Banting Memorial Lecture 1975 Diabetes and the Alpha Cell

The Banting Memorial Lecture 1975

Diabetes and the Alpha CellRoger H. linger, M.D., Dallas, Texas

It is appropriate that glucagon should be the sub-ject of a Banting Memorial Lecture, in that Bantingand Best were probably the first to observe thebiologic action of glucagon. In a letter written in1971 to Dr. Piero Foa, long a leader in the glucagonfield, Dr. Best reminisced about the historic 1921experiments in which crude pancreatic extracts wereinjected into depancreatized dogs.1 He wrote, "I havea very clear recollection of the immediate rise in bloodsugar to about 5 to 10 mg.%. This lasted about Vihour. As you may know, we thought this might havebeen due to epinephrine liberation and, for thisreason, we failed to investigate it thoroughly."

An explanation for their oversight is not necessary.At that historic moment in medical history, theycould hardly have been expected to concern them-selves with the initial upward deflection of the bloodsugar curve when their goal, the discovery of insulin,was in clear view. However, one year later, Murlinand Kimball reported that aqueous extracts of pan-creas raised blood sugar levels of depancreatized dogsby 200 mg./lOO ml. or more.2 They believed that thiswas due to a glucoregulatory hormone they named"glucagon," meaning "glucose-driving."

But, for most of the half century since its discovery,glucagon was regarded either as a hormone of trivialimportance or as a "nonhormone," an artifact of theextraction procedure for insulin. The possibility thatit might play a role in human disease was not consi-dered seriously. Ferner was one of the few who be-lieved that glucagon was pathogenically involved inthe metabolic derangements of diabetes mellitus, andhe even proposed that the diabetes caused by totalpancreatectomy was the result of gastrointestinal

Presented at the Thirty-fifth Annual Meeting of theAmerican Diabetes Association in New York on June 16,1975.

From the Department of Internal Medicine, Veterans Ad-ministration Hospital, and the University of Texas SouthwesternMedical School, Dallas, Texas.

Accepted for publication December 31, 1975.

glucagon,3 but his remarkable ideas were based oncontroversial morphologic evidence. The method-ologic capabilities essential to test the role ofglucagonfirst, the ability to measure glucagon inhuman plasma and, second, the ability to produce aglucagon-deficiency statehad not yet been de-veloped. Only within the past decade have both be-come available.

The ability to measure glucagon4 came as the first"spin-off' of the monumental methodologic break-through of Berson and Yalow5 that reshaped biomedi-cal science; that is, it was the second radioimmunoas-say to be developed.

In 1957, Anne Eisentraut and I had begun chal-lenging rabbits with glucagon in the hope of produc-ing antibodies for a hemagglutination-inhibition im-munoassay for glucagon. It became increasingly obvi-ous that we were wasting our time when, by chance, Ireread the 1956 Berson-Yalow paper describing thedetection of insulin antibodies with 131I-labeledinsulin.6 It occurred to me that by labeling theglucagon molecule with 131I instead of with a redblood cell, a far more sensitive and less capriciousimmunoassay might result. I telephoned Dr. Berson,then a total stranger, and explained to him the con-cept of the radioimmunoassay and its unlimited po-tential, not just for the assay of glucagon and insulin,but for other peptide hormones as well. He listenedpatiently and politely, but a lack of enthusiasm wasevident. However, he agreed to meet with me.

Some days later, in his office at the Bronx VAHospital, I learned the reason for his flat response. Heand Dr. Yalow had already developed a remarkablysensitive and reproducible radioimmunoassay for insu-lin. I was elated by this evidence of their achievementand asked if they would help me develop a similarassay for glucagon. Dr. Berson advised me not towaste my timethey had already tried it, and it didnot work because of inability to produce glucagonantibodies. But, at my insistence, they were kindenough to teach us the iodination and chromatoelec-

136 DIABETES, VOL. 2 5 , NO. 2

ROGER H. UNGER, M.D.

trophoresis technics with which we found that most ofour rabbits already had measurable glucagon an-tibodies. When I sent this good news to Dr. Berson, 1received in reply the gracious letter reproduced infigure 1.

VETERANS ADMINISTRATIONHOSPITAL

ISO WEST KINGSBRIOGC ROABRONX 68, NEW YORK

July 2I4, 1959>5O81/172

Dr. Roger H. OngorVA Hospitalli500 S. Uncastor RoadDallaa 16, Texas

Dew Dr. Unger:

Wo are delighted to hear of your success with the glucagonantibody. Oar own results In guinea pigs turned out to bespuriously optimistic. The material migrating with serunproteins did not Migrate with the garni globulin butrepresented damaged fractions. Oulnea pig serum^espedally srCundilutedtappears to be a particular nuisance In this respect.I-hope that you hare success on the assay for plasmaglucagon. We hare been doing a lot of work on the plasmaassay of insulin and enclose a manuscript which we submittedsome tine ago. As you probably appreciate^ the assay of plasmalerels i s considerably more difficult than that of the con-centrations of hormones which you can add In Titro. Shouldyou hare any diff icult ies we will be pleased to communicateto you some problems which we encountered in the a*age44&en *2of plasna insulin and sane of the tricks we hare used toIncrease sensitivity. We are able to assay as l i t t l e as10 Micro-alcrograns of insulin (l/b \i unit) in plasma.Rot joins e In sending you our best regards and wishes foryour continued success.

Sincerely yours,

wi K- -Solomon A. Berson, M.D.Chief, Radioisotope Serrlce

SAB: 88

FIG. 1. A letter from Dr. Berson. (Reprinted with permission ofMetabolism.)

The generous help of Drs. Berson and Yalow wereimportant factors in the development of the glucagonassay. As he had warned, the problems of measuringhormone in plasma were formidable. Although wepublished standard curves in 19594 and could measurechanges in endogenous glucagon in the pancreaticvenous effluent in 1961, thus permitting the firstphysiologic studies in animals,7 it was not until 1967that we could measure glucagon with confidence inthe peripheral blood of animals and man8 and thustest its physiologic and pathophysiologic roles.9

The second essential capability for the estimation ofglucagon's importance, namely, the production of aglucagon-deficiency state, was also a spin-off ofanother landmark achievementthe discovery,

purification, and synthesis of somatostatin in 1973 byDr. Roger Guillemin and his colleagues, Drs.Brazeau, Vale, Rivier, Burgus, and Ling at the SalkInstitute.10 The unexpected finding by Koerker andher associates in Seattle11 of dramatic suppression ofplasma glucagon during the infusion of somatostatinin baboons identified, for the first time, a powerfulglucagon-suppressing agent.

Today, a vast array of data concerning the physiol-ogy and pathophysiology of glucagon has been gener-ated by means of these methodologic tools. The fun-damental knowledge of the relationships of glucagonand insulin actions provided by the brilliant in-vitrostudies of Exton and Park12 and of Sokal andcolleagues13 and the more recent in-vivo studies ofVranic and colleagues14 and of Liljenquist, Chiasson,Cherrington, Lacy, and Jennings et al.,15 has createda substantial base upon which reasonable concepts ofthe function and malfunction of the alpha-beta cellunit can be constructed.

THE a- AND /3-CELLAS A FUNCTIONAL UNIT

The idea of a single bihormonal metabolic regulatorwas first expressed in 1907, at the very dawn of endo-crinology, by M.A. Lane.16 He reported that certainislet cells contained alcohol-precipitable granules andnamed them alpha cells and called the others betacells. He wrote: "One is led to the conviction that theislets of Langerhans are structures which, in all proba-bility, have a function of producing a two-fold sub-stance which, when poured into the bloodstream, hasan important effect on metabolism." In the early1950s, Ferner,3 in Germany, also a morphologist, andPincus,17 in the United States, stressed the an-tagonism of the secretory products of these adjacentcells. Today, however, the counterbalancing and op-posing biologic activities of glucagon and insulinupon the liver and other common target tissues arewell established. The over-all effects of insulin are

TABLE 1Demonstrated opposition on common targets: insulin vs. glucagon

Target CellLiver

Fat

EffectGlycogenesisGlycogenolysisGlyconeogenesisKetogenesisVLDL secretionLysosome formationLipolysis

Insulin*

Glucagon

tt

t

FEBRUARY, L976 137

DIABETES AND THE ALPHA CELL

anabolic and promote the biosynthesis of largemolecules, such as glycogen, fat, and protein, whilethose of glucagon are catabolic and promote thebreakdown of glycogen and fat into smaller energy-yielding components and the use of amino acids forgluconeogenesis at the expense of protein synthesis(table 1).

GLUCOREGULATORY FUNCTIONS

The unique biologic opposition of the two hor-mones endow the alpha-beta cell unit with the ability

NORMAL BASAL

GLUCAGON ,_^_ INSULIN

LIVER-lOg/hr

ECF

[GLUCOSE]

80 mg %

14g/hr

6g/hr

LIVER* FATMUSCLE

- BRAIN

BEXERCISE

|GLUCAGON _ _ | INSULIN

MUSCLE

LIVER

BRAIN

to vary glucose flux in a manner physiologically ap-propriate to the prevailing circumstances while main-taining extracellular glucose concentrations withinremarkably narrow limits, irrespective of those cir-cumstances. This concept is schematized in somewhatoversimplified form in figure 2. Levine first demon-strated that insulin is the hormone of glucose effluxfrom the extracellular space,18 and although, as Madi-son showed, insulin also restrains glucose influx,19normally glucagon is the dominant regulator of glu-cose influx. Obviously, if extracellular fluid (ECF)glucose concentration is to remain unchanged duringwide changes in glucose flux, glucose efflux and influxmust at all times remain approximately equal. Thiscritical balance appears to be in large part the result ofremarkably precise variation in the insulin-glucagonmixture. During violent exercise, for example (figure2B), efflux into muscle is markedly increased. Hypo-glycemia is prevented by a proportionate increase inglucose influx, partly because of a marked increase inglucagon, and adequate glucose delivery to the centralnervous system thus maintained. Conversely, during ameal, when exogenous glucose influx is increased (fig-ure 2C), glucose efflux is increased proportionately toprevent hyperglycemia through increased insulin se-cretion, glucose efflux rates often approaching the rateof influx. Consequently, plasma glucose concentrationalmost never exceeds 150 mg./lOO ml. in young nor-mal subjects after even the largest carbohydrate meal.

Throughout the life of a normal, healthy indi-vidual, ECF glucose concentration is confined withinthese narrow limits (figure 2), but whenever a criticalinjury or other serious stress is sustained, ECF glucosemust increase promptly for the purpose of maintain-

LIVER

MEAL

CARBOHYDRATE MEAL

{GLUCAGON ^ flNSULIN5^

50g/hr 6g/hr

[ LIVERMUSCLE

[ FAT* BRAIN

FIG. 2. Schematization of the glucoregulatory function of thealpha cell-beta cell couple in the normal basal state (A),during extreme exercise (B), and during the influx of in-gested glucose (C). The box labeled "ECF" represents theextracellular-fluid-glucose space.

STRESSAdrenergic

ECF

[GLUCOSE]

200 mg %

LIVERBRAIN

FIG. 3. Schematization of the glucoregulatory roles of the alphacell-beta cell during severe stress. The box labeled "ECF"represents the extracellular-fluid-glucose space.



FIG. 4A. Distribution of glucagon-producing cells in an islet ofLangerhans from an adult nondiabetic subject deter-mined by the indirect immunofluorescent technic. (X200)

ing cerebral glucose delivery in the face of a decliningcerebral blood flow (figure 3). To achieve stresshyperglycemia, insulin secretion declines andglucagon secretion increases, presumably by means ofadrenergic control of the islets, as the work of Porte etal.20 and of Frohman and his colleagues21 indicates,and stress hyperglycemia continues as long as thethreat to cerebral fuel delivery persists.

The (X and (3 cells of this remarkable and vital"organ of Langerhans" then serve the nutrient needs ofthe far-flung tissues of the body, directing the storageof nutrients when these are in abundance and retriev-ing them as required in time of famine, flight, fight,or injury, always in perfect accord with the needs ofthe moment. At all times, hyperglycemia and hypo-glycemia, hyperketonemia, and unnecessary nitrogenlosses are successfully avoided.

THE a- AND 0-CELLAS A STRUCTURAL UNIT

Lacy in this country22 and Orci and his associates inGeneva23 have advanced our understanding of isletstructure and function relationships. At the opticlevel, immunofluorescent staining of a human pan-creas with specific antiglucagon serum reveals the dis-tribution of a cells in the human islet (figure 4A).Similar studies using antisomatostatin as well as anti-

FIG. 4B. Islet of Langerhans in a chronic juvenile diabetic treatedwith the indirect immunofluorescent technic againstglucagon. (X 500)

glucagon and anti-insulin sera reveal in man and inthe rat an outer heterocellular rim containing most ofthe a and 5 cells, with the (3 cells in the centralregion.24 At the ultrastructural level Orci has ex-amined intercellular relationships between islet cells.He has observed junctional complexes of the nexustype, so-called gap junctions, low-resistance pathwaysof intercellular communication, connecting (3 and (3cells, a and a cells, and (3 and a cells (figures 5 and6). The same structures have been identified in thehuman islet (figures 7 and 8).

Orci has searched for such intercellular communica-tions between heterogeneous endocrine cells in theanterior pituitary gland; thus far, the a and (3 cells ofthe islets are the only endocrine cells with differentsecretory products in which such intercellular com-munications have been observed.25 The possibilitythat the islet cells may function as a synctitial networkwould help to explain the remarkably precise titrationof the "twofold substance" imagined by Lane some 68years ago to achieve the balance of glucose efflux andinflux.

Like insulin secretion, glucagon secretion also in-volves the process of exocytosis; as shown in figure 9,glucagon granules are extruded into the intercellularspace, and in figure 10 a freeze etching of a Chinesehamster a-cell provides a three-dimensional view ofsuch an event.25

FEBRUARY, 1976 139

hV?

V .--A . -V T(k


FIG. 5A, B. Isolated islets from normal rat stained en bloc with uranyl acetate. The junction between an a- and a /3-cell present in the(opposite framed area of figure 5A is shown at a higher magnification in figure 5B. In the encircled area, the outer leaflets of the

page) adjacent cell membranes undergo fusion, resulting in a pentalaminar structure, characteristic of a tight junction. (Figure 5A:X 38,000, figure 5B: X 124,000) (Reprinted with permission of the J. Clin. Invest.)

FIG. 5C. Pancreas treated with lanthanum hydroxide, which delineates the intercellular space in black. The area outlined by the(opposite rectangle is shown at high magnification in the inset. At.places indicated by arrows, the intercellular space between an

page) a- and fi-ce\\ appears considerably narrowed (presumably a gap junction). (X 31,500; inset: X 102,000) (Reprinted withpermission of the J. Clin. Invest.)

EXTRAPANCREATIC a-CELLS

It is now clear that o:-cells are not confined to theislets of Langerhans. In 1948, Sutherland and deDuvereported the presence of glucagon-like biologic activ-ity in the upper gastrointestinal tract of dogs,27 andFerner3 believed, on the basis of silver staining, thathe had identified a-cells in a distribution correspond-ing to that of Sutherland and deDuve's glucagon activ-ity. In 1968, Orci, using electron microscopy, firstdescribed "a-like cells" in the gastrointestinal tract of

the rat.28 More recently, a-cells in the stomach andduodenum have been carefully studied and found to beindistinguishable immunohistochemically and ultra-structurally from pancreatic a-cells. Using a specificantiserum for glucagon, glucagon-immunofluorescenta-cells have been identified in the oxyntic mucosa ofthe dog stomach, both at the optic and ultra-structural29 levels, and glucagon-immunoflu-orescent cells have also been observed at the optic lev-el in a human fundus30 (figure 11). Figure 12 revealsthe similarity of the human a-cell in the pancreas

FIG. 6A. Part of the periphery of an islet showing a poorly granulated /3-cell neighboring two well granulated a-cells. (X 13,000)(Reprinted with permission of the J. Clin. Invest.)

FEBRUARY, 1976 141


*5

FIG. 6B. Freeze-etch replica of a similar area. The fracture pro- FIG. 6C. Higher magnification of the framed area in figure 6B.cess has split the plasma membrane between two cells One can see the linear ridges of fibrils, characteristic oftentatively identified as 0- and a-cells on the basis of tight junctions (TJ), and the aggregates of particles,their content in secretory granules. (X 25,000) (Re- characteristic of gap junctions (arrows). (X 92,000) (Re-printed with permission of they. Clin. Invest.) printed with permission of the /. Clin. Invest.)

and that of the duodenum, an observation reportedpreviously by Sasagawa et al.31

Moreover, using extracts of hog duodenum pro-vided by Prof. Viktor Mutt, of Karolinska Institute,Stockholm, we have recently succeeded in separatingfrom the more abundant glucagon-like immunoreac-tivity or GLI of hog duodenum a material indisting-uishable from pancreatic glucagon with respect to itsmolecular weight, isoelectric point, immunometricratio, glycogenolytic activity, and binding affinityand adenylate-cyclase-stimulating activity in isolatedrat liver membranes3233 (table 2). GLI, by contrast,differed in all these parameters. The identity of pan-creatic and duodenal glucagon seems rather well estab-lished.

"DOUBLE-TROUBLE" HYPOTHESIS OF DIABETES:FUNCTIONAL CONSIDERATIONS

We have recently proposed the bihormonal abnor-mality hypothesis of the pathogenesis of the metabolicderangements of diabetes.34 This theory assigns topancreatic and/or extrapancreatic glucagon the role ofan essential comediator of the full-blown disorder in

carbohydrate metabolismin contradistinction to thetraditional unihormonal-deficiency hypothesis, whichattributes all of the metabolic derangements of dia-betes to hypofunction of the (3 cell. As shown in figure13, the "double-trouble" concept ascribes the under-utilization of glucose to insulin deficiency but attrib-utes to glucagon excess, either absolute or relative tothe reduced insulin level, most of the glucose over-production. McGarry and Foster have extended thebihormonal-abnormality hypothesis to the area ofdiabetic ketoacidosis by putting forth evidence thatketoacidosis requires both an increase in free fattyacids, the substrate for ketogenesis, and an additionalfactor that converts the liver to a "ketogenic mode."35Insulin deficiency causes the marked increase inlipolysis that provides the liver with ketogenic sub-strate, but it appears that glucagon is essential for theconversion of the liver into a ketone-producing organ(figure 14). Their findings are supported at the clini-cal level by the studies of Gerich et al.36 and of Al-berti and his colleagues37 demonstrating thatsomatostatin blockade of glucagon secretion can pre-vent ketoacidosis in insulin-deprived juvenile-typediabetes.

142 DIABETES, VOL. 25, NO. 2


FIG. 7A. The field shows the intercellular space () between a FIG. 7B. The freeze-fracture process has exposed a large area inhuman insulin-producing cell (/3-cell) and a glucagon- the plasma membrane of a human islet cell, the cyto-producing cell (a-cell). The intercellular space is nar- plasm of which can be seen in the upper left corner. Therowed at several points in which the two cell membranes freeze-fractured membrane contains a network of var-come into very close vicinity (circles). These regions may iegated fibrils (TJ) that characterizes the tight junction,be the site of specialized intercellular junctions of the The fibrils delimit various domains in the membrane () .tight or gap type. Their exact nature, however, cannot be (X 26,000) (Reprinted with permission of they. Clin.clearly determined with this technic. (X 42,000) (Re- Endocrinol. Metab.)printed with permission of the_/. Clin. Endocrinol. Metab.)

The concept of a bicellular abnormality has evolvedvery gradually since 1969, when we first reported thathyperglucagonemia was present in human diabetics,38particularly during meals39 or arginine infusion.9Assan,40 Buchanan,41 Felig,42 Gerich,43 Kalkhoff,44and their associates have made similar observations.We had also observed hyperglucagonemia in everyknown form of experimental diabetes45 with the ex-ception of totally depancreatized dogs well regulatedwith insulin. However, in 1973, Vranic, Pek, and

Kawamori,46 Matsuyama and Foa,47 and Mashiterand Field and associates48 made the key observationthat levels of circulating immunoreactive glucagonwere high in insulin-deprived depancreatized dogs,which we subsequently confirmed32 (figure 15). Thismeant that hyperglucagonemia was present in everyknown form of endogenous hyperglycemia. Moreover,when the rise in the plasma level of nonpancreaticglucagon following pancreatectomy was blocked bysomatostatin (figure 16), hyperglycemia was markedly

FEBRUARY, 1976 143


FIG. 8. Higher magnification of a freeze-fractured islet cellplasma membrane. One recognizes the characteristicnetwork of fibrils of the tight junction (TJ). In addition,localized domains in the network contain patches ofclosely packed globular subunits representing gap junc-tions (GJ). (X 65,000) (Reprinted with permission of they.Clin. Endocrinol. Metab.)

reduced, if not prevented.3249 It was the finding insuch dogs of a relationship between plasma nonpan-creatic glucagon and glucose rise49 that led us topropose that glucagon is the principal mediator of theendogenous hyperglycemia of diabetes mellitus.34Similar relationships between hyperglucagonemia andhyperglycemia have been demonstrated in insulin-deprived alloxan-diabetic dogs.50

Gastrointestinal glucagon may play a role not onlyin pancreatic diabetes but also in other forms of dia-betes, such as alloxan diabetesat least in theinsulin-deprived state.51 Simultaneous measurementsof glucagon in the gastric vein, the pancreatic vein,tand the vena cava in insulin-deprived alloxan-diabeticdogs reveal large basal glucagon gradients across bothpancreas and stomach. But the administration of insu-lin promptly turns off gastric glucagon secretion.Pancreatic glucagon secretion is also reduced by insu-lin, but only to the normal rate, not to zero. Thelower insulin requirements in diabetes following pan-createctomy may be due to the exquisite sensitivity toinsulin of the gastrointestinal a-cell in the absence ofany pancreatic glucagon secretion.

CLINICAL IMPLICATIONS OF THE"DOUBLE-TROUBLE" HYPOTHESIS

In both the adult and the juvenile types, the mostconsistent and striking abnormality in a-cell functionis the relative or absolute hyperglucagonemia that oc-curs during the ingestion of meals or with the infusionof arginine or alanine. Is this a-cell abnormality in theinherited forms of human diabetes merely secondaryto insulin lack stemming from the /3-cell disorder, asit must certainly be in experimental diabetes, or isthere an intrinsic a-cell defect that is independent ofand may, therefore, appear before, after, or simul-taneous to the /3-cell abnormality? While there is nodoubt that in human diabetes, just as in the previ-ously nondiabetic laboratory animal, insulin defi-ciency can cause hyperglucagonemia, the questionthat is still unclear pertains to an a-cell defect inde-pendent of insulin lack in the diabetic patient. In-deed, when insulin lack is absent or corrected withexogenous insulin an abnormality in diabetic a-cellfunction seems to remain. For example, as shown intable 3, in fasting human diabetics, glucagon levelscan be lowered significantly by intravenous insulininfusion in amounts that raise plasma insulin to onlyabout 30 /u,U. per milliliter, but they do not decline tothe nadir to which glucagon levels of nondiabetics canbe suppressed with a comparable insulin level and

FIG. 9. Field from an a-cell of rat pancreatic islet stimulated witharginine. The arrows point to two stages of exocytoticevents of a-granules. (X 42,000)

144 DIABETES, VOL. 25 , NO. 2


FIG. 10. Freeze-fracture replica of an a-cell from a diabetic (ketotic) Chinese hamster. The arrows point to several exocytotic (emiocytotic)stomata, three of which show protruding granule cores. For the problem of identification of a-cell in freeze-fracturing technic see"Morphological characterization of membrane systems in A- and B-cells of the Chinese hamster," by L. Orci, AA. Amherdt, F.Malaisse-Lagae, A. Perrelet, W. E. Dulin, G. C. Gerritsen, W. Malaisse, and A. E. Renold. Diabetologia 70:529-39, 1974. (X49,000)

even less marked hyperglycemia.52In adult-type diabetics the exaggerated glucagon

response to arginine53 and the abnormal response tocarbohydrate ingestion54 is not corrected by even su-praphysiologic doses of simultaneously infused insu-lin. This would agree with the report of Aronoff andassociates,55 who found that in diabetic Pima Indianswith endogenous hyperinsulinemia the glucagon re-sponse to arginine was as exaggerated as in those withsignificant hypoinsulinemiaand significantly great-er than in nondiabetic Pima controls, in whom insulin

levels were intermediate between the two diabeticgroups. Further evidence that the o;-cell derangementis independent of the circulating endogenous insulinlevel may be found in demonstrations of abnormala-cell function in first-degree relatives of diabetics56and in the concordant identical nondiabetic twins ofdiabetics.57

THERAPEUTIC IMPLICATIONS

At the therapeutic level, plasma glucagon and glu-

FEBRUARY, 1976 145


FIGURE 11

Fundic mucosa of human stomach ob-tained three hours after death. Positiveimmunoperoxidase reaction for antiglu-cagon 30K is present in several cells (ar-rows). (X 400)

cose were measured at two-hour intervals for up to 10days in juvenile-type diabetics during aggressive at-tempts to achieve "optimal" control with insulin, ir-respective of the amount and frequency of the injec-tions. In most such patients, a significant reduction ofplasma glucagon levels was observed as control im-proved, but this required from 90 to 210 U. of insulinper daycertainly far more than is secreted by

nondiabetics.58 Moreover, in most of the juvenilediabetics, wide-ranging fluctuations in plasmaglucagon were observed during the daytime, probablyrelated to the meals. Such fluctuations in glucagonwere infrequent in nondiabetic subjects similarlystudied58 (figure 17). Massive doses of insulin willultimately achieve normoglycemia in most such pa-tients but probably by maintaining a disproportion-

FIG. 12A, B. Notice the similarity of the granules of the human pancreatic a-cell and those of human duodenal endocrine cell (arrows).(X 26,000) (Unpublished document of D. Baetens, P. Loeb, and R. H. Unger, Geneva and Dallas.)



TABLE 2

Comparison of gastrointestinal glucagon,GLI, and pancreatic glucagon

NORMALPancreatic GIglucagon glucagon GLI

M. W.Isoelectric pointRatio 78J/30KGlycogenolytic activity:

per cent of 10 /xg. of glucagon70 per cent of maximum

adenylate cyclase stimulationAffinity for rat liver

membranes

3,4856.21.0

100

1O"8M

3,500 2,9006.2 >100.9 61

100 50

10"8M 10"7M

4 x 10'9 3 x 10'9 5 x 10"8

ately high rate of glucose efflux without appropriatesuppression of glucagon-mediated glucose influx. In-appropriate meal-time hyperglucagonemia, in thepresence of a fixed level of circulating exogenous insu-lin, may be the cause of the bursts of hyperglycemiathat are commonly observed. The a-cell of the diabet-ic patient ignores hyperglycemia that lowers the sec-retory activity of the nondiabetic a-cell and no longerfunctions as a glucose sensor guarding the limits ofextracellular fluid on glucose concentration.

"DOUBLE-TROUBLE" HYPOTHESIS:

MORPHOLOGIC CONSIDERATIONS

Very little is known about the pathology of dia-betes, and modern morphologic technics have onlyrarely been applied to man. Drs. Orci, Baetens,Rufener, Amherdt , Ravazzola, Studer, andMalaisse-Lagae59 have studied the pancreas of twojuvenile diabetics shortly^ after death, using both im-munofluorescent stains and electron microscopy. Isletswere generally sparse, as had been reported byGepts,60 and in addition to a complete absence of

DOUBLE-TROUBLE HYPOTHESIS OF DIABETES

GLUCAGON ~ I INSULIN

LIVER

6g/hrBRAIN

FIG. 13. Schematization of abnormal glucoregulation by thealpha cell and beta cell in diabetes mellitus. The boxlabeled "ECF" represents the extracellular-fluid-glucosespace.

KETONES

DIABETIC KETOACIDOSIS

KETONES

FIG. 14. Schematization of the ketogenic roles of the alpha andbeta cells under normal circumstances and in diabeticketoacidosis according to the hypothesis of McGarry,Wright, and Foster.35 Upper pane!: Normally the presence ofinsulin restrains lipolysis and limits the delivery to theliver of free fatty acids, the substrate for ketone produc-tion. Lower panel: In the absence of insulin, unrestrainedlipolysis provides sufficient free fatty acids for a highrate of ketone production, but the presence of glucagon issomehow required to convert the liver into a "ketogenicmode." Thus, diabetic ketoacidosis also requires the pre-sence of this glucagon as well as the absence of insulin.

insulin-immunofluorescent cells, an increase in bothglucagon-immunofluorescent a-cells, which made upmost of the islet (figure 4B), and somatostatin-immunofluorescent 8-cells was noted. Orci's grouphas, therefore, demonstrated by morphologic technicsa relative or absolute a:-cell increase in the islets of theonly two juvenile diabetics studied thus far.

FEBRUARY, 1976 147


Arginine

HZD-mg% (2mM/kg)400200

0-jull/ml

nlnsulindmU/kg/min.)-*-Glucose

GLUCOSE

INSULIN

pq/m

300

200

100

0-ng/ml

2.0-

1.0-

0-

GLUCAGON

~ > *

GLI

-10 0 20 50 80 110 140 170 200MINUTES

FIG. 15. Circulating glucagon is present in the plasma of this to-tally depancreatized, insulin-deprived dog, and in-creases during the administration of arginine. When in-sulin is infused, however, circulating glucagon rapidlydeclines to unmeasurable levels.

GLUCAGON /

PANCREASOUT

300 360 420 480MINUTES

18hrs

FIG. 16. Plasma glucose in a dog during and after total pan-createctomy, during the continuous intravenous infusionof somatostatin. Blockade of the rise in plasma glucagonprevents the development of hyperglycemia despite theabsence of measurable levels of plasma insulin. Whenplasma glucagon is elevated after discontinuation of thesomatostatin infusion, hyperglycemia is present.

TABLE 3

Comparison of mean nadirs of plasma glucagon during intravenousinsulin in:

Hyperglycemicnondiabetics(N=5)39 2

Adult-typediabetics(N= 10)54 1

Juvenile-typediabetics(N=7)53 4

p


metabolic normalization of the diabetic cannot bepredicted on the basis of currently available evidence.However, when science fails us, "biotheologic" infer-ence and common sense may provide the best availablebasis for a guess:

A. Nature's efforts are seldom purposeless.B. Nature makes a substantial effort to avoid

hyperglycemia throughout life.C. When nature's effort to avoid hyperglycemia is

successful, microangiopathy is rarely, if ever, present.D. When nature's effort to avoid hyperglycemia

fails, microangiopathy usually develops.E. Medical efforts to avoid hyperglycemia only

rarely succeed, and only rarely is microangiopathyprevented.

It would be the height of irresponsibility to suggestat this time that therapy directed at the hyperglu-cagonemia, as well as the hypoinsulinemia, would bemore successful than conventional therapeuticmethods of glucoregulation directed only at insulindelivery. But, on the other hand, it would be theheight of nihilism not to hope so, and the height ofindifference not to find out.

ACKNOWLEDGMENTS

The work was supported by VA Institutional Re-search Support Grant 549-8000-01; NIH Grant AM02700-16; Pfizer Laboratories, New York; BristolMyers Company, New York; Mead Johnson Center,Evansville, Ind.; Dr. Karl Thomae GmbH, Germany;Hoffman-LaRoche, Nutley, N.J . ; Hoechst Phar-maceutical Company, Somerville, N.J.; Ciba-GeigyCorporation, Ardsley, N.Y.; the Upjohn Company,Kalamazoo, Mich.; Eli Lilly, Indianapolis, Ind.; 30KRabbit Fund, and various donors.

The following postdoctoral fellows at the Univer-sity of Texas Southwestern Medical School performedthe studies upon which this review is based: Drs.Herman Ketterer, Akira Ohneda, DemetraRigopoulou, Isabel Valverde, Eugenio Aguilar-Parada, Jose Marco, Walter Muller, Ingolf Bottger,Fausto Santeusanio, Dalva M. Rocha, Jan Braaten, C.Alfred Lindsey, Boan Rubalcava, Hideo Sasaki,Yoshikuni Fujita, Enrique Blazquez, LucianoMunoz-Barragan, Hideo Sakurai, Gerald Faloona(subsequently Assistant Professor of Biochemistry atthe University of Texas Southwestern Medical Schooland Research Chemist at the Dallas Veterans Ad-ministration Hospital), and Richard E. Dobbs (cur-rently Assistant Professor of Physiology at the Univer-sity of Texas Southwestern Medical School and Re-

search Physiologist at the Dallas Veterans Administra-tion Hospital).

The author wishes to thank the following personsfor their outstanding technical work: Anna M. Eisen-traut, former Chief Technologist; Ms. Virginia Har-ris, Chief Technologist; Kay McCorkle, ResearchBiologist; Nancy Whitten, former Research Biologist;Margaret Bickham, Biology Technician; CathyMitchell, Biology Technician; Vicki Lupean, BiologyTechnician; Shirley Harvey, former Biology Techni-cian; Daniel Sandlin, Biology Technician; BrendaTower, former Biology Technician; Ava Marie Jones,former Biology Technician; Loretta Clendenen, Re-search Technician.

The morphologic material employed in this reviewwas generously provided by Prof. Lelio Orci, Chair-man of the Institut d'Histologie et Embryologie,University of Geneva School of Medicine, and his col-leagues, Drs. F. Malaisse-Lagae, M. Amherdt, M.Ravazzola, D. Baetens, and C. Rufener.

The author wishes to express his thanks to his se-cretaries, Ms. Billie Godfrey and Ms. Carol Tower, fortheir help in preparing this manuscript.

REFERENCES

'Banting, F. G., and Best, C. H.: The internal secretion of thepancreas. J. Lab. Clin. Med. 7:251-66, 1922.

2Kimball, C. P., and Murlin, J. R.: Aqueous extracts of pan-creas. III. Some precipitation reactions of insulin. J. Biol. Chem.58:337-46, 1923.

3Ferner, H.: The A- and B-cells of the pancreatic islets assources of the antagonistic hormones glucagon and insulin. Theshift of the AB-relation in diabetes mellitus. Am. J. Dig. Dis.20:301-06, 1953.

4Unger, R. H. , Eisentraut, A. M., McCall, M. S., Keller, S.,Lanz, H. C., and Madison, L. L.: Glucagon antibodies and theiruse for immunoassay of glucagon. Proc. Soc. Exp. Biol. Med.702:621-23, 1959.

5Yalow, R. S., and Berson, S. A.: Immunoassay of endogenousplasma insulin in man. J. Clin. Invest. 39:1157-75, I960.

6Berson, S. A., Yalow, R. S., Bauman, A., Rothschild,M. A., and Newerly, K.: Insulin I131 metabolism in humansubjects: Demonstration of insulin-binding globulin in the circu-lation of insulin treated subjects! J. Clin. Invest. 35:170-90,1956.

7Unger, R. H. , Eisentraut, A. M., McCall, M. S., and Madi-son, L. L.: Measurements of endogenous glucagon in plasma andthe influence of blood glucose concentration upon its secretion. J.Clin. Invest. 47:682-89, 1962.

8Eisentraut, A. M., Ohneda, A., Aguilar-Parada, E., andUnger, R. H.: Immunologic discrimination between pancreaticglucagon and enteric glucagon-like immunoreactivity (GLI) intissues and plasma. Diabetes 7 7 (Suppl. l):32l, 1968.

9Unger, R. H. , Aguilar-Parada, E., Muller, W. A., and Eisen-traut, A. M.: Studies of pancreatic alpha cell function in normal

FEBRUARY, 1976 149


and diabetic subjects. J. Clin. Invest. 49:837-48, 1970.10Brazeau, P., Vale, W. , Burgus, R., Ling, N . , Butcher, M.,

Rivier, J . , and Guillemin, R.: Hypothalamic polypeptide thatinhibits the secretion of immunoreactive growth hormone. Science179:77-79, 1973.

"Koerker, D. J . , Ruch, W., Chideckel, E., Palmer, J. ,Goodner, C. J . , Ensinck, J. , and Gale, C. C.: Somatostatin:Hypothalamic inhibitor of the endocrine pancreas. Science784:482-83. 1974.

12Park, C. R., and Exton, J. H.: The role of cyclic AMP in thecontrol of liver metabolism. Adv. Enzyme Regul. 6:391-407,1968.

13Mackrell, D. J., and Sokal, J. E.: Antagonism between theeffects of insulin and glucagon on the isolated liver. Diabetes78:724-32, 1969.

l4Cherrington, A. D., and Vranic, M.: Effect of arginine onglucose turnover and plasma free fatty acids in normal dogs. Dia-betes 22:537-43, 1973.

15Jennings, A. S., Cherrington, A. D. , Chiasson, J. L., Liljen-quist, J. E., and Lacy, W. W.: The fine regulation of basalhepatic glucose production. Clin. Res. 23:323A, 1975.

16Lane, M. A.: The cytological characters of the areas ofLangerhans. Am. J. Anat. 7:409-22, 1907.

17Pincus, I. J . , and Rutman, J. Z.: Glucagon, the hyper-glycemic agent in pancreatic extracts; possible factor in certaintypes of diabetes. Arch. Intern. Med. 92:666-77, 1953.

l8Levine, R.: Action of insulin: An attempt at a summary.Diabetes 27 (Suppl. 2):454-56, 1972.

19Madison, L. L., Seyffert, W. A., Jr. , Unger, R. H. , andBaker, B.: Effect of plasma free fatty acids on plasma glucagon andserum insulin concentrations. Metabolism 7 7:301-04, 1968.

20Porte, D. , Jr., Graber, A. L., Kuzuya, T., and Williams,R. H.: The effect of epinephrine on immunoreactive insulin levelsin man. J. Clin. Invest. 42:228-36, 1966.

2IFrohman, L. A., and Bernardis, L. L.: Effect of hypothalamicstimulation on plasma glucose, insulin and glucagon levels. Am.J. Physiol. 227:1596-1603, 1971.

22Lacy, P. E.: Beta cell secretionfrom the standpoint of apathobiologist . (Banting Memorial Lecture) Diabetes79:895-905, 1970.

23Orci, L.: A portrait of the pancreatic B-cell. Diabetologia70:163-87, 1974.

24Orci, L., and Unger, R. H.: Functional subdivision of isletsof Langerhans and possible role of D cells. Lancet 2:1243-44,1975.

25Orci, L., Stauffacher, W., Renold, A. E., and Rouiller, C :The ultra-structural aspect of A-cells of non-ketotic and ketoticdiabetic animals: indications for stimulation and inhibition ofglucagon production. Acta Isot. 70:171-86, 1970.

26Orci, L., Malaisse-Lagae, F., Ravazzola, M., Rouiller, C ,Renold, A. E., Perrelet, A., and Unger, R. H.: A morphologicalbasis for intercellular communication between a- and b-cells in theendocrine pancreas. J. Clin. Invest. 46:1066-70, 1975.

"Sutherland, E. W., and DeDuve, C : Origin and distributionof the hyperglycemic-glycogenolytic factor of the pancreas. J.Biol. Chem. 773:663-74, 1948.

28Orci, L., Pictet, R., Forssmann, W. C , Renold, A. E., andRouiller, C : Structural evidence for glucagon producing cells inthe intestinal mucosa of the rat. Diabetologia 4:56-67, 1968.

29Baetens, D., Rufener, C , Srikant, C. B., Dobbs, R. E.,Unger, R. H., and Orci, L.: Identification of glucagon-producingcells (A-cells) in dog gastric mucosa. J. Cell Biol. (In press).

30Munoz-Barragan, L., Rufener, C , Srikant, C. B., Shannon,A., Baetens, D., and Unger, R. H.: Immunohistologic identifica-tion of glucagon-containing cells in a human fundus. (Submittedfor publication).

31Sasagawa, T., Kobayaski, S., and Fujita, T.: Electron micro-scope studies on the endocrine cells of the human gut and pan-creas. In Gastro-Entero-Pancreatic Endocrine System. A CellBiological Approach. Fujita, T., Ed. Tokyo, Igahu Shoin, 1974,pp. 17-38.

32Dobbs, R., Sakurai, H., Sasaki, H., Faloona, G., Valverde,I., Baetens, D., Orci, L., and Unger, R. H.: Glucagon: Role inthe hyperglycemia of diabetes mellitus. Science 787:544-47,1975.

33Sasaki, H., Rubalcava, B., Baetens, D., Blazquez, E., Sri-kant, C. B., Orci, L., and Unger, R. H.: Identification ofglucagon in the gastrointestinal tract. J. Clin. Invest. 56:135-45,1975.

34Unger, R. H., and Orci, L.: The essential role of glucagon inthe pathogenesis of the endogenous hyperglycemia of diabetesmellitus. Lancet 7:14-16, 1975.

35McGarry, J. D., Wright, P., and Foster, D.: Hormonalcontrol of ketogenesis: Rapid activation of hepatic ketogeniccapacity in fed rats by anti-insulin serum and glucagon. J. Clin.Invest. 53:1202-09, 1975.

36Gerich, J. E., Lorenzi, M., Bier, D. M., Schneider, V.,Tsalikian, E., Karam, J. H., and Forsham, P. H.: Prevention ofhuman diabetic ketoacidosis by somatostatin. Evidence for an es-sential role of glucagon. N. Engl. J. Med. 292:985-89, 1975.

37Alberti, K. G. M. M., Christensen, N. J. , Iversen, J . , andOrskov, H.: Role of glucagon and other hormones in developmentof diabetic ketoacidosis. Lancet 7:1307-11, 1975.

38Aguilar-Parada, E., Eisentraut, A. M., and Unger, R. H.:Pancreatic glucagon secretion in normal and diabetic subjects.Am. J. Med. Sci. 257:415-19, 1969.

39Muller, W. A., Faloona, G. R., Aguilar-Parada, E., andUnger, R. H.: Abnormal alpha cell function in diabetes: Responseto carbohydrate and protein ingestion. N. Engl. J. Med.283:109-15, 1970.

40Assan, R., Hautecouverture, G., Guillemant, S., Douchy,F., Protin, P., and Derot, M.: Evolution de parametres hor-monaux (glucagon, cortisol, hormone somatotrope) etenergetiques (glucose, acides gras libres, glycerol) dans dixacido-cetoses diabetiques graves traitees. Pathol. Biol.77:1095-1105, 1969.

41Buchanan, K. D., and McCarrol, A. M.: Abnormalities ofglucagon metabolism in untreated diabetes. Lancet 2:1394-95,1972.

42Felig, P., Pozefsky, T., Marliss, E., and Cahill, G. F., Jr.:Alanine: Key role in gluconeogenesis. Science 767:1003-04,1970.

43Gerich, J. E., Langlois, M., Noacco, C , Karam, J. H., andForsham, P. H.: Lack of glucagon response to hypoglycemia indiabetes: Evidence for an intrinsic pancreatic alpha cell defect.Science 782:171-73, 1973.

44Kalkhoff, R. K., Gossain, V. V., and Matute, M. L.: Plasmaglucagon in obesity. N. Engl. J. Med. 289:465-67, 1973-

45Muller, W. A., Faloona, G. R., and Unger, R. H.: Theeffect of experimental insulin deficiency on glucagon secretion. J.Clin. Invest. 50:1992-99, 1971.

46Vranic, M., Pek, S., and Kawamori, R.: Increased glucagonimmunoreactivity (IRG) in plasma of totally depancreatized dogs.Diabetes 23:905-12, 1974.



47Matsuyama, T., and Foa, P: Plasma glucose, insulin, pan-creatic and enteroglucagon levels in normal and depancreatizeddogs (38288). Proc. Soc. Exp. Biol. Med. 747:97-102, 1975.

48Mashiter, K., Harding, P. E., Chou, M., Mashiter, G. D. ,Stout, ]., Diamond, D., and Field, J. B.: Persistent pancreaticglucagon but not insulin response to arginine in pancreatec-tomized dogs. Endocrinology 96:678-93, 1975.

4!)Sakurai, H., Dobbs, R. E., and Unger, R. H.: The role ofglucagon in the pathogenesis of the endogenous hyperglycemia ofdiabetes mellitus. Metabolism 24:1287-97, 1975.

50Sakurai, H . , Dobbs, R. E., and Unger, R. H.: So-matostatin-induced changes in insulin and glucagon secretion innormal and diabetic dogs. J. Clin. Invest. .54:1395-1402, 1974.

5lMunoz-Barragan, L., Blazquez, E., and Unger, R. H.: Gas-tric A-cell function in normal and diabetic dogs. Diabetes24(Suppl. 2) :4 l l , 1975.

52Raskin, P., Fujita, Y., and Unger, R. H.: Effect of insulin-glucose infusions on plasma glucagon levels in fasting diabeticsand nondiabetics. J. Clin. Invest. .56:1132-38, 1975.

53Raskin, P., Aydin, I., and Unger, R. H.: Effect of insulin onthe exaggerated glucagon response to arginine stimulation indiabetes mellitus. Diabetes 25:1976 (In press).

54Unger, R. H. , Madison, L. L , and Muller, W. A.: Abnor-

mal alpha cell function in diabetics, response to insulin. Diabetes27:301-07, 1972.

55Aronoff, S., Bennett, P., Miller, M., and Unger, R. H.:Evidence for the independence of the A-cell and B-cell dysfunctionin human diabetes. (Submitted for publication).

56Kirk, R. D., Dunn, P. J. , Smith, J. R.,Beaven, D. W. ,andDonald, R. A.: Abnormal pancreatic alpha-cell function in first-degree relatives of known diabetics. J. Clin. Endocrinol. Metab.40:913-16, 1975.

"Day , J. L., and Tattersall, R. B.: Glucagon secretion inunaffected monozygotic twins of juvenile diabetics. Metabolism24:145-51, 1975.

58Raskin, P., and Unger, R. H.: Unpublished observations.59Orci, L., Baetens, D., Rufener, C., Amherdt, M., Ravaz-

zola, M., Studer, P., Malaisse-Lagae, F., and Unger, R. H.:Hypertrophy and hyperplasia of somatostatin-containing D-cellsin diabetes. Proc. Natl. Acad. Sci. (In press).

60Gepts, W.: Pathologic anatomy of the pancreas in juvenilediabetes mellitus. Diabetes 74:619-33, 1965.

61Gerich, J. E., Lorenzi, M., Karam, J. H., Schneider, V.,and Forsham, P. H.: Abnormal pancreatic glucagon secretion andpostprandial hyperglycemia in diabetes mellitus. JAMA234:159-65, 1975.

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Banting Memorial Lecture 1975 Diabetes and the Alpha Cell

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