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Gluconeogenesis In The Kidney Cortex

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Gluconeogenesis In The Kidney Cortex

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Content GLUCONEOGENESIS IN THE KIDNEY CORTEX by Robert Alan Rognstad A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Cellular and Molecular Biology) February 1972 I 7 2 - 1 7 ,5 0 5 ROGNSTAD, R o b e r t A l a n , 1 9 3 0 - i GLUCONEOGENESIS IN THE KIDNEY CORTEX. University of Southern California, Ph.D., 1972 Biochemistry University Microfilms. A X E R O X . Company, Ann Arbor. Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED UNIVERSITY O F SO U T H E R N CALIFO RN IA TH E GRADUATE SC H O O L UNIVERSITY PARK LOS A N G ELES. C A LIFO R N IA 9 0 0 0 7 This dissertation, written by a ^ under the direction of h.IS.... Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Gradu ate School, in partial fulfillment of require ments of the degree of D O C T O R OF P H I L O S O P H Y f 'Tri Dean Date. /Vo v. 30 j (311 DISSERTATION COMMITTEE airman _ U) PLEASE NOTE: Some pages may have indistinct print. Filmed as received. University Microfilms, A Xerox Education Company Acknowledgements I should like to express my sincere appreciation to Dr. Joseph Katz who guided and encouraged me in this work. The financial support of the United States Public Health Service is gratefully acknowledged. ii Table of Contents Page I. Historical and Theoretical Introduction ... 1 A. General Aspects of Gluconeogenesis ... 1 B. Gluconeogenesis in the Kidney Cortex . . 2 C. Control Mechanisms in Gluconeogenesis . . 4 D. Quantitative Pathway Determination ... 11 E. Scope of Dissertation.....................13 II. Methods...................................... 15 A. Incubation Procedure .................. 15 B. Fractionation of Products .............. 16 C. Degradation Procedures ................ 18 D. Syntheses.................................19 1. 1 4 C Labeled Substrates................ 19 2. Tritium Labeled Substrates ........ 24 III. Results and Discussion.......................30 A. Inhibitor Studies ...................... 30 1. Aminooxyacetate...................... 30 2. n-Butylmalonate and p-Iodobenzyl malonate.......................... 35 3* y-Hydroxyglutamate, y-Hydroxy-a- fcetoglutarate, and Glyoxylate ... 39 4. 2,4-Dinitrophenol ...............44 5. Quinolinate.......................... 46 6. Fluorocitrate, Chlorosuccinate, and Malonate.......................... 48 iii Page 7. Phenylpyruvate, Oxalate, and Oxamate . 52 B. Pathways of Carbon.....................52 1. Computer Model.................... 52 2. Reversibility in the Whole Pathway . . 63 a. Relative Yields of 14C in Gluconeo genic and Glycolytic Products . 63 b. Effects of Non-homogeneous Nature of Gluconeogenic Organs .... 69 3 . Exchange of Malate between Mitochondria and Cytosol.....................73 C. Pathways of Hydrogen...................76 1. Tritium as a Tracer for Mitochondrial and Cytosolic Reducing Hydrogen . . 76 2. Shuttles for the Transfer of Reducing Equivalents..................... 89 a. Import Shuttles................... 89 b. Export Shuttles...................95 D. Control Mechanisms......................... 98 1. Effect of Added Cyclic AMP......... 98 2. Effect of Redox State............ 101 3 . ATP Balance ........................107 E. General Discussion ...................... 110 1. The Importance of the Determination of Carbon Flux in the Intact Cell . . . 110 iv Page 2. Future Application of Computer Models to More Complex Systems . . 112 3. Possible Regulatory Significance of Futile Cycles .................. 115 4. Possible Special Role of the Pyruvate, PEP Cycles........................ 119 5. The Role of Glycolysis in Gluco neogenic Tissues.................. 120 6. Control of Aspartate and Malate Efflux............................ 122 7. Distribution of the Dicarboxylic Acids between the Mitochondria and Cytosol.......................... 124 IV. Summary...................................... 130 V. List of References.......................... 153 VI. Appendices.................................. 143 A. Appendix I .............................. 143 B. Appendix I I .............................. 145 v List of Schemes Scheme Page 1. Model of Major Metabolic Pathways in the Kidney Cortex.................. 55 2. Cycles Involving Pyruvate, Oxalacetate and Phosphoenolpyruvate .............. 62 3. Proposed Import Shuttles .............. 91 4. Simplified Model for Gluconeogenesis in the Isolated Perfused Liver........ 113 5a. Hypothetical Figure Showing the Possible Effect of a Change of a Parameter on Enzymes Catalyzing a Reaction in Opposite Directions .................. 117 5b. Hypothetical Figure Showing Required Enzyme Response to Give Same Net Flux with No Futile Cycling .............. 117 6. Possible Compartmentation of Oxalacetate in the Mitochondria .... 125 vi List of Tables Table Page 1. Effect of Aminooxyacetate on Detritiation of L-Aspartate-2T During Gluconeogenesis from L-Lactate........................ 32 2. Effect of Aminooxyacetate on Detritiation of Amino Acids During Gluconeogenesis from Lactate or Pyruvate.....................34 3. Effect of n-Butylmalonate and p-Iodobenzylmalonate on Gluconeogenesis . . 37 4. Effect of p-Iodobenzylmalonate (IBM) on the Ratio of Isotopic Yields in Glucose and COg .......................... 38 5. Effect of y-Hydroxyglutamate, y-Hydroxy- a-Ketoglutarate and Glyoxylate on Gluconeogenesis .......................... 40 6. Effect of y-Hydroxyglutamate and y-Hydroxy- y-Ketoglutarate on l 4C02 Yield from Acetate-l-14C .............................41 7. Effect of y-Hydroxyglutamate and Glyoxylate on the Concentration of Intermediates During Gluconeogenesis from Pyruvate .... 43 8 . Effect of Dinitrophenol on Gluconeogenesis from Various Substrates .................. 45 vii Table Page 9. Effect of Quinolinate on Gluconeogenesis from Various Substrates .................... 47 10. Effect of Quinolinate on Gluconeogenesis, Oxidation and Recycling of Phosphoenolpyruvate ........................ 49 11. Effect of DL-Chlorosuccinate and DL-Fluorocitrate on Gluconeogenesis from Lactate and 1 4C02 Formation from Acetate-l-uC ............................ 50 12. Effect of Phenylpyruvate, Oxalate and Oxamate on Gluconeogenesis .............. 53 15. Rate of Formation of Major Metabolic Products from Pyruvate or Lactate in Kidney Cortex Slices .................. 57 14. Rates Calculated from Distribution of Radioactivity in Glucose, Lactate and Glutamate Formed from Pyruvate- 2-14C or L-Lactate-2-14C .................. 59 15. 1 4 C Yields During Glycolysis and Gluconeo genesis in the Kidney Cortex............ 64 16. 14C Yields in the Products of the Gluconeo genic and "Glycolytic" Pathways .......... 66 viii Table Page 17. UC Yields in the Products of the Gluconeogenic and "Glycolytic" Pathways . . 68 18. Yields of WC in Glucose and C02 from L-Y~Glycero 1-P-U-uC- 1-H and -ID........... 72 19. Ratio of 14C Yields in Glucose and C02 from Mitrochrondrial and Cytosolic Substrates............................... 75 20. Estimation of Rate of Exchange of Malate between Compartments .............. 77 21. Yields of Tritium in Glucose and Water and Distribution of Tritium in Glucose............................... 80 22. Specificity of NADH-linked Dehydro genases for A and B side of C-4 of Nicotinamide........................... 81 2 3. Fate of Tritium from Succinate 2,3-T Compared with L-2C1-Succinate-2T and -3T................................... 87 24. Effect of Aminooxyacetate on Lactate Oxidation................................. 93 25. Effect of DL-Glyceraldehyde-3P on Lactate Oxidation .................... 95 ix Table Page 26. Effect of Aminooxyacetate on Recycling of PEP to Pyruvate During Gluconeogenesis from Pyruvate . . . 97 27. Effect of the Concentration of Cyclic AMP on Gluconeogenesis.............. 99 28. Effect of 0.3mM Cyclic AMP on Gluconeo genesis from Various Substrates ........ 100 2 9. Effect of TMPD and PMS on Gluconeogenesis .............. ..... 105 50. Effect of Aeetaldehyde and L-Glyceraldehyde on Gluconeogenesis . . . 106 31. ATP Balance during Gluconeogenesis from Pyruvate and Acetate............ 109 x List of Abbreviations Symbol Meaning A.A.................Amino acids AcSCoA ............ Acetyl Coenzyme A AOA .............. Aminooxyacetate Asp.................Aspartate D .............. Deuterium DHAP .............. Dihydroxyacetone phosphate DNP ..............2,4 Dinitrophenol FGP .............. Fructose-6-phosphate FDP ..............Fructose-1,6-diphosphate G .............. Generally labeled (not necessarily uniformly) GAP .............. Glyceraldehyde-3-phosphate GDH .............. Glutamate dehydrogenase G l u t ..............Glutamate GOT .............. Glutamate-oxalacetate transaminase G6p .............. Glucose-6-phosphate aGP .............. L-a-Glycerol phosphate y-HO-a-Kg .......... y-Hydroxy-a-Ketoglutarate IBM .............. p-Iodobenzylmalonate aKg .............. a-Ketoglutarate Mit .............. Mitochondrial N.d.................Not determined OAA ..............Oxalacetate xi Symbol Meaning PMS .............. Phenazine methosulfate P .............. Phosphate Pi .............. Inorganic phosphate PEP .............. Phosphoenolpyruvate 5 PGA..............^Phosphoglycerate T .............. Tritium TMPD .............. N,N,N'jN'-Tetramethyl-p-phenylene- diamine TEA .............. Triethanolamine VAC, ..............Rate of formation of acetyl coenzyme A v ..............Rate of glucose synthesis from END endogenous substrates ..............Rate of fumarase exchange VGLU..............Rate of glucose synthesis ..............Rate of lactate synthesis V.r r > T r ..............Rate of malate dehydrogenase 1 , 1 ) 1 1 exchange VpD H ..............Rate of pyruvate dehydrogenase Vp K ..............Rate of pyruvate Kinase VpYR..............Rate of pyruvate utilization xii I. Historical and Theoretical Introduction A. General Aspects of Gluconeogenesis. The net result of gluconeogenesis from lactate is simply the reverse of anaerobic glycolysis. However, Krebs (1954) theorized that the pathway of glucose formation would differ from that of glucose degradation at three sites which are highly exergonic reactions of glycolysis: (1) glucose > glucose-6-P; (2) fructose- 6-P---» fructose diP; and (3) phosphoenolpyruvate > pyruvate. In addition to the thermodynamic arguments against a direct reversal of glycolysis, the enzymes catalyzing these glycolytic reactions have very limited catalytic effect in the reverse reactions. For example, pyruvate kinase catalyzes the conversion of pyruvate to PEP at about 1/600 of the maximal rate of conversion of PEP to pyruvate by this enzyme. Active specific phos phatases are involved in the reversal of steps (1) and (2), while the conversion of PEP to pyruvate is now known to involve at least two enzymes, pyruvate carboxylase and PEP carboxykinase, both reactions requiring high energy phosphate (Utter et al., 1964). These three steps at which gluconeogenesis differs from glycolysis are obvious likely sites of control in tissues having the capacity for 2 either glycolysis or gluconeogenesis. The step from pyruvate to PEP, being the first committed sequence in the pathway, is the most likely site of primary control. There is evidence that in most species this sequence in volves more than two enzymes. This follows from the dif ferences in location of pyruvate carboxylase, a mitochon drial enzyme, and PEP carboxykinase, which in most species is a cytosolic enzyme (Nordlie and Lardy, 1963)5 and from the fact that oxalacetate itself does not penetrate the mitochondrial inner membrane (Borst, 1 963). Thus depend ing on the redox status of the cytosol, either L-aspartate or L-malate is thought to act as a carrier of the oxalace tate moiety out of the mitochondria, with participation of the mitochrondrial and cytosolic isoenzymes of either glutamate-oxalacetate transaminase or malate dehydrogenase. B. Gluconeogenesis in the Kidney Cortex. The reported maximal rates of gluconeogenesis in the isolated perfused rat kidney (Bowman, 1970) on a weight basis are as high as those obtained with the perfused rat liver (Hems et al., 1966). The kidney cortexes, however, weigh only about 12$ as much as the liver; for this reason alone, the liver is plainly the major gluconeogenic organ. In addition hormones such as glucagon and the catechola mines which play major roles in controlling and directing the energy supplies of the body apparently affect gluconeo- genesis in the liver only (Bowan, 1970). While gluconeo genesis in the kidney (as in the liver) is stimulated by exogenous cyclic AMP, the only hormone yet known to raise the level of endogenous cyclic AMP in the kidney cortex is parathyroid hormone (Pagliari and Goodman, 1969). Gluconeogenesis in the kidney is apparently restricted to the cortex, the medulla and other regions being strictly glycolytic. The physiological significance of gluconeo genesis in the kidney as yet has not been well delineated. According to Cahill et al. (1968), under conditions of long fasting of very obese human subjects, about 50$ of total body gluconeogenesis is derived from the kidney. Under these conditions the major glucogenic substrates are amino acids arising from the breakdown of tissue pro teins . There are considerable differences in the effective ness of various substrates between the liver and kidney of the rat. While lactate, pyruvate, glycerol, and fructose are good substrates in both tissues, many organic acids including most of the Krebs cycle intermediates are rather poor glucogenic substrates in the liver but good substrates in the kidney. Restricted permeability of the hepatic cell to these intermediates is apparently the cause of their limited effectiveness (Ross et al., 1967). On the other hand L-alanine, a good glucogenic substrate 4 in the liver, is only weakly glucogenic in the rat kidney, probably because of much lower glutamate-pyruvate trans aminase activity. Thus there is some measure of comple mentarity in the response of these tissues to the range of substrates encountered. To date the pathways of glu coneogenesis have been found to be very similar in the kidney and liver in many regards, e.g. site of action of cyclic AMP (Exton and Park, 19^9] Pagliari and Goodman, 1 9 6 9)] mode of oxalacetate transfer from the mitochondria (Rognstad and Katz, 1970] Anderson et al.«1Q70); stimula tory effect of fatty acid oxidation (Williamson et al., 19685 Krebs, 1967)] extent of "futile” or "useless" re cycling (Rognstad et al, 1970, Friedmann et al. ,1971)] specific fructose metabolizing enzymes (Kranhold et al., 1 9 6 9)] and active glutamate dehydrogenases (Krebs and Veech, 1970). C. Control Mechanisms in Gluconeogenesis. As mentioned above, potential control sites in the gluconeogenic pathway are likely to be at those steps which are catalyzed by enzymes which operate much more effectively in one direction than the other. It appears that in most pathways enzymes can be grouped largely into two classes, those which maintain the reactants and 5 products close to equilibrium, and those which catalyze reactions in which the mass action ratio is more than an order of magnitude (and often several orders of magnitude) different from the equilibrium constant. Three main criteria have been used to characterize the "near equilibrium" class of enzymes: (1) maximal rate of the enzyme catalyzed reaction in a cell-free system greatly in excess of net rates of flow in the intact cell; (2) close correspondence between the mass action ratio, determined by analysis, and the equilibrium constant; and (3) isotopic exchange between reactants and products in several fold excess over net flow. Of these criteria, the second in principle is the most rigorous. However in practice it is not always easy to measure all the substrates of a given enzyme. Some, such as glyceraldehyde-3-P in the liver and kidney are in low concentration, while others, such as oxalacetate and 1,3 diphosphoglycerate, are both in low concentration and also are rather unstable. The problem of low concentra tion requires that large amounts of tissue are sampled and this generally means that whole organs are used. This in turn creates another problem (generally ignored) of heterogeneity of cell types. While perhaps 10% of the liver cells are the gluconeogenic parenchymal cells, it is conceivable that even a rather small proportion of 6 glycolytic cells (e.g. bile duct, Kupfer, or red blood cells) may contain much higher concentrations of certain intermediates, thus producing significant errors in mass action ratios based on the uniform cell assumption. Criterion (1) is certainly very informative. Although it is recognized that conditions in the cell can and will greatly diminish certain reaction rates as measured under optimum conditions in an extract, those enzymes which assay maximally more than an order of magnitude above net flux rates have seldom been found to be rate limiting in the intact cell. Criterion (5) has been widely used in this laboratory (e.g. landau and Katz, 1965)* While a rigorous quanti tative theory on the direct relation between the rate of isotopic exbhange and nearness to equilibrium remains to be developed, all cases so far investigated suggest that rapid isotopic exchange does indeed correlate with high enzyme titers and close agreement between mass action ratios and the quilibrium constant. This approach alone can give an estimate of the actual rate at which these near-equilibrium enzymes operate in the intact cell. The exact localization of the rate-limiting enzyme or enzymes in a given pathway, under fixed conditions and from a given substrate, is by no means a simple matter. While actually only one enzyme is likely to be rate- 7 limiting, it is certainly possible that one (or more) other enzymes may be also so close to their maximum poten tial under the given conditions that very slight changes in the conditions will make this the rate-limiting step; in such cases "rate-limiting enzymes" may be the appro priate term. The rate limiting reaction will be defined as that enzyme which, upon a small increase of its con centration in the cell, will cause a net increase in the flux of the pathway. If transport steps are included (as they should be) as potential rate-limiting steps, one should consider carrier sites as well as enzymes. In the cell, of course, an increase in net rate can be caused by affecting the rate-limiting step in many ways: (1) by an increase in the rate of synthesis (or decrease in degradation) of the enzyme leading to an increase in its concentration; (2) by a change in the concentration of allosteric effectors of the enzyme, which may increase the V^x* decrease the Km of a non-saturated substrate, or increase the 0f an inhibitory product; (5) by a conversion of the enzyme to a more active form, modulated by hormonal messengers; (4) by an increase in the concentration of the substrate or substrates of the reaction, provided that these are not already saturating the enzyme; and (5) by a decrease in the concentration of 8 the product or products of the reaction, providing that these products exert important inhibitory effects on the net reaction. The "crossover" analysis developed by Chance and Williams (1956) has been applied in attempts to delineate rate-limiting steps in metabolic pathways. This is a useful method for indicating sites of interaction in the pathway caused by altering conditions. However it has only recently been realized that this approach does not necessarily yield any information on rate controlling sites, and attempts at more rigorous treatments are being made. Both Williamson (1967) and Exton and ParK (1 9 6 9) interpreted earlier data showing crossover sites at glyceraldehyde-P dehydrogenase under certain conditions as indicating that this site had become a rate limiting step. The recognition that this enzyme, by all three criteria mentioned above, is in the near-equilibrium class in gluconeogenic tissues requires reinterpretation of the crossover data. Thus conditions which increase gluconeogenesis by an increase in the extramitochondrial NADH/NAD+ ratio will cause an increase in the GAP/1,3 PGA ratio of this near equilibrium reaction. Since, by all three criteria, aldolase, triose-P isomerase, P-glycerate Kinase, P-glycerate mutase and enolase are also near equilibrium reactions, this in effect means an 9 increase in the FDP/PEP ratio. Now whether the increase of FDP or the decrease of PEP is the cause of increased gluconeogenesis would depend on whether the fructose diphosphatase step or the pyruvate — — ► PEP sequence is rate-limiting. If, as is likely, the latter sequence is rate-limiting, the decrease in PEP concentration could increase gluconeogenesis in various ways, e.g. by stimulation of a rate-limiting PEP carboxykinase step via relief of product inhibition or decrease of back reaction; or by a decrease in the rate of recycling of PEP to pyruvate via pyruvate kinase, thus sparing more of the PEP for gluconeogenesis — this would apply to rate limitation at either PEP carboxykinase or pyruvate carboxylase. A valuable approach in the identification of rate- limiting sites is the use of substrates which enter the pathway at different levels. Thus since the maximal rate of gluconeogenesis in both liver and kidney is considerably greater from fructose than from lactate or pyruvate, it is likely that gluconeogenesis from lactate or pyruvate is not limited by the fructose diphosphatase or glucose-6- phosphatase reactions. While the logic is not totally rigorous — it is conceivable that some metabolite is formed from lactate or pyruvate, but not from fructose, which inhibits fructose diphosphatase -- in the absence 10 of other information this is probably at present the best method of locating the rate-limiting site. The differen tial effect of activators on gluconeogenesis from a range of substrates is also informative. Thus, in both liver and kidney, exogenous cyclic AMP stimulates glucose forma tion from lactate but not from fructose or glyceraldehyde. This again is most simply interpreted as an activation of cyclic AMP at a rate-limiting site between pyruvate and PEP during gluconeogenesis from lactate. A case based on this approach which is more ambiguous is the following. Exton and Park (1 9 6 7) found that while gluconeogenesis was about the same from lactate or pyru vate at high substrate concentrations, at low substrate concentrations more glucose was formed from pyruvate than from lactate. They interpreted this as indicating that lactic dehydrogenase had become rate-limiting because of its high for lactate. Granting that this is a plausible explanation, one can offer another interpretation which is at least as plausible (here simply to emphasize the lack of logical rigor to even this "best" approach). Assume that lactic dehydrogenase maintains a near-equilibrium condition, and that pyruvate carboxylase is rate-limiting from both substrates. It is certainly conceivable that the intracellular pyruvate concentration at low lactate sub strate concentrations becomes sufficiently low to decrease 11 pyruvate carboxylase flux, while, at higher substrate con centrations, pyruvate concentration from either substrate may be sufficient to saturate the enzyme. Considerable differences of opinion exist in the more exact pinpointing of the rate-limiting site somewhere be tween pyruvate and PEP. As noted above, several enzymes are involved but pyruvate carboxylase and PEP carboxykinase are the likely enzyme sites of control. In addition, the transport steps between the mitochrondria and the cytosol have been studied as potential rate-controlling steps (Williamson et al., 1970). D. Quantitative Pathway Determination. Previous studies have led to the development of isotopic techniques for the determination of pathways of carbon and hydrogen flow in the intact cell (Katz and Wood, 1958, 1963; Katz and Rognstad, 196 6, 1967). This approach has helped to establish the major function of cyclic pathways such as the pentose cycle (Landau and Katz, 1965) and has contributed to the discovery of other cycles such as the pyruvate cycle in adipose tissue (Rognstad and Katz, 1966; Rognstad, 1969). It has also provided the first quantitative balance studies of the formation and utilization of reduced nicotinamide nucleo tides in the cytosol and mitochondria of complex mammalian cells, as well as providing a balance which accounts for 12 most of the utilization of the ATP formed. Quantitative pathway determination is also essential to the problem of regulation. As an example, Krebs and Veech (1970) have recently used the assumption (in the calculation of mitochondrial ATP/ADP ratios) that pyruvate carboxylase is a near-equilibrium enzyme in rat liver, based on a comparison of maximal capacity of this enzyme of 10 (imoles/gm/min (Ballard and Hanson, 1967), versus maximal flux of 5 M-moles/gm/min of lactate converted to glucose. In this manuscript it will be shown that in the kidney cortex the rate of recycling of PEP to pyruvate can approach the rate of conversion of PEP to glucose. As a consequence net flux in the pyruvate carboxylase (and PEP carboxykinase) reaction would be twice that of the rate of glucose synthesis. If recycling rates are this high in the liver also (as the early results of Friedmann et al. [1971] suggest), it would appear that pyruvate carboxylase is operating at or very near capacity, rather than catalyzing a near equilibrium reaction. The approach used to determine pathways involves the assumption of metabolic and isotopic steady state, i.e. that the concentrations and specific activities of the intermediates of the pathway remain constant. The assumption should be reasonably valid in in vitro experi ments of at least one hour duration. Even if some changes occur in pathway fluxes, the results can be interpreted as average rates over the time period involved. It is essential only to exceed the initial non-steady state period which probably lasts from a few seconds to a few minutes at most. A model to be tested is constructed and algebraic relations equating inflow and outflow of radio activity from all unique carbon atoms of the model are- written. For simple models algebraic solution yields the relative specific activities of all carbon atoms in terms of the rates. For complex models algebraic solution is cumbersome, and the results from a computer program of the model, given values of known rates and ranges of values of unknown rates, are fit to experimentally determined specific activity ratios by a trial and error procedure. E. Scope of Dissertation. While most of the enzymic reactions involved in glu- coneogenesis are probably known, the control of the over all process is by no means established. It is now recog nized that the cell is not a simple "bag of enzymes" and the transfer of compounds between the various structures of the cell may be involved in regulatory systems. In addition, while the physiological specialization of the various cells of organs has been much studied, the bio chemical specialization of different cell types of an organ has in many cases received relatively little study. In this dissertation the focus of attention will be on the estimation of the fluxes of carbon, hydrogen and energy which occur in the intact kidney cortex cell while glu- coneogenesis is occurring. The transfer of compounds con taining these elements between the mitochondria and the cytosol must be examined in regard to their possible regu latory function. The exact molecular nature of hormonal control mechanisms is largely outside the scope of this dissertation. However, the elucidation of possible rate- limiting steps in gluconeogenesis from different substrates should prove valuable in establishing likely target sites of hormone action. 15 II. Methods A. Incubation Procedure. Wistar strain rats of either sex were used. They were either fed a normal rat diet or fasted for periods of 18 to 48 hours. They were killed by decapitation, the kidneys removed and kept in ice in a phosphate-salts buffer (Krebs at al.> 19^3) made as follows: 232 ml 0.154 M NaCl 8 ml 0.154 M KC1 6 ml 0.11 M CaCl2 2 ml 0.10 M MgSO^ 26 ml 0.10 M Na phosphate pH 7.4 The kidneys were cut in half lengthwise and the outer cortex cut away with a scissors. This was cut in thin slices (0.4 mm) with a Mickle chopper and the slices were washed several times with cold buffer. An alternative procedure based on the method of Guder and Wieland (1970) was used in some experiments in which the cortex strips were forced through a 30 mesh sieve, and the resulting segments were washed 3 times in cold buffer with centri fugation at about 100 X g for 2 minutes. 125 mg of slices or segments were incubated in a metabolic shaker for 2 hours at 38° C with 100# oxygen as the initial gas phase in 2 ml of the above buffer. Incubation was in a 25 ml Erlenmeyer flask with a rubber serum cap from which a 16 plastic hanging cup was suspended. Labeled and unlabeled substrates and activators or inhibitors were also present. Aliquots were taken before addition of tissue for deter mination of initial substrate concentration and initial radioactivity. The incubation was ended by injecting 0.3 ml of 4 N NaOH (COg free) or 0.3 ml of phenethylamine/ water (1/2) into the plastic cup and 0.5 ml of 1 N HgSO^ into the reaction mixture. The flasks were shaken for another 2 hours to collect CO,,. B. Fractionation of Products. The reaction medium was made to a total volume of 1 0 .0 ml. 8 ml of this was put on tandem ion exchange columns, the upper: 1 cm x 10 cm Amberlite CG-120 (H+ form, 100-200 mesh) and the lower: 1 cm x 11 cm Dowex-1 x 8 (acetate form, 100-200 mesh) and the columns washed with water until 30 ml of effluent was collected (neutral glucose fraction). Amino acids were eluted from the CG-120 column with 2 N NH^OH. Lactate was eluted from the Dowex-1 column with 2 N acetic acid. The amino acid fraction was taken to dryness in a rotary vacuum evapora tor, made to a 2 ml volume and put on a 1 cm x 20 cm Amberlite CG-4B column (acetate form, 200-400 mesh) which was washed with water. Glutamate was eluted with 0.5 N acetic acid, and aspartate with 1.0 N acetic acid. 17 Alanine was obtained from the dried neutral phase by paper chromatography. The lactate and glucose fractions (above) were also dried and chromatographed. Radioactive bands were located with x-ray film. When tritiated substrates were used, duplicate ali quots of the neutral glucose fraction were counted, one of which was dried (twice, with addition of water). The dried aliquot gives the tritium yield in glucose, while the non-dried aliquot gives the yield in glucose plus water. If a neutral tritium labeled substrate was used, glucose radioactivity was determined by treating an ali quot of the neutral fraction with ATP, Mg++ and hexokinase, trapping the g6p on an anion exchange column, and elution with 4 N formic acid. Analyses of the initial and final substrates and the major products (glucose, lactate) were carried out by enzymic procedures (Bergmeyer, 1965). Assay of radioactivity was done with a Packard model 3575 scintillation counter, with automatic external stand ard for quench correction. When 1 1 c labeled substrates were used, Jeffay (1961) scintillation fluid was used. With tritium or mixed tritium and 14C substrates either Wu (1964) scintillation fluid or Triton scintillation fluid (5 gm PPO, 16 mg POPOP, 250 ml Triton X-100, 18 750 ml toluene) was used, with standard crossover curves. C. Degradation Procedures. Glucose was degraded by either the procedure of Rognstad and Woronsberg (1968) or that of Schmidt et^ al. (1970). Lactate was degraded according to Katz et al. (1955) except that the initial step was carried out in 1 N H^POjj. for 18 hours at 4°C. C-l of glutamate was obtained with Chloramine-T (Mosbach et al., 1951) and C-5 of glutamate by the Schmidt reaction (Ehrensvard et al.,1951)« Tritium on C-6 of glucose was determined by periodate degradation according to Bloom (1962). A procedure for the total degradation of glutamate has been developed involving the following sequence: HN, 5 hooc-ch2-ch2-ch(nh2)-cooh C5 C4 C3 C2 Cl h2so4 ► KMnO^ co2 + h2n-ch2-ch2-ch(nh2)cooh C5 C4 C5 C2 Cl ► HNO, 2 co2 Cl + H2N-CH2-CH2-C00H C4 C5 C2 HAc ► HO-CHg-CHg-COOH 50^ HpSOn — -— ►CHg = CH-COOH NaClO^ OsO^ 19 NaI04 CHoOH-CHOH-COOH 1 » ► C0o + HCOOH + HoC0 . pn 04 C3 C2 ru4 C2 03 C4 The overall yield of 20# is about five-fold higher than that of previous procedures. D. Syntheses. 1. 1 4C-Labeled Substrates. a. Glucose-6-P-U-1 4C. Glucose-U-14C (10 (Jimoles), TEA-HC1 buffer, pH 7.4 (200 (imoles), ATP (30 p.moles), MgClg (60 M-moles) and 10 units of hexokinase (either F-300 or C-302 from Sigma, both generally free of phosphohexose isomerase) were incubated for 2 hours at room temperature in a final volume of 5 ml. The reaction mixture was put on tandem 1 cm x 5 cm CG-120 (H+) and 1 cm x 11 cm AG-1 (acetate) columns (hereafter denoted as the standard tandem column system), washed with water, and G6p eluted from the AG-1 column with 4 N formic acid. This was taken to dryness on a rotary vacuum evaporator and chromato graphed on Whatman 3 MM paper using n-propanol/conc. NH^OH/water (6/3/1, by volume). The yield was about 95$. b. Fructose-6-P-U-1 4C. From fructose-U-14C as in a. c. Fructose-1,6-diP-U-1 4C. As in a. with the addition of 20 units of phosphohexose isomerase and 20 10 units of fructose-6p kinase. After elution with 4 N formic acid as above, FDP-U-14C was eluted with 2 N formate pH 3 (made from 1 part ammonium formate plus 4 parts formic acid). Ammonium ion was removed by putting the radioactive fractions through a CG-120 (H+) column and the FDP-U-14C (free acid) was taken to dryness at 35° under vacuum to remove formic acid. Since it is quite unstable in acid form it was immediately brought to pH 6 with NH^OH (in which form it can be stored) and chromatographed as in a. The yield was about 90$. d. L-a-Glycerol-P-U-14C and 3-P-Glycerate-U-1 4C. These were prepared from glucose-U-14C essentially by the dismutation procedure of Schmidt ejt al. (1970) and purified on tandem columns. aGP-U-14C was eluted with 4 N formic acid, and 3PGA-U-14C with 1 N formate pH 3* (General reference for column chromatography is Bartlett, 1959)• These compounds were subsequently chromatographed on Whatman 3MM paper using ethyl acetate/acetic acid/water (3/3/1* by volume). The yields were about 85$. e. Phosphoenol pyruvate-U-1 4C. 3PGA-U-14C (36 nmoles), TEA-HC1 pH 7.4 (1000 nmoles), MgCl2 (200 nmoles), 2,3 diP-glycerate (0.5 nmoles) in a final volume of 10 ml was incubated for 90 minutes with 12 units of enolase and 50 units of phosphoglycerate mutase. Assay 21 showed 24 (xmoles of 3PGA, 2 (xmoles of 2PGA and 10 p-moles of PEP. 2- and 5PGA-U-14C were eluted from the lower of standard tandem columns with 1 N fornate pH while PEP-U-14C was eluted with 3 N formate pH 5* NH^+ was removed with CG-120 (H+) and formic acid by evaporation. Chromatography used the system described in d. The yield of PEP was 20 to 25$. f. D-Glyceraldehyde-U-^C. This was synthesized by a small scale modification of the method of Perlin (1962). Fructose-U-14C (40 (xmoles) was dissolved in 50 ml of water. 2 .5 ml of glacial acetic acid was added, and the solution placed in an l8° bath. 40 mg Pb(Ac)i|. (about a 10# excess) was added over a period of 10 minutes. The reaction mixture stood for another 20 minutes at room temperature and then was put through a 1 cm x 5 cm CG-120 (H+) column (to remove lead ions) which was washed with water to a volume of 10 ml. This was dried at 55° on a rotary vacuum evaporator. The glycolate ester of glyceraldehyde was hydrolyzed by adding 2 ml of 0.1 N HgSO^. and keeping at 40°C for 20 hours. It was then put through a 1 cm x 5 cm AG-1 (acetate) column to remove sulfate and glycolate-U-1 4C. The first 10 ml wash of the column contained about 50# of the original fructose activity. This was taken to dryness and chromatographed 22 on Whatman 3MM paper, using n-butanol/acetic acid/water (4/1/2, by volume). D-Glyceraldehyde-U-14C migrated with a high Rp in a rather wide band. Elution of this band from the paper with water required considerable time. Final yield of D-glyceraldehyde-U-14 C was about 60# of the theoretical. A well separated band of residual fructose-U-1 4C (about 5$ of initial activity) was the only other band. g. D-Glycerate-U-1 4C and Glycerol-U-1 4C. The corresponding phosphorylated compounds were incubated with 1000 (xmoles of acetate buffer pH 5*0 and 5 mg of potato acid phosphatase for 18 hours at room temperature. Using tandem columns glycerol-U-14C appears in the water wash while D-glycerate-U-14C was eluted from the AG-1 column with 2 N acetic acid. The yields were about 95$. h. L-Glycerate-U-1 4C. To 15 (imoles of L-serine- U-14C in 1 ml of water plus 1 ml of glacial acetic acid was added dropwise 0.2 ml of 5 N NaNOg. The reaction then stood for 5 hours at room temperature. Using tandem columns, residual L-serine-U-14C was eluted from the CG-120 column with 2 N NH4OH, while L-glycerate-U-14C was eluted from the AG-1 column with 2 N acetic acid. The yield was 90$. i. L-Lactate-2-14C (or -3^*C). About 5 (xmoles of pyruvate-2-1 4 C (or -3-1 4C) were incubated with TEA-HC1 pH 7.4 25 buffer (100 |imoles), NADH (7-5 (imoles) and 20 units of lactate dehydrogenase for 50 minutes. Using tandem columns, lactate-14C was eluted from the AG-1 column with 2 N acetic acid. This was taken to near dryness at 50°C in a stream of air, and then chromatographed using 95# ethano1/cone.NH^0H/H20 (160/10/50, by volume). Th e yield was about 70#. j. Citrate-5-uC (or -4-1 4C). Acetate-l-14C (or -2-1 4C) (15 (imoles), TEA-HC1 pH 8.0 (1000 (xmoles), ATP (50 (xmoles), MgClg (100 (xmoles), CoA (2 (xmoles), oxalacetate (150 (imoles), reduced glutathione (15 (xmoles) and 10 units of acetate kinase, 50 units of phospho- transacetylase, and 10 units of citrate synthase were incubated for 5 hours at room temperature. Using standard tandem columns, residual acetate-14C was eluted from the AG-1 column with 1 N acetic acid (less than 1# of orginal radioactivity) and citrate-^C was eluted with 4 N formic acid, dried and chromatographed on paper with M-butanol/ acetic acid/water (4/1/2, by volume). The yield was 95$. k. L-a-Glycerol-P-U-1 4C-lD. 5P-glycerate-U-14C (5 (xmoles), glucose-6p-lD, > 99$ deuterium on C-l, (10 ixmoles), TEA-HC1 pH 7.4 (500 (xmoles), ATP (10 (xmoles), MgCl2 (20 (imoles), PEP (10 (imoles), reduced glutathione 24 (5 (imoles), and NAD+ (2 (imoles) were incubated in 3 ml for 2 hours with 5 units of glucose-6-P dehydrogenase (from L. mesenteroides), 10 units of pyruvate kinase, 20 units of P-glycerate kinase and 6 units of glycer- aldehyde-P dehydrogenase. 0.1 ml of 10 N H2S0^ was added to inactivate the enzymes, the mixture again brought to pH 7.4 and incubated for 60 minutes with 10 (imoles of NADH, 5 units of triose-P-isomerase and 4 units of a-glycerol-P dehydrogenase. Using tandem columns L-a-glycerol-P-U-1 4C-lD was purified as in d. The yield was about 40$. 2. Tritium Labeled Substrates. a. L-Lactate-2T and Citrate-2T. DL-Malate-2T (40 (imoles), TEA-HC1 pH 7-4 (500 (imoles) and 50 units of fumarase were preincubated for 4 hours at room temperature. TEA-HC1 pH 8.0 (4000 (imoles), NAD+ (100 (imoles), acetyl-P (200 iimoles), CoA (2 (imoles) and 50 units of malate dehydrogenase, 50 units of phosphotransacetylase, and 10 units of citrate synthase were added in a final volume of 50 ml and incubated for 2 hours. 200 (imoles of pyruvate and 50 units of lactate dehydrogenase were added and the reaction continued for 30 minutes. The mixture was put on tandem 1 cm x 12 cm CG-120 (H+) and 1 cm x 30 cm AG-1 (acetate) columns. Prom the AG-1 column, L-lactate-2T 25 was eluted with 2 N acetic acid (25# yield); D-malate-2T was eluted with 1 N formic acid (50# yield); and citrate- 2T was eluted with 5 N formic acid (25# yield). b. L-Malate-2T. DL-Lactate-2T (10 [imoles), glycine-NaOH buffer pH 9*5 (1000 [imoles), NAD+ (50 [imoles), and L-glutamate (250 [imoles) were incubated with 50 units of lactate dehydrogenase and 40 units of glutamate-pyruvate transaminase in a 10 ml volume for 2 hours. 50 [imoles of oxalacetate and 20 units of malate dehydrogenase were added. D-Lactate-2T and L-malate-2T (40# yield) were separated as in a. c. Fumarate-2,5-T. L-Malate-2T (20 (imoles) was incubated with TEA-HC1 pH 7 A (200 [imoles) and 20 units of fumarase in a volume of 2 ml. Using standard tandem columns, L-malate-2,5-T was eluted from the AG-1 column with 2 N formic acid and fumarate-2,5-T was eluted with 6 N formic acid, in a 20# yield. d. NADP+ (4T). Glucose-6p-lT (15 [imoles), TEA-HC1 pH 7.4 (1500 [imoles), NADP+ (40 [imoles) and 50 units of glucose-6p dehydrogenase were incubated for 50 minutes at room temperature in a final volume of 45 ml. 500 [imoles of pyruvate and 50 units of lactate dehydro genase were added. After 50 minutes the reaction mixture was put on a 1 cm x 25 cm AG-1 (acetate) column. NADP+(4T) 26 was eluted with 2 N formic acid, taken to dryness in a rotary vacuum evaporator at 30°, and chromatographed on Whatman 3 MM using ethylacetate/acetic acid/water (3/3/1, by volume). The yield was about 90$. e. Isocitrate-2T. Glucose-6p (3 (imoles), Na phosphate pH 7*4 (200 (imoles), NaHCO^ (320 (imoles, gassed with 100$ C02), MgSO^ (10 (imoles), a-ketoglutarate (50 (imoles) and NADP+(4T) (4 (imoles) were incubated with 20 units of glucose-6p dehydrogenase ( salt free) and 20 units of isocitrate dehydrogenase (in 50$ glycerol) for 1 hour at room temperature under 100$ C02. Two further additions of 5 (imoles of glucose-6p were made at 30 minute intervals and the incubation continued for another 60 minutes. Using the standard tandem column procedure, isocitrate-2T was eluted with 3 N formic acid. Chroma tography on Whatman 3 MM used n-butanol/acetic acid/water (4/1/2, by volume).The yield was about 50$. f. D-f3-Hydroxybutyrate-3T. DL-Malate-2T (12 (imoles), TEA-HC1 pH 8.0 (1000 (imoles), NAD+ (1 (imole), acetyl P (50 (imoles), CoA (1 (imole), acetoacetate (100 limoles) together with 50 units of malate dehydrogenase, 20 units of phosphotransacetylase, 20 units of citrate synthase and 10 units of 0-hydroxybutyrate dehydrogenase were incubated for 150 minutes. Using tandem columns, 27 D-p-H0-butyrate-3T was elated with 1 N acetic acid, and D-malate-2T with 2 N formic acid. Chromatography used the solvent system 95# ethanol/conc.NH^OH/water (160/10/30, by volume).The yield was about 80#. g. L-p-Hydroxybutyrate-3T. DL-p-Hydroxybutyrate- 3T (12 [imoles), hydrazine-HCl pH 8.5 (10 mmoles), Tris- acetate pH 8.5 (1000 [imoles), NAD+ (50 [imoles) and 4 units of D-p-hydroxybutyrate dehydrogenase in a final volume of 15 ml were incubated for 8 hours at room temperature. 6 [imoles of unlabeled DL-p-hydroxybutyrate were added and the incubation continued for 8 hours. Using standard tandem columns, L-P-hydroxybutyrate-3T was eluted from AG-1 with 1 N acetic acid and dried. No D-p-hydroxy- butyrate was detectable by enzymic assay. The yield was about 90# of theory. h. L-glutamate-2T. (1) Low specific activty: L-glutamate (500 [imoles), Na phosphate pH 7.4 (100 [imoles), pyridoxal-P (0.5 ixmoles) and 50 units of glutamate- pyruvate transaminase were incubated in 1 ml of THO (specific activity about 10 |iC/[i at H) for 24 hours at room temperature. THO was removed and recovered by lyophilization. The dried sample was acidified with HgSO/j. and put on a 1 cm x 30 cm Amberlite CG-4B (acetate form, 200-400 mesh) column which was thoroughly washed 28 with water. (Strongly basic resins should be avoided since considerably decomposition of glutamate occurs on these columns.) L-Glutamate-2T was eluted with 0.5 N acetic acid and taken to dryness. (2) High specific activity: Glucose-6-P-lT (20 ixmoles), Na phosphate pH 7.6 (500 ixmoles), NAD+ (50 ixmoles), NH/j.01 (500 ixmoles), a-ketoglutarate (200 ixmoles) and 20 units of glucose-6p dehydrogenase (from L. mesenteroides) plus 10 units of glutamate dehydrogenase were incubated for 4 hours at room temperature in a volume of 10 ml. The mixture was put on a 1 cm x 15 cm CG-120 (H+) column and glutamate-2T eluted with 2 N NH^OH, dried and chromatographed using n-butanol/ acetic acid/water (4/1/2, by volume)jrhe yield was 95$. i. L-Aspartate-2T. (1) Low specific activity: As in h. with the substitution of L-aspartate and glutamate-oxalacetate transaminase. (2) High specific activity: As in h. with four-fold increase in phosphate and NH^Cl, replacement of a-ketoglutarate with 500 ixmoles of oxalacetate and increase of incubation time to 24 hours. The yield was about 80$. k. (3S)-L-aspartate-3T. DL-Malate-2T (80 ixmoles), Na phosphate pH 7.4 (200 ixmoles) and 80 units of fumarase were preincubated for 5 hours in 3 ml. Na phosphate pH 7.4 (1000 ixmoles), NAD+ (100 (xmoles), glutamate 29 (500 ixmoles), pyruvate (500 ixmoles) plus 200 units malate dehydrogenase, 100 units lactate dehydrogenase and 50 units glutamate oxalacetate transaminase were added and the volume brought to 10 ml. Incubation was for 5 hours at room temperature. (Longer incubations may decrease yields due to slow detritiation of oxalacetate-5T, in equilibrium with L-aspartate-5T.) Using standard columns, L-aspartate-5T was eluted from CG-120 with 2 N NH4OH. The yield was about 18# of initial activity. 1. L-2Cl-succinate-2T or (5S) L-2Cl-succinate-5T. To L-aspartate-2T or (5S)-L-aspartate-5T (40 ixmoles) in 5 ml of 8 N HC1 in a 25° water bath was added, dropwise, 1 ml of 1 N NaN02 over a period of 10 minutes. The reaction mixture stood another 60 minutes, and was then made to 10 ml and put on tandem 1 cm x 5 cm CG-120 (H+) and 1 cm x 50 cm AG-1 (acetate) columns which were washed with water. The AG-1 column was washed with 100 ml of 1 N formic acid (about 5# of the original activity, probably malate, appeared here) and then with 6 N formic acid to elute the tritiated chlorosuccinate. Evaporation and chromatography on Whatman 5 MM using n-butanol/acetic acid/water (4/1/2, by volume) gave a single band with a high Rp. The yield was about 80$. 3 0 III. Results and Discussion A. Inhibitor Studies. Specific inhibitors of enzymes or transport processes are extremely valuable tools for the determination of pathways and are also useful in the localization of rate controlling sites. However as yet the number of inhi bitors found which are reasonably specific for a single site are limited. We have tested a number of compounds for their effect on gluconeogenesis and the Krebs cycle in the kidney cortex. A number of substrates were used with each inhibitor in order to garner some preliminary idea of a possible specific site of action. Inhibitors which have been reported to be effective on isolated enzymes or transport steps in mitochondrial studies have been examined in the intact cell. Inhibitors which act at sites which are close to equilibrium in the intact cell must be very potent in order to reduce the capacity of the enzyme to less than the normal net flux through it. 1. Aminooxyacetate. Aminooxyacetate (AOA) has been reported to inhibit several transaminases, including glutamate-oxalacetate and glutamate-pyruvate transaminase (Hopper and Segal, 1962; Roberts et. al., 1964; Hotta, 1968). We have previously used this compound to test the role of the dual trans- 31 aminase mechanism for oxalacetate transfer in gluconeo- genesis from L-lactate (Rognstad and Katz, 1970). At a concentration of 0.1 mM, AOA inhibited glucose formation from L-lactate by about 90$, while this same concentration produced at most only minor inhibitory effects with all other substrates tested. Glutamate-oxalacetate trans aminase activity causes exchange of tritium from L- aspartate-2T and L-glutamate-2T with water (Wenzel et al., 1967). As an indicator of transaminase inhibition in the intact cell, we determined the effect of AOA on the detri- tiation of various various amino acids labeled with tritium on C-2. Aminooxyacetate has been reported to be a competitive inhibitor of the amino acid substrates of glutamate- pyruvate transaminase (Hopper and Segal, 1962). Table 1 shows the inhibitory effect of AOA on glutamate- oxalacetate transaminase when lactate is the gluconeogenic substrate. 0.1 mM AOA decreases the amount of detritiation of L- aspartate-2T from 99$ to 35$* while 0.5 mM AOA depressed detritiation to 14$. These values should not necessarily be considered to represent a quantitative picture of glutamate-oxalacetate transaminase inhibition, since AOA may inhibit to varying degrees a number of transaminases which labilize tritium from L-aspartate-2T. It is conceivable also that the two glutamate oxalacetate 32 TABLE 1.— Effect of Aminooxyacetate on Detritiation of L-Aspartate-2T During Gluconeogenesis from L-Laetate L-Lactate concentration was 10 mM. L-Aspartate-2T concen tration was 1 mM. 125 mg of kidney cortex slices were incubated in 2 ml buffer for 2 hours at 37°C. Aminooxyacetate Tritium Yield Concentration in Water (mM) (% of added T) 0 99 0 .1 35 0 . 5 14 oa 95 o.ia 88 o.5a 72 aPreincubated 30 minutes with tissue plus lactate, then washed out before L-aspartate-2T added. isoenzymes may be differently affected by AOA in the intact cell. Thus even though 35% detritiation occurred with 0.1 mM AOA, one or both of the glutamate-oxalacetate transaminases was sufficiently inhibited to cause approximately an 83% inhibition of glucose synthesis in this experiment. It should be pointed out that in the absence of AOA, nearly complete (> 95%) detritiation of 35 this amount of L-aspartate-2T has been found in only 15 minutes. 35# detritiation in a 2 hour experiment corresponds to about 5# detritiation per 15 minutes, indicating that 0.1 mM AOA does cause a fairly strong transaminase inhibition. When the tissue was preincubated with AOA and then washed free of the inhibitor, the inhibitory effect was largely removed (Table 1), indicating little irreversible inhibition of the transaminases by AOA. Table 2 shows the effect of changing the substrate from lactate to pyruvate on the detritiation of 5 transaminase substrates, L-glutamate-2T, L-aspartate-2T and L-alanine- 2,5-T. In contrast to glutamate-oxalacetate transaminase which labilizes only the hydrogen on C-2 of its amino acid substrates, glutamate-pyruvate transaminase causes complete exchange of the hydrogens on C-2 and C-5 of alanine with water. Aminooxyacetate, being a substituted hydroxylamine, would be expected to react to some extent with keto compounds such as pyruvate. This could lower the concentration of the inhibitor or completely remove it. Such an effect would be reflected by less detritiation of these amino acids. Indeed as shown in Table 2, the inhibition of detritiation caused by 0.1 mM AOA was less, with all three tritiated amino acids, when pyruvate 54 TABLE 2.— Effect of Aminooxyacetate on Detritiation of Amino Acids During Gluconeogenesis from Lactate or Pyruvate The concentration of the unlabeled substrate was 10 mM, while that of the tritiated substrate was approximately 1 mM. The amount of radioactivity added was about 2 x 10^ cpm* Incubation was 2 hours. Unlabeled Substrate Labeled Substrate AOA Cone. (mM) Tritium Yield in H20 ($ added) Glucose For mation (mmoles/ 125 mg/ 2 hr) L-Glutamate-2T 0 95 5-58 L-Glutamate-2T 0.1 45 1.04 L-Lactate L-Aspartate-2T 0 100 5.57 L-Aspartate-2T 0.1 57 1.05 L-Alanine-G-T 0 71 5.02 L-Alanine-G-T 0.1 18 0.95 L-Glutamate-2T 0 95 4.11 L-Glutamate-2T 0.1 59 4.08 L-Aspartate-2T 0 100 5.79 Pyruvate L-Aspartate-2T 0.1 85 5.88 L-Alanine-G-T 0 77 4.00 L-Alanine-G-T 0.1 25 5 .8 0 55 replaced lactate as the gluconeogenic substrate. This again was too long an experimental period (control = 100$ detritiation) for a good estimation of transaminase inhibition, especially for the glutamate oxalacetate substrates. However the rather mild effect of pyruvate on the AOA inhibition of L-alanine-G-T detritiation suggests that the amount of AOA which may have been removed by reaction with pyruvate was not sufficient to greatly lessen the inhibitory effect on this enzyme. However it would seem desirable, in any experiment in which AOA is employed to act as a transaminase inhibitor, to run short interval detritiation experiments to ensure that trans aminases are effectively being inhibited in the intact cell. Detritiation of L-alanine-G-T has been employed by Dunn at al. (1971) in vivo as a measure of glutamate- pyruvate transaminase activity in a study of possible rate-limiting sites in gluconeogenesis from L-alanine. 2. n-Butylmalonate and p-Iodobenzylmalonate. These compounds have been found to inhibit the malate-phosphate exchange reaction through the inner mitochondrial membrane, in studies by several groups on isolated mitochondria (Robinson and Chappell, 1967; Quagliariello et al., 1969). n-Butylmalonate has been employed in studies on gluconeogenesis in the perfused 36 rat liver, with the results being interpreted on the basis of a specific inhibitory action on this malate transport reaction (Williamson, 1970). Gluconeogenesis from both lactate and pyruvate was inhibited in the perfused liver. Inhibition from pyruvate would be expected if malate trans port is the site affected. However inhibition from lactate could only be rationalized as follows: the dual trans aminase mechanism involving aspartate outflow requires simultaneous a-ketoglutarate outflow; this can occur only by exchange of cytosolic malate into the mitochondria; to get this malate back out of the mitochondria requires the malate-phosphate exchange reaction, and this is blocked by n-butylmalonate (Williamson jet al., 1970). Table 3 presents the effects of these compounds on gluconeogenesis from several substrates in kidney cortex slices. Inhibition is nearly the same for all substrates, including L-malate. Since gluconeogenesis from L-malate occurs in the cytosol, there is no readily apparent reason why a specific block in malate transport between the mito chondria and cytosol should affect this process (although rather involved rationalizations can be invoked here also). A simpler explanation is that n-butylmalonate is not a specific inhibitor of malate-Pi exchange, but rather indeed has major inhibitory effects at another site, probably in 37 the Krebs cycle. Robinson and Williams (1969) have found that in experiments with isolated mitochondria n-butyl- malonate appears to inhibit at a site other than malate transport, and has proposed that p-iodobenzylmalonate may be a more specific inhibitor. However this compound, although more potent than n-butylmalonate, appears to act in a similar manner in the kidney cortex (Table 3). TABLE 3 .— Effect of n-Butylmalonate and p-Iodobenzylmalonate on Gluconeogenesis Substrate concentration was 10 mM. Expt. No. Substrate n-Butyl- malonate (mM) p-Iodo- benzyl- malonate (mM) Glucose Formation (M m ole s/ 125mg/2hr L-Lactate 0 5.31 L-Lactate 10 1.98 K-80 Pyruvate 0 4.22 Pyruvate 10 2.44 L-Malate 0 3.64 L-Malate 10 1.85 L-Lactate 0 3.52 L-Lactate 1 1.36 K-151 Pyruvate 0 2.94 Pyruvate 1 I .83 L-Malate 0 3.47 L-Malate 1 1 .6 0 58 The isotopic technique described below in Section III, B.3 was also used to determine if 1 mM p-iodobenzylmalonate had any inhibitory effect on the rate of exchange of malate between the cytosol and the mitochondria. As seen from the similarity of the glucose-1 4C/^CC^ ratio of yields from malate-U-14C and the mitochondrial substrates, succinate- U-14C and citrate-4,5-1 4C, there is no apparent decrease in the rapid isotopic rate of exchange (Table 4). Such a technique however might not rule out a block in a specific transport site for malate, among several other sites. TABLE 4.— Effect of p-Iodobenzylmalonate (IBM) on the Ratio of Isotopic Yields in Glucose and C02 The unlabeled substrates were 10 mM L-lactate plus 10 mM acetate. About 2 x 10 cpm and about 0.1 (imole of the labeled substrate were added. Labeled Substrate IBM Cone. (mM) 14C Yields {% of added 1 4C) Ratio of Yields (Glucose /C02) Glucose COg L-Malate-U-14C 0 28.4 39.4 0 .7 2 Succinate-U-14C 0 2 8 .3 40.4 0.70 Citrate-4,5-14C 0 27.7 37.2 0.74 L-Ma lat e - U- 1 4 C 1 22.0 45.3 0.49 Succinate-U-^C 1 2 2 .6 45.8 0.49 Citrate-4,5-”C 1 1 6 .9 32.3 0.52 39 3. •yHydroxyglutamate, y-Hydroxy-a-ketoglutarate and Glyoxylate. y-Hydroxyglutamate at a concentration of 5 mM produces a strong inhibition of gluconeogenesis from pyruvate and L-lactate, but has essentially no effect on gluconeogenesis from L-malate or the other Krebs cycle intermediates (Table 5)* The first interpretation of this data was that this compound, or a metabolite of it, was a very specific pyruvate carboxylase inhibitor. This inter pretation was incorrect. y-Hydroxyglutamate is a good substrate for glutamate-oxalacetate transaminase (and also glutamate dehydrogenase), forming Y~hydroxy-a-ketoglutarate (Goldstone and Adams, 1962; Maitra and Dekker, 1 9 6 3). This compound can be cleaved by an aldolase in liver and kidney to pyruvate plus glyoxylate. Y-Hydroxy-a-ketoglutarate has been reported to inhibit several enzymes of the Krebs cycle (Ruffo elt al., 1962; Payes and Laties, 1963). The similarity of the effects of the three title compounds suggests that perhaps Y-hydroxy-a-ketoglutarate may be the inhibitor produced in all cases. In the absence of a gluconeogenic substrate, or in the presence of malate as the gluconeogenic substrate, Y-hydroxyglutamate produces little effect on the Krebs cycle, as measured by the MC02 yield from acetate-14C (Table 6). However when pyruvate is the substrate, a TABLE 5 . - - E f f e c t o f ‘ Y rH yd roxyglu tam ate.,y - H y d r o x y - a - K e t o g lu t a r a t e and G ly o x y la t e on G lu c o n e o g e n e s is S u b s t r a t e c o n c e n t r a t i o n was 10 m M e x c e p t i n e x p e r im e n t K -38 w here i t was 5 mM. 10 m M a c e t a t e was a l s o p r e s e n t . E x p t. No. S u b s t r a t e y -Ho- G lu tam ate (mM) Y-HO O -K eto G lu t a r a t e (mM) G ly - o x y l a t e (mM) G lu c o s e F o rm a tio n ( p m o le s / I2 5 m g /2 h r) P y r u v a te — — — 2 .7 9 K-38 P y r u v a te 5 — — 0 .4 6 L - L a c t a t e — — — 3 .4 4 L - L a c t a t e 5 — — 0 .9 0 P y r u v a te — — — 4 .3 1 P y r u v a te 5 — — 0 .4 9 P y r u v a te — 2 — 0 .2 0 L - L a c t a t e — — — 4 .0 8 K - l l l L - L a c t a t e 5 — — 1 .2 6 L - L a c t a t e — 2 — 0.56 L -M a la te — — — 3 .5 5 L -M a la te 5 — - - 5 .0 2 L -M a la te — 2 — 4 . 2 9 & - K e t o g lu t a r a t e — — 4 .2 1 K -121 'QL - K e t o g lu t a r a t e 5 — — 5 .1 0 S u c c in a t e - - — — 3 . 3 4 S u c c in a t e 5 — — 3 .7 9 D-G1yc e r a ld e h y d e — — - - 4 .2 6 K -123 D -G ly c e r a ld e h y d e 5 — — 2 .8 5 D L -G ly c e r a te - - - - 2 .2 8 D L -G ly c e r a te 5 - - — 1 .2 7 D -F r u c to s e mm mm _ «, 1 0 .4 0 K-125 8 .1 2 D -F r u c t o s e 5 “ — “ - P y r u v a te - - — — 3 .5 2 P y r u v a te — — 1 1 .6 0 L - L a c t a t e _ _ — H 2 .9 4 K -151 0 .8 0 L - L a c t a t e - - - - 1 L -M a la te - - - - - - 3 .4 7 L -M a la te - - — 1 3 .9 7 K -198 P y r u v a te /M a la te - - - - - - 6 .6 7 P y r u v a te /M a la te 5 — - - 6 .3 2 41 TABLE 6.— Effect of 'y-Hydroxyglutamate or Y-Hydroxy-a- Ketoglutarate on UC02 Yield from Acetate-l-14C The concentration of the gluconeogenic substrate and that of acetate-l-uC was 10 mM. n.d. = not determined. Expt. No. Substrate y-ho gluta mate (mM) y-ho a-keto gluta- rate (mM) 1 4 C Yield in CO2 added 1 4C) Glucose Formation ([xmoles/ 125 mg/2hr) None 19-1 n.d. None 5 - (lost) n.d. None - 2 1 8 .8 n.d. Pyruvate - 2 7 .0 3.67 K-134 Pyruvate 5 - 9.8 0.37 Pyruvate - 2 7.4 0 .1 8 L-Lactate - - 41.0 4.52 L-Lactate 5 - 2 5 .6 O .56 L-Lactate - 2 15-2 0.27 None 3 6 .6 n.d. None 5 - 59*4 n.d. K-125 Pyruvate - - 3 2 .6 n.d. Pyruvate 5 - 6 .6 n.d. L-Malate - - 53.1 n.d. L-Malate 5 - 33.7 n.d. 42 strong depression of the Krebs cycle occurs. Prom substrates above PEP in the gluconeogenic pathway, y-hydroxyglutamate causes a moderate inhibition of gluco neogenesis, considerably less than that from pyruvate. At this stage one can only speculate on possible interpretations of these results. The effect of pyruvate in potentiating the inhibitory effect could possibly reflect an effect of the high pyruvate concentration in keeping the y-hydroxy-a-ketoglutarate aldolase equilibrium towards the presumed inhibitor, y-hydroxy-a-ketoglutarate • However lactate as substrate also induces a strong inhi bitory effect and the intracellular pyruvate concentration is probably at most one-tenth as great in this case. (Weidemann elk al., 1969)- Also in the presence of 10 mM L-malate, 10 mM pyruvate does not induce any major inhi bitory effects in combination with Y-hydroxyglutamate or glyoxylate (Table 5)* Table 7 shows the effects of y-hydroxyglutamate on the concentrations of various intermediates during gluconeo genesis from pyruvate. The accumulation of citrate and depression of a-ketoglutarate suggests that the major inhibition may occur at aconitase or isocitrate dehydro genase. It should also be noted that while glucose formation was strongly depressed, lactate synthesis was TABLE 7*— Effect of Y-Hydroxyglutam&te and Glyoxylate on the Concentration of Intermediates and Products During Gluconeogenesis from Pyruvate 20 mM pyruvate and 20 mM acetate were the substrates. 1 gm of kidney cortex slices were incubated for 60 minutes in a total volume of 10 ml. Analytical values are total |imoles per incubation flask. When both a-ketoglutarate and Y-HO-a-ketoglutarate were present, a-ketoglutarate was first estimated using excess alanine plus glutamate pyruvate transaminase, NADH, and lactate dehydrogenase. y-HO-a-ketoglutarate was then estimated using excess aspartate plus gluta&ate oxalacetate transaminase, NADH and malate dehydrogenase. Inhibitor Cone. (mM) Glucose Lactate Citrate aKg Malate y-hq a-Kg None - 18.7 1 6 .0 1.57 2 .1 2 1.04 - Y-Hydroxyglutamat e 5 1 .8 9.6 2.48 0.30 0 .2 6 2.50 Glyoxylate 2 0 .6 7.9 2 .6 2 0.15 0 .2 8 3 .0 2 • f * 44 only about 40# less in the presence of Y-hydroxyglutamate. In a separate but similar experiment using acetate-l-14C as the labeled substrate it was found that the specific activity of the lactate formed was only slightly depressed when Y-kydroxyglutamate was present. The labeling of lactate could occur because of recycling of PEP to pyruvate, suggesting that pyruvate carboxylase and PEP carboxykinase are still operating at a moderate rate. The lack of inhibition in the presence of malate and related intermediates may in some manner be related to an exclusion of the inhibitor from its site of action in the mitochondria because of the high extramitochondrial concentration of the anion involved. 4. 2,4-Dinitrophenol. 2,4-Dinitrophenol (DNP) is the classical uncoupler of oxidative phosphorylation and the mechanism of this proximate site of action will not be considered here. At low concentrations, DNP produces an increase in oxidation, but at higher concentrations it may become inhibitory to oxidation. The mechanism of this inhibitory action is not fully known. In the rat kidney cortex, dinitrophenol is inhibitory toward gluconeogenesis from all substrates (Table 8). At low concentrations of DNP, gluconeogenesis from L-malate 45 TABLE 8 .— Effect of Dinitrophenol on Gluconeogenesis from Various Substrates Substrate concentration was 10 mM. Expt. No. Substrate Dinitrophenol Concentration (mM) Inhibition of Glucose Formation (*) Lactate 9 Pyruvate 0.005 4 Malate 23 Lactate 17 Pyruvate 0.010 14 Malate 57 Lactate 26 Pyruvate 0.020 27 Malate 52 K-74 Lactate 34 Pyruvate 0 .0 3 0 40 Malate 84 Lactate 55 Pyruvate 0 .0 5 0 55 Malate 98 Lactate 87 Pyruvate 0.100 86 Malate 99 Succinate n m n 6 a-ketoglutarate U • UJ.U 22 Succinate 28 K-78 a-ketoglutarate 0 .0 3 0 62 Citrate 57 Succinate n i on 76 a-ketoglutarate U • J.VJU 98 46 is inhibited more markedly than that from pyruvate or L-lactate. This is somewhat surprising, since the energy requirement for the malate — ► glucose pathway is less than that for pyruvate—— ►glucose. For example at 0.05 mM DNP, gluconeogenesis from L-malate was completely abolished while that from pyruvate and lactate proceeded at about half the normal rate. In a separate experiment it was found that 0.05 mM DNP decreased 14C02 production from acetate-l-14C by about 80$, and decreased total C02 pro duction by about 60$, with malate as the substrate. This increased sensitivity to DNP is thus very probably related to the strong inhibition of the Krebs cycle. These results may give some support to the theory that oxalacetate removal, after its production from malate, is an energy requiring step (Tyler, 1955; Slater and Hulsmann, 1961), and that an insufficient rate of removal allows oxalacetate to accumulate and inhibit succinate dehydrogenase. With pyruvate or lactate as substrate, both production (via pyruvate carboxylase) and removal of oxalacetate should be depressed by the lower energy level. 5. Quinolinate. Quinolinate has been studied by Lardy and his coworkers who have proposed that its inhibition of gluco neogenesis in the liver is the result of an effect on PEP 47 carboxykinase (Veneziale et al., 1967). Crossover studies show a decrease in PEP concentration and increases in malate and OAA. Table 9 shows the effect of 2.5 mM quinolinate on gluconeogenesis in the kidney cortex. Marked inhibitions TABLE 9.— Effect of Quinolinate on Gluconeogenesis from Various Substrates Substrate concentration was 10 mM. 10 mM acetate was also present. Expt. No. Substrate Quinolinate Concentration (mM) Glucose Formation (p-moles/ 125mg/2hr) Pyruvate Pyruvate 0 2.5 4.06 1.05 K-106 L-Lactate L-Lactate 0 2.5 3.53 0.50 L-Malate L-Malate 0 2.5 3.19 1.99 D-Fructose D-Fructose 0 2 .5 9.49 8 .6 5 K-130 Glycerol Glycerol 0 2 .5 O J CO OJ • • p H p H DL-Glycerate DL-Glycerate r o 0 U l 2.59 2.37 48 are produced from pyruvate and L-lactate, somewhat less from L-malate, while glucose formation from glycerate, glycerol and fructose was only slightly affected. These results can be rationalized on the basis of a fairly specific inhibition at PEP carboxykinase, with possibly an additional inhibition at pyruvate carboxylase. Table 10 shows the effect of a higher concentration (4 mM) on gluconeogenesis, Krebs cycle, substrate oxidation and recycling of PEP, using 2-14C labeled substrates. Both gluconeogenesis and recycling are decreased, as would be expected if PEP carboxykinase (or also pyruvate carboxylase) were the inhibitory site. 6. Flurocitrate, Chlorosuccinate and malonate. These three compounds are all known to inhibit at sites in the Krebs cycle, fluorocitrate being a strong competitive inhibitor of aconitase, malonate a competitive inhibitor of succinate dehydrogenase, and L-chlorosuccinate a competitive inhibitor and also a substrate of succinate dehydrogenase. The product of this last reaction, chloro- fumarate, could also be a Krebs cycle inhibitor. In a single experiment, 5 mM malonate was found to produce a 44# inhibition of gluconeogenesis from 10 mM pyruvate, and a 52# inhibition from 10 mM L-mal&te. Table 11 shows the effects of a range of concentrations TABLE 10.-Effect of Quinolinate on Gluconeogenesis, Oxidation and Recyling of Phosphoenolpyruvate The substrate concentration was 10 mM. When pyruvate-2-“C was the substrate, the lactate formed was degraded; when L-lactate-2-14C was the substrate, alanine was degraded. The relationship of the randomization of 14C in lactate or alanine to recycling of PEP to pyruvate is described in Section III, B.l. Analytical values are M-moles/125 mg slices/2 hours. Quinolinate concentration when added was 4 mM. Labeled Substrate Quino linate Subst. Converted „,to Glucose Subst. Convert e AcboA Rate of * Krebs Cycle Lactate Formed Relative Specific Activity Lactate or Alanine C-l C-2 C-3 Pyruvate-2-1 4C - 6.5 9.6 20.2 1.4 10.4 100 10.6 Pyruvate-2-1 4 C + 1.4 12.1 17-7 5.2 4.5 100 2.7 L-Lactate-2-14C - 4.6 7.9 17.8 -------- 4.9 100 5.0 L-Lactate-2-14C + 0.5 8.7 15.9 -------- 1-7 100 1.6 vo 50 TABLE 11.-Effect of DL-Chlorosuccinate and DL-Fluorocitrate on Gluconeogenesis from Lactate and 14C02 Formation from Acet&te-l-14C Substrates were 10 mM L-Lactate jSlus 10 mM acetate. DL-Chloro succinate Cone. (mM) DL-Fluoro- citrate Cone. (mM) Glucose Formation (ixmoles/ 125mg/2hr) 14C Yield in C02 ($ added l 4C) 0 4.21 45.1 0.05 3-95 49.8 0 .1 0 3.13 46.6 0 .2 0 3 .0 0 5 2 .0 0 .5 0 2.27 51.4 1 .0 0 1.49 41.7 2 .0 0 1.14 39.1 5 .0 0 0 .6 7 5 1 .0 0 4.21 45.1 0 .0 0 2 5 3-97 48.8 0.005 2.54 39.7 0.010 1.51 31.6 0.025 0.45 2 5 .2 51 of DL-Cl-succinate and DL-F-citrate on gluconeogenesis and 14COg production from acetate-l-14C, with 10 mM L-lactate as the substrate. Both compounds produce inhibitions of glucose formation even at low concentrations. There is a slight increase in “COg production at these low concentra tions, which might suggest another gluconeogenesis inhibitory site somewhere outside the Krebs cycle. However this increase in 14C0g probably does not mirror the rate of the Krebs cycle. A decreased rate of gluconeogenesis means less dilution (by exchange) of oxalacetate specific activity with unlabeled carbon from the substrate. The higher yield in 14C02 could then be the result of a somewhat lower net rate of the Krebs cycle masked by a higher specific activity of the carboxyl carbons of the Krebs cycle intermediates. In the experiments with 0 and 2.0 mM DL-Cl-succinate the organic acids were separated on an anion exchange column (1 cm x 11 cm Permutit FF-1P, acetate form, 100-200 mesh). L-Cl succinate produced about a 60$ increase in succinate radioactivity and about a 40$ increase in citrate. However the major increase (> 300$) was in the malate fraction. Thus the major inhibitory site of action of Cl-succinate, or the Cl-fumarate produced from it, remains to be located. 52 In contrast to the nearly equal inhibitory effect of malonate on glucose formation from pyruvate or L-malate, fluorocitrate at a concentration of 0.1 mM gave a 35% inhibition of gluconeogenesis from pyruvate, but only a 20%> inhibition from L-malate. Similar (though even more pronounced) results were found with the Y-hydroxyglutamate series of inhibitors. 7. Phenylpyruvate, Oxalate, Oxamate. Phenylpyruvate inhibition of pyruvate carboxylase was reported by Krebs and De Gasquet (1964), while oxalate and oxamate inhibition was found by Seubert and Huth (1965). The latter group also tested these compounds for their site specificity by comparing their effects on gluconeogenesis from pyruvate and fumarate. As shown in Table 12, oxamate at a concentration of 5 mM appears (on the basis of this preliminary type of experiment) to be the most specific pyruvate carboxylase inhibitor. Oxamate is also a known inhibitor of lactate dehydrogenase. However only a very strong inhibition of the latter active enzyme would be likely to affect gluconeogenesis. B. Pathways of Carbon. 1. Computer Model. The model of gluconeogenesis to be tested is shown TABLE 12.-Effect of Phenylpyruvate, Oxalate and Oxamate on Gluconeogenesis. Substrate concentration was 10 mM. Substrate Phenyl Pyruvate Cone. (mM) Oxalate Cone. (mM) Oxamate Cone. (mM) Glucose Formation (limoles/ 125mg/2hr) Per Cent Inhibition - - - 5.09 - L-Lactate 5 - - 1.27 75 - 2 - 1.05 79 - - 5 1.45 72 - - - 4.65 - 5 - - 2.31 50 L-Malate - 2 - 3.67 21 - - 5 4.37 6 54 in Scheme 1. This involves an interacting system of glu coneogenesis, the tricarboxylic acid cycle, and the so- called "futile" or "useless" cycle. The substrates used to test the model are L-lactate-2-14C or pyruvate-2-1 4 C; the specific activity of C-2 of the substrate is set equal to one. The small letters below the carbon atoms denote specific activities. The rates of the various enzyme re actions are denoted by V with a subscript. The rates are usually expressed relative to a rate of gluconeogenesis (Vqlu) se^ eQual to one. While separate pools of oxal acetate and malate are shown, the data are as yet not ade quate for an independent determination of and (Scheme 1); thus in this paper will arbitrarily be set to a high value and will be calculated. Complete degradation of glutamate may permit separate evaluation of VMDH and VFUMJ and ^ is Possible that us actually the slower of these two back reactions. It should be noted that the rate of pyruvate carboxylase (VGLU + VpR) is the sum of the rate of glucose formation and the rate of re cycling. The algebraic equations written from the model are shown in Appendix I. The kidney cortex possesses certain advantages for the study of gluconeogenesis. Endogenous glycogen and glucose are negligible, and the rate of glucose synthesis 55 Scheme 1 . M odel o f M ajor M e ta b o lic Pathw ays i n th e K idney C o r te x Glucose V. GLU 0 I I ch3 c cooh v PYR 1* VPK 5 ch2 = c - COOH - ► ch3 c cooh V . LAC ch3 choh cooh m3 m2 ml P3 P2 PI CO r +V GLU PK COOH HOOC 04 01 +V GLU PK M DH -V -V GLU PK CHOH - COOH 'HOOC - CH. CO PDH PI AC CH0 C SCoA a2 CA CH2 - COOH Ja2 al HO - C - COOH 102 01 04 m4 m3 ml CH. FUM HOOC - CH = CH - COOH fo TCA CO. co2 01 04 5 6 from endogenous sources is generally less than 10% of that from added glucogenic substrates. The rate of substrate lactate (or pyruvate) oxidation, i.e. pyruvate dehydro genase, can be estimated from the rate of substrate util ization minus the rate of glucose and pyruvate (or lactate) formation. Total COg formed, determined manometrically, minus that produced in the pyruvate dehydrogenase reaction, provides a measure of the Krebs cycle. No bicarbonate is initially present in the buffer. Analytical data and rates derived from these data are shown in Table 13 for experi ments involving pyruvate-2-uC or lactate-2-14C as substrate in the absence or presence of DL-p-hydroxybutyrate. Iso topic data and the computer model (see below) are required to estimate other rates. The glucose, glutamate, and lactate (or alanine) formed in these incubations was purified and degraded. In preliminary experiments the top and bottom halves of glu cose were found to be symmetrical; therefore only the bottom half was usually degraded. This gives the relative specific activities of m^ (C-6 of glucose), m^ (C-5)* and m^ (C-4) (see scheme 1). To determine the rate of the fumarase back reaction (Vp^)* experimental values of VGLUa VTCA> VPDH* and a ran®e of values of VpK and VpuM are inserted in the computer program and a range of values TABLE 1 3 . — Rate of Formation of Major Metabolic Products from Pyruvate or Lactate in Kidney Cortex Slices V a lu e s e x c e p t w here o t h e r w is e in d i c a t e d a r e pm oles p er 125 mg t i s s u e (w et w e ig h t ) p e r 2 h o u r s . S u b s t r a t e c o n c e n t r a t i o n and D L -/3 -h y d r o x y b u ty r a te c o n c e n t r a t i o n , when ad d ed , was 10 mM. E x p t. No. S u b s t r a t e DL- /3 -HO B u t y r a t e S u b s t r a t e to G lu c o se Total to G lu c o se S u b s t r a t e U t i l i z e d Lactate o r P y r u v a te Formed S u b s t r a t e O x id iz e d (VPDH> CD 6 o CM o u o ■u < fl O CM O O OT O o < 3 > ° I s > 3 > ° " g 42 A P y r u v a te - 8 . 4 9 .2 1 7 .4 1 .3 7 .7 6 1 .8 2 7 .0 3 .0 0 0 .8 4 42 B P y r u v a te + 1 1 .2 1 2 .0 1 7 .4 1 .6 4 . 6 6 7 . 6 3 1 .5 2„ 60 0 .3 8 90 A P y r u v a te - 6 .5 7 .7 1 7 .5 1 .4 3 .6 5 0 .0 2 0 .2 2 .6 3 1 .2 5 90 B P y ru v a te + 1 1 .2 1 2 .4 1 8 .0 2 .2 4 .5 5 2 .5 2 4 .0 1 .9 3 0 .3 6 90 C L - L a c ta te - 4 . 6 5 .8 1 2 .8 0 .3 7 .9 4 3 . 4 1 7 .8 3 .0 7 1 .3 6 90 D L - L a c ta te + 7 .8 9 .0 1 0 .3 0 .3 2 .2 4 6 .2 2 2 .0 2 .4 5 0 .2 4 58 of m^/m2 are read out. It is found that this ratio is very insensitive to VpK and VpDH and these can be set to zero for initial estimates of VpuM. The experimental value of m^/m2 can now be used to graphically estimate VpuM. To determine the rate of the pyruvate kinase reaction, known values of VGLU, VTCA, VpDH, and VFUM and a range of values of VpK are fed to the computer and the resultant p-j/Pg ratios examined. Prom the experimental p^/pg ratio, provided by the degradation of lactate or alanine, one can estimate VpK. Two methods are now available to determine Vppp. which was previously estimated from analytical data. These in volve plotting the calculated ratios of a^/o^ or m^/m2 versus VpDII for a range of values of VpDjj, using the other known rates. The experimental a-j/o^ ratio is given by the C-5/C-1 ratio in glutamate, while the m^/nig ratio is given by the C-4/C-5 ratio in glucose. Essentially by these procedures the rates VpK, and have been calculated for the experiments of Table 13, and the results are shown in Table 14. This gives the experimental degradation patterns, and the values of the above rates which give a close fit to these experimental patterns. TABLE 1 4 . — R a te s C a lc u la t e d from D i s t r i b u t i o n o f R a d i o a c t i v i t y i n G lu c o s e , L a c t a t e and G lu tam ate Formed from P y r u v a t e - 2 - ,4C o r L - L a c t a t e - 2 - ,4C. The e x p e r im e n ts a r e t h o s e d e s c r ib e d i n T a b le 13 i n th e same o r d e r . The " c a lc u la t e d " v a l u e s o f th e d e g r a d a tio n p a t t e r n s a r e t h o s e w h ich a r e o b t a in e d u s in g t h e com puter program and th e v a lu e s o f th e r a t e s g iv e n i n t h e l a s t fo u r co lu m n s. When l a c t a t e - 2 - 14C was u s e d , a l a n i n e was d eg ra d ed . R e l a t i v e S p e c i f i c A c t i v i t y R a te s U sed For Compound D egraded: G lu co se L a c t a t e (o r A la n in e ) G lutam ate C a lc u l a t i o n (Vglu s e t ecL u a l to 1) E x p t. Carbon Number: C4 C5 C6 Cl C2 C3 Cl C5 vtca Vfum VPDH VPK No. S p . A c t i v . i n Scheme 1: ml m2 m3 PI P2 P3 04 a l 42 A E x p e r im e n ta l C a lc u la t e d 6 5 .5 6 5 .8 100 100 7 4 .2 7 5 .5 1 5 .0 1 4 .2 100 100 1 6 .4 1 6 .3 100 100 136 123 3 .0 12 1 .1 1 .0 42 B E x p e r im e n ta l C a lc u la t e d 4 0 .3 4 3 .2 100 100 7 1 .3 7 1 .4 7 .7 5 .3 100 100 8 .0 8 .8 100 100 82 78 2 . 6 10 0 .3 5 0 .5 0 90 A E x p e r im e n ta l C a lc u la te d 6 0 .0 6 1 .6 100 100 6 7 .1 7 1 .2 1 0 .4 9 .8 100 100 1 0 .6 1 0 .8 100 100 156 145 2 .6 3 8 1 .0 0 .6 0 90 B E x p e r im e n ta l C a lc u la t e d 3 4 .3 3 5 .8 100 100 7 3 .2 7 3 .4 6 .2 5 .8 100 100 1 0 .5 1 1 .9 100 100 48 64 1 .9 3 10 0 .2 0 0 .6 0 90 C E x p e r im e n ta l C a lc u la t e d 5 8 .7 6 0 .4 100 100 7 1 .1 7 1 .3 4 . 9 4 .5 100 100 5 .0 5 .3 100 100 179 135 3 .0 10 0 .8 0 0 .3 0 90 D E x p e r im e n ta l C a lc u la t e d 2 6 .6 3 3 .3 100 100 7 2 .2 7 2 .2 2 .5 2 .5 100 100 5 .2 5 .7 100 100 46 36 2 .4 5 10 0 .1 0 0 .3 0 U i VO 6o The rate of the fumarase exchange reaction ranged from about three to five times the rate of the forward Krebs cycle. The rate of pyruvate dehydrogenase was similar to the rate of gluconeogenesis in the absence of added p-hydroxybutyrate, but was markedly depressed in its presence. Most surprisingly, the rate of recycling of PEP to pyruvate was found to approach the rate of glucose synthesis, indicating extensive operation of the so called useless cycle, or modifications thereof. It is conceivable that the randomization in lactate may not be due to the recycling as shown in Scheme 1, but rather is the result of isotopic exchange reactions involving either pyruvate carboxylase or malic enzyme. However the rate of the pyruvate carboxylase reverse reaction is only 10$ of the forward reaction (Scrutton and Utter, 1965) even at high (1 mM) concentrations of oxalacetate, while mitochondrial oxalacetate concentrations are estimated to be at least two orders of magnitude below this (Krebs, 1967). Malic enzyme exchange is a more serious problem, but the fact that a non-bicarbonate buffer is used should minimize any such exchange since this enzyme has a high for C02. In the liver the capacity of malic enzyme in a cell free assay was found to be less than that of pyruvate carboxylase (Krebs and Veech, 1970); in 6l addition, the rate of the reverse reaction (pyruvate to raalate) proceeds at only about one-third that of the forward reaction (Lardy et al., 1964). Thus recycling via pyruvate kinase appears to be by far the most likely explanation for the randomization found. The magnitude of this cycle requires investigation of a possible function. The net result of the ’ Useless cycle"(Scheme 2A) is the dissipation of one high energy phosphate bond. However another cycle may be written involving malate rather than aspartate outflow from the mitochondria (Scheme 2B). The net result of this cycle is an energy-driven transfer of reducing equivalents from the mitoohondria to the cytosol. For the purposes of this paper we will call this cycle the "useful cycle". Such a transfer must occur during gluconeogenesis from pyruvate to provide for the amount of lactate formed. However the rate of recycling is greater than the rate of lactate synthesis. Further aspects of the possible roles of such cycles will be discussed below. While we have used the method of comparing degradation patterns in various products, other techniques could be applied to test the model proposed. Ratios of isotopic yields in various products (glucose, lactate, C02) from specifically labeled substrates (e.g. pyruvate-l-14C, -2-uC, 62 Scheme 2 . C y c le s I n v o lv in g P y r u v a te , O x a la c e t a t e and P h o sp h o e n o lp y r u v a te A. The " U s e le s s C y c le ATP A D P f _ CYTOSOL MITOCHONDRIA P y r u v a te PEP P y r u v a te GDP CO GTP ATP CO . ADP O x a la c e t a t e g lu t a m a t e O x a la c e t a t e aKg g lu ta m a te L - a s p a r t a t e aKg L - a s p a r t a t e B. The " U s e fu l C y c le CYTOSOL ATP MITOCHONDRIA ADP P y r u v a te PEP P y r u v a te CO , GDP 'ATP GTP C02 ADP O x a la c e t a t e O x a la c e t a t e NADH' NAD NADH L -M a la te NAD L -M a la te 65 -5--uC or acetate-l-14C and -2-14C) can be used. It is necessary to use ranges of values of typical rates and determine which ratios will be sensitive functions of a given rate. These techniques have been used in other tissues (Katz and Wood, 1962; Rognstad, 1969; Rognstad and Katz, 1 969). 2. Reversibility in the Whole Pathway. a. Relative Yields of 14C in Gluconeogenic and Glycolytic Products. The "useless"or'Useful"cycles of the previous sections demonstrate the partial reversibility of this portion of the gluconeogenic pathway. Since this tissue has the potential for both glycolysis and glueoneogenesis it might be anticipated that the entire pathway would show some reversibility. That this is indeed so is shown by the data in Table 15. Here duplicate flasks containing either glucose-U-14C or L-malate-U-14C were incubated with 10 p-moles of glucose and a range of values of malate of from 0 to 20 nmoles. In the absence of malate there was a small net utilization of glucose and increasing amounts of malate gave increasing rates of glucose synthesis. In spite of net glucose uptake, 14C from malate-U-14C appeared in glucose; and in spite of net and active gluconeogenesis, 14C from glucose-U-14C appeared in C02j with the amount not 64 TABUS 1 5 . - - ,4 C Y i e l d s D u rin g G l y c o l y s i s and G lu c o n e o g e n e s is in the Kidney Cortex Duplicate flasks, one with L-malate-U-14C and the other with D-glucose-U-14C, were incubated with the proporations of unlabeled glucose and L-raalate given. u- 1 4 c Labeled Substrate Initial Glucose (pmol) Initial Malate (pmol) Change in Glucose (pmol) 14C Yields Glucose (% Added , 4C) C02 L-Malate 10 0.01 -0.7 21.3 66.4 L-Malate 10 1 0 33.1 59.1 L-Malate 10 2 +0.8 33.7 55.9 L-Malate 10 4 +1.1 32.2 56.8 L-Malate 10 10 +3.0 37.7 52.6 L-Malate 10 20 +3.9 24.0 41.2 D-Glucose 10 0 -0.4 84.3 10.1 D-Glucose 10 1 +0.1 85.1 10.8 D-Glucose 10 2 +1.0 82.7 11.5 D-Glucose 10 4 +1.2 86.0 11.0 D-Glucose 10 10 +2.8 83.6 10.4 D-Glucose 10 20 +4.3 88.8 7.3 65 being greatly affected by the increasing extent of gluco- neogenesis. In principle one can measure the rate of reversibility at each step df the gluconeogenic pathway by specifically labeling the various intermediates with 14C and examining the isotopic yields at both ends of the pathway, i.e. glucose, the product of the gluconeogenic pathway, and COgf lactate and amino acids which are the major products of the exchange or backward pathway. Kidney cortex cells are unusually permeable with most phosphorylated compounds being capable of entering the cells to some degree. Table l6 shows an experiment in which U-14C labeled phosphorylated intermediates at tracer levels were added to a series of flasks in which gluconeogenesis was pro ceeding from L-malate and acetate as the main substrates. This gives a graded series of the ratios (R) of products in the order expected. Intermediates such as glucose-6p- U-14C and fructose-6p-U-14C, which would be expected to be close to isotopic equilibrium in the cell, should give essentially the same values of the product ratio and this is what is found. The closeness of the ratios between any two intermediates is a measure of the rapidity of isotopic exchange between these compounds. While this approach of adding the phosphorylated intermediates directly would be 66 TABLE 16.— “C Yields in the Products of the Gluconeogenic and "Glycolytic" Pathways The unlabeled substrates in all flasks were 10 mM L-malate plus 10 mM acetate. Tracer compounds were 0.02 to 0.05 mM. u->«c Tracer Substrate llC Yields (# of Added l 4C) Ratio of Yields [Glucose/ COo + Lac. + A 0 A. ] R Glucose C V I 0 0 Lactate Amino Acids Glucose-6p 84.2 1 2 .5 4.5 4.8 3.9 Pructose-6p 8 1 .1 1 1 .9 4.4 4.5 3.9 Fructose diP 7 6 .2 1 5 .2 4.7 5.7 3.0 a-Glycerol-P 6 7 .8 1 9 .0 4.8 5.2 2.3 Glycerate-3P 34.6 3 0 .6 9.9 11.0 O .67 P-Enolpyruvate 2 6 .8 2 8 .0 9-5 15.7 0.52 L-Lactate 22.5 5 1 .8 7.5 6.7 0.34 67 very useful, further experiments have shown that problems exist, more serious with certain compounds than with others. Thus dephosphorylation occurs to varying degrees with different compounds, e.g. fructose-6p is partially dephosphorylated but fructose diP only very slightly. Extracellular metabolism (due to enzyme leakage?) may occur, notably with 3PGA and PEP. Rather than using the phosphorylated intermediates as tracers one can add tracer amounts of non-phosphorylated compounds which enter the pathway at known sites (Table 17). D-Glyceraldehyde-U-14C in gluconeogenic tissues enters the pathway by phosphorylation via triokinase to D-glycer- aldehyde-3P-U-uC. D-Glycerate-U-14C enters the pathway via glycerate kinase, forming 2P-glycerate-U-14C. Thus the nearness of the product ratio with these two tracer com pounds suggests rapid isotopic reversibility of glycer- aldehyde-P-dehydrogenase, as well as P-glycerate kinase and P-glycerate mutase. In such experiments galactose-U-14C can be used to label intracellular glucose-6p-U-14C and mannose- U-14C to label fructose-6p-U-14C (experiments not yet carried out). However only FDP-U-14C itself can be used to label this intracellular intermediate. It is apparent that considerable isotopic reversibility is occuring even between the pairs glucose and glucose-6p, and fructose-6p and fructose diP. These are additional 68 sites for possible "futile" cycling because of the presence of enzymes catalyzing reactions in the opposite directions, ^ile this data is not yet sufficient for quantitative estimation of this recycling, it is evident that the glycolytic enzymes hexokinase and fructose-6p- kinase are not completely supressed during gluconeogenesis. TABLE IT.— 14C Yields in the Products of the Gluconeogenic and "Glycolytic" Pathways Conditions were as in Table 16. U-14C Tracer Substrate 14C Yields of Added 1 4 C) Ratio of Yields [Glucose/ CO2 + Lac. ■f A. A.] R Glu cose co2 Lactate Amino Acids D-Gljicose 71.4 8.8 3.2 2.1 5.1 D-Fructose 44.6 9.5 3.4 3.9 2.7 Glycerol 50.0 9.7 3.0 4.4 3.1 D-Glyceraldehyde 51.6 15.8 6.8 4.7 2.0 D-Glycerate 49-5 12.7 6.0 7.2 1-9 L-Aspartate 17.5 41.2 3.7 9.2 0.52 L-Alanine 16.6 55.7 7 .1 11.6 0.52 L-Malate 18.5 50.7 3.8 4.0 0.48 69 These experiments showing partial isotopic reversibility of the whole gluconeogenic-glycolytic pathway indicate that caution should be used in the interpretation of isotopic data. Even substantial isotopic yields in the products of either pathway do not necessarily indicate that this is the net direction of carbon flow. Thus in adipose tissue incorporation of 14C into triglyceride glycerol from pyruvate-14C has been interpreted as proving glycerol-neogenesis in this tissue (Ballard et al,, 1967). Proof of such a pathway however will have to exclude the operation of exchange reactions. b. Effects of the Non-homogenous Nature of Gluconeogenic Organs. The liver and kidney have a capacity for net glycolysis in addition to their capacity for net gluco- neogenesis. It is important in the study of control mechanisms to know whether, or how much of, the glycolytic function resides in the gluconeogenic cell types, or whether they are restricted to other cells with different functions. Thus what has been considered futile cycling could also conceivably be the result of gluconeogenesis in one cell type together with a slower rate of glycolysis in other cells. The most direct approach to resolution of this problem 70 is an attempt to isolate a pure fraction of active cells of the gluconeogenic type — the parenchymal cells of the liver or the tubular cells of the kidney cortex. Prepara tion of active clusters of kidney cortex tubular cells has been reported; however active single cells have not yet been prepared (Guder and Wieland, 1970). Berry and Friend (19&9) have prepared isolated rat liver parenchymal cells largely free of other cell types, with no fractiona tion procedure required. These cells synthesized glucose from lactate at rates considerably higher than those attained with liver slices, but about half the rate o# gluconeogenesis in the isolated perfused rat liver. These cells should be adequate for tests of futile cycle activity by the procedures of the previous section. An indirect test for the existence of two cell types with opposite pathways was made using a version of the technique developed by Rose (1961). This involves the use of a doubly (intramolecularly) labeled compound, labeled uniformly with 14C and with deuterium at a site in which an isotope discrimination effect occurs in a known enzyme reaction. If such a compound can be metabolized in different directions, one involving an isotope effect and the other not, isotope discrimination will cause a higher isotopic yield in the product of the non-discrimina- 71 tory pathway. If however the isotope discrimination step occurs in a unidirectional pathway, no decrease will occur in the product of this pathway. A rapid increase in the concentration of the substrate discriminated against will occur, after which the net rate through the step will resume its normal value (i.e. the rate which occurs with a non-deuterium labeled compound). Examples of the effects of isotope discrimination on branched and non-branched pathways have been described (Katz e£ al., 1966). This method can be applied to the problem of hetero geneity. Thus if we have two separate and opposite pathways in different cell types, both of which metabolize the labeled compound but in opposite directions, to glucose or to glycolytic products, isotope discrimination effects would not be expected to be evidenced. But if both gluconeogenesis and "glycolysis" occur in the same cell, at the same time, isotope discrimination should change the ratio of yields in the separate ends of the pathway. Since net gluconeogenesis is occurring, by "glycolysis" in this case we are referring to isotopic exchange reactions which result in labeling of what are normally glycolytic products such as lactate, C02 and amino acids. An example is given in Table 18. Here L-a-glycerol-P- frAP U-1 4C-ID and L-a-glycerol-P-U-14C were the two tracer 72 compounds compared. The major substrates in this experi- GAP ment were fructose and acetate. L-a-Glycerol-P-ID indicates the species of a-glycerol-P which is converted enzymically via a-glycerol-P-dehydrogenase and triose-P isomerase to glyceraldehyde-JP-lD. This compound gives a slower rate in the glyceraldehyde-P dehydrogenase reaction than the non-deuterated species. Thus if the single cell assumption is correct, the deuterated compound should give a higher ratio of the yield of 14C in glucose to that in C02. This ratio was indeed about 20% higher. More data will be required to establish the significance of this effect, and other doubly labeled compounds should also be employed. TABLE 18.-Yields of 14C in Glucose and CO2 from L-a- Glycerol-P-U-1 4C-lH and -ID Unlabeled substrates were 10 mM D-fructose and 10 mM acetate. Labeled Substrate 14C Yields of added 1 4C) Glucose CO2 Ratio of Yields [Glucose/ C02] a-Glycerol-P-U-1 4C-lH 46.5 10.1 4.6 a-Glycerol-P-U-1 4C-lD 42.8 7 .8 5.5 73 3. Exchange of Malate between Mitochondria and Cytosol. Another aspect of reversibility is the flow of metabolites between compartments in the cell. It is important to know the rates of these exchange reactions for the proper interpretation of isotopic data. Also transport steps are potential sites of rate control and it is therefore of interest to determine whether a given isotopic flow is unidirectional across a given membrane or whether flux is rapid in both directions. The latter case suggests (but does not absolutely prove) the absence of Important rate-limitation at the transport step. We have previously shown (Rognstad, 1970) that the apparent rate of malate exchange between the ctyosol and the mitochondria is rapid, i.e. at least several times the rate of net gluconeogenesis. These experiments were carried out using substrate (10 mM) levels of L-malate-U-^C, using a technique of comparing the experimental and theoretical specific activities of the glucose formed. On the other hand Anderson et al. (1970) in studies on the perfused rat liver have concluded that the rate of malate exchange is not rapid and therefore this is considered to be an important potential control site. A possible criticism of our earlier work is that the high substrate malate concentrations used increased 74 markedly the rate of exchange, which might be much slower when gluconeogenesis was occurring from a more physiological substrate. Experiments were thus performed in which lactate or fructose was the gluconeogenic substrate together with tracer levels of L-malate-U-1 4C, succinate-U-uC or citrate-4,5_ 1 4C. The ratio of isotopic yields in glucose and CO2 was determined. Since both succinate-U-14C and citrate-4,5-14C will form mitochondrial malate-U-14C (before any loss of 14C occurs) while added malate-U-14C will first label cytosolic malate-U-14C (upon entrance into the cell), any difference in the product ratio will reflect incomplete or slow exchange of malate through the inner mitochondrial membrane. Table 19 shows that the ratios of yields in glucose and C02 were virtually identical for all three labeled compounds. This suggests that rapid malate exchange occurs during gluconeogenesis in this tissue. The interpretations of the previous paragraphs have assumed malate to be the only dicarboxylic acid exchanging between the compartments. Mitochondrial studies generally have shown that malate readily enters these organelles. Oxalacetate is generally considered to be a non-penetrant. However via the mitochondrial and cytosolic glutamate- oxalacetate transaminases, oxalacetate transfer could in TABLE 19*— Ratio of 14C Yields in Glucose and COg from Mitochondrial and Cytosolic Substrates The concentration of the gluconeogenic substrate was 24 mM in experiment K-170 and 10 mM in experiment K-I8 5. Acetate at 10 mM was also present. Expt. No. Glucogenic Substrate Labeled Substrate 14C Yields Added 1 4C) Glucose C02 Ratio of Yields ("Glucose- ] L C02 J L-Malate-U-14C 3 8 .2 42.7 0.90 K-170 L-Lactate Succinate-U-14C 39-1 41.8 0.93 Citrate-4,5-14C 3 1 .8 34.7 0.92 L-Malate-U-14C 41.3 43.3 0.95 L-Lactate Succinate-U-14C 3 8 .2 39-9 0.96 K-I85 Citrate-4,5-14C 34.0 35.6 0.95 L-Malate-U-14C 62.3 2 3 .2 0.37 D-Fructose Succinate-U-14C 60.3 2 2 .0 0.37 Citrate-4,5-14C 51.9 19.7 0 .3 8 76 principle occur via aspartate which may penetrate the mitochondrial membrane. If aspartate flow is rapid in both directions, this rather than rapid malate exchange could account for the experimental data. However Table 20 shows that aminooxyacetate, a transaminase inhibitor, did not measurably affect the rapid rate of exchange between compartments. This experiment was based on the method published earlier (Rognstad, 1970) and requires analytical data and a simple algebraic model. The experiment does not rule out the possibility that both malate and aspartate exchanges are rapid, since exchange rates, once about five-fold in excess above net fluxes, produce little effect upon further increase in rate. Net aspartate outflow from the mitochondria has been proven to occur in gluconeo genesis when lactate is the substrate (Rognstad and Katz, 1970). C. Pathways of Hydrogen. 1. Tritium as a Tracer for Mitochondrial and Cytosolic Reducing Hydrogen. Gluconeogenesis from most substrates involves a number of oxidation-reduction reactions. The coupling of these reactions is complicated by the fact that they do not all occur in the same space, some talcing place in the cytosol and others in the mitochondria. Depending upon the TABLE 20.— Estimation of Rate of Exchange of Malate between Compartmets The model used is given in Rognstad (1970). The rates VGLU, VTCA, VpDH and VEND (rate of endogenous glucose formation) are obtained from analytical data. Rexp is defined as the ratio: specific activity of glucose___________ . specific activity of C-1,2,3 of malate Rtheoret is caicuia-fced from the expression (derived on the assumption of a single malate pool): ^ 2 (VGLU + vppH) theoret 2V„ttt + V + V™ . + 2V _ GLU + PDH TCA END The rate of exchange of malate between the mitochondria and cytosol (VE) can in principle be calculated from the data. However when Rexp and R-kheoret are nearly the same, VE is large (> 5 vglu^s no accura' fce calculation can be made. Labeled Substrate (10 mM) Amino- Oxyacetate Cone. (mM) Rates (VGLU = = 1) R_ exp VTCA VPDH VEND ^theoret L-Malate-U-14C 0 0 .6 5 1 .7 0 0.35 0 .0 9 0.64 L-Malate-U-uC 0 .1 0.64 I .69 0 .2 9 0 .1 0 0 .6 2 -3 78 gluconeogenic substrate, a net inflow or outflow of reducing equivalents to or from the mitochondria may be required. Tritiated substrates have been used previously to follow the coupling of redox systems in the cytosol of various tissues and cells (Hoberman and D'Adamo, i9 6 0; Kemp and Rose, 1964; Katz and Rognstad, i9 6 0). Coupling of mitochondrial redox systems has been very little studied by this method, perhaps because of the belief that exchange reactions with water in the mitochondria are so rapid that water will be the only labeled product (Bucher, 1970). There is evidence that the outflow of reducing hydrogen from the mitochondria involves the mitochondrial and cytosolic malate dehydrogenases (Lardy at al., 19^5} Rognstad and Katz, 1970). Since these enzymes are quite active and since, as we have seen, malate exchange between the mitochondria and cytosol appears to be rapid, one would expect a certain degree of isotopic mixing of the NADH pools in these compartments even in the absence of any net flow. Thus tritium from those mitochondrial substrates which form NADTmit, in enzyme reactions which occur in the same mitochondrial space as malate dehydro genase, should appear in glucose. Also tritium from cytosolic substrates which form NADTcyt will appear in 79 water as a result of exchange with NADH1 1 1 ^ and subsequent mitochondrial oxidation of NADHmit. Since there are also sites in the cytosolic pathway at which carbon bound tritium will exchange with water, it is difficult to estimate separately the mitochondrial pathway. However since this pathway involves cytosolic labeling of L-malate-2T, with subsequent formation of L-malate-2,3-T in the mitochondria via the active fumarase in this space, the extent of labeling of C-6 of glucose (derived from C-3 of malate) gives some measure of the extent of this pathway. Kidney cortex slices were incubated with an unlabeled gluconeogenic substrate together with tracer levels of various tritiated substrates which form NADT either in the cytosol or the: mitochondria, and the tritium yields in water and in glucose were determined (Table 21). In some experiments the distribution of tritium in glucose was also determined. Hoberman and D'Adamo (1960a,b) have previously found that C-4 and C-6 were essentially the sole sites of tritium or deuterium labeling of glucose from various substrates. In the interpretation of this data it is essential to note the specificity of the NAD enzymes involved in regard to whether they add hydrogen from the substrate to 80 TABLE 2 1 . — Y i e l d s o f T r itiu m i n G lucose and Water and D i s t r i b u t i o n o f T r itiu m i n G lucose U n la b e le d s u b s t r a t e and a c e t a t e c o n c e n t r a t io n were 10 mM. T r i t i a t e d s u b s t r a t e s were l e s s than 0 . 1 mM. n .d . = n o t determ ined. U n la b eled T r i t i a t e d AOA Cone. T r itiu m Y ie ld (% Added T) S p e c i f i c Y ie ld in G lucose 7o T in C-6 o f S u b s t r a t e S u b s t r a t e (mM) Water G lucose (7o) G lucose L-Glutam ate-2T ----- 9 2 .8 0 0 n .d . L-Glutamate-2T 0 .2 4 5 .5 0 .2 0 . 4 n. d. L -M alate L-Glutam ate-2T 2 . 0 3 3 .2 0 . 6 1 .8 n .d . L -N o rv a lin e-2 T ----- 7 1 .1 0 0 n .d . L -N o rv a lin e-2 T 1 .0 5 .2 0 .1 1 2 .1 n .d . D-/3-H0- B u ty ra te-3 T 1 .0 6 8 .7 0 . 4 0 . 6 n. d. D- L- /3 -HO B u tyrate-3T 1 .0 5 0 .8 1 .2 2 .3 n .d . F r u c to s e L-Glutamate-2T 1 .0 7 2 .1 0 .9 1 .2 n. d. C itr a te -2 T 1 .0 8 1 .4 4 . 0 4 .7 n .d . L-M alate-2T 1 .0 8 0 .7 7 .6 8 . 6 n. d. L -L a cta te-2 T 1 .0 7 3 .9 7 .9 9 .6 n. d. L- /3 -H0- B u tyrate-3T — 2 6 .2 0 . 6 2 .2 1 8 .6 C it r a t e - 2 T - - - 64.3 1 .4 2 .2 3 7 .7 L -L a c ta te S u c c i n a t e - 2 , 3T ----- 7 2 .8 4 . 9 6 .3 4 3 .3 Fum arate-2,3T ----- 7 5 .0 6 .3 7 .7 4 4 .0 L-M alate-2T ----- 7 8 .4 6 .2 7 .3 4 5 .9 L -L a c ta te -2 T — 3 4 .8 3 .0 8 .1 2 3 .4 C itr a te -2 T ----- 6 9 .9 1 .8 2 .5 3 8 .2 S u c c in a t e - 2 ,3 T — 7 1 .3 6 .0 7 .7 4 1 . 4 L -L a c ta te L-M alate-2T — 7 0 .8 6 .8 8 .7 4 0 .3 L -L a cta te-2 T -- 5 5 .6 5 .5 9 .0 2 0 .7 L- O t - G ly c e r o l P-2T — 5 3 .7 8 .1 1 3 .1 8 .2 C itr a te -2 T _____ 5 8 .0 1.9 3 .2 3 7 .6 S u c c in a t e - 2 ,3 T — 7 3 .2 7 .6 9 . 4 3 8 .9 P yru vate L-M alate-2T — 7 2 .3 8 .0 1 0 .0 3 7 .8 L -L a cta te-2 T — 4 9 .5 5 .7 10.3 19 .5 L - a - G lycerol-P 2T — 4 7 .1 9 .5 16 .8 5 .7 L -L a c ta te I s o c i t r a t e - 2 T — 3 0 .4 0 .8 2 .6 n .d . P yru vate I s o c i t r a t e - 2 T — 5 1 .8 1 .3 2 .5 n .d . 81 the A or B side of C-4 of the nicotinamide moiety of the nucleotide. The specificity of these enzymes is listed in Table 22. In the transfer of tritium from mitochondrial substrates to the cytosol, malate dehydrogenase is probably the obligatory route, and this enzyme is of the A type. On the other hand the NADH dehydrogenase of the respiratory chain is of the B type. Thus tritiated substrates which form 4A-NADTmit should give relatively higher specific yields in glucose than those which form AB-NADT1 1 1 ^. TABLE 22.--Specificity of NADH-linked Dehydrogenases for A and B Side of C-4 of Nicotinamide Dehydrogenase Speci ficity Reference Isocitrate A Vennesland, i960 Glutamate B Vennesland, i960 Lipoamide B Drysdale, 1966 L-Malate A Hoberman and Prosky, 1967 NADH B Hoberman and Prosky, 1967 Glyceraldehyde-P B Levy ejt al, 1962 a-Glycerol-P B Levy et al., 1962 L-Lactate A Levy et al., 1962 Alcohol A Levy et al., 1962 D-P-Hydroxybutyrate B Hoberman and Prosky, 1967 82 4B-NADTmit, in order to form 4A-NADTmit, will have to undergo an oxidation by an A type enzyme NAD NAD+ (4T) J , followed by reduction by a B type enzyme. Thus tritium yields in glucose from citrate-2T or isocitrate-2T, reaction, are greater than those from L-glutamate-2T or respective dehydrogenases. The incorporation of tritium on C-4 of glucose proceeds through the B-type glyceraldehyde-P dehydrogenase reaction in the cytosol, while incorporation on C-6 of glucose requires intervention of the A-type malate dehydrogenase. Thus tritiated cytosolic substrates which form 4B-NADTcyt should give more labeling of C-4 of glucose, and give higher yields of tritium in glucose, than cytosolic substrates which form 4A-NADTcyi:. Comparison of the results using L-a-glycerol-P-2T, which gives 4B-NADTCyt in the a-glycerol-P dehydrogenase reaction, and L-lactate- 2T, which yields 4A-NADTcyt in the lactate dehydrogenase reaction, shows agreement with these predictions. The amount of tritium appearing in glucose from citrate-2T ranges from about 2 to 5 per cent of the substrate utilized. In these and similar experiments, mapping of the fluxes of carbon and hydrogen (see Rognstad which form 4A-NADTmit in the isocitrate dehydrogenase D-p-hydroxybutyrate-JT, which form 4B-NADTrni^ by their 83 and Katz, 1970) shows that the cytosolic NADH flux leading to glucose synthesis is about 10 per cent of the total NADH turnover in the mitochondria plus cytosol. Thus if complete equilibration of the two NADH pools were assumed, one might expect about 10 per cent of the tritium to appear in glucose. This figure however must be reduced by one-half, since the DHAP-1T formed from GAP-IT loses its tritium to water in the aldolase reaction, while the tritium from C-3 of DHAP is lost to water in the hexose-P isomerase reaction (Rose and Rieder, 1955; Rose et al., 1969)* Thus the maximal expected yield in glucose would be approximately 5 per cent. In view of the unlikelihood of complete equilibration of NADH via the malate dehydro genases and malate exchange, and in view of possible cytosolic exchange reactions with water [^e.g. aldolase, triose-P isomerase catalyzed exchange, (Katz and Rognstad, release to water via mitochondrial exchange reactions may be rather limited or even absent. Thus while water is the main labeled product, this is to be expected on the basis of the high net rate of NADH oxidation via the respiratory chain and the known occurrence of labilization of tritium in the cytosol. Substrates of glutamate dehydrogenase, L-glutamate-2T 1966; Rose and Warms that tritium 84 and L-norvaline-2T, yield tritium in glucose only in the presence of rather high concentrations of the transaminase inhibitor, aminooxyacetate (Table 21). Active trans aminases such as glutamate-oxalacetate transaminase cause rapid exchange of tritium on C-2 of their substrates with water. Thus transaminase activity must be nearly totally inhibited in order to follow the fate of this tritium atom in reductive reactions. It should be pointed out that results using L-p-hydroxy butyrate-JT cannot be completely rationalized by the schemes invoked to explain the other data. This substrate after activation should be metabolized by the mitochondrial enzymes which oxidize fatty acids, i.e. by L-3-hydroxy- acyl CoA dehydrogenase. While the relative tritium yields in water and glucose suggest a mitochondrial site of metabolism, the tritium distribution in glucose corresponds more closely to that obtained from a cytosolic substrate such as L-lactate-2T. It is possible that this substrate may be partially metabolized by the p-L-hydroxyacid dehydrogenase described by Smiley and Ashwell (1961). The use of either L-lactate or pyruvate as the gluconeogenic substrate causes very little difference in the tritium yields in glucose from the various mito chondrial substrates. This occurs in spite of the fact 85 that net malate efflux from the mitochondria occurs during gluconeogenesis from pyruvate, while net aspartate efflux occurs during glucose synthesis from L-lactate. This again is consistent with rapid malate exchange between compart ments largely masking any difference in net flux that occurs with these substrates. Incorporation of tritium from succinate-2,3-T into products of reductive biosynthesis has been interpreted as indicating production of reduced tritiated flavin in the succinic dehydrogenase reaction, transfer of tritium to NADH by reverse oxidative phosphorylation, with subsequent transfer from NADT into the product (Whereat, 1965)* Hoberman and Prosky (1967) in studies on liver mitochondria have demonstrated that the tritium incorpora tion into the reduced products very likely is not derived from succinic dehydrogenase but rather Reflects NADT produced in the subsequent malate dehydrogenase reaction from L-malate-2,3-T. The experiments in Table 23 indicate that this interpretation fully applies to the kidney cortex. Thus while succinate-2,3-T produces considerable tritium labeling in glucose, L-2Cl-succinate-2T and (3S)-L-2Cl-succinate-3T yield tritium only in water. L-Cl-succinate has been shown to be a good substrate for succinic dehydrogenase, with a about twice that for 86 succinate (Gawron et al., 1963). The 3S and 2 hydrogens are removed from L-2C1-succinate in the succinate dehydrogenase reaction (Gawron et al., 1966). Thus one or the other of these hydrogens should be incorporated (at least transiently) into the reduced flavin of the enzyme. The fact that these substrates only label water indicates that none of the glucose labeling caused by succinate- 2, j5-T came about via the succinic dehydrogenase reaction. The site of the labilization of tritium from the tritiated chlorosuccinates is not known, but coUld involve exchange at the flavin level, or possibly at ubiquinone or cytochrome b (Hoberman and Prosky, 1967). In contrast to the complete exchange of the tritium involved in succinic dehydrogenase, our previous results suggested that exchange of tritium from mitochondrial NADT with water was not extensive in the intact kidney cortex cell. These divergent results suggest that the respiratory chain is probably not in a near-equilibrium status. This would be in accord with "kinetic" control of the respiratory chain, as proposed by Lardy (1956) and Chance and Williams (1956), rather than "thermodynamic" control, as held by Klingenberg (1965). Klingenberg1s concept rests largely on the phenomenon of "reverse oxidative phosphorylation" which occurs with TABLE 25.— Fate of Tritium from Succinate-2,3T Compared with L-2C1-Succinate-2T and -3T Unlabeled Substrates (concentration in brackets) Labeled Substrate Tritium Yield {% of Added UE) Specific Yield of T in Glucose m Water Glucose L-Lactate (20 mM) plus Butyrate (20 mM) Succinate-2,JT 72.8 8.7 1 0 .7 L-2C1-Succinate-2T 6 7 .0 0 0 Pyruvate (20 mM) plus Acetoacetate (20 iriM) Succinate-2 3yi 84.8 4.5 4 .8 L-2C1-Succinate-2T 9 7 .2 0 0 L-Lactate (20 mM) plus Acetate (10 mM) Succinate-2a3T 73-5 7.6 9 .4 L-2C1-Suecinate-2T 8 3 .3 0 0 (5S) L-2C1-Succinate- 3T 6 9 .5 0 0 88 isolated mitochondria under certain unusual conditions (i.e. high succinate concentrations, no cycling in the Krebs cycle). This of course does not represent or even approximate conditions which prevail in the intact functional mammalian cell. There is no evidence that net flow in the respiratory chain ever proceeds backwards in intact mammalian cells. In such biosynthetically active tissues as adipose tissue and liver, the amount of mitochondrially generated NADH which is used at times for reductive biosynthesis is never more than a small fraction of that which is oxidized by the respiratory chain to generate ATP (Rognstad and Katz, 196 6; Williamson et al., 1970). Reverse oxidative phosphorylation operating in a space separate from the respiratory chain would make even less sense, since this would require construction of a separate compartment to catalyze an exchange reaction. The results of the experiments with tritiated substrates have shown that most of the compounds can be grouped into two classes: those which are metabolized initially either in the mitochondria or the cytosol. It should now be feasible to apply this method to determine where other compounds (e.g. acetaldehyde using acetaldehyde-lT) are metabolized. While these experiments with tritiated substrates have 89 produced useful Information, they provide as yet little definitive evidence on the routes and net fluxes of hydrogen transport between the mitochondria and cytosol. Other approaches are described in the next section. 2. Shuttles for the Transfer of Reducing Equivalents, a. Import Shuttles. Since the discovery by Lehninger (1951) that mitochondria are impermeable to NADH, various shuttle mechanisms have been proposed for the transport of reducing equivalents from the cytosol into the mitochondria. In insect flight muscle there is strong evidence for the operation of the a-glycerophosphate shuttle (Zebe et al., 1957)* In any tissue the prerequisite for a shuttle is the presence of two enzymes catalyzing a given redox reaction in the separate compartments. The capacity of the enzymes must be commensurate with the flux of hydrogen transport in the cell. In mammalian cells the two most likely candidates for transfer of cytosolic reducing hydrogen into the mitochondria (an "import" shuttle) are generally held to be (a) the a-glycerophosphate shuttle; and (b) the malate-oxalacetate shuttle. Since oxalacetate itself does not penetrate the mitochondrial membrane, Borst (1963) proposed that two transamination reactions participate in the shuttle with aspartate being the 90 the compound that flows out of the mitochondria. The two proposed import shuttles are shown in Scheme 3. The kidney cortex contains a sufficient quantity of enzymes of both pairs of the a-glycerophosphate shuttle and the Borst cycle to permit either in principle to function as the import shuttle in this tissue (Lee and Lardy, 1965)* Since the a-glycerophosphate shuttle catalyzes a transfer of reducing equivalents from cytosolic NADH to mitochondrial flavin, it might be considered energetically more favorable than the Borst cycle which effects the transfer from cytosolic NADH to mitochondrial NAD+. Indeed the latter cycle as written above would be energetically impossible in the kidneyoortex or liver under normal conditions, since the NADH/NAD+ ratio in the cytosol is considerably lower than that in the mitochondria (Krebs, 1 9 6 7). However there is some evidence that an energy input step may occur at some site in this cycle, and this would make the system energetically feasible. Slater and Hulsman (1961), Tager (1963) and others have proposed that oxalacetate may be transferred from one mitochondrial compartment to another by an energy requiring mechanism, while La Noue and Williamson (1970) have suggested that aspartate transport out of the mitochondria may require energy. 91 Scheme 3 . Proposed Import S h u t t l e s A. The a - G ly c e r o p h o s p h a t e S h u t t l e : NADH NAD+ v y D ih y d ro x y a ceto n e-P w m m m ^ — ► a - G ly c e r o l- P c y t o s o l \ I m ito ch o n d rio n D ih y d ro x y a ceto n e-P FH2 fh ■ a-G lycerol-P B. The B o rst Cycle: L-Glutamate O x a la c e ta te / p a - k e t o g l u t a r a t e NAD L -A sp a r ta te L-M alate c y t o s o l mi tochondr ion L -A sp a r ta te L-M alate a - k e t o g lu t a r a t e ' NAD > S s' ^ .L -G lu ta m a te NADH O x a la c e t a t e 92 In order to test for the operation of these shuttles in the kidney cortex, we have attempted to employ inhibitors which block a given enzyme in these cycles. However only for the Borst cycle has a specific and potent inhibitor yet been found. Here we have again used the effective transaminase inhibitor, aminooxyacetate. As yet only a few situations are known to occur in the kidney cortex in which a marked (and therefore readily measurable) excess of cytosolic reducing equivalents is produced. Ethanol is rather poorly metabolized in the kidney cortex and thus cannot be used as readily for this purpose as it has been in the liver. The process of gluconeogenesis from L-lactate involves no excess or deficit of cytosolic NADH. However if L-lactate is used as a substrate in the absence of added fatty acids, the amount of lactate which is oxidized to COg is found to be nearly the same as the amount which is converted to glucose. The oxidative pathway involves production in the cytosolic lactate dehydrogenase reaction of NADH which is not used in reductive synthesis and which therefore must be shuttled into the mitochondria. Table 24 shows that aminooxyacetate does not significantly inhibit the transfer of this excess of reducing equivalents into the mitochondria under normal conditions in the kidney cortex, providing 93 TABLE 24.— Effect of Aminooxyacetate on Lactate Oxidation Lactate concentration was 10 mM. No acetate or fatty acids were added. Expt. No. Aminooxy acetate Cone. (mM) Dinitro- phenol Cone. (mM) Lactate to Glucose (ixmole/ 2 hr) Lactate to C02 (p-mole/ 2 hr) 0 0 6.6 6.5 0.1 0 0.4 5.9 K-129 0 0.2 0 9.6 0.1 0.2 0 4.2 0 0 3.8 8.2 0.1 0 0.2 6.6 K-131 0 0.2 0 7-0 0.1 0.2 0 4.5 0 0 5-2 4.0 K-150 0.1 0 0.8 4.3 0 0.2 0 7.7 0.1 0.2 0 4.5 0 0 6.2 6.4 0.1 0 0.6 7.0 K-139 0 0.2 0.4 11.2 0.1 0.2 0 5.4 K-184 0 0 6.9 8.9 1.0 0 0 7.9 94 evidence against the operation of a Borst cycle. In the presence of dinitrophenol, on the other hand, amino oxyacetate partially inhibits the import of reducing equivalents, suggesting that a Borst cycle may participate under these unusual conditions. By elimination, the a-glycerophosphate shuttle should operate as the normal import shuttle in the kidney cortex. However the possibility of other shuttles yet unfound makes a more direct test desirable. The mitochondrial flavin linked a-glycerophosphate dehydrogenase has generally been found to be the enzyme of least capacity in the a-glycerophosphate shuttle. A purified mito chondrial a-glycerophosphate dehydrogenase from pig brain was found to be inhibited by DL-glyceraldehyde-3-P, with _IL a of 3.5 x 10 for the L-isomer and 5 x 10 for the D-isomer (Dawson and Thorne, 19^9)• Since the kidney cortex has a certain permeability to phosphorylated compounds, these compounds might prove to be effective in the intact cell. Table 25 shows that 2 mM DL- glyceraldehyde-5P produced a moderate inhibition of apparent lactate oxidation in the kidney cortex. However controls were not run in this experiment to determine' if any of the glucose was formed from the inhibitor. L-Glyceraldehyde-3P, which is unlikely to form glucose 95 and which is the more potent inhibitor, would be preferable to the DL-mixture. TABLE 25.— Effect of DL-Glyceraldehyde-3P on Lactate Oxidation Analytical data are in jj.moles/125 mg. tissue/2 hours. DL-Glyceraldehyde- P Concentration (mM) Lactate Used Lactate to Glucose Lactate to CO2 0 15-6 5.6 10.0 0.2 15.5 5.4 8.1 e.o 11.4 6.6 4.8 b. Export Shuttles. In gluconeogenesis from substrates which do not generate sufficient NADH in the cytosol for the glyceraldehyde phosphate dehydrogenase reaction, there is good evidence that export of mitochondrial malate can provide both the carbon and reducing hydrogen required (Lardy et al., 1965; Rognstad and Katz, 1970). However - the term "shuttle" implies a cycle or back and forth pathway, and a shuttle for hydrogen transport in the 96 absence of net carbon flow may be required in addition to net outflow of carbon and hydrogen via malate. For an export shuttle the major candidates are (a) a reverse Borst cycle, or (b) the "useful cycle" (see Section III, B.l). A minimum estimation cf net export flux can be made from balance studies. Thus during gluconeogenesis from substrate pyruvate, there is also a net synthesis of lactate (until most of the pypuvate has been utilized, at which time lactate becomes the gluconeogenic substrate). The amount of lactate synthesis is a minimum estimate of the export shuttle. It must also be considered whether there is some simultaneous operation of both export and import shuttles. This will not appear in balance sheets of cytosolic NADH generation and utilization. In principle a test of the relative roles of the reverse Borst cycle and the"useful cycle"(and also the 'Useless cycle") can be made by examining the effects, of a transaminase inhibitor such as aminooxyacetate. Both the reverse Borst cycle and the useless cycle would involve oxalacetate transfer via the dual transaminase mechanism, although net flow would be in opposite directions. As shown in Table 26, 0.1 mM aminooxyacetate gives actually a slight increase in recycling of PEP to pyruvate 97 during gluconeogenesis from pyruvate, and also a small increase in the amount of lactate formed. At face value this would rule out both the reverse Borst cycle and the it i i useless cycle. However this ignores the chemistry of the inhibitor. Aminooxyacetate is a substituted hydroxylamine, and can undergo a slow reaction with keto compounds such as pyruvate. Thus pyruvate at high substrate concentrations may slowly react with and deactivate the inhibitor. These experiments should be repeated at higher aminooxyacetate concentrations, or possibly using an alternative transaminase inhibitor such as difluorooxalacetate (Kun, 1963) . TABLE 2 6.--Effect of Aminooxyacetate on Recycling of PEP to Pyruvate During Gluconeogenesis from Pyruvate Pyruvate-3-“C concentration was 10 mM. The lactate produced was purified and degraded. Aminooxy acetate Concentra tion Glucose Formed (pmoles/ 2 hr) Lactate Formed (lxmoles/ 2 hr) Relative Specific Activity In Lactate ($ of C-3) (mM) C-3 C-2 C-l 0 4.39 1 .8 0 100 9.0 14.8 0.1 4.24 2 .3 6 100 9.9 17.5 98 D. Control Mechanisms. 1. Effect of Added Cyclic AMP. 3 1 5* Cyclic AMP stimulates gluconeogenesis in the liver and kidney whether added exogenously or produced intracellularly by hormone action (Exton and Park, 1968; Bowman, 1970). The specific site and mechanism of action is still not resolved. One possible effect is the well known lipolytic action, which then might stimulate pyruvate carboxylase as a result of the increased acetyl CoA produced (Friedmann et al., 1967). There is, however, evidence for a separate site of action, localized in the liver in the region between pyruvate and PEP (Exton and Park, 1968). Table 27 shows the effect of cyclic AMP at a range of concentrations on gluconeogenesis in the kidney cortex. Above 0.5 mM inhibitory effects become apparent. Generally 0.1 to 0.3 mM has been found to give a maximum stimulation of gluconeogenesis. The effect of 0.3 mM cyclic AMP on gluconeogenesis from a number of different substrates is shown in Table 2 8. No stimulatory effect is found with substrates which enter the gluconeogenic pathway above PEP, e.g. with D-glycerate, D-glyceraldehyde, glycerol or D-fructose. Also there is only a very slight stimulation from pyruvate of doubtful 99 TART.F. 2 7 . - - E f f e c t o f t h e C o n c e n tr a tio n o f C y c lic AMP on G lu c o n e o g e n e s is S u b s t r a t e C y c lic AMP C o n c e n tr a tio n (mM) G lu c o s e Formed ((j.mole/125 mg/2 h r ) P y r u v a te 0 4 . 1 6 P y r u v a te 0 .0 1 4 .2 8 P y r u v a te 0 .1 0 4 .2 7 L - L a c t a t e 0 3 .4 0 L - L a c t a t e 0 .0 1 3 .2 6 L - L a c t a t e 0 .1 0 4 .1 4 L -M a la te 0 4 .1 4 L -M a la te 0 .0 1 5 .0 8 L -M a la te 0 .1 0 5 .4 4 P y r u v a te 0 4 .0 0 P y r u v a te 0 .1 0 4 .1 7 P y r u v a te 0 .5 0 4 .0 5 P y r u v a te 1 .0 0 2 .1 9 L - L a c t a t e 0 3 .0 3 L - L a c t a t e 0 .1 0 3 .4 4 L - L a c t a t e 0 .5 0 2 .9 8 L - L a c t a t e 1 .0 0 2 .5 9 L -M a la te 0 3 .8 5 L -M a la te 0 .1 0 4 .4 6 L -M a la te 0 .5 0 3 .8 3 L -M a la te 1 .0 0 2 .5 2 D -F r u c to s e 0 9 .1 4 D -F r u c t o s e 0 .1 0 8 .4 5 D - F r u c t o s e 0 .5 0 7 .1 1 D -F r u c t o s e 1 .0 0 6 .5 5 ! | j 100 significance. There is a constant and reproducible stimu latory effect of approximately 40$ from malate and other Krebs cycle intermediates. TABLE 28.— Effect of 0.3 mM Cyclic AMP on Gluconeogenesis from Various Substrates Substrate Number of Experi ments Effect Of Cyclic AMP On Glucose Formation (Average $ Increase) Pyruvate 7 + 3 L-Lactate 7 + 16 L-Malate 7 + 38 Fumarate 3 + 45 Succinate 3 + 43 a-Ketoglutarate 1 + 30 Isocitrate 1 + 38 D-Glyceraldehyde 4 + 1 D-Fructose 4 - 2 D-Glycerate 3 + 4 Glycerol 2 0 101 These results suggest that cyclic AMP might increase the rate of PEP carboxykinase. Another possibility might be an inhibition of pyruvate kinase. The lack of stimula tion from pyruvate suggests that pyruvate carboxylase is the rate limiting enzyme during gluconeogenesis from this substrate, under the conditions employed. Cyclic AMP moderately stimulates glucose formation from L-lactate. Absolute rates of gluconeogenesis are generally somewhat less from L-lactate than from pyruvate in the kidney cortex, and recycling of PEP to pyruvate may also be less (see Section III, B.l). Thus pyruvate carboxylase may be operating at somewhat under capacity in gloconeogenesis from L-lactate, in the absence of the maximal cyclic AMP stimulus. In the kidney cortex caffeine does not potentiate the stimulatory effect of cyclic AMP. At 0.5 mM, caffeine produces about a 15# inhibition of gluconeogenesis from pyruvate. 0.5 mM cyclic AMP partially removes its inhi bitory effect. 2. Effect of Redox State. Glucose synthesis from L-lactate and L-malate is in cytosolic redox balance, but gluconeogenesis from pyruvate requires a mitochondrial source of NADH. If the transfer of NADH from the mitochondria to the cytosol is a process 102 of unlimited capacity, gluconeogenesis from pyruvate would be no more sensitive to treatments which tend to deplete cytosolic NADH than would gluconeogenesis from lactate. The reverse Borst cycle involving the active malate dehydrogenases and glutamate oxalacetate transaminases, if energetically feasible, should be a process of great capacity. On the other hand if NADH transfer involves the "useful cycle", limited capacity would be expected since this cycle involves pyruvate carboxylase and PEP carboxy- kinase. Glucose synthesis from L-lactate or L-malate could be affected by an increase or decrease in cytosolic NADH/NAD+ ratios in different ways at different sites, and the overall result cannot be always predicted. Thus an increase in this ratio would (as described in the introduction) lower the PEP concentration, leading possibly to an increase in PEP carboxykinase activity or a decrease in PEP conversion to pyruvate. On the other hand an increased NADH/NAD+ ratio would lower the concentration of the oxidized substrate of the active, near-equilibrium, lactate and malate dehydrogenases. Lowering of the concentration of pyruvate or oxalacetate could then lower the rate of, respectively, pyruvate carboxylase or PEP carboxykinase if the concentrations are in the region of the (typical 103 values: KPyruva,ke for pyruvate carboxylase from chicken liver mitochondria is 0.44 mM [Scrutton and Utter, 1965a^; O A A for PEP carboxykinase from pig liver is 0.15 mM [Chang et al., 19663). Depending on what enzyme is rate- limiting, and on effects at other sites, an effect on net glucose synthesis may appear. Treatments which decrease the NADH level in the cytosol are readily available, either through autoxidizable dyes which react non-enzymatically with NADH, e.g. methylene blue or phenazine methosulfate (PMS), or via compounds such as N,N,N',N'-tetramethylphenylenediamine (TMPD), which are thought to be capable of effecting transfer of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain, possibly at a site on the outer surface of the inner mitochondrial membrane (Robinson and Halperin, 1970). Treatments which raise the cytosolic NADH level are available but have not been fully developed in the case of the kidney cortex. Ethanol and acetaldehyde are both considered to be oxidized by cytosolic NAD-linked enzymes, but rigorous proof of this is not yet available in the kidney. Sorbitol and xylitol are apparently metabolized by a variety of NAD- and NADP-linked enzymes in both the mitochondria and cytosol (Touster, 19^9)> and the major 104 enzyme used In the kidney is again not known. The level of alcohol dehydrogenase in the rat kidney cortex is only one-tenth that in rat liver (Krebs et al., 1969). The effect of treatments which tend to deplete cyto solic NADH is shown in Table 2 9. There occur only very small effects with L-lactate as substrate (possibly even a slight stimulation at high lactate concentrations), but addition of the same concentrations of TMPD or PMS causes a definite decrease in glucose formation from pyruvate and from L-malate. Higher concentrations of TMPD or PMS become inhibitory from all substrates. Only a limited amount of data is available on conditions which may lead to an increase in cytosolic NADH. The effect of acetaldehyde and L-glyceraldehyde is shown in Table 30. That these compounds form cytosolic NADH is suggested by the increased production of lactate from pyruvate. Both compounds stimulate gluconeogenesis from L-malate, but, at high concentrations, depress gluconeo genesis from L-lactate and pyruvate. The inhibitory effect of NADH depletion on gluconeo genesis from pyruvate is in accord with a limited system for export of mitochondrial reducing equivalents, such as the "useful cycle". One can speculate also on the opposite effect of NADH 105 TABLE 2 9 . — E f f e c t o f TMPD and PMS on G lu c o n e o g e n e s is Expt. No. Substrate Cone. (mM) TMPD Cone. (mM) PMS Cone. (mM) Glucose Forma tion (pinole / 2 hr) % Change L-Lactate 24 0 __ 5 .5 0 4- L-Lactate 24 0 .1 0 5 .8 2 T U K -169 L-Malate 24 0 3 .5 8 -2 3 L-Malate 24 0 .1 0 2 .7 6 L-Lactate 24 0 — 5 .1 0 +19 L-Lactate 24 0 .2 0 — 6 .0 9 K -162 Pyruvate 24 0 — 6„ 41 - 4 4 Pyruvate 24 0 .2 0 3 .5 8 L-Lactate 10 — 0 3 .5 8 - 4 L-Lactate 10 — 0 .0 0 5 3 .4 2 K -100 Pyruvate 10 — 0 3 .9 9 - 2 9 Pyruvate 10 - - 0 .0 0 5 2 .8 3 L-Malate 10 — 0 3 .3 9 -2 7 L-Malate 10 — — 0 .0 0 5 2 .4 9 L-Lactate 12 0 — 5 . 2 4 - 8 L-Lactate 12 0 .1 0 - - 4 .8 4 L-Lactate 24 0 - - 5 .9 1 + 6 L-Lactate 24 0 .1 0 — 6 .2 7 K -157 Pyruvate 12 0 — 4 .9 5 - 2 4 Pyruvate 12 0 .1 0 — 3 .7 8 Pyruvate 24 0 — 6 .9 3 - 3 1 Pyruvate 24 0 .1 0 — 4 .7 8 106 TABLE 3 0 . - - E f f e c t o f A c e ta ld e h y d e and L -G ly c e r a ld e h y d e on G lu c o n e o g e n e s is S u b s t r a t e c o n c e n t r a t i o n was 10 mM. n . d . = n o t d e te r m in e d . Substrate Acet aldehyde Cone. (mM) L-Glycer- aldehyde Gone. (mM) Lactate Formed ((j.mole/2 hr) Glucose Formed ((j.mole/2 hr) L-Lactate 0 — n.d. 4 .9 1 L-Lactate 10 — n. d. 3 .6 8 Pyruvate 0 — 1 .5 9 4 .9 8 Pyruvate 10 — 3 .5 8 3 .4 3 L-Malate 0 — n.d. 3 .6 3 L-Malate 10 — n.d. 4 .9 8 L-Lactate — 0 n. d. 4 .0 0 L-Lactate — 20 n. d. 3 .7 6 L-Lactate — 40 n.d. 1 .6 0 Pyruvate — 0 n. do 3 .7 8 Pyruvate — 20 n.d. 3 .4 4 Pyruvate — 40 n.d. 1 .7 3 L-Malate — 0 n. d. 4 .5 0 L-Malate — 20 n.d. 5 .6 9 L-Malate — 40 n. d. 5 .7 2 Pyruvate — 0 2 .2 6 4 .1 5 Pyruvate — 20 4 .2 2 3 .4 1 L-Malate — 0 n.d. 3 .8 0 L-Malate — 20 n.d. 5 .0 7 107 depletion on glucose synthesis from lactate and malate. In the case of lactate, the additional amount of NADH required could be furnished by exporting malate rather than aspartate from the mitochondria, with no increase in the rate of pyruvate carboxylase or PEP carboxykinase being required. On the other hand with malate as substrate, export of mitochondrial reducing equivalents would require (if our hypothesis is correct) operation of the useful cycle. This would entail increased flux through PEP carboxykinase, but this cannot occur because this enzyme is probably rate-limiting in gluconeogenesis from L-malate (Section III, D.l). 3. ATP Balance. We have previously made balance studies of ATP formation and utilization in adipose tissue (Rognstad and Katz, 1966, 1969). The amount of ATP required for synthe tic functions accounted for most (i.e. >80$) of the amount of ATP formed, calculated assuming theoretical P/0 ratios. The "excess" which presumably includes the re quirement for maintenance of the tissue involves such pro cesses as cation transport and protein synthesis. In the kidney cortex this basal energy metabolism is very high and exceeds greatly the amount of extra energy used for gluco neogenesis (Krebs et al., 196 3). This is not an artifact 108 of the slice technique, since the perfused whole kidney likewise shows high rates of oxygen uptake in the absence of a gluconeogenic substrate (Bowman, 1970). Probably transport processes are particularly involved in this high energy requirement. While no overall ATP balance can thus be made, it may be possible to compare the ATP requirement for glucose synthesis with the amount of extra oxygen uptake caused by addition of the gluconeogenic substrate. Table J>1 shows the data obtained with 10 mM pyruvate and 10 mM acetate as the gluconeogenic substrate mixture. The amount of ATP formed, even when corrected for the amount of ATP required for acetate activation, is considerably greater than the amount of ATP required for glucose synthesis itself. However if recycling of PEP to pyruvate is assumed to be equal to the rate of glucose synthesis, this could entail the loss of two additional ATP’s per triose, if malate outflow is involved. With the many assumptions involved the close agreement between extra ATP productiqn and utilization is undoubtedly fortuitous. However it is evident that exact balance studies will require estimations of recycling, not only involving PEP and pyruvate, but also between the pairs fructose-6P:fructose diP and glucose: glucose-6p. TABLE 31.— ATP Balance during Gluconeogenesis from Pyruvate and Acetate Oxygen uptake was measured in Dixon-Keilin flasks. All values are in iimoles per 125 mg tissue per 70 minutes. Extra oxygen uptake was total oxygen uptake minus the oxygen uptake of a flask which contained 10 mM acetate only. All values are the average of duplicate flasks. Glucose synthesis is corrected for that in the endogenous case which was 0.21 [jimoles. Column A for ATP formation assumes all oxidation to generate 6 p,moles of ATP per p.mole of Og. Column B assumes all ATP generation was from oxidation of acetate. Each acetyl oxidized in the cycle generates 12 ATP's, but activation of acetate requires 2 ATP1s. Column C assumes each pyruvate converted to glucose requires 5 ATP's. Column D arbi trarily assumes a recycling of PEP to pyruvate equal to the rate of glucose synthesis. Total Oxygen Uptake Extra Oxygen Uptake Glucose Formed A ATP Formed B ATP Formed C ATP Used D ATP Used 17.46 A.54 2.18 2 6 .2 21.9 13.1 21.8 H o M3 110 E. General Discussion. In this section we will discuss the contributions of this dissertion to the study of the pathways and control mechanisms of gluconeogenesis. We will try to point out some of the major unsolved problems and attempt to suggest general approaches or possible specific techniques for their solution. It should be stated that a major aspect of the control of gluconeogenesis, that is, the exact mole cular nature of hormonal action on these tissues, is largely outside the scope of this work. 1. The Importance of the Determination of Carbon Fluxes in the Intact Cell. Since the advent of the concept of feedback or allosteric control of enzymes, practically every enzyme study carried out has been made with at least some regard to possible regulatory mechanisms controlling the rate of the given enzyme. Often the regulatory effects found on the given enzyme were automatically assumed to be an important control mechanism in the intact cell. The re sult is a rather bewildering array of potential control systems with little indication of what might be of major importance, minor importance (fine tuning) or simply of no importance at all in the intact cell. Even an attempt at a quantitative estimation of actual Ill rates in the intact cell is thus of value in the resolution of the important overall rate control of a given pathway. Thus enzymes showing rapid isotopic reversibility in the intact cell are unlikely candidates for control sites. Also the measurement of the actual flux through a given enzyme in the intact cell can be compared with the maximal rate of this enzyme in a cell free system. The case of pyruvate carboxylase has been discussed. While insufficient data is yet available to resolve the question, it may be possible by the isotopic techniques illustrated in this thesis to determine whether the mechanism of action of cyclic AMP involves a stimulation of PEP carboxykinase or an inhibition of pyruvate kinase. Certainly no technique or approach should be relied on exclusively. Indeed the major insights into regulation have come from studies of isolated enzymes. Studies of enzyme levels in cell free systems, of control effects in isolated mitochondria as well as studies with isolated cells or the intact animal all make contributions to the problem of regulation, and it will be an advantage to any investigator to broaden his range to include as many such areas as possible. 112 2. Future Application of Computer Models to More Complex Systems. In the in vitro system used in this paper it is possible to measure certain rates rather exactly using analytical methods and then to compare rates estimated by modeling techniques with these data. In more complex systems, e.g. in perfused whole organs or in vivo, analytical methods become less reliable or even unavailable. It will thus be necessary to attempt to measure most rates by isotopic techniques. It is hoped that the results obtained in the in vitro system, where several independent checks can be used to compare rates, will help to establish the validity of rate estimation in the more complex systems, where the data is necessarily more limited. An example of a possible simplified model will be described for use with the isolated perfused liver. In this system highly accurate measurement of oxygen uptake is difficult, and measurement of total COg production is not possible since a bicarbonate buffer is normally used. Thus measurement of the Krebs cycle will generally require an isotope method. A somewhat simplified model is shown in Scheme 4. Here pyruvate is the gluconeogenic substrate, while acetate-l-14C or acetate-2-uC is the labeled substrate. The model uses as simplifying 113 Scheme 4 . S i m p l i f i e d M odel f o r G lu c o n e o g e n e s is in t h e I s o l a t e d P e r f u s e d L iv e r G lu c o s e P y r u v a te CEL V GLU OP C - COOH V . PYR PK 0 I I V . LAC CH3 - C - COOH P2 PI CO PDH r +V GLU PK 01 CO , CO, P I +V GLU PK AC CH- C SCoA HOOC - CH 01 02 COOH 02 01 HOOC - CH = CH - COOH TCA CH, TCA HO CH, CO CO. 01 01 L a c t a t e COOH 1 A c - 1 - ,4C 0 A c - 2 - ,4C - COOH a l COOH 01 - COOH 01 114 approximations the assumptions of complete equilibration of the dicarboxylic acids via fumarase and also that the C02 fixed in the pyruvate carboxylase reaction is unlabeled, because of the large amount of unlabeled COg in the buffer. The algebraic representation and necessary solutions are shown in Appendix II. Analytical data on glucose and lactate formation and pyruvate utilization should be available. This again should provide an estimate of pyruvate oxidation, i.e. pyruvate dehydrogenase. Comparison of the specific activities of lactate and glu cose gives a measure of recycling of PEP to pyruvate. Using this data and the ratio of specific activities of C-l to C-5 in glutamate, obtained by degradation, one can calculate the rate of the Krebs cycle. When acetate-2-14C is the labeled substrate, to solve the equations derived in Appendix II requires data both from the degradation of glucose and glutamate. Appendix II also indicates a simple procedure for estimating the Krebs cycle in an in vivo experiment, when only a glucose or glutamate degradation may be available. Estimation of possible errors due to neglect of recycling are made. These simplified models can provide estimates of the major pathways of carbon flow. More accurate determina 115 tions of carbon flow can be made using complex models and the digital computer, with solution by trial and error or by an iteration procedure. We have extended the computer model of this paper to a 67 equation system, which now includes intra- and extramitochondrial pools of malate and oxalacetate, outflows into amino acids, the reversible enzyme reactions in the glycolytic region, glycogen synthesis and breakdown, as well as the system shown in Scheme 1. With sufficient experimental data this should eventually permit quantitative establishment of the major carbon flow in gluconeogenic organs in any in vitro or in vivo system. 3 . Possible Regulatory Significance of "Futile" Cycles. There are indications resulting from these studies that the activity of glycolytic enzymes are not completely inhibited during active gluconeogenesis. These results are contrary to general expectations (Atkinson, 1966); thus the preliminary nature of the evidence should be stressed. One possibility previously mentioned is that there are two populations of cells, one strictly gluconeo genic and the other glycolytic. There is suggestive evidence for reversibility in essentially a single cell population, but proof will require experiments with iso lated cell types. 116 The possible relationship of these apparently wasteful opposing pathways to control must then be considered. The ideal control mechanism is one that is highly efficient, accurate, and rapidly responsive to changing conditions. It is possible to overemphasize the importance of absolute efficiency. Thus one can estimate maximal rates for futile cycling in the kidney cortex at roughly the following: rate of pyruvate kinase = rate of triose conversion to glucose; rate of phosphofructokinase and hexokinase = one-half the net rate of glucose synthesis. Such recycling would increase the energy requirement for glucose formation about 50%• However the energy require ment for gluconeogenesis in the kidney cortex is only about one-fourth the total energy requirement of the tissue. Thus the inefficiency is about 12% on the basis of the total energy generated by these cells. One can speculate on possible advantages to the cell in the operation of opposing pathways in control systems. A sharper response to slight alterations in conditions would occur if opposite effects on the enzymes of the two pathways took place, as shown in a hypothetical example in Scheme 5A. Here, for example, one might imagine the opposite responses of pyruvate kinase and PEP carboxy- kinase to decreasing concentrations of PEP along the 117 Scheme 5A. H y p o t h e t i c a l F ig u r e Showing th e P o s s i b l e E f f e c t o f a Change o f a P a ra m eter on Enzymes C a t a ly z in g a R e a c t io n i n O p p o s ite D i r e c t i o n s G lu c o n e o g e n ic Enzyme 4 3 Glycolytic Enzyme 2 1 0 1 Net Flux 2 3 4 Scheme 5B. H y p o t h e t ic a l F ig u r e Showing R e q u ir e d Enzyme R esp o n se to G ive Same N et F lu x w it h No F u t i l e C y c lin g 4 Gluconeogenic Enzyme 3 Glycolytic Enzyme 2 1 0 1 Net Flux 2 3 4 KEY Absolute Enzyme Activity Net Response 118 abcissa. Ideally such a net result could also occur if the enzymes responded as in Scheme 5B, together with complete lack of wasteful opposing fluxes. However this idealized situation does not correspond to the typical sigmoid type curves found for most regulatory enzymes. Another possible advantage is the availability of more sites of control if two (or more) enzymes are involved in the net flux through a given step. The possible existence of opposing cycles in other pathways has not been extensively studied. For example, are the enzymes of glycogen synthesis and breakdown simul taneously active under certain conditions? There is little doubt that under drastic conditions pathways become essentially unidirectional. Thus under anoxia glycogen breakdown and glycolysis proceed in the absence of glycogen synthesis and gluconeogenesis; or in the presence of an uncoupler fatty acid oxidation proceeds in the absence of fatty acid synthesis. The question is what conditions prevail during the transitions which will occur periodically between net catabolism and net synthesis in a given pathway. In adipose tissue there is evidence that fatty acid oxidation is largely suppressed under conditions leading to high rates of fatty acid synthesis. However it might be pointed out that the isotopic techniques used 119 generally would not detect such opposing pathways if the newly synthesized fatty acids were reoxidized before mixing with the large unlabeled triglyceride pool. 4. Possible Special Roles of the Pyruvate, PEP Cycles. We have postulated earlier that a possible role of this type of cycle is in the transfer of reducing equi valents from the mitochondria to the cytosol. Such a transfer may be necessary under certain conditions to maintain a sufficiently high NADH/NAD+ ratio in the cytosol to permit efficient gluconeogenesis. There are indications that this cycle may operate more rapidly when the cytosolic NADH/NAD+ level is lower, e.g. when pyruvate rather than lactate is the substrate. However the cycle may also operate with aspartate outflow in which case the net result is the use of one high energy phosphate. To what extent each of these two types of the cycle operates would probably depend upon the NADH/NAD+ status in the cytosol. In these tissues glycolysis will not occur until flux through pyruvate kinase exceeds the reconversion of pyruvate to PEP. In view of the probable site of action of cyclic AMP at PEP carboxykinase, it is likely that in the liver hormones such as glucagon stimulate gluconeo genesis or repress glycolysis mainly their action in raising cyclic AMP levels, in turn affecting this enzyme. 120 On the other hand insulin may cause a lowering of the cyclic AMP level, inhibiting PEP carboxykinase and thereby promoting glycolysis by blocking the opposing pathways. This postulated effect of insulin in inhibiting the production of PEP and cytosolic NADH, both of which are also glycolytic products, might be involved in the intra cellular action of insulin which has been demonstrated in various tissues (Leonards and Landau, i9 6 0; Jungas, 1970; Halperin, 1970). 5- The Role of Glycolysis in Gluconeogenic Tissues. Net gluconeogenesis is thought to be the usual condition in the liver and kidney cortex. Net glycolysis is thought to occur only when blood sugar, insulin and liver glycogen is high and when levels of glucagon, catecholamines and of glucogenic substrates are low. While such an expectation is reasonable enough, our results suggest that measurement of apparent glycolysis in these tissues by isotopic means is probably not valid. Indeed uC-labeled glucose will produce “COg in these tissues even when high rates of gluconeogenesis are occuring. Careful analytical techniques should be used to measure glycolysis in these tissues. There are high levels of glycogen in the liver under an expected glycolytic situation, and high levels of glucose should also favor glycolysis. Thus 121 measurements of glycolysis will require careful deter mination of rather small differences between fairly large values. Possibly such studies have been carried out in the past.; however recent studies have used mainly isotopic techniques. In species such as the rat and the mouse the glycolytic capacity of the liver is rather limited and even if net glycolysis occurs under cettain conditions, there is at least some doubt as to whether this is ever a major pathway. In other species such as birds and possibly humans the glycolytic capacity of the liver is much higher. Also in these species there are indications that the main site of fatty acid synthesis may be in the liver. Liver glycolysis in these species might then become a major pathway in the energy metabolism of the body. However even in these species the extent of liver glycolysis should be verified by experiment rather than left to teleology. It is conceivable that the major carbon source for fatty acid synthesis could be lactate produced in extrahepatic tissues and transported to the liver via the blood, rather than pyruvatfe produced by glycolysis in the liver itself. 122 6. Control of Aspartate and Malate Efflux from the Mitochondrion. Krebs (19^7) has estimated the NADH/NAD+ ratios in the cytosol and mitochondria of the isolated rat liver during gluconeogenesis from lactate. The hundred-fold higher NADH/NAD+ ratio in the mitochondria suggests that the mitochondrial oxalacetate concentration (in that space which contains the malate dehydrogenase) is about hundred fold lower than that in the cytosol, if one assumes equili bration of malate between these compartments. Yet we know from studies with the inhibitor aminooxyacetate (Rognstad and Katz, 1970) that oxalacetate is transferred (via aspartate) from the mitochondria to the cytosol in gluco neogenesis from lactate. Either there is an unknown energy input step involved or the assumption of equal malate concentrations in these spaces is wrong. While the evidence is by no means conclusive, what we have shown in this paper suggests malate equilibration. It is plain that some type of restricted or one way permeability, energy input, or compartmentation must be involved, or else the active malate dehydrogenases and glutamate-oxalacetate transaminases in the mitochondria and cytosol would rapidly equalize the NADH/NAD+ ratio between these spaces. 125 To study the rate of aspartate inflow or outflow from the mitochondria, it will be necessary to develop a potent inhibitor for the active malate exchange reaction(s) or else a specific inhibitor of the active cytosolic malate dehydrogenase enzyme. The fluoromalates and chloromalates are possible candidates. To study the problem of possible oxalacetate compart- mentation in the mitochondria, more degradation data are required from experiments using specifically labeled substrates such as pyruvate-2-14C. Thus one can sample the oxalacetate which exists in the space containing citrate synthase by a total degradation of glutamate or citrate. Degradation of aspartate will sample the oxalacetate in the spaces containing both glutamate-oxalacetate transaminases. A possibility here is to use difluorooxalacetate, a potent glutamate-oxalacetate transaminase inhibitor (Kun et al., 1965)» which may by virtue of impermeability to the mitochondria be a specific inhibitor of the cytosolic isoenzyme. Degradation of glucose will sample the carbon pattern in mitochondrial malate. If the degradation patterns show significant differences, some evidence of compartmentation may be suggested. It should be stated that mitochondrial fractionation studies have not furnished evidence for oxalacetate compartmentation. Malate 124 dehydrogenase, citrate synthase, glutamate oxalacetate transaminase and pyruvate carboxylase are all found to fractionate in a similar manner, and have all been pre sumed to be in the mitochondrial matrix (Marco and Sols, 1970).. A model of possible oxalacetate compartmentation is shown in Scheme 6. This is based on the work and hypothe ses of Slater, Tager and coworkers (Slater and Hulsman, 1961; Tager, 1963; Oestreicher ejt al., 1969). Pyruvate carboxylase is assumed to be located in compartment I. 7. Distribution of the Dicarboxylic Acids Between the Mitochondria and the Cytosol. A number of studies have appeared in which attempts are made to calculate absolute concentrations of intermediates in the mitochondria and cytosol. The main assumption under lying these calculations is that certain enzymes are suffi ciently active to maintain near equilibrium between their reactants. Of course no enzyme is likely to maintain a re action exactly at equilibrium, but it is widely believed that very active enzymes such as malate dehydrogenase or glutamate oxalacetate transaminase keep their reactions close enough to equilibrium so that the assumption of actual equilibrium used in the calculations causes only minor errors. By this approach it has been found that the NADH/NAD+ couple is nearly two orders of magnitude more 125 Scheme 6. P o s s i b l e C om p artm en tation o f O x a la c e t a t e i n t h e M ito c h o n d r ia CYTOSOL MITOCHONDRIAL SPACE I I CYT A s p a r t a t e I I A c e t y l CoA A s p a r t a t e CYT I I I I O x a la c e t a t e O x a la c e t a t e C i t r a t e O x a la c e t a t e CYT P y r u v a te M a la te M a la te MITOCHONDRIAL SPACE I CYTOSOL 126 reduced in the mitochondria of liver than in the cytosol. The early apparent successes of this approach have prompted further extensions of it in efforts to calculated concentrations of metabolites in various compartments of of cells. These calculations however have sometimes involved the use of near-equilibrium approximations with enzymes which are not of great capacity, sometimes with enzymes which assay maximally in homogenates only at perhaps rates two to three-fold in excess of net rates of a pathway in the intact cell. It is not surprising then that these approaches can give discrepant results depending upon which assumptions are used. The approach still could be valuable if independent methods yielded values which agreed fairly well (at least within an order of magnitude). However, as shown below, discrepancies of more than two orders of magnitude can occur with the use of different assumptions. Anderson and Garfinkel (1971) have attempted to cal culate the cytosolic and mitochondrial concentrations of malate, oxalacetate, aspartate, glutamate and a-keto- glutarate. For this purpose they used the known total concentrations of these metabolites, the NADH/NAD+ ratios calculated from independent data, the known pyruvate and alanine concentrations, and then calculated the five 127 remaining unknowns using the five equations involving cytosolic and mitochondrial malate dehydrogenases and glutamate oxalacetate transaminases together with the cytosolic glutamate pyruvate transaminase. One result of these calculations is that the concentration of aspartate in the mitochondria is greater than that of mitochondrial 4 oxalacetate by a facor of about 4 x 10 . If one uses a similar approach and similar data, except that glutamate dehydrogenase rather than glutamate- pyruvate transaminase is used as the fifth near-equilibrium enzyme, one can calculate again a ratio of mitochondrial aspartate and oxalacetate concentrations, using the following equations: kG0T _ (asp)mlt(aKg)I ll i^ _ g g e< l (oaar^glut)”1* mit = 3.9 x 10"5 mM Using the data from Anderson and Garfinkel (1971), NADHmit = 1___ } NH4+ = 3 mM NAD+ 20.5 and rearranging the above equations gives K ™ = (aKg)mlt(MADH)m:Lt(BHa+) (glut)mlt(HAD+)mlt 128 g£L- (aKg)mit (glut)mit ■GOT eq _ NADH mit (NH4+)*it ^ 09-9. j GDH NAD+ 6.6 5 mM 20.5 250 5.9 x lCf^mM This is more than two orders of magnitude less than the value calculated using glutamate-pyruvate transaminase as the fifth equation. Anderson and Garfinkel recognize that their calculated results are consistent with a glutamate dehydrogenase near equilibrium only if it is assumed that this enzyme reacts essentially only with mitochondrial NADP+ rather than NAD+, as has been suggested in some laboratories (Klingenberg and Pette , 1962; Tager and Papa, 19^5). Krebs and Veech (1970) have reported that in the liver glutamate dehydrogenase is about twenty-fold higher in optimal activity than is glutamate pyruvate transaminase. In addition this transaminase has very high K^'s for alanine (Km=54mM) and glutamate (K^^mM). Since the total intracellular concentrations are considerably lower than the Kjjj's, the assumption of near equilibrium becomes more tenuous. Thus while the determination of the relative distribu- 129 tion of metabolites between the mitochondria and cytosol would be very valuable, present approaches and experimental techniques have not as yet been made reliable enough to warrant any firm conclusions to be drawn. 150 IV. Summary A quantitative study of the major pathways of carbon flow in the kidney cortex has been made. This tissue carries out active glucose synthesis from a wide variety of substrates. Using a computer solution of steady state models of kidney cortex metabolism, we estimated the rates of glucose synthesis, the tricarboxylic acid cycle, the so-called useless cycle" as well as the rates of many exchange reactions or transport systems. The rate of recycling of phosphoenolpyruvate to pyruvate was found to approach the rate of glucose synthesis. Evidence is presented showing that, under certain conditions, malate rather than aspartate outflow from the mitochondria may be involved in this recycling, the net result being an energy-driven transfer of reducing equivalents from the mitochondria to the cytosol. The entire gluconeogenic pathway was found to be isotopically reversible. A method is described which will permit determination of the rate of isotopic exchange at most steps of the pathway, providing further information on potential rate-limiting steps. The rate of malate transfer between the mitochondria and the cytosol was found to be rapid, suggesting that this is not a site of kinetic control. The yields of tritium in glucose and water were compared from substrates which form NADT in the cytosol or the mitochondria. The results suggest that mitochondrial NADT does not rapidly lose tritium to water by exchange reactions. This casts some doubt on the hypothesis that the respiratory chain exists in a near-equilibrium state. While tritium from succinate-2, j5-T appears in glucose, tritiated chlorosuccinates label only water, indicating that none of the tritium in glucose from succinate-2,5-T was derived from the succinic dehydrogenase reaction. Using aminooxyacetate as a transaminase inhibitor, it was shown that a malate-aspartate shuttle is probably not normally involved in transferring excess cytosolic reducing equivalents into the mitochondria. Numerous compounds were tested for their ability to inhibit at specific sites of the metabolic pathways studied. n-Butylmalonate, supposedly a specific inhibitor of the malate-phosphate transport system, apparently is inhibitory at other sites. y-Hydroxyglutamate inhibits glucose synthesis from pyruvate almost completely, while that from L-malate is unaffected. The high sensitivity of gluconeogenesis fi*om L-raalate to inhibition by low concen trations of dinitrophenol lends some support to the hypothesis that energy-linked removal of oxalacetate is 132 necessary to prevent inhibition of succinic dehydrogenase. Added cyclic AMP stimulates gluconeogenesis from L-malate and other intermediates of the Krebs cycle, but not from D-glycerate, D-glyceraldehyde or D-fructose, suggesting that PEP-carboxykinase is the site affected. Under the conditions used, cyclic AMP does not stimulate glucose formation from pyruvate, indicating that pyruvate carboxylase is the rate-limiting step in gluconeogenesis from this substrate. Treatments which tend to deplete cytosolic NADH affect gluconeogenesis from pyruvate and L-malate more critically than that from L-lactate. 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Appendix I. The model is shown in Scheme 1. Metabolic and isotopic steady state is assumed. Thus the amount of radioactivity entering a given species of carbon atom equals the amount of activity leaving that species. The specific activity of carbon 2 of the 2-14C labeled sub strate is set equal to 1. The rate of glucose synthesis (VGLU) is also set equal to 1 for purposes of computation. The following equations obtain when 2-uC-pyruvate is the labeled substrate: C-5 of pyruvate VPK m2 = (VGLU + VPK + VPDH + VLAc) P5 C-2 of pyruvate VPYr (1) + VPKm2 = (VGLU + VPK + VPDH + VLAC^ P2 C-l of pyruvate VPK ml = (VGLU + VPK + VPDH +VLAC^ P1 C-2 of acetyl CoA VPDH P3 = VTCA a2 144 C-l of acetyl CoA VPDH P2 “ VTCA al C-l and C-4 of pyruvate a-i + 0-z \ I 1 114 + m-, VTCA I 5 J + VPUM ( 5 I = ^VTCA + V5W fo C-2 and C-3 of fumarate fal + 0?\ / + ”gl VTCA \ 2 I + VFim I 2 I = (VTCA + VFDM^ fi C-4 of malate (VTCA + VFUM^ fo + VMDH °4 = (VTCA + VFUM + VMDH^ m4 C-3 of malate (VTCA + VFUM^ fi + VMDH °3 = (VTCA+ VFUM + VMDH^ m3 C-2 of malate (VTCA + VFUM^ fi + VMDH °2 = (VTCA + VFUM + VMDH^ ”2 C-l of malate (VTCA + VFUM^ fo + VMDH °1 * (VTCA + VFUM + VMDH^ ml C-4 of oxalacetate (VTCA+VMDH"VGLlfVPK ^ m4 + (VGLU + VPK^ C = (VTCA + VMDH^ °4 C-3 of oxalacetate (VTCA+VMDH“VGLU-VPk ) m3 + (VGLU + VPK^ P3 = ^VTCA+VMDH^ °3 C-2 of oxalacetate (VTCA+VMDH~VGLU-VPK) m2 + (VGLU + VPK^ P2 = ^VTCA+VMDH^ °2 C-l of oxalacetate (VTCA+VMDH~VGLU~VPK ^ ml + (VGLU + VPK^ P1 = ^VTCA+VMDH^ °1 co2 VTCA^°l+04) + (VGLU+VPk) m4 + VPDH P1 = ^ 2VTCA+VPDH^ C B. Appendix II. The model is shown in Scheme 4. This is a simplified scheme applicable to the isolated perfused liver or liver in vivo, and illustrates a method of obtaining estimates of the major pathways from limited data. Such preliminary estimates can also serve as starting points for more 146' accurate estimation using complex models and computer solution. We define X and Y as follows: X = Ze e and Y vtca vpyr+vfk V = V PK PYR (*> Also with pyruvate as the glucogenic substrate V = V + V + V PYR GLU LAC PDH Thus if analytical data are available, calcula-ted from VPBH = VPYR " ^VGLU + VLAC^‘ We again set the specific activity of the labeled carbon atom of the labeled substrate equal to one. 1. Case I: Acetate-l-14C as the labeled substrate. C-l of Acetyl CoA Va c*1) = ai vtca or ai = VAC VTCA C-l or C-4 of OAA (VGLU+VPk) + VTCA ~ = °1 ^ VGLU+VPK+VTCA ) 147 C-l of pyruvate VPK °1 “ P1 (VPK + VPYR^ P1 " Y ° 1 Y = On (specific activity of lactate) 1 /2 (specific activity of glucose) Solving these equations yields (VGUJ + VPk) ( 2 “ Y) + 2VTCA TCA or V, TCA <VGLU + Vptt) ( 2 " Y) PK; ra-^ AC-1-14C - 2 (eq. IA) Y is available from the specific activities of glucose and lactate as shown above. VGLU and VpYR are obtained by analysis. VpK is obtained from Y and VpYR. a^ is given Ol by the C-5/C-1 ratio in glutamate, obtained by degradation. 2. Case II: Acetate-2-14C as labeled substrate. C-l of Acetyl CoA VPDH P2 “ al VTCA al = X p2 148 C-2 of Acetyl CoA VPDH P2 + ^C^1) “ a2 VTCA a2 “ X P2 + V, VAC TCA C-l or C-4 of OAA P. I aa+o2 (VGLU+VPk) 21 + VTCa( 2 ) 0l (VGLU+VPK+VTCa) C-2 or C-3 of OAA (VGHJ+VPk) P2 + VTCA| — — -] = °2 (VGLU+VPK+VTCA^ C-l of pyruvate VPK °1 = P1 (VPYR + VPk) P1 Y °1 C-2 of pyruvate VPK °2 = P2 (VPYR + VPK^ P2 Y °2 Solving these equations yields r TCA Q \AC-2-C (1 +XY) °2/ (2"T) (VGLU+VPK^ + 2VTCA ' a A AC- ^ ‘C 1 / a, o - 2) or X = Y ^ 149 1 /2 (specific activity glucose) (specific activity lactate) V, TCA (2-Y) (VGLU + VpK) Oi AC-2-14C °2 (eq. IIIA) 1 + XY ~ 2 AC-2-“C Alternatively, if analyses are available VTCA = VPYR ~ (VGHJ + VLAC^ X 3. Case III: Acetate-l-uC or -2-14C as the labeled substrate in an in vivo system. Here we assume that only glucose or glutamate degradation data are available. In this case, to obtain preliminary estimates, it is necessary to set the recycling term Y equal to zero. Then eq. IA becomes V 17 a-j \ AC-1-“C"| pi) J 2 (eq. IIIA) and eq. IIA becomes 2 V glu fi o2 AC-2-14C V, TCA (eq. IIIB) 150 To estimate the amount of error the use of these equations may involve, assume the following set of data: VPYR = i-0* VGHJ = VLAC = VPDH “ °‘2> VPK 0 .4, Q ^ A C - 2-»C = 0m2^ Q i y c - 1- c = 5>57j X = 0.167, Y = 0.286. Prom either eq. IA or eq. IIA, VTCA = 1,2* Prom eq* IIIA> VTCA = 1-°> whj-ch is a 17# underestimation. Prom eq. IIIB, VTCA = 0.9* which is a 25$ underestimation.

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Creator Rognstad, Robert Alan (author)

Core Title Gluconeogenesis In The Kidney Cortex

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Degree Doctor of Philosophy

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Publisher University of Southern California (original), University of Southern California. Libraries (digital)

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Advisor Katz, Joseph (committee chair), Dunn, Arnold S. (committee member), Harding, Boyd W. (committee member), Shugaram, Peter M. (committee member)

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