Hormones and substrates in the regulation of gluconeogenesis in fasting man

HORMONES IN THE

REGULATION

AND

SUBSTRATES

OF GLUCONEOGENESIS

IN FASTING

MAN

E. MARLISS,T. T. AOKI,P. FELIG,T. POZEFSKYand G. F. CAHILL,JR.* Joslin Diabetes Foundation and the Department of Medicine, Harvard Medical School and the Peter Bent Brigham Hospital, Boston, Massachusetts

INTRODUCTION

IN previous volumes of this series, data have been presented in which energy balance was quantified and aspects of its control demonstrated in man subjected to prolonged fasting (1, 2). Man adapts to the fasted state by an accelerated consumption of fat and its breakdown products as fuel and a sparing of body protein. That the protein conservation is the result of a marked attenuation of hepatic gluconeogenesis was suggested by the observation of diminished urinary nitrogen excretion (3), and confirmed by the measurement of splanchnic substrate exchange (4). The reduced rate of gluconeogenesis is related to the adaptation of the brain to utilization of acetoacetate and B-hydroxybutyrate as principal substrates in place of glucose (5). Recent studies of the individual amino acids as gluconeogenic substrates have shown that alanine is quantitatively the most important (6). The marked diminution in hepatic alanine uptake in prolonged fasting is due to diminished plasma concentration inasmuch as its fractional extraction remains unchanged. Furthermore, exogenous alanine administration results in a prompt hyperglycemic response due to its conversion to glucose (7), suggesting that hepatic gluconeogenic mechanisms remain intact. Studies of forearm muscle metabolism from this laboratory have demonstrated that the decrease in plasma alanine concentration in starvation results from a marked decrease in its release from muscle, the body's principal protein store (8). Figure 1 demonstrates the forearm arteriovenous differences of amino acids in the postabsorptive and prolonged-fasted states. Of particular interest is that those amino acids showing significant forearm release in both states correspond in pattern and magnitude to the splanchnic uptake previously described (6). *Supported in part by U.S. Public Health Service Grants AM--05077, AM-09584, AM-09798, FR-31-05 and the John A. Hartford Foundation Inc., New York. B

3

E. MARLISS, T. T. AOKI~ P. FELIG~ T. POZI~SKY AND G. F. CAHILL, JR. POSTABSOflPTtVE .k4(

~

~ONG~O STAnVATIOfl

-t2C -tO( -8C A-DV ~.Mt~e/L GC

FiG. 1 Amino acid balanco across forearm muscle tissue in the post-absorptive state and after 4-6 weeks starvation. A-DV=arterio-deep venous difference.

Thus, the rate-limiting step in the control of hepatic gluconeogenesis in prolonged starvation is seen to lie in the release of precursor substrate from the periphery. This occurs despite a substrate-hormone milieu well-documented as favoring hepatic gluconeogenesis: elevated free fatty acid levels, lowered insulin levels, and the associated increase in the activity of key gluconeogenic enzymes. The roles of other hormones in influencing the flux of substrates and rate of gluconeogenesis have been investigated. Glucagon has long been known to augment hepatic gluconeogenesis in intact man (9) and in liver studied in vitro (10). Technical difficulties in the measurement of circulating levels have thus far yielded conflicting estimates of its response in the fasted state (11-13). No data have previously been obtained in prolonged fasting. Similarly, growth hormone by virtue of its known adipokinetic, anabolic and glucoregulatory effects has been proposed as an important fasting hormone (14). Though no significant elevation in serum growth hormone levels occurs in fasted obese subjects, an increased sensitivity to its effects has not thus been excluded (15). To further examine the possible physiological roles of glucagon and growth hormone in prolonged-fasted man, we have administered them to such subjects and have investigated the metabolic responses.

MATERIALS

AND METHODS

Ten obese nondiabetic subjects between the ages of 23 and 52 years (five males and five females) were admitted to the Clinical Research Center of the Peter Bent Brigham Hospital for voluntary prolonged therapeutic starvation. All the patients fasted five to six weeks, during which time their daily oral

GLUCONEOGENESIS IN FASTING MAN

intake was 1500 ml of water, 17 mEq NaCI, 13 mFxt KCI and one multivitamin tablet. Techniques of urine collection, blood sampling and analysis are described elsewhere (4, 6).

METABOLIC

16-

R E S P O N S E TO P R O L O N G E D IN O B E S E SUBJECTS

URINE NITROGEN

STARVATION

(MS. 23yrs J)

1412I0gin/Z4 hrs.

86420 IMMUNOREACTIVE GLUCAGON (Meon-+S E., 3subjects)

160q

SERUM GROWTH HORMONE (Meon-.+SE, II subjecfsl

mpg/ml.

IMMUNOREACTIV£ INSULIN ( M e o n ± S £ . 9 subjecfs} 50 pU/ml.

|

f

5

~o

~5

2o

z5

30

35

40

45

DAYS OF STARVATION

FtG. 2

Metabolic response to prolonged starvation in obese subjects. Daffy urine nitrogen excretion is shown for a typical patient. Values for immunoreactive 81ucason and growth hormone during fasting are not significantly different from postabsorptive. (Immunoreactive glucagon determinations performed by Dr. R. H. Unger.)

6

E. MARLISS,T. T. AOKI, P. FELIG, T. POZEFSKY AND G. F. CAHILL, JR.

Six patients received glucagon (crystalline, Eli Lilly and Company) by constant intravenous infusion in an albumin-containing solution over 48 hr during the fifth week of fasting. Daily dose was 10 mg in three patients, 1 mg in two patients, and 0.I mg in one patient. Immunoreactive glucagon determinations were kindly performed by Dr. Roger H. Unger, by a recent procedure developed to exclude non-pancreatic glucagon immunoreactivity (16). Four further patients received human growth hormone (generously supplied by Dr. M. S. Raben) for three days in a daily dose of 10 mg, administered as 5 mg intramuscularly every 12 hr.

RESULTS AND DISCUSSION Figure 2 shows the metabolic response of obese patients to prolonged fasting. The urine nitrogen excretion drops from 11-15 g/day to a plateau of 4--6 g/day, representing marked curtailment of protein mobilization from 70-90 g to 25-35 g daily. Serum immunoreactive glucagon shows a small increase from postabsorptive values (p < 0.05, paired t). Serum growth

(Meon volues. 4 subjects)

[]

, NITROGEN AMMONIA

I FOR I E_CONTROL1 ~GLUCAGO~ N! t I

•

, UREA NITROGEN

POST-CONTROL ~

gm/24ht

OAYS OF STARVATION

FIG. 3 Influence of glucagon on urinary ammonia and urea nitrogen excretion during prolonged starvation. The increase in total nitrogen is due to a significant increase in the urea nitrogen fraction only (days 35 through 40 vs. mean of forecontrol, paired t).

GLUCONEOGENF~ISIN FASTING MAN hormone concentrations do not change, but immunoreactive insulin falls significantly, as previously documented (4). In the glucagon-infused subjects a steady-state elevation of glucose and insulin was induced, which in the 10 mg/day studies was 42 + 9 mg/100 ml for glucose and 27 :t: 5 /zU/ml for insulin. Figure 3 demonstrates the marked increase in nitrogen excretion which occurred due entirely to the urea fraction, with no alteration in ammonia. That the increment in gluconeogenesis thus induced is due to augmented hepatic gluconeogenic activity is further suggested by the striking fall in plasma amino acid concentrations. All amino acids measured showed a decrease from baseline at one or more of the sampling intervals during glucagon infusion. Figures 4 and 5 show those amino acids for which a significant decrease (p < 0.05, paired t) in concentration was found. Most showed a trend toward baseline values by 48 hr. Figure 4 shows those amino acids whose nadir in concentration was at 24 hr, Fig. 5 those at 8 hr. These alterations are similar to those recently reported after subcutaneous glucagon injection in the postabsorptive state (17). To be noted in Fig. 5 is that with the exception of taurine, lysine and histidine, the remainder are the amino acids which are known to be most sensitive to ambient insulin concentration (18). With the smaller glucagon doses no

250

[ ] 0 lime •

200

24 hours

[ ] 48 hours .

-'ii/o tO0f 50 0

THR

SER

PRO

GLY ALA AMINO ACID FIG.

.~

ORN

ARG

4

Plasma amino acid response to continuous glucagon infusion over 48 hr (10 rng/24 hr). 0 time values are typical of those reported in prolonged-fasted man (6). Amino acids whose maximum decrease in concentration occurred at 24 hr are shown. (Mean +SE.)

E. MARLISS, T. T. AOKI, P. FELIG, T. POZEFSKY AND G. F. CAHILL, JR. 20C

I ~ 0 time

i

8 hours

[]

48 hours

15C uM/L

TAU

a-NH z BUT

VAL

MET

ILE

LEU

TYR

PHE

LYS

HIS

AMINO ACID

FtG. 5 Plasma amino acid response to continuous 81ucagon infusion over 48 hr (10 m8/24 hr). Amino acids whose maximum decrease in concentration occurred at 8 hours are shown. (Mean -t- SE.)

increment in insulin was observed and no diminution in this group of amino acids occurred, though the rest of the amino acids followed the pattern shown in the Figures, and urine urea excretion was augmented. Thus, at least part of the response shown was likely due to insulin-modulated inhibition of muscle release of these amino acids. Of interest is the fact that the greater-than-doubled urea excretion cannot be accounted for by an increase in alanine gluconeogenesis alone. In the physiological situation this amino acid is quantitatively the most important glucose precursor. This could persist after glucagon only if both muscle and liver blood flow were greatly increased, because alanine concentration is reduced by 50~o to a mean of 69/~M/L; this latter concentration approximately equals the observed forearm and hepatic arteriovenous differences previously reported (6, 8). Neither splanchnic blood flow nor limb flow have been reported to change in man after single injections or short infusions of glucagon (19, 20). On the other hand, the increased urine urea excretion could be accounted for by a more "unselective" increase in hepatic amino acid extraction and conversion to glucose, as suggested by the data of Fig. 4. Of particular note is that the level of glycine, which is uniquely and markedly increased in prolonged fasting but n o t significantly extracted by the splanchnic circulation (6), diminished to 65 ~o of its preinfusion concentration. Though glycine is a favored substrate for renal gluconeogenesis-amoniagenesis, the absence of an increase in ammonia excretion suggests that it, too, was extracted by the liver.

GLUCONEOGENESISIN FASTINGMAN

9

Though further studies to quantify hepatic and muscle handling of individual amino acids after glucagon are required to confirm that the striking diminution in peripheral plasma concentrations is due to a primary stimulation of hepatic extraction, this conclusion is suggested by several other observations. Studies in postabsorptive man (19) and experimental animals (21) have shown augmented hepatic amino nitrogen uptake after glucagon. Recent studies by Mallette, Exton and Park (22) in the isolated, perfused rat liver have demonstrated both increased alanine transport and gluconeogenesis from alanine with glucagon in the perfusion medium, even with greater than physiological concentrations of alanine. Further studies are required to clarify its role in modulating the substrate flow to liver, the most important step in the diminution of gluconeogenesis in prolonged fasting. That it acts in concert with the diminished insulin levels in determining the fraction of substrate presented to the liver which is extracted cannot, however, be ruled out. The metabolic response of the patients who received growth hormone is shown in Fig. 6. Administration of this hormone resulted in a 50% increase in blood B-OH butyrate and acetoacetate (to 9 and 2.4 mM/L respectively) and a threefold increase in urine ketone acid excretion to 300 mM/day 05). Although urine urea nitrogen fell by 50~o to l g/day, ammonia nitrogen

(Meon volues, 4 subjects) R • NITROGEN AMMONIA UREA • • NITROGEN l FORE-CONTROL t aF

I HGH (SmQq 12hrs.)~ 1 I

POST-CONTROL

I

I

gm/24hr2

0Avs OF STARVAT=ON F~O. 6 Influence of human growth hormone on urinary ammonia and urea nitrogen excretion during prolonged sta_rvation. The oppositely dLrected sigmficam changes

in both ammoniaand urea nitrogenmitigateany change in total nitrogen(days 35 through 41 vs. mean of fore-control,paired t).

~

]0

E. MARLISS, T. T. AOKI, P. FELIG, T. POZEFSKY AND G. F. CAHILL, JR.

excretion increased by 50 ~o to 3 g/day (obligated by the increased metabolic acidosis) and resulted in no net decrease in total nitrogen excreted. The increased ketogenesis coincided with elevated plasma free fatty acids and blood glycerol. Therefore, any net protein conservation produced by growth hormone was mitigated by the accelerated lipolysis and ketogenesis. The latter necessitated mobilization o f amino acid as the source of urinary a m m o n i a to maintain acid-base equilibrium. SUMMARY Attenuation o f hepatic gluconeogenesis in prolonged-fasted man as a mechanism o f protein conservation is achieved by diminished presentation of precursor substrate (mainly alanine). This has been shown to result from decreased release o f amino acids from their stores in skeletal muscle. Exogenous growth h o r m o n e fails to enhance conservation o f b o d y protein. I n a s m u c h as the endogenous levels o f growth h o r m o n e are not changed, the data presented suggest that it is not the prime agent minimizing protein breakdown. G l u c a g o n lowers plasma amino acids and though hepatic gluconeogenesis is augmented an influence on muscle amino acid release is not exclued. Synergism between normal or elevated endogenous glucagon and low insulin m a y determine the proportion o f substrate extracted by the liver. REFERENCES 1. G. F. CAHILL, JR., O. E. OWENand A. P. MORGAN,The consumption of fuels during prolonged starvation, Advances in Enzyme Regulation 6, 143-150 (1968). 2. P. FELIG, E. MARLISS,O. E. OWEN and G. F. CAHILL, JR., Role of substrate in the regulation of hepatic gluconeogenesis in fasting man, Advances in Enzyme Regulation, 7, 41-46 (1969). & F. G. BLmr.Dlc'r, A study of prolonged fasting, Carnegie Institute of Washington, publication 203 (1915). 4. O. E. OWEN, P. FELIG, A. P. MORGAN, J. WAHRENand G. F. CAHILL, JR., Liver and kidney metabolism during prolonged starvation, J. Clin. Investig. 48, 574-583 (1969). 5. O. E. OW~N,A. P. MORGAN,H. G. KEMP,J. M. SULLIVAN,M. G. HeRRL~L~,and G. F. CAHILL, JR., Bra.ill metabolism during fasting, J. Clin. Investig. 46, 1589-1595 (1967). 6. P. FEUG, O. E. OWEN, J. WAHRENand G. F. CAHILL,JR., Amino acid metabolism during prolonged starvation, J. Clin. lnvestig. 48, 584-594 (1970). 7. P. Fl~uo, E. MARLtSS,T. POZEFSKYand G. F. CAHILL, JR.) Amino acid metabolism in the regulation of gluconeogenesis in man, Am. J. Cli,. Nutr., in press (1970). 8. P. FELIG, T. POZEFSKY, E. MARLISS and G. F. CAHILL, JR., Alanine: key role in gluconeogenesis, Science 167, 1003-1004 (1970). 9. J. M. SALTER,C. EZRIN, J. C. LAIDLAWand A. G. CORNALL, Metabolic effects of glucagon in human subjects, Metabolism 9, 753-768 (1960). 10. L. L. MILLER,Glucagon: a protein catabolic hormone in the isolated perfused rat liver, Nature 185, 248 (1960). 11. E. AGUILAR-PARADA,A. M. EISENTRAUTand R. H. UNGER,Pancreatic glucagon levels in starvation in man, Diabetes 18, suppl. I, 327-328 (1969). 12. J. E. VANCE,K. D. BUCHANANand R. H. WILLIAMS,Effect of starvation and refeeding on serum immunoreactive glucngon and insulin levels, J. Lab. Clin. Med. 72, 290-297 (1968).

GLUCONEOGENESIS IN FASTING MAN

11

13. A. M. LAWRENCE,Radioimmunoassayable glucagon levels in man: effects of starvation, hypoglycemia and glucose administration, Proc. Natl. Acad. Sci. 55, 316-320 (1960). 14. M. S. RAnEN, Growth hormone; anabolic and anticatabolic agent, Diabetes 14, 374 (1964). 15. P. FELIG, E. MARLISSand G. F. CAHILL,JR., Interaction ofadipokinetic and proteinsparing effects of human growth hormone in fasting man, p. 35 in Abstracts of the Fifty-first Meeting of the Endocrine Society (1969). 16. E. AGLrILAR-PARADA,A. M. EISENTRAUTand R. H. UNGER, Pancreatic glucagon secretion in normal and diabetic subjects, Am. J. Med. Sci. 257, 415-419 (1969). 17. R. L. LA~a>AUand K. LUG1BIHL,Effect of glucagon on concentration of several free amino acids in plasma, Metabolism 18, 265-276 (1969). 18. H. H. ZIENEMAN,F. A. NUTrALL and F. C. GOETZ, Effect of endogenous insulin on human amino acid metabolism, Diabetes 15, 5-8 (1966). 19. R. F. IOnLER, W. J. TAYLORand J. D. MEYERS,The effect of glucagon on net splanchnic balances of glucose, amino acid nitrogen, urea, ketones, and oxygen in man, J. Clin. lnvestig. 43, 904-915 (1964). 20. P. K. Bor,a~y and L. R. CARDILLO,The effect of glucagon on carbohydrate metabolism in normal human beings, J. Clin. Investig. 35, 494-501 (1956). 21. W. C. SHOEMAKERand T. B. VAN ITALUE, The hepatic response to glucagon in the unanesthetized dog, Endocrinology 66, 260--268 (1960). 22. U E. MALLErrE, J. H. EXTONand C. R. PAaK, Control of gluconeogenesis from amino acids in the perfused rat liver, J. BioL Chem. 244, 5713-5723 (1969).