Oxidation of plasma glucose

Plasma glucose oxidation showed an identical pattern to that of the Ra and Rd glucose. Lo-Glu caused a 44 % increase in plasma glucose oxidation compared with Fast and Hi-Glu produced a 126 % increase. The rate of disappearance ([6,6-²H₂]-glucose tracer) and the rate of oxidation ([U-¹³C]-glucose tracer) during the second hour of exercise are displayed in Fig. 5. Plasma oxidation was not significantly different from Rd glucose.

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Figure 5. Rd glucose versus plasma glucose oxidation Values are means ± S.E.M. The rate of disappearance of glucose (Rd) and the rate of plasma glucose oxidation (Rox) during the 60-120 min period with ingestion of water (Fast; open circles and squares), a low dose of glucose (Lo-Glu; shaded circles and squares) or a high dose of glucose (Hi-Glu; filled circles and squares). The Rd presented is calculated from measurements of [6,6-2H2]-glucose with GC-MS.

Muscle glycogen oxidation

Furthermore, whole body muscle glycogen oxidation (total CHO oxidation minus plasma glucose oxidation) was not reduced by CHO ingestion. Figure 6 describes the shifts in substrate utilization as a result of glucose ingestion. It should be noted that glucose feedings, markedly reduced fat oxidation and increased plasma glucose oxidation but did not alter the rate of muscle glycogen (and lactate) oxidation.

		View larger version [in this window] [in a new window]
Figure 6. The energetic contribution of substrates Values are means ± S.E.M. The energetic contribution of substrates during the 60-120 min period of exercise at 50 % VO2,max with water ingestion (Fast), a low dose of glucose (Lo-Glu) or a high dose of glucose (Hi-Glu).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Effect of carbohydrate ingestion on muscle glycogen oxidation

Here we report that muscle glycogen oxidation during exercise, measured using an indirect tracer approach, was not reduced by glucose ingestion. Even when large amounts of glucose were ingested (180 g h^-1) and plasma glucose and insulin levels were slightly, but significantly elevated, glycogen oxidation was similar to the fasted condition. This is in agreement with several direct measurements of muscle glycogen concentrations in muscle biopsies before and after continuous cycling exercise with or without carbohydrate ingestion (Fielding et al. 1985; Coyle et al. 1986; Flynn et al. 1987; Hargreaves & Briggs, 1988; Bosch et al. 1994). However, studies in runners showed that carbohydrate ingestion reduced the net glycogen breakdown in type I fibres and this might have been responsible for the observed improvements in endurance capacity (Tsintzas et al. 1995, 1996). Tsintzas & Williams (1998) suggested that continuous cycling would cause less marked changes in plasma glucose concentration and insulin concentrations compared with running and that these differences might play an important role in the effect on glycogen utilization to be observed. However, in the present study we observed a large insulin response to the carbohydrate feedings and also plasma glucose concentration was increased. Yet, this did not result in 'glycogen sparing' as proposed by Tsintzas & Williams (1998). Therefore, we feel that factors other than insulin are responsible for the discrepant findings in running (Tsintzas et al. 1995, 1996) and cycling (Fielding et al. 1985; Coyle et al. 1986; Flynn et al. 1987; Hargreaves & Briggs, 1988; Bosch et al. 1994).

It could be argued the exercise intensity in the present study was too low to increase glycogen breakdown and therefore the effects of glucose ingestion on glycogenolysis were small and not measurable. However, other studies (Coyle et al. 1986; Bosch et al. 1994) showed that exercise intensities of 70-75 % O₂,max did not result in glycogen sparing with glucose ingestion. In addition, higher exercise intensities might have abolished the insulin effect and, therefore, it would be more likely that glycogen sparing at lower exercise intensities was observed.

Percentage of Rd oxidized

Another important finding of this study was that the rate of disappearance (Rd) of glucose was closely matched by the plasma glucose oxidation rate (Figs 4 and 5). The percentages of Rd glucose oxidized were very high in the present study (96-100 %), but are in agreement with studies by Coggan et al. (1991, 1992), who observed that 93 % of Rd glucose was oxidized during exercise at 70 % O₂,max (Coggan et al. 1991). Others have found that lower percentages of Rd glucose were oxidized during exercise at intensities comparable with the intensity in the present study (50 % O₂,max) (Colberg et al. 1994; Roberts et al. 1996; Friedlander et al. 1997). For instance Roberts et al. (1996) observed that only 33-38 % of Rd glucose was oxidized and Colberg et al. (1994) reported 33-50 % of Rd was oxidized. However, these studies were performed at lower absolute exercise intensities and did not prime the bicarbonate pool as was done in the present study and it is thus possible that a fair amount of ¹³CO₂ was temporarily trapped in the bicarbonate pool which may have caused a marked underestimation of the true plasma glucose oxidation. Other studies in which a bicarbonate prime was given showed values more comparable with those in the present study (88-94 %) (Coggan et al. 1992).

It has been reported that when using a ¹³C-tracer for studying fatty acid metabolism part of the tracer may be trapped in exchange reactions with the tricarboxylic acid (TCA) cycle (Sidossis et al. 1995; Schrauwen et al. 1998). For example, some ¹³C-label may be incorporated into the glutamate-glutamine pool via -ketoglutarate, or into phosphoenolpyruvate via oxaloacetate. This label fixation results in a decreased recovery of label in the expired gases and in order to correct for this loss, the acetate correction factor has been proposed (Sidossis et al. 1995). The label loss is dependent on metabolic rate and at high oxygen uptakes (38-39 ml kg^-1 in the present study) recovery of the [1-¹⁴C]-acetate label has been shown to be 85-90 % (Sidossis et al. 1995). Besides that, [U-¹³C]-glucose has six labelled carbons of which two will appear directly in ¹³CO₂ and, therefore, do not enter the TCA cycle. Only two-thirds (66 %) of the glucose carbons is subject to label fixation. Therefore the recovery of carbons from [U-¹³C]-glucose will be higher than the recovery of [1-¹³C]-palmitate (Sidossis et al. 1995) or [U-¹³C]-palmitate (Schrauwen et al. 1998), explaining why we find that 96-100 % of the infused glucose tracer is oxidized. The small difference from 100 % may be explained by the acetate correction factor implying that all glucose molecules disappearing from the plasma might have been oxidized both in the presence and absence of glucose ingestion.

Plasma glucose turnover and the rate of ingestion

Plasma glucose has been shown to be an important substrate during exercise, especially during the later stages when muscle glycogen levels are reduced (Coggan, 1991). Here we observed that plasma glucose contributed 19 % to energy expenditure during fasting and this value increased to 42 % during Hi-Glu ingestion (Fig. 6). During Hi-Glu ingestion, plasma glucose was the most important substrate from a quantitative point of view.

Glucose turnover was markedly increased by the glucose ingestion as evidenced by increased Ra and Rd glucose, increased plasma glucose oxidation and metabolic clearance rate (MCR). The Lo-Glu ingestion increased Ra glucose by 44 % and Hi-Glu ingestion increased Ra glucose by 126 %. However, whereas glucose ingestion during Hi-Glu was five times larger than during Lo-Glu, the Ra glucose increased only less than twofold (72 µmol kg^-1 min^-1 during Lo-Glu and 109-113 µmol kg^-1 min^-1 during Hi-Glu). Mean ingestion during the second hour of exercise was 0·3 g min^-1 in the Lo-Glu and 1·5 g min^-1 in the Hi-Glu trials. The total rates of appearance of glucose were 0·84 and 1·36 g min^-1, respectively, suggesting that with the Lo-Glu feeding the liver was still producing glucose whereas with the Hi-Glu feeding it is theoretically possible that hepatic glucose production was completely suppressed. Dual tracer studies with infusion of one glucose tracer and ingestion of another are needed to distinguish between gut-derived Ra and hepatic glucose output. Without glucose ingestion the hepatic glucose production was approximately 0·5 g min^-1 indicating that the liver during the second hour of exercise may have produced about 30 g glucose. This is a relatively large amount since it is believed that the liver contains approximately 80 g of glycogen (Hultman & Nilsson, 1971). Glucose ingestion during exercise may reduce hepatic glucose production (i.e. spare liver glycogen) at 70 % O₂,max as shown by Bosch et al. (1994) and McConell et al. (1994).

Limitations of exogenous carbohydrate oxidation

These results also indicate that, with large doses of ingested glucose (360 g in the 2 h exercise period), not all the glucose appears in the plasma. This is in agreement with previous suggestions that the rate of exogenous glucose oxidation may be limited by the rate of digestion, absorption, maximal hepatic glucose output and subsequent transport of glucose into the systemic blood supply rather than by glucose uptake and oxidation by the muscle (Hawley et al. 1994). Several studies showed that oral carbohydrate oxidation is limited to about 1 g min^-1 (Hawley et al. 1992). Even when large amounts of carbohydrate were ingested oxidation rates did not exceed 1 g min^-1 (Rehrer et al. 1992; Wagenmakers et al. 1993).

Effects of carbohydrate ingestion on fat and carbohydrate oxidation

Glucose ingestion also markedly suppressed fat oxidation compared with fasting and this effect was dose dependent; Hi-Glu suppressed fat oxidation more than Lo-Glu. The lower rates of fat oxidation may be at least in part insulin mediated (Fig. 1). Insulin has been shown to be a potent inhibitor of lipolysis and Ra FFA (Montain et al. 1991; Campbell et al. 1992; Horowitz et al. 1997). Here, higher insulin levels after glucose ingestion reduced whole body lipolysis as indirectly indicated by lower plasma glycerol and FFA concentrations (Fig. 1). Decreased FFA availability and higher plasma insulin concentrations along with an increased glycolytic flux have been shown to reduce FFA oxidation at rest (Sidossis & Wolfe, 1996) and during exercise (Coyle et al. 1997). Although in this study we observed that glucose ingestion during exercise increased total CHO oxidation, McConell et al. (1994) and Bosch et al. (1994) did not observe increased CHO oxidation rates with CHO ingestion during exercise. This may, in part, be explained by the differences in the exercise intensity and possibly the difference in plasma insulin concentration, which may have been higher in the present study. Whereas in their studies the exercise intensities were 70 % O₂,max, we employed an exercise intensity of 50 % O₂,max. At low to moderate exercise intensities (50 % O₂,max) glucose utilization may be enhanced by CHO ingestion whereas during more intense exercise ( > 70 % O₂,max) exogenous CHO simply seems to substitute for endogenously produced glucose, with no overall increase in CHO oxidation (Bosch et al. 1994; McConell et al. 1994).

Gluconeogenesis (glucose carbon recycling)

When measured by GC-IRMS [U-¹³C]-glucose carbon may recycle during the process of gluconeogenesis and this may lead to an underestimate of the Ra of glucose. The reason for this is the following. One molecule of uniformly labelled glucose produces two molecules of uniformly labelled pyruvate. This pyruvate may be converted into lactate or alanine (predominantly lactate) which are precursors for gluconeogenesis and this may lead to the incorporation of label into newly synthezised glucose. Fifty percent of the carbon atoms will be ¹³C in the newly synthezised glucose. In GC-combustion-IRMS analysis the whole molecule is oxidized to CO₂ prior to the enrichment measurement and therefore the increase in ratio ¹³C/¹²C of all carbons of the glucose molecule is measured. Tracer dilution measurements by GC-combustion-IRMS analyses of this tracer therefore exclude gluconeogenesis and consequently represent a possible underestimation of the true Ra of glucose. It is believed that a more appropriate method of measuring total Ra glucose is the use of a [6,6-²H₂]-glucose tracer which measures total glucose production when the enrichment is measured by GC-MS as the tracer atoms do not recycle during gluconeogenesis (Wolfe, 1992). To compare the above mentioned methods we also infused a [6,6-²H₂]-glucose tracer. The difference between the two methods theoretically gives an estimate of glucose carbon recycling and a rough estimate of gluconeogenesis (Tserng & Kalhan, 1983). In this study we found that Ra and Rd glucose were similar, regardless of the tracer used (i.e. [U-¹³C]-glucose measured by GC-IRMS or [6,6-²H₂]-glucose measured by GC-MS) (Table 3). In Fig. 4, Ra glucose calculated from the [6,6-²H₂]-glucose tracer is plotted against the Ra glucose calculated from the [U-¹³C]-glucose tracer (total enrichment). This figure shows that, generally, Ra glucose calculated from the [U-¹³C]-glucose tracer gave slightly lower numbers, which is in agreement with the expectations. The recycling rate of ¹³C was low (4-10 % of total Ra glucose) and was not reduced by the glucose feedings, suggesting that independently of the amount of glucose ingested, gluconeogenesis was minimal. These low rates of recycling may have resulted from low precursor availability (i.e. low lactate concentrations as observed in previous studies with similar designs (Jeukendrup et al. 1996a, c, 1997b). Gluconeogenesis may also be lower in trained individuals (Coggan, 1997) and since the training level of the subjects in the present study was very high, the rates of gluconeogenesis may have been very low. Friedlander et al. (1997) also suggested that adrenoceptors were downregulated in response to endurance training and this may be a mechanism for the blunted gluconeogenesis. With the high training level of the athletes in the present study this could be an explanation for the low levels of glucose carbon recycling as well.

General overview: effect of carbohydrate feedings on substrate metabolism

The results of the present study indicate that glucose ingestion during exercise leads to a decreased fat oxidation, partly because of an inhibition of lipolysis (Horowitz et al. 1997) and an increased carbohydrate oxidation. The increased carbohydrate oxidation was explained by an accelerated plasma glucose turnover. The appearance of glucose in the blood stream increased with carbohydrate feedings and this increase was dose dependent. However, at very high ingestion rates the appearance of glucose in plasma levelled off.

The increased rate of appearance of glucose did not lead to a large increase in the plasma glucose concentration because the rate of disappearance is also increased. Higher glucose concentrations may have been partly responsible for the increased glucose uptake during Hi-Glu, simply by mass action. However, the augmented glucose uptake was mainly explained by an increased clearance of glucose from the bloodstream. This seems to suggest that glucose transport was activated by mechanisms involving GLUT4 translocation, activation of the oxidative enzymes (phosphofructokinase (PFK) and/or activation of the pyruvate dehydrogenase (PDH) complex).

The glucose that disappeared from the plasma (and most likely was taken up by active skeletal muscle) was oxidized and not used for glycogen synthesis. Muscle glycogen oxidation was not reduced by the glucose feedings. Gluconeogenesis in the liver was negligible in all conditions.

In summary, a method was developed to measure Ra glucose and plasma glucose oxidation by using small amounts of a [U-¹³C]-glucose tracer and measuring plasma tracer enrichment with GC-IRMS. The method seems to be valid to make estimates of glucose turnover and for indirect estimates of muscle glycogen consumption at whole body level. The results of this study indicate that carbohydrate feeding during cycling exercise at 50 % O₂,max in highly trained subjects decreased fat oxidation and increased the Ra of glucose, the MCR and plasma glucose oxidation. Muscle glycogen oxidation was not reduced even when large amounts of glucose were ingested and non-oxidative glucose disposal was negligible. A large percentage (96-100 %) of glucose disappearing from the plasma was oxidized. Comparison of Ra glucose calculated from [6,6-²H₂]- and [U-¹³C]-glucose tracers revealed that only a small quantity of glucose carbons is recycled in the process of gluconeogenesis.

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Acknowledgements

This study was supported by a NOVARTIS nutrition research grant (NOVARTIS Nutrition, Bern, Switzerland). We also greatly appreciate the co-operation of the subjects and the excellent technical and analytical support of Mr Frank van de Vegt and Mr Joan Senden.

Corresponding author

A. E. Jeukendrup: School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

Email: A.E.Jeukendrup{at}bham.ac.uk

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				G. K McConell, R. S Lee-Young, Z.-P. Chen, N. K Stepto, N. N Huynh, T. J Stephens, B. J Canny, and B. E Kemp Short-term exercise training in humans reduces AMPK signalling during prolonged exercise independent of muscle glycogen J. Physiol., October 15, 2005; 568(2): 665 - 676. [Abstract] [Full Text] [PDF]