Biomedical subjects
Publications and source records attributed to B E Wright.
The results of using metabolic control theory versus biochemical systems theory to analyze a model of the tricarboxylic acid cycle in Dictyostelium are described. The two types of analysis give essentially the same results, except that smaller responses to perturbations are observed for the model analyzed by metabolic control theory. The basis for this difference is discussed.
Dehydroepiandrosterone (DHEA) reduces food intake in obese Zucker rats. To study the role of taste aversion on this process, we used two approaches. First, we presented increasing concentrations of DHEA in chow to lean and obese Zucker rats, either in competition with unadulterated chow, or alone. Second, we examined energy intake following parenteral DHEA administration. Both lean and obese rats always preferred nonadulterated chow to DHEA-supplemented chow. However, lean rats required a higher DHEA concentration (0.06%) than obese rats (0.015%) to achieve the same degree of aversion. When DHEA-supplemented chow was presented alone, only high concentrations (0.3 and 0.6% DHEA) decreased food intake. Rats given DHEA by IP injection (200 mg/kg/day) also decreased their energy intakes. The results demonstrate that although DHEA can cause taste aversion at low concentrations in Zucker rats, it does not alter energy intake until high concentrations are given. In addition, nonoral DHEA also decreases energy intake in these animals. These results suggest that DHEA's antiobesity effect is not mediated by taste aversion.
Oral dehydroepiandrosterone (DHEA) causes weight loss in the obese Zucker rat. To study this process, we fed lean and obese female Zucker rats either control chow diets alone or diets containing 0.6% DHEA for 4 weeks. DHEA treatment led to a significant increase in the caloric intake of lean-treatment rats and a significant decrease in obese-treatment rats compared to their respective controls. These phenotype-specific divergent effects began acutely and were sustained. The energy intake changes with DHEA treatment were significant after correcting for body weight. Divergent effects of DHEA were also observed in body weight changes and in the food efficiency ratios of the animals; DHEA affected obese rats but not lean ones. The results of the present study suggest that the appetite component of DHEA's antiobesity effect in the Zucker fatty rat cannot be discounted.
A steady-state model of the tricarboxylic acid cycle was constructed using a dynamic systems analysis computer program, METASIM. The model was based on radioactive tracer analyses which provided flux relationships and compartmented metabolite concentrations. Ten of the enzymes modeled were purified and characterized from Dictyostelium discoideum. Although experimentally determined enzyme mechanisms and constants were used in the model, Vmax values were found to be unreliable, i.e. they did not reflect enzyme activity in vivo. This value was therefore calculated as the only unknown in each enzyme kinetic equation and called Vvivo, to distinguish it from Vmax determined in vitro.
A steady-state computer model of the tricarboxylic cycle in Dictyostelium discoideum was analyzed using metabolic control theory. The steady state had variations of less than 0.04% over the last half of the simulation for both metabolite concentrations and fluxes. Metabolite and flux control coefficients were determined by varying enzymatic activities within 2% of their initial values and simulating the responses of metabolite concentrations and fluxes to these changes. Under these conditions, summation properties were met for most metabolite and all flux control coefficients. Maximum flux control coefficients were found for succinate dehydrogenase (0.35), malic enzyme (0.24), and malate dehydrogenase (-0.18). Comparable control was found for the reaction supplying pyruvate (0.14) and for the sum of the input amino acids (0.43), which serve as an energy source for D. discoideum. The time-dependent processes by which a new steady state was established were examined after increasing malic enzyme or malate dehydrogenase activities. This provided a method for an analysis of the mechanisms by which the observed control coefficients were generated. In addition, the effects of increasing the stimuli within 5-20% of the original enzyme activity were examined. Under these conditions, more typical of experimental stimuli and measurable responses, the metabolic model failed to return to steady state, and thus summation properties were not met. Whether "true" steady states ever occur or whether metabolic control theory can be applied in vivo is discussed.
Human serum contains an inhibitor of leukotriene D4 (LTD4) dipeptidase which was separated from the enzyme by ultrafiltration (Amicon, YM-10). Removal of the inhibitor resulted in a 3- to 5-fold increase in total LTD4-dipeptidase activity in the material retained by the filter. Inhibitor activity (which was assayed with a partially purified LTD4-dipeptidase) was recovered in the filtrate. Ultrafiltration of serum using YM-3, YM-1, and YC-05 membranes suggested an inhibitor molecular weight of less than 500. Elution of inhibitor activity from a Bio Gel P2 gel filtration column was identical to the elution pattern of pure carbonate. The inhibitor was heat stable (95 degrees C, 30 min), stable in 0.1 N NaOH, but rapidly inactivated by 0.1 N HCl at both 4 degrees C and 30 degrees C. Partially purified LTD4-dipeptidase was inhibited by carbonate and phosphate but not by nitrate, sulfate, or chloride. Based on these observations it was concluded that the inhibitor of LTD4-dipeptidase in human serum either was carbonate or required carbonate. The relative concentrations of LTC4, LTD4, and LTE4 appear to be important parameters in determining the duration and intensity of LT mediated reactions. The relative concentration of carbonate in serum or extracellular fluids might, therefore, be a factor in modulating localized LT mediated responses.
The acute effect of dehydroepiandrosterone (DHEA) and its conjugate, DHEA-sulfate (DHEA-S) on glucocorticoid action was tested in vivo using male Swiss-Webster mice. The authors found that DHEA and DHEA-S significantly inhibited induction of hepatic tyrosine aminotransferase activity, although the former was more potent. This inhibition was dose- and time-dependent and was not demonstrable with other steroids. The same inhibitory effect of DHEA was seen with kidney tyrosine aminotransferase induction, as well as with liver and kidney ornithine decarboxylase enzyme activity, another glucocorticoid-induced enzyme. The conclusion is that DHEA acts acutely as an antiglucocorticoid and exerts its effect in different glucocorticoid-sensitive systems.
Dehydroepiandrosterone (DHEA) reduces weight gain in the hypercorticosteronemic Zucker fatty rat, an animal model of genetic obesity. However, the mechanism of action of DHEA is still unclear. We propose that DHEA acts as an antiglucocorticoid in the Zucker fatty rat. To test this hypothesis we examined DHEA's ability to block the activation of the glucocorticoid-inducible enzymes tyrosine aminotransferase (TAT) and ornithine decarboxylase (ODC) by dexamethasone (i.p. 5 micrograms/100 g body weight) in hepatic tissue of 6-10 week old Zucker rats. Injections of DMSO, the vehicle, served as a control. DHEA alone did not affect TAT, but when DHEA (500 micrograms/100 g b.w.) was administered simultaneously with dexamethasone, activation did not occur. Similar results were seen using a second tissue (kidney). We conclude that DHEA can act acutely as an antiglucocorticoid in the young obese Zucker rat and hypothesize that its chronic anti-obesity effect may reflect, at least in part, a chronic antiglucocorticoid activity.
This review describes increasingly complex kinetic models that simulate carbohydrate metabolism in a simple eucaryotic system which undergoes differentiation. Dynamic models of complex metabolic networks serve to organize and analyze the many interdependent variables involves and to define the rate-limiting events controlling metabolism in vivo. Since the ultimate justification for and test of any model are its predictive values, a series of predictions and related experiments will be described.
The activity of crude and pure enzyme preparations as well as the molecular weight of these enzymes were obtained from the literature for several organisms. From these data enzyme concentrations were calculated and compared to the concentration(s) of their substrates in the same organism. The data are expressed as molar ratios of metabolite concentration to enzyme site concentration. Of the 140 ratios calculated, 88% were one or greater, indicating that in general substrates exceed their cognate enzyme concentrations. Of the 17 cases where enzyme exceeds metabolite concentration, 16 were in glycolysis. The data in general justify the use of enzyme kinetic mechanisms determined in vitro in the construction of dynamic models which simulate in vivo metabolism.
The relative rates of cysteinyl-leukotriene metabolism were analyzed in fresh human and mouse serum. Human serum contained higher gamma-glutamyl-transpeptidase activity than mouse serum, and a higher percentage of the metabolized leukotriene C4 was recovered as leukotriene D4 in the human serum than in the mouse serum. The results suggest that the patterns of metabolism of the cysteinyl-leukotrienes could be an important factor in determining the relative sensitivity of an animal to the development of hypersensitivity reactions.
The in vivo rate of the reaction catalyzed by the pyruvate dehydrogenase complex in Dictyostelium discoideum was measured by exposing cells to [1-14C]alanine and relating the rate of 14CO2 evolution to the specific radioactivity of the isolated [1-14C]pyruvate. The rate obtained, 0.33 mumol/min per ml packed cell volume, was very similar to that determined by independent calculations based upon the rate of utilization of endogenous protein as an energy source in this system.
The 2-oxoglutarate dehydrogenase complex was isolated from the cellular slime mould, Dictyostelium discoideum, and purified 113-fold. The enzyme exhibited Michaelis-Menten kinetics and the Km values for 2-oxoglutarate, CoA, and NAD were 1.0 mM, 0.002 mM, and 0.07 mM, respectively. The Ki value for succinyl-CoA was determined to be 0.004 mM and the Ki for NADH was 0.018 mM. AMP had positive effects whereas ATP had negative effects on the enzyme activity. The kinetic constants determined in this study and the reaction mechanism suggested can now be incorporated into a transition model of the tricarboxylic acid cycle during differentiation of D. discoideum.
Young sorocarps of Dictyostelium discoideum were incubated in the presence of 50 mM [14C]glucose, and nine metabolites were isolated over a period of 60 min to determine their specific radioactivity. The program TFLUX was used to construct models consisting of 17 metabolite pools and 40 reactions (excluding external pools). Net glucose uptake was 10% or less in the two experiments chosen for extensive analysis, and a single steady-state model was adequate to describe the data in both cases. Despite differences in metabolite levels, flux, and labeling kinetics, the models of glucose-perturbed metabolism confirm earlier conclusions regarding metabolic compartments.
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Young sorocarps (consisting of a mass of spore cells resting on a stalk) were exposed to low levels of [U-14C]glucose and the spore cells were rapidly separated from stalk cells. Metabolites were isolated from spores and their specific radioactivities compared to these metabolites isolated from the whole organism; i.e. spore plus stalk cells. Based on these data, known reaction rates, and metabolite concentrations, highly constrained steady state models of metabolism in spore and stalk cells were constructed. Direct evidence has been obtained which substantiates earlier predictions regarding cell permeability, the distribution of specific metabolites, and the location of reactions in vivo.
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The constraints in the parameters in models of the spore and stalk cells in Dictyostelium discoideum have been examined. It was found that the relative sizes of the two cellular glucose pools are not very critical, i.e. they can be varied in the models over a fairly wide range and still allow simulations which are compatible with the data. In contrast, the following model parameters are highly constrained, and must fall within narrow limits: flux through the glycogen cycle; the fraction of glycogen present which actually participates in glycogen turnover; the net rate of glycogen degradation; the concentration of exogenous labelled glucose which actually participates in cellular metabolism; the rates of exchange of this exogenous glucose with the two cellular glucose pools; the concentration of the spore glucose-6-phosphate pool, and the rate of exchange of stalk glucose-1-phosphate and stalk glucose-6-phosphate.