Glycolysis is independent of oxygenation state in stimulated human skeletal muscle in vivo

We tested the hypothesis that the cytoplasmic control mechanism for glycolysis is affected by the presence of oxygen during exercise. We used a comparison of maximal twitch stimulation under ischaemic and intact circulation in human wrist flexor and ankle dorsiflexor muscles. 31P magnetic resonance spectroscopy followed the phosphocreatine (PCr), Pi and pH dynamics at 6–9 s intervals. Glycolytic PCr synthesis was determined during stimulation from pH and tissue buffer capacity, as well as the oxidative phosphorylation rate. Ischaemic vs. aerobic stimulation resulted in similar glycolytic fluxes in the two muscles. The onset of glycolysis occured after fifty to seventy stimulations and the extent of glycolytic PCr synthesis was directly proportional to the number of stimulations thereafter. Two-fold differences in the putative feedback regulators of glycolysis, [Pi] and [ADP], were found between aerobic and ischaemic stimulation. The similar glycolytic fluxes in the face of these differences in metabolite levels eliminates feedback as a control mechanism in glycolysis. These results demonstrate that glycolytic flux is independent of oxygenation state and metabolic feedback, but proportional to muscle activation. These results show a key role for muscle stimulation in the activation and maintenance of glycolysis. Further, this glycolytic control mechanism is independent of the feedback control mechanism that governs oxidative phosphorylation. Glycolysis is commonly described as an anaerobic process providing ATP in the absence of oxidative phosphorylation. However, glycolytic flux in anoxic resting muscle is typically low and often does not generate sufficient ATP synthesis to meet ATP demands (Blei et al. 1993; Yamada et al. 1993). In contrast, far higher rates of glycolysis are found in aerobic muscle during exercise than in anaerobic muscles at rest. This high flux provides between 50 and 100 % of the substrate needed for oxidative phosphorylation in exercising muscle of humans (Brooks & Mercier 1994; Connett & Sahlin 1996). Clearly, glycolysis is not simply an anaerobic process and the mechanisms responsible for elevating flux under resting conditions cannot account for the high glycolytic flux found under aerobic exercising conditions. Muscle stimulation is a key difference between resting and exercising muscle that is related to the control of glycolysis and may account for the high glycolytic flux during exercise. Glycogenolytic and glycolytic fluxes vary with muscle stimulation rate under ischaemic or anoxic conditions in animal and human studies and are much larger than in unstimulated muscle (Karpatkin et al. 1964; Ozand & Narahara, 1964; Conley et al. 1997). Calcium activation of glycogenolysis is a well-known example of how substrate mobilization is related to muscle activation rate (see Connett & Sahlin, 1996). Similarly, a rate limiting enzyme in glycolysis, phosphofructokinase (PFK), may also undergo a reversible phosphorylation with muscle stimulation which decreases the Km, increases Vmax and facilitates binding to cytoskeletal elements (Luther & Lee, 1986). If Ca2+ activation governs both glycogenolysis and glycolysis, then glycolytic flux should be proportional to stimulation rate independent of oxygenation state or the rate of oxidative phosphorylation. Such an independent control of glycolysis and oxidative phosphorylation would account for the finding of lactate generation in fully aerobic animal and human muscle (Connett & Sahlin, 1996). An important barrier to characterizing the mechanisms governing glycolysis has been quantifying flux during exercise. Muscle biopsies are limited in the number of variables measured, the time resolution and the muscles that can be examined. The advent of non-invasive 31P magnetic resonance spectroscopy has overcome many of these limitations (Kemp et al. 1994). We have previously shown that 31P spectra contain sufficient information to characterize glycogenolytic and glycolytic flux with high time resolution under ischaemic conditions in human muscle in vivo (Conley et al. 1997). In this paper, we show how to extract the same information under aerobic exercise conditions in two muscle groups that are typically not biopsied. The purpose of this study was to test the mechanism of glycolytic control by comparison of glycolytic flux during aerobic and ischaemic stimulation in two muscle groups differing in contractile and oxidative properties. The first muscle group was a set of wrist flexors composed of primarily fast-twitch fibres (Johnson et al. 1973) and our measurements were centred over the flexor digitorum superficialis, as previously described (Blei et al. 1993; Conley et al. 1997). The second muscle group lies in the anterior leg compartment and has predominantly slow-twitch, aerobic fibres (Johnson et al. 1973). Our measurements were centred over the tibialis anterior. These experiments allowed us to determine whether muscle activation controls glycolytic flux independent of the oxygen content of the muscle and of oxidative phosphorylation.