Role of changes in insulin and glucagon in glucose homeostasis in exercise.

This experiment was performed to determine if plasma glucose homeostasis is maintained in normal human volunteers during light exercise (40% maximal oxygen consumption [VO2 max]) when changes in insulin and glucagon are prevented. Hormonal control was achieved by the infusion of somatostatin, insulin, and glucagon. Glucose kinetics and oxidation rates were determined with stable isotopic tracers of glucose, and by indirect calorimetry. Two different rates of replacement of insulin and glucagon were used; in one group, insulin was clamped at 19.8 +/- 2.6 microU/ml (high-insulin group), and in the other group insulin was clamped at 9.2 +/- 1.3 microU/ml (low-insulin group). Glucagon was maintained at 261 +/- 16.2 and 124 +/- 6.4 pg/ml, respectively, in the high-insulin and low-insulin groups. Without hormonal control, plasma glucose homeostasis was maintained during exercise because the increase in glucose uptake was balanced by a corresponding increase in glucose production. When changes in insulin and glucagon were prevented, plasma glucose concentration fell, particularly in the high-insulin group. Glucose uptake increased to a greater extent than when hormones were not controlled, and glucose production did not increase sufficiently to compensate. The increase in glucose uptake in the hormonal control groups was associated with an increased rate of glucose oxidation. When euglycemia was maintained by glucose infusion in the hormonal control subjects, the modest increase in glucose production that otherwise occurred was prevented. It is concluded that during light exercise there must be a reduction in insulin concentration and/or an increase in glucagon concentration if plasma glucose homeostasis is to be maintained. If such changes do not occur, hypoglycemia, and hence exhaustion, may occur.

Substrate cycling between gluconeogenesis and glycolysis in euthyroid, hypothyroid, and hyperthyroid man.

Substrate, or futile cycles, have been hypothesized to be under hormonal control, and important in metabolic regulation and thermogenesis. To define the role of thyroid hormones in the regulation of substrate cycling in glycolysis and gluconeogenesis, we measured rates of cycling in normal (n = 4), hypothyroid (n = 5), and hyperthyroid (n = 5) subjects employing a stable isotope turnover technique. Glucose labeled with deuterium at different positions (2-D1-, 3-D1-, and 6,6-D2-glucose) was given as a primed-constant infusion in tracer doses, and arterialized plasma samples were obtained and analyzed by gas-chromatography mass-spectrometry for the steady state enrichment of glucose that was labeled at the various positions. The rate of appearance (Ra) was then calculated for each isotopic tracer. The difference between the Ra determined by 2-D1-glucose (Ra2) and the Ra determined by 3-D1-glucose (Ra3) represents the substrate cycling rate (SCR) between glucose and glucose-6-phosphate. The difference between the Ra determined by 3-D1-glucose (Ra3) and the Ra determined by 6,6-D2-glucose (Ra6) represents the SCR between fructose-6-phosphate and fructose-1,6-diphosphate. The difference between Ra2 and Ra6 represents the combined SCR of both cycles. In normal subjects (serum thyroxine [T4] = 8.4 +/- 1.2 microgram/dl (all expressions, mean +/- SD), n = 4), the rates of appearance for Ra2, Ra3, and Ra6 were 3.23 +/- 0.56, 2.64 +/- 0.50, and 2.00 +/- 0.27 mg/kg X min, respectively, whereas those in the hypothyroid subjects (T4 = 1.0 +/- 0.8 microgram/dl; n = 5) were 1.77 +/- 0.56 (P less than 0.01), 1.52, 1.57 +/- 0.31 (P less than 0.05) mg/kg X min, respectively. Conversely, the rates of appearance for Ra2 and Ra6 in the hyperthyroid subjects (T4 = 23.9 +/- 3.6 micrograms/dl) were 3.94 +/- 0.43 (P less than 0.05) and 2.54 +/- 0.22 (P less than 0.02), respectively, compared with the normal subjects. On the basis of these data, we noted that the normal subjects had a combined SCR of 1.23 +/- 0.35 mg/kg X min. In contrast, the hypothyroid patients had a significantly decreased combined SCR, 0.20 +/- 0.54 mg/kg X min (P less than 0.02). The hyperthyroid patients had a combined SCR of 1.39 +/- 0.23 mg/kg X min (P less than NS). To determine whether these cycles responded to thyroid hormone treatment, these same hypothyroid subjects were acutely treated for 1 wk with parenteral 50 micrograms/d sodium L-triiodothyronine and chronically with 100-150 micrograms/d L-thyroxine. After 7 d, their mean oxygen consumption rate and carbon dioxide production rate increased significantly from 102+/-13 micromol/kg.min, to 147+/-34 micromol/kg.min (P<0.05), and from 76+/-13 micromol/kg.min to 111+/-19 micromol/kg.min (P<0.05), respectively. The combined SCR (Ra(2)--Ra(6) remained unchanged at 0.07+/-0.37 mg/kg.min. However, after 6 mo of oral L-thyroxine therapy (T(4)=9.5+/-1.4 microgram/kl) the treated hypothyroid patients had increased their combined SCR (Ra(2)--Ra(6)) to 0.86 +/-0.23 mg/kg.min (P<0.02), a value not significantly different from the combined SCR of normal subjects. We conclude that substrate cycling between glucose and glucose-6-phosphate and between fructose-6-phosphate and fructose-1,6-diphosphate occurs in man and is affected by thyroid hormone. Substrate cycles may represent a mechanism by which thyroid hormone alters the sensitivity of certain reactions to metabolic signals.

Hormonal control of substrate cycling in humans.

Recent studies have established the existence of substrate cycles in humans, but factors regulating the rate of cycling have not been identified. We have therefore investigated the acute response of glucose/glucose-6P-glucose (glucose) and triglyceride/fatty acid (TG/FA) substrate cycling to the infusion of epinephrine (0.03 microgram/kg.min) and glucagon. The response to a high dose glucagon infusion (2 micrograms/kg.min) was tested, as well as the response to a low dose infusion (5 ng/kg.min), with and without the simultaneous infusion of somatostatin (0.1 microgram/kg.min) and insulin (0.1 mU/kg.min). Additionally, the response to chronic prednisone (50 mg/d) was evaluated, both alone and during glucagon (low dose) and epinephrine infusion. Finally, the response to hyperglycemia, with insulin and glucagon held constant by somatostatin infusion and constant replacement of glucagon and insulin at basal rates, was investigated. Glucose cycling was calculated as the difference between the rate of appearance (Ra) of glucose as determined using 2-d1- and 6,6-d2-glucose as tracers. TG/FA cycling was calculated by first determining the Ra glycerol with d5-glycerol and the Ra FFA with [1-13C]palmitate, then subtracting Ra FFA from three times Ra glycerol. The results indicate that glucagon stimulates glucose cycling, and this stimulatory effect is augmented when the insulin response to glucagon infusion is blocked. Glucagon had minimal effect on TG/FA cycling. In contrast, epinephrine stimulated TG/FA cycling, but affected glucose cycling minimally. Prednisone had no direct effect on either glucose or TG/FA cycling, but blunted the stimulatory effect of glucagon on glucose cycling. Hyperglycemia, per se, had no direct effect on glucose or TG/FA cycling. Calculations revealed that stimulation of TG/FA cycling theoretically amplified the sensitivity of control of fatty acid flux, but no such amplification was evident as a result of the stimulation of glucose cycling by glucagon.

Investigation of kinetics of integrated metabolic response to adrenergic blockade in conscious dogs.

We have used multiple isotope infusions to study the integrated response of glucose, fat, and protein metabolism to combined alpha + beta-adrenergic blockade in conscious, unstressed, fasting (15 h) dogs. The response to the blocking agents was evaluated both with and without control of the glucoregulatory hormones. The hormones were controlled at the basal level by infusions of somatostatin and metyrapone to block their secretion, and by the infusion of insulin, glucagon, growth hormone, and cortisol at physiological rates. We found that adrenergic blockade markedly inhibited lipolysis, as reflected by falls in glycerol and plasma FFA appearance. The decrease in fat mobilization after blockade resulted in a proportionate shift from fat as an energy substrate toward carbohydrate. Glucose production and oxidation were both enhanced after blockade. The source of the increased glucose production was presumably hepatic glycogen because urea production was presumably hepatic glycogen because urea production was unaffected and glycerol uptake was decreased. These results are consistent with the interpretation that basal adrenergic activity plays an important role in the mobilization of fat in fasting dogs. A secondary consequence of that action is apparently a diminution of glucose production and oxidation, although the mechanism responsible for the latter response is not clear.

Effect of thermal injury on energy metabolism, substrate kinetics, and hormonal concentrations.

We have used a chronic dog model to study the kinetic aspects of the metabolic response to thermal injury. In order to interpret the kinetic data in the context of the overall response to injury, we also determined certain cardiovascular parameters as well as selected hormonal concentrations. We used a paired study design: each animal was studied twice before injury and then at 5 and 7 days following injury. The burn was induced while the animals were deeply anesthetized with sodium pentobarbital (30-35 mg/kg) but all studies were done in awake, unrestrained dogs using tracer methodology and both radiolabeled and stable isotopes. The burn induced a hyperdynamic state, with significant elevations in cardiac output, heart rate, and metabolic rate. The free fatty acid (FFA), glycerol and glucose flux rates were all elevated, as was the rate of production of urea. The increase in resting metabolic rate was due to comparable increases in the rates of oxidation of carbohydrate, fat, and protein. We found the responses of this canine burn model to closely resemble the human response to injury, and thus this model will be useful as a tool to elucidate some of the mechanisms responsible for the response to injury.

Isotopic approaches to the estimation of protein requirements in burn patients.

Determination of the protein requirement of the severely burned patient has been hampered by the lack of an appropriate means of evaluating the effects of various levels of intake. We have used two new isotopic techniques in an attempt to assess the effects of two levels of protein intake in six severely burned adult patients. A crossover experimental design enabled us to study each patient at the end of two 3-day dietary regimens. All diets were isocaloric and provided approximately 25% more calories than the measured energy expenditure (means = 40 Kcal/kg X day). In one regimen, each patient received 2.2 g protein/kg X day; during the other treatment period they received 1.4 g protein/kg X day. The patients were studied in the fed state and after 10-12 h of fasting. Leucine kinetics were determined by means of the primed-constant infusion of (1-13C)-leucine. We were able to distinguish the oxidation of plasma leucine from the oxidation of leucine derived from intracellular protein at the site of the deamination of leucine (predominantly muscle) by simultaneously determining both leucine and alpha-ketoisocaproic acid enrichment. Also, rates of whole-body protein synthesis and catabolism were calculated by means of another stable-isotope technique involving the infusion of (15N2)-urea. Finally, a third means of estimating net protein catabolism based on urinary N-excretion data was used at the same time that the isotopic studies were performed. The 13C-leucine data and the N-excretion data indicated that a balance between protein synthesis and catabolism could be achieved with a protein intake of 1.4 protein/kg X day. When protein intake was increased to 2.2 g protein/kg X day, neither isotopic method indicated a further beneficial effect on net protein synthesis, although the absolute rates of protein synthesis and catabolism were stimulated. The N-excretion data, however, indicated a significant improvement in net protein synthesis with higher protein intake. Regardless of the level of protein intake, the underlying alterations in protein metabolism that occurred as a response to burn injury persisted.

Response to glucose infusion in humans: role of changes in insulin concentration.

We have used the primed-constant infusion of stable isotopes of glucose ([6,6-d2]glucose), alanine([3-13C] alanine), and urea ([15N2]urea) to investigate their kinetic interrelationships in normal volunteers in the postabsorptive state and during the infusion of unlabeled glucose at two rates. Each glucose infusion was tested with and without the simultaneous infusion of somatostatin (S), insulin (I), and glucagon (G) to clamp those hormonal levels. When glucose was infused at 1 mg X kg-1 X min-1, endogenous glucose production was suppressed almost exactly 1 mg X kg-1 X min-1, regardless of whether S plus I plus G were infused. The 4 mg X kg-1 X min-1 glucose infusion suppressed endogenous glucose production, both with and without hormonal control. The plasma concentration of glucose also increased to the same extent during the 4 mg X kg-1 X min-1 infusion in both protocols, which indicated that the spontaneous insulin response to the glucose infusion (an increase from 11 +/- 2 to 24 +/- 3 microU/ml) did not stimulate the peripheral clearance of glucose. The high rate of glucose infusion, both with or without hormonal control, stimulated alanine flux and inhibited urea production. These results indicate that glucose, per se, is an important direct controller of normal metabolic interactions of endogenous alanine, glucose, and urea kinetics.

Response of protein and urea kinetics in burn patients to different levels of protein intake.

The effects of two levels of protein intake on protein metabolism in six severely burned adult patients were studied (means of 70% BSA burned). A crossover experimental design enabled the authors to study each patient at the end of two three-day dietary regimens. All diets were isocaloric and provided approximately 25% more calories than the measured energy expenditure (means = 40.8 Kcal/kg X day). In one regimen, each patient received 2.2 g protein/kg X day, while during the other treatment period they received 1.4 g protein/kg X day. The patients were studied in the fed state and after 10 to 12 hours of fasting. Leucine kinetics were determined by means of the primed-constant infusion of [1--13C]--leucine. The authors were able to distinguish the oxidation of plasma leucine from the oxidation of leucine derived from intracellular protein at the site of the deamination of leucine (predominantly muscle) by simultaneously determining both leucine and alpha-ketoisocaproic acid enrichment. Also, rates of whole-body protein synthesis and catabolism were calculated from the leucine flux and oxidation data. Net protein synthesis was also calculated by means of another stable-isotope technique involving the infusion of [15N2]--urea. Finally, a third means of estimating net protein catabolism based on urinary N-excretion data was used at the same time that the isotopic studies were performed. The 13C leucine-data and the N-excretion data indicated that a balance between protein synthesis and catabolism could be achieved with a protein intake of 1.4 protein/kg X day. When protein intake was increased to 2.2 g protein/kg X day, neither isotopic method indicated a further beneficial effect on net protein synthesis, although the absolute rates of protein synthesis and catabolism were stimulated. The N-excretion data, on the other hand, indicated a significant improvement in net protein synthesis with higher protein intake. Regardless of the level of protein intake, the underlying alterations in protein metabolism that occurred as a response to burn injury persisted.

Effect of substrate intake and physiological state on background 13CO2 enrichment.

The natural enrichment of 13C in energy substrates varies, and this variation must be taken into account when stable isotopic tracers are used in metabolic studies. This is conventionally accomplished by measuring background samples taken before the tracer infusion begins and subtracting these values from postinfusion values. Whereas this approach is satisfactory if no perturbation occurs between the collection of the background samples and the collection of postinfusion sample, the data presented in this paper show that any change in the metabolic state can significantly alter the background enrichment of expired CO2. This study not only confirmed that the introduction of natural energy sources may alter the background enrichment of CO2, but we also found that changes in substrate oxidation induced by different physiological states, such as exercise, can cause significant changes in expired CO2 enrichments. Conclusions from studies in which oxidation of substrates were measured by means of a 13C tracer but potential changes in background enrichments were not accounted for must, therefore, be reassessed.

Isotopic determination of amino acid-urea interactions in exercise in humans.

We recently reported that in light exercise (30% VO2max) the oxidation of [1-13C]leucine was significantly increased but the rate of urea production was unchanged (J. Appl. Physiol: Respirat. Environ. Exercise Physiol. 52: 458-466, 1982). We have therefore tested three possible explanations for this apparent incongruity. 1) We infused NaH13CO3 throughout rest and exercise and found that, although altered bicarbonate kinetics in exercise resulted in greater recovery of 13CO2, the difference between rest and recovery was small compared with the increase in the rate of 13CO2 excretion during exercise when [1-13C]leucine was infused. 2) We infused [15N]leucine and isolated plasma urea N to determine directly the rate of incorporation of the 15N. During exercise there was no increase in the rate of 15N incorporation. Simultaneously, we infused [2,3-13C]alanine and quantified the rate of incorporation of 15N in alanine. We found that [15N]alanine production from [15N] leucine more than doubled in exercise, and by deduction, alanine production from other amino acids also doubled. 3) We tested our previous assumption that [1-13C]leucine metabolism in exercise was representative of the metabolism of other essential amino acids by infusing [1-13C] and [alpha-15N]lysine throughout rest and exercise. We found that the rate of breakdown of lysine during exercise was not increased in a manner comparable to that of leucine. Thus, these data confirm our original findings that leucine decarboxylation is enhanced in light exercise but urea production is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Leucine and urea metabolism in acute human cold exposure.

Four healthy males voluntarily underwent acute cold exposure at 10 degrees C. Metabolic rate doubled for the 100-min exposure. [1-13C]leucine and [15N2]urea were used as tracers of protein metabolism via a primed constant infusion. Total and plasma transported leucine oxidation approximately doubled, but the oxidation of leucine derived from protein in the tissue where oxidation occurred ("intracellular oxidation") did not change as it did when the same subjects underwent mild exercise. Rate of appearance of urea and leucine in plasma were not significantly different between control and cold. Although the rate of protein synthesis calculated from the leucine data did not change, the rate of catabolism increased. Net protein catabolism based on the urea data agreed well with the leucine data at rest but did not exhibit a significant increase during exposure. However, net protein catabolism based on the leucine data did increase significantly during acute cold exposure. Further, there appears to be a qualitative difference in the protein catabolism associated with the voluntary muscular activity of exercise and muscular shivering aimed at thermogenesis.

Isotopic analysis of leucine and urea metabolism in exercising humans.

We have used the primed constant infusion of di-[15N]urea and [1-13C]leucine to determine the effects of mild exercise (approx 30% Vo2max for 105 min) on urea production and leucine metabolism in human subjects. The oxidation of plasma leucine was distinguished from the oxidation of leucine that never entered the plasma pool ("intracellular" leucine) by means of determining the enrichment of alpha-ketoisocaproic acid (alpha-KICA). Total leucine oxidation increased from 0.38 +/0 0.05 to 1.41 +/- 0.14 micromol . kg-1 . min-1 during exercise due to increases in the oxidation of plasma leucine (150%) and intracellular leucine (600%). Plasma leucine flux decreased slightly, but not significantly (0.1 greater than P greater than 0.05), and the percent of alpha-KICA derived from plasma leucine dropped significantly (P less than 0.05) from 79.5 +/- 4.3 at rest to 62.0 +/- 5.3% over the last 30 min of exercise. Despite the increase in leucine oxidation during exercise, urea concentration and production did not change. Thus in exercise urea production does not accurately reflect all aspects of amino acid metabolism.

Urea, glucose and alanine kinetics in man: effects of glucose infusion.

1. The simultaneous effects of an intravenous glucose infusion on plasma urea, glucose and alanine kinetics were investigated in normal post-absorptive man. 2. The primed constant intravenous infusion of compounds labelled with stable isotopes, [15N2]urea, [6-2H]glucose and [3-13C]alanine, was used. 3. The rate of appearance of glucose and urea in the plasma was rapidly reduced by the 17.7 mumol min-1 kg-1 glucose infusion. 4. In contrast, during the glucose infusion there was an increased rate of appearance of alanine in the plasma, and an increased percentage of glucose carbon atoms derived from alanine. 5. Reduced production of glucose and urea during the glucose infusion was not due to decreased gluconeogenesis from alanine.