Glucagon concentrations ranging from 1·16 to 300-0μg/0·1 ml diluent were injected into the yolk of chick embryos on incubation days 8,10, and 12. Studies of survival rates, embryo weights, blood sugars, liver and tibiotarsus glycogen histochemistry, and pancreatic alpha and beta tissue histogenesis were undertaken during the 9- to 16-day incubation period. Glucagon dosages of 37·5 and 150·0μg/0·1 ml diluent gave the best survival rates. Glucagon caused an increase in embryo weight, increased liver glycogen storage, a chondrocyte glycogen storage pattern which correlated with blood sugar levels, an increase in pancreatic beta tissue and a decrease in pancreatic alpha tissue. Studies of blood sugars following glucagon treatment showed that most concentrations caused an initial (first 16h) hyperglycemia. Following this, two general patterns were exhibited: (1) the lower glucagon concentrations caused hypoglycemia after about 24 h, and (2) the higher concentrations caused a more prolonged hyperglycemia when administered on incubation day 10 but caused hypoglycemia when administered on days 8 and 12. Interpretation of these results is based on the contribution of three factors to the expression and duration of the glucagon effect: (1) concentration of glucagon administered, (2) insulin secretion, and (3) levels of glycogen storage at the incubation stage of administration.

While a number of studies of the teratogenic influences of insulin on avian embryogenesis have been reported, fewer studies (referenced below) have been reported on the effects of glucagon on the developing chick embryo. The interrelated actions of glucagon and insulin offer the possibility that glucagon may contribute to the formation of some of the abnormalities which develop after insulin treatment. This possibility is emphasized by the work of Kalliecharan & Gibson (1972) on the effects of insulin on the histogenesis of the pancreatic islets in the chick embryo. They reported a delay in the differentiation of beta tissue suggesting a delay in the elaboration of endogenous insulin. They also reported an increased activity of alpha tissue suggesting an increased glucagon production. The present study was undertaken, therefore, to describe some of the changes caused by the administration of glucagon to chick embryos.

Fertile White Leghorn chick eggs (Shaver’s Starcross 288) were obtained from a commercial hatchery and incubated at 38°C for use in this study. The embryos were studied for body weights, blood sugar levels, histochemically for liver and chondrocyte glycogen storage, and histologically for amounts of pancreatic islet tissue.

The eggs were divided into three groups: the untreated series, the glucagon-treated series in which the eggs were injected with various concentrations (see below) of glucagon suspended in diluent, and the control series in which the eggs were injected with diluent only. Eggs of the latter two series were injected on one of incubation days 8, 10, or 12, and specimens were collected for study on each incubation day following treatment until day 16 or until mortality prevented further study. In addition, for the blood sugar study, embryos were collected at the 2 h, 4 h, 8 h, 12 h, and 16 h stages following glucagon treatment.

The diluent described by Thommes & Firling (1964) was used. It consisted of 2·8 gm lactose, 1·6 ml glycerine, 0·2 ml phenol, and 98·2 ml distilled water. The glucagon was obtained from Sigma Chemical Company (lot. no. AZ 2013), Orangeburg, New York. The concentrations of glucagon used were 1·16, 4·65, 9·37, 37·5, 150·0 and 300·0 μg in 0·1 ml of diluent. Treatment consisted of a single injection into the yolk mass of 0·1 ml of diluent, or of diluent plus glucagon.

Blood sugar levels were measured by the colorimetric method of Teller (1956) using Glucostat Reagent (Worthington Biochemical Corporation, Freehold, New Jersey, U.S.A.). The absorbance was measured with a Spectronic 20 spectrophotometer at a wavelength of 450 μm.

Glycogen storage in the liver and tibiotarsus was visualized histochemically using tissues fixed in cold Rossman’s fluid and prepared with the periodic acid-Schiff reagent (PAS) method of McManus (Pearse, 1961). Companion sections were treated with malt diastase to distinguish the glycogen from other PAS-positive substances. An assessment based on staining intensities was made of the relative amounts of glycogen stored in the liver. Since all sections were prepared in an identical manner, the intensity of the staining reaction was taken as evidence of the amount of glycogen present, and major changes in staining intensity were assumed to provide evidence of changes in the amounts of glycogen stored. The staining intensities were calculated from photocytometric recordings of the light transmitted through the tissue sections before and after staining. .

Pancreas was fixed in Bouin’s fluid and prepared with Gomori’s chrome alum hematoxylin-phloxine method (Lillie, 1965) to demonstrate the alpha and beta cells. The method of Chaikey (1943) was used to determine the relative quantity of the alpha and beta tissues. An ocular micrometer was moved across the field in units of 100 scale divisions and readings were recorded at every second stop of the scale. At each reading, the cell (acinar, connective tissue, alpha, and beta) underlying every 10th scale division was identified and recorded as a ‘hit’. A total of 600 ‘hits’ were recorded for each animal studied, and the amounts of alpha and beta tissue are expressed as a percentage of this total.

Survival data

Table 1 shows the percent surviving embryos on each incubation day following treatment. Administration of diluent increased mortality but not as severely as the administration of glucagon. Administration of glucagon caused severe mortality within the first 24 h, after which the surviving embryos showed three general patterns. The lower concentrations, with exceptions on days 8 and 10, gave high mortalities; the intermediate concentrations gave lower mortalities; and the highest concentration used (300 μg) gave the highest mortality.

Table 1

Survival data (%) for glucagon-treated and diluent-treated chick embryos

Survival data (%) for glucagon-treated and diluent-treated chick embryos
Survival data (%) for glucagon-treated and diluent-treated chick embryos

Embryo weights

The diluent caused a decrease in body weight (Table 2), necessitating a comparison between diluent-treated and glucagon-treated embryos to determine the actual glucagon effect. Compared to the diluenttreated embryos, the glucagon-treated embryos showed an increased body weight. However, in only a few instances did the glucagon-treated embryos exhibit body weights which were significantly higher than those of the untreated embryos. In these instances, the higher concentrations administered at the later incubation stages had the more prolonged effect.

Table 2

Weights (g) of untreated, diluent-treated, and glucagon-treated chick embryos on the incubation stages studied

Weights (g) of untreated, diluent-treated, and glucagon-treated chick embryos on the incubation stages studied
Weights (g) of untreated, diluent-treated, and glucagon-treated chick embryos on the incubation stages studied

Blood sugar

The blood sugar levels during the first 16 h following the injection of glucagon show that most of the glucagon concentrations used caused hyper-glycemia (Table 3).

Table 3

Blood glucose (mg%) levels for untreated and glucagon-treated embryos measured at 2 and 4 h intervals following treatment

Blood glucose (mg%) levels for untreated and glucagon-treated embryos measured at 2 and 4 h intervals following treatment
Blood glucose (mg%) levels for untreated and glucagon-treated embryos measured at 2 and 4 h intervals following treatment

The blood sugar levels measured at 24 h intervals following treatment are given in Table 4. Most glucagon treatments caused an initial hyperglycemia, after which the blood sugars showed two general patterns: (1) the lower glucagon concentrations caused hypoglycemia after about 24 h, and (2) the higher concentrations caused a more prolonged hyperglycemia when administered on day 10 but caused hypoglycemia when administered on days 8 and 12.

Table 4

Blood glucose (mg%) levels for untreated, diluent-treated, and glucagon-treated embryos measured at daily intervals following the stage of treatment

Blood glucose (mg%) levels for untreated, diluent-treated, and glucagon-treated embryos measured at daily intervals following the stage of treatment
Blood glucose (mg%) levels for untreated, diluent-treated, and glucagon-treated embryos measured at daily intervals following the stage of treatment

Liver glycogen

Liver sections from untreated embryos contained scattered glycogen granules on incubation days 9 and 10, with sites of accumulation around the major blood vessels. On incubation days 11 to 15, the staining was more evenly distributed throughout the lobules. The photocytometric measurements (Table 5) show: (1) a progressive increase in glycogen storage during the period studied, except for a stage of reduced storage on incubation day 13; and (2) a major increase in glycogen storage between days 14 and 15.

Table 5

Percent staining intensity of glycogen in liver from untreated and glucagon-treated embryos

Percent staining intensity of glycogen in liver from untreated and glucagon-treated embryos
Percent staining intensity of glycogen in liver from untreated and glucagon-treated embryos

All glucagon concentrations used, regardless of the day of injection, resulted in significant increases in glycogen staining (Table 5) for most of the period studied. On incubation day 15, embryos treated with 1·16 and 150·0 μg glucagon on day 10 exhibited reduced glycogen staining correlating with the hyperglycemic blood shown by these embryos at this stage.

Tibiotarsus glycogen

In tibiotarsi from untreated embryos on incubation day 9, the chondrocytes of the epiphyses possessed a few glycogen granules. The early proliferative zone showed little staining, the late proliferative zone stained more intensely, the early hypertrophic zone gave the most intense reaction, and the late hypertrophic zone contained little glycogen. The glycogen distribution on incubation days 10 to 15 was similar and showed a progressive increase in staining intensity.

In glucagon-treated embryos, changes in the glycogen-staining pattern were consistently observed only in tibiotarsi from the more severely affected embryos. These variations, with few exceptions, mirrored changes in the blood sugar levels. That is, when blood sugar levels increased (e.g. day-9 tibiotarsi treated with 9·37 μg on day 8), there was also increased glycogen storage in the early and late hypertrophic zones. Conversely, when blood sugar levels decreased (e.g. day-12 tibiotarsi treated with 150 μg on day 10), both the epiphyses and early hypertrophic zones showed glycogen depletion.

Pancreas

The study of the relative amounts of alpha and beta tissue was limited to those treatments which had a severe effect on blood sugar levels. Table 6 shows that the amounts of alpha tissue in the treated embryos were either similar to or reduced below the values for the untreated pancreas on most of the stages studied. Two of the three glucagon treatments used (37·5 μg day 10, and 150·0μg day 12) caused increases in the amounts of beta tissue, and one treatment (150·0 μg day 10) caused decreased amounts of beta tissue on most of the stages studied. By incubation day 15, the values for both the alpha and beta tissues were similar to those of the untreated pancreas.

Table 6

Relative amounts (percentage) of pancreatic islet tissue in untreated and glucagon-treated chick embryos

Relative amounts (percentage) of pancreatic islet tissue in untreated and glucagon-treated chick embryos
Relative amounts (percentage) of pancreatic islet tissue in untreated and glucagon-treated chick embryos

Survival rates

The survival patterns described (Table 1) correlate with changes in the blood sugar levels (Tables 3 and 4). The initial high mortality, within the first 24 h, correlates with the period of hyperglycemia. This hyperglycemia was most severe following treatment with the 300 μg concentration and no embryo receiving this concentration survived the first 24 h. Embryos receiving the other glucagon concentrations also showed an initial hyperglycemia but this was reduced within the first 24 h to control or to hypoglycemic levels. It is suggested: (1) that the initial hyperglycemia together with glucagon’s insulinogenic role stimulates the elaboration and release of insulin, and (2) that this insulin counteracts the effects of the glucagon and lowers the blood sugars to below lethal or to hypoglycemic levels. Furthermore, it may be this insulin-induced hypoglycemia, and not the initial glucagon-induced hyperglycemia, which contributes to the continuing mortality rate.

Embryo weight

The decrease in body weight caused by the diluent may be related to its lactose component. Rutter, Krochevsky, Scott & Hansen (1953) studied the effects of a lactose diet on post-hatched Columbian chicks and reported a reduction in body weight as well as other abnormalities.

Some glucagon treatments caused significant weight increases when compared with the untreated embryos. Thus, the glucagon acted as a growth stimulant, more than simply blocking the depressant effects of the diluent. In contrast, no significant weight changes following glucagon administration were reported in rats and rabbits (Root, 1953), White Leghorn chick embryos (Elrick, Konigsberg & Arai, 1958), or 21- to 28-day broilers (Cavora & Kondra, 1970). Cavallero (1956), however, reported that glucagon administered to White Leghorn chick embryos caused significant weight increases.

Liver glycogen

The pattern of liver glycogen storage for the untreated embryos, including the decrease at day 13, is comparable to those described in previous reports (Dalton & Hanzal, 1940; Jenkins, 1955; Leibson, Zheludkova, Plisetskaya & Strabrovsky, 1961a; Daugeras, 1968). While the pancreatic beta cells show insulin granules at earlier incubation stages, Kalliecharan & Gibson (1972) demonstrated a major increase in the staining intensity of these cells, suggesting increased insulin elaboration, beginning on incubation day 13. Also, Leibson et al. (1961 b) have reported that insulin is present in increased amounts in the blood plasma on days 11–14. This increased insulin production may contribute to the major increase in liver glycogen storage between days 14 and 15.

Previous studies have reported initial decreases in liver glycogen storage following glucagon treatment in chick embryos (Grillo, 1961; Thommes & Firling, 1964; Korec, 1967; and Verne & Hebert, 1968). Grillo studied liver glycogen during the first 8 h following glucagon injection and reported a significant decrease during the first 3 h and a return to control values during the remaining 5 h. In the present study, all liver sampling was undertaken at 24 h intervals following glucagon injection and missed the short term effects described by Grillo. The present study is, consequently, a continuation of the previous work and has shown that the initial glycogen loss is followed by a more prolonged period of increased storage. Pincus & Snedecor (1956) stated that glucagon’s hyperglycemic effect resulted from its glycogenolytic activity. Thus, liver glycogenolysis accounts for the initial glycogen loss from the liver contributing to the hyperglycemic blood. The important suggestion here is that this initial reaction appears to be counteracted by the secretion of insulin. Thus, the initial hyperglycemia is reduced to untreated or to hypoglycemic levels, and the liver glycogen is increased to a storage pattern above that of the untreated embryos. Ui, Claus, Exton & Park (1973) have shown that glucagon stimulates the transamination of glutamate to L-ketoglutarate and also increases phos-phoenolypyruvate activity. Thus, the protein reserves in the yolk sac may be mobilized by glucagon in the form of glucose, and this gluconeogenic effect of glucagon, as well as its insulinogenic effect, may contribute to the concurrent appearance of hyperglycemia and increased liver glycogen staining.

Blood sugars

The blood sugar levels of the untreated embryos reported in this study are in general agreement with those reported by Arsenault, Gibson & Mader (1975) and Zwilling (1948).

Most of the glucagon treatments caused an initial hyperglycemia. This has also been reported by Thommes & Firling (1964). Following this initial hyperglycemia, the blood sugar studies illustrate the contribution of three factors to the expression and duration of the glucagon effect. (1) The concentration of the glucagon administered influenced the blood sugar levels. For example, 24 h after the day-10 injections, the lower glucagon concentrations caused hypoglycemia and the higher concentrations caused hyperglycemia. The latter observation indicates that sufficient glycogen reserves were present for mobilization. The lower concentrations, therefore, were not sufficiently concentrated to mobilize these reserves. (2) The secretion of insulin also influenced the expression of the glucagon effect. Leibson et al. (1976) showed that insulin is released before incubation day 12 and Table 6 shows that the beta tissue is often increased in amount by the administration of glucagon. Thus, it is suggested that the initial hyperglycemia is reduced by the release of additional insulin and that it is the increased insulin that maintains the hypoglycemia. (3) The time of treatment, and the glycogen reserves available at that time, influenced the glucagon effect. This is illustrated by the observation that the same concentration administered on different incubation days elicited varying responses. For example, the 150 μ g treatment caused hypoglycemia after 24 h when administered on days 8 and 12 but a more prolonged hyperglycemia when administered on day 10. The liver and yolk sac are two important sites of glycogen storage. Studies of untreated liver tissue (Table 5) showed a low level of glycogen storage on incubation days 9 and 13, and a high level on days 11 and 12. Juurlink & Gibson (1973) demonstrated a weak glycogen reaction in the yolk sac on day 13.

Tibiotarsus -glycogen

The glycogen staining pattern described for the untreated tibiotarsus is similar to that reported by Ho & Gibson (1972) and Rabinovitch & Gibson (1972). In the glucagon-treated embryos, only the more severely affected tibiotarsi showed variations from the untreated glycogen pattern and these variations usually mirrored the changes in the blood sugar levels.

Pancreatic islets

In untreated embryos, during the 12- to 15-day incubation period, the pattern of increasing amounts of alpha and beta tissues is similar to that described by Kalliecharan & Gibson (1972) and Arsenault & Gibson (1974). In glucagon-treated embryos, the changes in the amounts of alpha and beta tissue showed no consistent variation which could be correlated with hypoglycemic or hyperglycemic blood. However, many of the embryos studied did show a reduction in the amount of alpha tissue and an increase in the amount of beta tissue. The major exception to this pattern was the reduction in beta tissue shown by those embryos treated with 150 μ g of glucagon on day 10 (Table 6). These were the embryos in which the blood returned to hyperglycemic levels (Table 4). Peterson & Hellman (1963) reported increased beta tissue and decreased A2 cells in older rats following prolonged glucagon treatment.

It is suggested: (1) that the exogenous glucagon, which has an insulinogenic role (Crockford, Porte, Wood & Williams, 1966; Devrum & Recant, 1966; and Samols, Marn & Marks, 1966), caused an increased development of beta tissue, and (2) that it reduced the release of endogenous glucagon and delayed the histogenesis of the alpha tissue.

One reason for the present study was to examine the possibility that increased glucagon release might contribute to those abnormal patterns of development that appear following the administration of insulin. Rumplessness, beak deformities, micromelia, etc. did not develop after the glucagon treatment. Abnormalities in skeletal histogenesis (Rabinovitch & Gibson, 1972; and others) were not observed in the glucagon-treated embryos. Insulin treatment causes prolonged hypoglycemia (Zwilling, 1948; Arsenault et al. 1975); whereas, glucagon caused an initial hyperglycemia followed by hypoglycemia. Insulin treatment causes a delay in the histogenesis of the beta islets and increased activity of the alpha islets (Kalliecharan & Gibson, 1972); whereas, glucagon treatment frequently led to the opposite effect. The present study suggests, therefore, that exogenous glucagon and insulin-stimulated endogenous glucagon may have somewhat different roles. Exogenous glucagon stimulates insulin secretion and it is the increased insulin that contributes to many of the abnormal patterns described in this paper. Endogenous glucagon, stimulated as the result of insulin treatment, possibly functions more to counteract the effects of the exogenous insulin, and does not appear to contribute to those gross morphological abnormalities described as the insulin syndrome.

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

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