INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

DURING HIGH-INTENSITY physical exercise, splanchnic blood flow decreases by ~70% of the resting value, whereas hepatic glucose production can increase by 5- to 10-fold over resting rates (26, 31). The reduction in splanchnic blood flow is thought to reflect increased local vascular resistance mediated by increased sympathetic nervous activity (25). However, the evidence for this is very limited and based on correlations between a marked reduction in splanchnic blood flow and a rise in plasma norepinephrine and heart rate rather than cause-effect relationships (25). Although an effect of sympathetic nerves in decreasing splanchnic blood flow has been demonstrated at rest by direct stimulation of the sympathetic nerves to the gut (24), during exercise pharmacological blockade of the sympathetic nervous activity to the splanchnic area did not influence the exercise-induced increase in hepatosplanchnic vascular resistance (18). Furthermore, exercise in subjects with sympathetic denervation due to primary autonomic failure is also associated with a full, albeit slower, decrease in splanchnic blood flow compared with control subjects (22). These findings strongly point at other mechanisms involved in the regulation of the hepatosplanchnic blood flow during exercise.

The role of ANG II in splanchnic blood flow regulation has been highlighted with the use of angiotensin-converting enzyme (ACE) inhibitors during lower body negative pressure (23) and head-up tilt in humans (29). In these conditions the use of ACE inhibitors suppressed the reduction of splanchnic blood flow normally associated with both of these stimuli. Furthermore, just like heart rate and plasma norepinephrine, the angiotensin plasma concentration increases during exercise in an intensity-dependent manner (7). Thus angiotensin is a likely candidate for regulation of splanchnic flow during exercise. In line with this, Symons and Stebbins (30) demonstrated that ANG II had an important role in the reduction of splanchnic blood flow during exercise in miniswine by using ANG II-receptor antagonists. However, there is still no evidence for this regulatory mechanism to be effective in humans during dynamic prolonged exercise.

In addition to this, the rise in hepatic glucose production during exercise cannot be completely accounted for by already described hormonal mechanisms such as the rise and fall of glucagon and insulin concentrations, respectively. Even when counterregulatory hormones are kept constant, epinephrine is absent, and the sympathetic nervous activity to the liver is blocked or the liver is denervated, the rise in hepatic glucose production during exercise still occurs (3, 6, 14, 18, 28, 33). Interestingly, ANG II has been shown to acutely increase gluconeogenesis (5, 34) and liver glycogenolysis (8, 13), but its role in endogenous glucose production during exercise in humans has yet to be determined. On the basis of these findings, it was hypothesized that the renin-angiotensin system is important for the decrease in hepatosplanchnic blood flow and the increase in glucose production during exercise in humans. Accordingly, in the present study, human subjects were exercised either with or without an infusion of an ACE inhibitor (enalapril) both at mild and moderate exercise levels during which simultaneous measurements of hepatosplanchnic blood flow and glucose production, as well as hormonal and metabolic changes across the splanchnic bed, were performed.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects. Eight young men [mean age, 25 yr (range 23-30 yr); height, 182 (174) cm; weight, 79 (67-95) kg; and maximal O₂ consumption (O_2 max), 50 (46-60) ml · kg¹ · min¹] gave informed written consent to participate in this study. The protocol was approved by the Ethics Committee of Copenhagen. The O_2 max for each individual was determined 4-8 days before the experimental study using a protocol of incremental workloads (2 min at each workload) to exhaustion on a modified electromagnetically braked Krogh cycle ergometer. While the subjects cycled, the upper part of the body formed an angle of 45° with the horizontal plane. O_2 max was the highest O₂ consumption (O₂) attained during the latter stages of the test and was accompanied by a respiratory exchange ratio of more than 1.1, a heart rate close to the age-predicted maximum (220 beats/min  age), and a leveling-off phenomenon of O₂. None of the subjects was taking medications or had any history of endocrine disease. All subjects refrained from training, smoking, and drinking alcohol the day before the experiment. Seven subjects were nonsmokers, whereas one subject smoked in social gatherings. His results did not differ from the others.

Procedures. For this crossover study, subjects arrived at the laboratory at 8:00 A.M. on two separate occasions with an interval of 2-3 wk between randomized control or ACE-blockade experiments. For the purpose of taking blood samples, a cannula was inserted in the radial artery of the nondominant arm of each subject. A venous line was inserted in a forearm vein for infusion and indocyanine green (ICG) dye. Additionally, a separate catheter was inserted into another forearm vein for infusion of radiolabeled [3-³H]glucose to compare the radioisotopic dilution method with the arteriohepatic venous balance technique for determination of endogenous glucose production during exercise as described in our previous paper (2). In all subjects a catheter was introduced into a femoral vein and advanced, under fluoroscopy, into a right-sided hepatic vein approximately 3-4 cm from the wedge position. Its location was verified after exercise by ultrasonography (2). Patency of the catheter was maintained by flushing with heparinized-isotonic saline solution (10 U heparin/ml).

Tilt-table experiment. After completion of the ergocycle exercise protocol, subjects were moved to a tilt table, on which they lay horizontally for 1 h. The subjects were then submitted to an orthostatic challenge by raising the table to a 30% head-up angle for a period of 10 min to test for the effectiveness of the drug on blocking the angiotensin response. Blood pressure and heart rate were monitored throughout this time interval (every minute), and blood sampling was performed immediately before and at the end of the orthostatic challenge before the subjects were brought back to a horizontal lying position. Values reported were obtained after 10 min of tilting. To minimize the venous return from the lower limbs during this experiment, subjects were held in position by a bicycle saddle and were told not to contract their leg muscles.

Analytical methods. The estimated splanchnic blood flow (ESBF) was measured by the ICG dye excretion method (26) as described in detail previously (2). Briefly, ICG clearance is calculated from infusion rate and plasma concentrations of ICG. Assuming that ICG is only eliminated in the liver and that hepatic extraction is complete, splanchnic blood flow can be calculated from ICG clearance and hematocrit (2). Hormonal and metabolite exchange data measured across the splanchnic bed were the product of estimated splanchnic plasma flow and the arteriohepatic venous difference in plasma concentration. Plasma glucose and lactate concentrations were immediately determined with an automatic glucose analyzer (YSI 23AM; Yellow Springs Instruments). Catecholamine concentrations were determined by a single-isotope radioenzymatic method (1) evaluated previously (21). The concentrations of insulin, glucagon, growth hormone, cortisol (10), adrenocorticotropic hormone (ACTH) (9), C peptide (12), and ANG II (16) were determined by radioimmunoassay as described previously. Free fatty acids, glycerol, -hydroxybutyrate, and alanine were measured by enzymatic fluorimetric methods. Hematocrit was measured by the microhematocrit method. Blood oxygen saturation and hemoglobin were measured by an automated gas and acid-base balance microanalyzer (ABL 4, Acid Base Laboratory, Radiometer, Bagsvaerd, Denmark) and an OSM3-hemoxymeter immediately after samples had been drawn. Interassay and intra-assay coefficients of variation were 3 and 4%, respectively, for oxygen saturation and 2% for hemoglobin.

Statistical analysis. Statistical analysis was performed using a two-way ANOVA with repeated-measures design. Tukey tests were used for post hoc analysis in the event of significant effect of treatment or time. A 95% level of confidence (2-tailed testing) was accepted for all comparisons. All data are reported as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

ANG II concentrations. ANG II plasma levels at rest were similar in both groups (Fig. 1). However, ACE blockade resulted in decreased plasma ANG II concentrations 30 min after the infusion of enalapril (30 min, 8.31 ± 1.20 vs. 0 min, 2.61 ± 0.37 pg/ml; P < 0.05). Subjects completed 40 min of cycling at 50.9 ± 1.5 and 50.4 ± 1.5% O_2 max [not significant (NS)] followed by 30 min of cycling at 68.8 ± 2.4 and 69.0 ± 2.2% O_2 max (NS) in the enalapril-infused (ACE blockade) and the control groups, respectively (Table 1). In the control trial, plasma concentrations of ANG II increased by approximately 4- and 12-fold during dynamic exercise at 50 and 70% O_2 max, respectively. In contrast, ACE blockade efficiently inhibited the exercise-induced elevation of ANG II (Fig. 1). One hour after exercise, before raising the subjects to a 30° angle on a tilt table, ANG II plasma concentrations were more elevated in the control group compared with the ACE-blockade trial; the values were, respectively, 10.7 ± 2.6 and 4.6 ± 0.9 pg/ml (P < 0.05). The infusion of the ACE inhibitor was successful in preventing a rise in ANG II concentration in response to the passive 10-min head-up 30% tilt, whereas hormone concentrations were significantly increased in the control group (17.9 ± 3.6 pg/ml; P < 0.05) when blood was withdrawn while subjects were still in the tilted position.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Arterial plasma concentrations of ANG II from human male subjects (n = 8) studied twice either with or without (control) angiotensin-converting enzyme (ACE) blockade (enalapril, 10 mg iv) administered 30 min before exercise. Measurements were performed at rest and during exercise on a cycle ergometer for 40 min at 50% of maximal oxygen consumption (O2 max) and 30 min at 70% O2 max. Inset: ANG II data obtained before and at the end of a 10-min passive head-up tilt performed 1-h postexercise. Values are means ± SE. * Data from both ACE-blockade conditions at 0 min is significantly different from corresponding 30-min value (P < 0.05).  Significantly different from corresponding ACE-blockade value (P < 0.05). § Significantly different from corresponding pretilt data (P < 0.05).

Heart rate, blood pressure, and respiratory exchange ratio at rest and exercise. O₂, respiratory exchange ratio, heart rate, and systolic blood pressure increased during exercise (P < 0.05), and significant differences between the two experiments were found neither at rest nor during exercise. Mean arterial pressure remained stable, whereas diastolic blood pressure decreased similarly in both groups throughout the course of exercise compared with resting values. The perceived rate of exhaustion (Borg scale) increased similarly in both groups during exercise.

Splanchnic O₂ uptake and blood flow. ESBF at rest was not significantly different between ACE-blockade and control groups (Fig. 2). ESBF and splanchnic vascular resistance, respectively, decreased and increased progressively in both groups, reaching significant difference compared with rest during exercise intensity of 70% O_2 max (P < 0.05). No significant differences of ESBF and splanchnic vascular resistance were found between the two experimental conditions. Splanchnic O₂ uptake rates were similar under resting conditions in ACE-blockade and control groups and increased during exercise (P < 0.05) (Table 1). Hematocrit values rose, respectively, from 45.4 ± 0.7 at rest to 47.3 ± 0.7 and 47.7 ± 1.1% in the control group and from 45.8 ± 0.5 to 48.0 ± 0.5 and 48.2 ± 0.8% in the ACE-blockade group during mild and moderate exercise. There were no significant differences between the two conditions.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Estimated splanchnic blood flow (ESBF) from human male subjects (n = 8) studied twice either with or without ACE blockade (enalapril; 10 mg iv) administered 30 min before exercise. Measurements were performed at rest and during exercise on a cycle ergometer for 40 min at 50% of O2 max and 30 min at 70% O2 max. Values are means ± SE. * Data from both ACE-blockade and control conditions are significantly different from corresponding resting values (P < 0.05).

Arterial metabolites and hormone plasma concentrations. Both cortisol and epinephrine plasma concentrations increased during exercise. The concentrations of cortisol were higher during ACE blockade compared with the control trial throughout the duration of the protocol (P < 0.05), whereas the difference in epinephrine levels did not attain statistical significance (Table 2 and Fig. 3, respectively). Other counterregulatory hormones fluctuated similarly in both ACE-blockade and control groups (Fig. 4 and Table 2). Insulin and C peptide concentrations decreased progressively and similarly during exercise in both ACE-blockade and control groups (Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Arterial epinephrine plasma concentrations from human male subjects (n = 8) studied twice either with or without ACE blockade (enalapril; 10 mg iv) administered 30 min before exercise. Measurements were performed at rest and during exercise on a cycle ergometer for 40 min at 50% of O2 max and 30 min at 70% O2 max. Values are means ± SE. * Data from both ACE-blockade and control conditions are significantly different from corresponding resting values (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Arterial norepinephrine plasma concentrations from human male subjects (n = 8) studied twice either with or without ACE blockade (enalapril; 10 mg iv) administered 30 min before exercise. Measurements were performed at rest and during exercise on a cycle ergometer for 40 min at 50% of O2 max and 30 min at 70% O2 max. Values are means ± SE. * Data from both ACE-blockade and control conditions are significantly different from corresponding resting values (P < 0.05).

Splanchnic exchange. Splanchnic glucose output nearly doubled during mild exercise (50% O_2 max) and further rose to 250-300% of initial resting values during moderate exercise (~70% O_2 max) (Fig. 5). Splanchnic glucose output at rest did not differ between the ACE-blockade group and the control group. Furthermore, the increased splanchnic glucose output observed during exercise was not affected by the absence of a normal rise in plasma ANG II concentrations in the ACE-blockade group compared with the control group. Similar values were also found both at rest and during exercise in the ACE-blockade and control groups when endogenous glucose production was assessed using the radioisotopic dilution method (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Splanchnic glucose output (SGO) from human male subjects (n = 8) studied twice either with or without ACE blockade (enalapril; 10 mg iv) administered 30 min before exercise. Measurements were performed at rest and during exercise on a cycle ergometer for 40 min at 50% of O2 max and 30 min at 70% O2 max. Values are means ± SE. * Data from both ACE-blockade and control conditions are significantly different from corresponding resting values (P < 0.05).

Head-up tilt. During the short orthostatic challenge, mean arterial blood pressures were similar in both groups and did not change significantly. Mean arterial blood pressures before and during the orthostatic challenge were 78.3 ± 0.6 and 80.5 ± 0.5 mmHg and 77.1 ± 1.0 and 77.5 ± 0.5 mmHg, respectively, in ACE-blockade and control groups. After subjects had lain in the horizontal position for 1 h, the mean resting heart rate in the ACE-blockade and control groups was similar (77.9 ± 2.7 vs. 75.6 ± 2.5 beats/min, respectively). Heart rate increased significantly (P < 0.01) in both ACE-blockade (93.4 ± 1.6 beats/min) and control groups (94.4 ± 2.0 beats/min) when subjects were brought up to a 30° angle.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The objective of the present study was to test the putative implication of the renin-angiotensin system in the regulation of splanchnic blood flow and glucose output during prolonged dynamic exercise in humans. The injection of enalapril, an ACE inhibitor, 30 min before exercise reduced the plasma concentration of ANG II at rest and abolished the increase in angiotensin during exercise. In the absence of an elevation in plasma ANG II concentration during mild and moderate exercise, the normal decrease in estimated splanchnic blood flow and the rise in glucose production were maintained, indicating that the renin-angiotensin system plays a minor role in the regulation of splanchnic blood flow and glucose output during dynamic exercise in humans.

After the exercise, we tested the response of the renin-angiotensin system to a passive head-up tilt to a 30° angle. The rise in plasma ANG II known to occur during such a procedure (27) was prevented during the enalapril trial. These data, along with the absence of an increase in plasma ANG II concentrations during exercise, demonstrate that we successfully inhibited the release of this vasoconstrictive agent into the circulation. Therefore, the lack of effect of an ACE inhibitor on splanchnic blood flow and glucose output cannot be explained by a failure of inhibiting the renin-angiotensin system during exercise. The present data contrast with a report from Symons and Stebbins (30) that demonstrated a role for ANG II receptors in the regulation of splanchnic blood flow during exercise. However, that study was performed on miniswine, and the difference between those data and the data in the present study might be based on species difference. Alternatively, the difference might reflect that ACE inhibition interferes less specifically with the angiotensin system than angiotensin blockers, inhibition of vasodilator mechanisms possibly counteracting inhibition of the renin-angiotensin system during ACE inhibitor infusion. Another possibility is that compensatory mechanisms, e.g., increases in cortisol and epinephrine concentrations (Table 2 and Fig. 3), blunted the effect of ACE blockade in the present study.

Aside from ANG II, major hormones responding to exercise were also measured to verify if a compensating mechanism could have masked a potential role of ANG II in the decrease in splanchnic blood flow and the rise in glucose output. As previously described and observed again in the present experiment, norepinephrine, epinephrine, and growth hormone arterial concentrations increased in a stepwise fashion during mild and moderate exercise, and ACTH and glucagon were significantly increased during moderate exercise, whereas insulin and C peptide concentrations decreased progressively (18, 19). However, hormonal arterial concentrations did not differ during ACE blockade compared with the control trial, with the exception of cortisol levels, which were slightly higher in the former condition (Table 2). Cortisol is an integral part of the counterregulatory response to exercise in humans, and it may contribute to the elevation of hepatic glucose production during exercise (17, 32). Furthermore, cortisol may increase splanchnic vascular resistance by enhancing the arteriolar response to norepinephrine (11). However, such effects cannot be expected to occur within the first hour of elevated cortisol production and can, accordingly, not account for findings during exercise in the present study.

In physiological concentrations, epinephrine is thought to have a minor role in the regulation of hepatic glucose production during exercise in humans (18). Supporting this contention is the recent demonstration that during 60 min of exercise on a cycle ergometer, adrenalectomized patients who are epinephrine deficient have a normal hepatic glucose production and successfully maintain euglycemia (15). Similarly, the effect of epinephrine concentrations on the reduction of splanchnic blood flow during exercise is minimal. Kjær et al. (18) have reported that, in experiments with celiac ganglion blockade, splanchnic blood flow decreased normally during exercise whether or not epinephrine was infused. Therefore, it is not likely that the slightly higher epinephrine and cortisol concentrations during ACE blockade could have masked a role for ANG II in the regulation of splanchnic blood flow and glucose production during exercise, although this possibility cannot be completely ruled out. Glucagon rose with exercise despite a decrease in splanchnic release (Tables 2 and 5). This is a new observation and must most likely be due to a decreased clearance by the kidney. Insulin release decreased more than the drop in C peptide release from the splanchnic area (Table 5). This indicates an exercise-induced increase in splanchnic clearance of the hormone.

Arterial concentrations of plasma lactate were slightly higher during blockade of the renin-angiotensin system compared with when subjects were studied under the control condition (Table 3). In the present study, one could speculate, on the basis of rat studies showing the stimulating effect of ANG II on gluconeogenesis (5, 33), that the increased plasma lactate concentrations in the ACE-blockade trial were due to reduced hepatic gluconeogenesis, for which lactate is a precursor. However, this possibility is not likely because there was a similar splanchnic extraction of all major gluconeogenic precursors, i.e., lactate, glycerol, and alanine both at rest and during exercise either with or without the normal increase in ANG II concentrations (Table 4). Also, the hepatic supply and extraction of free fatty acids, which may influence hepatic glucose production, were similar in the two experiments (Table 4). The higher plasma lactate concentration observed during ACE blockade is most likely to be due to slightly greater anaerobic glycolysis from the active muscles under these conditions.

In conclusion, the present study shows that ACE blockade using enalapril does not alter the normal splanchnic blood flow and glucose output during mild and moderate prolonged cycle ergometer exercise in humans. These data suggest that ANG II does not play an important role in the regulation of these physiological responses and point to other neurohumoral factors that are yet to be determined. In addition, the study has shown that during prolonged exercise splanchnic release of both insulin and glucagon may decrease, the decrease in splanchnic release of insulin reflecting both decreased pancreatic secretion and increased hepatic clearance.

Perspectives