Development of Glucagon Sensitivity

in Neonatal Rat Liver

FRANKVINICOR, GARYHIGDON, JULIA F. CLARK, and CHARLEs M. CLAR, JR.

From the Veterans Administration Hospital and Departments of Medicine andPharmacology, Indiana University Medical Center, Indianapolis, Indiana 46202

A B S T R A C T The ontogenesis of the hepatic glucagon-sensitive adenylate cyclase system has been studied inthe rat. With a partially purified liver membrane prepa-ration, fetal adenylate cyclase was less responsive toglucagon than the enzyme from neonatal or adult livers.Similar results were obtained in gently prepared liverhomogenates, suggesting that destruction of essentialcomponents of the fetal liver membrane did not accountfor the relative unresponsiveness of the adenylate cy-clase enzyme to glucagon.

Investigation of other factors that might account fordiminished fetal hepatic responsiveness to glucagon in-dicate (a) minimal glucagon degradation by fetal mem-branes relative to 8-day or adult tissue; and (b) avail-able adenylate cyclase enzyme, as suggested by a 13-fold increase over basal cyclic AMPformation with NaFin fetal liver membranes. These results indicate thatneither enhanced glucagon degradation nor adenylatecyclase enzyme deficiency accounts for the relative in-sensitivity of the fetal hepatic adenylate cyclase systemto glucagon.

In early neonatal life, hepatic adenylate cyclase re-sponsiveness to glucagon rapidly developed and wasmaximal 6 days after birth. These changes were closelyparalleled by a fivefold increase in glucagon binding andthe kinetically determined V.ax for cyclic AMPforma-tion.

These observations suggest that (a) fetal hepatic un-responsiveness to glucagon may be explained by a lim-ited number of glucagon receptor sites; (b) during theneonatal period, the development of glucagon binding isexpressed primarily as an increase in adenylate cyclase

This study was presented in part at the 35th AnnualMeeting of the American Diabetes Association, June 1975.

Dr. Vinicor is a Research Associate of the VeteransAdministration Hospital, MRIS 1445-01.

Received for publication 16 August 1975 and in revisedform 11 May 1976.

Vma.; (c) the ontogenesis of hepatic responsiveness toglucagon may be important in the resolution of neonatalhypoglycemia.

INTRODUCTION

In the adult, glucagon plays a central role in the regula-tion of hepatic glycogenolysis and gluconeogenesis byactivating the membrane-bound enzyme, adenylate cy-clase, and increasing cyclic AMPconcentrations (1, 2).During the perinatal period, however, the exact roleof the hepatic adenylate cyclase system in the control ofglucose homeostasis has not been clearly defined.

In the last third of gestation, fetal animals store gly-cogen in a variety of organs (3, 4). At birth in the rat,there is an immediate increase in plasma glucagon anda decrease in plasma insulin concentrations (5, 6), aswell as a shift in hepatic carbohydrate metabolism fromglycogen synthesis to glycogen breakdown and gluco-neogenesis (3, 7).

While alterations in the hormonal environment appearessential in the regulation of carbohydrate metabolism,the capacity of the liver to respond to these hormonalchanges may be equally necessary in the maintenanceof euglycemia during the neonatal period. Other investi-gators have demonstrated that before birth, fetal hepaticglycogenolytic and gluconeogenic enzyme systems arerelatively insensitive to glucagon, but not to dibutyrylcAMP (8, 9). In contrast, the neonatal hepatic adenylatecyclase system in the rat is very responsive to glucagon(10-12), suggesting that the development of hepaticsensitivity to glucagon may be important in the estab-lishment of neonatal glucose homeostasis.

In the present report, we have investigated the devel-opment of hepatic adenylate cyclase sensitivity to glu-cagon in partially purified membranes from fetal, neo-natal, and adult rats.

The Journal of Clinical Investigation Volume 58 September 1976 -571-578 571

TABLE IIncreases in MeIembrane Alarker Enzyme

5 '-Nucleotidase

Fetal Adult

nmol/gI/20 min

Homogenate 0.22±40.04 0.60±0.1Partially purified membrane 2.00 ±0.23 5.90 ±0.4Increase in specific activity 9.1 9.8

Mean±SEM of two fetal or three adult experiments, eachwith a different tissue preparationi.

METHODS

[a-3P]ATP (10-16 Ci/mmol) was purchased from NewEngland Nuclear (Boston, Mass.); [3H]cAMP (15-20 Ci/mmol) was obtained from ICN Pharmaceuticals Inc. (LifeSciences Group, Cleveland, Ohio); glucagon (crystalline)was a gift from Eli Lilly and Company (Indianapolis, Ind.)(Dr. William Bromer) ; carrier-free [I251]iodide was ob-tained from Union Carbide Corp. (New York); all un-labeled chemicals were analytical grade.

Tissue preparationWhole homogenates. Pregnant rats (Charles River

Breeding Laboratories, Inc., Wilmington, Mass.) of knowngestational ages were stunned, the fetuses removed andlivers rapidly excised, and placed in an ice-cold solution of0.25 M sucrose, 10 mMTris-HCl, pH 7.6. Adult females(> 60 days of age; 200-250 g) and neonatal livers wereobtained similarly. Minced liver tissue was homogenized at4°C with one plunge of a loose-fitting Teflon-glass homoge-nizer, filtered through cheesecloth, and centrifuged for 5min at 600 g. Pellets were resuspended in the sucrose-Trisbuffer to a protein concentration of 3-5 mg/ml.

Hepatic membranes. Livers from rats of known agewere removed and placed in ice-cold 1 mMv NaHCO3. At4°C, partially purified plasma liver membranes were pre-pared by the method of Neville et al. (13) as modifiedby Pohl et al. (14). The preparation sequence was rigidlyfollowed for membranes of all ages, and increases in thespecific activity of the membrane marker enzyme. 5'-nucleo-tidase, were similar (15) (Table I). Membranes were storetlat - 60'C for up to 3 mo without decrease in adenylate cy-clase activity.

Adenylate cyclase assayAdenylate cyclase activity was measured by a modification

of the method of Pohl et al. (14) as previously described(16). Unless specified otherwise, the assay medium (50Al final volume) contained the following reactants: 0.4 mM[3P]ATP (10-20 dpm/mmol); 4.5 mM MgC12; 1 mMEDTA; 8 mMtheophyline; 20 mMTris-HCl, pH 7.6; anATP-regenerating system (20 mMphosphoenolpyruvate and4 uU/ml of pyruvate kinase); human serum albumin orglucagon diluted in human serum albumin; and 20-40 ,ugof protein (partially purified membrane or homogenate)diluted in 0.25 M sucrose, 10 mMTris-HCl, pH 7.6. Re-actions were initiated by the addition of 10 IAI of membraneand continued for 5 min at 30'C. The adenylate cyclase re-action was terminated by the addition of 50 ul of recoverymix (40 mMATP, 12.5 mM ['H]cAMP [30,000-50,000

cpm] and boiling for 3.5 min. [32P]cAMP was separatedby the method of Salomon et al. (17) and [3H] and [3'P]were measured by liquid scintillation counting. Final valuesfor cAMP formed were calculated from the efficiency ofcounting, recovery of [3H]cAMP, subtraction of "blanks"(cAMP formed in absence of membrane), and specific

activity of ['3P]ATP. Protein was determined fluorometri-cally (18) and adenylate cyclase activity was expressed aspicomoles of cAMP formed per milligram protein per 5-min incubation.

lodination of glucagonGlucagon was iodinated with [r"Il iodide by the chlora-

mine-T method of Hunter and Greenwood (19). To a 12X 75-mm test tube containing 10 4l (16 ,ug) of glucagonin 0.05 N HCl, the following were added in rapid succes-sion: 75 4l of 0.5 Mf NaPO4, pH 7.4, 5 4l (1-2 mCi) of[1251]iodide in 0.01 N NaOH, 20 4u of freshly preparedchloramiine-T (16 mg in 4 ml of 0.5 'M NaPO, pH 7.4)with slow mixing for 30 s, and 100 4l of sodium metabi-sulfite (12 mg in 5 ml of 0.5 M NaPO., pH 7.4). 1 ml of1 mMNaPO4, pH 7.4, was added and separation of `1-glucagon was carried out oy modification of the method ofRosselin et al. (20). The entire reaction volume was adde(dto 30 mg of microfine silica (QUSO-G-32, PhiladelphiaQuartz Co., Philadelphia, Pa.), mixed, and centrifuged for10 min at 1,000 g. The supernate was withdrawn and thepellet thoroughly resuspended in 2 ml of 1 mMNaPO4,pH 7.6, and centrifuged for 10 min at 1,000 g, and the super-natant was discarded. The pellet was again resuspendedin 1.5 ml of 10 mNI NaPOI, 1% albumin, pH 10, andcentrifuged for 10 mim at 1,000 q. The supernate, whichcontained 125I-glucagon, was saved. The iodinated hormone(sl) act 100-200 ,Ci/Ag) was chemically characterized bytrichloroacetic acid precipitability (> 90% precipitation) andpaper counterelectrophoresis (> 95% remained at the ori-gin). Molar concentrations of 'I-glucagon were determinedby activation of adenylate cyclase in adult, partially purifiedliver membranes, wvith unlabeled glucagon as standards.Yields of labeled glucagon were 30-50%. Aliquots of theiodinated hormone were frozen and used within 2 wk.

Glucagon binding assayBindling of I251-glucagon to liver membranes was assayed

by a mo(lification of the method of Roclbell et al. (21).Liver membranes were incubated with 1251-glucagon underconditions idlentical to the adenylate cyclase assay (seeMethods). At the appropriate time, 50-,l aliquots wereremoved and layered on top of 300 4l ice-cold 2.5% albuminin 20 mM Tris-HCl, pH 7.6, in plastic microcentrifugetubes. These tubes were centrifuged for 1 min at 10,000 gin a Beckman microcentrifuge (Beckman Instruments, Inc.,Spinco Div., Palo Alto, Calif.), the supernate was aspirated,the pellet was carefully washed with 300 4l of ice-cold 10%sucrose, and the supernatant fluid was again aspirated. Thetips of the centrifuge tubes were carefully cut off just abovethe pellet and the "SI-radioactivity remaining in the tipswas determined. With each experiment, two controls wererun: (a) incubation as above with I251-glucagon in the ab-sence of liver membrane; the amount of radioactivity inthe tip was always <1% of 22I counts added; (b) incuba-tion as above, except that in addition to lI5I-glucagon, a1,000-fold excess of cold glucagon was added; the differencebetween "I counts bound without and with excess coldglucagon represents nonspecific binding and was usually

572 F. Vinicor, G. Higdon, J. F. Clark, and C. M. Clark, Jr.

TABLE I IAdenylate Cyclase Activity in Liver Homogenates

18-day fetal 1-day neonatal 6-day neonatal Adult

pmol cA JIP formed /mg protein /5 min

Basal 24.9 +2.8* 43.4±+0.5 62.24±2.6 43.1±i3.1Glucagon (0.4 ,iM) 58.9i3.6 § 146.9±4.511 233.9±7.211 173.642.511Percent activity over basal 240 340 375 400

* Mean+±SEM of two separate experiments, performed in triplicate, each with different mem-brane preparations for each age.I Compared to basal, P < 0.01 by Student t test.§ Compared to 1- and 6-day neonatal and adult, P < 0.001 by Student I test.11 Compared to basal, P < 0.001 by Student t test.

< 5% of the 'I counts bound. Nonspecific binding was sub-tracted from total binding in calculating picomoles of glu-cagon bound.

Since the biological activity of chloramine-T-preparedmonoiodinated glucagon is similar to the unlabeled hor-mone (22, 23), binding and adenylate cyclase activity weredetermined in the same test tube. As an example, underadenylate cyclase assay conditions as previously described,'I-glucagon was incubated with liver membranes in 200

,IA final volume. At the appropriate time, two 50-Al aliquotswere rapidly removed for determination of l"I-glucagonbinding and the remaining reaction mix was immediatelyassayed for adenylate cyclase activity. This procedure al-lowed examination of the relationship between glucagonbinding and activation of adenylate cyclase under identicalconditions.

Glucagon degradationReaction A: degradation of 125I-glucagont by liver mem-

branes. 'I-Glucagon was incubated with 150 jig of livermembrane under adenylate cyclase assay conditions (seeMethods) (24). At 5 min, the entire reaction volume (300IAl) was rapidly transferred to a microfuge tube and centri-fuged for 1 min at 10,000 g. 20-IAl aliquots of the supernate(supernate A) were counted for 'I and identical aliquotswere assayed for adenylate cyclase activation in reaction B.

Reaction B: adenylate cyclase activationt by ["..II] -gluca-gon. In fresh adult liver membranes, activation of adenylatecyclase by varying concentrations of '5I-glucagon was de-termined and the results plotted as counts of '5I versuspicomoles of cAMP formed. 20-,dl aliquots of supernate Awere also assayed for adenylate cyclase activity in the sameexperiment If the 20-,ul aliquots produced less adenylatecyclase activation than an identical number of counts of1"I-glucagon, it was assumed that glucagon had been de-graded in reaction A. The percent of glucagon degradedwas calculated by dividing the number of 'I counts nolonger associated with biologically active 'I-glucagon bythe total number of 12'I counts added to reaction A.

Adenylate cyclase kineticsIn partially purified liver membranes from animals of

different ages, cyclic AMP formation was determined atvarying concentrations of ATP in basal and glucagon-stimu-lated conditions. To insure linear cAMP formation at ATPconcentrations less than 0.4 mM (data not shown), thereaction was allowed to proceed for only 4 min. Km andVmax were determined by the Wilkinson formula (25), and

results were confirmed by least-squares analysis (26) anddouble-reciprocal plots (data not shown).

RESULTSEffects of glucagon concentration on adenylate cylase

activity. In experiments summarized in Fig. 1, adenyl-ate cyclase activity in partially purified hepatic mem-branes from 20-day fetal, 8-day neonatal, and adultanimals was determined in the presence of varying glu-cagon concentrations. Whereas the glucagon concentra-tion giving half-maximal stimulation of adenylate cy-

Glucagon Concentration+20 day Fetal 4.7 nM

400- -A8day Neonatal 5.2 nM

U-U Adult 4.8 nM

Ec

i 300-0

cn/E

Ea 200-

E0

L.a. ~100* *

l" l l 1 l0 0.01 0.1 1 10 100 1000

Glucagon (nM)

FIGURE 1 Effects of glucagon concentration on adenylatecyclase activity. Partially purified liver membranes (20-40,ug protein/tube) were incubated for 5 min at 30°C fordetermination of cAMP formation. Points are means±+SEMof triplicate determinations in three experiments on threedifferent membranes for each age. * Concentration of glu-cagon giving initial significant stimulation of hepatic aden-ylate cyclase over basal activity. tConcentration of gluca-gon giving half-maximal stimulation of hepatic adenylatecyclase.

Glucagon Sensitivity in Neonatal Rat Liver 573

E

o Adulta 3.0-

0

0 2.5-

E 16dayn 2.0- Neonatal

'0

1.5-

8dayc0 1.0 Neonatal01

20 day-

Fetal

FIGURE 2 Effects of age on degradation of ['SI]glucagon.In the adenylate cyclase assay, partially purified hepaticmembranes (150 Ag of protein) were added to incubationmix containing ['I] glucagon (30 nM). After 5 min at300 C, the amount of glucagon degraded was determined(see Methods). Bars represent means±SEM of two experi-ments performed in triplicate with different membrane prep-arations for each age.

clase is remarkably similar for the three ages, around 5nM, the fetal membrane was less responsive than the8-day neonatal or adult hepatic membrane at any glu-

c 1500-

InEtoc

o 1200-L-

E

E 900-

4)

E

1-

uo 600-a.

TABLE I I IEffects of Glucagon and NaF on Adenylate

Cyclase Activity

20-day 8-dayfetal neonatal Adult

pmol cAMPformed/mg protein/S minBasal (HSA) 53±4* 66411 64 47Glucagon (2,uM) 147415t 201i12 478±17Fluoride (15 mM) 699 ±21 § 4784±22 493 4±29Glucagon (2 ,M)

+fluoride (15 mM) 690±13 490428 529±41

* Mean±SEMof two experiments, each performed in quad-ruplicate with different membrane preparations at each age.t Compared to neonatal and adult, P < 0.001 by Student'st test.§ Compared to neonatal and adult, P < 0.01 by Student'st test.

cagon concentration greater than 10 pM. This differencewas greater at the higher glucagon concentrations. Fur-thermore, while fetal hepatic adenylate cyclase activitywas significantly activated at a glucagon concentrationof 1 nM, activation of adenylate cyclase in the 8-day

., 1500c

E

0)

o 900-

L.

E

E

0

CL.Io A%

A,

-1II IlI I

-4 -2 2 4 6 8 12 16Birth

-4-2 T 2 4 6 8 12 16Birth (days)

I ,,

30 Adult

Age (days)

FIGuRE 3 Effects of age on adenylate cyclase activity.Partially purified hepatic membranes (20-40 g) were in-cubated for 5 min at 30°C in the absence (--- ) or presence( ) of ['I]glucagon (77 nM) and cAMP formationwas determined. Points are means± SEM of three experi-ments, each performed in triplicate with three differentmembrane preparations for each age.

30 Adult

FIGuRE 4 Effects of age on adenylate cyclase activity andglucagon binding. Under adenylate cyclase assay conditions,partially purified hepatic membranes (20-40 Mg) were in-cubated for 5 min at 30°C in the presence of [MI]glucagon(77 nM), and cAMP formation ( ) and glucagon bind-ing (--- -) were simultaneously determined (see Methods).Points are means± SEM of three experiments, each per-formed in triplicate with three different membrane prepara-tions for each age.

574 F. Vinicor, G. Higdon, J. F. Clark, and C. M. Clark, Jr.

t

c

L

EE~0

C

0

1-

U

Sl

and adult hepatic membrane occurred at 0.1 nM gluca-gon, close to the physiologic concentration of this hor-mone (1).

Effects of tissue preparation on adenylate cyclase ac-tivity. To investigate the possibility that some essentialcomponent of the fetal adenylate cyclase system was de-stroyed in the preparation of partially purified hepaticmembranes, adenylate cyclase activity was determined ingently prepared liver homogenates from animals ofvarying age (Table II). While glucagon significantlyincreased cAMP formation over basal at all ages stud-ied, the 18-day fetal tissue was significantly less respon-sive than at other ages.

Effects of age on degradation of "'I-glucagon. Toexamine the possibility that enhanced glucagon degrada-tion by the fetal hepatic membrane accounts for the ap-parent insensitivity of this tissue to this hormone, glu-cagon inactivation was studied (Fig. 2). There ap-peared to be an age-dependent increase in the capacityof the liver membrane to inactivate glucagon: in con-trast to the fetal tissue, which inactivated only 0.22±0.01pmol/150 ,og protein per 5 min, or about 2% of glucagonadded, the 8-day, 16-day, and adult hepatic membranedegraded 0.78±0.02 (6.5%), 1.8±0.05 (15.1%), and2.99±0.05 (25%) pmol of glucagon/150 iAg protein per5 min under these conditions.

Effects of glucagon and NaF on adenylate cyclase ac-tivity. To investigate whether an insufficient quantity ofthe adenylate cyclase enzyme itself may account for therelative insensitivity of the fetal hepatic membrane toglucagon, adenylate cyclase activity was determined in

response to maximal stimulating concentrations of glu-cagon, NaF, or both (Table III). While the 20-dayfetal membrane responded minimally to glucagon com-pared to the 8-day neonatal and adult tissue, the pres-ence of 15 mMNaF increased cAMPformation 13 timesbasal in fetal tissue, greater than in either the 8-day(7.5-fold) or the adult (7.7-fold) animals. Previousstudies by Birnbaumer et al. indicate that in the adultliver, glucagon and fluoride act on the same adenylatecyclase enzyme (27). Similar results were obtained inthese studies, in which maximal concentrations of fluo-ride and glucagon failed to stimulate this enzyme morethan fluoride alone.

Effects of age on adenylate cyclase activity and "I-glucagon binding. In Fig. 3, studies of hepatic adenylatecyclase activity in the presence and absence of glu-cagon are summarized. Whereas glucagon increasedadenylate cyclase activity 2.5-fold over basal in the18-day fetal membrane, the youngest tissue examined(11±0.5 to 29±2 pmol/mg protein per 5 min, P < 0.01),by the 6th day of life, glucagon stimulated cAMP forma-tion 6 times basal to 1,472±37 pmol/mg protein per 5min. For unexplained reasons, hepatic responsiveness toglucagon then decreased in later neonatal life to 730±6(8 days) and 544±3 (16 days) pmol/mg protein per 5min.

Experiments in which specific 'I-glucagon bindingwas simultaneously compared to glucagon-stimulatedadenylate cyclase activity are summarized in Fig.4. A close age-dependent relationship existed be-tween these two parameters: as hepatic adenylate cyclase

0-O Control*-* Glucagon

cn

E

.E 6000-

0

-W0L.

Ea.4000-4u

E

X(2000-E

1 1 f2-Age4612 16 30 AdultBrhAge (days)

&A_Z_-v a v X X X . I I * v . .I t I I I I I I-22 4 6 8 12 16Bith Age (days)

FIGURE 5 Adenylate cyclase kinetics. cAMP formation was determined at 30°C for 4 minin the absence (0-O) and presence (- 0) of glucagon (0.18 iLM) and varying con-centrations of ATP. Km and Vms. were determined by the formula of Wilkinson (25). Pointsare means+SEM of two separate experiments performed in triplicate.

Glucagon Sensitivity in Neonatal Rat Liver 575

24 -

20-

I1.6-

a.

1.2-E

Q8 -

O4 -

30 Adult

responsiveness to glucagon developed in early neonatallife, there was a 4.5-fold increase in glucagon bindingfrom 0.43+0.06 (18-day fetal) to 1.82±0.08 (6-day neo-natal) pmol/mg protein per 5 min. Similarly, as cAMPformation decreased in later neonatal life at 8 and 16days, glucagon binding decreased to 1.28+0.06 and 0.92-'-0.11 pmol/mg protein per 5 min. In adult hepatic mem-branes, 'I-glucagon binding again increased to 1.5±0.05pmol/mg protein per 5 min.

Adenylate cyclase kinetics. Under our assay condi-tions, there were no statistical differences among theKm's for ATP in fetal and neonatal partially purifiedmembranes (Fig. 5). In contrast, development of glu-cagon binding was expressed primarily as an increasein maximum velocity of cAMPformation: 986±85 (20-day fetal) vs. 5,028+403 (6-day neonatal) pmol cAMPformed/mg protein per 4 min.

DISCUSSIONBefore birth, fetal gluconeogenic and glycogenolytic ca-pacities are severely limited (9, 28) ; fetal plasma glu-cose concentrations are maintained by transfer of glu-cose from mother to fetus (3). To maintain euglycemiaat birth, the animal must (a) store a potential glucosesource, e.g. glycogen, before birth for use during theinitial neonatal period; (b) alter the hormonal environ-ment so that endogenous glucose formation is maximal;and (c) develop the hepatic capacity for glycogenolysisand gluconeogenesis in response to hormonal stimulation.Previous investigations have documented prenatal gly-cogen storage (3, 4) and a postnatal catabolic hormonalmilieu (5, 6, 29). The development of the capacity ofthe neonatal liver to respond to one of these catabolichormones, glucagon, is the subject of the present report.

The results of our studies indicate that whereas the18-day fetal hepatic adenylate cyclase system can re-spond to glucagon at a concentration greater than 1 nM,this response is quite limited when compared to that ofneonatal and adult animals, both in quantity of cyclicAMPformed and sensitivity to glucagon. Relative fetalhepatic adenylate cyclase insensitivity to glucagon hasbeen observed by others in liver homogenates (11, 30),particles (10), and slices (10), and in vivo (10, 12).This lack of response to glucagon, together with a highinsulin-glucagon ratio (31), would insure glycogen stor-age during the last third of gestation.

Several reasons could explain the relative unrespon-siveness of the fetal hepatic adenylate cyclase to glu-cagon. Friedman et al. have indicated that purification offetal lamb myocardial tissues damages the glucagon-re-sponsive adenylate cyclase system (32). Destruction ofa component of the fetal adenylate cyclase system dur-ing preparation of partially purified membranes seemsan unlikely explanation from our results, however, be-

cause similar observations of fetal hepatic unresponsive-ness to glucagon have been made in gently prepared liverhomogenates (Table II) (11), in intact livers (10), andin liver slices (10), conditions in which tissue damageshould be negligible.

Differences in the cellular composition of the fetalliver may explain the relative unresponsiveness of thistissue to glucagon compared to neonatal liver. Very fewhematopoietic and reticulo-endothelial cellular elementsare present in the liver in the late gestational andearly newborn period. However, and at most, there is atwo- to fourfold increase in hepatocytes after birth(33, 34). In contrast, changes in adenylate cyclase re-sponsiveness to glucagon in the perinatal period are 50-fold (Fig. 3), suggesting real changes in the capacity ofthe liver membrane to respond to the hormone.

Pohl et al. have documented the existence of a po-tent hepatic glucagon-degradation system (24). Fur-thermore, evidence exists that the hepatic adenylate cy-clase and glucagon degradation systems are independentprocesses (35, 36). Studies of glucagon degradation(Fig. 2). however, indicate that enhanced hormone deg-radation by the fetal liver does not occur and thus can-not explain the relative unresponsiveness of this mem-brane to glucagon.

An insufficient quantity of adenylate cyclase enzymeitself might account for the insensitivity of the fetalhepatic membrane to glucagon. As noted by others (10,11), however, the fetal adenylate cyclase system is veryresponsive to NaF and, in our system, more sensitivethan in neonatal or adult tissue. Since fluoride and glu-cagon appear to act on the same adenylate cyclase en-zyme in the liver (27, Table III), we conclude that inthe fetal liver, this enzyme system is potentially very ac-tive and the relative inability of glucagon to stimulatethis enzyme must reflect delayed development of a com-ponent other than the enzyme itself.

When comparisons were made between glucagon-stimulated adenylate cyclase activity and glucagon bind-ing, there was a close age-dependent relationship be-tween these two properties. Furthermore, alterations inhormone binding appeared to account in large part forthe ontogenesis of hepatic adenylate cyclase responsive-ness to glucagon in the early neonatal period. The 18-day fetal membrane bound very little glucagon and theadenylate cyclase system was relatively insensitive to thehormone. As noted by others in different tissue prepa-rations (10-12), hepatic adenylate cyclase stimulationby glucagon then peaked during the 1st wk of neonatallife and in our experiments was closely paralleled by anincrease in specific glucagon binding to neonatal hepaticmembranes (Fig. 4). Furthermore, these changes inglucagon-binding were accompanied primarily by in-creases in the maximum velocity of cAMP formation

576 F. Vinicor, G. Higdon, J. F. Clark, and C. M. Clark, Jr.

(Fig. 5), perhaps due to activation of more adenylatecyclase enzyme molecules.

Recent investigations that indiacte (a) a dissociationbetween rates of glucagon-stimulated hepatic adenylatecyclase activation and 'I-glucagon binding (37); and(b) binding of hormones to nonreceptor materials (38)suggest that one must be exceedingly careful in the in-terpretation of hormone binding studies. Similarly, theapplication of our results to other species or diseasestates must be done with caution. Nevertheless, possibleconsequences of delayed hepatic responsiveness to glu-cagon deserve comment. Offspring of diabetic mothersare prone to develop severe neonatal hypoglycemia (39),presumably due to hyperinsulinemia (40), and an inade-quate glucagon response to this hypoglycemia (41).Experimental evidence suggests that hormone inductionof gluconeogenic and glycogenolytic enzymes can beblocked by hyperglycemia (9, 42). If glucagon's effecton these enzymes is mediated via cAMP, hyperglycemiamay delay the normal development of hepatic adenylatecyclase responsiveness to glucagon and thus contrib-ute to the profound hypoglycemia observed in offspringof diabetic mothers.

"Small-for-gestation" infants are also at risk for thedevelopment of hypoglycemia (43, 44). This "hypogly-cemic potential" appears not to be due to defects in hor-mone secretion, including glucagon (45, 46) or lack ofpotential glucose substrates (45). Rather, hepatic en-zyme induction by glucagon may be limited in thesesmall-for-gestation infants (45). In normal-weight new-borns, gluconeogenic (47), hyperglycemic, and urinarycAMP responses (48) to pharmacological doses of glu-cagon do not reach maturity until 3-4 days of life. Thissame immaturity of hepatic responsiveness may be ex-aggerated in small-for-gestational infants and reflect de-layed development of glucagon-stimulated hepatic adenyl-ate cyclase system. Clearly, further work is necessary tovalidate this hypothesis.

Our findings suggest that the development of hepaticadenylate cyclase responsiveness to glucagon may be im-portant in the normal developmental pattern of carbohy-drate homeostasis: in the fetal period, a relative lack ofadenylate cyclase responsiveness to glucagon wouldfavor glycogen storage; in the neonatal period, develop-ment of glucagon binding is correlated with heightenedsensitivity of adenylate cyclase to glucagon, a situationoptimal for endogenous glucose formation via glyco-genolysis and gluconeogenesis.

The cellular factors involved in the ontogenesis ofhepatic responsiveness to glucagon, the signal for thedevelopment of functional hepatic glucagon receptors,the influence of environmental factors (e.g. hypergly-cemia) on neonatal adenylate cyclase sensitivity to glu-

cagon, and the relationship of these events to humanphysiology and disease will require further investigation.

ACKNOWLEDGMENTSWe thank Mrs. Roberta Bergman for secretarial assistanceand Drs. Norman Bell, Sherry Queener, and T. K. Li forhelpful criticism of this paper.

This work was supported in part by grants from theAmerican Heart Association and from the American HeartAssociation, Indiana Affiliate.

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578 F. Vinicor, G. Higdon, J. F. Clark, and C. M. Clark, Jr.