohydrate re-feeding. Most re-feeding studies have used prolonged fasting as a perturbation, and very little work has been done in human skeletal muscle. In rodents, early studies in cardiac muscle indicated that re-feeding following 6 h starvation recovered PDHa activity to ~75% of normal levels in as little as 1–2 h. However, as the duration of the starvation period increased, the time course of the response to re-feeding was longer, in such that after 48 h of starvation, PDHa activity recovered to only ~25% of control values after 4 h . In later rodent studies, this increasing resistance to PDH complex activation was accompanied by increased PDK activity, which correlated with the duration of the fast or high-fat diet [25, 26]. Following 48 h starvation and re-feeding, PDK activity and PDK4 protein in skeletal muscle decreased ~50–60% in approximately 4 h of re-feeding . However, little is known about the time course reversion of PDK activity and PDK isoform expression following a LCD in human skeletal muscle.
In human skeletal muscle, Pilegaard et al.  recently examined changes in PDK4 mRNA concentrations in human skeletal muscle following fasting and re-feeding. Subjects fasted for 20 h and then were given either a high-carbohydrate meal or a high-fat meal. In muscle biopsies taken 1 h after the re-feeding meal, these authors found increased transcription rate and mRNA concentration of the PDK4 isoform regardless of the composition of the meal. Based on rodent studies of PDK activity, these data were unexpected, since it would be expected that the high-carbohydrate meal would suppress PDK4 gene expression. These data suggested that the skeletal muscle PDK 4 gene was very sensitive to metabolic disturbances. However, without measurement of PDK or PDH activity, the study gave little information regarding how quickly the fasting-induced increase in PDK activity was reversed with carbohydrate re-feeding. A recent study examining carbohydrate re-feeding following a 6 d LCD indicates that PDK activity is rapidly reversed and PDHa activity has fully recovered in as little as 3 h in resting human skeletal muscle . Thus, the adaptive change in PDK activity observed in human skeletal muscle is rapidly reversed with re-feeding of carbohydrates, regardless of potential changes in PDK4 mRNA expression .
There is little information regarding the skeletal muscle adaptation on "the fat side" to a LCD. In human skeletal muscle, most studies restrict their measurements to gene expression or mRNA concentrations of the pertinent enzymes involved in fat oxidation, and very few have measured the more physiologically relevant concentrations of enzyme activity or protein concentration. Still, there is evidence in human skeletal muscle for increased activities of several regulatory enzymes and proteins in skeletal muscle fatty acid uptake and oxidation following high fat diets or LCD. Key steps include delivery of fatty acids to the muscle through muscle lipoprotein lipase (LPL), sarcolemmal fatty acid transporters and plasma membrane fatty acid binding proteins (FAT/CD26 and FABPpm respectively), mitochondrial uptake and oxidation through carnitine palmitoyl transferase I (CPT I), fatty acid beta-oxidation (marker enzyme β-hydroxy acyl CoA dehydrogenase (β-HAD)), and general oxidative capacity (marker enzyme citrate synthase (CS)).
In response to a 4 week adaptation to a high fat (~62% fat) moderate LCD (~20% CHO), skeletal muscle LPL activity increased almost 2-fold, increasing fatty acid availability to the muscle and increasing intramuscular triglyceride content significantly . In terms of muscle fatty acid uptake, there is evidence that the FAT/CD36 protein and mRNA were increased modestly (1.25-fold) after only 5 d on a moderate LCD (20% CHO), while FABPpm gene expression and protein content were unaffected by the diet . In general, muscle uptake of fatty acids and very low density lipoprotein triglycerides, as well as plasma fatty acid oxidation were higher during exercise following a fat-rich LCD (21% CHO) when exercise training was combined with the diet perturbation .
In human studies, skeletal muscle CPT I is unaffected by LCD. This was demonstrated at the level of maximal enzyme activity following a 6 d LCD (~3% CHO) , and mRNA levels following a 5 d LCD (19% CHO) . However, in skeletal muscle of rats fed a high-fat diet, CPT I enzyme activity capacity was increased up to 1.3 to 2-fold at 10 weeks, depending on the fatty acid composition of the diet . In another rat study, increased gene expression of CPT I mRNA appears to be restricted to type I slow oxidative muscle fibers, since a significant increase was only documented in the soleus muscle, and not the extensor digitorum longus following 8 weeks of a high-fat LCD (0% CHO) . Taken together, these studies suggest that the short term 5–6 d LCD perturbation may not be prolonged enough to evoke a significant change in activity or gene expression of this enzyme which regulates transport of fatty acids into the mitochondria for oxidation. This is further supported by the fact that in well trained human subjects, maximal CPT activity was modestly increased following a prolonged (4 week) very low LCD (<20 g CHO), although it is not clear from this data whether this measurements included CPT I and CPT II activity together .
Increased activity of a key marker enzyme for fatty acid beta-oxidation has been observed in human skeletal muscle during prolonged LCD perturbations as well. Although a 6 d LCD (3% CHO) did not alter β-HAD activity , Helge et al.  observed increased β-HAD activity following a 4 week LCD (20% CHO) perturbation in untrained subjects. However, they found no increase in either whole body VO2max or CS activity, suggesting that the increase in beta-oxidation was specific rather than a generalized increase in oxidative capacity. Similarly, a more carbohydrate restricted diet (3% CHO) for 6 d did not alter CS activity in human skeletal muscle . In contrast, results from some rat studies have demonstrated modest increases in CS activity of approximately 20% [37–39], with the largest increases demonstrated in type IIb fibers . Although the increase in β-HAD activity in human skeletal muscle and possibly CS in rat muscle could potentially suggest an increase in oxidative capacity, recent research has demonstrated that there was no difference in human skeletal muscle mitochondrial density (as determined by electron microscopy), even though there was an increase in fat oxidation at rest and during incremental exercise following a 5 week high fat LCD (25–30% CHO) .
In summary, following a 6 d LCD in human subjects, PDHa activation is attenuated during intense exercise and this is due at least in part to increased PDK activity and PDK4 gene expression. This decreased activation of PDHa decreases carbohydrate and increases fat oxidation during exercise. PDK activity increases in as little as 24 h on a LCD, and PDK activity increases linearly over the 6 d. Impaired glucose clearance in response to an oral glucose tolerance test was observed in healthy subjects following only 56 h of LCD, but this may be dependent on the fatty acid composition of the diet. With re-feeding of carbohydrates, PDK activity drops to pre-diet levels in 3 h, although this does not appear to correlate with mRNA concentration. If intense exercise is restricted and muscle glycogen stores and utilization rates are preserved during the LCD, the activation of the PDH complex is similar to that following a mixed diet.
The up-regulation of enzymes involved in muscle fatty acid uptake and fat oxidation appears to be slower to response to a LCD perturbation. In addition, these adaptations appear to be of a smaller magnitude. In human studies there is evidence that muscle uptake of fatty acids is up-regulated by LCD through increased maximal activity of LPL and increased FAT/CD36. However, the maximal rate of mitochondrial transport of fatty acids through CPT I appears to be resistant to adaptive changes in response to the diet. In addition, although increased maximal β-HAD activity has been documented in response to LCD, there is no evidence that the overall oxidative capacity is elevated following a LCD in human skeletal muscle.
S.J. Peters is supported by the National Science and Engineering Research Council (NSERC), and P.J. LeBlanc is supported by an NSERC post-doctoral fellowship.
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