Lars Hagenfeldt

Substrate Turnover during Prolonged Exercise in Man: SPLANCHNIC AND LEG METABOLISM OF GLUCOSE, FREE FATTY ACIDS, AND AMINO ACIDS

Arterial concentrations and substrate exchange across the leg and splanchnic vascular beds were determined for glucose, lactate, pyruvate, glycerol, individual acidic and neutral amino acids, and free fatty acids (FFA) in six subjects at rest and during 4 h of exercise at approximately 30% of maximal oxygen uptake. FFA turnover and regional exchange were evaluated using (14)C-labeled oleic acid. The arterial glucose concentration was constant for the first 40 min of exercise, but fell progressively thereafter to levels 30% below basal. The arterial insulin level decreased continuously, while the arterial glucagon concentration had risen fivefold after 4 h of exercise. Uptake of glucose and FFA by the legs was markedly augmented during exercise, the increase in FFA uptake being a consequence of augmented arterial levels rather than increased fractional extraction. As exercise was continued beyond 40 min, the relative contribution of FFA to total oxygen metabolism rose progressively to 62%. In contrast, the contribution from glucose fell from 40% to 30% between 90 and 240 min. Leg output of alanine increased as exercise progressed. Splanchnic glucose production, which rose 100% above basal levels and remained so throughout exercise, exceeded glucose uptake by the legs for the first 40 min but thereafter failed to keep pace with peripheral glucose utilization. Total estimated splanchnic glucose output was 75 g in 4 h, sufficient to deplete approximately 75% of liver glycogen stores. Splanchnic uptake of gluconeogenic precursors (lactate, pyruvate, glycerol, alanine) had increased 2- to 10-fold after 4 h of exercise, and was sufficient to account for 45% of glucose release at 4 h as compared to 20-25% at rest and at 40 min of exercise. In the case of alanine and lactate, the increase in precursor uptake was a consequence of a rise in splanchnic fractional extraction. It is concluded that during prolonged exercise at a low work intensity (a) blood glucose levels fall because hepatic glucose output fails to keep up with augmented glucose utilization by the exercising legs; (b) a large portion of hepatic glycogen stores is mobilized and an increasing fraction of the splanchnic glucose output is derived from gluconeogenesis; (c) blood-borne substrates in the form of glucose and FFA account for a major part of leg muscle metabolism, the relative contribution from FFA increasing progressively; and (d) augmented secretion of glucagon may play an important role in the metabolic adaptation to prolonged exercise by its stimulatory influence on hepatic glycogenolysis and gluconeogenesis.

Effect of protein ingestion on splanchnic and leg metabolism in normal man and in patients with diabetes mellitus.

The inter-organ flux of substrates after a protein-rich meal was studied in seven healthy subjects and in eight patients, with diabetes mellitus. Arterial concentrations as well as leg and splanchnic exchange of amino acids, carbohydrate substrates, free fatty acids (FFA), and ketone bodies were examined in the basal state and for 3 h after the ingestion of lean beef (3 g/kg body wt). Insulin was withheld for 24 h before the study in the diabetic patients. In the normal subjects, after protein ingestion, there was a large amino acid release from the splanchnic bed predominantly involving the branched chain amino acids. Valine, isoleucine, and leucine accounted together for more than half of total splanchnic amino acid output. Large increments were seen in the arterial concentrations of the branched chain amino acids (100-200%) and to a smaller extent for other amino acids. Leg exchange of most amino acids reverted from a basal net outut to a net uptake after protein feeding which was most marked for the branched chain amino acids. The latter accounted for more than half of total peripheral amino acid uptake...

Splanchnic and leg exchange of glucose, amino acids, and free fatty acids during exercise in diabetes mellitus.

The influence of exercise on leg and splanchnic exchange of substrates was examined in eight insulin-dependent diabetics 24 h after withdrawal of insulin and in eight healthy controls studied at rest and after 40 min of bicycle ergometer exercise at 55-60% of maximal capacity. In four of the diabetic subjects, basal arterial ketone acid levels were 3-4 mmol/ liter (ketotic diabetics) and in the remainder, below 1 mmol/liter (nonketotic diabetics). ,ree fatty acid (FFA) turnover and regional exchange were evaluated with 14-C- labeled oleic acid. Leg uptake of blood glucose rose 13-18 fold during exercise in both the diabetics and controls and accounted for a similar proportion of the total oxygen uptake by leg muscles (25-28%) in the two groups. In contrast, leg uptake of FFA corresponded to 39% of leg oxygen consumption in the diabetic group but only 27% in controls. Systemic turnover of oleic acid was similar in the two groups. Splanchnic glucose output increased during exercise 3-4 fold above resting levels in both groups. In the diabetics, splanchnic uptake of lactate, pyruvate, glycerol, and glycogenic amino acids rose more than twofold above resting levels and was fourfold greater than in exercising controls. Total precursor uptake could account for 30% of the splanchnic glucose output in the diabetic group. In contrast, in the controls, total splanchnic uptake of glucose precursors was no greater during exercise than in the resting state and could account for no more than 11% of splanchnic glucose output. The augmented precursor uptake during exercise in the diabetics was a consequence of increased splanchnic fractional extraction as well as increased peripheral production of gluconeogenic substrates. The arterial glucagon concentration was unchanged by exercise in both groups, but was higher in the diabetics. In the diabetic subjects with ketosis in the resting state, exercise elicited a rise in arterial glucose and FFA, an augmented splanchnic uptake of FFA, and a 2-3 fold increase in splanchnic output of 3-hydroxybutyrate. Uptake of 3-hydroxybutyrate by the exercising leg rose more rapidly than splanchnic production, resulting in a fall in arterial levels of 3-hydroxybutyrate. It is concluded that (a) glucose uptake by exercising muscle in hyperglycemic diabetics is no different from that of controls; (b) splanchnic glucose output rises during exercise to a similar extent in diabetics and controls, while uptake of gluconeogenic substrates is markedly higher in diabetics and accounts for a greater proportion of total splanchnic glucose output; (c) exercise in diabetic patients with mild ketosis is associated with a rise in blood glucose and FFA levels as well as augmented splanchnic production and peripheral uptake of ketone bodies.

Uptake of individual free fatty acids by skeletal muscle and liver in man

Arterial-venous concentration differences for individual free fatty acids (FFA) were measured across the deep tissues of the forearm, the splanchnic vascular bed, and the kidney in healthy, postabsorptive subjects. In addition, arterial-portal venous FFA differences were determined in five patients undergoing elective cholecystectomy. The differences in fractional uptake among the individual FFA across the forearm were small and not statistically significant. Splanchnic fractional uptake was high for FFA with short chain lengths and rose with increasing degree of unsaturation. Small, negative arterial-portal venous differences for individual FFA were observed, indicating that arterial-hepatic venous FFA differences mainly reflect hepatic uptake. When the arterial FFA concentration was reduced to approximately 25% of the control values by the administration of nicotinic acid, net uptake of total FFA ceased but there was release of stearic acid and uptake of lauric, myristic, and palmitoleic acid to the splanchnic region. Muscle and liver uptakes of individual FFA were both dependent on their arterial concentrations with the exception of the splanchnic uptake of stearic acid. There was no uptake of free arachidonic acid by either muscle or liver, nor was there significant uptake of any of the free fatty acids by the kidney. It is concluded (a) that there are important quantitative differences between the net exchanges of individual FFA across the splanchnic vascular bed, (b) that tracer studies of FFA metabolism require the determination of individual FFA specific activities, (c) that palmitic and oleic acid appear to be suitable tracers for the entire FFA fraction in most instances.

Human Forearm Muscle Metabolism during Exercise II Uptake, release and oxidation of individual FFA and glycerol

The uptake, release, and oxidation of individual free fatty acids (FFA) in the human forearm were studied during a 60-min exercise period. Muscular uptake of FFA rose with the arterial concentration. The muscle showed a slight preference for linoleic and oleic acid compared to palmitic acid. The forearm RQ indicated a constant fat oxidation during the period of exercise. The uptake of FFA covered about 50 per cent of the fat oxidation. On the average 60 per cent of the muscular FFA-14C uptake was oxidized to 14CO2. The remaining radioactivity left the muscle as water-soluble metabolite(s). β-Hydroxybutyrate leaving the muscle was shown to be labeled. No release of glycerol accompanied the release of FFA during exercise. Glycerol-1-14C was oxidized by the exercising muscle. Glycerol dehydrogenase activity was demonstrated in human skeletal muscle.

Influence of somatostatin on splanchnic glucose metabolism in postabsorptive and 60-hour fasted humans.

Cyclic somatostatin was administered intravenously (10 mug/min for 60 min) to 10 healthy overnight fasted (postabsorptive) subjects and to 5 healthy 60-h fasted subjects. In both groups, arterial insulin and glucagon fell 50% and splanchnic release of these hormones was inhibited. In the overnight fasted subjects splanchnic glucose output fell 70%, splanchnic uptake of lactate and pyruvate was unchanged, alanine uptake fell by 25%, and glycerol uptake rose more than twofold in parallel with an increase in arterial glycerol. In the 60-h fasted group splanchnic glucose output was less than 40% of that observed in the overnight fasted subjects. Somatostatin led to a further decrease (--70%) in glucose production. Splanchnic uptake of lactate and pyruvate fell by 30-40%, amino acid uptake was unchanged, while uptake of glycerol rose fivefold. Total uptake of glucose precursors thus exceeded the simultaneous glucose output by more than 200%. Splanchnic uptake of FFA rose fourfold during somatostatin while output of beta-hydroxybutyrate increased by 75%. Estimated hepatic blood flow fell 25-35% and returned to base line as soon as the somatostatin infusion ended. It is concluded that (a) somatostatin-induced hypoglucagonemia results in inhibition of splanchnic glucose output in glycogen-depleted, 60-h fasted subjects as well as in postabsorptive subjects, indicating an effect of glucagon on hepatic gluconeogenesis as well as glycogenolysis; (b) the glucagonsensitive step(s) in gluconeogenesis affected by somatostatin involves primarily intra-hepatic disposal rather than net hepatic uptake of glucose precursors; (c) splanchnic uptake of fatty acids and ketone output are increased in the face of combined insulin and glucagon deficiency; and (d) diminished splanchnic blood flow may contribute to some of the effects of somatostatin on splanchnic metabolism.

N-acetylaspartic aciduria due to aspartoacylase deficiency — a new aetiology of childhood leukodystrophy

We describe a male infant with psychomotor retardation and leukodystrophy who excretes large quantities ofN-acetylaspartate in his urine. A high CSF/plasma concentration ratio ofN-acetylaspartate indicates that this substance originates in the brain. Fibroblasts from the patient are deficient in aspartoacylase activity. It is proposed that the dysmyelination in the patient may be due to failure ofN-acetylaspartate to serve as a carrier of acetyl groups from mitochondria to the cytosol for lipogenesis.

Turnover and splanchnic metabolism of free fatty acids and ketones in insulin-dependent diabetics at rest and in response to exercise.

Nine insulin-dependent diabetics and six healthy controls were studied at rest, during, and after 60 min of bicycle exercise at a work load corresponding to 45% of their maximal oxygen intake. The catheter technique was employed to determine splanchnic and leg exchange of metabolites. FFA turnover and regional exchange was evaluated using [14C]oleate infusion. Basal glucose (13.8 +/- 1.1 mmol/l), ketone body (1.12 +/- 0.12 mmol/l), and FFA (967 +/- 110 mumol/l) concentrations were elevated in the diabetics in comparison with controls. In the resting state, splanchnic ketone acid production in the diabetics was 6-10-fold greater than in controls. Uptake of oleic acid by the splanchnic bed was increased 2-3-fold, and the proportion of splanchnic FFA uptake converted to ketones (61%) was threefold greater than in controls. In contrast, splanchnic fractional extraction of oleic acid was identical in diabetics and controls. A direct relationship was observed between splanchnic uptake and splanchnic inflow (plasma concentration X hepatic plasma flow) of oleic acid that could be described by the same regression line in the diabetic and control groups. During exercise, splanchnic ketone production rose in both groups. In the control group the increase in ketogenesis was associated with a rise in splanchnic inflow and in uptake of oleic acid, a rise in splanchnic fractional extraction of oleate, and an increase in the proportion of splanchnic FFA uptake converted to ketone acids from 20-40%. In the diabetic group, the increase in ketogenesis occurred in the absence of a rise in splanchnic inflow or uptake of oleic acid, but was associated with an increase in splanchnic fractional extraction of oleic acid and a marked increase in hepatic conversion of FFA to ketones, so that the entire uptake of FFA was accountable as ketone acid output. Splanchnic uptake of oleic acid correlated directly with splanchnic oleic acid inflow in both groups, but the slope of the regression line was steeper than in the resting state. Plasma glucagon levels were higher in the diabetic group at rest and during exercise, while plasma norepinephrine showed a twofold greater increment in response to exercise in the diabetic group (to 1,400-1,500 pg/ml). A net uptake of ketone acids by the leg was observed during exercise but could account for less than 5% of leg oxidative metabolism in the diabetics and less than 1% in controls. Despite the increase in ketogenesis during exercise, a rise in arterial ketone acid levels was not observed in the diabetics until postexercise recovery, during which sustained increments to values of 1.8-1.9 mmol/l and sustained increases in splanchnic ketone production were observed at 30-60 min. The largest increment in blood ketone acids and in splanchnic ketone production above values observed in controls thus occurred in the diabetics after 60 min of recovery from exercise. We concluded that: (a) In the resting state, increased ketogenesis in the diabetic is a consequence of augmented splanchnic inflow of FFA and increased intrahepatic conversion of FFA to ketones, but does not depend on augmented fractional extraction of circulating FFA by the splanchnic bed. (b) Exercise-induced increases in ketogenesis in normal subjects are due to augmented splanchnic inflow and fractional extraction of FFA as well as increased intrahepatic conversion of FFA to ketones. (c) When exercise and diabetes are combined, ketogenesis increases further despite the absence of a rise in splanchnic inflow of FFA. An increase in splanchnic fractional extraction of FFA and a marked increase intrahepatic conversion of FFA to ketones accounts for the exaggerated ketogenic response to exercise in the diabetic. (d) Elevated levels of plasma glucagon and/or norepinephrine may account for the increased hepatic ketogenic response to exercise in the diabetic. (e) Ketone utilization by muscle increases during exercise but constitutes a quantitatively minor oxidative fuel for muscle even in the diabetic. (f) The accelerated ketogenesis during exercise in the diabetic continues unabated during the recovery period, resulting in an exaggerated postexercise ketosis.

Benefits of Neonatal Screening for Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency) in Sweden

Objectives. The aim of this study was to evaluate the benefits of neonatal screening for congenital adrenal hyperplasia (CAH). Methods. All children with CAH born in Sweden from January 1989 to December 1994 were subjected to a systematic follow-up. Clinical symptoms were recorded and laboratory data collected. The clinical diagnosis versus diagnosis by screening was investigated. The results were compared with those of a retrospective study of all patients diagnosed during 1969–1986 (before the introduction of neonatal screening). Results. The prevalence of CAH in Sweden was 1:9800 with screening. Patients with CAH were identified earlier by screening. Half of the infants (47%) were not diagnosed at the time of recall, which was 8 days (median). In the study population, 25% of the girls and 73% of the boys were diagnosed by screening alone. The median age at the time of the definite diagnosis in boys was 21 days before screening as compared with 9 days (median) during the last part of the screening period. During the screening period, only 1 boy had a severe salt loss crisis, which occurred at the age of 8 days. Before screening, (1969–1986) 2 boys had died in the neonatal period because of an adrenal crisis. The lowest serum sodium recorded at the time of diagnosis was 124 mmol/L (median; range, 93–148) before, as compared with 134 mmol/L (median; range, 115–148) after the introduction of screening. The number of girls who were initially considered to be boys was not reduced by screening (17% vs 18%). The period of uncertainty regarding gender attributable to virilization was shortened considerably, as well as the time it took to make a correct gender assignment: 23 days (median) before screening versus 3 days (median) with screening. The maximum time it took to make the correct gender assignment was 960 days before screening and 14 days with screening. The number of patients diagnosed late, ie, after the first year of life, decreased considerably after the introduction of screening. The false-positive rate (when a new filter paper blood sample was requested or when a child was referred to a pediatrician for follow-up) was <0.05% and in about 60% of the cases, it was attributable to preterm infants. The cost of screening was US dollar 2.70 per screened infant. Conclusion. The main benefits of screening were avoidance of serious salt loss crises, earlier correct gender assignment in virilized girls, and detection of patients who would have otherwise been missed in the neonatal period. Deaths in the neonatal period were prevented by screening. The aim of the screening program was to identify patients with the severe forms of CAH. Nevertheless, it must be considered a distinct benefit that a number of patients with milder forms of CAH were detected earlier, because earlier therapy results in decreased virilization, normalized growth and puberty, and, in all probability, an improved psychosocial situation for these children. We conclude that, in the Swedish health care system, the benefits of screening for CAH outweigh the costs.

Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples.

A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.