The Evolution of Pancreatic Islets: Proceedings of a Symposium Held at Leningrad, September 1975, under the Auspices of the Academy of Sciences, Leningrad

Epple,     U.S.A.

INTRODUCTORY REMARKS

L. Leibson,     Chairman of the Organizing Committee

Ladies and Gentlemen, dear colleagues.

On behalf of the Organizing Committe of the Symposium, I welcome you to Leningrad to the Academy of Sciences, the oldest research centre in our country.

The symposium to be opened today is sponsored by two institutions of the Academy: the Scientific Council on the Combined Problems of Physiology of Man and Animals, which coordinates the research in the field of Physiology, and Sechenov Institute of Evolutionary Physiology and Biochemistry. The Institute was founded by academician Orbeli in 1956.

During all his life academician Orbeli insisted on applying evolutionary principle of physiology, as the base for correct interpretation of the body functions. On the other hand, he believed that the knowledge of functional evolution can add much to further development of the evolution theory.

According to Orbeli, the evolutionary physiology should be built on the results due to comparative study of functions of animals belonging to different levels of evolution and due to developmental study of functions. He also pointed out that even when studying the functions of higher animals, the fact that their organs belong to different evolutionary levels should always be taken into account. At last, he stressed the necessity to study the evolution of functions by joined efforts of physiologists, biochemists and morphologists.

These ideas of Orbeli are the foundation stones of the Institute.

As you can see, the program of the Symposium is also worked out according to these ideas. It contains reports on the phylogeny and ontogeny of pancreatic islets; and specialists in different fields - physiology, biochemistry and morphology - participate in the Symposium.

Such a complex approach to the study of functions finds more and more application nowadays. In particular, it found its way in the conferences held every two years by the European Society of Comparative Endocrinology and International Symposia on Comparative Endocrinology held every three years.

It is a characteristic feature of our symposium that it is dedicated to the evolution of one endocrine organ only, namely pancreatic islets. The importance of such so-to-say, narrow symposia in addition to conferences on a large scale is evident. Science is advancing so fast that it is not possible to fully discuss the results concerned with one field even, let alone many fields.

Recently the study of evolution of pancreatic islets made a great success due to the work of prominent specialists. It gives real pleasure to see many of them here. I should like to stress that systematic study of the above problem began as long ago as 15 - 20 years only. This success is accounted for by a very quick progress of endocrinology as a whole, which, in its turn, is due to the discovery of basically new methods in biochemistry, genetics, cytology, immunology and other biological sciences.

It would be unfair to say that the comparative approach to the study of pancreatic islets did not attract attention before recently. Some interesting publications appeared many years ago. It is enough to mention the works of Diamare and Kuliabko who as early as 1901 studied the effect of extracts of fish Brockmann bodies on carbohydrate metabolism, as well as the works of Macleod, MacCormick and others who in the early twenties studied insulin isolated from fish pancreatic islets. However, only recently have real achievements in the study of evolution of pancreatic islets as well as of their ontogenetic development been made.

The study of both problems is very important not only from the theoretical point of view but for practical medicine also. Diabetes is a most widely-spread endocrine disorder. The cause of this disorder is not disclosed yet despite many efforts. There is no doubt that the evolutionary approach to the study of one of the most important organs in regulating the metabolism, the endocrine pancreas, will throw a new light on the nature of diabetes.

The idea to hold this Symposium occurred two years ago at the VII Conference of the European Society of Comparative Endocrinology in Budapest when Professor Epple from the U.S.A., Dr. Thorpe, from England, Dr. Trandaburu, from Rumania and myself agreed that a special symposium on evolution of pancreatic islets would be very urgent.

Dr. Epple proposed for a symposium to be held in Leningrad. I regarded it as appreciation of our work and believed that such a symposium in this country could stimulate investigation in this field and provoke interest among a wider circle of researchers here in our laboratories. I consulted the Head of Department of Physiology of the Academy of Sciences, academician Kreps. He supported the idea, and we all should express our gratitude for his assistance. I also wish to thank the Presidium of the Academy of Sciences, Director of our Institute, Professor Govirin, and all those who made it possible for the Symposium to be held here. We are grateful to Professor Epple and all our colleagues who so willingly agreed to take part in our Symposium and regret that not all of them could come.

I hope the work of the Symposium will be fruitful. I wish you all, our guests from abroad as well as from the Soviet Union, to spend time successfully and to enjoy your stay in our town.

PART I

THE ONTOGENETIC ASPECTS OF THE STRUCTURAL AND FUNCTIONAL EVOLUTION OF PANCREATIC ISLETS

Outline

Chapter 2: THE ONTOGENY OF MAMMALIAN INSULAR FUNCTION

Chapter 3: THE EMBRYONIC DEVELOPMENT OF PANCREATIC ISLETS OF OFFSPRING OF ALLOXAN RATS

Chapter 4: NEW DATA CONCERNING EARLY POST-NATAL DEVELOPMENT HUMAN PANCREATIC ISLETS

Chapter 5: DEVELOPMENT OF THE FUNCTION OF ENDOCRINE PANCREAS IN THE HUMAN FOETUS

Chapter 6: IMMUNOCYTOLOGICAL STUDY OF ENDOCRINE PANCREAS ONTOGENY IN THE CHICK EMBRYO: NORMAL DEVELOPMENT AND PANCREATIC POTENTIALITIES IN THE EARLY SPLANCHNOPLEURE

Chapter 7: BIOSYNTHESIS OF PROINSULIN AND INSULIN BY THE CHICK EMBRYO PANCREAS FROM LABELLED AMINO ACID

Chapter 8: THE SECRETION AND THE ROLE OF INSULIN IN CHICK EMBRYOS AND CHICKENS

THE ONTOGENY OF MAMMALIAN INSULAR FUNCTION*

Piero P. Foà, Enrique Blázquez, Jean-Claude Sodoyez and Francoise Sodoyez-Goffaux,     Department of Research, Sinai Hospital of Detroit, Detroit, Michigan, USA

Publisher Summary

The metabolic development of the mammalian organism may be divided arbitrarily into six major periods. In the first period, representing about one-third of the intrauterine life, glucose and other nutrients are utilized for energy and growth and need not be stored by the embryo as they are provided in practically unlimited amounts by placental transfer. During the second period, large deposits of glycogen and fat are formed and the fetus prepares itself for the interval between the abrupt loss of placental support and the start of oral nutrition. In the third period, most newborn lose weight and the preservation of euglycemia takes precedence over growth. During the fourth period, the glucagon to insulin ratio remains relatively high so that the animal, which consumes a high triglyceride, low carbohydrate diet, can obtain needed fuel from lipolysis. The fifth phase represents a period of transition, during which many of the endocrine and metabolic processes characteristic of the third and fourth phases are reversed. This is a period of accelerated growth leading to the sixth and final phase when the animal is fully adapted to the high carbohydrate mixed diet typical of the adult.

The metabolic development of the mammalian organism may be divided arbitrarily into six major periods. In the first period, representing about one-third of the intrauterine life, glucose and other nutrients are utilized for energy and growth (8) and need not be stored by the embryo since they are provided in practically unlimited amounts by placental transfer. As gestation progresses, with the development of the liver and of the adipose tissue, there begins a second period during which increasingly large deposits of glycogen and fat are formed (2, 15, 16, 35) and the fetus prepares itself for the interval between the abrupt loss of placental support and the start of oral nutrition. During this third period of physiologic fast, lasting about 1 or 2 hours in the rat and 12 to 24 hours in man, these deposits are mobilized, in part as a result of a high glucagon to insulin ratio and thus provide the endogenous substrates necessary to sustain the high metabolic activities of the newborn and the glucose required by the brain and other glucose-dependent tissues. In this period, most newborn lose weight and the preservation of euglycemia takes precedence over growth which resumes only when the emergency is over and nutrients become available once again in the form of milk. This moment marks the beginning of the fourth or nursing period during which even though the secretion of insulin increases, the glucagon to insulin ratio remains relatively high, so that the animal which consumes a high triglyceride, low carbohydrate diet can obtain needed fuel from lipolysis and needed glucose from gluconeogenesis. The fifth or weaning phase represents a period of transition during which many of the endocrine and metabolic processes characteristic of the third and fourth phases are reversed: the glucagon to insulin ratio decreases, lipolysis gives way to lipogenesis, glycogenolysis to glycogen syntheses and gluconeogenesis to protein synthesis. This is a period of accelerated growth leading to the sixth and final phase when the animal is fully adapted to the high carbohydrate mixed diet typical of the adult.

The transition from one period of metabolic development to the next requires profound, sometimes abrupt, changes in the activity of many enzymes, in turn, heralded by changes in the relative abundance of their respective regulatory hormones. This paper will review the contributions to embryologic endocrinology published by our laboratory during the last 15 years and will make only tangential reference to the work of other investigators. Our initial experiments were the outcome of an unsuccessful attempt to find a method of measuring insulin-like activity in biologic materials, an attempt made when the epididymal fat pad and the radioimmunoassay were not available and the rat diaphragm method was beset with technical difficulties. The search for a suitable object led to the chick embryo heart, a muscle preparation which, unlike the diaphragm, can be removed from the animal without injury and which, unlike the adult heart, has a rudimentary coronary system and, therefore, can be incubated without perfusion (31; Fig. 1). Although it became apparent very soon that the sensitivity of the chick embryo heart to insulin was not sufficient for biologic measurements, the preparation turned out to have other interesting properties. Among them, the development of an insulin-sensitive glucose transport system which, on or about the 7th day of incubation, displaces intracellular phosphorylation as the rate-limiting step of glucose uptake (12, 16, 17, 19-23, 25; Fig. 2 and 3). The apparent coincidence of this phenomenon with the appearance of granules in the pancreatic β cells (36) suggested the interesting hypothesis that the ontogeny of hormones is related to that of the enzymes that mediate their action. Measurements of the pairs glycogen synthetase (UDPG-glycogen transferase)-insulin and phosphorylase-glucagon-like materials in chick and rat embryo tissus at various stages of development lend support to this hypothesis. For example, it could be demonstrated that in the tissues of the rat embryo, glucagon synthetase activity starts approximately when insulin and glycogen synthesis appears and increases until birth as they are known to increase (1, 2, 3, 4, 5, 30; Figs. 4 and 5). This accumulation of insulin suggests that the fetal rat pancreas, under favorable intrauterine conditions, dependent in part on the level of maternal blood glucose, (30, 34) may secrete an amount of insulin adequate for growth and glycogen synthesis. Indeed, between the 20th and 22nd day of gestation the insulin concentration in the fetal blood exceeds that of the maternal blood (11) and insulin, probably of fetal origin, appears in the amniotic fluid (9). At about the same time, the adipose tissue (24), the liver (26), the diaphragm (11) and the heart (10) become sensitive to insulin. Thus, during the last period of gestation, in the presence of a constant provision of maternal nutrients, lipids are deposited in adipose tissue (35), liver glycogen accumulates at an accelerated rate and, just before delivery, reaches values that greatly exceed those found in the liver of the adult rat (5). This phenomenon was described by Claude Bernard more than a century ago (3) and by other investigators after him (for references see 5). At birth these anabolic processes are abruptly reversed and the newborn enters a period of accelerated glycogenolysis, gluconeogenesis and lipolysis. The increase in phosphorylase activity and cyclic AMP content of the liver (6, 29) and in the glucagon-to-insulin ratio occurring at birth (5, 14, 27, 32, 33) provides the enzymatic and endocrine environment favorable to these metabolic changes (Fig. 6 and Tables I and II) and to the preservation of normal serum glucose levels in an animal whose only supply of carbohydrate is milk.

Table I

Glucose, free fatty acids (FFA), insulin, total and pancreatic glucagon in the serum of human babies at and after birth. Ave. ± SEM. Number of observations in parentheses

ap <0.05 Note that while the concentration of insulin decreases after birth, that of glucagon increases.

bp <0.02 This reversal of the insulin to glucagon ratio is conductive to lipolysis, indicated by the rise in serum FFA level and to the prevention of fasting hypoglycemia. The increase in total glucagon

cp <0.01 24 hours after birth is the result of the baby’s first meal.

dp <0.001 1Total glucagon was measured with antiglucagon serum AGS 10; pancreatic glucagon with AGS 18 (28).

Table II

Concentration of cyclic AMP in the liver of fetal and newborn rats. Average ± SEM. Number of observations in parentheses

ap <0.001 vs. day 19

bp <0.01 vs. day 22

cp <0.001 vs. day 22

Fig. 1 Rate of contraction, incidence of irregularities and oxygen consumption of embryonic chick hearts incubated in a cartesian diver ( 31).

Fig. 2 ) or in the presence (• – – •) of insulin. The hearts were incubated at 37.5 C in Krebs-Henseleit bicarbonate buffer, pH 7.4, in an atmosphere of 95% O2 and 5% CO2. Initial glucose concentration, 8 × 10− M. Insulin concentration, 0.01 U/ml. Arrow points to a value of 489 µmoles at 120 min. Each point represents the mean of 3 experiments ± SE.

Fig. 3 ) insulin. The hearts were incubated for 1 hour at 37.5°C in Krebs-Henseleit buffer, pH 7.4, in an atmosphere of 95% O2 and 5% CO2. Osmolarity of the medium, 0.306. Insulin concentration, 0.01 U/ml. Each point represents the mean of 5 experiments ± SE.

Fig. 4 UDPG-glycogen synthetase activity in the developing liver of rat embryo.

Fig. 5 Insulin content and concentration of the fetal and neonatal rat pancreas.

Fig. 6 Perinatal changes in liver glycogen and in the serum levels of insulin and glucogon in the rat.

The insulinogenic response to nutritional stimuli reappears shortly after birth when the animal begins to nurse and increases sharply 10 to 12 days later (Fig. 7). Indeed, up to 10 days after birth the release of insulin by the nursing rat following an oral load of glucose or of a mixture of amino acids is delayed and the animal is in a state of relative glucose intolerance which has an additional glucose sparing effect (7). During this period, the animals’ respiratory quotient decreases from the fetal value of 1.0 to 0.7, indicating an increased utilization of fatty acids. As nursing progresses, the synthesis of proinsulin, the conversion of proinsulin to insulin and its release by isolated pancreatic tissue increase (Fig. 7), reaching full maturity after the third week of extrauterine life, when the animal switches from a milk diet, rich in fat and protein and relatively poor in carbohydrate, to a high carbohydrate commercial rat food.

Fig. 7 Effect of glucose on the release of insulin in vitro by fetal and neonatal rat pancreas.

REFERENCES

1. Adam, P. A.J. Control of glucose metabolism in the human fetus and newborn infant. Adv. Metab. Dis. 1971; 5:183.

2. Ballard, F. J., Oliver, I. T. Glycogen metabolism in embryonic chick and neonatal rat liver. Biochim. Biophys. Acta (Amst.). 1963; 71:578.

3. Bernard, C. M. De la matière glycogène considérée comme condition de développement de certains tissus, chez le foetus avant l’apparition de la fonction glycogénique du foie. C. R. Acad. Sci. (Paris). 1859; 48:673.

4. Blázquez, E., Montoya, E., Lopez-Quijada, C. Relationship between insulin concentrations in plasma and pancreas of foetal and weanling rats. J. Endocr. 1970; 48:553.

5. Blázquez, E., Sugase, T., Blázquez, M., Foà, P. P. The ontogeny of metabolic regulation in the rat, with special reference to the development of insular function. Acta Diabetol. Latina. 1972; 9:13.

6. Blázquez, E., Sugase, T., Blzáquez, M., Foà, P. P. Neonatal changes in the concentration of rat liver cyclic AMP and of serum glucose, free fatty acids, insulin, pancreatic and total glucagon in man and in the rat. J. Lab. Clin. Med. 1974; 83:957.

7. Blázquez, E., Lipshaw, L. A., Blázquez, M., Foà, P. P. The synthesis and release of insulin in fetal, nursing and young adult rats: Studies invivo and invitro. Ped., Res. 1975; 9:17.

8. Burch, H. B., Lowry, O. H., Kuhlman, A. M., Skerjaner, J., Diamant, E. J., Lowry, S. J., von Dippe, P. Changes in patterns of enzymes of carbohydrate metabolism in the developing rat liver. J. Biol. Chem. 1963;