ABSTRACT
Insulin, in varying concentrations, has been applied to the chick embryo explanted by New’s technique after 24 hours incubation at stages 3–8 and cultured for 22–24 hours in vitro.
It has been demonstrated that, in whatever manner insulin is applied to the embryo, its effects are most frequently manifest in the brain and neural tube. Somites are inhibited to a much lesser extent and heart, except with the higher doses, is unaffected.
Application of either sodium pyruvate or nicotinamide with the insulin has very little protective action and larger doses of both these substances potentiate the inhibitory action of insulin.
Diphosphopyridine nucleotide in the oxidized form given with the insulin provides complete protection, but the reduced form of DPN has no such effect.
Previous work by Spratt and others on the metabolic characteristics of the early chick embryo, by Landauer on the teratogeni caction of insulin, and recent investigations (e.g. by Randle & Smith) are considered in the light of our findings.
Like Duffey & Ebert (1957) we hesitate to accept Spratt’s (1950) hypothesis for the dependence of brain on oxidative mechanisms and the heart on glycolysis. We suggest, instead, that the heart, in contrast to the brain, can if necessary survive for a period of time under anaerobic conditions but, where possible, it makes full use of aerobic metabolic pathways.
The complete protection afforded by DPN but not by DPNH leads us to believe that the site of action of insulin lies in the reoxidation of the pyridine nucleotides, i.e. in a hydrogen transport system.
INTRODUCTION
The list of effective teratogenic agents has become embarrassingly long: physical agents, metabolic inhibitors, vitamin deficiency, hypervitaminosis, and hormones, to mention only a few, are all known to produce congenital abnormalities. These substances have been administered at different times and in varying doses to nearly every group of animals, from amphibians to mammals, but there has been little effort to examine their mode of action. All too often, investigation ceases with the demonstration of a statistically significant number of abnormalities, enough to label the agent teratogenic. Sometimes a section of the work describes the histology of the lesions but does not indicate the primary action of the teratogen. Occasionally a drug has a well-recognized metabolic or chemical effect, e.g. cyanide on cytochrome oxidase, iodoacetate on triosephosphate dehydrogenase (Spratt, 1950), and we learn, by deduction, a little of the normal metabolism of development. Relatively few substances fall into this category, however. Lack of efficient techniques may have been the cause of the deficiencies, but, with the reliable modern culture methods now available, closer examination by observation and by experiment of the action of these drugs or physical agents in producing abnormalities is not only possible but relatively easy.
One of the best-known teratogenic agents is insulin, which has been used extensively as such in the chick embryo. When implanted into the yolk sac at various stages of development it readily induces abnormalities closely resembling certain mutant conditions. Landauer (1945) produced rumplessness in chicks by injecting insulin into the yolk sac at 24 hours of incubation. Later Landauer (1947) found abnormalities of the beak, extremities, and eyes after giving it at 5–6 days of incubation. Duraiswami (1950) also found abnormalities of the beak, eyes, and limbs with insulin treatment of the chick embryo.
In spite of so much recorded work, there is still no agreement or clear understanding of the mechanism and site of the inhibitory action of insulin in tissue metabolism. Landauer (1948) and Landauer & Rhodes (1952) claimed that nicotinamide protected insulin-treated embryos from micromelia and beak defects and that sodium pyruvate likewise prevented rumplessness induced by insulin. The authors suggested that such protection was mediated via diphosphopyridine nucleotide (DPN), but their results with DPN treatment were negative.
Until now experimental procedures, in general, have involved the embryo in ovo. However, using the New (1955) technique for the cultivation of chick embryos in vitro, we can now study more accurately the effect of insulin in the early chick embryo and correlate the effects observed with the metabolic processes underlying normal development.
In the first group of these experiments, the embryos were treated with insulin alone in varying concentrations. This was followed by a series of protection experiments in which an effective dose of insulin was administered with one of the following substances : sodium pyruvate, nicotinamide, DPN, and its reduced form DPNH.
MATERIALS AND METHODS
All the eggs were obtained from a Brown Leghorn × Light Sussex cross. After an initial incubation period of 24 hours at 37° C. they were explanted according to the New technique (1955), and then incubated in vitro for a further 22–24 hours. The embryos were staged according to Hamburger & Hamilton (1951).
The insulin employed in these experiments was of therapeutic standard, Insulin A.B., in a concentration of 40 units per c.c. (Allen & Hanburys Ltd., London). The sodium pyruvate (C. F. Boehringer & Son, Germany) was prepared as a molar solution in sterile phosphate buffer to bring the pH to 7-7-5. Nicotinamide, obtained from Hopkin & Williams Ltd., Essex, England, was dissolved in sterile Howard’s modified chick Ringer saline in molar concentration. Similarly, molar solutions were prepared, in sterile phosphate buffer, of diphosphopyridine nucleotide in the oxidized form, i.e. Coenzyme 1, and the reduced disodium salt of diphosphopyridine nucleotide (British Drug Houses, Poole, England). All solutions were stored in the refrigerator.
The insulin was diluted with three parts of sterile chick Ringer solution and the final amounts injected were as follows : 0·075 ml. = 1 unit; 0·150 ml. = 2 units; 0·225 ml. = 3 units; 0·300 ml. = 4 units.
Insulin was applied either dorsally or ventrally to the embryos, i.e. into the albumen on the dorsal surface or directly on to the ventral surface of the embryo. When pyruvate, nicotinamide, DPN, or DPNH were used together with insulin, the insulin was given on the dorsal surface and the supplement on either the ventral or the dorsal surface. Embryos were fixed in 10 per cent, neutral formalin, dehydrated in Dioxan or in graded alcohols, embedded in paraffin wax, cut and stained with haematoxylin and eosin.
RESULTS
Insulin, alone (Table 1)
The insulin was administered in doses of 1–4 units on either the ventral or the dorsal surface. There was no difference in effect between the two methods of application.
One unit of insulin produced little or no visible change; 2 or 3 units caused inhibitory effects in the brain and neural tube only, the latter usually remaining widely open along most of its length and the brain showing little or no differentiation into vesicles (Plate 1, fig. A). When 4 units were applied to each embryo, the somites developed abnormally, being smaller and poorly defined compared with the controls and with a tendency for them to crowd together near the anterior end of the embryo (Plate 1, fig. B). Only in a few cases was any effect noted on the heart, and this only with the highest dose of insulin. Two cases of cardia bifida were recorded.
In several of the embryos receiving higher doses of insulin considerable lysis and fragmentation of tissue developed around the primitive streak (Plate 1, fig. B).
Serial sections of control and treated embryos confinned that the neural tube in the treated embryos was wide open throughout most of its length. The neural folds had formed but failed to meet and fuse in the midline (Plate 2, fig. E). Sometimes the medullary plate was quite flat (Plate 2, fig. F). There was, however, no necrosis of cells in any of the inhibited tissues.
Insulin+ sodium pyruvate (Tables 2 and 3)
Concentrations of 10–1 M to 5× 10–1 M sodium pyruvate were given with 4 units of insulin, both on the dorsal surface. The smallest concentration of pyruvate provided no protection and the inhibition was typical of insulin treatment alone. 3·33 × 10–1 M sodium pyruvate and insulin inhibited the embryo much more than insulin alone, particularly somite formation; in most cases there were no somites, only two strands of undifferentiated paraxial mesoderm (Plate 1, fig. C). Even when this concentration of sodium pyruvate was given alone there was slight inhibition of the embryos. With 5×10–1 M sodium pyruvate and 4 units of insulin the embryos failed to develop at all, the blastoderm did not expand, and the embryos were generally at the same stage as when they were explanted. Similar results were obtained with the same dose of pyruvate alone.
The effect of sodium pyruvate given alone and with insulin; sodium pyruvate on the ventral surface, insulin on the dorsal surface

Concentrations of sodium pyruvate between 5 ×10–2 M and 2× 10–1 M were given on the ventral surface along with insulin on the dorsal surface. In this series the lowest concentrations of pyruvate, i.e. 5 × 10–2 M and 10–1 M, had no protective action but a dose of 2 × 10–1 M plus 3 units of insulin did provide slight protection. When the insulin dose was reduced to 2 units and supplemented with 10 1 M sodium pyruvate this protection was slightly greater, i.e. in most cases the neural tubes were closed, but the embryos were much smaller than the controls.
Insulin+nicotinamide (Table 4)
The lowest concentration of nicotinamide (2 × 10–2 M) ventrally, together with insulin dorsally, gave slight protection, the embryos generally being smaller than normal but in most cases with a completely closed neural tube. 5 × 10−2 M and 10–1 M nicotinamide plus insulin caused very severe damage, much more than with insulin alone; the neural tube was wide open, the somites poorly developed, and the heart tubes could be seen rising in front of the brain. These higher concentrations of nicotinamide when administered without insulin produced the same effects (Plate 1, fig. D).
Insulin+DPN (Table 5)
Two concentrations of DPN were used: 2·5× 10–3 M and 5× 10–3 M, both on the ventral surface. With each of these concentrations plus 3 units of insulin the embryos were completely protected and in every respect resembled the controls (Plate 2, figs. G, H).
The effect of DPN given alone and with insulin; DPN on the ventral surface, insulin on the dorsal surface

When the insulin was given on the dorsal surface 4 hours prior to the DPN on the ventral surface, just under 50 per cent, of the embryos were normal. The remaining embryos, however, were not severely affected. If, however, the experiment was reversed, the same concentration of DPN being given 4 hours before the insulin, complete protection was obtained.
A concentration of 2·5 × 10–3 M DPN on the ventral surface did not completely protect the embryos from the effects of 4 units of insulin on the dorsal surface, but the residual inhibition was only slight.
Insulin+DPNH (Table 6)
The effect of DPNH given alone and with insulin; DPNH on the ventral surface, insulin on the dorsal surface

5 × 10−3 M DPNH and 2·5 × 10–3 M DPNH, ventrally, gave no protection from 3 units of insulin dorsally. The inhibition was typical of insulin treatment alone, viz. wide-open neural tubes and in a few cases slight inhibition of somite formation.
DISCUSSION
These experiments show clearly : (a) that for the explanted 24–48-hour chick embryo the teratogenic effect of 2–3 units of insulin is mainly inhibition of the neural tube, and that 4 units or more inhibit the mesodermal derivatives as well as neural tissue; and (b) that DPN in its oxidized form provides protection for the embryo from the teratogenic effects of 2, 3, or 4 units of insulin.
The significance of these observations depends largely on our knowledge of the metabolism of the chick embryo between 24 and 48 hours of incubation and also on the interpretation of the many conflicting views on the mechanism of action of insulin.
Although there is general agreement that carbohydrate is essential for the early chick embryo (Needham, 1931; Spratt, 1948 a, b, 1949), the carbohydrate metabolic pathways used by the embryo and its integral parts are still not clear. The evidence of Needham & Nowinski (1937), Needham (1942), Burrows (1921), and DeHaan (1956) points to a high glycolytic activity in embryonic tissues during the first few days, falling rapidly to a low level by the 5th day; Laser (1936), quoted by Needham, showed, however, that appreciable oxidation occurs in the blastoderm at days of incubation, although this too decreases rapidly by the 5th day and thereafter rises again.
Spratt’s (1950) work with fluoride, iodoacetate, and cyanide led him to conclude that heart, inhibited by fluoride, was dependent primarily on glycolysis for its development, and that brain, inhibited by iodoacetate and cyanide, required respiratory mechanisms; but Duffey & Ebert (1957) cautioned against too ready an acceptance of this theory, for iodoacetate and fluoride, in spite of Spratt’s results, can interfere with both glycolysis and respiration (see Fuhrman & Field, 1943; Judah & Williams-Ashman, 1951; Potter, Le Page, & Klug, 1948; Slater & Bonner, 1952). Accordingly, both brain and heart can be inhibited with higher concentrations of these drugs. Furthermore, McKenzie & Ebert (1960) have shown that the effects of antimycin A on the chick embryo depend largely on the method of its administration—if applied to the ventral surface in critical doses the heart alone is damaged and if given dorsally brain and spinal cord are affected. We thought it would be of interest to try this experiment with fluoride, for Spratt applied the fluoride to the ventral surface of the embryo only. A preliminary series of experiments using the New technique and administering the fluoride either dorsally or ventrally indicates that fluoride has no specificity of action, for both heart and brain were inhibited, and in some instances the brain more severely so. However, these experiments will have to be carried out in more detail.
It is doubtful, therefore, whether we can still accept the hypothesis that the heart depends primarily on glycolysis and the brain on oxidative mechanisms. The explanation may be that the heart, while normally making full use of the aerobic metabolic pathways (when these are possible), has retained the faculty, resident in the whole of the early blastoderm, of surviving for a certain period and at a lower level of metabolism under anaerobic conditions.
In ovo experiments with the chick have been the most popular means of investigating the teratogenic action of insulin. Landauer (1945), having determined that insulin will produce rumplessness when injected into the egg during the first 2 days, and micromelia and beak defects if injection is delayed until the 4th or 5th day, tested the effects of supplementing the insulin with intermediate carbohydrate metabolites. Among the more significant findings in relation to the present experiments were the action of pyruvic acid, lactic acid, and nicotinamide. Initially, nicotinamide was reported as reducing the incidence of rumplessness and, to an even greater degree, the micromelia and beak defects of the insulin-treated chick (Landauer, 1948). Later, however, it became clear that nicotinamide in larger doses ‘proved to have considerable toxicity’ (Landauer & Rhodes, 1952); e.g., at 96 hours, 5 mg. nicotinamide reduced the insulin mortality but 10 mg. increased it even with a reduced dose of insulin. Pyruvic acid and lactic acid, although reducing the mortality from insulin in the early embryos, resulted in high mortality rates in the later ones.
These results form an interesting comparison with the present findings in the explanted chick embryo: lower doses of nicotinamide give slight protection against insulin but higher doses not only potentiate the action of insulin but even per se cause severe inhibition. Pyruvate affords no protection at the lower dosage, with the possibility of slight protection when increased fivefold but marked toxicity when the dose is further increased 3–5 times. Landauer & Rhodes (1952) suggest that the differences, (a) between the anomalies produced by insulin injection in the first 48 hours and those by injection on the 4th or 5th day and (b) between the effects of the supplementary intermediates (i.e. pyruvic and lactic acids) when given early and when given later, were, in both instances, the results of the transition from anaerobic glycolysis of the first 2 days to oxidative metabolism by the 4th or 5th day. Furthermore, they suggested that the insulin during the 4th and 5th days of incubation interferes with codehydrogenase activity as the only established function of nicotinamide is the formation of Coenzyme I.
In our experiments only the brain is affected at low doses; overdosage damages the heart and other mesodermal derivatives as well. Thus brain, the more sensitive tissue compared with heart in regard to lack of oxygen, is also more sensitive to insulin although neither is completely resistant.
It may be deemed wiser to treat the teratogenic action of insulin quite separately from its therapeutic effects in the body, but this may not be entirely necessary. The role of insulin in intermediate metabolism has recently been the subject of intensive discussion and experimentation, with the evolution of laudable but conflicting hypotheses. Randle & Smith (1958 a, b) drew an interesting comparison between the action of insulin and of substances such as 2:4 dinitrophenol, which are known to inhibit oxidative phosphorylation of carbohydrates. The anoxic-like effect of such substances is to stimulate the glucose metabolism, at least in a tissue like muscle—the Pasteur effect—whereby the smaller amount of energy-rich phosphate derived from glycolysis requires an increased uptake of glucose. The inference is that insulin itself may act as an inhibitor of oxidative phosphorylation, although this is denied by Stadie (1954). Yet our experiments suggest an inhibitory action by insulin first on brain and, with larger doses, on heart and other mesodermal tissues. Furthermore, DPN but not DPNH is capable of reversing the insulin effect.
If we must provide a hypothesis to explain our results, an inhibition of the oxidative phosphorylation of carbohydrates is a distinct possibility, the effect occurring in both brain and heart and being accompanied by the differential response arising from the greater susceptibility of brain to anaerobic or anaerobic-like conditions, or alternatively, the greater resistance of heart muscle to the end-products of glycolysis. These toxic end-products of glycolysis may reasonably be identified as pyruvate or lactate, especially as Landauer (1952) found that pyruvic acid and lactic acid were toxic when given at 96 hours of incubation and, as the present work reveals, an inhibition of brain and later of heart by higher doses of pyruvate with or without insulin. Chain (1960), however, disagrees with many other workers who claim that insulin activity will increase the production of CO2 and lactic acid.
The effect of nicotinamide under the circumstances of the present experiments is perhaps even more obscure, but the slight protection afforded by the smaller doses may be the result of the extra DPN synthesized on the addition of nicotinamide. On the other hand, the toxicity of larger doses may be the result of depletion of ATP reserves or even interference with the basic resources following its incorporation with nicotinamide in the formation of DPN. These side-issues are important and require further investigation.
As for the exact mode of action of insulin, at least from these experiments, it is barely enough to claim that it may inhibit the phosphorylative mechanisms of the citric acid cycle associated with DPN. We can go further and suggest that it must interfere with the reoxidation of the reduced form of DPN (which has no protective effect)—in other words, the site of action lies in a hydrogen transport system, but in which system or where deserves more study along the same lines as the present experiments.
RÉSUMÉ
L’action inhibitrice de l’insuline sur le jeune embryon de Poulet
1. On a administré de l’insuline, à des concentrations variées, à l’embryon de poulet explanté selon la technique de New, après 24 heures d’incubation, aux stades 3-8, et cultivé 22 à 24 heures in vitro.
2. On a démontré que, quelle que soit la manière selon laquelle l’insuline est administrée à l’embryon, ses effets se manifestent le plus fréquemment dans l’encéphale et le tube neural. Les somites sont inhibés à un degré moindre et le cœur n’est pas affecté, sauf aux doses les plus élevées.
3. L’administration de pyruvate de sodium ou de nicotinamide, conjointement à celle d’insuline, n’a qu’une très faible action protectrice, et des doses plus élevées de ces deux substances renforcent l’action inhibitrice de l’insuline.
4. Le diphosphopyridinenucléotide sous sa forme oxydée, administré avec l’insuline, fournit une protection complète, mais la forme réduite du DPN n’a pas d’effet.
5. A la lumière de ces résultats, les auteurs considèrent les travaux antérieurs de Spratt et d’autres auteurs sur les caractéristiques métaboliques du jeune embryon de poulet, de Landauer sur l’action tératogène de l’insuline et les récents travaux de Randle et Smith, par exemple.
6. Comme Duffey & Ebert (1957) les auteurs hésitent à admettre l’hypothèse de Spratt (1950) sur la dépendance du cerveau à l’égard de la glycolyse. Ils suggèrent, à la place, que le cœur, à la différence du cerveau, peut, s’il est nécessaire, survivre quelque temps dans des conditions d’anaérobiose, mais, lorsque c’est possible, il utilise à plein les voies métaboliques aérobies.
7. La protection complète apportée par le DPN mais non par le DPNH tend à faire admettre que le point d’action de l’insuline se trouve dans la réoxydation des nucléotides pyridiniques, c’est-à-dire dans un système transporteur d’hydrogène.
ACKNOWLEDGEMENTS
We wish to thank Professor R. D. Lockhart for the facilities of his department and for reading the manuscript.
Miss Patricia Barron gratefully acknowledges the receipt of an award from the Medical Research Council which enabled her to carry out this work.
REFERENCES
EXPLANATION OF PLATES
Fig. A. Explanted at stage 5; 3 units of insulin applied to dorsal surface and cultured for 22 hours.
Fig. B. Explanted at stage 4; 4 units of insulin applied to dorsal surface and cultured for 22 hours; showing lysis round the primitive-streak region and shortening of the somite region.
Fig. C. Explanted at stage 5; 4 units insulin + 3·33 × 10–1 M sodium pyruvate applied to the dorsal surface and cultured for 22 hours.
Fig. D. Explanted at stage 5; 3 units insulin applied dorsally+5×10–2 M nicotinamide ventrally and cultured for 22 hours.
Fig. A. Explanted at stage 5; 3 units of insulin applied to dorsal surface and cultured for 22 hours.
Fig. B. Explanted at stage 4; 4 units of insulin applied to dorsal surface and cultured for 22 hours; showing lysis round the primitive-streak region and shortening of the somite region.
Fig. C. Explanted at stage 5; 4 units insulin + 3·33 × 10–1 M sodium pyruvate applied to the dorsal surface and cultured for 22 hours.
Fig. D. Explanted at stage 5; 3 units insulin applied dorsally+5×10–2 M nicotinamide ventrally and cultured for 22 hours.
Fig. E. Transverse section of embryo explanted at stage 4 and given 3 units insulin on ventral surface; cultured for 22 hours.
Fig. F. Transverse section of embryo explanted at stage 4 and given 4 units insulin on dorsal surface; cultured for 22 hours.
Fig. G. Explanted at stage 5; 3 units insulin applied to dorsal surface and 5×10’3 M DPN on ventral surface; cultured for 22 hours.
Fig. H. Explanted at stage 4; 3 units insulin applied to dorsal surface and 2·5 ×103 M DPN on ventral surface; cultured for 22 hours.
Fig. E. Transverse section of embryo explanted at stage 4 and given 3 units insulin on ventral surface; cultured for 22 hours.
Fig. F. Transverse section of embryo explanted at stage 4 and given 4 units insulin on dorsal surface; cultured for 22 hours.
Fig. G. Explanted at stage 5; 3 units insulin applied to dorsal surface and 5×10’3 M DPN on ventral surface; cultured for 22 hours.
Fig. H. Explanted at stage 4; 3 units insulin applied to dorsal surface and 2·5 ×103 M DPN on ventral surface; cultured for 22 hours.