classical experiments

STUDIES OF ANTERIORPITUITARY TISSUE IN VITRO: EFFECTS OF INSULINANDEXPERIMENTALDIABETES MELLITUS UPON

CARBOHYDRATEMETABOLISM*

By CHARLESJ. GOODNERt AND NORBERTFREINKEL

(From the Thorndike Memorial Laboratory and Second and Fourth (Harvard) MedicalServices, Boston City Hospital, and the Department of Medicine, Harvard

Medical School, Boston, Mass., and the Howard Hughes MedicalInstitute, Miami, Fla.)

(Submitted for publication June 24, 1960; accepted October 7, 1960)

Since the classical experiments of Houssay andBiasotti (1), Evans, Meyer, Simpson and Reichert(2), Young (3), and Long and Lukens (4), thediabetogenic potential of pituitary hormones hasbeen amply documented (5). However, the me-tabolism of glucose by the pituitary has receivedbut little examination (6-9), and the authors havebeen unable to find any published studies of thehormonal determinants of hypophyseal carbohy-drate economy. Because of the potential con-tribution of these factors to the endocrine functionof the pituitary, experiments were initiated to de-fine the role of insulin. The studies have demon-strated that the assimilation and disposition of glu-cose by surviving preparations of anterior pitui-tary from normal rats and beef may be enhancedby insulin in vitro. Moreover, it has been shownthat glucose metabolism is significantly reducedin anterior pituitaries excised from rats renderedinsulin-deficient by total pancreatectomy or al-loxanization, and that these hypophyseal derange-ments can be prevented by replacement therapywith insulin in vivo.

METHODS

I. Preparation of tissues for incubation

A. Calf pituitary. Pituitary glands were obtainedfrom male and female calves at the abattoir within 5 to15 minutes after the death of the animals by exsanguina-tion. The whole glands, left in their fascial investments,were placed in plastic bags and transported to the labora-

* This investigation was supported in part by ResearchGrant A-1571 and Training Grant 2A-5060, National In-stitute of Arthritis and Metabolic Diseases, Bethesda, Md.Portions of this work have been published in abstractform: Clin. Res. 1960, 8, 25; J. clin. Invest. 1960, 39, 991.

t Research Fellow of the National Institute for Ar-thritis and Metabolic Diseases, U. S. Public HealthService.

tory in an insulated container at a temperature of 4 to10° C. Upon arrival at the laboratory, the capsules andadherent diaphragma sellae were removed with scissorsand the glands were separated into anterior (AP) andposterior (PP) lobes by blunt dissection in Krebs-Ringer bicarbonate (KRB) at room temperature. Adefinite cleavage plane between the two portions (APand PP) facilitated separation. This was documentedhistologically, and in calf the pars intermedia wvas in-cluded with the AP.

Slices of 0.7 to 1.0 mmthickness were prepared free-hand from calf AP which was held loosely betweenplastic blocks and supported on filter paper moistenedwith KRB. Although it was appreciated that gaseousdiffusion into the center of the slice would probably belimited, thick slices were utilized for this study on thepremise that insulin responsiveness in vitro might be en-hanced by minimizing the cut surface area per unitwveight of tissue. While this work was in progress, areport appeared which supported this assumption in stud-ies of the effect of insulin upon rat spinal cord in vitro(10). Care was taken to pair the slices in control andexperimental vessels with respect to gland topographysince the various adenohypophyseal cell types are notuniformly distributed in the calf pituitary (11, 12).Calf AP was divided into hemilobes which were cut intofour slices. The two slices representing the outer layersof the hemilohes were smaller in diameter, whereas twolarger slices wvere derived from the central portion. Tofill four incubation vessels from a single AP, one outerand one inner slice from opposite hemilobes were intro-duced into each vessel. The total calf AP averaged393 ± 27 mg1 and tissue weights in AP vessels rangedfrom 76 to 140 mg.

B. Rat pituitary. Male albino rats weighing 200 to350 g were obtained from the Charles River BreedingLaboratories (Sprague-Dawley descendants) and main-tained in this laboratory on Purina laboratory pellets. Inall experiments, pituitaries were excised within 1 min-ute following sacrifice of animals by stunning and de-capitation. Individual glands were placed directly inKRB at room temperature and AP and PP were teasedapart with the aid of a dissection microscope (20X).Histological examination demonstrated complete separa-

1 Mean ± standard error (SE).

261

CHARLESJ. GOODNERAND NORBERTFREINKEL

Sal/8AT/ONMEDIA

(ajoomL..)

KR8 L - Rubber Spocer

FIG. 1. MODIFIED REACTION VESSEL FOR INCUBATION OF

SINGLE RAT ANTERIOR PITUITARY. For description, see

text.

tion of AP and PP. The pars intermedia was presentas adherent fragments about the posterior lobe.

For studies of pituitaries from normal fed donor rats,tissues from multiple animals were pooled. Each APwas divided at the midline with a sharp blade and eachhemilobe was a) gently rolled on gauze, b) weighed on a

10 mg torsion balance accurate to 0.01 mg, and c)placed in either a "control" or "experimental" vessel(see below) for incubation. To fill two such paired ves-

sels, a total of 6 to 10 rats was sacrificed serially. Thus,individual vessels contained the equivalent of 3 to 5 an-

terior pituitaries (16.4 to 20.0 mg). In paired vessels,the tissue weights were within 1 mg. On the otherhand, for studies of hypophyseal carbohydrate metabo-lism in rats which had been subjected to experimentaldiabetes or other manipulations prior to sacrifice (seebelow), single anterior pituitaries were incubated indi-vidually. Herein, the intact AP was employed and hemi-section was not instituted.

II. Incubation technique and media composition

In all of the studies, tissues were incubated for 3 hoursat 38° C in a Dubnoff metabolic shaker (shaking rate96 cycles per minute). The suspending medium con-

sisted of KRB, pH 7.4, supplemented with unlabeled (Na-tional Bureau of Standards) and C14-labeled glucose tofinal concentrations of 1.0 to 2.0 mg glucose per ml, and0.8 to 3.0 fuc C14 per ml. The gas phase consisted of 95per cent O, 5 per cent CO2. The various preparationsof radioactive glucose, [i.e., labeled uniformly (C-U) ; or

in the first (C-1) or sixth (C-6) carbon atoms] were

obtained chromatographically pure from several com-

mercial sources.2 For studies of insulin action in vitro

2 Glucose-U-C14 purchased from Tracerlab and New

England Nuclear Corporation, Boston, Mass. Glucose-1-C14 and glucose-6-C14 purchased from Nuclear-Chicago,Ill.

upon calf slices and pooled hemisected AP from normalrats, media were further supplemented with either 1.0 Uper ml (40 jug) of "glucagon-free" crystalline beef in-sulin 3 ("experimental vessels") or 40 jug per ml of crys-talline bovine albumin 4 ("control vessels"). In a fewinstances, albumin was omitted from the "control" sys-tems (see below).

Incubations were conducted in 25 X 50 mmglass weigh-ing bottles sealed with a rubber stopper (13). Tworubber-capped glass vents in the stopper permitted in-troduction of solutions or gases into the sealed vesselsthrough needles. The stoppers also supported smallplastic cups into which NaOH was introduced at theend of incubation for collection of C402 as outlined be-low. In the experiments with calf pituitary slices andpooled hemisected rat AP, the total volume of incubationmedium consisted of 1.2 ml which was directly introducedinto the weighing bottle. For incubation of individualrat anterior pituitaries, the flasks were modified as shownin Figure 1. A small glass cup measuring 14 X 16 mm(inner chamber, Figure 1) was placed in the larger bottle.A rubber ring (cut from rubber tubing) was slippedaround the small vessel to prevent capillary migration offluid between the inner and outer chambers. Intact singleadenohypophyses were incubated in the inner chamberand suspended in a total volume of 0.3 ml KRB. Tominimize evaporative losses in this system and to pro-mote conduction of heat from the Dubnoff bath to theinner chamber, an additional 1.0 ml of isosmotic KRBwas placed into the outer chamber.

Incubation was terminated by introducing 0.2 ml 5 NNaOH into the plastic cups, and thereafter either a)introducing 0.1 ml 1 N HCl directly into the 1.2 ml ofsuspending medium, or b) introducing 1.0 ml of 0.03 NHCl in isotonic NaCl into the inner chamber and 0.1 ml1 N HCl into the outer chamber. Two hours was al-lowed for evolution of C1402 after acidification of themedia.

III. General analytical techniques

A. Glucose. Protein-free filtrates were prepared fromplasma or whole blood by precipitation with Ba(OH) 2+ZnSO4 (14). Plasma was obtained from heparinizedblood collected from the severed neck vessels at sacrifice.In some experiments, whole blood was obtained from thetail vein prior to sacrifice. The filtrates were analyzedfor glucose by the enzymatic calorimetric method ofHuggett and Nixon (15). Aliquots of suspending mediawhich had been incubated both with and without tissueswere similarly precipitated with Ba(OH)2 +ZnSO4, andthe protein-free filtrates were employed for duplicateassay of glucose concentration.

B. Radioactive carbon dioxide. C14O2 evolved duringthe incubation period was precipitated quantitatively with

3 Crystalline zinc insulin (lot no. 499667) assaying25 U per mg was generously provided by Dr. 0. K.Behrens and Dr. C. W. Pettinga of Eli Lilly Company.

4 Armour lot no. T 68204.

262

INSULIN AND ANTERIOR PITUITARY METABOLISM

barium chloride fronm sodium hydroxide and assayed induplicate as barium carbonate, as (lescril)c(l previously(16).

C. Glycogen C14. At the end of incubation, calfAP slices were placed directly in 1 ml of 30 per centKOHand 1 ml of 95 per cent enthanol and autoclavedfor one hour (at 15 lbs 120° C). Carrier glycogen, 12.0mg, was added and glycogen was precipitated by themethod of Good, Kramer and Somogyi (17). The gly-cogen was precipitated a second time and then dissolvedin 2.5 ml of distilled water for 24 hours of dialysis at 40C against three changes of 100 vol of 0.001 M phosphatebuffer (pH 7.4). The dialyzed glycogen solutions werethen decanted and duplicate 1.0 ml aliquots were de-posited on weighed cup planchets for radioactive assayin a gas-flow counter. Mass absorption corrections wereapplied.

The dialysis method was validated for calf anteriorpituitary by comparing the specific activity of gly-cogen obtained by this method with the specific activityof thrice recrystallized glucosazones, prepared from anacid hydrolysate of the parent glycogen solution. Infour experiments, the glucosazone and dialysis meth-ods yielded similar results. Control studies also indi-cated that glycogen radioactivity was not measurablycontaminated with cerebrosides (18).

D. Lipid C14. In experiments with pooled AP tissuefrom normal rats, total chloroform: methanol-extractablelipids (CHCl3: MeOHlipid C"4) (19) were obtained forradioactive assay as follows. After incubation, the tis-sues were placed directly into chloroform: methanol 2: 1(vol/vol) and homogenized. The mixture was centri-fuged, the supernatant fluid decanted, and the sedimentre-extracted in warm (60'C) chloroform: methanol.The combined extracts were washed three times withisotonic NaCl (19) and assayed at infinite thinness oncup planchets in a gas-flow counter. For calf AP and asmall number of experiments with rat AP, tissue lipidswere fractionated into nonsaponifiable lipids, and longchain fatty acids (20). The nonsaponifiable lipid extractwas reduced in volume and quantitatively plated for ra-dioactive assay in a micromil-window gas-flow counter.The fatty acids were evaporated under reduced pressureand redissolved in toluene, 0.4 per cent 2,5-diphenyl-oxazole (PPO) and 0.005 per cent 1,4-bis-2- (5-phenyl-oxazolyl)-benzene (POPOP)5 solution for radioactiveassay in a Packard Tri-Carb liquid scintillation counter.6

IV. Expression of results

Net glucose assimilation in micrograms of glucoseper milligram of tissue was calculated from the initialtissue wet weight and the total micrograms of glucosedisappearing from the media during 3 hours of incuba-tion. The latter value was calculated on the basis of thedifference between the final content of glucose in ex-

5 Purchased from Pilot Chemical Co., Watertown,Mass.

6 Packard Instrument Company, Inc., La Grange, Ill.

perimental vessels and in vessels which had been incui-bated without tissue.

Disposition of glucose-C14 was expressed as microgramsof glucose-C14 per milligram of initial wet tissue weightand calculated on the basis of the specific activity of theglucose in the initial suspending medium and the recov-ered C14. Initial specific activity was determined for eachexperiment by separate chemical and radioactive assayof the media in duplicate blank vessels. Sufficient countswere collected to reduce random counting error to lessthan ± 1 per cent for standards, C1402, and glycogen-C'4,and to less than + 3 per cent for the lipid fractions.Since the authors were unable to demonstrate glucose-6-phosphatase activity in calf anterior pituitary tissue (21),the values for glucose assimilation and initial specific ac-tivity of the medium could be employed to assess netassimilation of glucose radioactivity.

The statistical significance of the effects of insulin invitro was evaluated by testing the ratios of the observedvalues in paired "control" and "experimental" vessels ofindividual experiments for deviation from a ratio of unity.The common logarithms of the ratios were employed andStudent's t values were calculated from the mean andstandard error of the mean of the log form of these ra-tios (22). Such analysis of relative changes effected byinsulin made it possible to pool data from multiple ex-periments conducted with varying concentrations of glu-cose in the medium.

TABLE I

The effect of insulin in vitro upon glucose assimilation byslices of calf anterior pituitary

Reaction mixture: 76 to 140 mg tissue slices suspended in 1.2 ml KRBcontaining 2.0 mg glucose per ml and either 1.0 U, 40 Ag, insulinper ml (+) or 40 Mg crystalline bovine albumin per ml (-).

Incubation: 3 hours, 380 C.Glucose assimilation

Gland (-) (+)

9g/mg1 3.282 4.073 5.694 4.735 8.496 6.207 5.508 5.449 1.34

10 5.7411 1.5312 3.2713 3.7614 4.7615 5.0016 29717 3.79

3.654.797.257.947.587.817.016.352.085.931.495.005.314.886.484.004.30

Insulin effect

+/ -X10o*111.3117.7127.4167.9

89.3126.0127.5116.7155.2103.3

97.4152.9141.2102.5129.6134.7113.5

Mean + SE 124.4 i 4.5p 0.001

* Insulin effect: results observed in insulin-containingsystems (+) expressed as a percentage of the values incontrol (-) vessels. By this convention, 100 denotes noeffect.

263


TABLE, 1I

Mietabolism of glucose- U- C11 by ut/ianterior pituitary

Reaction mixture: as in Table I; glucose 2.0 mg per ml labeled with0.8 to 1.2 Asc glucose-U-C'4 per ml.

Incubation: as in Table I.

Glucose-U-C04 (jsg/mg) to:C02 Glycogen Non-sap. lipid Fatty acid

Glandno.* (-) (+) (-) (+) (-) (+) (-) (+)

6 1.12 1.35 0.72 1.01 0.0028 0.0040 0.025 0.417 0.76 1.17 0.64 0.91 0.0024 0.0045 0.036 0.0428 0.81 1.17 0.77 0.83 0.0027 0.0040 0.044 0.0589 0.24 0.33 0.14 0.21 0.0006 0.0007 0.0026 0.0031

10 0.85 0.85 0.76 0.82 0.0032 0.0029 0.021 0.03111 0.41 0.36 0.20 0.21 0.0011 0.0008 0.0048 0.0061

* Gland numbers as in Table I. Values for total glucose assimila-tion from the suspending medium in the absence (-) and presence ( +)of insulin are shown in Table I.

RESULTS

I. Eff ects of added insulin upon carbohydratemetabolism of surviving anterior pituitary tis-sue from normal animals

A. Calf. Seventeen separate experiments wereperformed in which slices of adenohypophysesfrom slaughterhouse calves were incubated for 3hours in 1.2 ml of media containing 2 mg glucoseper ml (Table I). Under these conditions, netassimilation of glucose from the suspending mediaaveraged 4.44 + 0.43 I.tg per mg tissue in "control"vessels. With 13 of the 17 glands, glucose up-take was increased at least 10 per cent above con-trol values by the addition of 1.0 U of insulin perml. For all experiments, the induced augmenta-tion of glucose assimilation averaged 24.4 per centand was highly significant (p = 0.001, Table I).

In 12 of the 17 glands, intracellular dispositionof some of the assimilated glucose was evaluatedwith glucose-C14. Uniformly labeled glucose wasused in six instances (i.e., glands 6 through 11).Herein, C1402 was employed as an index of oxi-dative degradation, and anabolic utilization was as-sessed on the basis of the incorporation of radio-activity into glycogen, nonsaponifiable lipids, andfatty acids. Results are summarized in Table II.In the four experiments in which insulin promotedglucose uptake (i.e., 6 through 9), oxidative de-carboxylation, glycogenesis and lipogenesis werealso augmented. Contrariwise, in glands 10 and11, in which insulin did not significantly affect theassimilation of glucose, uniform effects upon thedisposition of glucose-U-C'4 were not observed.

In six experiments (i.e., glands 12 through 17),differentially labeled preparations of radioactive

glucose were emnployed (Trable LII). The exist-ence of the phosphogluconic acid pathway for glu-cose oxidation in anterior pituitary tissue wasdemonstrated by the routine evolution of greaterquantities of C1402 from glucose labeled in thefirst carbon than from glucose labeled in the sixthposition. In control vessels, C-1/C-6 ratios forC1402 averaged 2.04 + 0.17. Addition of insulincaused an average 14.8 per cent increase in C1402from glucose-i-C14 (p = 0.1) and an average50.9 per cent increase in C140, from glucose-6-C14 (p 0.01, Table III).

B. Rats. Eighteen experiments were performedwith pooled tissues from normal fed rats. Pairedgroups of hemisected anterior pituitaries were in-cubated for 3 hours in 1.2 ml of media containing1.0 to 2.0 mg of glucose per ml and 1.1 to 2.6 ,tcglucose-U-C14 (Table IV). Results from all ex-periments were combined in order to assess thestatistical significance of changes effected by sup-plementing suspending media with 1.0 U of insu-lin per ml. In the presence of insulin, the meanassimilation of glucose was 16.7 per cent greaterthan control values (p = 0.02, Table IV). Con-comitantly, the evolution of C1402 from glucose-U-C14 was increased an average of 6.4 per cent(p = 0.02) and the mean incorporation of radio-activity into total chloroform-methanol extractablelipids was augmented 19.0 per cent (p = 0.01).

II. (Carbohydrate metabolism of surviving anteriorpituitary tissue from insulin-deficient rats

In this series of experiments, rats were sub-jected to various manipulations in vivo designed

TABLE III

Ml'etabolism of glucose-i-C'4 and glucose-6-C'4 bycalf anterior pituitary

Reaction mixture: as in Table I; glucose 2.0 mg per ml labeled with 1.1and 1.3 1c per ml of glucose-l-CI4 and glucose-6-C'4, respectively.

Incubation: as in Table I.Control (-) Insulin (+)

C'402 (;Ag/mg) C'402 (Jsg/mg)Gland Ratio Ratio

no.* C-1 C-6 C-1/C-6 C-1 C-6 C-1/C-6

12 2.04 1.34 1.52 2.74 2.39 1.1413 2.64 1.55 1.70 3.31 2.10 1.5814 1.62 0.686 2.37 1.77 1.12 1.5815 1.90 0.811 2.34 2.02 1.57 1.2916 1.01 0.552 1.83 1.49 0.794 1.8817 1.68 0.679 2.48 1.34 0.737 1.82

Mean+SE 2.04 40.17 1.55 40.12

C-1 (-)vs C-1 (+) p>0.1C-6 (-) vs C-6 (+) p <0.01C-1/C-6 (-) vs C-1/C-6 (+) p <0.02

* Gland numbers as in Table I.

264


TABLE IV

The effect of insulin in vitro upon the metabolism of glucose-U-CUC by rat anterior pituitary

Reaction mixture- 16 to 20 mg of paired, hemisected anterior pituitaries from normal fed rats suspended in 1.2 ml KRBcontaining 1.0 to 2.0 mgglucose per ml. Glucose labeled with 1.1 to 2.6 ,uc of glucose-U-C"4. Media further supplemented with either 1.0 U, 40 Mg, insulin per ml (-+)or 40 Msg crystalline bovine albumin per ml (-).

Incubation: 3 hours, 380 C.Glucose

assimilation

( -) ( +)

CHC13-MeOHInsulin* C1402 Insulin* lipid C'4

effect effect(+/-X100) (-) (+) (+/-X100) (-) (+)

Insulin*effect

(+/-X 100)

?ng,l^nl1.0 it 7.2

2t 6.83t 9.14t 10.45 10.26 10.3

1.1 7 11.88 10.7

1.5 9 13.510 13.1

2.0 11 20.412 19.813 16.714 15.315 21.116 8.217 21.218 11.4

g, fmg9.0 125.0

10.7 157.410.9 119.810.5 101.0

9.2 90 210.4 101.014.7 124.614.6 136.416.6 123.013.9 106.116.6 81.416.9 85.4

1 25.7 153.916.9 110.521.5 101.918.8 229.324.3 114.6

t 12.4 108.8

TMean i SE 116.7 ± 6.0p 0.02

* Insulin effect: results observed its insulin-containing sysvessels (-).

t Albumin omitted from control vessels.t Fatty acids.

to alter levels of circulating insulin. Thereafter,anterior pituitaries from individual rats were ex-

cised and separately incubated in 0.3 ml KRBcontaining 2.0 mg glucose per ml, 3.0 ptc glucose-U-C14 per ml and no supplemental albumin or in-suilin. Adenohypophyses were examined in theintact state, and were not hemisected as in theabove studies with pooled tissues from normalanimals.

A. Rats with total pancreatectomy. Total (99.5per cent) pancreatectomy was performed as de-scribed by Scow (23) upon rats weighing be-tween 220 and 330 g and fasted from 12 to 20hours prior to operation. Comparable rats,matched by weight in individual experiments, were

subjected to either: a) sham pancreatectomy, con-

sisting of laparotomy and exposure of stomach,liver, duodenum and spleen to air for a similar pe-

riod of time; or b) partial pancreatectomy, remov-

ing only the "first portion" (23) of the pancreas.

Additional control groups were prepared byremoving a short section of bile duct to producebile peritonitis or by removing 4.0 ml of blood

temns (+) expressed as a percentage of the values in control

from the aorta and injecting this blood into theperitoneal cavity to produce conditions of intra-abdominal hemorrhage with shock.

All rats were given 5.0 ml of saline and 12,000U of crystalline penicillin subcutaneously afteroperation. The rats were maintained in indi-vidual metabolic cages until sacrifice, 20 hoursfollowing surgery. At that time, anterior pitui-taries were excised and incubated individually.

To preclude the effects of fasting in the post-operative period, and hypoglycemia in animalswhich were treated with insulin (see below), glu-cose was administered to all of the rats subcutane-ously. A total dose of 0.5 g glucose per 100 g

body weight was administered as 10 per cent glu-cose in water in three divided doses at 6-hour in-tervals during the 20-hour postoperative period.The last injection was routinely administered 1 to2 hours prior to sacrifice.

Some of the pancreatectomized animals were

treated with insulin during the postoperative pe-

riod. Therapy consisted of 0.5 U of protaminezinc insulin and 0.5 U of regular insulin subcu-

Mediumglucoseconcen- Experi-tration ment

Ag/mg1.60 1.461.30 1.432.14 2.572.12 2.122.45 2.163.14 3.103.09 3.012.76 3.462.52 2.571.98 2.234.14 4.873.76 3.793.78 3.843.81 3.862.91 3.403.11 3.593.65 3.813.19 3.89

91.3110.0120.1100.0

88.298.797.4

125.4102.0112.6117.6100.8101.6101.3116.8115.4104.4121.9

106.4 ± 2.40.02

Mg/mg

0.0431 0.056T0.039t 0.055T0.165 0.1930.146 0.1800.186 0.2050.220 0.2520.224 0.3000.251 0.3520.212 0.2610.220 0.1800.200 0.2730.192 0.2010.219 0.2440.179 0.2010.200 0.2570.210 0.2210.181 0.2330.203 0.234

130,2141.0117.0123.3110.2114.5133.9140.2123.1

81.8136.5112.3111.4112.3128.5105.2128.7115.3

119.0 :1 3.30.01

265


SHAMPoncreotectomy Ponc.

TOTALPanc.

+ Insulin P,

00

PARTIALPonc.

FIG. 2. TOTAL ASSIMILATION OF GLUCOSEAND MICRO-GRAMS PER MILLIGRAM OF GLUCOSE-U-C14 OXIDIZED TOC"O°2DURING INCUBATION OF SINGLE ANTERIOR PITUI-TARIES REMOVEDFROM RATS 20 HOURS AFTER SURGICALMANIPULATION. Mean glucose assimilation is indicatedby the total heights of the bars; the shaded areas de-note mean values for glucose oxidation to CO2. Indi-vidual values for these parameters are indicated by theopen and closed symbols, respectively. The single circledvalue in the totally pancreatectomized, insulin-treatedgroup represents the result in the animal in whom nor-moglycemia was not restored by therapy (see text).Mean values for the percentile reductions in glucose uti-lization by the totally pancreatectomized rats and thestatistical significance of these changes are indicated onthe illustration. Numbers in parentheses indicate thenumber of animals in each category.

taneously at 5 hours and 0.1 U of regular insulinat 18 hours after operation.

Measurements of plasma glucose were employedto assess the severity of the experimental diabetesand the adequacy of the replacement therapy withinsulin. Values were obtained 5 hours followingsurgery (i.e., prior to the administration of thefirst dose of glucose and before the exhibition ofinsulin in the insulin-treated group) and at sacri-fice (i.e., following three doses of glucose and in-sulin therapy in some animals). Plasma glucoseaveraged 272 ± 33 mg per 100 ml [4]7 in un-treated pancreatectomized rats 5 hours after oper-ation and 762 + 110 mg per 100 ml [15] at sacri-fice following three doses of glucose. In five ofsix similar pancreatectomized rats (mean plasma

7 Throughout this paragraph, the blood sugar valuesgiven are expressed as the mean ± standard error in therespective groups. Brackets denote the number of ani-mals in each experimental group from which blood was

sampled. Five-hour specimens of blood were obtainedfrom the tail vein. Bloods at sacrifice were collectedfrom the neck following decapitation of the animals.

glucose 270 + 29 [6], 5 hours postoperatively),the exhibition of insulin in association with glu-cose reduced blood sugar to an average of 75 ± 11mg per 100 ml [5] at sacrifice. Insulin therapywas inadequate in the sixth animal and final bloodsugar was 187 mg per 100 ml. Significant ab-normalities in blood sugar were not observed atany time intervals in the partial or sham pancrea-tectomized groups. Respective values for bloodsugar in these groups were 84 mg per 100 ml [2]and 58 ± 4 mg per 100 ml [5] 5 hours followingsurgery, and 107 ± 5 mg per 100 ml [4] and102 + 6 mg per 100 ml [13] at sacrifice.

Patterns of carbohydrate metabolism observedduring the in vitro incubations of individual an-terior pituitaries are summarized in Figures 2 and3. In untreated pancreatectomized rats, total as-similation of glucose, oxidation of glucose-U-C14(Figure 2) and conversion of glucose-U-C14 tofatty acids (Figure 3) by AP, were significantlyreduced as compared to values obtained with AP

9g /mg.08F 0o Fnttv -1.07 -

.06F

.05 F.041-

.03 F

.02 F

(6)

(6)

8

0

.01 F

rui l y mauabNon-saponifiable Lipid

i. .0

SHAM TOTALPANCREATECTOMY PANCREATECTOMY

FIG. 3. INCORPORATION OF GLUCOSE-U-C1 INTO NON-SAPONFIABLE LIPIDS AND FATTY ACIDS DURING INCUBATIONOF SINGLE ANTERIOR PITUITARIES REMOVEDFROMRATS 20HOURSAFTER SURGICAL MANIPULATION. The total heightsof the bars represent the sum of the micrograms per milli-gram of glucose-U-C14 recovered as fatty acids (diagonallines) and nonsaponifiable lipids (cross-hatched). Meanvalues for the percentile reductions in lipogenesis in to-tally pancreatectomized rats and the statistical significanceof these changes are indicated on the illustration. Num-bers in parentheses denote the number of animals in eachcategory.

266

48%'A .% ^


TABLE V

The effect of severe operative trauma upon the glucose metabolism of rat anterior pituitary

Reaction mixture: single intact APobtained from rats 20 hours after operation and suspended in 0.3 ml KRBcontaining 2.0 mgglucose per ml labeledwith 3.0 jsc glucose-U-C14 per ml.

Incubation: 3 hours, 380 C.Operative group Rat Glucose assimilation C1402

A. Pancreatectomized ratsPancreatectomized +

ComplicationsPancreatectomized +

No complications

;sg/mg

Mean i4: SE 5.63 + - 1.23 [7]*

Mean i SE 4.51 + - 1.65 [4]

ag/mg

2.16 i 0.14 [10]

2.32 ±t 0.15 [5]

1234

In tra-abdomninalhemorrhage

Sham

1

234

1

23

9.788.57

11.259.09

Mean i: SE 9.67 + 0.58

11.268.75

10.0011.46

Mean ± SE 10.37 + 0.63

9.538.45

10.24Mean i SE 9.41 + 0.52

* Number of animals in brackets.

from sham-operated animals. The values for thesethree parameters of glucose metabolism were re-

duced to 71 (p = 0.01), 58 (p = 0.001) and 48(p = 0.01) per cent of the control values, respec-

tively. Reduction of glucose metabolism was notobserved with AP derived from the partially pan-

createctomized rats or the five pancreatectomizedrats in which replacement therapy with insulinhad been adequate (Figure 2).

Many of the pancreatectomized animals dis-played operative complications consisting of bileperitonitis or intra-abdominal hemorrhage. Al-though these phenomena did not appear to alterAP metabolism in pancreatectomized animals(Table V, A), separate experiments were per-

formed to assess the effects of operative complica-tions independent of disturbances in carbohydratemetabolism. Bile peritonitis or intra-abdominalhemorrhage with shock was produced in normalanimals, and, after 20 hours, AP glucose metabo-lism was compared with that of sham-operatedrats (Table V, B). Despite oliguria and prostra-tion comparable in severity to that seen in pan-

createctomized animals with operative complica-tions, the AP metabolism of glucose was indistin-guishable from that of the concurrently examinedsham-operated control population. Thus, the ob-served depression in the metabolism of glucose byAP of pancreatectomized rats cannot be ascribedto operative trauma or shock.

The occurrence of operative complications intwo-thirds of the pancreatectomized rats also af-forded a means of assessing whether the derange-ment of pituitary metabolism was a primary con-

sequence of hypophyseal insulin deprivation or

whether it was secondary to generalized metabolicdisturbances (i.e., acidosis, ketosis, triglyceridemiaand so forth) resulting from insulin deprivationelsewhere. Gross stigmata of aberrant lipid me-

tabolism (24) were not so pronounced in theanimals with operative complications and surgicalshock (25). Correlations were attempted byvisually grading 0 to 4 +: a) fatty infiltration ofthe liver, b) turbidity of plasma, and c) semi-quantitative chemical estimation of plasma "aceto-acetic acid and acetone" (26). The metabolism of

B. Control ratsBile peritonitis 4.48

4.313.993.924.18 i 0.13

3.883.984.174.144.04 i 0.06

3.254.113.783.71 i 0.25

267


glucose by excised AP did not correlate in severitywith any of these indices of generalized metabolicabnormalities.

In the above studies of experimental diabetes,values for glucose assimilation by AP from sham-operated control animals (Figure 2 and Table V)were considerably lower than the glucose uptakesobserved during incubation of pooled hemisectedAP from normal fed rats in media similarly con-

taining 2 mg per ml of glucose (Experiments 11-18, Table IV). The reasons for this are notimmediately apparent, especially since comparabledifferences did not obtain in the evolution of C1402(Figure 2 and Table V vs Table IV). However,in any analysis; it should be noted that experi-mental conditions were not identical. Thus, thestudies differed not only in the dietary and manip-ulative preparation of donor animals but also inthe volume of suspending medium (i.e., 0.3 vs 1.2ml), in the nature of suspending medium (i.e.,plain KRB vs KRB+ 40 ,/g albumin per ml),and in the status of the tissues (i.e., intact vs hemi-sected AP). That such subtle factors might in-fluence assimilative function is suggested by thedemonstration by Roberts and Keller (7) of ap-

preciable anaerobic glycolysis in rat AP. In any

event, the findings would suggest that normalvalues for glucose assimilation by surviving prep-

arations of AP can only be defined within a rigidframework of given in vitro experimental con-

ditions.B. Alloxanized rats. Chronic diabetes mellitus

was induced in rats by the subcutaneous adminis-tration of 125 mg alloxan monohydrate per kgbody weight after a 24-hour fast. Six weeks afteradministration of alloxan, 12 diabetic rats were

paired by weight and fasting blood glucose con-

centration (Table VI). Thereafter, all rats were

allowed food ad lib. and one member of each pairwas treated with insulin for the 7 days precedingsacrifice. Treatment consisted of single daily in-jections of 1.0 to 3.0 U of protamine zinc insulinper 100 g body weight adjusted to prevent urineglucose concentrations above 0.5 per cent. Twohours prior to sacrifice, the treated rats were given

4.0 U of regular insulin subcutaneously. Theuntreated rats gained little weight and exhibiteduniformly high plasma glucose levels at sacrifice.The treated animals gained an average of 10 g

body weight per day and had normal or moder-ately hypoglycemic levels of blood glucose at sacri-fice (Table VI).

At the end of the treatment period, the anteriorpituitary glands from all of the rats were incubatedindividually with glucose-U-C14. The mean glu-cose assimilation and oxidation of glucose-U-C14

TABLE VI

The metabolism of glucose by anterior pituitary in untreated and treated alloxan-diabetic rats

Reaction mixture: as in Table V.Incubation: as in Table V.

Plasma glucose Metabolism of glucose-U-C14 by APBody wt

Initial Sacrifice GlucoseGroup Pair Initial Sacrifice (fasting) (fed) assimilation C1402

g g mg% mng% pg/mg pg/mgAlloxan:untreated 1 230 235 452 754 6.34 2.84

2 325 375 358 498 8.20 2.713 270 310 340 723 11.14 2.954 330 325 312 558 6.71 3.115 355 385 180 526 6.73 2.826 370 385 161 481 6.41 3.08

Mean i SE 313 336 301 590 7.59 -+ 0.76 2.92 ±t 0.07

Alloxan:treated 1 300 375 423 72 11.95 4.06

2 250 335 370 40 11.84 3.083 360 400 336 49 9.62 3.834 225 330 292 74 7.20 3.845 335 400 231 36 9.49 3.606 355 425 148 38 8.34 3.57

Mean =1: SE 304 378 300 52 9.74 i 0.77 3.66 i 0.14

Untreated as %of treated 78 80p 0.1 0.001

268

INSULIN ANDANTERIOR PITUITARY METABOLISM

to C1402 by AP in untreated rats were 78 and 81per cent, respectively, of the corresponding valuein the treated group. The difference in C140production was statistically significant (p = 0.001)

In this, as in all other studies, metabolic activitiin vitro has been related to a unit of mass, i.e.milligrams of anterior pituitary. The validity ocomparing glands of different weight in this fashion was examined by plotting oxidation of glucoseto C1402 as a function of total AP weight (Figur(4). This indicated that activity per unit masswithin each experimental group remained rela-tively constant despite wide variations in pituitarymass. Thus, at any single level of AP weight, thetotal activity of AP from treated or sham-operatedanimals exceeded the values observed in untreateddiabetic rats. Since AP: total body weight ratiosin the animals with experimental diabetes mellitusdid not significantly deviate from normal, it maybe inferred that reduction of activity per unit massrepresented a valid index of reduction in totalpituitary activity.

DISCUSSION

In establishing that a tissue is responsive toinsulin and that the responsiveness is of physio-logical significance, multiple criteria should befulfilled. First and ideally, one should be able toelicit a direct effect with insulin in vitro. Thisdesideratum need not always obtain. For ex-ample, although liver slices do not usually respondto added insulin (27, 28), mounting evidencewould indicate that the hormone can directlymodify hepatic function in the intact animal (29,30) or in the perfused liver (31). Furthermore,even when an action of insulin can be demon-strated in vitro, the relationship between dose andresponse need not equate with sensitivity to insulinin vivo. Many tissues contain soluble insulin-degrading enzymes which are leached into thesuspending medium during incubation of cut prep-arations (32, 33). Recent work in this laboratoryhas indicated that the resultant extracellular pro-teolysis of the added insulin diminishes its mani-fest effectiveness upon the incubated tissue (34).Moreover, besides such leaching artifacts, onemight anticipate that the response of an insulin-sensitive tissue to exogenous insulin in vitro couldbe inversely conditioned by its exposure to endo-genous insulin prior to excision from the donor

* Sham Op.x Alloxan Diabetes+ Insulin %

(;14 v Alloxan Diabetes02 o Total Pancreatectomy

(sig/mg )

5.0 _

4.0

3.0

2.0

1.0

* 0 * .Jx * Mean (*,X)

0 A X

o ° V Mean (o)_O

00

0

0 0

0

3 5 7 9 11TOTAL WETWGT. OF ANT PITUITARY (mg)

FIG. 4. THE RELATIONSHIP BETWEENTOTAL ANTERIORPITUITARY WEIGHT AND OXIDATION OF GLUCOSE-U-C1 TOC'°2 PER MILLIGRAM OF INCUBATED ANTERIOR PITUITARYTISSUE. Symbols denote individual values in the sham-operated, diabetic, and treated diabetic categories. Hori-zontal lines denote mean values for glucose oxidation inthe various groups.

animal. In the present studies, experimental de-sign was influenced by these considerations. Thus,because "insulinase" (35) and other proteases(36) are present in anterior pituitary, and becausehypophyseal vascularity may be sufficient to effectnear maximal delivery of insulin to AP in theintact animal, "supramaximal" doses of 1 U perml were employed for investigation of insulinaddition in vitro. Precise dose-response relation-ships were not examined. Rather, attempts weremade to assess only whether anterior pituitary isor is not capable of directly responding to insulin.Within this framework, unequivocal effects wereobtained with slices of calf AP, and smaller, al-though still statistically significant, changes wereobserved with pooled hemisected AP from normalfed rats. Insofar as added insulin definitely af-fected the carbohydrate metabolism of survivingpituitary tissue, the in vitro studies demonstratedthe existence of adenohypophyseal componentscapable of direct hormonal modification. How-ever, direct response to pharmacological quantitiesof insulin need not connote physiological signifi-cance nor justify extrapolations concerning thecontribution of insulin to the normal function ofthat tissue in the intact animal. Such conclusionsrequire the demonstration of metabolic defects inthe "insulin-responsive" tissue following with-drawal of insulin in vivo.

269


Accordingly, to fulfill this second criterion, pitu-itaries from animals with experimental diabeteswere examined. Carbohydrate metabolism wasreduced 30 to 50 per cent below control values inanterior pituitaries derived from rats renderedacutely aninsulinemic by total pancreatectomy.The magnitude of this derangement approachesthe degree of impaired glucose utilization whichhas been observed in such classically insulin-responsive tissues as diaphragm (37, 38) oradipose tissue (39) from diabetic rats. Further-more, the defect correlated best with the degreeof insulin lack rather than of operative trauma.Thus, it was not observed in partially pancreatec-tomized animals or in rats with surgically in-duced shock or hypovolemia in which pancreaticstructure was preserved. Moreover, in the al-loxanized animals in which absolute insulin de-ficiency was not achieved (as judged by theirlimited inanition and their survival in the un-treated state), reduction of pituitary carbohydratemetabolism was not as profound. It would seemunlikely that the hypophyseal "biochemical les-ions" in the diabetic animals represented merelysecondary consequences of ketonemia, acidosis orlipemia, since significant differences could not bedemonstrated between animals in which total pan-createctomy was uncomplicated, and animals inwhich circulatory collapse precluded the full devel-opment of post-pancreatectomy ketogenesis (25)or lipid mobilization. Indeed, the manifest asso-ciation between insulin deprivation in vivo anddeterioration of pituitary carbohydrate metabolismreinforces the significance of the small effectswhich were obtained by directly adding insulin tonormal rat AP in vitro.

Theoretically, for any given tissue, the thirdcriterion of physiological insulin-responsivenessshould consist of the correction of metabolic de-fects by the in vivo administration of insulin toinsulin-deficient animals. In the present studieswith anterior pituitary, this objective was alsofulfilled. Exhibition of exogenous insulin withinhours following total pancreatectomy completelyprevented the decline of hypophyseal carbohydrateeconomy. Moreover, in animals with chronichypoinsulinism, i.e., the alloxanized rat, therapywith insulin enhanced the metabolic performanceof pituitary tissue. Although precise temporalrelationships were not fully documented, the re-

versibility would indicate that permanent altera-tions in the carbohydrate metabolism of the ante-rior pituitary did not result from 6 weeks ofpartial insulin deprivation.

Thus, by the most rigid criteria, it would appearthat the anterior pituitary must be included in thegrowing list of insulin-responsive tissues (40).Indeed, the present demonstration that intrahypo-physeal utilization of glucose will respond to in-sulin in vitro, deteriorates following insulin with-drawal in vivo, and can be restored by insulinadministration to insulin-deficient animals in vivo,would suggest that the anterior pituitary must beconsidered "diabetic" in the syndrome of diabetesmellitus. For the moment, translation of the find-ings into more definitive implications for normalhomeostasis within the pituitary or pathologicalaberrations in spontaneous diabetes mellitus mustbe deferred. First, since the cellular populationof the anterior pituitary is not homogeneous, itremains to be defined which cell types are respon-sive to insulin. Possibly, only a limited proportionmight be affected or response-thresholds of variouselements might differ considerably. That somecellular components may be altered is suggestedby the reported occurrence of basophilic changesin the pituitaries of totally pancreatectomized dogs(41). Second, the precise relationship betweenhypophyseal carbohydrate metabolism and intra-pituitary turnover of hormones remains to beelucidated. By analogy to other systems (42-44),the pathways which have been described in thepresent studies for the storage of glucose, themobilization of glycogen, and the oxidation ofglucose-6-phosphate within the pituitary couldsubserve important functions in selective or gen-eralized hormonogenesis. Derangement of thesepathways and diminished energy generationthrough impaired oxidation of glucose might notonly alter the balanced biosynthesis of knownpituitary hormones, but could even promote theformation of abnormal, biologically potent proteinor peptide principles. However, elaboration ofanterior pituitary hormones also entails storage,release, and responsiveness to peripheral as wellas to suprahypophyseal regulators. It is conceiv-able (45) that any or all of these processesmight be conditioned by pituitary carbohydratemetabolism.

At present, there seems to be no compelling

270

INSULIN ANDANTERIOR PITUITARY METABOLISM

clinical evidence for derangement of pituitar3function in spontaneous diabetes mellitus. On theother hand, the relatively crude methods whichhave been available for such evaluation do notconstitute compelling reasons for discarding thepossibility. The present studies with laboratoryanimals highlight the critical need for re-examina-tion of the possible interrelationships and afford afeasible experimental approach for this type ofinquiry.

SUMMARY

1. Pathways of carbohydrate metabolism havebeen examined in surviving preparations of nor-mal calf and rat anterior pituitary. Mechanismsfor active glycogen turnover and glucose oxidationvia the hexose-monophosphate shunt in vitro havebeen demonstrated. Anterior pituitaries fromboth species responded to the direct addition ofinsulin to the suspending media.

2. Micro-methods have been developed for in-cubation of single rat anterior pituitaries. Withthis technique, it has been demonstrated thathypophyseal carbohydrate metabolism is signifi-cantly reduced in anterior pituitaries excised fromrats rendered acutely insulin-deficient by totalpancreatectomy or chronically diabetic by alloxan-ization. The phenomenon could not be attributedto such systemic manifestations of insulin depriva-tion as ketoacidosis and could not be duplicated bycomparable degrees of surgical trauma in animalsin which pancreatic function was preserved. Thederangement of hypophyseal glucose utilization inrats with experimental diabetes could be preventedor reversed by treatment with replacement quan-tities of insulin in vivo.

3. The data indicate that the anterior pituitaryis responsive to insulin and that intrahypophysealcarbohydrate metabolism in the intact animal maybe conditioned by the availability of insulin. Thesignificance of insulin dependency in terms of thenormal endocrine function of the anterior pituitaryand pituitary status in spontaneous diabetes mel-litus remains to be elucidated.

ACKNOWLEDGMENTS

The excellent technical assistance of Mr. Edward C.Saef and Miss Martha F. McMullen are gratefullyacknowledged. Calf pituitary glands were obtainedthrough the kind cooperation of the New England Dressed

y Meat and Wool Co., Somerville, Mass. The authors aregreatly indebted to Dr. Robert 0. Scow for his patientinstruction in the technique of total pancreatectomy in therat

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