Differential effects of insulin on peripheral and visceral tissue protein synthesis in neonatal pigs.

We recently demonstrated in neonatal pigs that, with amino acids and glucose maintained at fasting levels, the stimulation of protein synthesis in longissimus dorsi muscle with feeding can be reproduced by a physiological rise in insulin alone. In the current report, we determine whether the response of protein synthesis to insulin in the neonatal pig is 1) present in muscles of different fiber types, 2) proportional in myofibrillar and sarcoplasmic proteins, 3) associated with increased translational efficiency and ribosome number, and 4) present in other peripheral tissues and in viscera. Hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs infused with 0, 30, 100, or 1,000 ng. kg(-0.66). min(-1) of insulin to reproduce insulin levels present in fasted, fed, refed, and supraphysiological conditions, respectively. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. Insulin increased protein synthesis in gastrocnemius muscle and, to a lesser degree, masseter muscle. The degree of stimulation of protein synthesis by insulin was similar in myofibrillar and sarcoplasmic proteins. Insulin increased translational efficiency but had no effect on ribosome number in muscle. All of these insulin-induced changes in muscle protein synthesis decreased with age. Insulin also stimulated protein synthesis in cardiac muscle and skin but not in liver, intestine, spleen, pancreas, or kidney. The results support the hypothesis that insulin mediates the feeding-induced stimulation of myofibrillar and sarcoplasmic protein synthesis in muscles of different fiber types in the neonate by increasing the efficiency of translation. However, insulin does not appear to be involved in the feeding-induced stimulation of protein synthesis in visceral tissues. Thus different mechanisms regulate the growth of peripheral and visceral tissues in the neonate.

Validation of a [13C]bicarbonate tracer technique to measure neonatal energy expenditure.

The use of a stable isotope-labeled [13C]bicarbonate infusion to measure energy expenditure is advantageous, as a complete collection of expired air is not required. This technique allows for facile measurements of energy expenditure in intubated neonates. The aim of the present study was to determine the accuracy of energy expenditure estimates in postsurgical neonates by using the [13C]bicarbonate method compared with the current standard, indirect calorimetry. Eight neonates who were receiving total parenteral nutrition [98 +/- 21 (SD) kcal x kg(-1) x d(-1); 3.1 +/- 0.7 (SD) protein g x kg(-1) x d(-1)] were studied on postoperative d 15.5 +/- 11.9. A primed continuous 3-h intravenous infusion of NaH13CO3 and indirect calorimetry were performed simultaneously. Energy expenditure was calculated separately from the Weir equation and from the dilution of 13CO2 in the breath in combination with the individual energy equivalents of CO2 from the diet. The rate of CO2 appearance and energy expenditure calculated from the bicarbonate method (0.725 +/- 0.021 mol x kg(-1) x d(-1); 89.5 +/- 2.5 kcal x kg(-1) x d(-1)) highly correlated (r = 0.94 and 0.98, respectively) with the CO2 excretion and energy expenditure determined by indirect calorimetry (0.489 +/- 0.016 mol x kg(-1) x d(-1); 60.2 +/- 2.0 kcal x kg(-1) x d(-1)) when analyzed nonproportionately to weight. Bland-Altman analysis demonstrated the 95% confidence interval to be +/- 8.2 kcal x kg(-1) x d(-1). Linear regression analysis revealed a highly statistically significant equation relating the two energy expenditures: Indircal (kcal/d) = -9.341 + [0.705 x Bicarb (dcal/d)]; p < 0.001, r2 = 96.4%. We conclude that energy expenditure in neonates can be accurately determined using the [13C]bicarbonate method and a regression equation. Therefore, the bicarbonate method may be useful for determining energy expenditure in neonates not readily accessible to indirect calorimetry, such as those being mechanically ventilated or on extracorporeal life support.

Improved inpatient billing metrics by physician-directed billing software.

Billing may not be prominent on academic physicians' lists of priorities because of other demands and commitments for their time. The authors developed and examined the use of an electronic inpatient billing tool that also facilitated physician rounding responsibilities. The results show that use of an electronic tool was more acceptable to physicians than their original card system, and it significantly decreased charge-processing lag time.

Combination growth hormone and estrogen increase bone mineralization in girls with Turner syndrome.

Turner syndrome is characterized by osteopenia and impaired skeletal growth. Neither feature is normalized by current modes of hormone therapy. The purpose of this study was to determine whether GH would increase protein anabolism and provide additional benefit to a regimen of estrogen replacement on calcium metabolism in girls and women with Turner syndrome. Using stable isotopes of calcium and leucine, we determined calcium absorption, urinary calcium loss, calcium retention, deposition into bone, leucine rate of appearance from protein, leucine incorporation into protein, and leucine oxidation in seven girls (10-17 y of age) and four adult females (16-34 y of age) with Turner syndrome, before and after 3 mo of GH treatment. All adults were treated with estrogen (ethinyl estradiol, 50 micrograms/d) and progesterone before and throughout the study. Three girls received no estrogen, and four girls were treated with low-dose estrogen (ethinyl estradiol, 5 micrograms/d) in combination with GH. The addition of estrogen to GH treatment resulted in a significant increase in calcium absorption and deposition in girls. GH did not affect calcium kinetics in adults already receiving estrogen/progesterone replacement therapy, nor did GH alone affect calcium kinetics in girls, and neither GH nor estrogen affected protein metabolism. These data suggest that the addition of low-dose estrogen to a regimen of GH improves bone deposition and calcium metabolism in girls with Turner syndrome and that estrogen is facultative for GH effects on bone.

Response of skeletal muscle protein synthesis to insulin in suckling pigs decreases with development.

The elevated rate of muscle protein deposition in the neonate is largely due to an enhanced stimulation of skeletal muscle protein synthesis by feeding. To examine the role of insulin in this response, hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs. Pigs were infused with 0, 30, 100, or 1,000 ng . kg-0.66 . min-1 of insulin to mimic the plasma insulin levels observed under fasted, fed, refed, and supraphysiological conditions, respectively. Whole body amino acid disposal was determined from the rate of infusion of an amino acid mixture necessary to maintain plasma essential amino acid concentrations near their basal fasting levels. A flooding dose of L-[4-3H]phenylalanine was used to measure skeletal muscle protein synthesis. Whole body amino acid disposal increased progressively as the insulin infusion rate increased, and this response was greater in 7- than in 26-day-old pigs. Skeletal muscle protein synthesis was stimulated by insulin, and this response was maximal at a low insulin infusion rate (30 ng . kg-0.66 . min-1). The stimulation of muscle protein synthesis by insulin was also greater in 7- than in 26- day-old pigs. These data suggest that muscle protein synthesis is more sensitive to insulin than whole body amino acid disposal. The results further suggest that insulin is a central regulatory factor in the elevated rate of muscle protein deposition and the increased response of skeletal muscle protein synthesis to feeding in the neonate.

Developmental changes in the relationship between IGF-I and body composition during puberty.

To examine the hypothesis that anthropometric measures and physical fitness influence circulating insulin-like growth factor-I (IGF-I) during puberty, we measured IGF-I, free IGF-I, IGFBP-1, and IGFBP-3 concentrations in 156 healthy girls (9-16 years old) characterized by aerobic capacity (VO2max), fat-free mass (FFM), percent fat mass and pubertal development. IGF-I, free IGF-I and IGFBP-3 increased with pubertal development while IGFBP-1 declined. Percent fat mass correlated inversely with IGFBP-1 (r = -0.57) and directly with insulin (r = 0.50), while VO2max correlated inversely with percent fat mass (r = -0.63), body mass index (BMI, r = -0.57), and FFM (r = -0.40). When subdivided by Tanner stage, IGF-I correlated directly with weight, height, BMI, and FFM in pre-pubertal girls, but these relationships all diminished or disappeared completely by late puberty. Inverse correlations between IGF-I and percent fat mass, and direct correlations between IGF-I and VO2max as observed previously in adults, were not seen until late puberty. These data suggest that in pre-pubertal and early pubertal girls, IGF-I concentrations in blood reflect overall somatic size. This relationship between IGF-I and body size diminishes with sexual maturation, while correlations between IGF-I and both fitness and fatness emerge.

Insulin does not stimulate protein synthesis acutely in prepubertal children with insulin-dependent diabetes mellitus.

Insulin treatment in adult type I diabetic patients decreases protein loss primarily by inhibiting protein breakdown without stimulating protein synthesis. In young growing rodents, insulin treatment has been reported to stimulate protein synthesis. We examined whether insulin stimulates protein synthesis in normally growing prepubertal children with insulin-dependent diabetes mellitus. Five prepubertal children with insulin-dependent diabetes mellitus (aged 8.6-11.25 yr) were studied in the postabsorptive state on two occasions: once during insulin deprivation (I-; blood glucose, 325 +/- 67.8 mg/dL; mean +/- SD) and once during insulin administration for 4 h (I+; blood glucose, 96 +/- 23.6 mg/dL). Leucine kinetics were measured using a 4-h primed continuous infusion of L-[1-13C]leucine. Serum insulin concentrations were lower (I- vs. I+, 0.6 +/- 0.3 vs. 7.5 +/- 4.3 microU/mL; mean +/- SD; P = 0.02), whereas serum beta-hydroxy-butyrate (I- vs. I+, 3.4 +/- 0.5 vs. 0.9 +/- 0.5 mg/dL; P < 0.001) and free fatty acid concentrations (I- vs. I+, 2.9 +/- 0.4 vs. 0.9 +/- 0.4 mEq/L; P < 0.001) were higher in the insulin-deprived state than during insulin administration. Leucine Ra, an index of protein breakdown (I- vs. I+, 200.5 +/- 23.4 vs. 167 +/- 17 mumol/kg.h; P = 0.008), and leucine oxidation (I- vs. I+, 56.5 +/- 20.7 vs. 29.6 +/- 9.3 mumol/kg.h; P = 0.03) were reduced by insulin treatment. Nonoxidative leucine disposal, an index of protein synthesis, was not affected by insulin treatment (I- vs. I+, 144 +/- 20.8 vs. 137.5 +/- 13.5 mumol/kg.h; P = 0.4). We conclude that the acute decline in net protein loss during insulin treatment in growing prepubertal children, like that in adults, is due primarily to an inhibition of protein breakdown without stimulation of protein synthesis.

The efficiency of dietary protein utilization is increased during puberty.

We investigated whether the efficiency of dietary protein utilization for growth increases during the pubertal growth spurt in both nondiabetic and diabetic subjects. We measured leucine oxidation and retention (intake minus oxidation) in orally fed nondiabetic (n = 9) and diabetic (n = 9) human subjects, aged 7-17 yr. Eight subjects were Tanner stage I, and 10 were Tanner stages III-V; groups were not matched for gender. After 3 days of consuming a diet containing approximately 1 g/kg. day protein, subjects drank a commercial liquid nutrition formula, containing L-[1(-13)C]leucine, every 30 min for a total of 6 h to provide 1 g protein/kg. day. Isotopic enrichment of CO2 was used to calculate the fractional leucine oxidation rate and, together with alpha-ketoisocaproate isotopic enrichment, to calculate total leucine oxidation. Leucine oxidation rates decreased with puberty in both nondiabetic subjects (36.0 +/- 10.4 vs. 23.9 +/- 4.2 mumol/kg fat-free mass (FFM).h, prepubertal and pubertal, respectively; P < 0.05) and diabetic (33.6 +/- 4.9% vs. 27.3 +/- 3.4 mumol/kg FFM.h, prepubertal and pubertal, respectively; P < 0.1) subjects. Leucine retention increased with puberty in both nondiabetic (0.27 +/- 3.2 vs. 15.7 +/- 5.3 mumol/kg FFM.h, prepubertal and pubertal, respectively; P < 0.001) and diabetic (1.9 +/- 4.9 vs. 13.2 +/- 4.4 mumol/kg FFM.h, prepubertal and pubertal subjects, respectively; P < 0.05) subjects. The data suggest that the pubertal growth spurt is associated with a marked increase in the efficiency of dietary protein utilization for growth.

Insulin-stimulated amino acid utilization during glucose and amino acid clamps decreases with development.

Neonatal animals utilize their dietary amino acids for protein accretion with high efficiency, and this efficiency declines during early life. The factors responsible for this developmental change are unknown. Our objectives were to determine whether amino acid (AA) utilization is stimulated by insulin in the neonate and whether this response changes during the suckling period. Two hyperinsulinemic-euglycemic clamp infusion studies, using 10-2,000 ng insulin.kg-0.66.min-1, were performed in 7- and 26-day-old pigs. In study I, no AA were provided during the infusion, and the resultant decline in plasma AA levels was defined. In study II, plasma AA were clamped at near-fasting levels, and whole body utilization of exogenous AA was determined by measuring the rate of infusion of an AA mixture necessary to maintain basal plasma lysine concentrations. In study I, the half-maximal effective dose (ED50) for the fall in AA concentrations with increasing plasma insulin concentration was lower in 7- than in 26-day-old pigs, and the nadir in AA concentration was achieved by only 20 microU/ml insulin. In study II, the utilization of exogenous AA during hyperinsulinemic-euglycemic AA clamps exhibited a higher maximum response (Rmax) (49 vs. 26 mumol AAtotal.min-1.kg-1) and a lower ED50 (18 vs. 45 microU insulin/ml) in 7- than in 26-day-old pigs. Plasma urea nitrogen concentrations did not rise with increasing insulin and AA infusion rates. These results indicate that insulin stimulates the utilization of exogenous AA in neonatal pigs and that both the insulin sensitivity and responsiveness of AA utilization decline over the suckling period. The infused AA were likely utilized for protein accretion.

Spectrophometric assay for measuring branched-chain amino acid concentrations: application for measuring the sensitivity of protein metabolism to insulin.

Plasma amino acid concentrations fall during insulin infusion. Amino acid concentrations can be maintained using an infusion of amino acids if their plasma concentration can be determined within a few minutes. We developed a spectrophometric assay which determines the total concentration of all three branched-chain amino acids in plasma within 1 min. The enzyme leucine dehydrogenase oxidatively deaminates leucine, isoleucine, and valine, with stoichiometric reduction of NAD that is measured using a spectrophotometer. The assay was developed in both a kinetic and end-point format. For the kinetic assay the buffer conditions were formulated to obtain equivalent rates with all three amino acids so that it could be used in samples containing unknown mixtures. For the end-point assay additional enzyme was added so that an end-point could be reached within 1 min. The application of the kinetic assay for "clamping" the branched-chain amino acids during hyperinsulinemic euglycemic clamps in humans is demonstrated.

Corticosterone has independent effects on tissue maturation and growth in the suckling rat.

Glucocorticoids are associated with reduced weight gain when used to improve pulmonary function in premature infants. However, tissue maturation is stimulated during normal development by an increase in serum glucocorticoids. We evaluated the effects of glucocorticoid treatment on tissue weight gain and the activity of specific enzymes in the suckling rat, with the hypothesis that these processes are independently regulated. Before the ontogenic surge in corticosterone, 6-d-old rat pups were implanted with a pellet to release corticosterone continuously at 0 (placebo), 48, 120, 240, or 360 micro g/d. We killed the pups at 7, 9, or 12 d of age and measured tissue weights and activities of sucrase and glutamine synthetase. Serum corticosterone concentrations were elevated with dose. Tissue weight gain was proportional to ln(e) serum corticosterone at all ages. In contrast, enzyme indices of tissue maturation did not respond to corticosterone until d9. Also, intestinal tissue was more sensitive than muscle to the effects of corticosterone on weight but less sensitive to its effects on maturation. We conclude that the immediate response, in terms of weight versus the delayed response of the enzymes and their reciprocal sensitivity in muscle and gut, indicates that these processes are independently regulated.

Valine oxidation: the synthesis and evaluation of L-[3-3H]valine as a tracer in vivo.

The suitability of L-[3-3H]valine for measuring valine oxidation was studied by comparing its oxidation rate with that of L-[1-14C]valine in rats and pigs. L-[3-3H]valine was synthesized by removal of the tritium on carbon-2 of L-[2,3-3H]valine by acetylation. The acetyl group was removed enzymatically using pig renal acylase 1 (EC 3.5.1.14) and the product was purified by ion-exchange and paper chromatography. For the first rat experiment L-[3-3H]valine was synthesized in our laboratory; for the subsequent experiments it was produced by Amersham International plc. In the first experiment in rats the two tracers were given by injection and 14CO2 was collected for 2 h. The oxidation of tritiated valine was significantly higher than that of L-[1-14C]valine. In a second experiment there was no difference. This was probably due to the higher purity of the labelled valine which, for the second experiment, was shown by nuclear magnetic resonance to contain only one tritium atom. In a study with pigs in which the two tracers were given by continuous infusion there was no significant difference between them in flux or oxidation. The results of this experiment were used to evaluate a model to estimate amino acid requirements. With pigs given a methionine-limiting diet a reduction in methionine intake, by reducing protein accretion, increased valine oxidation by the same proportion.

Synergistic interactions between piracetam and dihydroergocristine in some animal models of cerebral hypoxia and ischaemia.

In pharmacological screening tests for activity against the cerebral insults of hypoxia and ischaemia induced by MgCl2 or decapitation in mice, the combination of piracetam and dihydroergocristine has been shown to produce synergistic effects in prolonging the survival time. This was not the case in the model of histiocytic anoxia induced by KCN. Using an optimal combination of piracetam and dihydroergocristine (533:1, Diemil) significant increases in cerebral resistance to hypercapnic anoxia and reductions in the duration of the ensuing electrical silence on the electrocorticogram have been demonstrated in the rat. The same combination was also effective in antagonizing the memory ablating effects of anoxia in rats subjected to electric footshocks during a standard passive avoidance response. The absence of clear effects on gross cerebral blood flow and metabolism, together with considerations of the known pharmacological properties of the two components of the combination and the effects of standard drugs in the models used, lead to the conclusion that the explanation of the observed synergism probably lies in complimentary actions at the level of the cerebral neurones and is independent of simple vasodilation.

Pharmacological studies with almagate, a potent new antacid compound.

Pharmacological screening tests have been done in order to provide an initial assessment of the new antacid compound almagate (aluminium-magnesium hydroxycarbonate hydrate, Al2Mg6(OH)14(CO3)2 X 4 H2O, Almax). In rats with pyloric ligatures, almagate (125-500 mg/kg) was significantly more potent than aluminium hydroxide in raising the pH and reducing the total acidity of the gastric juice produced, without affecting the volume secreted. Pepsin activity in the gastric juice was also significantly inhibited by almagate even after adjustment to the optimal enzyme pH 2, a phenomenon not demonstrable with aluminium hydroxide. Almagate in oral doses up to 3 g/kg was without effects on the central, autonomic and somatic nervous systems in mice, nor at 500 mg/kg did it influence the cardiovascular system or blood pressure responses to agonist drugs in anaesthetised cats. The results confirm almagate to be a potent antacid drug devoid of systemic pharmacological or toxicological effects.

Efficacy in vivo of almagate in neutralising histamine-induced acid secretion in a rat re-perfused stomach preparation.

The efficacies of almagate (hydrated aluminium-magnesium hydroxycarbonate, Al2Mg6(OH)14(CO3)2 X 4H2O, Almax) and aluminium hydroxide to neutralise histamine-induced gastric acid hypersecretion were compared in a closed-circuit perfused stomach preparation modified for pH-stat titration in the intact, anaesthetised rat. After intravenous injection of histamine (0.75 mg/kg), the amount of antacid automatically added to the closed system to maintain the stomach perfusion fluid at pH 4 was measured during 60 min. The results obtained show that the acid neutralising capacity of almagate in this hypersecretion model was some 8 times greater than that of aluminium hydroxide. Analysis of the titration curves obtained also demonstrates that the velocity of neutralisation to pH 4 at the start of the experiment was considerably more rapid with almagate. It is concluded that almagate will be a rapid acting, potent drug to reduce acidity of the gastric contents in conditions of acid hypersecretion.

Protective action of almagate against bile-facilitated gastric ulceration in the pylorus-ligated (Shay) rat.

In man, the reflux of duodenal contents into the stomach leads to gastritis and produces a mucosa susceptible to ulceration. This effect, predominantly due to bile acids, was studied in an animal model in which the oral administration of bile (ox bile powder containing a minimum of 45% cholic acid) markedly increased the gastrolesive action of subcutaneous indomethacin in the pylorus-ligated rat. Oral administration of the new antacid almagate (hydrated aluminium-magnesium hydroxycarbonate, Al2Mg6(OH)14 (CO3)2 X 4 H2O, Almax) gave a significant dose-related reduction in the severity of bile-facilitated gastric lesions whereas aluminium hydroxide gel did not. The antiulcer activity of almagate in this model is presumed to be attributable to its ability to sequester and inactivate bile acids at the pH of the stomach contents, an effect which should beneficially contribute to its acid-neutralising property in the treatment of gastritis, peptic ulcer and associated conditions.