In vivo NMR studies of glycogen metabolism: a new look at a time honored problem.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
E J Barrett
Publications and source records attributed to E J Barrett.
Myocardial amino acid transport by canine sarcolemma vesicles.
The transport of L-alanine and L-leucine into membrane vesicles isolated from mature canine ventricular myocardium was studied. Transport was assessed in purified sarcolemma and in vesicles differentially enriched either for sarcolemma or sarcoplasmic reticulum to further localize these transport systems. An imposed inward gradient of a NaNO3 stimulated uptake of L-alanine but not L-leucine by these vesicles. Amino acid uptake by these vesicles occurred into an osmotically active space. The stimulatory effect of Na+ on alanine transport was most striking in the purified sarcolemma vesicles, where Na+-stimulated alanine flux was 45 +/- 14 pmol X mg-1 X min-1. Furthermore, Na+-dependent alanine transport activity appeared to copurify with Na+-K+-ATPase activity, which served as a marker for sarcolemma membrane when these activities were compared in the three different membrane preparations. Leucine transport by sarcolemma was not altered by an imposed Na+ gradient. However, leucine uptake was a saturable function of extravesicular leucine and was inhibited by valine. In contrast, in sarcoplasmic reticulum membrane vesicles leucine uptake increased proportionately with increasing media leucine and was unaffected by valine. Our results demonstrate the feasibility of directly studying the transport of naturally occurring amino acids in membrane vesicles from mammalian heart, and the presence of Na+-dependent alanine transport system and a Na+-independent leucine transporter in the sarcolemma but not in sarcoplasmic reticulum of canine ventricular myocardium.
Alanine↗
Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man.
Although insulin stimulates protein synthesis and inhibits protein breakdown in skeletal muscle in vitro, the actual contribution of these actions to its anabolic effects in man remains unknown. Using the forearm perfusion method together with systemic infusion of L-[ring-2,6-3H]phenylalanine and L-[1-14C]leucine, we measured steady state amino acid exchange kinetics across muscle in seven normal males before and in response to a 2-h intraarterial infusion of insulin. Postabsorptively, the muscle disposal (Rd) of phenylalanine (43 +/- 5 nmol/min per 100 ml forearm) and leucine (113 +/- 13) was exceeded by the concomitant muscle production (Ra) of these amino acids (57 +/- 5 and 126 +/- 9 nmol/min per dl, respectively), resulting in their net release from the forearm (-14 +/- 4 and -13 +/- 5 nmol/min per dl, respectively). In response to forearm hyperinsulinemia (124 +/- 11 microU/ml), the net balance of phenylalanine and leucine became positive (9 +/- 3 and 61 +/- 8 nmol/min per dl, respectively (P less than 0.005 vs. basal). Despite the marked increase in net balance, the tissue Rd for both phenylalanine (42 +/- 2) and leucine (124 +/- 9) was unchanged from baseline, while Ra was markedly suppressed (to 33 +/- 5 and 63 +/- 9 nmol/min per dl, respectively, P less than 0.01). Since phenylalanine is not metabolized in muscle (i.e., its only fates are incorporation into or release from protein) these results strongly suggest that in normal man, physiologic elevations in insulin promote net muscle protein anabolism primarily by inhibiting protein breakdown, rather than by stimulating protein synthesis.
Adolescent↗
Chronic thyroxine ingestion leading to thyroid storm and accelerated thyroxine turnover.
Explore the source record for details and available documents.
Aged↗
Effect of age and muscle type on regeneration of neuromuscular synapses in mice.
A silver stain was used to investigate the regeneration of nerve terminals in mouse diaphragm, superior gluteus and tensor fasciae latae (TFL) muscles following nerve crush injury at different ages (9-530 days). The development of myelinated terminal branches and the development and elimination of terminal sprouts were little affected by age or muscle type following reinnervation. However, multiple axonal innervation developed on up to 50% of the gluteus and TFL muscle fibres, and this was subsequently eliminated only in the youngest animals.
Aging↗
Amino acid and glucose metabolism in the postabsorptive state and following amino acid ingestion in the dog.
Amino acid and glucose metabolism was studied in nine awake 18-hour fasted dogs with chronic portal, arterial, and hepatic venous catheters before and for three hours after oral ingestion of amino acids. The meal was composed of a crystalline mixture of free amino acid, containing neither carbohydrate nor lipid. Following the amino acid meal, plasma glucose concentration declined slowly and this occurred despite a rise in hepatic glucose release. Portal plasma insulin rose transiently (30 +/- 7 to 50 +/- 11 microU/mL, P less than 0.05) while the increase in portal glucagon was more striking and persisted throughout the study (162 +/- 40 to 412 +/- 166 pg/mL). Over the three hours following amino acid ingestion, the entire ingested load of glycine, serine, phenylalanine, proline, and threonine was recovered in portal blood as was 80% of the ingested branched chain amino acids (BCAA). The subsequent uptake of these glucogenic amino acids by the liver was equivalent to the amount ingested, while hepatic removal of BCAA could account for disposal of 44% of the BCAA absorbed; the remainder was released by the splanchnic bed. During this time, ongoing gut production of alanine was observed and the liver removed 1,740 +/- 170 mumol/kg of alanine, which was twofold greater than combined gut output of absorbed and synthesized alanine. In the postcibal state, the total net flux of alanine and five other glucogenic amino acids from peripheral to splanchnic tissues (1,480 mumol/kg 3 h) exceeded the net movement of branched chain amino acids from splanchnic to peripheral tissues (590 mumol/kg/3 h).(ABSTRACT TRUNCATED AT 250 WORDS)
Absorption↗
Effect of free fatty acids on blood amino acid levels in human.
Raised plasma free fatty acid (FFA) levels effectively impede glucose uptake in vivo, thereby conserving plasma glucose and sparing glycogen. To test whether FFA have any effect on blood amino acid levels, we infused Intralipid plus heparin or saline into healthy volunteers under four different experimental conditions: A) overnight fast; B) euglycemic hyperinsulinemia (approximately 100 microU/ml); C) hyperglycemic (approximately 200 mg/100 ml) hyperinsulinemia (approximately 50 microU/ml); and D) hyperglycemic (approximately 300 mg/100 ml) normoinsulinemia (approximately 20 microU/ml). In the fasting state (A), lipid infusion was associated with lower blood levels of most amino acids, both branched chain and glucogenic. This effect, however, could not be ascribed to lipid infusion alone, because plasma insulin levels were also stimulated. The clamp studies (B, C, and D) allowed to assess the influence of lipid on blood amino acid levels at similar plasma insulin and glucose levels. It was thus observed that lipid infusion has a significant hypoaminoacidemic effect of its own under both euglycemic (B) and hyperglycemic (C) conditions; this effect involved many glucogenic amino acids (alanine, glycine, phenylalanine, serine, threonine, and cystine) but none of the branched-chain amino acids (leucine, isoleucine, and valine). In marked contrast, normoinsulinemic hyperglycemia (D), with or without lipid infusion, caused no change in the blood level of any measured amino acid. We conclude that lipid infusion has a hypoaminoacidemic action. We also suggest that this action is permitted by insulin and may involve specific metabolic interactions (e.g., reduced availability of glucose-derived pyruvate or glycerophosphate) as well as enhanced uptake by the liver.
Adolescent↗
Characterization of glutamine transport by liver plasma membrane vesicles.
Plasma membrane vesicles were prepared from livers of fed normal and diabetic rats and used to characterize the membrane transport process responsible for glutamine uptake by the liver cell. In vesicles from normal rats the initial velocity of glutamine uptake was fourfold more rapid (0.20 +/- 0.02 vs. 0.05 +/- 0.02 nmol X mg protein-1 X 10 s-1) when Na+ replaced K+ in the extravesicular buffer. In the presence of a Na+-gradient glutamine uptake by vesicles was saturable, with a Km of 1.3 +/- 0.5 mM and a Vmax of 10 +/- 2.3 nmol X mg-1 X min-1. Lithium could fully substitute for Na+ in stimulating glutamine entry. In the presence of an imposed K+-gradient glutamine uptake was a linear function of its extravesicular concentration. In accord with the sodium-stimulated uptake of glutamine occurring via a sodium symport process, we observed that glutamine stimulated the initial rate of 22Na+ entry into vesicles by four- to fivefold. We further observed that glutamine entry was more rapid when lipophilic anions accompanied sodium in the incubation buffer, suggesting that Na+-glutamine flux is electrogenic. Preloading of vesicles with glutamine did not effect subsequent entry of labeled glutamine (no transstimulation), whereas intravesicular alanine did enhance alanine but not glutamine entry. Alloxan diabetes, which is known to stimulate the Na+-alanine cotransporter in these vesicles did not increase glutamine entry at any concentration tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine↗
Hepatic and peripheral insulin resistance following streptozotocin-induced insulin deficiency in the dog.
Insulin resistance and insulin deficiency are both present in many patients with diabetes mellitus. We tested the hypothesis that insulin resistance can evolve from a primary lesion of the beta-cell secretory function. Insulin-mediated glucose uptake (insulin clamp), endogenous glucose production, and glucose-stimulated insulin secretion (hyperglycemic clamp) were measured in awake dogs before and four to six weeks after streptozotocin-induced diabetes mellitus. Streptozotocin (30 mg/kg) resulted in a significant rise in the mean fasting plasma glucose concentration from 104 +/- 2 mg/100 mL to 200 +/- 34 mg/100 mL, (P less than 0.05), and a slight decrease in the mean fasting plasma insulin concentration (from 21 +/- 2 microU/mL to 15 +/- 2 microU/mL). Under conditions of steady-state hyperglycemia (+75 mg/100 mL hyperglycemic clamp, insulin secretion was reduced by 75% in the streptozotocin-treated dogs (P less than 0.025), and the total amount of glucose metabolized decreased from 13.56 +/- 1.04 to 4.74 +/- 0.70 mg/min X kg (P less than 0.001). In the postabsorptive state, endogenous glucose production was slightly, although not significantly, higher in the diabetic dogs (3.05 +/- 0.46 v 2.51 +/- 0.22 mg/min . kg), while the glucose clearance rate was 35% lower (P less than 0.001). When the plasma insulin concentration was increased to approximately 45 microU/mL (insulin clamp) while holding plasma glucose constant at the respective fasting levels (99 +/- 1 and 186 +/- 30 mg/100 mL), endogenous glucose production was completely suppressed in control dogs but suppressed by only 51% (1.46 +/- 0.37 mg/min . kg, P less than 0.025) in diabetic animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Hepatic and extrahepatic splanchnic glucose metabolism in the postabsorptive and glucose fed dog.
In awake dogs we measured the glucose balance across the liver and extrahepatic splanchnic tissues in the postabsorptive state and during two hours of IV infusion of glucose or for three hours following ingestion of oral glucose and during four hours of sequential intraportal followed by oral glucose. The IV glucose infusion rate was adjusted to maintain a steady state glucose concentration of either euglycemic levels (insulin clamp, group 1, N = 4), 125 mg/100 mL above the postabsorptive glucose concentration (+125 mg glucose clamp, group 2, N = 3) or 200 mg/100 mL above basal glucose levels (+200 mg glucose clamp, group 3, N = 7). Oral glucose was given at a dose of either 1.5 g/kg (group 4, N = 7) or 2.5 g/kg (group 5, N = 12). In dogs that received IV glucose, basal gut glucose uptake (0.5 +/- 0.1 mg/min X kg) was stimulated by hyperglycemia (1.5 +/- 0.5 and 1.4 +/- 0.1 mg/min X kg for group 2 and 3, respectively, P less than 0.05). In these same animals basal hepatic glucose output (-2.7 +/- 0.3 mg/min X kg) was promptly suppressed and net hepatic glucose uptake occurred (2.8 +/- 0.2 and 2.4 +/- 0.5 mg/min X kg in group 2 and 3 respectively). Euglycemic hyperinsulinemia (group 1) suppressed postabsorptive hepatic glucose release but did not enhance glucose removal by either the liver or gut tissues. After oral glucose gut tissues released absorbed glucose into portal blood. Over three hours following the glucose meal 74% and 59% of the ingested glucose was absorbed in group 4 and 5, respectively. As with IV glucose, postabsorptive hepatic glucose production was suppressed and over the first two hours after feeding the liver took up glucose (3.4 +/- 1.0 and 3.1 +/- 0.7 mg/min X kg groups 4 and 5, respectively) at a rate similar to that seen with IV glucose. To further examine the effect of the route of glucose administration on liver glucose handling, hepatic glucose balance was measured serially over four hours in three dogs that received IV glucose into a mesenteric vein to produce portal hyperglycemia (+125 mg/dL portal glucose clamp N = 3). Oral glucose (2.5 mg/kg) was given at two hours, and the rate of the mesenteric glucose infusion adjusted to maintain portal glycemia constant. The hepatic glucose balance averaged 5.5 mg/min X kg over the 0 to 2 hour period and 4.2 +/- 1.0 mg/min X kg over the 2 to 4 hour time.(ABSTRACT TRUNCATED AT 400 WORDS)
Alanine↗
Nuclear magnetic resonance spectroscopy: its evolving role in the study of myocardial metabolism.
Explore the source record for details and available documents.
Animals↗
Regulation of myocardial amino acid balance in the conscious dog.
The effects in vivo of physiologic increases in insulin and amino acids on myocardial amino acid balance were evaluated in conscious dogs. Arterial and coronary sinus concentrations of amino acids and coronary blood flow were measured during a 30-min basal and a 100-min experimental period employing three protocols: euglycemic insulin clamp (plasma insulin equaled 70 +/- 11 microU/ml, n = 6); euglycemic insulin clamp during amino acid infusion (plasma insulin equaled 89 +/- 12 microU/ml, n = 6); and suppression of insulin with somatostatin during amino acid infusion (plasma insulin equaled 15 +/- 4 microU/ml, n = 6). Basally, only leucine and isoleucine were removed significantly by myocardium (net branched chain amino acid [BCAA] uptake equaled 0.5 +/- 0.2 mumol/min), while glycine, alanine, and glutamine were released. Glutamine demonstrated the highest net myocardial production (1.6 +/- 0.2 mumol/min). No net exchange was seen for valine, phenylalanine, tyrosine, cysteine, methionine, glutamate, asparagine, serine, threonine, taurine, and aspartate. In group I, hyperinsulinemia caused a decline of all plasma amino acids except alanine; alanine balance switched from release to an uptake of 0.6 +/- 0.4 mumol/min (P less than 0.05), while the myocardial balance of other amino acids was unchanged. In group II, amino acid concentrations rose, and were accompanied by a marked rise in myocardial BCAA uptake (0.4 +/- 0.1-2.6 +/- 0.3 mumol/min, P less than 0.001). Uptake of alanine was again stimulated (0.9 +/- 0.3 mumol/min, P less than 0.01), while glutamine production was unchanged (1.3 +/- 0.4 vs. 1.6 +/- 0.3 mumol/min). In group III, there was a 4-5-fold increase in the plasma concentration of the infused amino acids, accompanied by marked stimulation in uptake of only BCAA (6.8 +/- 0.7 mumol/min). Myocardial glutamine production was unchanged (1.9 +/- 0.4-1.3 +/- 0.7 mumol/min). Within the three experimental groups there were highly significant linear correlations between myocardial uptake and arterial concentration of leucine, isoleucine, valine, and total BCAA (r = 0.98, 0.98, 0.92, and 0.97, respectively); P less than 0.001 for each). In vivo, BCAA are the principal amino acids taken up by the myocardium basally and during amino acid infusion. Plasma BCAA concentration and not insulin determines the rate of myocardial BCAA uptake. Insulin stimulates myocardial alanine uptake. Neither insulin nor amino acid infusion alters myocardial glutamine release.
Amino Acids↗
An assessment of insulin action in hyperosmolar hyperglycemic nonketotic diabetic patients.
The effect of insulin treatment on the rate of decline of plasma glucose concentration was determined in nine patients with hyperosmolar hyperglycemic nonketosis [HHNK; mean plasma glucose, 999 +/- 59 (+/- SEM) mg/dl] and in six normal subjects rendered hyperglycemic by a combined infusion of somatostatin and glucose (mean plasma glucose, 653 +/- 28 mg/dl). Both the fractional glucose turnover and the half-time of the fall in plasma glucose during low dose (5-10 U/h) insulin treatment were reduced 10-fold (P less than 0.001) in the diabetic patients compared with the hyperglycemic normal subjects. In the hyperosmolar patients, the mean glucose clearance during insulin treatment was only 7% that in the normal subjects (P less than 0.001). The rate of plasma glucose decline in our hyperosmolar patients after hydration and insulin administration was 80 +/- 7 mg/dl X h. This decline is comparable to the results reported in other series, although in striking contrast to the 508 +/- 32 mg/dl X h decline in normal subjects (P less than 0.001). Our findings do not support the clinical impression that HHNK patients are insulin sensitive. We conclude that marked resistance to infused insulin delays the correction of hyperglycemia during treatment of HHNK and suggest that resistance to the normal basal insulin levels encountered in some HHNK patients may contribute in part to the development of the hyperosmolar state.
Adult↗
Diabetic ketoacidosis: diagnosis and treatment.
Explore the source record for details and available documents.
Bicarbonates↗
Regulation by insulin of myocardial glucose and fatty acid metabolism in the conscious dog.
In vivo small doses of insulin inhibit lipolysis, lower plasma FFA, and stimulate glucose disposal. Lowering of plasma FFA, either in the absence of a change in insulin or during combined hyperglycemia and hyperinsulinemia, promotes glucose uptake by heart muscle in vivo. In the isolated perfused heart, large doses of insulin directly stimulate heart glucose uptake. To assess the effect of physiological elevations of plasma insulin upon myocardial glucose and FFA uptake in vivo independent of changes in plasma substrate concentration, we measured arterial and coronary sinus concentrations of glucose, lactate, and FFA, and coronary blood flow in conscious dogs during a 30 min basal and a 2 h experimental period employing three protocols: (a) euglycemic hyperinsulinemia (insulin clamp, n = 5), (b) euglycemic hyperinsulinemia with FFA replacement (n = 5), (c) hyperglycemic euinsulinemia (hyperglycemic clamp with somatostatin, n = 5). In group 1, hyperinsulinemia (insulin = 73 +/- 13 microU/ml) stimulated heart glucose uptake (7.3 +/- 4.4 vs. 28.2 +/- 2.8 mumol/min, P less than 0.002), lowered plasma FFA levels by 80% (P less than 0.05), and decreased heart FFA uptake (28.4 +/- 4 vs. 1.5 +/- 0.9, P less than 0.01). When the fall in plasma FFA was prevented by FFA infusion (group 2), hyperinsulinemia (86 +/- 10 microU/ml) provoked a lesser (P less than 0.05) stimulation of glucose uptake (delta = 8.2 +/- 4.2 mumol/min) than in group 1, and there was no significant change in FFA uptake (25.3 +/- 16 vs. 16.5 +/- 4). Hyperglycemia (plasma glucose = 186 +/- 8 mg/100 ml) during somatostatin infusion resulted in only a small rise in plasma insulin (delta = 12 +/- 7 microU/ml), and although plasma FFA tended to decline, heart glucose uptake did not rise significantly (delta = 5.5 +/- 3.2 mumol/min, P = NS). There was no significant change in coronary blood flow during any of the three study protocols. We conclude that, in the dog, insulin at physiologic concentrations: (a) stimulates heart glucose uptake, both directly and by suppressing the plasma FFA concentration, and (b) does not alter coronary blood flow. Hyperglycemia per se has little effect on heart glucose uptake.
Animals↗
In vivo 31P-NMR studies of myocardial high energy phosphate metabolism during anoxia and recovery.
31P NMR spectra of heart in-situ in live guinea pigs were obtained continuously in 20.5 s time blocks during 3 min of anoxia, during subsequent reoxygenation and, in separate animals, during terminal anoxia. Reversible anoxia resulted in rapid degradation of phosphocreatine (t 1/2 = 54.5 +/- 2.5 s) which recovered fully during reoxygenation. Heart Pi increased during anoxia and returned to basal levels after oxygen was restored. During 3 min of anoxia, no significant changes in ATP levels or pH were detected. The results demonstrate that it is feasible to measure rapid fluxes of high energy phosphates by 31P NMR in intact animals during and after anoxic stress to the myocardium.
Animals↗
In vivo carbon-13 nuclear magnetic resonance studies of heart metabolism.
Guinea pig heart metabolism was studied in vivo by 13C NMR at 20.18 MHz. High-quality proton-decoupled 13C NMR spectra with excellent signal-to-noise ratios and resolution could be obtained in 6 min. Natural-abundance spectra showed resonances that could be assigned to fatty acids, but glycogen was not seen. During intravenous infusion of D-[1-13C]glucose and insulin, the time course of myocardial glycogen synthesis was followed serially for up to 4 hr. Anoxia resulted in degradation of the labeled glycogen within 6 min and appearance of 13C label in lactic acid. Infusion of sodium [2-13C]acetate resulted in incorporation of label into the C-4, C-2, and C-3 positions of glutamate and glutamine, reflecting "scrambling" of the label expected from tricarboxylic acid cycle activity. Examination of the 31P NMR spectrum of the guinea pig heart in vivo demonstrated no change in the high-energy phosphates during the time periods of the 13C NMR experiments. Our studies indicate that 13C NMR is a unique non-destructive tool for the study of heart metabolism in vivo.
Acetates↗
Effect of fatty acids on glucose production and utilization in man.
Since the initial proposal of the glucose fatty acid cycle, considerable controversy has arisen concerning its physiologic significance in vivo. In the present study, we examined the effect of acute, physiologic elevations of FFA concentrations on glucose production and uptake in normal subjects under three controlled experimental conditions. In group A, plasma insulin levels were raised and maintained at approximately 100 microU/ml above base line by an insulin infusion, while holding plasma glucose at the fasting level by a variable glucose infusion. In group B, plasma glucose concentration was raised by 125 mg/100 ml and plasma insulin was clamped at approximately 50 microU/ml by a combined infusion of somatostatin and insulin. In group C, plasma glucose was raised by 200 mg/100 ml above the fasting level, while insulin secretion was inhibited with somatostatin and peripheral glucagon levels were replaced with a glucagon infusion (1 ng/min X kg). Each protocol was repeated in the same subject in combination with a lipid-heparin infusion designed to raise plasma FFA levels by 1.5-2.0 mumol/ml. With euglycemic hyperinsulinemia (study A), lipid infusion caused a significant inhibition of total glucose uptake (6.3 +/- 1.3 vs. 7.4 +/- 0.6 mg/min X kg, P less than 0.02). Endogenous glucose production (estimated by the [3-3H]glucose technique) was completely suppressed both with and without lipid infusion. With hyperglycemic hyperinsulinemia (study B), lipid infusion also induced a marked impairment in glucose utilization (6.2 +/- 1.1 vs. 9.8 +/- 1.9 mg/min X kg, P less than 0.05); endogenous glucose production was again completely inhibited despite the increase in FFA concentrations. Under both conditions (A and B), the percentage inhibition of glucose uptake by FFA was positively correlated with the total rate of glucose uptake (r = 0.69, P less than 0.01). In contrast, when hyperglycemia was associated with relative insulinopenia and hyperglucagonemia (study C), thus simulating a diabetic state, lipid infusion had no effect on glucose uptake (2.9 +/- 0.2 vs. 2.6 +/- 0.2 mg/min X kg) but markedly stimulated endogenous glucose production (1.4 +/- 0.5 vs. 0.5 +/- 0.4 mg/min X kg, P less than 0.005). Under the same conditions as study C, a glycerol infusion producing plasma glycerol levels similar to those achieved with lipid-heparin, enhanced endogenous glucose production (1.5 +/- 0.5 vs. 0.7 +/- 0.6 mg/min X kg, P less than 0.05). We conclude that, in the well-insulinized state raised FFA levels effectively compete with glucose for uptake by peripheral tissues, regardless of the presence of hyperglycemia. When insulin is deficient, on the other hand, elevated rates of lipolysis may contribute to hyperglycemia not by competition for fuel utilization, but through an enhancement of endogenous glucose output.
3-Hydroxybutyric Acid↗