The Structure and Metabolism of the Pancreatic Islets: A Centennial of Paul Langerhans' Discovery

Sweden

OPENING ADDRESS

S. FALKMER, B. HELLMAN and I.-B. TÄLJEDAL,     Departments of Histology and Pathology, University of Umeå, Umeå, Sweden

Six years ago a symposium entitled The Structure and Metabolism of the Pancreatic Islets was held in Uppsala and Stockholm. The positive response to this first international meeting entirely devoted to the pancreatic islets has encouraged us to again invite prominent representatives of diabetes research from laboratories all over the world to participate in a second islet symposium. Before the 18th of February 1869 no author seems to have perceived in the pancreas other glandular elements than the exocrine acini. With the present meeting we would therefore like to commemorate the passing of exactly one hundred years since Paul Langerhans defended his thesis Beiträge zur Mikroskopischen Anatomie der Bauchspeicheldrüse at Friedrich Wilhelms Universität in Berlin.

Initially the members of the organizing committee felt somewhat hesitant about arranging a symposium in the month of February near the arctic circle. We have, however, adhered to our idea of giving the second international islet symposium the nature of a centennial of Langerhans’ discovery. This is to a great deal due to the enthusiasm expressed by the Chancellor of this University and the generous support forwarded by the Swedish Medical Research Council and the Swedish Diabetes Association as well as from pharmaceutical companies interested in diabetes. We gratefully recognize the great participation at this symposium and are particularly honoured by the acceptance of Professor Bernardo Houssay to act as our Honorary President.

At the time of the first international islet symposium, 6 years ago, refined and more elaborate techniques for studies of the islets such as microdissection, radioimmunoassays of the hormones of the islets and ultramicrochemical analyses of their enzymes and glucose intermediates were still rather unexploited. Since then, however, considerable progress has been made. This is to a great degree due to the fact that so many new and skilful research workers have realized the importance of this field of diabetes research. A part of this attraction may be explained by the accumulating evidence of a deficient β-cell function as a common denominator for both the juvenile and senile type of human diabetes. We sometimes feel that more secrets have been revealed about the pancreatic islets over the last 6 years than during the whole previous part of the century which has elapsed since the time of Paul Langerhans’ fundamental discovery.

Ladies and gentlemen, we have now the honour of declaring that Paul Langerhans’ memorial symposium is formally in session. May our discussions be carried out in a fruitful spirit of friendship and initiate further scientific progress in the forthcoming second century of islet research!

PAUL LANGERHANS A MEMORIAL LECTURE

A. LOUBATIÈRES,     Laboratory of Pharmacology and Pharmacodynamics, Faculty of Medicine of Montpellier

OVER and above his own personal qualities, PAUL LANGERHANS was fortunate enough to benefit from the excellent intellectual environment in which he lived. He was born on 25 July 1847 in Berlin to a family of doctors. His father, Paul Langerhans (Fig. 1), of the Protestant faith, practised in Berlin where he was very well known. His mother, née Keibel, was the cousin of Franz Keibel, an eminent histologist. He had two brothers: the first Robert (1859–1904), who was Virchow’s assistant for a long time, rose to the rank of Professor of Pathology. The second, Richard, practised medicine in Berlin and obtained the honorary title of Sanitätsrat (health consultant).

FIG. 1 PAUL LANGERHANS (standing in the middle) in 1884. Sitting: Dr. Paul Langerhans senior. Standing: Dr. Richard Langerhans (left) and Dr. Robert Langerhans (right)

After completing his secondary education at the Zum Grauen Kloster in Berlin (1858–65), PAUL LANGERHANS JR., enrolled successively at the universities of Berlin and Iena as early as Easter 1865. At Jena he regularly attended lectures given by Gegenbaur. He studied at Berlin, under Bardeleben, Du Bois-Raymond, Cohnheim, Frerichs, von Langenbeck, Traube, and especially Rudolph Virchow who directed his inaugural thesis (Fig. 2) for the degree of Doctor of Medicine entitled: Beiträge zur mikroskopischen Anatomie der Bauchspeicheldrüse (Contribution to the microscopic anatomy of the pancreas). This work was upheld before the Medical Faculty of Berlin (Friedrich Wilhelms Universität) on 18 February 1869. This thesis was published by Gustav Lange.

FIG. 2 PAUL LANGERHANS’ thesis

PAUL LANGERHANS completed his final national examination in February 1870. Endowed with the title of Doctor of Medicine, he was not immediately concerned with beginning to practise medicine. His first desire was to know the world and people.

The Kieperts, father and son, who were both geographers, were preparing a journey to explore North Africa and Asia Minor. PAUL LANGERHANS followed them. Thus he visited Egypt, Syria, Palestine, and especially Jerusalem. He thus had the opportunity to become interested in the various manifestations of leprosy (1870) as well as leproseries. He also applied himself to the study of anthropology and ethnography; traces may be found in the notes he published concerning the mensurations of the skulls of Bedouins and Fellaheen. However, this voyage only lasted a few months. Upon his return, he found his country at war with France. Thus he enlisted in the German army at the age of 23 and took part in most of the military campaigns, administering his care in turn at the Berlin hospitals or in the ambulances at the front.

Once the war was over, PAUL LANGERHANS returned directly to the University. He worked for some time in Crede’s laboratory and then with Ludwig at Leipzig. In 1871 he became prosector in pathology at the University of Freiburg in Breisgau. He had to write a second thesis before he was promoted to the position of lecturer (Habilitationsschrift). He devoted this work to the study of the cells of the sympathetic ganglions (1871).

It was at Freiburg that he spent the longest part of his university career. All his publications appeared from then on in Virchow’s Archives (Archiv für pathologische Anatomie und Physiologie und für klinische Medizin). They dealt with the histology of the heart (1873), the accessory glands of the genital organs (prostate, vas deferens, seminal vesicles, Cowper and Bartholin glands) (1874), the architecture of the spongy tissue of the vertebral column (1874). PAUL LANGERHANS was then promoted to the position of full Professor at Freiburg, but he had to interrupt his university career because of serious tuberculosis which appeared in 1874; he was at that time 27 years old.

At that time the essential role of climate in the treatment of this illness was in vogue. This explains his successive stays at Silvaplana, Badenweiler, and then at Capri, where he stayed after having worked in Naples at the Dhorn Institute. In the autumn of 1875 he settled first on the isle of Teneriffe, then at Funchal, capital of the isle of Madeira. There he devoted himself to zoological studies of different marine animals (1875–77). He published some works on the treatment of leprosy (1881), on the etiology of tuberculosis as well as on the diffusion of the tuberculosis bacillus in the organism (1888). He also edited a Manual of Madeira (1885) in which he described the climatic and curative riches of the island. Madeira proved to be so good for his health that he decided to practise medicine there. In 1885 he married Marguerite Ebart (née Jordan) who gave him three years of indescribable happiness. A daughter was born of their union.

Unfortunately in the month of February 1887 he was inflicted with severe nephritis contracted after a chill. This caused his death, which occurred unexpectedly at the age of 41 on 20 July, 1888. PAUL LANGERHANS was buried at Madeira profoundly regretted by his family, his patients, and his numerous friends.

There are few documents left concerning his character and his temperament, but those that knew him describe him as a good, courteous, and cheerful man, generous to all. Throughout his short life he had an ardent desire to learn and to increase his knowledge. It was only much later on that his genius was recognized: he was, in fact, gifted with exceptional faculties of perception.

His aptitude for research study manifested itself very early, while he was still a student. It was at that time that he published a study of the nerves of human skin (1868) by using Cohnheim’s gold-chloride technique. In this work he described, amongst others, two structures which carry his name: Langerhans’ granular layer, a layer of epidermal cells above the rete mucosum (Malphigian germinative layer); and the Langerhans’ stellate corpuscles, which he interpreted as nerve fibre endings in the rete mucosum of the epidermis. In 1869 he published a work on the state of the tactile corpuscles in diseases of the central nervous system and the skin. In collaboration with Hoffman he studied the behaviour of cinnabar (red mercuric sulphide) in the rabbit and the guinea-pig, the substance being injected intravenously; he showed, particularly, the capturing and fixation of this substance by the cells of connective tissue (1869).

PAUL LANGERHANS was 22 years old when he upheld his thesis for the title of Doctor of Medicine (his inaugural dissertation) (Contribution to the microscopic anatomy of the pancreas) on 18 February 1869 (thus just one hundred years ago) before his professors at the Friedrich Wilhelms University in Berlin. He had begun his studies on the fine structure of the pancreas during the summer of 1867 under the direction of Virchow in the Institute of Pathology. It was to this eminent master that he dedicated his thesis. However, in his introduction he also expressed his gratitude to Doctor V. Kühne, first assistant at the Institute of Pathology, who guided him and put his own research apparatus at his disposal. After 3 months of research he had to interrupt his work and was not able to come back to it until a year later, in October 1868. He thus devoted about 8 months to the elaboration and writing of his thesis, which is relatively little.

Reserved and modest, unaware of the renown that scientific history was to reserve for him later on, PAUL LANGERHANS expressed himself as follows:

With regret, I must begin my communication by declaring that I cannot present any conclusive results; I can, at most, advance several isolated observations, which suggest that the pancreas has a far more complicated structure than was thought to be the case until now. The purpose of these remarks is thus to draw attention to the pancreas more than has been done by anatomists until now.

In his work he also expressed his surprise that the brilliant results obtained in the field of pancreatic physiology, notably by Claude Bernard in his Memoir on the pancreas and on the role of the pancreatic juice in digestive phenomena (Fig. 3) were not accompanied by an equal development in the histological knowledge of this gland. This is one of the reasons, he said, why he decided to devote himself to its study. Claude Bernard, assuredly, had described the glandular lobule. The blood vessels irrigating the pancreas were also known; these had been demonstrated by intra-aortic injections of colouring substances. But before PAUL LANGERHANS no one had yet had the idea of injecting coloured liquid up-stream into the excretory ducts of the gland to facilitate their examination.

FIG. 3 Claude Bernard’s work on pancreas secretion, which motivated PAUL LANGERHANS to start this morphological study of the gland

Very scrupulous, doubtful of the importance of his discoveries, he asks the indulgence of his peers and writes: I will have to be excused if these lines fall into the hands of a specialist in microscopy and if he finds in this work too many things concerning what is already known, and not enough about what is unknown. Great innovators always have the impression of being pygmies perched on the shoulders of giants.

Knowing well the anatomy and histology of the pancreas in man, in the guinea-pig, in the dog, in the pigeon, in the snake, in the frog, and in the triton which have a compact pancreas, he excluded them from his experimental material. He chose the pancreas of the rabbit, this animal, he wrote possesses a pancreas whose structure is favourable for investigation. It is flat, and spreads out between the mesenteric folds in such thin layers that microscopic observation can be made of fragments simply sectioned by scissors without any other preparation. It is, furthermore, this same technique that is used today to study insulin secretion in vitro.

The description of the pancreas given to us by PAUL LANGERHANS shows him to be a poet as well as scientist. Scientists and poets often follow the same mental proceedings. Let us appreciate the manner in which he describes the canalicular system: If we observe a piece of the pancreas of a recently sacrificed rabbit under a microscope under low power, the canalicular system looks like the branches of a tree which has lost its leaves and is formed of fine yellow brilliant granules. He called this tree Körnchenbaum, which means a tree composed of granules. This tree, he continued, branches out finely and shows in the final lobulations a band which passes in the centre of the lobule. If it is examined under high power it becomes apparent that in the centre there is an empty space, very fine …. He noticed that the granulations are situated at the interior apical portion of the cells, immediately adjacent to the lumen of the acinus. These were the zymogen granules that were later identified by Heidenhain as the pancreatic enzymes.

In his study, PAUL LANGERHANS described nine types of cells: (1) epithelial cells of the peritoneal covering, (2) connective cells of the mesenterium, (3) smooth cells of the vessels, (4) blood cells, (5) non-myelinic fibrils as well as cells of the ganglions, (6) real secretory exocrine cells, (7) epithelial cells of the large excretory ducts, (8) spindle-shaped cells or centro-acinar cells, (9) small cells, homogenous in content, and polygonal in form, with a round nucleus containing no nucleoli and which are found in groups of two or several. The last two types of cells (8) and (9) had never been described before: they are the centro-acinar cells, and cells which, not until 1893, were given the name of cells of the islets of LANGERHANS. The spindle-shaped centro-acinar cells were so called because they were seen in the centre of each acinus and in contact with the secretory exocrine cells.

The close resemblance between these new cells and those of the epithelium of the terminal ducts and their continuous arrangement, suggested to LANGERHANS that the centro-acinar cells are among the constituents at the beginning of the excretory ducts and that they were intermediary between the acinar cells and the canalicular cells. He did not give any final interpretation of their role and wrote the following sentence the centro-acinar cells are placed between the two other kinds of cells (acinar cells and duct cells) like a bothersome intermediary. It is surprising that this interpretation is still valid today, as the real role of the centro-acinar cells, if suspected, is nevertheless not yet finally demonstrated.

The description of this last group of cells (9) constitutes the essential of his discovery. Let us quote LANGERHANS‘ original text:

I mentioned above, when describing the varied structure which the pancreas shows after maceration in Müller’s fluid, a cell form not yet described. This cell is a small irregular polygonal structure. Its cytoplasm is brilliant and free of any granules, its nucleus distinct, round, and of moderate size. Its diameter is around 0.0096–0.012 mm while that of its nucleus 0.0075–0.008 mm. These cells lie together, generally in considerable numbers, diffusely scattered in the parenchyma of the gland. If a pancreas, after being kept in Müller’s fluid for 2 or 3 days is examined under low power, these groups of cells are seen scattered throughout the gland as vivid yellow specks. Under high power these specks are seen to consist entirely of our cells. They are gathered in round masses, 0.12–0.24 mm in diameter, distributed at regular intervals in the parenchyma, and can easily be seen in preparations of fragments of the fresh gland or in those treated for a short time with iodized serum. Like all fresh cells, they are completely round, but do not differ in any way from the cells described above, their contents are particularly shiny.

LANGERHANS can offer no explanation as to the nature of these cells. He continues:

If the answer that I have given as to the nature of the centro-acinar cells was insufficient, the same thing is unfortunately true of these cells [the cells of the islets]. If I was able, at least to a certain extent, to formulate a hypothesis concerning the centro-acinar cells, I must admit that for these I have no possible explanation.

As we will see later on, this confession of ignorance gave birth to a fervent desire to know. But further studies published arrived at no useful interpretation until that brought forth by Laguesse (1893). Neither could LANGERHANS draw any conclusions from his observations of the nerves or lymphatics of the pancreas. However, he implied the existence of a connection between his cells and the nervous apparatus of the gland.

I have seen relatively often a little bundle of pale nerves above our groups of cells, without being able, however, to establish the relationship between them; in the same way, our groups of cells were sometimes situated near a ganglion, and in certain cases, they seem to be very close to the nerve bundle.

LANGERHANS description is minute. Unfortunately it is not accompanied by any iconographic document. This is surprising since at the time when his thesis was presented the printing of iconographs was already very advanced. We also notice that LANGERHANS never used the term islet but the expression group of cells. It is the discovery and the description for the first time of this group of cells scattered here and there in the parenchyma of the pancreas (and which in weight represent a little less than one-hundredth of the gland) which made the name of LANGERHANS renowned the world over.

In fact from 1869, when he wrote his thesis, to 1893 the recognition of the exactitude of LANGERHANS‘ observations came only slowly. Scientific history shows that to bring about a change in the ways of thinking of people one must overcome a certain heavy inertia. Innovators always come up against strong resistance. It is relatively more difficult to overcome and convince people than to overcome things.

In 1893 Edouard Laguesse, an eminent French histophysiologist, became particularly interested in the groups of cells that he found in abundance in the human pancreas. He identified them as those which were earlier described by LANGERHANS in 1869 and gave them in reverent and generous homage the name of Islets of LANGERHANS (Fig. 4). In a note which appeared in Les Comptes Rendus de la Société de Biologie in 1893, Laguesse indeed wrote:

FIG. 4 Work by Edouard Laguesse in which the expression islets of Langerhans was first suggested

LANGERHANS was the first, in 1869, to point out small masses or groups of special cells in the adult pancreas, scattered from place to place, between the acini, and of unknown significance…. In the pancreas of the adult man [executed] I rediscover these numerous and large islets [in the meantime I will call them the islets of LANGERHANS].

The history of science shows that it is easier for those who are themselves of exceptional merit to do justice to the merits of others.

In fact, Laguesse had the merit to be the first person to have suggested that the islets of LANGERHANS were the site of internal secretion. It seems logical that here we are in the presence of an internal secretion, a secretion which continues throughout existence.

It is from this remarkable hypothesis, based on no less remarkable histophysiological studies, that the works and discoveries that constitute the fundamental basis of our knowledge and concept of diabetes blossomed out.

Such are, for example, among the most remarkable, the physiological demonstrations respectively by Oscar Minkowski (1889) and by Emmanuel Hedon (1890–92) that the pancreas liberates into the blood that flows through it, a hormone (named insulin by De Meyer in 1909) that plays an essential role in the regulation of the blood sugar level and the metabolic equilibrium; the differentiation by Lane (1907) of the α- and β-cells of the islets of LANGERHANS, as well as the remarkable histological studies by Bensley (1911); the description by the pathologist Weichselbaum of the alteration of the islets in most cases of diabetes (1910–11); and, finally, the extraction of insulin from the pancreas by Frederik Banting and Charles Best (1922). May we pass over in silence, but without really forgetting them, the names which come to mind to each one of us at this moment. They have all contributed generously to the blooming of our knowledge of diabetes and of its treatment. We can render homage more justly and more generously by our thoughts and intentions than by a list which might not be complete.

What really counts in a discovery is not only the discovery itself but also its ability to give rise to a multitude of ideas and new discoveries. Our presence today at this anniversary symposium is the purest and truest token of gratitude that we could render to the discovery, to the pioneer work, as well as to the memory of PAUL LANGERHANS.

The reader interested by the publications of Paul Langerhans will find a list in BARDELEBEN (1888) and MORRISON (1937).

REFERENCES

BARACH, J.H. Paul Langerhans 1847–1888. Diabetes. 1952; 1:411.

BARDELEBEN, K. Paul Langerhans. Anat. Anz. 1888; 3:850.

BERNARD, C. (1856) Mémoire sur le pancréas et sur le rèle du suc pancréatique dans les phénomènes digestifs (Baillère, J. B., Ed.), Paris, p. 187.

LAGUESSE, E. Sur la formation des îlots de Langerhans dans le pancréas. C. R. Soc. Biol., Paris. 1893; 5:819.

LANGERHANS, P. (1869) Beiträge zur mikroskopischen Anatomie der Bauchspeicheldrüse. Inaugural-Dissertation zur Erlangung der Doctorwürde in der Medicin und Chirurgie vorgelegt der Medicinischen Facultät der Friedrich-Wilhelms-Universität zu Berlin und öffentlich zu vertheidigen an 18. Februar 1869. Gustav Lange, Berlin.

LAZARUS S.S., VOLK B.W., eds. The Pancreas in Human and Experimental Diabetes. Grune Stratton: New York, 1962:1–13 Part I, Historical Review

LOUBATIÈRES, A. (1953), La part des recherches d’Emmanuel Hedon dans nos connaissances sur le diabète, Le Diabéte, No. 2.

MORRISON, H. Contributions to the microscopic anatomy of the pancreas by Paul Langerhans (Berlin 1869). Bull. Hist. Med. 1937; 5:259.

WARREN S., LECOMPTE P.M., LEGG M.A., eds. Historical considerations. Lea & Febiger: Philadelphia, 1966:9–18. [The Pathology of Diabetes Mellitus Chap. I].

PART I

DIFFERENTIATION AND GROWTH OF THE ENDOCRINE PANCREAS

ISLET CELL PROLIFERATION IN EXPERIMENTAL AND GENETIC DIABETES*

J. LOGOTHETOPOULOS, G. BROSKY and H.F. KERN,     Banting and Best Department of Medical Research, University of Toronto, Canada

SUMMARY

The proportion of β-cells incorporating tritiated thymidine was estimated in experimental conditions in which the β-cells are exposed to stimulation. All results indicated that β-cells have a limited potential for mitotic divisions during their life span. It is in the nature of the problem that absolute histological proof cannot be obtained in the adult animal which could exclude the formation of β-cells from precursor cells or duct cells. The experimental results with alloxan diabetic mice make such a process unlikely.Severe loss of β-cells can only partly be compensated for by limited proliferation of differentiated β-cells. This proliferative capacity may differ from species to species. In a particular animal it may be determined by whether previous stimulations have occurred which have utilized the mitotic potential. The rate of β-cell loss becomes therefore the important factor in maintaining or reducing a population of β-cells.

MULTIPLE injections of insulin antibodies provided a convenient method to stimulate β-cells of the pancreatic islets. Mice so treated have been kept for periods up to 3 weeks in good health with blood glucose levels maintained continuously above 300 mg%. The ultrastructural changes and the mitotic response of the β-cells studied by autoradiography following an injection of ³H-thymidine have been reported (Logothetopoulos and Bell, 1966).

The main characteristics of the stimulated mitotic response of the β-cells are summarized as follows:

1. A latent period, between 24 and 36 h, precedes the rise of the proportion of β-cells incorporating ³H-thymidine into DNA (radioactive index). Maximum values, 4 to 10 times of those found in control mice, are established by 72 h.

2. After cessation of the antibody injections on the second day, the mitotic response persists on the 3rd and 4th day in mice that have reverted to normoglycemia.

3. Actinomycin D blocks the response.

4. The radioactive index progressively declines after the first week in spite of maintained stimulation and persisting hyperglycemia.

5. β-cells which had been stimulated by a short (3 day) period of insulin antibody injections show a decreased response to a second stimulation 15 days later. An immune inactivation of the insulin antibody was eliminated as a contributing factor to the decreased response.

6. Evidence for a stem-cell type of β-cell was not obtained.

In view of the results of the experiments described, the regenerative pattern of a population of β-cells restricted by the injection of a diabetogenic compound acquired a special interest. The increased β-cell mass in the obese-hyperglycemic mouse also raised certain problems on β-cell proliferation.

MATERIAL AND METHODS

Mice

Inbred C57/6 Jax male mice obtained from the R. B. Jackson Memorial Laboratories were used throughout the study. Mice were fed with breeder’s chow ad libitum. One part of Locke’s solution was added to seven parts of drinking water. Obese-hyperglycemic mutant mice together with their lean littermates were obtained from the same source.

Injections of Alloxan or Streptozotocin

Solutions were made immediately prior to injection in normal saline of pH 3.5. Seventy milligrams of alloxan monohydrate per kilogram body weight in a volume of 0.15 to 0.20 ml were injected intravenously into a lateral tail vein.

Streptozotocin was similarly dissolved and injected at a dose of 100 mg/kg body weight. All mice were fasted for 12–16 h prior to injections. Over 95 % of the injected mice developed severe diabetes with practically no immediate mortality or symptoms of general toxicity.

Injections of ³H-Thymidine

Tritiated thymidine (sp. activity 6.7 c/mM) was injected subcutaneously at a dose of 0.5 μc/g body weight in a volume of 0.2 ml of sterile saline solution. Obese-hyperglycemic mice and their lean littermates received one or two (9 p.m. and 9 a.m.) injections and were killed 1 h later. Diabetic mice and their controls received 8 subcutaneous injections of tritiated thymidine at 12 h intervals. This procedure had the following advantages:

(a) In the diabetic mice with a very low density of β-cells, valid proportions of labelled cells were obtained by counting a smaller number of total β-cells.

(b) Differences between diabetic and control mice were magnified and diurnal variations eliminated.

Autoradiographic Techniques

The stripping film technique of Pelc was applied to 4 μ paraffin sections, of the splenic part of the pancreas fixed in Bouin’s fluid. Sections were stained with aldehyde-fuchsin or PAS before the application of the emulsion. Both stainings resisted the process of photographic development. A weak hematoxylin solution was used as a nuclear stain following the photographic development.

The proportion of cells with labelled nuclei was estimated, under oil immersion, in acinar, duct, and islet cells (α and β) until statistically valid proportions were established. Random areas equally distributed across the sections were used for acinar and duct cells.

Insulin Treatment

Protamine zinc insulin was diluted in protamine zinc vehicle. Treated diabetic mice received 0.1–0.2 units in the morning and 0.4–0.5 units in the late afternoon.

EXPERIMENTS AND RESULTS

A Mitotic Activity of Islet Cells Following Alloxan or Streptozotocin Diabetes

A total of 8 injections of ³H-thymidine was given during a period of 4 consecutive days to groups of diabetic and control mice on the following intervals after the injection of the diabetogenic compound: 3rd to 6th day; 8th to 11th day; 57th to 60th day; 87th to 90th day; 117th to 120th day. Separate experiments were spread throughout the year. Each diabetic group had its own control group of mice of the same age group and from the same shipment lot. After the last injection of tritiated thymidine diabetic mice were treated repeatedly with a mixture of protamine zinc insulin and insulin Toronto until the urine became free of glucose. This increased the granule content of the previously stimulated β-cells and greatly facilitated their identification in aldehyde-fuchsin stained autoradiographs.

All mice were killed 36 h after the last injection of ³H-thymidine. The proportion of labelled nuclei in β- and α-cells of the islets and of acinar and duct cells of the exocrine pancreas are shown in Table 1. The greatest number of labelled β-cells was found during the first week after alloxan administration. Values declined during the second week of diabetes but remained significantly higher than control values. By the end of 2 months the proportions of labelled nuclei in diabetic and control mice overlapped. By the 3rd and 4th month, there was no difference between diabetic and control animals. The same broad pattern was found in the streptozotocin diabetic mice (Fig. 1).

TABLE 1

Proportions of Labelled Islet, Acinar, and Duct Cells (³H-Thymidine Index) of the Pancreas in Diabetic (D) and Control (C) Mice Mean and range are given. Each group consists of 5–7 mice

aH-thymidine was injected twice daily at 12 h intervals during the 4 day period.

bLabelled cells per 500 cells. Over 1000 β-cells were counted in control mice but the numbers stated refer to 500 cells for proper comparison. The error of the estimated ³H-thymidine indices in controls is therefore smaller than in diabetics.

cLabelled cells per 1000 cells.

FIG. 1 ³H-thymidine index of β-cells during streptozotocin diabetes

The mitotic activity of α-cells did not show any fluctuations at any period. Differentiation of labelled α-cells from labelled endothelial or fibroblastic cells was not always possible. When in doubt, labelled cells were included as α-cells. The total number of α-cells counted per mouse did not exceed 500 cells. For the reasons stated the estimate of the ³H-thymidine index of the α-cells contains a larger error than that for the other cells. Strikingly high proportions of duct cells incorporating ³H-thymidine were found in the diabetic mice during the first and second week after the induction of diabetes (Table 1). Radioactive indices at 2,3, and 4 months were within the range found in controls with the exception of two mice at 2 months showing very high values.

Labelled duct cells were always found scattered, often in pairs, throughout the circumference of the ducts in complete alignment with the non-labelled cells. Budding of labelled duct cells was in no circumstance observed.

B Recovery from Diabetes in Insulin-treated and Untreated Diabetic Mice

A hundred mice 13–15 weeks old were made diabetic with alloxan as described. One month after the injection of alloxan the few which showed severe ketonuria or had reverted to intermittent glucosuria were eliminated. The others were divided randomly into two groups. One group was treated with protamine zinc insulin twice daily, as described in Materials and Methods. The second group was injected with the protamine zinc vehicle only. Urine was tested for glucose and ketones by Testape® daily.

The insulin-treated mice gained weight and looked healthy. When glucose-free urine was found for three consecutive days, the insulin injections were stopped. They were resumed in case the mouse showed again progressively increasing glucosuria. In general, most of the treated mice showed periods of glucosuria between the daily injections of insulin. The untreated mice did not gain weight and showed persistent severe glucosuria and occasionally positive tests for ketone bodies.

Table 2 shows the fate of mice in both groups by the end of 10 months. Deaths in the insulin-treated group were mainly due to hypoglycemia. The cause of death in the untreated diabetic mice was severe wasting or infection. Reversions to urine permanently free of glucose were uncommon in both groups. No significant difference in the number of recoveries from diabetes between the two groups was evident.

TABLE 2

Recoveries from Diabetes of Insulin Treated and Untreated Alloxan Diabetic Mice Observed for 10 Months

aUntreated mice received injections of protamine zinc vehicle only.

bTreated mice died from hypoglycemic episodes; untreated mice died mainly from infections or dehydration.

C Proliferative Potential in the Obese-Hyperglycemic Mouse

The proportions of β-cells incorporating thymidine in 9-week-old obese-hyperglycemic mice were higher, as expected, than those found in lean littermates of the same age. All obese mice were glucosuric and hyperglycemic. The mitotic activity declined with age. The radioactive indices in 10-months old obese mice were similar to those found in lean littermates (Table 3). This decreased mitotic activity could be easily correlated with the decline of hyperglycemia. Non-fasting blood glucose levels in this older group of obese mice exceeded slightly the values obtained in their lean littermates. The question remained open whether the enlarged population of β-cells in the ageing obese mouse would respond to an acute stimulation.

TABLE 3

Blood Glucose and ³H-Thymidine β-Cell Index in 2½ and 10-month-old Obese-hyperglycemic Mice and their Lean Littermates

aMice killed 1 h after a subcutaneous injection of ³H-thymidine.

Obese mice, 14 months of age, were injected with concentrated antiinsulin γ-globulins every 6–8 h. The volume injected was adjusted according to tests for ketone bodies and glucose in the urine. Lean littermates required about one-third of the antibody that was injected into the obese for sustained glucosuria. Two injections of ³H-thymidine were given at 48 and 60 h after the first injection of antibody. Mice were killed 2 h after the second ³H-thymidine injection. As Fig. 2 shows, very few β-cells with labelled nuclei were found the obese mice.

FIG. 2 ³H-thymidine index in obese and lean mice 14 months old after insulin antibody injection

DISCUSSION

The experiments with multiple injections of antibody have demonstrated that the mitotic activity of the β-cells cannot be maintained continuously at a high level by this procedure: mitotic activity declines rapidly in spite of persisting hyperglycemia. A population of β-cells, even after a long period of rest, responded poorly to a second stimulation with insulin antibody as shown by a lower ³H-thymidine index. The β-cells of the obese-hyperglycemic mutant mouse are exposed from the first postnatal weeks to stimulation by hyperglycemia. When ageing obese-hyperglycemic mice which had reverted to normoglycemia were subjected to a period of injection with insulin antibody, their β-cells, in contrast to those of their lean littermates, showed very low ³H-thymidine indices. All these experiments clearly indicate that the potential for multiple divisions of the β-cells in the mature mouse is limited.

The autoradiographic results with mice in which the number of β-cells was acutely reduced by a diabetogenic dose of alloxan or streptozotocin conformed to the same basic pattern. During the first week a very large proportion of β-cells incorporated ³H-thymidine. The ³H-thymidine index declined during the second week. It should be mentioned that Cavallero demonstrated in 1947 the same declining mitotic activity of β-cells in the alloxanized rat by metaphase counts after colchicine during the first 2 weeks of diabetes.

An approximate calculation based on the rapid degradation of circulating ³H-thymidine and on a mean duration of DNA synthesis (S-phase) in the β-cell of 4–5 h makes very probable a 50% increase in the number of β-cells during the first week after alloxanization. The diabetic mice at 2, 3, and 4 months showed low ³H-thymidine indices within the range of the control groups. Islet growth in the rodent occurs continuously throughout adult life (Hellman et al., 1961). New β-cells therefore contribute to an expanding population and do not only compensate for β-cell turnover. To what extent this model applies to a small residual population of β-cells exposed to continuous hyperglycemia is an open question. The low rates of proliferation of β-cells after the initial short wave of activity following the injection of alloxan or streptozotocin would indicate that the evolution of experimental diabetes (deterioration or amelioration) would be mainly determined by the following factors:

(a) The number of β-cells which survived the initial cytotoxic death.

(b) The subsequent rate of β-cell loss.

(c) The minimal β-cell mass compatible with glucose homeostasis.

The significance of this model would obviously depend on whether β-cells in the mature mouse can arise out of differentiated cells of the exocrine pancreas or from potent precursor β-cells. The finding of a widespread increased mitotic activity of predominantly duct cells during the first week after alloxanization could be taken as evidence for this process of β-cell neogenesis. If this were so, one would expect to find (a) a high density of scattered, small agglomerations of labelled β-cells with specific granules throughout the pancreas, and (b) a high rate of prompt recoveries from diabetes in alloxanized mice kept for a long period. Neither of these conditions was fulfilled. The density of islets in the diabetic mice was much lower than in controls. Many sections had to be screened in order to establish valid ³H-thymidine indices for β-cells. The regranulation of β-cells by the intense insulin treatment after the injection of ³H-thymidine permitted detection of even a few pairs of β-cells.

During the first weeks, islets and ducts with a high proportion of labelled cells were often encountered in the frequently described relationship of close apposition. Whereas labelled cells in the duct were located throughout its circumference as part of the lining cells, within the islet, labelled β-cells were randomly scattered. No connecting bridges of labelled cells were encountered.

Whatever the cause for the regenerative wave of duct cells following alloxanization it has no apparent connection with the formation of new β-cells. Persisting insulin treatment of alloxan diabetic mice did not result in a significant increase in the number of recoveries from manifest diabetes as compared with non-treated mice. The treatment with insulin injections was started 1 month after alloxanization and did not extend beyond the 10th month. In view of the results of Lazarow (1952) on the late spontaneous recoveries of diabetic and subdiabetic rats the possibility remains that prolongation of treatment might have shown a significant effect.

DISCUSSION

FALKMER: You said that there were no new β-cells in the ducts, but are there not already in the normal animal some granulated β-cells in the ducts? Do not the labelled nuclei in the ducts indicate multiplication of those β-cells? Could you not also see cell renewal by amitotic cell division, e.g. as shown by Pehlemann in the stimulated interrenal gland in Rana temporaria (Z. Zellforsch.,84, 516, 1968)?

LOGOTHETOPOULOS: I agree fully with you. Differentiated β-cells can be close to the duct or be found between duct cells. The incidence differs from species to species. Obviously they will respond to a mitotic stimulus. This model has to be clearly distinguished from that in which specialized duct cells turn into β-cells in the adult animal. As far as amitotic divisions are concerned, I would assume that duplication of DNA would precede also this type of division.

GEPTS: I have been very interested in Dr. Logothetopoulos’ paper because he has been able to reproduce experimentally a condition similar to that in acute juvenile diabetes in the human. It is unfortunate that we have so little electron microscopical data on the pancreas in human diabetes. With the light microscope, neoformation of β-cells is frequently seen in the pancreas of the adult diabetic or not. The newly formed β-cells seem to arise from centro-acinar cells, but one can also find them in the epithelial lining of larger ducts with budding of endocrine cells into the surrounding connective tissue. In islet cell tumours transition between duct-like structures and endocrine tissue are also frequent. In chronic juvenile diabetics we have never seen β-cell neoformation, but in juvenile cases, which died shortly after the clinical onset of diabetes, very active islet neoformations can be found. Since they are located around tubules, we feel that they are derived from tubular epithelium. So there seems to be little doubt that transformation of duct cells into islet cells does