Influence of uremia and hemodialysis on theturnover and metabolic effects of glucagon.

R S Sherwin, … , R Hendler, P Felig

J Clin Invest. 1976;57(3):722-731. https://doi.org/10.1172/JCI108330.

To evaluate the mechanism and role of hyperglucagonemia in the carbohydrate intoleranceof uremia, 19 patients with chronic renal failure (12 of whom had undergone chronichemodialysis for at least 11 mo) and 35 healthy control subjects were studied. Plasmaglucagon, glucose, and insulin were measured in the basal state, after glucose ingestion(100 g), after intravenous alanine (0.15 g/kg), and during a 3-h continuous infusion ofglucagon (3 ng/kg per min) which in normal subjects, raised plasma glucagon levels into theupper physiological range. Basal concentrations of plasma glucagon, the increment inglucagon after infusion of alanine, and post-glucose glucagon levels were three- to fourfoldgreater in uremic patients than in controls. The plasma glucagon increments after theinfusion of exogenous glucagon were also two- to threefold greater in the uremics. Themetabolic clearance rate (MCR) of glucagon in uremics was reduced by 58% as comparedto controls. In contrast, the basal systemic delivery rate (BSDR) of glucagon in uremics wasnot significantly different from controls. Comparison of dialyzed and undialyzed uremicsshowed no differences with respect to plasma concentrations, MCR, or BSDR of glucagon.However, during the infusion of glucagon, the increments in plasma glucose in undialyzeduremics were three- to fourfold greater than in dialyzed uremics or controls. When theglucagon infusion rate was increased in controls […]

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Influence of Uremia and Hemodialysis on the Turnoverand Metabolic Effects of Glucagon

ROBERTS. SHERWIN, CHRISTINE BASTL, FREDRIC 0. FINKELSTEIN,MERRICKFISHER, HENRYBLAcK, ROSAHENDLER,and PHmiP FELIG

From the Department of Internal Medicine, Yale University School of Medicine,NewHaven, Connecticut 06510

A B S T R A C T To evaluate the mechanism and role ofhyperglucagonemia in the carbohydrate intolerance ofuremia, 19 patients with chronic renal failure (12 ofwhom had undergone chronic hemodialysis for at least11 mo) and 35 healthy control subjects were studied.Plasma glucagon, glucose, and insulin were measuredin the basal state, after glucose ingestion (100 g),after intravenous alanine (0.15 g/kg), and during a 3-hcontinuous infusion of glucagon (3 ng/kg per min)which in normal subjects, raised plasma glucagon levelsinto the upper physiological range.

Basal concentrations of plasma glucagon, the incre-ment in glucagon after infusion of alanine, and post-glucose glucagon levels were three- to fourfold greaterin uremic patients than in controls. The plasma gluca-gon increments after the infusion of exogenous glucagonwere also two- to threefold greater in the uremics. Themetabolic clearance rate (MCR) of glucagon in uremicswas reduced by 58% as compared to controls. In con-trast, the basal systemic delivery rate (BSDR) of glu-cagon in uremics was not significantly different fromcontrols.

Comparison of dialyzed and undialyzed uremicsshowed no differences with respect to plasma concen-trations, MCR, or BSDRof glucagon. However, duringthe infusion of glucagon, the increments in plasma glu-cose in undialyzed uremics were three- to fourfoldgreater than in dialyzed uremics or controls. Whenthe glucagon infusion rate was increased in controlsto 6 ng/kg per min to produce increments in plasmaglucagon comparable to uremics, the glycemic responseremained approximately twofold greater in the un-dialyzed uremics. The plasma glucose response to glu-

Dr. Felig is the recipient of a Research Career Develop-ment Award (AM 70219) from the National Institutes ofHealth.

Received for publication 2 September 1975 and in revisedf orm 23 October 1975.

cagon in the uremics showed a direct linear correlationwith oral glucose tolerance which was also improvedwith dialysis. The glucagon infusion resulted in a 24%reduction in plasma alanine in uremics but had noeffect on alanine levels in controls.

It is concluded that (a) hyperglucagonemia in uremiais primarily a result of decreased catabolism rather thanhypersecretion of this hormone; (b) sensitivity to thehyperglycemic effect of physiological increments in glu-cagon is increased in undialyzed uremic patients; and(c) dialysis normalizes the glycemic response to gluca-gon, possibly accounting thereby for improved glucosetolerance despite persistent hyperglucagonemia. Thesefindings thus provide evidence of decreased hormonalcatabolism contributing to a hyperglucagonemic state,and of altered tissue sensitivity contributing to thepathophysiological action of this hormone.

INTRODUCTIONChronic renal failure is characterized by an increasedincidence of glucose intolerance (1-3). The role of in-sulin resistance in the pathogenesis of the carbohydrateintolerance in uremia has recently been emphasized (4).Decreased sensitivity to the hypoglycemic effects ofendogenous (2, 5, 6) and exogenous (3, 5-8) insulinhas been observed in uremic man, whereas basal andglucose-stimulated insulin concentrations are normalor increased (1-3, 5, 6, 9). After chronic dialysis, glu-cose tolerance generally improves in association withincreased tissue sensitivity to insulin (5, 9-13). Themechanism of uremia-induced insulin resistance and theimprovement in insulin sensitivity after dialysis has notbeen established. Recently, Bilbrey et al. (14) ob-served increased levels of circulating glucagon, a knowninsulin antagonist, in chronic renal failure. The patho-physiological significance of these findings was ques-tioned however, since dialysis resulted in marked im-

The Journal of Clinical Investigation Volume 57 March 1976. 722-731722

provement in glucose tolerance despite unchanged levelsof plasma glucagon (14). The possible role of alteredtissue responsiveness to physiological increments ofglucagon in uremia and the effect of dialysis on gluca-gon sensitivity have not been previously examined.Furthermore, whether hyperglucagonemia in uremia isa consequence of increased secretion or is solely dueto reduced hormone catabolism has not been determined.

The present study was consequently undertaken toexamine the turnover of plasma glucagon and the tissueresponsiveness to physiological increments in glucagonin uremia. In the evaluation of glucagon kinetics andsensitivity, special attention was given to the influenceof dialysis on these parameters. The data indicate thathyperglucagonemia in uremia is a result of decreasedturnover rather than hypersecretion. Furthermore, ure-mia is associated with increased sensitivity to the hyper-glycemic effects of glucagon. This increased tissue sen-sitivity to glucagon returns to normal after chronicdialysis.

METHODS

Subjects

Two groups of subjects were studied. The control groupconsisted of 35 healthy, nonobese volunteers (18 males and17 females) within 15%o of ideal body weight (based on1959 Metropolitan Life Insurance Company tables). Thesubjects ranged in age from 18 to 50 yr. They consumedweight-maintaining diets containing at least 200 g of carbo-hydrate and were taking no drugs. All had negative primaryfamily histories for diabetes mellitus and none had anelevated fasting plasma glucose. 20 of the subjects receivedan oral glucose tolerance test (100 g) and all demonstrateda normal response (15).

The uremic group consisted of 19 ambulatory patients(12 males and 7 females) with chronic renal failure, aged20-63 yr (44+4 yr, mean±-SE). Seven of the patients hadnot undergone treatment with dialysis before study (un-dialyzed group). 12 patients had undergone chronic hemo-dialysis for at least 11 mo in the dialysis unit of the Yale-New Haven Hospital before study. None of the uremicpatients had evidence of an acute medical illness or periph-eral edema at the time of study. None were receiving cor-ticosteroids. The medications received by dialyzed and un-dialyzed groups were similar with the exception that 9 of12 dialyzed subjects and 2 of 7 undialyzed subjects receivedparenteral androgens. All but three uremic subjects werewithin 15% of ideal body weight (based on 1959 Metro-politan Life Insurance Company tables). The patients con-sumed weight-maintaining diets containing at least 250 g ofcarbohydrate for 3 days before study. Dialyzed patients con-sumed approximately 80 g of protein, while undialyzedpatients were restricted to 50-60 g of protein. Pertinentlaboratory data in the uremic group included: serum creati-nine, which ranged from 7.5 to 25.1 mg/100 ml (15.6±+1.2,mean+±SE); serum potassium, 4.0-6.9 meq/liter (5.3+0.2,mean+SE); serum bicarbonate, 11.5-25.0 meq/liter (18.6+1.0, mean+SE); serum calcium, 6.3-10.4 mg/100 ml (8.6+0.2, mean+SE); and serum phosphate 3.2-14.4 mg/100ml (6.5+0.6, mean+SE). There were no significant differ-

ences with respect to these laboratory values, ages, orweights between the dialyzed and undialyzed patients.

Procedures

All subjects were studied in the postabsorptive state aftera 12-15-h overnight fast. Uremic subjects were instructedto omit all medications on the day of study. Dialysis patientswere studied just before their next dialysis. An indwellingcatheter was inserted in an antecubital vein for blood sam-pling. An additional catheter was inserted in the contra-lateral vein or ateriovenous fistula during the glucagon-in-fusion studies. Subjects were recumbent for at least 20-30min before study. At least two control blood samples wereobtained at 10-15-min intervals before glucose, alanine, orglucagon administration. Control values presented in eachstudy represent the mean of the base-line determinations.Three types of procedures were employed.

Oral glucose tolerance. A 100-g oral glucose tolerancetest was performed in 20 normal and 10 uremic subjects. Inthis study, blood samples were drawn at 30-60-min intervalsfor 3 h after ingestion of 100 g glucose dissolved in 200 mlof water.

Alanine infusion. L-Alanine (Nutritional BiochemicalCorp., Cleveland, Ohio) was given intravenously (0.15 g/kgbody weight) over 2-4 min to 21 normal and 9 uremicsubjects. Methods used in the preparation of the alanineinfusate have previously been described (16). Blood sampleswere obtained at 10-30-min intervals for 90 min after theinfusion of alanine.

Glucagon infusion. Crystalline beef and pork glucagon(Eli Lilly and Co., Indianapolis, Ind.) was administered as

a continuous infusion via a peristaltic pump (ExtracorporealMedical Specialties, Inc., King of Prussia, Pa.) to 21normal and 10 uremic subjects. Before administration, theglucagon was initially diluted in sterile, pyrogen-free watercontaining 300 mg/100 ml of human serum albumin (Ar-mour Pharmaceutical Co., Chicago, Ill.). This solution wasfurther diluted with physiological saline and an aliquot ofthe subject's whole blood (4 ml of whole blood per 100 mlof infusate, to prevent adherence of the glucagon to glass-ware or tubing) so as to achieve a final glucagon concen-tration in the infusate of 500 ng/ml. The continuous in-fusion dose was 3 ng/kg body weight per min, administeredfor 150-180 min. In eight additional healthy controls, glu-cagon was infused at a dose of 6 ng/kg body weight permin. Blood samples were obtained at 15-30 min intervalsduring the infusion.

Analyses and calculations

Glucose was measured by the glucose oxidase technique(17). The methods employed for the determination of plasmaimmunoreactive insulin, plasma immunoreactive glucagon(using Unger antibody 30K), and plasma acidic and neu-tral amino acids have been described previously (18).

The metabolic clearance rate of glucagon (MCRG),' thevolume of plasma completely and irreversibly cleared ofglucagon per minute, was calculated according to theformula (19):

MCRG=Glucagon infused per minute

Glucagon concentration at equilibrium - basal concentration'

1 Abbreziations used in this paper: BSDRG, basal systemicdelivery rate of glucagon; MCRG, metabolic clearance rateof glucagon.

Influence of Uremia and Hemodialysis on Glucagon 723

300 - Uremics

200 _

Glucagon(pg/ml)

Normals100 _\-IP 0.05

* P< 0.05

FIGURE 1 P1after the oraland uremic sthe changes frDespite the fithe uremic gnificantly higthroughout thwere noted withe data forin the figure.

In this calcuglucagon secrstudy; (b) eindistinguishalglucagon remstudied. Recerhave, in fact,a wide ranglevels similaringestion of Enous glucagoglucagon con(glucagon alorof endogenouhormone (20gon (BSDR(leased into tIlated as thetration and Isince our calcnic glucagonthe general cit

Statistical;test (the pairegression anthe text are

' Fisher, MThe kineticspreparation.

RESULTS

Glucagon concentrations in the basal state and afterglucose and alanine administration. In accordance withthe observations of Bilbrey et al. (14), the postabsorp-tive glucagon concentration was increased fourfold inuremic patients (349±32 pg/ml) as compared to con-trols (90±8 pg/ml, P < 0.001). Basal glucagon levelsin uremics were not significantly different in dialyzed(326±34 pg/ml) or undialyzed patients (384±65 pg/ml). Hyperglucagonemia was also observed in theuremic group after glucose (Fig. 1) or alanine ad-ministration (Fig. 2). After glucose ingestion, glucagonconcentration in the uremics fell by 20-25% below post-absorptive levels; however, the postglucose values inthe uremics remained three- to fourfold greater than inthe controls (Fig. 1). After intravenous alanine, plasma

o 30 60 9'0 120 150 180 glucagon concentration increased in both normal andTm(m0n6090 120 150

uremic subjects (Fig. 2). The mean maximal glucagonTime (min) increment (401±135 pg/ml) and the area under theasma glucagon levels in the basal state and glucagon-response curve (15,880+4,875 pg/ml X min)

administration of 100 g of glucose to normal glca onrespon curvea(e,804,7 p/ x mm)ubj ects. P values refer to the significance of were three- to fourfold greater than in the controls*om the basal state (0 time) within each group. (106±13 pg/ml [P < 0.05], and 4,581-+639 pg/ml Xall in plasma glucagon induced by glucose in mm [P < 0.05], respectively). More striking, however,roup, glucagon concentrations remained sig- was th pst e fh uarespons i hev'her in the uremic group than in controls was the persistence of the glucagon response in theie period of study (P < 0.001). No differences uremics. Glucagon increments 50 min after alanine wereith respect to dialyzed vs. nondialyzed uremics; markedly increased in the uremics (221+66 pg/ml vs.both uremic groups are consequently combined

300 _

lation it was assumed that: (a) endogenous-etion remains at basal levels throughout each 250 Uremicsxogenous beef and pork glucagon turnover is Able from that of endogenous origin; and (c) looval is linear over the range of concentrations /IUem1nt studies from our laboratory in normal man 200 I . I

shown that glucagon removal is linear over I '1re of physiological concentrations (including

to those observed in uremia).2 In addition,3lucose during a continuous infusion of exoge- Glucagon IS_n results in a significant decline in plasma- (pg /ml)centration (as compared to subjects receiving-e), thus suggesting continued basal secretion 100s glucagon during the infusion of exogenous). The basal systemic delivery rate of gluca-G), the quantity of endogenous glucagon re- 'Ihe systemic circulation each minute, is calcu- 50product of the basal plasma glucagon concen- No'lMCRG (19). The term BSDRG was chosen Normalsculations exclude that portion of total splanch-secretion removed by the liver before entering 0 30 60 90rculation.analyses were performed with the Student's t Time (min)red t test was used when applicable), linear FIGURE 2 Changes in plasma glucagon after the infusionalysis, and analysis of variance (21). Data in of alanine (0.15 g/kg) in normal and uremic subjects. Glu-

cagon levels were significantly greater in the uremic groupat 50 min (P <0.02) and at 60 min (P <0.05). No differ-

L., R. S. Sherwin, R. Hendler, and P. Felig. ences were noted with respect to dialyzed vs. nondialyzedof glucagon in man: Effects of starvation. In uremics; the data for both uremic groups are consequently

combined in the figure.

724 Sherwin, BastI, Finkelstein, Fisher, Black, Hendler, and Felig

900

Boo

700

600

500

Uremics

Glucagon(pg/m) )

400 I Normals

300 -

200 -

100100 GLUCAGONINFUSION

(3ng/kg/min)

0 30 60 90 120 150 180Time (min)

FIGURE 3 Plasma glucagon concentrations during the in-fusion of exogenous glucagon (3 ng/kg per min) to normaland uremic subjects. The response was the same in non-dialyzed and dialyzed uremics; the data for both uremicgroups are consequently combined in the figure.

40±7 pg/ml in controls, P < 0.02). The prolonged ele-vation of plasma glucagon is compatible with decreasedhormone turnover in uremia. The plasma glucagon re-sponses to glucose and alanine administration were notsignificantly different in the dialyzed as compared tothe undialyzed patients.

Glucagon kinetics in uremic and normal subjects.The continuous infusion of glucagon in the control anduremic patients resulted in stable plasma glucagon con-centrations after 15-45 min (Fig. 3). However, theincrement in glucagon in the uremic group (581±48pg) was two- to threefold greater than in controls (233±13 pg/ml, P < 0.001). Furthermore, the time requiredto reach a stable plateau after glucagon administrationwas longer in the uremics (45 min) than in controls(15 min), thus suggesting decreased hormone degrada-tion. The calculated MCRGwas 5.8±0.6 ml/kg per minin the uremics, which is 58% below that observed incontrols (13.8±0.9 ml/kg per min P <0.001). In con-trast, the BSDRG was not significantly different inuremic patients (1.78±0.20 ng/kg per min) as com-pared to healthy controls (1.34±0.17 ng/kg per min,P > 0.1 ). Chronic dialysis had no effect on the incre-ment in plasma glucagon induced by the glucagon in-fusion (569+41 pg/ml in the dialyzed group and 593

±93 in the undialyzed group), nor did it affect thecalculated MCRG(5.9+0.4 ml/kg per min in dialyzeduremics and 5.8±1.3 in undialyzed uremics). Similarly,BSDRGwas not significantly different in dialyzed (2.03+0.34 ng/kg per min) and undialyzed uremics (1.53±0.21 ng/kg per min, P > 0.1).

Glucose and insulin concentrations in the basal stateand in response to glucose ingestion. The postabsorp-tive glucose concentration was increased in the uremicpatients (93±4 mg/100 ml) as compared to controls(78±1 mg/100 ml, P<0.001). Basal glucose concen-tration was not significantly different in dialyzed (89±4 mg/100 ml) and undialyzed (96+5 mg/100 ml)uremics. Similarly, postabsorptive insulin concentrationwas increased in uremic patients (24±3 vs. 14+1 tU/ml in controls, P<0.005). However, when uremic pa-tients were subdivided into undialyzed (30±6 ,uU/ml)and dialyzed (19±3 AU/ml) subjects, only the undia-lyzed uremics demonstrated significant basal hyperin-sulineimia (P < 0.02).

After oral glucose administration, the plasma glucoseconcentration was significantly increased in the uremicgroup as compared to healthy controls (Fig. 4A). Whenuremics were subdivided on the basis of dialysis, un-dialyzed uremics demonstrated significantly increasedglucose values when compared to dialyzed uremics(Fig. 4B). The improved glucose tolerance in the dia-

lyzed group is also indicated by the fact that the glucosevalues in this group differed significantly from the con-trol subjects only at 120 and 180 min (P < 0.05) (Fig.4B). Plasma insulin concentrations after glucose inges-tion closely paralleled the glucose response. Uremic pa-tients demonstrated significantly increased insulin levelswhen compared to controls (Fig. 4C). However, where-as the glucose-stimulated insulin response was 2- to 10-fold above control levels in the undialyzed uremic groupthroughout the 3-h study, the dialyzed uremics showedonly a delayed, 2- to 3-fold increment in insulin (Fig.4D).

Glucose and insulin responses to intravenous alanine.After alanine administration, the mean maximal incre-ments in glucose and insulin for the uremic group asa whole (7+2 mg/100 and 27+7 AU/ml, respectively),were not significantly different from normal subjects(6±1 mg/100 ml and 15±2 AU/ml, respectively). How-ever, when uremics were subdivided according to dialy-sis, the mean maximal increments in glucose in theundialvzed uremics (12±3 mg/100 ml) tended to begreater than in controls (6±1 mg/100 ml, P< 0.1) orin dialyzed uremics (3±1 mg/100 ml, P < 0.05). Simi-larly, the mean maximal increment in insulin in theundialyzed uremics (43±12 ,uU/ml) was significantlygreater than in controls (15+2 ,uU/ml, P < 0.05). orin dialyzed uremics (11+2 AU/ml, P < 0.05).

Influence of Uremia and Hemodialysis on Glucagon 72.5

A

UREMICS

I. 'L. NORMALSI I .j

-_

I~~~~~

0 60 120 180

TIME (min)

B UNDIALYZEDUREMIC S

1U\// DIALYZED

UREMICS

NORMALS

DUNDIALYZED

UREMICS

DIALYZEDUREMICS

V/ NORAMAALS-..a

0 60 120

TIME (min)ISC

FIGURE 4 Plasma glucose and insulin response to ingestionof 100 g glucose in normal and uremic subjects. In panelsA and C, the responses of the dialyzed (n = 6) and un-

dialyzed (n = 4) uremics are combined, while in panels Band D the two uremic groups are shown separately. Theglucose response of the undialyzed uremics was significantlygreater than the dialyzed group at 60 (P <0.02), 90 (P <0.01), and 120 min (P < 0.05). Similarly, the insulin re-sponse in the undialyzed uremics was greater than in thedialyzed group at 30 (P < 0.02), 60 (P < 0.05), 90 (P <0.005), 120 (P < 0.005), and 180 min (P < 0.02).

Glucose and insulin response to glucagon administra-tion. The increment in plasma glucose concentrationafter intravenous glucagon infusion was two- to four-fold greater in the uremics as a whole, as compared tocontrols (Fig. 5A). However, when the effect of dialy-sis on glucagon-induced hyperglycemia was examined,the undialyzed uremics account for the differences ob-served. Despite comparable increments in plasma glu-cagon in the dialyzed and undialyzed subjects (seeabove), the glucose response to glucagon was increasedabove control levels only in the undialyzed uremics(Fig. 5B). The mean maximal glucose increment inundialyzed uremics (37±9 mg/100 ml) was three- tofourfold greater than in dialyzed uremics (10±1 mg/100 ml, P < 0.02) or controls (10±1 mg/100 ml, P< 0.02). Furthermore, the area under the glucose-response curve in the undialyzed uremics (2,922+882mg/100 ml X min) was significantly increased whencompared to dialyzed uremics (730±215 mg/100 mlX min, P < 0.05) or controls (577±+150 mg/100 mlX min, P < 0.02). For the entire uremic group, a sig-

nificant direct linear correlation was observed between

the maximal glucose response to glucagon administra-tion and the 2-h plasma glucose concentration duringthe oral glucose tolerance test (r = 0.82, P < 0.05).

Changes in plasma insulin after glucagon administra-tion were similar to those demonstrated for glucose. In-creases in the plasma insulin response to glucagon inuremic subjects were accounted for by hyperinsulinemiain the undialyzed group only (Fig. 5C and D). Meanmaximal insulin increments in undialyzed uremics (27±6 ALU/ml) exceeded those of controls (9±1 AU/ml,P < 0.01) and dialyzed uremics (5±1 /LU/ml, P <0.01).

In addition to the 3 ng/kg per min infusion of glu-cagon, the healthy control subjects received infusions

E0

E

w

Co

0

0

CD)

cn

-J

a.

Em

z

-J 15

Cl)

z

4

C,)

-J

a-

o 30 60 90 120 150 0 30 60 90 120 150TIME (min) TIME (min)

FIGURE 5 Changes in plasma glucose and insulin in re-

sponse to the infusion of glucagon (3 ng/kg per min) innormal and uremic subjects. In panels A and C, the re-

sponses of the dialyzed (n = 5) and undialyzed (n = 5)uremics are combined, while in panels B and D, the twouremic groups are shown separately. The increments inplasma glucose were greater in the undialyzed uremics thanin the dialyzed uremics or healthy controls at 45 (P <0.005), 60 (P < 0.05), 90 (P < 0.05), and 120 min (P <0.05). Similarly, the insulin response was greater in un-dialyzed uremics than in dialyzed uremics or normal con-trols at 45 (P < 0.02), 60 (P < 0.02), 90 (P < 0.01), and150 min (P < 0.02).

726 Sherwin, Bastl, Finkelstein, Fisher, Black, Hendler, and Felig

i 25000

a 200E

o 150

-JCD

4 100

.4-JtL 50

500

7E, 375

z-1 250

z

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n4-JIL

k

D

at a rate of 6 ng/kg per min. This rate of infusionresulted in increments in plasma glucagon in the normalgroup which were equivalent to those observed inuremics during the 3 ng/kg per min infusion (Fig. 6).Despite comparable hyperglucagonemia, the glucose risein the undialyzed uremics exceeded that of healthy con-trols (Fig. 6). The mean maximal glucose incrementin the control subjects receiving the 6 ng/kg per minglucagon infusion (16±3 mg/100 ml) was less thanhalf that observed in the undialyzed uremic group re-ceiving the 3 ng/kg per min infusion (37±9 mg/100ml, P < 0.05). The maximal insulin increment in thecontrols during the 6 ng/kg per min infusion (12+3/LU/ml) was significantly below that observed in theundialyzed uremics during the 3 ng/kg per min infusion(27±6, P < 0.05).

Plasma amino acids in the basal state and after gluca-gon administration. Plasma amino acid concentrationsin the basal state and the changes observed after glu-cagon infusion are shown in Table I. Uremic subjectsdemonstrated threefold increases in basal levels of citrul-line and cystine (P < 0.001), as well as elevated taurine(P < 0.05), glycine (P < 0.01), and proline (P <0.001) concentrations. Basal valine (P < 0.005), tyro-sine (P < 0.02), and a-aminobutyrate (P < 0.05) con-centrations were significantly reduced. No differenceswere observed between dialyzed and undialyzed subjectsexcept in the case of glycine. Glycine concentration inthe dialyzed group (548+90 umol/liter) was twice thatobserved in the undialyzed group (249±+18, P< 0.01)or in normal controls (207±16, P < 0.005).

UNDIALYZED UREMICS( 3 no lg /k in )

o 30 60 90 120 150

TIME (min)

O 40 UNDIALYZED UREMICSO (3ngq/kq/min)

30 6 s

U)

0

D 20

NIORMALS4 %I (6ng/kg/min)

a.

0 30 60 90 120 150

TIME (min)

FIGURE 6 Changes in plasma glucagon and glucose in un-dialyzed uremics and in normal controls during the infusionof glucagon at rates causing equivalent increases in plasmaglucagon. Glucagon was infused at a rate of 3 ng/kg permin in the uremics (n = 5) and at a rate of 6 ng/kg permin in the normal controls (n = 8). The glucose responseover the course of the study was significantly greater in theuremics as compared to controls (P <0.02, analysis ofvariance). In addition, the mean maximal increment in glu-cose in the uremics (37±9 mg/100 ml) was significantlygreater than in controls (16±3 mg/100 ml, P < 0.05).

The glucagon infusion (3 ng/kg per min) failed toproduce any consistent changes in plasma amino acidconcentrations in the normal control group, except fora 7% decline in serine (Table I). In contrast, a sig-nificant fall in virtually all amino acids was demon-strable in the uremic group (Table I). The magnitudeof the decrease in alanine (24±2%) exceeded that ofall other amino acids. The amino acid response to glu-cagon was the same in dialyzed and undialyzed uremicpatients. When the glucagon infusion rate was increasedto 6 ng/kg per min in normals to achieve increments inplasma glucagon comparable to the uremics (Fig. 6),plasma alanine concentration still failed to decline inthe normal group (Fig. 7). With the exception of small(10-19%) reductions in glycine, serine, and threonine,the other amino acids were also unchanged by thehigh-dose glucagon infusion.

DISCUSSION

The current data demonstrate that chronic renal failureis associated with hyperglucagonemia in the basal state,after glucose suppression, and after alanine stimulation.Similar results have been previously reported with re-spect to basal and postglucose values (14). Bilbrey etal., however, failed to observe augmented glucagonlevels after protein ingestion (14). Whether the differ-ences observed between protein ingestion and intrave-nous alanine are related to altered protein absorptionin uremia (22), or are due to the different nature ofthe aminogenic stimulus remains to be determined. Thepresent findings extend previous observations, (a) byproviding data on the relative contributions of alteredhormone catabolism and secretion to increased plasmaglucagon levels in uremia, and (b) by evaluating theeffects of uremia and dialysis on the glycemic andamino acid responses to physiological increments inplasma glucagon.

In the uremic group, the MCRGwas decreased by58% as compared to healthy subjects. In contrast, theBSDRGwas unchanged from controls. These data thussuggest that hyperglucagonemia in uremia is a conse-quence of decreased hormonal catabolism rather thanhypersecretion. The techniques employed in the currentstudy cannot exclude the possibility of increased hepaticextraction of glucagon and associated hypersecretion inthe uremic group. However, previous observations inintact man have demonstrated a portal-peripheral gra-dient for glucagon which is no greater than 1.3: 1, in-dicating that the liver is a minor site of glucagondegradation (23). Thus, the comparable BSDRGin theuremic and normal subjects are likely to be a closeapproximation of the true pancreatic secretory rates.

It should be noted that the calculations employed indetermining MCRGassume ongoing secretion of en-

Influence of Uremia and Hemodialysis on Glucagon 727

TABLE IPlasma Concentrations of Amino Acids during Intravenous Infusion of Glucagon

(3 ng/kg body weight per min) in Normal and Uremic Subjects*

Amino acid Condition 0 30 min 60 min 120 min 150-180 mint

Taurine

Threorine

Serine

Proline

Citrulline

Glycine

Alanine

a-Amniobutyrate

Valine

Cystine

Methionine

Isoleucine

Leucine

Tyrosine

Phenylalanine

N§ 33--2Ull 49--6

N 109±t6U 1324±14

N 1014-4U 1004±12

N 158±t16U 285--11

N 29--5U 86±-11

N 207±+16U 399±-65

N 295 ±t22U 309±t25N 16±t2U 8--3

N 203±l11U 150±12

N 99±12U 283 ±-27

N 28±-1U 26±t3N 60±-3U 56--5

N 112--7U 90+9

N 45--3U 33--3

N 49--3U 55±-5

* Data presented(paired t).

as mean ±SE. P values refer to significance of difference from preinfusion values

I Indicates that amino acids were measured at 150 min in uremics and 180 min in normal subjects.§ Indicates normal subjects (n = 10).11 Indicates uremic subjects (n = 10).

Indicates P < 0.05.** Indicates P < 0.01.$1 Indicates P < 0.001.

dogenous glucagon. This assumption is based on indirectevidence in normal subjects infused with glucagon fol-lowed by glucose (20). If this assumption were not

valid, the values for MCRawould be lower both in thecontrols (9.5+0.4 ml/kg per min) and the uremics(3.6±0.4). However, the significant differences be-tween these groups (P < 0.001) would not be appreci-ably altered. On the other hand, if one assumes that

endogenous glucagon secretion is suppressed in controls

but not in uremics, the metabolic clearance rate in theuremics remains significantly reduced when comparedto controls (P < 0.001).

The demonstration that hyperglucagonemia in uremiais associated with decreased hormonal catabolismstrongly implicates the kidney as a principal site ofglucagon degradation. As noted above, the small portal-

728 Sherwin, Bastl, Finkelstein, Fisher, Black, Hendler, and Felig

32 ±t246--6

111±I7132 ±t17

101 ±487 ±t12

161 ±t15282 ± 15

29--484--13

209 ± 17386 ±72307 --31295 ±+31

18--28±t3

2064-12151±+-1394 - 19

283 --23

27±t225 ±t3

59--459--6

111 ±-887 -9

44±t332 -3

48±t356±t6

33±-245--7

110l±7117 416¶101 ±5

83 ±9¶

158±-15264-±-16

29--583 ± 13

210--17353 lt61**

307 --29280--23

16±-27±42

200 ± 12141± 10

91±-16278±-29

28±-222 ±2

58-±353±4-6

109±-881±t7

44±t329±t248±-350±t51¶

31±t247 ±7

1094-8114±-16¶

99 ±i587±ll¶

155± 16261 -16¶

26±483-± 13

207±t16365 -±60¶

306±t27270±-24¶

16±t28±t3

196-± 12133 -l12 T

87±114**267 ±t28

29±-120±t2

54±4550-±7

105 ± 1078i8¶

46±I326±2¶48±t347 -t4

30±t243±O7

104-±7106±14**

94±t5 ¶80 -t±14**

146 ± 14251 ±17¶

23±-t274±-t10**

204 ± 163364-5911294 ±22237 ±21tt

15 ±28 ±3

190 ±l1127 4l31199±49

254+25

30±119 ±3

55 ±444±6tj

106t±975+10**

43+226±+3**

46±244±5tj

Normals(3 ng/kg/mir

- 20-

a

-80-*P05

**P< 0.001

30 60 90 120Time ( mi)

FIGURE 7 Changes in plasma alanine in r4infusion of glucagon in uremic and normalnormal subjects were studied during the inftgon at rates of 3 and 6 ng/kg per min. Theithe uremics was 3 ng/kg per min. The alanidialyzed and undialyzed uremics was the samthe data for the two uremic groups are cofigure. P values refer to the significance cfrom the basal concentration. In the normaboth infusion rates ) there were no significaalanine levels during the infusion.

peripheral gradient for glucagon (23, 24)with an extrahepatic degradative site. Gludative enzymes have, in fact, recently beenin kidney tissue (25 ). Consistent renalglucagon has been observed in the autotrackidney (26). Recent studies in our labora'fractional extraction ratio of glucagon acroin the intact dog and rat of 40%.3 After therenal failure by 75% nephrectomy, a consisttake of glucagon is no longer observed.3renal artery clamping (27) and ureteral ligdogs result in a rapid rise in plasma glucatrast, a decrease in circulating glucagonafter successful renal transplantation inThese data thus indicate that renal functtaken into account in interpreting plasrlevels in a variety of pathological states.gonemia associated with traumatic shockmyocardial infarction (30), diabetic ketoaeand hyperosmolar coma (32) may, in part,by altered renal hemodynamics associated

a.

conditions.Of particular interest were the effects of

dialysis on the glycemic response to exogei

tFisher, M., C. Bastl, R. Sherwin, F. FHayslett, and P. Felig. Unpublished data.

dogenous glucagon. As compared to normal controls,n)7 undialyzed uremics demonstrated significantly increased~: ~i glucose responses to physiological increments in plasma

glucagon, induced by infusion of this hormone. In con-trast, dialyzed uremics responded to exogenous glucagon

/ kg/mi) in a manner comparable to controls (Fig. 5). Further-more, when the glucagon infusion rate was increasedin normals (6 ng/kg per min) so as to achieve incre-ments in plasma glucagon comparable to the uremics,the glucose response remained two- to threefold greaterin the undialyzed uremics (Fig. 6). In addition to theresponse to exogenous glucagon, the glycemic responseto alanine-stimulated secretion of endogenous glucagonwas also greater in undialyzed uremics than in controlsor dialyzed uremics. These data thus suggest that ure-mia is characterized by increased sensitivity to the

15080 hyperglycemic effects of physiological increments inglucagon. This augmented sensitivity to glucagon re-

esponse to the turns to normal after dialysis.subjects. The The importance of altered tissue responsiveness to

ision of gluca- glucagon in uremia is underscored by the differentialinfusion rate in effects of dialysis on glucose tolerance and plasma glu-ne response ine consequently cagon levels. Although, in according with previousmbined in the reports (5, 9-12), dialysis was associated with an im-f the changes provement in glucose tolerance (Fig. 4), plasma glu-L1 subjects (at cagon levels remained unchanged. On the other hand,.nt changes in

a direct linear correlation was observed between the 2-hplasma glucose response to glucose ingestion and the

is compatible glycemic response to glucagon infusion. These observa-cagon degra- tions suggest that increased tissue sensitivity to thedemonstrated glycemic effects of glucagon is an important factor inextraction of uremia-induced glucose intolerance. Improvement inasplanted dog glucose tolerance after chronic dialysis is associatedtory reveal a with a return of glucagon sensitivity to normal. Ittss the kidney should be noted that previous studies involving bolus

induction of injections of 0.15-1.0 mg of glucagon have shown atent renal up- variable glucose response to glucagon in uremic pa-Furthermore, tients (1, 2, 5, 33). However, the data on the BSDRGation (14) in glucagon (less than 2 ng/kg per min in both theLgon. In con- uremics and controls) indicate that such bolus injec-

is observed tions represent pharmacological rather than physiologi-man (28). cal doses of glucagon.

tion must be Regarding the mechanism of augmented glucagonna glucagon sensitivity in uremia, recent studies have demonstratedHypergluca- increased sensitivity to glucagon-induced hyperglycemia(29), acute in circumstances of acute insulin deficiency (34) and

cidosis (31), in insulin-withdrawn diabetics (20). On the other hand,be explained physiological increments in glucagon do not alter glu-

I with these cose tolerance in normal subjects, chemical diabetics,or in juvenile-onset diabetics receiving insulin (20).

F uremia and The current findings of hyperinsulinemia in the undia-nous and en- lyzed uremics (Fig. 5) indicate that hypersensitivity to

xinkelstein, J. glucagon in uremia cannot be ascribed to insulin de-ficiency. The insulin response to oral glucose (Fig. 4),

Influence of Uremia and Hemodialysis on Glucagon 729

intravenous alanine, and exogenous glucagon (Fig. 5)was increased in the undialyzed uremics, yet was asso-ciated with a deterioration in glucose tolerance, sug-gesting insulin resistance. Whether insulin resistance isa cause or a consequence of augmented tissue respon-siveness to glucagon in undialyzed uremics remains to bedetermined. The current data do not exclude the possi-bility that uremia results in alterations in hepatic glu-cagon receptors or in steps subsequent to receptor bind-ing, independent of altered tissue responsiveness toinsulin.

Regarding amino acid concentrations in uremia, inaccordance wvith previous observations (22, 35-37),uremic subjects demonstrated increases in plasma, gly-cine, cystine, citrulline, proline, and taurine, and de-creases in tyrosine, valine, and a-aminobutyrate. Theamino acid pattern in uremia differed from prolongedstarvation in which circumstance hypoalaninemia is astriking find (38), thus it suggests that the alterationsobserved in uremia are not primarily the result ofcaloric deprivation. While increased glycine and prolinelevels are seen in protein malnutrition (39, 40), thethreefold increases in citrulline and cystine in uremicsubjects are not observed in protein deficiency (39, 40).Furthermore, dialyzed uremics were maintained on adiet of normal protein content (80 g). These data sug-gest that nondietary factors contribute to the changesobserved in amino acids in uremia. The increased citrul-line concentration may reflect alterations in urea cycleactivity; elevated arginine levels have also been observedin uremic patients (22, 35-37). Particularly note-worthy are the normal levels of serine in the uremicgroup (Table I). Studies in normal subjects have shownthat the kidney is the principal source of plasma serinewhich is extracted by the liver as well as muscle (41).The failure to observe a fall in serine in uremics sug-gests either a compensatory reduction in extrarenal up-take, stimulation of extrarenal production, or residualrenal release in the uremic group.

Although the infusion of glucagon in the normal sub-jects failed to alter plasma amino acid levels, theuremics demonstrated reductions in virtually all aminoacids (Table I). The glucagon-induced reduction inplasma alanine in the uremic patients exceeded that ofall other amino acids. These results suggest increasedsensitivity to the gluconeogenic effects of glucagon inuremia. Increased amino acid incorporation into glucosehas been observed in perfused rat livers after nephrec-tomy (42). It should be noted that while dialysis nor-malized the glucose response to glucagon (Fig. 5), thehypoalaninemic response to glucagon was comparable indialyzed and nondialyzed patients. These observationssuggest that the glycogenolytic and gluconeogenic re-sponses to glucagon may be independently influenced bythe uremic state.

ACKNOWLEDGMENTSWethank William Sweet, Ralph Jacob, and Andrea Belousfor their technical assistance and Tina Spinelli for her sec-retarial assistance. We are grateful to Kathi Hunt and thestaff of the Clinical Research Center at Yale-New HavenHospital for their efforts in caring for our patients.

This work was supported in part by National Institutesof Health contract NO1-AM-42221, and by National Insti-tutes of Health grants AM 13526 and RR 125.

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Influence of Uremia and Hemodialysis on Glucagon 731