Dietary regulation of galactose-metabolizing enzymes: adaptive changes in rat jejunum.

The effects of dietary galactose, sucrose, fructose, glucose, casein, and fasting upon the activity of four galactose-metabolizing enzymes (galactokinase, galactose-1-phosphate uridyltransferase, uridine diphosphate galactose 4-epimerase, and galactose dehydrogenase) were studied in the jejunum of rats. Galactose produced the greatest increase in enzyme activity, fructose and sucrose produced effects intermediate between galactose and glucose, and casein produced a greater activity increase than fasting, but less than the sugars.

Control of jejunal sucrase and maltase activity by dietary sucrose or fructose in man. A model for the study of enzyme regulation in man.

The specific effect of dietary sugars on jejunal disaccharidase activity in seven normal nonfasted male volunteers was studied. The sugars tested were sucrose, maltose, lactose, glucose, fructose, and galactose. Comparisons were made of the effects of each sugar in an isocaloric liquid diet. In all subjects, sucrose feeding, as compared to glucose feeding, significantly increased jejunal sucrase (S) and maltase (M) activities, but not lactase (L) activity. The S/L and M/L ratios increased to a significant degree. Fructose feeding, in two subjects, gave results similar to sucrose when comparing fructose and glucose diets. One subject was fed lactose, galactose, and maltose. These sugars, compared to glucose, did not increase disaccharidase activity. Fructose appears to be the active principle in the sucrose molecule. These results demonstrate that specific dietary sugars can alter enzyme activity in the small intestine of man in a specific fashion. Sucrose and fructose are able to regulate sucrase and maltase activity. Dietary alteration of intestinal enzymes may represent a suitable system for studying the regulation of enzyme activity in man.

Regulation of human jejunal glycolytic enzymes by oral folic acid.

The effect of oral folic acid on jejunal glycolytic enzyme activity in five fasting obese patients and in three normal male volunteers on a constant 3000 cal diet was studied. The glycolytic enzymes, fructokinase, hexokinase, glucokinase, fructose-1-phosphate aldolase, and fructose diphosphate aldolase, and the disaccharidases, sucrase, maltase, and lactase were measured. In both the fasting patients and the normal volunteers, oral folic acid significantly increased the jejunal glycolytic enzyme activities but had no effect on disaccharidase activity. When oral folic acid was discontinued in the normal volunteers, the glycolytic enzyme activities returned to control values. In the obese patients, refeeding and folic acid caused a further increase in glycolytic enzyme activities above that seen with fasting and folic acid. In contrast to oral folic acid, intramuscular folic acid, oral vitamin B(12), and oral tetracycline had no effect on glycolytic enzyme activities. These studies demonstrate that oral folic acid which is neither a substrate nor a coenzyme of these enzymes, increases human jejunal glycolytic enzyme activity in a specific fashion. This would appear to be an action of oral folic acid which has not been recognized previously.

The rapid changes of hepatic glycolytic enzymes and fructose-1,6-diphosphatase activities after intravenous glucagon in humans.

Glucagon (0.04-0.09 mg/kg/min) was given intravenously for either 2 or 3 min to eight patients with fasting-induced hypoglycemia. One child had hepatic phosphorylase deficiency, two children had glucose-6-phosphatase deficiency, two children had debrancher enzyme (amylo-1,6-glucosidase) deficiency, and two children and one adult had decreased hepatic fructose-1,6-diphosphatase (FDPase) activity. Liver biopsy specimens were obtained before and immediately after the glucagon infusion. The glucagon caused a significant increase in the activity of FDPase (from 50+/-10.0 to 72+/-11.7 nmol/mg protein/min) and a significant decrease in the activities of phosphofructokinase (PFK) (from 92+/-6.1 to 41+/-8.1 nmol/mg protein/min) and pyruvate kinase (PK) (from 309+/-39.4 to 165+/-23.9 nmol/mg protein/min). The glucagon infusion also caused a significant increase in hepatic cyclic AMP concentrations (from 41+/-2.6 to 233+/-35.6 pmol/mg protein). Two patients with debrancher enzyme deficiency who had biopsy specimens taken 5 min after the glucagon infusion had persistence of enzyme and cyclic AMP changes for at least 5 min. One child with glucose-6-phosphatase deficiency was given intravenous glucose (150 mg/kg/min) for a period of 5 min after the glucagon infusion and biopsy. The plasma insulin concentration increased from 8 to 152 muU/ml and blood glucose increased from 72 to 204 mg/100 ml. A third liver biopsy specimen was obtained immediately after the glucose infusion and showed that the glucagon-induced effects on PFK and FDPase were completely reversed. The glucagon infusion caused an increase in hepatic cyclic AMP concentration from 38 to 431 pmol/mg protein but the glucose infusion caused only a slight decrease in hepatic cyclic AMP concentration (from 431 to 384 pmol/mg protein), which did not appear to be sufficient to account for the changes in enzyme activities. Hepatic glucose-6-phosphatase and fructose-1,6-diphosphate aldolase activities were not altered by either the glucagon or the glucose infusion in any patients. Cyclic AMP (0.05 mmol/kg) was injected into the portal vein of adult rats and caused enzyme changes similar to those seen with glucagon administration in humans. Our findings suggest that rapid changes in the activities of PFK, PK, and FDPase are important in the regulation of hepatic glycolysis and gluconeogenesis, respectively, in humans and that cyclic AMP may mediate the glucagon- but probably not the glucose-insulin-induced changes in enzyme activities.

Calcium accumulation by chick intestinal mitochondria. Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3.

We have the evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca2+ accumulation by chick intestinal mitochondria. Ca2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na+-labile pool followed by an ATP-dependent accumulation into an Na+-resistant pool. Ca2+ accumulation is extensive at free Ca2+ concentrations greater than 3 . 10(-6) M in the presence of ATP. Ruthenium red and dinitrophenol block Ca2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca2+, or the rate and extent of ATP-supported Ca2+ accumulation. Intestinal cytosol stimulated Ca2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca2+-binding protein. 30 ng/ml 1,25(OH)2D-3 stimulated Ca2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 greater than 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca2+ concentration from 3 . 10(-6) to 1 . 10(-5) M increased Ca2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)2D-3 in vitro increased Ca2+ accumulation less than 2-fold, we conclude that 1,25(OH)2D-3 influences mitochondrial accumulation of Ca2+ in vivo primarily by altering cytosol concentrations of free Ca2+.

Acute effects of oral and intravenous ethanol on rat hepatic enzyme activities.

1. Oral administration of ethanol (3 ml) of 95% in 12 ml total volume over a two day period) significantly decrease plasma glucose and insulin levels and the activities of two key gluconeogenic enzymes, pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1) and fructose diphosphatase, (D-Fru-1,6-P2 1-phosphohydrolase, EC 3.1.3.11), and one glycolytic enzyme, fructose-1,6-P2 aldolase (Fru-1,6-P2 D-glyceraldehyde-3-P lyase, EC 4.1.2.13). In each instance, the administration of 2400 mug daily of oral folate in conjuction with the ethanol prevented these alterations in carbohydrate metabolism. 2. Intravenous injection of ethanol produced a rapid decrease (within 10--15 min) in the activities of hepatic phosphofructokinase, (ATP:D-fructose-6-phosphate 6-phosphotransferase, EC 2.7.1.11), pyruvate kinase, (ATP:pyruvate phosphotransferase, EC 2.7.1.40), fructose diphosphatase and fructose-1,6-P2 aldolase. 3. Intravenous ethanol significantly increased hepatic cyclic AMP concentration approximately 60% within 10 min, while oral ethanol did not alter hepatic cyclic AMP concentrations. 4. These data confirm the known antagonism ethanol and folate and suggest that oral folate might offer a protective effect against hypoglycemia in rats receiving ethanol.

Calcium potentiates the cyclic nucleotide and phosphaturic response to parathyroid hormone infusion.

To determine whether calcium modulates the action of PTH, we measured the cyclic nucleotide and phosphaturic response to PTH following a 4-h infusion of glucose (day 1) and calcium (day 2). The 12 subjects were selected to provide a range of low, normal, and high endogenous PTH function. PTH stimulated nephrogenous cAMP [185 +/- 31 nmol/100 ml glomerular filtrate (GF)], cyclic guanosine monophosphate (0.44 +/- 0.09 mumol/g creatinine), and phosphate (367 +/- 59 mg P/g creatinine) excretion. Calcium infusion stimulated nephrogenous cAMP excretion in the hypoparathyroid subjects (1.42 +/- 0.35 nmol/100 ml GF) but reduced it in subjects with normal parathyroid function (-2.22 +/- 0.46 nmol/100 ml GF). Calcium infusion stimulated cGMP (0.64 +/- 0.1 mumol/g creatinine) and phosphate (113 +/- 48 P/g creatinine) excretion in all subject groups. Calcium infusion led to a 2-fold increase in the cyclic nucleotide and phosphaturic response to PTH in the normal and hypoparathyroid subjects, but had little effect on the PTH response in hyperparathyroid subjects. The extent to which calcium potentiated the ability of PTH to stimulate nephrogenous cAMP excretion correlated negatively with the basal nephrogenous cAMP excretion (r = -0.685, P less than 0.01). We conclude that calcium potentiates the acute effects of PTH on renal cyclic nucleotide and phosphate excretion. This effect is modified by the basal levels of PTH stimulation of the kidney such that it is reduced in magnitude when basal PTH stimulation is increased.

Effects of carbohydrate restriction on glucose tolerance of normal men and reactive hypoglycemic patients.

Normal individuals ingesting a low carbohydrate diet frequently develop an impairment of glucose tolerance as measured by the oral glucose tolerance test; most of these diets, however, have been high in fat content. Our present studies demonstrate that a low carbohydrate diet (57 g/day) did not impair the glucose tolerance of normal men if the fat content was similar to values on a standard (301 g/day) carbohydrate diet. However, a low carbohydrate diet did lead to impaired glucose tolerance when the fat content was 16 per cent higher than on the control diet. Thus our present and previous studies demonstrate that normal men maintain normal glucose tolerance on low carbohydrate diets and suggest that the deterioration of the glucose tolerance observed on high fat diets is related to the increased fat content rather than to the reduced carbohydrate content of these diets. Seven patients with reactive hypoglycemia were exquisitely sensitive to carbohydrate deprivation. Whereas the glucose tolerance tests of normal men were not altered by a low carbohydrate, high protein diet, a significant deterioration of glucose tolerance occurred when reactive hypoglycemic patients were changed from control diets to low carbohydrate, high protein diets. These hypoglycemic patients also showed an exaggerated deterioration of the glucose tolerance after a 48-hour fast when compared tothe response of normal men. Our observations suggest that a low carbohydrate, high protein diet is not the best therapeutic diet for certain patients with reactive hypoglycemia because this diet does not provide symptomatic improvement and, in addition, leads to impaired glucose tolerance.

Comparison of the adaptive changes in disaccharidase, glycolytic enzyme and fructosediphosphatase activities after intravenous and oral glucose in normal men.

Seven subjects were fed a 3,000 kcal defined formula diet daily for 19 days. Except for one 5-day period, 50% of the total caloric intake was provided as either oral or intravenous glucose. The study was divided into four periods as follows: period I lasted 5 days and provided 50% of calories as glucose; period II lasted 5 days and provided no carbohydrate (70% fat and 30% protein); period III lasted 4 days and provided 50% of calories as intravenous glucose and 50% of calories as oral fat plus protein; period IV lasted 5 days and provided 50% of calories as oral glucose. Intestinal biopsy specimens were taken on days 3 and 5 of each period, except period III when biopsies were done only on day 4. No change in intestinal morphology occurred during the study. The carbohydrate-free diet caused the alpha-glucosidase (maltase and sucrase) activities to decrease significantly from that seen with the glucose diet. Sucrase decreased from 14.4 +/- 1.0 to 7.1 +/- 0.9 mumoles/min per g tissue and maltase decreased from 56.1 +/- 3.4 to 30.0 +/- 2.1 mumoles/min per g tissue. Glycolytic enzyme activities decreased during the carbohydrate-free period (pyruvate kinase decreased from 236 +/- 12 to 78 +/- 8, fructose 1-phosphate aldolase decreased from 147 +/- 6 to 53 +/- 4, fructose-1,6-diphosphate aldolase decreased from 151 +/- 8 to 55 +/- 3, and hexokinase decreased from 21 +/- 3 to 7 +/- 1 nmoles/min per mg protein, respectively). Intravenous glucose caused no change in disaccharidase activities. The enzyme activities during periods I and IV were identical and significantly higher than during period II with the exception of fructose-1,6-diphosphatase which increased during period II as compared with periods I and IV. These findings provide an explanation for the transient period of decreased tolerance to dietary sugars when patients are weaned from total parenteral feedings to enteral feedings.

Thiamin requirement of the adult human.

Young adult male subjects maintained on a metabolic ward were fed diets providing controlled intakes of thiamin and either 2800 or 3600 kcal. The higher level of calories was attained by an increased intake of carbohydrates. Constant weights were maintained by the subjects by adjusting daily activity and exercise schedules. Thiamin requirements were evaluated in terms of erythrocyte transketolase activity and urinary excretion of the vitamin. The results of the study revealed that a relationship exists between thiamin requirement and caloric intake and expenditure. Thus, when the calories being utilized were derived primarily from carbohydrate sources, the minimum adult male requirement for thiamin appeared to be 0.30 mg of thiamin per 1000 kcal. Urinary excretion of thiamin and erythrocyte transketolase activity appear to be reasonably reliable reflections of thiamin intakes and thiamin nutritional status. The use of these measurements in nutrition surveys appears justified. The microbiological assay (Lactobacillus viridescens) for measuring thiamin levels in urine samples appears to be a somewhat more sensitive but valid procedure as an alternate for the thiochrome method. Judged from the results of this study, the recommended intake for the adult human of 0.40 mg of thiamin per 1000 kcal by FAO/WHO and the recommended allowance of 0.5 mg per 1000 kcal by the Food and Nutrition Board of the NAS-NRC appear reasonable and amply allow for biological variations and other factors that may influence the requirement for this vitamin.

Work-induced muscle hypertrophy in vitamin D-deficient rats.

To determine if vitamin D deficiency would retard the ability of muscle to hypertrophy in response to mechanical stress, we severed the gastrocnemius tendon on one leg of rats in each of three groups, the treatment of which differed only in the amount of vitamin D in the diet. After 1 week the increased size of the soleus and plantaris in the leg in which the gastrocnemius was severed relative to that of the sham operated leg, was determined for each rat. Despite differences in body weight and serum calcium among the groups, we found no difference in the percent of muscle hypertrophy. We conclude that muscle hypertrophy can occur in response to local mechanical forces despite a deficient hormonal environment that otherwise retards growth.

Improvement in jejunal enzyme adaptation in obese adult-onset diabetic patients following a 30-day fast.

The adaptive responses of gastrointestinal enzymes, glucose tolerance, and plasma insulin to diet, folic acid, and insulin of five obese adult-onset diabetic patients were studied before and after a 30-day fast. Their data were compared to the adaptive responses of gastrointestinal enzymes to diet, folic acid, and insulin of 15 normal male volunteer subjects, ages 18 to 24. Each group during each testing period received a carbohydrate diet (50% calories as carbohydrate consisting of 1/2 glucose and 1/2 fructose) and a noncarbohydrate diet (70% of calories as corn oil and 30% as sodium caseinate) each without and with folic acid (5 mg three times per day). The effect of insulin was studied only on the carbohydrate diet plus folic acid. Our data demonstrate that obese adult-onset diabetic patients have an impaired adaptive response of jejunal carbohydrate-metabolizing enzyme activities (hexokinase, pyruvate kinase, fructose-1-6-diphosphate aldolase, fructosediphosphatase) to dietary carbohydrate, oral folic acid, and insulin when compared to normal subjects and nondiabetic obese patients. Following a 30-day fast, the obese diabetic patients showed an improvement in glucose tolerance, hyperinsulinemia, and the adaptive response of the jejunal carbohydrate-metabolizing enzyme activities to dietary carbohydrate, folic acid, and insulin. The greatest improvement in the adaptive response of the jejunal enzyme activities occurred on the carbohydrate diet.

Effects of insulin, tolbutamide, and glucagon on activities of jejunal carbohydrate-metabolizing enzymes in humans.

The activities of jejunal carbohydrate-metabolizing enzymes show adaptive drugs, and sex hormones. To learn whether insulin, tolbutamide, and glucagon had effects on these enzymes, we performed serial peroral jejunal biopsies in normal young men and in obese patients, before and after treatment with these agents. Jejunal mucosa was assayed for glycolytic enzyme activities, pyruvate kinase (PK), hexokinase (HK), and fructose-1,6-diphosphate aldolase (FDPA), and the nonglycolytic enzyme activity, fructose diphosphatase (FDPase). Insulin significantly increased the activity of jejunal PK (+48% change from control) and HK (+6%), decreased the activity of FDPase (-36%),and had no effect on FDPA. Glucagon had opposite effects; the activity of PK was decreased (-33%) and FDPase was increased (+50%). Tolbutamide significantly increased the activities of PK (+47%), HK (+14%), and FDPA (+7%), and decreased the activities of FDPase (-36%). The results of tolbutamide on glycolytic enzyme activities were independent of endogenous insulin. The data support the concept that jejunal carbohydrate-metabolizing enzymes in man respond to hormones and drugs similar to responses observed in rat liver. This is important because it now gives us a means of studying the actions of these hormones directly in human tissue.