Quantitative analysis of amino acid oxidation and related gluconeogenesis in humans.

Significant gaps remain in our knowledge of the pathways of amino acid catabolism in humans. Further quantitative data describing amino acid metabolism in the kidney are especially needed as are further details concerning the pathways utilized for certain amino acids in liver. Sufficient data do exist to allow a broad picture of the overall process of amino acid oxidation to be developed along with approximate quantitative assessments of the role played by liver, muscle, kidney, and small intestine. Our analysis indicates that amino acids are the major fuel of liver, i.e., their oxidative conversion to glucose accounts for about one-half of the daily oxygen consumption of the liver, and no other fuel contributes nearly so importantly. The daily supply of amino acids provided in the diet cannot be totally oxidized to CO2 in the liver because such a process would provide far more ATP than the liver could utilize. Instead, most amino acids are oxidatively converted to glucose. This results in an overall ATP production during amino acid oxidation very nearly equal to the ATP required to convert amino acid carbon to glucose. Thus gluconeogenesis occurs without either a need for ATP from other fuels or an excessive ATP production that could limit the maximal rate of the process. The net effect of the oxidation of amino acids to glucose in the liver is to make nearly two-thirds of the total energy available from the oxidation of amino acids accessible to peripheral tissues, without necessitating that peripheral tissues synthesize the complex array of enzymes needed to support direct amino acid oxidation. As a balanced mixture of amino acids is oxidized in the liver, nearly all carbon from glucogenic amino acids flows into the mitochondrial aspartate pool and is actively transported out of the mitochondria via the aspartate-glutamate antiport linked to proton entry. In the cytoplasm the aspartate is converted to fumarate utilizing urea cycle enzymes; the fumarate flows via oxaloacetate to PEP and on to glucose. Thus carbon flow through the urea cycle is normally interlinked with gluconeogenic carbon flow because these metabolic pathways share a common step. Liver mitochondria experience a severe nonvolatile acid load during amino acid oxidation. It is suggested that this acid load is alleviated mainly by the respiratory chain proton pump in a form of uncoupled respiration.(ABSTRACT TRUNCATED AT 400 WORDS)

The association between students' research involvement in medical school and their postgraduate medical activities.

The authors examined the impact of students' research involvement during medical school on their postresidency medical activities. The three medical schools involved--The Pennsylvania State University College of Medicine (PSU), The University of Connecticut School of Medicine (UCONN), and The University of Massachusetts Medical School (UMASS)--have nearly indistinguishable applicant, matriculant, and curriculum profiles. However, at PSU a research project is a curriculum requirement for students who did not do medical research prior to entering medical school. Questionnaires were sent to all graduates from the classes of 1980, 1981, and 1982. A total of 567 graduates completed the questionnaires, an overall response rate of approximately 76%. Medical school research experience was reported by 83% (183) of the PSU graduates, 34% (52) of the UCONN graduates, and 28% (54) of the UMASS graduates. When compared on a school-by-school basis, the graduates from the three schools did not differ with respect to residency specialty training, fellowship training, academic appointments, career practice choices, or postgraduate research involvement. However, when all the graduates studied were examined as a single group, medical school research experience was found to be strongly associated with postgraduate research involvement.

Interaction of insulin with its receptor. I. Possible role of a histidine-arginine interaction.

The interaction of beef and pork insulin with its receptors on rat liver plasma membranes has been studied as a function of pH in tris buffer. The dissociation binding constant decreased from 6.5 to 1.2 nM as the pH was increased from 6.8 to 7.8. Analysis indicated that this was the result of the deprotonation of a single residue with a pK'A of 7.62 at 20 degrees C. The enthalpy change associated with this deprotonation was estimated to be -7,500 cal/mol. On the basis of these parameters it is suggested that this group is a histidine residue on the surface of the insulin receptor. The positively charged group on the insulin molecule which interacts with this histidine was not either of the N-terminal residues, nor the lysine at position B-29; by elimination, it appears to be the B-22 arginine residue.

Regulation of urea synthesis by acid-base balance in vivo: role of NH3 concentration.

The purpose of this study was to clarify how changes in acid-base balance influence the rate of urea synthesis in vivo. Since ureagenesis was increased by an ammonium infusion into rats, regulation seemed to be a function of the blood ammonium concentration. The rate of urea synthesis was constant at a fixed rate of ammonium infusion and independent of the conjugate base infused, chloride or bicarbonate. The steady-state blood ammonium concentration was higher in the rats that developed metabolic acidosis. Thus it appeared that regulation was not directly mediated by this ammonium concentration per se. The rate of urea synthesis was also independent of the blood pH. Accordingly, the rate of urea synthesis was examined as a function of the plasma NH3 concentration. The rate of ureagenesis was found to be directly proportional to the plasma NH3 concentration. Assuming that plasma NH3 levels reflect those in mitochondria, the NH3 concentration yielding half-maximal rates of urea synthesis (close to 2 microM) was in the same range as Km for the rate-limiting step in ureagenesis, carbamoyl phosphate synthetase (EC 6.3.4.16). These results suggest that, at a constant ammonium concentration, the decreased rate of ureagenesis caused by a pH fall in vitro could reflect an acidosis-induced decline in the concentration of true substrate (NH3) for this pathway.

Is urea formation regulated primarily by acid-base balance in vivo?

Large quantities of ammonium and bicarbonate are produced each day from the metabolism of dietary protein. It has recently been proposed that urea synthesis is regulated by the need to remove this large load of bicarbonate. The purpose of these experiments was to test whether the primary function of ureagenesis in vivo is to remove ammonium or bicarbonate. The first series of rats were given a constant acid load as hydrochloric acid or ammonium chloride; individual rats received a constant nitrogen load at a time when their plasma acid-base status ranged from normal (pH 7.4, 28 mM HCO3) to severe metabolic acidosis (pH 6.9, 6 mM HCO3). Urea plus ammonium excretions and the blood urea, glutamine, and ammonium concentrations were monitored with time. Within the constraints of non-steady-state conditions, the rate of urea synthesis was constant and the plasma glutamine and ammonium concentrations also remained constant; thus it appears that the rate of urea synthesis was not primarily regulated by the acid-base status of the animal in vivo over a wide range of plasma ammonium concentrations. In quantitative terms, the vast bulk of the ammonium load was converted to urea over 80 min; only a small quantity of ammonium appeared as circulating glutamine or urinary ammonium. Urea synthesis was proportional to the nitrogen load. A second series of rats received sodium bicarbonate; urea synthesis was not augmented by a bicarbonate load. We conclude from these studies that the need to dispose of excess bicarbonate does not primarily determine the rate of ureagenesis in vivo. The data support the classical view that ureagenesis is controlled by the quantity of ammonium to be removed.

Regulation of the maximum rate of renal ammoniagenesis in the acidotic dog.

Metabolism of glutamine results in the net production of ATP; however, cells cannot sustain an ATP production rate greater than their rate of ATP utilization. The purpose of these studies was to determine whether the rate of ATP turnover in the kidney could set an upper limit on renal glutamine metabolism and thereby renal ammoniagenesis. The acidotic dog kidneys extracted glutamine, lactate, citrate, and oxygen from the arterial blood and added ammonium and alanine to the venous blood. Renal glutamine metabolism was responsible for almost all the ammonium production. Renal ATP production was estimated from the rate of oxygen consumption and appeared to be derived roughly equally from the oxidation of glutamine and lactate. There was no apparent renal glucose production from ATP balance calculations and this impression was supported when the inhibitor of gluconeogenesis, 3-mercaptopicolinate, did not inhibit ammoniagenesis. Approximately 90% of the ATP synthesized was utilized to reabsorb sodium. When the amount of ATP utilized for sodium reabsorption in the proximal convoluted tubule (assumed to be 60% of filtered sodium) was compared with the amount of ATP produced from glutamine metabolism, the values were similar despite the fact that the glomerular filtration rate in individual dogs varied more than fourfold. When the quantity of ATP expended for sodium reabsorption was decreased by the infusion of ouabain or by the constriction of one renal artery without reducing glutamine delivery, the kidney lowered its rate of ammoniagenesis to a quantitatively predictable amount.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic acidosis in the alcoholic: a pathophysiologic approach.

The purpose of this paper is to review the acid-base abnormalities in patients presenting with metabolic acidosis due to acute ethanol ingestion and to review the theoretical constraints on ethanol metabolism in the liver. Alcohol-induced acidosis is a mixed acid-base disturbance. Metabolic acidosis is due to lactic acidosis, ketoacidosis and acetic acidosis but the degree of each varies from patient to patient. Metabolic alkalosis is frequently present due to ethanol-induced vomiting. However, it could be overlooked because of an indirect loss of sodium bicarbonate (as sodium B-hydroxybutyrate in the urine). Nevertheless, the accompanying reduction in ECF volume may play an important role in the pathogenesis of alcoholic acidosis because it could lead to a relative insulin deficiency. Treatment of alcohol acidosis should include sodium, chloride, potassium, phosphorus, magnesium and thiamine replacements along with attention to concomitant clinical problems. Unless hypoglycemia is present, glucose need not be given immediately. We feel that insulin should be withheld unless life-threatening acidemia is present or expected. Lastly, alcohol need not be detected on admission to make the diagnosis of this metabolic disturbance. However, when present, it could contribute directly to the lactic, acetic and B-hydroxybutyric acidoses. With respect to the theoretical constraints on ethanol metabolism, it appears that "overproduction" of NADH in the liver is best averted by converting ethanol to B-hydroxybutyric acid.

Effects of insulin on CO2 fixation in adipose tissue. Evidence for regulation of pyruvate transport.

Insulin was found to double the rate of incorporation of H14CO3- into protein by segments of rat epididymal adipose tissue provided the incubation medium contained a suitable energy substrate such as fructose. Overall protein synthesis was increased by insulin to a lesser extent, one-third as measured by tritiated water indicating that insulin also increased CO2 fixation into amino acids. The latter could be demonstrated only when the tissue amino acid pools were expanded by the addition of aspartate to the incubation medium. The pattern of labeling observed in the amino acids indicated that CO2 fixation occurred primarily at the pyruvate carboxylase step. Addition of pyruvate to the incubation medium also increased CO2 fixation and this effect was not additive with that of insulin, suggesting that insulin acted by increasing the availability of pyruvate to the carboxylase. No change in carboxylase activity could be measured. Mitochondria isolated from tissue exposed to insulin retained a higher capacity to fix CO2 into acid-soluble products provided they were not freeze-thawed or sonicated. Uptake of pyruvate by mitochondria incubated 1 min at 2 degrees C or 5 s at 15 degrees C was doubled by prior insulin treatment of the tissue. It is concluded that insulin increases the flux through pyruvate carboxylase in adipose tissue in part by increasing the transport of pyruvate through the inner mitochondrial membrane.

A quantitative analysis of renal ammoniagenesis and energy balance: a theoretical approach.

Many theories have been proposed to explain the regulation of renal ammoniagenesis during chronic metabolic acidosis but none of these is entirely satisfactory. Since the activity of each of the enzymes in this pathway greatly exceeds the maximum rate of ammonium production in vivo, even when physiological substrate concentrations are used in this calculation, it follows that ammoniagenesis must be inhibited in the intact animal. We shall present a novel hypothesis for the regulation of the maximum rate of ammoniagenesis which emphasizes the fact that ATP is a product of this pathway and that a limited rate of ATP utilization could control its maximum velocity during chronic metabolic acidosis. To test the validity of our hypothesis, a quantitative analysis of the pathways of ATP production and utilization in the kidney will be reviewed. This approach is similar to one already proposed for the regulation of the maximum rate of ketogenesis in the liver.

Evidence for a dual mechanism of lipolysis activation by epinephrine in rat adipose tissue.

Whole homogenates prepared from tissue previously exposed to epinephrine displayed a 3-fold increased rate of lipolysis of endogenous substrate. When the aqueous infranatant phase of such homogenates was collected by centrifugation and assayed against exogenous triolein emulsions, no hormone effect could be demonstrated. Treatment of such infranatants with cAMP-dependent protein kinase prepared from muscle increased their lipase activity against exogenous triolein by 80%. Employing [3H]triolein emulsions as exogenous substrate, rates of lipolysis of both endogenous and exogenous glycerides were measured simultaneously in whole tissue homogenates. Prior treatment of the tissue with epinephrine increased the rate of lipolysis of endogenous glycerides an average of 3-fold but had no effect on the hydrolysis of exogenous triolein. By contrast, treatment of whole homogenates with protein kinase accelerated lipolysis of exogenous triolein without altering the rate of hydrolysis of endogenous glycerides. The data suggest that a second pathway of lipolysis activation occurs in response to epinephrine in addition to that involving a cAMP-mediated increase in the state of phosphorylation of the hormone-sensitive lipase.

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.

Regulation of pyruvate dehydrogenase in isolated rat liver mitochondria. Effects of octanoate, oxidation-reduction state, and adenosine triphosphate to adenosine diphosphate ratio.

Factors which influence the distribution of pyruvate dehydrogenase between its active, unphosphorylated form (PDHa) and its inactive, phosphorylated form (PDHb) have been examined in isolated rat liver mitochondria. A rapid freezing method was developed for the extraction of pyruvate dehydrogenase from incubated mitochondria which prevented interconversions between PHDa and PDHb which normally occur when mitochondria are collected by centrifugal methods. The intramitochondrial ATP:ADP ration was varied over a 100-fold range by the addition of dinitrophenol, oligomycin, or both substances to mitochondria oxidizing 2-oxoglutarate. PDHa activity was found to be inversely proportional to the intramitochondrial ATP:ADP ratio but was not closely correlated with the extramitochondrial adenine nucleotide levels. When mitochondria were incubated in State 4 with succinate and rotenone, the addition of pyruvate increased PDHa activity more than 10-fold without appreciably altering the mitochondrial ATP:ADP ratio. These observations are most readily explained by the known inhibitory effects of pyruvate and ADP on PDHa kinase. PDHa activity could be maintained at a high level by incubating mitochondria in a condition resembling State 3 by the addition of succinate, glucose, and hexokinase. The further addition of octanoate reduced PDHa activity by 60% without appreciably altering the ATP:ADP ratio. Rotenone had a sililar effect. When added in the presence of octanoate, rotenone further decreased PDHa activity whereas 4-pentenoate led to an increase in activity. The effects of octanoate on PDHa activity were not seen when mitochondria were incubated in the presence of high levels of pyruvate, though pyruvate oxidation was till diminished by over 50%. The data suggest that octanoate addition favors the PDHa kinase reaction leading to inactivation of PDHa, and in addition causes the accumulation of NADH and acetyl-CoA which are recognized competitive inhibitors of pyruvate dehydrogenase.

Influence of luteinizing hormone and adenosine 3':5'-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumours.

1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-(3)H]cholesterol (50-100muCi per animal). Single-cell suspensions were prepared from the tumours 5-9 days after the injection of [(3)H]cholesterol and were incubated at 37 degrees C in foetal calf serum supplemented with 50mm-Tris-HCl, pH7.4. At various times after the start of incubation cells were collected by filtration of portions of the suspension and their sterols analysed. Within 10min after LH (5mug/ml) or 3':5'-cyclic AMP (20mm) was added to the cell suspensions an increased conversion of ester cholesterol into free cholesterol could be demonstrated. 2. To observe this rapid effect of LH it was necessary to incubate the cells for 60min before addition of hormone. 3. The specific radioactivity of testosterone produced was approximately equal to that of the intracellular cholesterol regardless of the presence or absence of LH. 4. The amount of free cholesterol produced in response to LH was far greater than that needed for steroid synthesis. 5. Free cholesterol, but not esterified cholesterol, was released into the incubation medium linearly with time and this release was unaffected by LH. LH may stimulate steroidogenesis in part by increasing the concentration of free cholesterol within the cell.

Metabolism of free and esterified cholesterol by Leydig-cell tumour mitochondria.

1. Experiments were designed to localize intracellularly the enzymes and sterol substrates required for steroidogenesis in Leydig-cell tumours. Subcellular fractions were prepared by differential centrifugation of tumour homogenates. Both free and esterified cholesterol were associated primarily with the fractions sedimenting at 1400g(av.) and the lipid layer floating on the surface of the isolation tubes; they were not found in the mitochondria, where the conversion of cholesterol into pregnenolone occurred. 2. Hydrolysis of esterified cholesterol was required before it could be oxidized to pregnenolone. 3. An enzyme capable of hydrolysing cholesterol esters was located external to the mitochondria. 4. Mitochondria were subfractionated by allowing them to swell in 0.02m-phosphate buffer (pH7.2) and separating the inner and outer membranes by sedimentation in sucrose gradients. The outer membrane, identified by its content of monoamine oxidase, contained most of the cholesterol associated with the mitochondria. The inner membrane, identified by its content of succinate dehydrogenase, contained the cholesterol side-chain-cleaving enzyme and very little cholesterol. 5. Accumulation of sterols by the mitochondria was studied by incubating this fraction with labelled free and esterified cholesterol suspended in lipid-free bovine serum albumin. Two phases of cholesterol accumulation were observed. The first phase, requiring 10-15min, was independent of the incubation temperature, and was inhibited by the presence of bovine serum albumin in the incubation medium. The second phase of accumulation was independent of the serum albumin content of the medium but was inhibited by low incubation temperature. 6. Esterified cholesterol was not accumulated by the mitochondria after the initial rapid binding phase. 7. The findings suggest that cholesterol was not rapidly accumulated by the mitochondrial fraction in vitro and that mechanisms may be required to facilitate cholesterol transport into mitochondria in intact tumour cells during the periods in which steroidogenesis is stimulated maximally.