Amino Acid Metabolism in the Regulation of Glucone ogenesis in Man1 2. T HE FACTORS INFLUENCING gluconeogenesis

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1 THE AMERICAN JOURNAL OF CLINICAL NUTRITION Vol. 23, No. 7, July, 1970, pp Printedin U.S.A. Amino Acid Metabolism in the Regulation of Glucone ogenesis in Man1 2 PHILIP FELIG,3 M.D., ERROL MARLISS,4 M.D., THOMAS POZEFSKY,5 M.D., AND T HE FACTORS INFLUENCING gluconeogenesis have been the subject of extensive investigative efforts in the past decade. A variety of hormones, enzymes, and cofactons have been shown to be of regulatory significance, primarily on the basis of in vitro studies involving experimental animals. In the present discussion we shall review some recent studies in intact man, demonstrating time importance of alterations in time availability and metabolism of endogenous amino acids in the regulation of gluconeogenesis. Altimough it is recognized that several noncarbohydrate moieties, sucim as lactate, pyruvate, and glycerol, may also serve as glucose precursors, gluconeo- 1 From the Joslin Diabetes Foundation, Inc., Dcpartment of Medicine, Harvard Medical School, and Peter Bent Brigham Hospital, Boston, Massachusetts. 2 Supported in part by Public Health Service Grants AM-05077, AM-09584, AM-09748, and FR-31, and the Adler Foundation, Inc., Rye, New York. 3 Assistant Professor of Medicine, Yale University School of Medicine (formerly Research Fellow, Elliott P. Joslin Research Laboratory, Department of Medicine, Harvard Medical School and Peter Bent Brigham Hospital). Recipient, Public Health Service Special Postdoctoral Fellowship 5 F3-AM Research Fellow, Elliott P. Joslin Research Laboratory, Department of Medicine, Haryard Medical School and Peter Bent Brigham Hospital. Recipient, Medical Research Council of Canada Fellowship. Research Fellow, Elliott P. Joslin Research Laboratory, Department of Medicine, Harvard Medical School and Peter Bent Brigham Hospital. Recipient, Public Health Service Special Postdoctoral Fellowship F-03-AM #{176} Associate Professor of Medicine, Harvard Medical School; Physician. Peter Bent Brigham Hospital; and Director, Elliott P. Joslin Research Laboratory. GEORGE F. CAHILL, JR.,#{176} M.D. genesis will be considered in the context of conversion of body protein to glucose in this discussion. Accordingly, time term substrate will be restricted to precursor amino acids. The role of endogenous amino acids will be examined in two situations in which modulation of gluconeogenesis is readily apparent: prolonged starvation and following infusion of glucose. Time thesis to be developed is that in the former circumstance diminished substrate availability consequent to decreased amino acid release from penipimeral protein stores is of paramount importance as a regulatory facton. In contrast, time inhibition in gluconeogenesis induced by glucose infusion (a situation analogous to feasting as opposed to fasting ) is primarily dependent on alterations in hepatic gluconeogenic mechanisms with substrate presentation renmaining adequate. During periods of starvation, the obligate requirements of certain tissues for some level of glucose consumption, coupled with the nminimal body stores of preformed carbohydrate, necessitate continuous formation of new glucose from endogenous precursors (1). On the other hand!, time potentially fatal consequences of dissolution of one-third to one-half time body protein stores underscore time need for conservation of protein in circumstances of calorie deprivation (2). Although one may dispute the teleologic basis for the limitation of gluconeogenesis ilm starvation, recent studies have clearly d!ocumented a marked decrease in imepatic glucose output after a 5- to 6-week fast (Table I). Whereas 986

2 Amino Acid Metabolism and Gluconeogenesis 987 in the postabsorptive state glucose is produced at a rate of g/day (3-5), total glucose formation is reduced to g/day following a prolonged fast (6). Moreover, the kidney assumes a quantitatively greater role during starvation by contributing 45% of the total output (6). Reflecting this diminution in glucose production from endogenous precursors is the progressive decrease in urine nitrogen excretion to ultimate levels of 3-5 g/day, representing a daily dissolution of g protein (7). In considering time possible role of substrate in effecting this limitation of gluconeogenesis in starvation, we may begin by examining the concentrations at which various amino acids circulate in plasma and thus are made available to the liver. It has been well established in the last 15 years that amino acids in postabsorptive man are normally present in plasma in a predictable pattern, with the concentration of individual amino acids varying from levels as high as 350 to 600 moles/liter for alanine and glutamine, respectively, to less than 25 moles/liter for methionine (Table II). These differences in plasma concentration are further accentuated when one studies, by means of simultaneous arterial and hepatic vein catheterization, the relative rates at which individual amino acids are extracted by time splanchnic circulation (Fig. 1). Of 20 amino acids measured, a TABLE Glucogenesis in man in the postabsorptive state and after a prolonged fasto I Values are mean ± SE. A-HI LMde/L 4 2 ID B 60 TABLE Plasma concentrations of amino acids in normal postabsorptive subjects (n = 10) Amino acid mole/liter Alanine 344 ± 29 Glycine 215 ± 8 Valine 212 ± 8 Proline 175 ± 13 Lysine 164 ± 9 Threonine 134 ± 10 Leucine 112 ± 4 Serine 109 ± 7 HaIf-cystine 92 ± 5 Histidine 73 ± 4 Arginine 69 ± 8 Ornithine 67 ± 9 Isoleucine 59 ± 2 Tyrosine 54 ± 4 Taurine 51 ± 3 Phenylalanine 49 ± 2 Tryptophan 39 ± 6 Citrulline 30 ± 3 Methionine 24 ± 1 a-nh2 butyrate 20 ± 2 Glutamine Glutamate significant exchange was demonstrable only for those amino acids shown (8). Of particular interest is the fact that uptake of alanine, exceeding that of all other amino acids, accounts for approximately 50% of the total net amino acid consumption. T 0 OVERNIGHT M$TlPO$T-G8$CTlVtI B 5-6 WEEXS 51405*11014 II Glucose production 41 II Rate, g/day Postabsorptive to 6-Week fast 86 a Data from references 6 and 7. Source Liver Kidney >90% <10% 55% 45% 0 SEP THR PRO vr TYR PHt MET FIG. 1. Splanchnic amino acid extraction in subjects in the postabsorptive state and after prolonged (5-6 weeks) starvation. A-HV = arteriohepatic yenous difference. Based on the data of Felig et al. (8). Or

3 DAYS 988 Felig et al. A-Iv I PR0I066D ALALYS SLY PRO T6 AR VAL L(U as T 5(6 FIG. 2. Amino acid balance across forearm muscle tissue in subjects in the postabsorptive state and after 4-6 weeks starvation. A-Dy = arterio-deep venous difference. Based on the data of Felig et al. (9). Colnplementing timis preferential hepatic utilization of alanine is the pattern of amino acid release from muscle tissue (9). As indicated in Fig. 2, examination of the deep venous effluent, draining primarily the muscle of the human forearm, reveals that alanine is released to a greater extent than all other amino acids. There is, in fact, remarkably good agreement between the relative rates of hepatic uptake and peripimeral release of the individual amino acids. Thus, while the enzymatic potential for conversion to glucose is available for all amino acids except leucine (10), alanine occupies quantitatively the most important position in terms of endogenous substrate excharmge between muscle (the major peripheral reservoir of body protein) and liver. In prolonged! starvation, however, measurement of total alpha-amino nitrogen reveals a relatively modest decline, which assurnes statistical significance only after 2 weeks of fastiimg (Fig. 3). However, analysis of the specific amino acids indicates that time indlividual components respond in varying magnitudes and directions. The timree primary patterns of response (8) are depicted in Fig. 3 by single representative amino acids. Specifically, a transient rise as evid!enced by valine, the progressive dedine demonstrated by alanine, and finally a delayed increase as manifested by glycine, are observed in subjects fasted 5-6 weeks (8). 51:05 FIG. 3. Influence of prolonged starvation on concentrations of total a-amino nitrogen and selected amino acids (8). The transient hyperaminoacidemia seen in time first 7-10 days of starvation involves not only valine, but eacim of time brancimed!- chain amino acids plus methionine and alpha-aminobutyrate (8). Since, as will be shown later, these amino acids are particularly sensitive to altered systemic levels of insulin, it is postulated that this early transient hyperaminoacidemia is probably related to the concurrent rapid! fall in serum insulin, characteristic of starvation. As anticipated from time cimanges in alpha-amino nitrogen, most amino acids ultimately decline in starvation. Time extent of this decline, imowever, is quite variable. Timus, altimougim 13 of 20 amino acids decrease in concentration, the magnitud!e of timis diminution in both relative and absolute terms is greatest for alanine (8). In view of the cardinal role of alanine in the flux of substrate from peripimery to liver in postabsorptive man, the importance of examining splancimnic amiimo acid! exchange after a prolonged! fast is read!ily apparent. As shown in Fig. 1, after a 5- to 6- week fast, alanine remains time primary amino acid extracted by the liver. However, in concert witim the decreased! rate of giuconeogenesis, the rate of hepatic alanine uptake is decreased by 50%, a decline timat cannot be ascribed to altered flow. A striking diminution in glycine uptake is also demonstrated.

4 Amino Acid Metabolism and Gluconeogenesis 989 Wlmen one examines the cimanges in hepatic alanine metabolism in greater detail, it is apparent that early in starvation timere is a primary stimulation in alanine uptake, as evidenced! by an augmented extraction ratio (8). On the other imand, the decline in alanine uptake denmonstrated after a 5- to 6-week fast is not due to a diminisimed extraction ratio, but is consequent solely to the decreased arterial levels (8). Tlmerefore, time data suggest that time ra)id!ity and magnitude of the fall in plasnma alanine in starvation are due to preferential, and! initially augmented, utilization of timis amino acid by time liver. Furtimermore, the low alanine concentration is of particular importance in that it, in turn, serves to limit the rate of precursor uptake by time liver. Accordingly, maintenance of imypoalaninemia is a crucial step in time regulatory mecimalmism whereby protein conversion to glucose is minimized. Supporting timis conclusion is time blood! glucose 1-esponse to infusion of alalmine in fasted subjects. Thus, a significalmt increment in blood! glucose was readily denmonstrable after alanine administration to subjects fasted 3-4 weeks (1 1). Since these data suggested rather than proved timat time administered alanine raised time blood glucose by acting as substrate for gluconeogenesis, subjects were given 50 Ci of uniformly labeled! 14C alanine along with 10 g unlabeled! alanine. As simowlm in Fig. 4, the level of 14C incorporation into glucose aftei a prolonged fast was equal to or sliglmtly greater than that noted in the postabsorptive state. Furthermore, the 25-35% peak level of alanine incorporation into glucose in the fasted state agreed closely with the rate of conversion estimated froln time maximum rise in blood glucose concentration of about 14 mg/ 100 ml, confirming the fact that alanine raised the blood sugar level by functioning as substrate for gluconeogenesis (12). These observations, therefore, substantiate the I000 4 C Incorporation Into Blood Glucose 600 DPM/cc M.S ,? % Administered C, 0, Recovered as,., Blood Glucose :, _o TIME (MINUTES) Fic. 4. Incorporation of C into blood glucose from L-alanine 14C in a subject studied in the postabsorptive state (solid line) and after 41 days of starvation (dotted line, open circles). Ten grams of unlabeled L-alanine and 50 4C-L-alanine (uniformly labeled) were administered intravenously over a 3-mm period. Blood samples were obtained at the intervals shown after the completion of the infusion (12). conclusion that time restraining influence in starvation is not consequent to intrinsic or imormonally induced cimanges in time liver, but is, in fact, substrate modulated. Returning to time pi-oblem of consei-vation of body protein in fasting man, we note that a decrease in plasma concentiation of alanine and otimer amino acidis does not necessarily imply a red!uction in their mobilization from body protein stores. Conceivably, increased! turnover at some extrahepatic site could result in augmented amino acid! release despite diminished splanchnic extraction. To resolve this question, forearm amino acid exchange was studied in time fasted group and compared with the values seen in postabsorptive subjects (Fig. 2). After a 4- to 6-week fast, mobilization of alanine from muscle fell by 75% (9). Furthermore, for each of those amino acids for which a decrease in circulating systemic levels was

5 990 Felig Ct al. noted, muscle output declined significantly, falling in several instances to levels that did not differ significantly from zero. Thus, it is clear that at least with regard to muscle, mobilization of endogenous amino acids is markedly curtailed by prolonged fasting (9). The third and final pattern of plasma amino acid response to starvation is that of a delayed increase demonstrated by glycine, timreonine, and, to a lesser extent, serine (8). The increase in glycine can be accounted for by the striking decline in its uptake by the liver as noted previously. In adidition, it is the only amino acid for which a significant decline in muscle release could not be demonstrated after a prolonged fast (Fig. 2). The pertinence of the hyperglycemia to a consideration of the regulation of gluconeogenesis derives from the fact that not only the rate, but also the site of gluconeogenesis is altered in starvation with the kidney assuming a quantitatively significant role. In this regard, it is noteworthy that glycine is one of time few amino acids, in addition to giutamine, consistently extracted by the renal circulation (13). Moreover, Pitts and Pilkington (14) have demonstrated that renal uptake and incorporation of glycine into ammonia increase pan pasu as systemic glycine levels are elevated. Consistent with their findings is the four- to six-fold increase in glycine uptake by time kidney observed after prolonged fasting (8). Since urinary excretion of glycine is not increased in starvation (8), it is likely that the glycine extracted is being utilized to provide the carbon skeletons and amino groups necessary for the augmented level of renal gluconeogenesis and ammoniagenesis characteristic of starvation. To consider gluconeogenesis by the liver once again, it is well known that insulin effectively lowers plasma amino acid levels ( 15). One may question: does insulin reduce hepatic gluconeogenesis in a manner analogous to timat observed in prolonged starvation, namely, by diminishing substrate availability? To answer this question, one must examine time effects of insulin on systemic levels of specific amino acids rather than on the group as a whole, particularly in view of time marked variation in the rates at which individual plasma amino acids are extracted by the splanchnic circulation. After stiniulation of endogenous insulin secretion by glucose, one cannot demonstrate a significant decline in the plasma levels of all amino acids. As mentioned previously, the concentrations of the branched-chain amino acids and tyrosine, and phenylalanine are most sensitive to fluxes in systemic insulin levels (16). On the other hand, alanine, which is the primary gluconeogenic substrate extracted by the liver, is not significantly reduced; in fact, in 5 of 1 1 patients alanine was increased after glucose infusion (16). To examine more directly the influence of insulin on hepatic anmino acid uptake, simultaneous arterial and imepatic venous samples were obtained before and after administration of glucose (12). The glucose was infused at a rate and concentration that resulted in reversal of the normal, basal glucose output by the liver to an uptake of 21 mg/ 100 ml. Peripheral serunm insulin levels rose appropriately. Following glucose administration, a 25% decrease in total amino acid uptake involving alanine, primarily, was noted (12). This observation is in good agreement with the 25% diminution in urea production induced by insulin in perfused liver (17). Particularly noteworthy is the fact that arterial alanine levels showed no significant change at a time when splanchnic alanine uptake was significantly reduced! (12). Thus, insulinor glucose-mediated effects, or both, on gluconeogenesis could! imot be ascribed to

6 Amino Acid Metabolism and Gluconeogenesis 991 SUBSTRATE AND HORMONAL INFLUENCES ON GLUCONEOGENESIS IN MAN LIVER MUSCLE AT ALANINE GLUCOSE OTHER PLASMA AA S si?es of reguiohj [1oPosed Fn.. Postulated substrate and hormonal influences on gluconeogenesis in man. diminished substrate availability. Despite all increase in arterial lactate concentratioim, lmepatic lactate extraction was reduced to insignificant levels (12). Thus, it is likely tlmat time enzymatic site of blockade of gluconeogenesis induced by glucose infusioim is at a step beyond transamination. Althouglm time precise locus cannot be defined from these data, it is clear that reductioim in splanchnic alanine uptake ind!uced! by glucose is not consequent to altered! peripheral alanine release. On the otimer imand, to the extent that insulin does block release of other amino acids by flhllsc!c tissue (18) contributing to gluconeogenesis, a substrate effect cannot be totally excluded. Iii summary (Fig. 5), data have been preseimted demonstrating that in postabsorptive man there is a net flux of amino acids from muscle to liver. Alanine is qua imtitatively time primary gluconeogenic plcctlrsor participating in this transfer of endogenous substrate. In circumstances in which conservation of body protein is vital to man s survival, namely in prolonged fasting, limitation of time conversion of body protein to glucose is achieved by means of decreased! substrate presentation to the liver in the face of unimpaired imepatic mechanisms of gluconeogenesis. In contrast, the restriction of hepatic glucose production engendered by glucose administration, found in feasting ratimer than fasting man, has been shown to depend on a primary blockade of precursor uptake by time liver rather than altered amino acid availability. REFERENCES 1. FELIG, P., 0. E. OwEN, A. P. MORGAN AND G. F. CAHILL, JR. Utilization of metabolic fuels in obese subjects. Am. I. Clin. Nutr. 21: 1429, GARROW, J. S., K. FLETCHER AND D. HALLIDAY. Body composition in severe infantile malnutrition. J. Clin. Invest. 44: 417, BONDY, P. K., D. F. JAMES AND B. W. FARRAR. Studies on the role of the liver in human carbohydrate metabolism by the venous catheter technique. I. Normal subjects under fasting conditions and following the injection of glucose. J- Gun. Invest. 28: 328, MYERS, J. D. Net splanchnic glucose production in normal man and in various disease states. J. Clin. Invest. 29: 1421, BEARN, A. G., B. H. BILLING AND S. SHERLOCK. Hepatic glucose output and Imepatic insulin sensitivity in diabetes mellitus. Lancet 2: 698, OwEN, 0. E., P. FELIG, A. P. MORGAN, J. WAHREN AND G. F. CAHILL, JR. Liver and kidney metabolism during prolonged starvation. J. Clin. Invest. 48: 574, FELIG, P., E. MARLISS, 0. E. OWEN AND G. F. CAHILL, JR. Blood glucose and gluconeogenesis in fasting man. Arch. Internal Med. 123: 293, FELIG, P., 0. E. OWEN, J. WAHREN AND G. F. CAHILL, JR. Amino acid metabolism during prolonged starvation. J. Clin. Invest. 48: 584, FELIG, P., T. POZEFSKY, E. MARLISS AND G. F. CAHILL, JR. Alanine: key role in gluconeogenesis. Science 167: 1003, WHITE, A., P. HANDLER AND E. L. SMITH. Principles of Biochemistry. New York: McGraw-Hill, 1964, p FELIG, P., E. MARLISS, 0. E. OWEN AND G. F.

7 992 Felig et a!. CAHILL, JR. Role of substrate in the regulation of hepatic gluconeogenesis in fasting man. Advan. Enzyme Reg. 7: 41, FELIG, P., E. MAp.uss AND G. F. CAHILL, JR. Feasting and fasting: alternation of insulin and substrate in the regulation of gluconeogenesis in man. J. Gun. Invest. 48: 2la, OwEN, E. E., AND R. R. ROBINSON. Amino acid extraction and ammonia metabolism by the human kidney during the prolonged administration of ammonium chloride. I. Clin. Invest. 42: 263, Prrrs, R. F., AND L. A. PILKINGTON. The relation between plasma concentration of glutamine and glycine and utilization of their nitrogens as sources of urinary ammonia. I. Clin. Invest. 45: 86, LUCK, J., G. MomusoN AND L. F. WILBUR. The effect of insulin on the amino acid content of blood. I. Biol. Chem. 77: 151, FELIG, P., E. MARLISS AND G. F. CAHILL, JR Plasma amino acid levels and insulin secretion in obesity. New Engi. I. Med. 281: 811, EXTON, J. H., I. S. JEFFERSON, JR., R. W. BUTCHER AND C. R. PARK. Gluconeogenesis in the perfused liver. The effects of fasting, alloxan diabetes, glucagon, epinephrine, adenosine 3, 5 - monophosphate and insulin. Am. I. Med. 40: 709, POZEFSKY, T., P. FELIG, J. D. T0BIN, J. S. SOELDNER AND G. F. CAHILL, JR. Amino acid balance across tissues of the forearm in postabsorptive man. Effects of insulin at two dose levels. I. Clin. Invest. 48: 2273, 1969.