GLUCOGENIC SUBSTRATE IN FASTING MAN
LEVELS
T. T. AOKI*, C. J. TOEWS, A. A. ROSSINI, N. B. RUDERMAN~"and G. F. CAMEL,JR. E. P. Joslin Research Laboratory and the Department of Medicine, Harvard Medical School at the Peter Bent Brigham Hospital, Boston, Massachusetts 02215
INTRODUCTION
AS MAN passes from the fed to the prolonged fasted state, his body makes a series of metabolic adjustments designed to maintain the fuel supply of brain, to conserve vital protein, and, in essence, to establish a metabolic near-steady state. These adjustments range from changes in levels of hormones and various substrates to changes in tissue sensitivity to these same hormones. Some of these adaptations have been reported in previous volumes (1-3) and elsewhere (4, 5). It was previously noted that the lactate/pyruvate ratio in blood does not change during the course of a prolonged fast whereas the fl-hydroxybutyrate/ acetoacetate ratio increases (5). Since these ratios in a given tissue are generally regarded as reflecting the redox states of the cytosol and mitochondria, respectively, it seemed reasonable to assume that their ratios in blood reflected a mean redox state of the cytosol and mitochondria in one or more tissues. If these assumptions are tenable, the mitochondria of these tissues in fasting man would be more reduced than in the fed state whereas the redox state of the cytosol would be unchanged. This conflicts with animal work (6), which suggests that both cell compartments become more reduced during the course of a fast. Recent modifications of existing enzymatic assays for lactate and pyruvate now permit a more rapid and accurate determination of these two substrates; therefore, the changes in levels of these fuels during the course of a fast were re-investigated. In this Symposium last year, we reported that the blood cells of postabsorptive man appear to play a passive role in amino acid metabolism (7). Following the ingestion of a meat meal, however, blood cells of both man (8) and dog (9-11) appear to function actively in amino acid transport. To date, * Fellow, The Medical Foundation, Boston, Massachusetts. I" Research Career Development Award, NIH. Supported in part by USPHS Grants AM-15191 and AM-05077. 329
330
T.T. AOKIet aL
there have been no reports concerning the role of the blood cells in amino acid metabolism during prolonged starvation. For this reason, using specific enzymatic assays we have also evaluated the role of the blood cells in the metabolism of alanine, glutamine and glutamate in fasting man, particularly since these substrates are closely related to levels of pyruvate.
M A T E R I A L S A N D METHODS
Twelve obese (178 + 6~o, Mean -4- S.E.M. ideal body weight, Metropolitan Life Tables) females ranging in age from 21 to 50 yr (28 4- 2, Mean 4- S.E.M.) were admitted to the Clinical Research Center of the Peter Bent Brigham Hospital. Prior to any investigational procedures, informed consent was obtained from each subject. The patients were maintained on a balanced 2500 calorie diet for 4-7 days and, immediately following the equilibration period, were fasted for 6-8 weeks. During the fast they were urged to drink at least 2000 ml of water each day and were required to ingest one multivitamin capsule together with small quantities (17 mEq) of NaCI and KCI. They were also constantly exhorted to remain physically active. Venous blood was withdrawn without staffs from an antecubital vein after the subjects had been recumbent for at least 1 hr, on days 0, 3, 5, 7, 10, 14, 21 and 28 of the fast. When arterial blood was needed, it was withdrawn via a needle inserted percutaneously into either the radial or brachial artery. The blood was immediately deproteinized with an equal volume of ice-cold 30yo perchloric acid. Lactate, pyruvate, a-ketoglutarate, alanine, glutamate and glutamine were assayed enzymatically on an American Instruments Company Fluorometer Model 4-7103 equipped with a waterjacketed sample changer and a mercury vapor lamp assembly. Details of the glutamine and glutamate assays have been previously reported (8) while the rest of the assays were all modifications of methods described in the new Bergmeyer text (12). Of interest, stability and recovery studies have shown that pyruvate and a-ketoglutarate are stable for at least 4--6 weeks when stored as an acid filtrate at --20°C.
RESULTS
During a fast, the concentrations of pyruvate in both arterial and venous blood declined sharply (venous > arterial) (Fig. 1); however, there was no significant change in blood lactate. As a result, the lactate/pyruvate ratios in both arterial and venous blood were increased (venous > arterial) by day 7, and remained elevated for the durat'ion of the fast. The concentration of a-ketoglutarate declined significantly in venous but not in arterial blood. There was a steady and significant decline in circulating levels of glutamine
331
SUBSTRATE LEVELS IN FASTING MAN .SUBSTRATE LEVELS 80(
SUSSTRATE RAT/08
LACTATE ( n = 5 )
LACTATE { n , [ 4 ) _ PYRUVATE
30
arterial). Thus, the mean cytosolic and mitochondrial redox states appear related, and they are both more reduced in fasting man. Measurements of arterio-venous differences across various organs suggest that muscle is more reduced than brain, kidney, or splanchnic bed and that it, in large part, accounts for the observed changes in blood. This more reduced state of muscle would favor the diminution of the irreversible oxidative decarboxylation of the ketoacid derivatives of the branchedchain amino acids. Whether this is the mechanism by which protein catabolism in muscle is diminished during a prolonged fast remains to be determined. ACKNOWLEDGEMENTS
We wish to thank Mines Dzidra Rumba, Patricia Hatch, Velta Ramolins, and Adaeie Allen, without whose help this investigation could not have been performed.
REFERENCES 1. P. F~uo, E. B. M~LLSS, O. E. OWEN and G. F. CAHILL, JR., Role of substrate in the regulation of hepatic gluconcogcnesis in fasting man, Advances in Enzyme Regulation 7, 41-46 (1969). 2. E. B. M~LLSS, T. T. AOg& P. FEUG, T. POZT~SKYand G. F. CAHILL, JR., Hormones and substrates in the regulation of gluconeogenesis in fasting man, Advances in Enzyme Regulation 8, 3-11 0970). A.E,R.--M
336
T.T. AOKI et aL
3. T. T. AOKI,W. A. MULLERand G. F. CAHILL,JR., Hormonal regulation of glutamine metabolism in fasting man, Advances in Enzyme Regulation 10, 145-151 (1972). 4. P. FELIG, O. E. OWEN, J. WAHRENand G. F. CAHILL,JR., Amino acid metabolism during prolonged starvation, J. Clin. Investig. 48, 584-594 (1969). 5. G. F. CAHILL,JR., M. G. HERRERA,A. P. MORGAN,J. S. SOELDNER,J. STEINKE,P. L. LEvY, G. A. REICrlARD,JR. and D. M. KIP~S, Hormone-fuel interrelationships during fasting, J. Clin. Investig. 45, 1751-1769 (1966). 6. H. A. Ka~ns, The redox state of nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver, Advances in Enzyme Regulation 5, 409-434 (1967). 7. T. T. AOKI, i . F. BRENNAN,W. A. MOLLERand G. F. CAHILL,JR., Amino acid levels across normal forearm muscle: whole blood vs. plasma, Advances in Enzyme Regulation 12, 3-11 (1974). 8. T. T. AOKI, W. A. MOLLER, M. e. BRENNANand G. F. CAmLL, JR., Blood cell and plasma amino acid levels across forearm muscle during a protein meal, Diabetes 22, 768-775 (1973). 9. D. H. ELWYN,Distribution of amino acids between plasma and red blood cells in the dog, Federation Proc. 25, 854-961 (1966). 10. D. O. ELWYN,H. C. PARIKHand W. C. SHOEMAKER,Amino acid movements between gut, liver, and periphery in unanesthetized dogs, Am. J. Physiol. 215,1260-1275 (1968). 11. D. H. ELWYN, W. J. LAUNDER,H. C. PARmH and E. M. WISE, Roles of plasma and erythrocytes in interorgan transport of amino acids in dogs, Am. J. Physiol. 222, 1333-1342 (1972). 12. H. U. BERGMEYER,Methods of Enzymatic Analysis, Academic Press, New York and London (1974), 2nd Eng. edn. 13. O. E. OWEN,A. P. MORGAN,H. G. KEMP,J. i . SULLIVAN,i . G. HERRERAand G. F. CAHILL,JR., Brain metabolism during fasting, J. Clin. Investig. 46, 1589-1595 (1967). 14. O. E. OWEN, P. FELtG, A. P. MORGAN,J. WAHRENand G. F. CAHILL,JR., Liver and kidney metabolism during prolonged starvation, J. Clin. Investig. 48, 574-583 (1969). 15. O. E. OWENand G. A. REICHARO,JR., Human forearm metabolism during progressive starvation, ./. Clin. Investig. 50, 1536-1545 (1971). 16. A. J. GARBER,P. H. MENZ~L, G. BODENand O. E. OWEN, Hepatic ketogenesis and gluconeogenesis in humans, J. Clin. lnvestig. 54, 981-989 (1974). 17. D. G. SAPIR,O. E. OWEN,T. POZErSKYand M. WALSER,Nitrogen sparing induced by a mixture of essential amino acids given chiefly as their keto-anaiogues during prolonged starvation in obese subjects, J. Clin. Investig. 54, 974-980 (1974).
LEVELS
T. T. AOKI*, C. J. TOEWS, A. A. ROSSINI, N. B. RUDERMAN~"and G. F. CAMEL,JR. E. P. Joslin Research Laboratory and the Department of Medicine, Harvard Medical School at the Peter Bent Brigham Hospital, Boston, Massachusetts 02215
INTRODUCTION
AS MAN passes from the fed to the prolonged fasted state, his body makes a series of metabolic adjustments designed to maintain the fuel supply of brain, to conserve vital protein, and, in essence, to establish a metabolic near-steady state. These adjustments range from changes in levels of hormones and various substrates to changes in tissue sensitivity to these same hormones. Some of these adaptations have been reported in previous volumes (1-3) and elsewhere (4, 5). It was previously noted that the lactate/pyruvate ratio in blood does not change during the course of a prolonged fast whereas the fl-hydroxybutyrate/ acetoacetate ratio increases (5). Since these ratios in a given tissue are generally regarded as reflecting the redox states of the cytosol and mitochondria, respectively, it seemed reasonable to assume that their ratios in blood reflected a mean redox state of the cytosol and mitochondria in one or more tissues. If these assumptions are tenable, the mitochondria of these tissues in fasting man would be more reduced than in the fed state whereas the redox state of the cytosol would be unchanged. This conflicts with animal work (6), which suggests that both cell compartments become more reduced during the course of a fast. Recent modifications of existing enzymatic assays for lactate and pyruvate now permit a more rapid and accurate determination of these two substrates; therefore, the changes in levels of these fuels during the course of a fast were re-investigated. In this Symposium last year, we reported that the blood cells of postabsorptive man appear to play a passive role in amino acid metabolism (7). Following the ingestion of a meat meal, however, blood cells of both man (8) and dog (9-11) appear to function actively in amino acid transport. To date, * Fellow, The Medical Foundation, Boston, Massachusetts. I" Research Career Development Award, NIH. Supported in part by USPHS Grants AM-15191 and AM-05077. 329
330
T.T. AOKIet aL
there have been no reports concerning the role of the blood cells in amino acid metabolism during prolonged starvation. For this reason, using specific enzymatic assays we have also evaluated the role of the blood cells in the metabolism of alanine, glutamine and glutamate in fasting man, particularly since these substrates are closely related to levels of pyruvate.
M A T E R I A L S A N D METHODS
Twelve obese (178 + 6~o, Mean -4- S.E.M. ideal body weight, Metropolitan Life Tables) females ranging in age from 21 to 50 yr (28 4- 2, Mean 4- S.E.M.) were admitted to the Clinical Research Center of the Peter Bent Brigham Hospital. Prior to any investigational procedures, informed consent was obtained from each subject. The patients were maintained on a balanced 2500 calorie diet for 4-7 days and, immediately following the equilibration period, were fasted for 6-8 weeks. During the fast they were urged to drink at least 2000 ml of water each day and were required to ingest one multivitamin capsule together with small quantities (17 mEq) of NaCI and KCI. They were also constantly exhorted to remain physically active. Venous blood was withdrawn without staffs from an antecubital vein after the subjects had been recumbent for at least 1 hr, on days 0, 3, 5, 7, 10, 14, 21 and 28 of the fast. When arterial blood was needed, it was withdrawn via a needle inserted percutaneously into either the radial or brachial artery. The blood was immediately deproteinized with an equal volume of ice-cold 30yo perchloric acid. Lactate, pyruvate, a-ketoglutarate, alanine, glutamate and glutamine were assayed enzymatically on an American Instruments Company Fluorometer Model 4-7103 equipped with a waterjacketed sample changer and a mercury vapor lamp assembly. Details of the glutamine and glutamate assays have been previously reported (8) while the rest of the assays were all modifications of methods described in the new Bergmeyer text (12). Of interest, stability and recovery studies have shown that pyruvate and a-ketoglutarate are stable for at least 4--6 weeks when stored as an acid filtrate at --20°C.
RESULTS
During a fast, the concentrations of pyruvate in both arterial and venous blood declined sharply (venous > arterial) (Fig. 1); however, there was no significant change in blood lactate. As a result, the lactate/pyruvate ratios in both arterial and venous blood were increased (venous > arterial) by day 7, and remained elevated for the durat'ion of the fast. The concentration of a-ketoglutarate declined significantly in venous but not in arterial blood. There was a steady and significant decline in circulating levels of glutamine
331
SUBSTRATE LEVELS IN FASTING MAN .SUBSTRATE LEVELS 80(
SUSSTRATE RAT/08
LACTATE ( n = 5 )
LACTATE { n , [ 4 ) _ PYRUVATE
30
arterial). Thus, the mean cytosolic and mitochondrial redox states appear related, and they are both more reduced in fasting man. Measurements of arterio-venous differences across various organs suggest that muscle is more reduced than brain, kidney, or splanchnic bed and that it, in large part, accounts for the observed changes in blood. This more reduced state of muscle would favor the diminution of the irreversible oxidative decarboxylation of the ketoacid derivatives of the branchedchain amino acids. Whether this is the mechanism by which protein catabolism in muscle is diminished during a prolonged fast remains to be determined. ACKNOWLEDGEMENTS
We wish to thank Mines Dzidra Rumba, Patricia Hatch, Velta Ramolins, and Adaeie Allen, without whose help this investigation could not have been performed.
REFERENCES 1. P. F~uo, E. B. M~LLSS, O. E. OWEN and G. F. CAHILL, JR., Role of substrate in the regulation of hepatic gluconcogcnesis in fasting man, Advances in Enzyme Regulation 7, 41-46 (1969). 2. E. B. M~LLSS, T. T. AOg& P. FEUG, T. POZT~SKYand G. F. CAHILL, JR., Hormones and substrates in the regulation of gluconeogenesis in fasting man, Advances in Enzyme Regulation 8, 3-11 0970). A.E,R.--M
336
T.T. AOKI et aL
3. T. T. AOKI,W. A. MULLERand G. F. CAHILL,JR., Hormonal regulation of glutamine metabolism in fasting man, Advances in Enzyme Regulation 10, 145-151 (1972). 4. P. FELIG, O. E. OWEN, J. WAHRENand G. F. CAHILL,JR., Amino acid metabolism during prolonged starvation, J. Clin. Investig. 48, 584-594 (1969). 5. G. F. CAHILL,JR., M. G. HERRERA,A. P. MORGAN,J. S. SOELDNER,J. STEINKE,P. L. LEvY, G. A. REICrlARD,JR. and D. M. KIP~S, Hormone-fuel interrelationships during fasting, J. Clin. Investig. 45, 1751-1769 (1966). 6. H. A. Ka~ns, The redox state of nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver, Advances in Enzyme Regulation 5, 409-434 (1967). 7. T. T. AOKI, i . F. BRENNAN,W. A. MOLLERand G. F. CAHILL,JR., Amino acid levels across normal forearm muscle: whole blood vs. plasma, Advances in Enzyme Regulation 12, 3-11 (1974). 8. T. T. AOKI, W. A. MOLLER, M. e. BRENNANand G. F. CAmLL, JR., Blood cell and plasma amino acid levels across forearm muscle during a protein meal, Diabetes 22, 768-775 (1973). 9. D. H. ELWYN,Distribution of amino acids between plasma and red blood cells in the dog, Federation Proc. 25, 854-961 (1966). 10. D. O. ELWYN,H. C. PARIKHand W. C. SHOEMAKER,Amino acid movements between gut, liver, and periphery in unanesthetized dogs, Am. J. Physiol. 215,1260-1275 (1968). 11. D. H. ELWYN, W. J. LAUNDER,H. C. PARmH and E. M. WISE, Roles of plasma and erythrocytes in interorgan transport of amino acids in dogs, Am. J. Physiol. 222, 1333-1342 (1972). 12. H. U. BERGMEYER,Methods of Enzymatic Analysis, Academic Press, New York and London (1974), 2nd Eng. edn. 13. O. E. OWEN,A. P. MORGAN,H. G. KEMP,J. i . SULLIVAN,i . G. HERRERAand G. F. CAHILL,JR., Brain metabolism during fasting, J. Clin. Investig. 46, 1589-1595 (1967). 14. O. E. OWEN, P. FELtG, A. P. MORGAN,J. WAHRENand G. F. CAHILL,JR., Liver and kidney metabolism during prolonged starvation, J. Clin. Investig. 48, 574-583 (1969). 15. O. E. OWENand G. A. REICHARO,JR., Human forearm metabolism during progressive starvation, ./. Clin. Investig. 50, 1536-1545 (1971). 16. A. J. GARBER,P. H. MENZ~L, G. BODENand O. E. OWEN, Hepatic ketogenesis and gluconeogenesis in humans, J. Clin. lnvestig. 54, 981-989 (1974). 17. D. G. SAPIR,O. E. OWEN,T. POZErSKYand M. WALSER,Nitrogen sparing induced by a mixture of essential amino acids given chiefly as their keto-anaiogues during prolonged starvation in obese subjects, J. Clin. Investig. 54, 974-980 (1974).
