To our knowledge, this is the first study to investigate the acute effect of insulin on muscle ATP turnover rate, and to track the time course of ATP turnover rate during physiological hyperinsulinaemia. Insulin is responsible for increasing whole body glucose disposal mainly by stimulating glycogen synthesis in the transition from fasted to fed state (23
). Carbon-13 magnetic resonance spectroscopy studies have shown physiological hyperinsulinaemia to increase muscle glycogen concentration in as little as 15 min in healthy individuals (24
), which is reflected in the rapid increase in glucose disposal rate observed in our experiments.
The glucose infusion rate data shown in , illustrate that the highest rate of change in glucose disposal occurs in the first 30–60 min of euglycaemic hyperinsulinaemia. The ATP turnover rate was unchanged during the first 20–50 min of the clamp, with an 8% increase after 50 min. There are notable differences with other reported measurements of ATP turnover rate during physiological hyperinsulinaemia. The single most important difference lies in the timing of the measurements. The original report of mitochondrial dysfunction associated with diabetes measured ATP turnover rate averaged over 30–150 min of euglycaemic hyperinsulinaemia (10
) while subsequent studies compared the later effects (120–350 min) of insulin stimulation of ATP turnover rate (8
). The long scan duration limited the time resolution and prevented examination of time course. However, in order to determine the relationship between parameters it is important to measure ATP turnover rate during the onset of insulin action, as early as 15 min of insulin stimulation (24
). Other processes, separate from insulin's effect on muscle glycogen synthesis, such as insulin's effect on mitochondrial fusion and proliferation (26
) and mitochondrial protein synthesis (28
), may affect ATP turnover rate on a timescale of 4–8 h of insulin stimulation. These processes are temporally dissociated from and not relevant to the early metabolic effects of insulin. They are likely to affect data on ATP turnover rates in the longer studies of prolonged hyperinsulinaemia.
In the present study insulin stimulation for 150 min brought about an 8% increase in ATP turnover rate, less than the 11–90% previously observed in normal control subjects in the literature (2
). The report of a 90% increase in ATP turnover rate in insulin-sensitive subjects with insulin stimulation (2
) related to extreme phenotypes of insulin sensitivity. Additionally, the insulin resistant offspring were reported to have a low basal resting ATP turnover rate. This has not been confirmed in subsequent studies comparing subjects with and without type 2 diabetes when subjects have been matched for habitual physical activity (9
). During a 240 min period of hyperinsulinaemia, Szendroedi et al.
reported a 10.5% increase in ATP turnover rates in the diabetic subjects, 10.6% in the age matched controls, and a 26% increase in ATP turnover rate in the young controls (11
). It has been widely postulated within the MR and diabetes communities that defects in mitochondrial function may underlie the reduced glucose disposal rate during hyperinsulinaemia in type 2 diabetes (11
). Although the data suggest a relationship between insulin sensitivity and ATP turnover rate, this does not indicate cause and effect. Hence, the extrapolation of hypothesis from the original observations (2
) to the pathophysiology of type 2 diabetes itself has not been well justified.
In addition to the shorter period of insulin stimulation examined in the present study as explanation for observing a lesser magnitude of increase of ATP turnover rate with insulin, other factors such as surface coil placement, coil diameter and acquisition sequences could affect the results in comparing between studies. The relative composition of the r.f. coil's sensitive volume of gastrocnemius and soleus muscles requires consideration as soleus muscle, which has predominantly Type I muscle fibres, has been shown to have higher resting ATP turnover rate than gastrocnemius (29
). We used a larger coil than used by Szendroedi et al.
) (14 cm diameter compared to 10 cm) and thus would expect to have a higher percentage of contribution of signal from the soleus muscle.
ATP turnover rate is calculated from both the intramyocellular Pi
concentration and the measured rate constant k1
. To ensure our estimates of ATP turnover rate were as accurate as possible during the rapidly changing Pi
concentration in the first hour after insulin stimulation, the average of the Pi
concentrations before and after the 27-min saturation transfer measurement was used. We observed insulin-stimulated Pi
concentration to increase by 14% until the end of the euglycaemic hyperinsulinaemic clamp, in agreement with previous studies (11
). The concentration of Pi
in the muscle tissue may be an important mechanism by which insulin regulates ATP synthesis (31
): there was no change in measured reaction rate, k1
across the two hour duration of this clamp, or in any of the parameters measured to calculate k1
. Such changes were implied by the early work in this field on young insulin sensitive volunteers (2
Measurements of ATP turnover rate by 31
P saturation transfer method report flux of ATP through Pi
incorporation into ATP, and are thus comprised of ATP generated by glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase reactions as well as by ATP synthase. This could result in a mismatch between oxygen consumption and ATP synthesis (32
), as demonstrated in studies performed in aerobic yeast suspensions Saccharomyces cerevisiae
) and perfused rat myocardium (33
). However, arterio-venous balance studies across the leg have shown that there is no net export of lactate from human muscle following insulinisation in either type 2 diabetic subjects or leaner normal controls such as used in the present study (34
). This net measurement does not exclude the possibility of a change in redundant cycling of 3-carbon compounds through the glycolytic exchange flux, but the extent to which this cycling contributes to the observed rate of overall ATP turnover still remains to be determined (35
A hallmark of muscle physiology is the ability to support a high energy demand for muscle function by phosphocreatine buffering of ATP, and by aerobic and anaerobic respiration. Thus postulation that muscle glycogen synthesis may be limited by ATP production, especially in resting muscle where ATP capacity is high, seems at odds with muscle function. In support of this, more recent reconsideration of the data supporting mitochondrial dysfunction as pathogenic for type 2 diabetes has concluded that the cause-and-effect relationship between mitochondrial dysfunction and insulin resistance is not supported by all data (9
For the first time, the early time course of skeletal muscle ATP turnover in response to insulin has been quantified in young, healthy controls. There was no increase in ATP turnover rate during the first hour of administering a high physiological concentration of insulin, the timescale where glycogen synthesis is initiated. ATP turnover rate was observed to increase only after 1 h, indicating that glycogen synthesis is not limited by ATP availability in healthy controls, with implications for hypotheses about the origins of defective insulin sensitivity in type 2 diabetes.