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Carbohydrate metabolism in insulin-treated chick embryos
Carbohydrate Metabolism in Insulin-Treated Chick Embryos1 Edgar Zwilling From the Stows Agricultural Received
Experiment March
Station, Stows, Connecticlct 15, 1951
Landauer (1,2) has shown that insulin, when injected into the yolk sacs of chick embryos, may consistently produce particular abnormalities depending on the age of the embryo when it is treated. Defects in the caudal region (rumplessness) are the chief consequence of insulin injections during the first 3 days of incubation (with a maximum incidence at 30 hr.), while disproportionate dwarfing of the hind limbs (micromelia) occurs when 4-7-day embryos are treated (with maximum incidence at 5 days). Both types of anomalies bear close resemblance to known gene-mediated conditions of chickens, i.e., they are phenocopies. We have preferred to concentrate on the insulin-induced micromelia for a study of possible relationship between the abnormalities and any derangement in physiology for the following reasons: (a) It may be produced consistently in large numbers; (b) the chief action leading to the condition occurs in relatively large embryos to which analytical procedures may be applied; (c) the condition closely resembles those produced by a number of other experimental procedures as well as at least six mutations. This last provides us with the possibility of producing the micromelia by other experimental procedures for comparisons aimed at discovering common pathways and processes which may be involved. The existence of mutations which produce similar syndromes may enable us to ascertain whether any of the information obtained from a study of the experimentally induced terata can be 1 This investigation was aided by grants from the International Baby Chick Association and from the American Cancer Society on the recommendation of the Committee on Gr0wt.h of the Sational Research Council. 228
CARBOHYDRATE
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applied to the gene-mediated conditions in an attempt to reveal the mechanism of action of these particular genes. Thus far there have been two procedural approaches to the problem of revealing which physiological systems may be disturbed by the action of insulin and related to the morphological derangements. One of these has been to observe the effects of a number of other substances which have been injected either simultaneously with, shortly before, or after the insulin. By these means Landauer (3,4) has discovered that cortin, which by itself causes a uniform dwarfing of the embryo, exaggerates the insulin effects; nicotinamide and, to a lesser extent, oc-ketoglutaric acid are effective in eliminating the insulin-induced micromelia. There are other results which shall not be discussed here (2-5). The other approach has been to perform assays for various metabolites and, by comparisons with untreated embryos, attempt to relate any deviations to the anomalous development. It has been found (6,7) that insulin-treated embryos become hypoglycemic and that there is a very suggestive relationship between the hypoglycemia and micromelia: the more pronounced and prolonged the hypoglycemia, the more extreme is the leg shortening. In addition, factors which exaggerate or relieve the micromelia also tend to accentuate or abolish the hypoglycemia. Two sets of problems are opened by these observations: the first’ concerns possible mechanisms through which the hypoglycemia may produce alterations of embryonic development (6,8), i.e., whether such derangements are a more or less direct result of the lowered available carbohydrate or whether they are due to some other effect of insulin. The second, with which this paper is concerned, involves the mechanism through which insulin produces hypoglycemia in the embryonic system. The latter problem has certain very interesting aspects for students of insulin physiology since the embryos which are subjected to insulin presumably have none of the hormones usually associated with insulin activity in the adult, and this condition persists for at least several days following the treatment. Pituitary gland activity is not noted until the 10th day of development (9); the formation of the adrenal cortex begins between the 4th and 6th days (10) but there is no certainty about the beginning of it’s secretory activity. Insulin itself does not appear in detectable amounts in the embryo until the 11th day (11). A rather curious situation, first brought to attention by Claude Bernard in 1858, exists in bird embryos. The glycogen storage functions are performed by t,he clrt’racmbryonic yolk-sac membrane
230
EDGATZ ZWILLING
(which Bernard called the “‘joie transitoire”) during the first, week or so of development. For this reason we have performed separate assays for carbohydrates (free reducing substances, glycogen, and “total carbohydrate”) upon the embryo and its yolk-sac membrane. The data obtained from these assays reveal that the glycogen content of the yolk-sac membranes of insulin-treated embryos is considerably greater than that of control yolk sacs. The data further indicate that t,he augmented glycogen of the yolk-sac membrane accounts for the embryonic hypoglycemia. All of the carbohydrate components of the embryo proper are diminished after the injection of insulin. MATERIAL
AND
METHODS
All of the eggs used in these experiments were from unselected (5) White Leghorn hens. They were injected at 5 days with 5 units of Iletin (insulin, Lilly)2 through holes drilled in the shell at the center of the blunt end of the egg. The holes were sealed with nail polish after the injection. One-inch 20 gage needles were used to insure the insulin’s reaching the yolk sac. The eggs were incubated in a forced-draft incubator on their sides, both prior to and following the injection. Controls consisted of unt.rrated embryos. At the proper stages the eggs were opened and the embryos, after being freed of membranes, etc., were rapidly removed to a 0.9% NaCl solut,ion. The rest of the egg was immersed in saline solution, and the yolk sac, freed of all other membranes, was removed. The exact procedure varied a bit depending on the age of the embryos. Wit,h g-day embryos, in which the vascularised yolk sac has not yet encroached upon the entire yolk, the membrane was removed by incising a short distance beyond the outermost blood vessel (sinus terminalis). At later stages, where the yolk-sac membrane encompassed all of the yolk, the incisions were made at a region where the yolk sac makes contact with the albumen residue. It was not, always possible to obtain intact membranes from the later stages; pieces were frequently lost. The yolk sacs were washed free of yolk and placed in clean saline. It was quite simple to free the yolk-sac membrane of yolk at the stages used in these experiments; by 13 or 14 days, however, the yolk is so viscous that this is almost impossible. The mashed tissue (either intact embryo or yolk-sac membrane) was then allowed to drain on toweling paper, weighed on a Roller-Smith balance and placed in the appropriate reagent for the determination to be carried out. In all instances controls were handled in a similar manner and the sequence of preparing control and injected tissues for assay was varied so that this could not affect the results. Apparently relatively little glycogenolysis occurs when the yolk-sac membranes arc kept in 0.0% KaCl solution for a short time. This was established by a series of glycogen assays of yolk-sac membranes (from 7Q-day embryos) which were placed in KOH immediately after removal from the egg and compared with membranes kept in unbuffered saline at room temperature for 15 min. Five membranes from untreated * Furnished Peck.
by the 1,illy Research
Laboratories
through
the courtesy
of Dr. F. R.
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embryos and five from insulin-injected embryos were in each group. The controls averaged: immediate assay, 5.88 mg./g.; 15-min. delay, 5.25 mg./g. Insulin-treated: immediate assay, 8.97 mg./g.; 15-min. delay, 9.57 mg./g. With such small numbers t,hrbse differences are not significant.. \\‘c selected 5 units of insulin becausc~ thca incid<~nce 01’ clst,reme response (micromcllia) is 111uc.hgycv~tc~ t II:~II with lower doses. In all cases lvhere the embr,vos were old c~nough so t,h:tt thcl Inorpllological c~ffe(*ts of the insulin mere evident, the most extreme WS,Y were scJ~t (~1 i’ol, assay. It, is peltinent to add i hat embryos treated with this tlosc~ of insulin a~ colrsistently smaller and slightly more retarded than controls (Table III!. The yolk-sac memi)ranc:s are also smaller and have fewer folds on their inrrclr surface (Table I). Howcvt~, \VC compared, in all cases, embryos of the same c*hronological age; in each set of tlctolmi~~atio~ls WV included controls and injected embryos which had been incubattstl on t.hc same tray. Our determinations were not cxrried out on rnhyms older than 12 days since we have shown that normal bloodsugar levels are restored by the 14th day in all insulin-injected embryos. For the most part standard procedures, with relatively slight modifications, were employed for these assays. Our greatest problem resulted from the increase in size of the chick embryos as older cmhryos were used. Since some components increased at a relatively greater rate than tha amount of tissue (in terms of wet weight), this required the use of larger fluid volumes and relatively greater final dilutions to bring the concent,ration of reducing substances a-it)hin the range of the technique employed. In some instances scvcral embryos or yolk sacs were pooled for a single determination. Somogyi’s (12) copper-iodometric reagent (modified to contain 140 g./l. Na$Oh) was used for all of t,he final determinations of reducing substance. All reactions were carried out on 2-ml. samples of unknown with 2-ml. samples of Somogyi’s reagent. These were placed in loosely stoppered 22 X 175 mm. test tubes, mixed, and heated in a boiling water bath for 10 min. After cooling, 1 ml. of 39?..ICI was run down the side of each tube and this was followed by 1 ml. of 2 N H,SOa run in rapidly with immediate agitation. Titration was performed in the same tubes with 0.008 N sodium thiosuliat,e to a starch end point. In all instances a glucose standard (1 or 0.5 mg./2-ml. sample) and reagent blank were carried through with each set of determinations. For the determination of free sugar (no attempt was made to ascertain to what, oxtent, ot,h(,r reducing substances might have? heen present) the following procedure was cmplo,x-ed: embryos and yolk sacs were placed in 1-2 ml. of distilled wat,er and ground to a uniform consistency in a motor-driven Lucite-in-glass homogenizer. Immediately after this the proteins were precipitated with equal amounts of 0.3 N Ba(OI& and 57, ZnS04.7H,0 [adjusted according to the directions of Somogyi (13)] which were added t,o the tube with stirring. Either 3 or 4 ml. of each of these reagents was added to each t’ube depending on the size of the embryos. The final dilution with distilled water was either to 10 or l-1 ml. After centrifuging the preparation, duplicate 2-ml. samples of the supernatant R-ere pipetted off and assayed as outlined above. The standard was a glucose solution adjusted in volume and concentration so that a 2-ml. sampIca of the final dilution contained 1 mg. glucose. Standard and blank were treated n-ith the samr amount, of the reagents used in the tubes with the tissue. When embryos or yolk sacs were treated differently as to amounts of reagent and final dilution a standard and blank mere carried through for each of the treatments.
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EDGAR ZWILLING
Glycogen was det,ermined according t,o the procedure of Good, Kramer, and Somogyi (14), with two modifications. Prior to the addition of alcohol to the KOII digested material 0.5 ml. of saturated xapSOa K-as added for each 4 ml. of digest, and the centrifuged glycogen precipitate was resuspended twice and brought to the boiling point in 60% ethyl alcohol in chloroform (959;. alcohol diluted to 60y0 with chloroform). This latter was done to eliminate the fatty material which was present, especially in the yolk sacs. The recovered purified glycogen was hydrolyzed with 4 ml. of 0.6 N HCl in a boiling water bath for 3 hr. After cooling, the hydrolyzatc was neutralized with KOH. Final dilution was made with water to 5, 10, or 20 ml. (In some instances the final dilution was made prior to neutralization and the final 2-ml. sample was neutralized prior to addition of the reducing solution. There was no apparent difference in the two procedures.) Duplicate 2-ml. samples were assayed for reducing substances as already indicated. The standard glucose solution was diluted, first with 4 ml. of 0.6 N HCl, then dist,illed water. The blank was 4 ml. of the IICl and enough water for the final dilution. “Total carbohydrate” (i.e., all reducing substances prosent after the hydrolysis described) was determined by hydrolyzing the weighed tissue in from 7 to 15 ml. (depending on the amount of tissue) of 0.6 N HCl for 4 hr. The hydrolyzates were then cooled and neutralized to phenol red with strong KOH. Protein was precipitated with enough Ba(OH)2 and ZnSOd to give a clear nonfoaming supernatant (samples at the various dilutions were tested with Millon’s reagent in the preliminary trials). Enough distilled water was added to give final dilutions of from 24 to 50 ml. Duplicate 2-ml. samples were assayed for reducing substances as indicated. All calculations were performed in a similar manner. The amount of glucose/ml. thiosulfate (blank - standard) X amount of thiosulfate needed to titrate unknown (blank -unknown) X dilution factor = mg. of reducing substance in terms of glucose in each sample assayed. All of the data are presented in terms of equivalent glucose reduction. All data are given as mg./g. tissue, and a chart (Fig. 1) based on mg./ embryo is included. Means are presented with their standard errors. Differences between means were not considered significant unless they exceeded twice the “stand: ard error of the differences”. “Bound carbohydrate”3 was calculated by subtracting the sums of the average values for glycogen and free sugar from the average value obtained for “total carbohydrate” for each stage.
RESULTS
In the yolk-sac membranes from untreated embryos the largest carbohydrate fraction for the period investigated is glycogen (Table I). This reaches a maximum at 8 days and then diminishes somewhat by represents is not clear. Seedham (11) 3 Exactly what the “bound carbohydrate” considers this fraction to be carbohydrate which is bound to protein. In the embryo proper he considers this to be mostly “mucoprotein” and in t.he rest of the egg “ovomucoid” substances. This fraction may also represent, some other forms of unhydrolyzed carbohydrate. If, as iYeedham speculates, it is important for the development of connective tissues and bones then the changes which we describe may be of significance. Further investigation of this fraction is required.
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233
METABOLISM
the 12th day. Total carbohydrate at first follows a similar course but t’hen comes down at 10 days only to rise again at 12 days. This behavior is (see below) related to a sharp decline in “bound carbohydrate” at 10 days followed by its augmentation at 12 days. Free sugar remains fairly constant throughout these stages. The data which we have obtained from our assays of yolk-sac membranes indicate clearly that there is an increase in glycogen and in total carbohydrate while there is a diminution in free sugar after insulin treatment (Table I). Virtually all of the increase in total carbohydrate can be accounted for by the augmented amount of glycogen on days TABLE
I
Carbohyzlrates in Yolk-Sac Membrane (In mg. glucose)
Carbohydrate component
d
Average ‘mg./g. yolk sac with .E. of mean)
Rang.!
4veraae wt. of mlk sacs Q.
15
8:% 1.366 2.079 0.556 0.697 0.807 1.284
: Insulin
Glycogen Control
Insulin
Ii: 6 5 6 24 11 ; 24 12 fl 14 : 3 12 9 i
“Bound carbohydrate” (calculated) Control Insulin
4.63 7.84 7.37 6.04 6.11 13.04 11.43 9.88
f f f f f f 3~ f
0.86 0.86 1.08 0.84 0.69 0.43 0.59 0.85
2~ ,021 zt .103 zt ,069 dc .102 z!z ,042 f .051 zt ,087 * ,193
3.28 2.21 0.76 3.64 3.74 2.79 0.69 0.78
.20 .42 .63 .54 .33 .65 .59 .43
3.15- 6.79 6.21-10.65 5.08-10.04 4.32- 8.80 4.77- 8.79 7.94-16.37 8.79-14.35 7.52-12.14 0.74Xl.93 0.37-1.21 0.86-1.29 0.64-1.07 0.49488 0.19XI.62 0.254.86 0.38-1.15
0.564 0.862 1.200 1.956
Q.
0.5384.681 0.7994.972 1.246-1.520 1.838-2.332 0.514Xl.620 0.609-0.818 0.758-0.862 1.068-1.284
X 0.901 1.237
0.357-0.777 0.760-1.087 1.072-1.285 1.516-2.262 0.358-0.701 0.538-0.898 0.740-1.082 0.949-1.423
ES 1.252 1.875 0.618 0.728 0.853 1.396
0.53OAI.626 0.7574.964 1.014-1.444 1.521-2.238 0.548-0.671 0.672-0.825 0.762-0.930 0.998-1.885
234
EDGAR
ZRILLING
6, 8, and 10 in the experimental yolk sacs. On day 12, however, the difference between the total carbohydrate content of controls and experimentals is not significant while that in regard to glycogen is highly significant. This is reflected in a drop in “bound carbohydrate”. The ‘lbound carbohydrate” behaves curiously at the IO-day stage. In both control and treated membranes it falls very low; then it reappears again on day 12. The fact that the same thing occurs in both sets of observations argues against its being an artifact. It seems as though glycogen synthesis occurs largely at the expense of the bound carbohydrate at this time. Apparently the reestablishment of the “bound carbohydrate” fraction is interfered with by the insulin. It is interesting to note that glycogen and total carbohydrate follow roughly the same TABLE
II
Carbohydrate Content of Yolk-Sac Membranes (Per cent of total carbohydrate) co1nponent 6 days “gY 10 days YO c/o
12 days %
Control Glycogen Free sugar “Bound carbohydrate”
52.8 9.8 37.4
71.8 7.9 20.2
80.0 11.7 8.2
57.4 8.0 34.6
Glycogen Free sugar “Bound carbohydrate”
57.9 6.5 35.5
80.2 2.6 17.1
89.9 4.7 5.4
85.8 7.4 6.7
Insulin
pattern after the 8th day. Apparently the differences are not fully established until this time. There is no significant difference in the amount of free sugar per gram of tissue in the various stages in the controls; in treated membranes this component drops significantly and then returns to normal levels by day 12. In terms of the per cent of total carbohydrate (Table II) it can be seen that the glycogen accumulates at the expense of the free and “bound carbohydrate” fractions, which are both relatively diminished in the insulin-treated yolk sacs. When the amount of carbohydrate per embryo is plotted (Fig. 1) it is seen that all of the constituents in the control embryos show a steady rise and follow, roughly, a similar course. Glycogen is the smallest fraction in all of the stages assayed. Free sugar is the largest com-
CARBOHYDRATE
METABOLISM
235
ponent at first, but by the 10th day the “bound carbohydrate” is predominant. Considered in terms of the amount of carbohydrate per gram of embryo (Table III) the picture is somewhat different. The greatest difference is that the free sugar per unit of tissue is relatively unaltered through this interval of development. Glycogen, which still is the smallest fraction, increases at first but then decreases between days 10 and 12. The increase in total carbohydrate (drop at day 12 probably due to the drop in glycogen) is reflected in t,he rise in (‘hound carbohydra,te”.
FIG.
1. Carbohydrate components in control and insulin-treated embryos calculatetl on the basis of carbohydrate per embryo (given in glucose equivalents).
The effect of the insulin on the entire embryo (Fig. 1) was not very marked on day 6 (24 hr. after the injection) except on free sugar, but] became increasingly evident by the smaller amounts of all of the carbohydrates present in the embryo. Except for the free sugar in insulin-treated embryos the course of accumulation of the fractions was essentially the same as in the controls, but in diminished amounts. The free sugar remained at about the same value until day 12. In terms of mg./g. embryo (Table III) the difference in glycogen is just barely significant while t’hat for free sugar is very significant on day 6. The “bound carbohydrate” is altered very little. Probably the diminution of free sugar accounts for most of the decrease in total carbohydrate. The amount of free sugar per unit of tissue actually decreases
236
EDGAR
until the 10th day in stant in the controls. embryos had no free our assay procedure.
ZWILLING
the treated embryos while it remains fairly conActually about + of the treated 8- and IO-day sugar at all; that is, it could not be detected by By the 12th day, however, there is no significant TABLE Carbohydrates
III in Embryo
(In mg. glucose) Carbohydrate component
Average (ma./9.
1NO. No. of xnbrvo sd ete
embryo, wit1 3. of mean
Range
\VWC%ge wt. of ,mbryos Q.
, , Insulin
, , , ,
8.
1.43 1.80 2.86 2.81 0.91 0.84 1.41 1.91
* f f * f f f *
.13 .13 .13 .lB .06 .09 .18 .14
0.73-2.02 1.34-2.24 2.54-3.36 2.36-3.26 0.33-1.10 0.45-1.10 0.76-2.01 1.42-2.30
0.468 1.310 2.567 4.991 0.410 0.999 1.690 3.071
.420-0.543 .262-1.371 .476-2.726 .756-5.399 .358-0.454 ‘~8 O-1.122 .4 9 3-1.874 .198-3.758
0.27 0.48 0.78 0.68 0.29 0.34 0.56 0.85
i + * f i i f f
,009 .Ol .05 .04 ,007 .Ol .03 .08
0.22-0.43 0.43-0.67 0.67-1.04 0.48-0.87 0.184.38 0.284.44 0.424.69 0.50-1.13
0.363 1.106 2.122 4.416 0.340 0.894 1.533 2.512
1.324-0.412 .045-1.172 .802-2.521 ,.678-4.974 1.277-0.418 1.805-1.061 .116-2.021 1.005-3.048
0.75 0.79 0.85 0.75 0.20 0.19 0.09 0.62
f * f & f rt f i
.0x .07 .lO .07 .06 .OQ .04 .07
0.62-0.91 0.59-1.28 0.58-1.36 0.61-0.96 0.00-0.53 0.00-0.70 0.004.29 0.44-0.77
0.424 1.174 2.463 4.909 0.419 1.042 1.715 2.975
1.339-0.466 .lll-1.239 1.085-2.694 .509-5.191 1.370-0.462 1.953-1.145 .415-1.929 1.153-3.631
Glycogen Control
Insulin
44 14 ii 44 16 9 8
II
II
I I I
“Bound carbohydrate” (calculated) Control
Insulin
0.41 0.53 1.23 1.38 0.42 0.31 0.76 0.44
difference in regard to free sugar. All of the other components are decreased significantly on days 8 and 10. On the 12th day the “bound carbohydrate,” as in the yolk-sac membranes, was relatively more diminished than at the earlier stages. On this day the glycogen in the treated embryos is actually greater than in the controls. This increase
CARBOHYDRATE
237
METABOLISM
is just barely significant when the sbandard error of the differences is considered (gd = .087, difference of means = .172). When these data are considered in terms of what proportion each fraction is of the total carbohydrate present (Table IV) several significant differences may be noted between the control and experimental animals. The free sugar is diminished and the glycogen is increased at all stages after insulin treatment. The tendency, in the controls, for free sugar to decline and (Lbound carbohydrate” to become augmented is no longer noted in the treated embryos. In the latter the free sugar remains fairly constant except for day 10. (Even the elimination of those cases in which free sugar could not be detected would not alter these percentages very much. This treatment for day 10 would give us TABLE Carbohydrate
IV
Content of Embryos
(Per cent of total carbohydrate) co1nponent
6 days %
8 $YS
10 days 70
12 days
/a
%
Control Glycogen Free sugar “Bound carbohydrate”
18.9 52.4 28.6
26.6 43.9 29.4
27.2 29.7 43.0
24.2 26.9 49.1
Insulin Glycogen Free sugar “Bound carbohydrate”
31.8 22.0 46.1
40.4 22.6 36.9
39.7 6.4 53.9
44.5 32.4 23.0
10% free sugar and 51% “bound carbohydrate”.) “Bound carbohydrate” fluctuates but remains high at all but the la-day stage. Glycogen is, as in the controls, relatively constant after day 6. The differential in glycogen content between the control and experimental embryos remains fairly uniform at all stages. At the time that the investigation of blood sugar levels (6) was under way we made some measurements for free sugar in the fluid yolk of normal and injected embryos. These data have not been presented previously but may be pertinent to the present discussion. Assays were performed on either O.Ol-ml. or O.l-ml. samples which were obtained from the living intact embryo by means of a cannula which pierced the yolk-sac membrane at a point adjacent to the umbilical stalk (the Folin-Malmros technique was used). In the course of these studies it
238
EDGAR
ZWILLING
TABLE
V
Free Sugar in Fluid
Yolk
Age
days
No. of citses
Av. value in mg.-6X glucose (with S.E. of mean)
6 8 10 12
13 11 11 5
168 139 112 112
f f f f
6 8 10 12
14 10 4 4
146 126 126 113
fi 11.65 f 9.7 f 16.6 f 8.3
Range
Controls 11.1 12.9 10.4 7.6
86-242 60-197 70-163 100-143
Insulin 63-198 74-164 67-149 94-127
became evident that the fluid (digested) yolk was by no means homogeneous (note the large standard errors). Samples taken at different points on the same yolk-sac membrane gave widely divergent values. A more precise method would involve removing all of the yolk, centrifuging it, and assaying the supernatant fluid. Since we were interested in having our embryos survive this was not done. However, the values which we present here (Table V) do indicate that insulin has relatively little effect on the free sugar content of the digested yolk. Other data for days 7, 9, and 13 and some which we have for yolk from embryos treated with 2 units of insulin indicate the same thing. DISCUSSION
The most striking fact to emerge from this study is that the glycogen content of the yolk-sac membrane is greatly increased by the action of insulin. When this is considered together with the fact that the blood in the vitelline veins is hypoglycemic and that all of the carbohydrates in the embryo are diminished it is very unlikely that any reaction of the embryo itself causes the lowered blood sugar. It is much more likely that the reduced embryonic blood sugar results from the failure of the yolk-sac membrane to release carbohydrates in normal quantities. Such a reaction of the yolk-sac membrane to insulin is compatible with the observations that insulin inhibits glycogenolysis (especially in the presence of glucose) in mammalian liver (15) and that the liver may gain glycogen after insulin treatment when the proper levels
CARBOHYDRATE
METABOLISM
239
of blood sugar are maintained (16). The unaltered free sugar in the fluid yolk in our experiments provides the high sugar concentration requisite for these effects. It is likely, therefore, that the insulin, in our experiments, inhibits glycogenolysis in the yolk-sac membrane. Adrenal cortical extract produces a proportionate dwarfing of chick embryos. Together with insulin this extract exaggerates the teratogenic effects of the latter (3). The cortical extract alone produces a hyperglycemia and with insulin exaggerates the hypoglycemia (6). The effect of the hormone by itself (i.e., dwarfing and raised blood sugar) indicates that it interferes with embryonic carbohydrate utilization. It is likely that in the presence of the insulin effect a superimposed interference with the utilization of the smaller amounts of carbohydrate reaching the embryo may increase the inhibition of glycogenolysis in the yolk-sac membrane. If the cortical extract has a direct antiglycogenolytic effect on the yolk-sac membrane as found in liver (17), then it is marked only in the presence of insulin. Evidently nicotinamide and cu-ketoglutaric acid interfere with the insulin’s inhibition of glycogenolysis, since they tend to alleviate the hypoglycemia (7). Thus far only two hormones have been reported in the yolk of early chick embryos: an estrogen (18) and an insulin-like substance (19). It is unlikely that the former exerts any control over carbohydrate metabolism and doubtful whether the latter exists. E. H. Lang, at the Wellcome Research Laboratories, could not confirm Shikinami’s work (unpublished tests made in 1947). In the absence of the hormones usually associated with carbohydrate control it is likely that carbohydrate levels in the embryo are maintained by a relatively simple balance between sugar levels in the yolk and in the yolk-sac membrane until the embryonic hormones begin to function. This material provides experimental confirmation of Claude Bernard’s now classical concept of the embryonic “transitory liver”. According to this idea the placenta in mammals and the yolk-sac membrane in birds serve as glycogen storage structures until the fetal liver can assume this function. Unlike that of the yolk-sac membrane of the chick embryo the glycogen in mammalian placentas is thought to be quite refractory to the action of insulin (20,21). In general mammalian embryos are refractory to insulin (20-24). Some individual experiments in these reports indicate a fall in one 01 another of the carbohydrate components, and Corey (28) has demon-
240
EDGAR
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&rated that insulin may cross the placenta in rates and cause hypoglycemia. The failure of response in many of these experiments may be due to exchange with the maternal blood, since sugar can cross the placenta in dogs (25), but may also be due to the fact that most of the experiments were of short duration and involved late stages, in which the fetal hormones could affect the results. Certainly chick embryos, at the stages treated, do respond to insulin.4 All of the carbohydrate components are diminished. This may reflect, wholly or in part, the decreased amount of carbohydrate reaching the embryo. The return of free sugar and glycogen to normal levels by day 12 (Table III) probably indicates that the embryonic hormones are beginning t,o function. Whether insulin has a more direct effect on the embryo cannot’ be determined from these data. In an attempt to decide this point me injected insulin into the allantoic sacs of a group of G-day embryos (younger ones fail to survive this treatment). These did indeed become hypoglycemic and did show the same morphological effects as injections into the yolk. However, the yolk-sac membranes had an increased amount of glycogen after this t’reatment too, and we are unable to make any decision in this regard. It is interesting to speculate in view of the demonstrations by Soskin and his group (26) that insulin enhances sugar penetration (and possibly utilization) and that it is most effective at low sugar levels, that it is the insulin itself which al10ms the embryo to survive at hypoglycemic levels. We should like to emphasize that the chick embryo may be a very valuable object for fut’ure work on problems of carbohydrat’e metabolism. In effect, during the early stages, it is an eviscerated and glandless animal which gets all of its nutrients via the yolk-sac membrane and this membrane responds to insulin and other substances. 4 Mme. Guelin-Schedrina has reported (27) that the early embryo is refractory to insulin since there was no precocious appearance of glycogen in livers of embryos subjected to insulin. Her experiments consisted of injecting lo-20 mg. insulin (220-440 units!) into the blood stream of 5- and B-day embryos and fixing these 6-24 hr. afterwards for histochemical examination of glycogen. In other experiments she grafted 3-day embryos to the chorioallantoic membrane of ‘J-day hosts, in an effort to influence the early embryo by “embryonic ” insulin. These showed no difference (other than a loss in cardiac glycogen) after as long as 6 days on the host membrane. These experiments are certainly not conclusive since on the one hand an extremely toxic dose of insulin was employed and on the other the proper host stage for t)esting the action of embryonic insulin was not used,
CARBOHYDRATE
METABOLISM
241
I should like, to acknowledge nay indebtedness to the follobving for their helpful of Connecticut; Dr. suggestions and criticisms: Dr. Walter Landauer, University Heinrich Wselsrh, K. T. State Psychiatric Institute; ant1 Dr. Salomc CluccksohnWaelsch, Columbia IJniversity.
1. Free sugar, glycogen, and total carbohydrate have been assayed in insulin-treated and control chick embryos and yolk-sac membranes at 6, 8, IO, and 12 days (5 units of insulin injected into the yolk sac at 5 days). “Hound carbohydrate” n-as calculated. 2. The glycogen content and tot,al carbohydrate of the yolk-sac membranes increased after insulin treatment, and the other carbohydrate components were diminished. All carbohydrate fract,ions decreased in the embryo proper. 3. Free sugar in the fluid yolk was not altered by the insulin. 4. It is concluded from these data that insulin which is injected into the yolk sac at 5 days produces its hypoglycemic effect’ by inhibiting glycogenolysis in the yolk-sac membrane. 5. It is not certain that the lowered carbohydrat’e content of the embryo results solely from the hypoglycemia or from an additional direct effect of the insulin. 6. These data are discussed with reference to the phenomenon of the “transitory liver” of Claude Bernard and to the action of insulin in the apparent absence of other hormones. REFEREKCES 1. LANDALTER,u'., J. Exptl. Zaol. 98, 65 (1945). 2. LANDAUER, W., J. Exptl. 2001. 105, 145 (1947). 3. LANDAUER, W., Endocrinology 41, 489 (1947). 4. LANDAUER, W., J. Exptl. 2001. 109, 283 (1948). 5. LANDAUER, Growth 12, 171 (1948). 6. ZWILLING, E., J. Exptl. Zool. 109, 197 (1948). 7. ZWILLING, E., hoc. Sot. Exptl. Biol. Med. 71, 609 (1949). 8. ZWVILLING,E., AND DERELL, J. T., J. Exptl. Zool. 115, 59 (1950). 9. RAHN, H., J. Morphol. 64, 483 (1939). 10. LILLIE, F’. R., The Development of the Chick. Henry Holt & Co., 19lY. Cambridge Univ. Press, 1931. Il. NEEDHAM, J., Chemical Embryology. 12. SOILIOGYI,hf., J. Biol. Chem. 160, 61 (1945). 13. SOrvfOGYI,hl., J. Biol. Chem. 160, 69 (1945). 14. GOOD,C. A., KRARIER, H., AND SORIOGYI,N., J. Biol. Chem. 100, 485 (1933).
242
EDGAR
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15. TAWENHAUS, M., LEVINE, R., AND SOSKIN, S., Proc. Sot. Exptl. Biol. Med. 42, 693 (1939). 16. BOUCKAERT, J. P., AND DE DUYE, CHR., Physiol. Revs. 27, 39 (1947). 17. COREY, E. L., AND BRITTON, S. W., Am. J. Physiol. 131, 783 (1941). 18. RIBOULLEAU, J., Compt. rend. sot. biol. 129, 914 (1938). 19. SHIKINAMI, Y., TGhoku J. Exptl. Med. 10, 1 (1928). 20. HUGGETT, A. ST. G., J. Physiol. (London) 67, 360 (1929). 21. COREY, E. L., Am. J. Physiol. 113, 450 (1935). 22. BRITTON, S. W., Am. J. Physiol. 95, 178 (1930). 23. SCHLOSSMANN, H., J. Physiol. (London) 92, 219 (1938). 24. PASSMORE, R., AND SCHLOSSMANN, H., J. Physiol. (London) 92, 459 (1938). 25. SCHLOSSMANN, H., Arch. exptl. Path. Pharmakol. 159, 213 (1931). 26. SOSKIN, S., AND LEVINE, R., Carbohydrate Metabolism. University of Chicago Press, 1946. 27. GUELIN-SCHEDRINA, A., Compt. rend. sot. biol. 121, 144 (1936). 28. COREY, E. L., Physiol. Zool. 5, 36 (1932).
Experiment March
Station, Stows, Connecticlct 15, 1951
Landauer (1,2) has shown that insulin, when injected into the yolk sacs of chick embryos, may consistently produce particular abnormalities depending on the age of the embryo when it is treated. Defects in the caudal region (rumplessness) are the chief consequence of insulin injections during the first 3 days of incubation (with a maximum incidence at 30 hr.), while disproportionate dwarfing of the hind limbs (micromelia) occurs when 4-7-day embryos are treated (with maximum incidence at 5 days). Both types of anomalies bear close resemblance to known gene-mediated conditions of chickens, i.e., they are phenocopies. We have preferred to concentrate on the insulin-induced micromelia for a study of possible relationship between the abnormalities and any derangement in physiology for the following reasons: (a) It may be produced consistently in large numbers; (b) the chief action leading to the condition occurs in relatively large embryos to which analytical procedures may be applied; (c) the condition closely resembles those produced by a number of other experimental procedures as well as at least six mutations. This last provides us with the possibility of producing the micromelia by other experimental procedures for comparisons aimed at discovering common pathways and processes which may be involved. The existence of mutations which produce similar syndromes may enable us to ascertain whether any of the information obtained from a study of the experimentally induced terata can be 1 This investigation was aided by grants from the International Baby Chick Association and from the American Cancer Society on the recommendation of the Committee on Gr0wt.h of the Sational Research Council. 228
CARBOHYDRATE
METABOLISM
229
applied to the gene-mediated conditions in an attempt to reveal the mechanism of action of these particular genes. Thus far there have been two procedural approaches to the problem of revealing which physiological systems may be disturbed by the action of insulin and related to the morphological derangements. One of these has been to observe the effects of a number of other substances which have been injected either simultaneously with, shortly before, or after the insulin. By these means Landauer (3,4) has discovered that cortin, which by itself causes a uniform dwarfing of the embryo, exaggerates the insulin effects; nicotinamide and, to a lesser extent, oc-ketoglutaric acid are effective in eliminating the insulin-induced micromelia. There are other results which shall not be discussed here (2-5). The other approach has been to perform assays for various metabolites and, by comparisons with untreated embryos, attempt to relate any deviations to the anomalous development. It has been found (6,7) that insulin-treated embryos become hypoglycemic and that there is a very suggestive relationship between the hypoglycemia and micromelia: the more pronounced and prolonged the hypoglycemia, the more extreme is the leg shortening. In addition, factors which exaggerate or relieve the micromelia also tend to accentuate or abolish the hypoglycemia. Two sets of problems are opened by these observations: the first’ concerns possible mechanisms through which the hypoglycemia may produce alterations of embryonic development (6,8), i.e., whether such derangements are a more or less direct result of the lowered available carbohydrate or whether they are due to some other effect of insulin. The second, with which this paper is concerned, involves the mechanism through which insulin produces hypoglycemia in the embryonic system. The latter problem has certain very interesting aspects for students of insulin physiology since the embryos which are subjected to insulin presumably have none of the hormones usually associated with insulin activity in the adult, and this condition persists for at least several days following the treatment. Pituitary gland activity is not noted until the 10th day of development (9); the formation of the adrenal cortex begins between the 4th and 6th days (10) but there is no certainty about the beginning of it’s secretory activity. Insulin itself does not appear in detectable amounts in the embryo until the 11th day (11). A rather curious situation, first brought to attention by Claude Bernard in 1858, exists in bird embryos. The glycogen storage functions are performed by t,he clrt’racmbryonic yolk-sac membrane
230
EDGATZ ZWILLING
(which Bernard called the “‘joie transitoire”) during the first, week or so of development. For this reason we have performed separate assays for carbohydrates (free reducing substances, glycogen, and “total carbohydrate”) upon the embryo and its yolk-sac membrane. The data obtained from these assays reveal that the glycogen content of the yolk-sac membranes of insulin-treated embryos is considerably greater than that of control yolk sacs. The data further indicate that t,he augmented glycogen of the yolk-sac membrane accounts for the embryonic hypoglycemia. All of the carbohydrate components of the embryo proper are diminished after the injection of insulin. MATERIAL
AND
METHODS
All of the eggs used in these experiments were from unselected (5) White Leghorn hens. They were injected at 5 days with 5 units of Iletin (insulin, Lilly)2 through holes drilled in the shell at the center of the blunt end of the egg. The holes were sealed with nail polish after the injection. One-inch 20 gage needles were used to insure the insulin’s reaching the yolk sac. The eggs were incubated in a forced-draft incubator on their sides, both prior to and following the injection. Controls consisted of unt.rrated embryos. At the proper stages the eggs were opened and the embryos, after being freed of membranes, etc., were rapidly removed to a 0.9% NaCl solut,ion. The rest of the egg was immersed in saline solution, and the yolk sac, freed of all other membranes, was removed. The exact procedure varied a bit depending on the age of the embryos. Wit,h g-day embryos, in which the vascularised yolk sac has not yet encroached upon the entire yolk, the membrane was removed by incising a short distance beyond the outermost blood vessel (sinus terminalis). At later stages, where the yolk-sac membrane encompassed all of the yolk, the incisions were made at a region where the yolk sac makes contact with the albumen residue. It was not, always possible to obtain intact membranes from the later stages; pieces were frequently lost. The yolk sacs were washed free of yolk and placed in clean saline. It was quite simple to free the yolk-sac membrane of yolk at the stages used in these experiments; by 13 or 14 days, however, the yolk is so viscous that this is almost impossible. The mashed tissue (either intact embryo or yolk-sac membrane) was then allowed to drain on toweling paper, weighed on a Roller-Smith balance and placed in the appropriate reagent for the determination to be carried out. In all instances controls were handled in a similar manner and the sequence of preparing control and injected tissues for assay was varied so that this could not affect the results. Apparently relatively little glycogenolysis occurs when the yolk-sac membranes arc kept in 0.0% KaCl solution for a short time. This was established by a series of glycogen assays of yolk-sac membranes (from 7Q-day embryos) which were placed in KOH immediately after removal from the egg and compared with membranes kept in unbuffered saline at room temperature for 15 min. Five membranes from untreated * Furnished Peck.
by the 1,illy Research
Laboratories
through
the courtesy
of Dr. F. R.
CARBOHYDRATE
METABOLISM
231
embryos and five from insulin-injected embryos were in each group. The controls averaged: immediate assay, 5.88 mg./g.; 15-min. delay, 5.25 mg./g. Insulin-treated: immediate assay, 8.97 mg./g.; 15-min. delay, 9.57 mg./g. With such small numbers t,hrbse differences are not significant.. \\‘c selected 5 units of insulin becausc~ thca incid<~nce 01’ clst,reme response (micromcllia) is 111uc.hgycv~tc~ t II:~II with lower doses. In all cases lvhere the embr,vos were old c~nough so t,h:tt thcl Inorpllological c~ffe(*ts of the insulin mere evident, the most extreme WS,Y were scJ~t (~1 i’ol, assay. It, is peltinent to add i hat embryos treated with this tlosc~ of insulin a~ colrsistently smaller and slightly more retarded than controls (Table III!. The yolk-sac memi)ranc:s are also smaller and have fewer folds on their inrrclr surface (Table I). Howcvt~, \VC compared, in all cases, embryos of the same c*hronological age; in each set of tlctolmi~~atio~ls WV included controls and injected embryos which had been incubattstl on t.hc same tray. Our determinations were not cxrried out on rnhyms older than 12 days since we have shown that normal bloodsugar levels are restored by the 14th day in all insulin-injected embryos. For the most part standard procedures, with relatively slight modifications, were employed for these assays. Our greatest problem resulted from the increase in size of the chick embryos as older cmhryos were used. Since some components increased at a relatively greater rate than tha amount of tissue (in terms of wet weight), this required the use of larger fluid volumes and relatively greater final dilutions to bring the concent,ration of reducing substances a-it)hin the range of the technique employed. In some instances scvcral embryos or yolk sacs were pooled for a single determination. Somogyi’s (12) copper-iodometric reagent (modified to contain 140 g./l. Na$Oh) was used for all of t,he final determinations of reducing substance. All reactions were carried out on 2-ml. samples of unknown with 2-ml. samples of Somogyi’s reagent. These were placed in loosely stoppered 22 X 175 mm. test tubes, mixed, and heated in a boiling water bath for 10 min. After cooling, 1 ml. of 39?..ICI was run down the side of each tube and this was followed by 1 ml. of 2 N H,SOa run in rapidly with immediate agitation. Titration was performed in the same tubes with 0.008 N sodium thiosuliat,e to a starch end point. In all instances a glucose standard (1 or 0.5 mg./2-ml. sample) and reagent blank were carried through with each set of determinations. For the determination of free sugar (no attempt was made to ascertain to what, oxtent, ot,h(,r reducing substances might have? heen present) the following procedure was cmplo,x-ed: embryos and yolk sacs were placed in 1-2 ml. of distilled wat,er and ground to a uniform consistency in a motor-driven Lucite-in-glass homogenizer. Immediately after this the proteins were precipitated with equal amounts of 0.3 N Ba(OI& and 57, ZnS04.7H,0 [adjusted according to the directions of Somogyi (13)] which were added t,o the tube with stirring. Either 3 or 4 ml. of each of these reagents was added to each t’ube depending on the size of the embryos. The final dilution with distilled water was either to 10 or l-1 ml. After centrifuging the preparation, duplicate 2-ml. samples of the supernatant R-ere pipetted off and assayed as outlined above. The standard was a glucose solution adjusted in volume and concentration so that a 2-ml. sampIca of the final dilution contained 1 mg. glucose. Standard and blank were treated n-ith the samr amount, of the reagents used in the tubes with the tissue. When embryos or yolk sacs were treated differently as to amounts of reagent and final dilution a standard and blank mere carried through for each of the treatments.
232
EDGAR ZWILLING
Glycogen was det,ermined according t,o the procedure of Good, Kramer, and Somogyi (14), with two modifications. Prior to the addition of alcohol to the KOII digested material 0.5 ml. of saturated xapSOa K-as added for each 4 ml. of digest, and the centrifuged glycogen precipitate was resuspended twice and brought to the boiling point in 60% ethyl alcohol in chloroform (959;. alcohol diluted to 60y0 with chloroform). This latter was done to eliminate the fatty material which was present, especially in the yolk sacs. The recovered purified glycogen was hydrolyzed with 4 ml. of 0.6 N HCl in a boiling water bath for 3 hr. After cooling, the hydrolyzatc was neutralized with KOH. Final dilution was made with water to 5, 10, or 20 ml. (In some instances the final dilution was made prior to neutralization and the final 2-ml. sample was neutralized prior to addition of the reducing solution. There was no apparent difference in the two procedures.) Duplicate 2-ml. samples were assayed for reducing substances as already indicated. The standard glucose solution was diluted, first with 4 ml. of 0.6 N HCl, then dist,illed water. The blank was 4 ml. of the IICl and enough water for the final dilution. “Total carbohydrate” (i.e., all reducing substances prosent after the hydrolysis described) was determined by hydrolyzing the weighed tissue in from 7 to 15 ml. (depending on the amount of tissue) of 0.6 N HCl for 4 hr. The hydrolyzates were then cooled and neutralized to phenol red with strong KOH. Protein was precipitated with enough Ba(OH)2 and ZnSOd to give a clear nonfoaming supernatant (samples at the various dilutions were tested with Millon’s reagent in the preliminary trials). Enough distilled water was added to give final dilutions of from 24 to 50 ml. Duplicate 2-ml. samples were assayed for reducing substances as indicated. All calculations were performed in a similar manner. The amount of glucose/ml. thiosulfate (blank - standard) X amount of thiosulfate needed to titrate unknown (blank -unknown) X dilution factor = mg. of reducing substance in terms of glucose in each sample assayed. All of the data are presented in terms of equivalent glucose reduction. All data are given as mg./g. tissue, and a chart (Fig. 1) based on mg./ embryo is included. Means are presented with their standard errors. Differences between means were not considered significant unless they exceeded twice the “stand: ard error of the differences”. “Bound carbohydrate”3 was calculated by subtracting the sums of the average values for glycogen and free sugar from the average value obtained for “total carbohydrate” for each stage.
RESULTS
In the yolk-sac membranes from untreated embryos the largest carbohydrate fraction for the period investigated is glycogen (Table I). This reaches a maximum at 8 days and then diminishes somewhat by represents is not clear. Seedham (11) 3 Exactly what the “bound carbohydrate” considers this fraction to be carbohydrate which is bound to protein. In the embryo proper he considers this to be mostly “mucoprotein” and in t.he rest of the egg “ovomucoid” substances. This fraction may also represent, some other forms of unhydrolyzed carbohydrate. If, as iYeedham speculates, it is important for the development of connective tissues and bones then the changes which we describe may be of significance. Further investigation of this fraction is required.
CARBOHYDRATE
233
METABOLISM
the 12th day. Total carbohydrate at first follows a similar course but t’hen comes down at 10 days only to rise again at 12 days. This behavior is (see below) related to a sharp decline in “bound carbohydrate” at 10 days followed by its augmentation at 12 days. Free sugar remains fairly constant throughout these stages. The data which we have obtained from our assays of yolk-sac membranes indicate clearly that there is an increase in glycogen and in total carbohydrate while there is a diminution in free sugar after insulin treatment (Table I). Virtually all of the increase in total carbohydrate can be accounted for by the augmented amount of glycogen on days TABLE
I
Carbohyzlrates in Yolk-Sac Membrane (In mg. glucose)
Carbohydrate component
d
Average ‘mg./g. yolk sac with .E. of mean)
Rang.!
4veraae wt. of mlk sacs Q.
15
8:% 1.366 2.079 0.556 0.697 0.807 1.284
: Insulin
Glycogen Control
Insulin
Ii: 6 5 6 24 11 ; 24 12 fl 14 : 3 12 9 i
“Bound carbohydrate” (calculated) Control Insulin
4.63 7.84 7.37 6.04 6.11 13.04 11.43 9.88
f f f f f f 3~ f
0.86 0.86 1.08 0.84 0.69 0.43 0.59 0.85
2~ ,021 zt .103 zt ,069 dc .102 z!z ,042 f .051 zt ,087 * ,193
3.28 2.21 0.76 3.64 3.74 2.79 0.69 0.78
.20 .42 .63 .54 .33 .65 .59 .43
3.15- 6.79 6.21-10.65 5.08-10.04 4.32- 8.80 4.77- 8.79 7.94-16.37 8.79-14.35 7.52-12.14 0.74Xl.93 0.37-1.21 0.86-1.29 0.64-1.07 0.49488 0.19XI.62 0.254.86 0.38-1.15
0.564 0.862 1.200 1.956
Q.
0.5384.681 0.7994.972 1.246-1.520 1.838-2.332 0.514Xl.620 0.609-0.818 0.758-0.862 1.068-1.284
X 0.901 1.237
0.357-0.777 0.760-1.087 1.072-1.285 1.516-2.262 0.358-0.701 0.538-0.898 0.740-1.082 0.949-1.423
ES 1.252 1.875 0.618 0.728 0.853 1.396
0.53OAI.626 0.7574.964 1.014-1.444 1.521-2.238 0.548-0.671 0.672-0.825 0.762-0.930 0.998-1.885
234
EDGAR
ZRILLING
6, 8, and 10 in the experimental yolk sacs. On day 12, however, the difference between the total carbohydrate content of controls and experimentals is not significant while that in regard to glycogen is highly significant. This is reflected in a drop in “bound carbohydrate”. The ‘lbound carbohydrate” behaves curiously at the IO-day stage. In both control and treated membranes it falls very low; then it reappears again on day 12. The fact that the same thing occurs in both sets of observations argues against its being an artifact. It seems as though glycogen synthesis occurs largely at the expense of the bound carbohydrate at this time. Apparently the reestablishment of the “bound carbohydrate” fraction is interfered with by the insulin. It is interesting to note that glycogen and total carbohydrate follow roughly the same TABLE
II
Carbohydrate Content of Yolk-Sac Membranes (Per cent of total carbohydrate) co1nponent 6 days “gY 10 days YO c/o
12 days %
Control Glycogen Free sugar “Bound carbohydrate”
52.8 9.8 37.4
71.8 7.9 20.2
80.0 11.7 8.2
57.4 8.0 34.6
Glycogen Free sugar “Bound carbohydrate”
57.9 6.5 35.5
80.2 2.6 17.1
89.9 4.7 5.4
85.8 7.4 6.7
Insulin
pattern after the 8th day. Apparently the differences are not fully established until this time. There is no significant difference in the amount of free sugar per gram of tissue in the various stages in the controls; in treated membranes this component drops significantly and then returns to normal levels by day 12. In terms of the per cent of total carbohydrate (Table II) it can be seen that the glycogen accumulates at the expense of the free and “bound carbohydrate” fractions, which are both relatively diminished in the insulin-treated yolk sacs. When the amount of carbohydrate per embryo is plotted (Fig. 1) it is seen that all of the constituents in the control embryos show a steady rise and follow, roughly, a similar course. Glycogen is the smallest fraction in all of the stages assayed. Free sugar is the largest com-
CARBOHYDRATE
METABOLISM
235
ponent at first, but by the 10th day the “bound carbohydrate” is predominant. Considered in terms of the amount of carbohydrate per gram of embryo (Table III) the picture is somewhat different. The greatest difference is that the free sugar per unit of tissue is relatively unaltered through this interval of development. Glycogen, which still is the smallest fraction, increases at first but then decreases between days 10 and 12. The increase in total carbohydrate (drop at day 12 probably due to the drop in glycogen) is reflected in t,he rise in (‘hound carbohydra,te”.
FIG.
1. Carbohydrate components in control and insulin-treated embryos calculatetl on the basis of carbohydrate per embryo (given in glucose equivalents).
The effect of the insulin on the entire embryo (Fig. 1) was not very marked on day 6 (24 hr. after the injection) except on free sugar, but] became increasingly evident by the smaller amounts of all of the carbohydrates present in the embryo. Except for the free sugar in insulin-treated embryos the course of accumulation of the fractions was essentially the same as in the controls, but in diminished amounts. The free sugar remained at about the same value until day 12. In terms of mg./g. embryo (Table III) the difference in glycogen is just barely significant while t’hat for free sugar is very significant on day 6. The “bound carbohydrate” is altered very little. Probably the diminution of free sugar accounts for most of the decrease in total carbohydrate. The amount of free sugar per unit of tissue actually decreases
236
EDGAR
until the 10th day in stant in the controls. embryos had no free our assay procedure.
ZWILLING
the treated embryos while it remains fairly conActually about + of the treated 8- and IO-day sugar at all; that is, it could not be detected by By the 12th day, however, there is no significant TABLE Carbohydrates
III in Embryo
(In mg. glucose) Carbohydrate component
Average (ma./9.
1NO. No. of xnbrvo sd ete
embryo, wit1 3. of mean
Range
\VWC%ge wt. of ,mbryos Q.
, , Insulin
, , , ,
8.
1.43 1.80 2.86 2.81 0.91 0.84 1.41 1.91
* f f * f f f *
.13 .13 .13 .lB .06 .09 .18 .14
0.73-2.02 1.34-2.24 2.54-3.36 2.36-3.26 0.33-1.10 0.45-1.10 0.76-2.01 1.42-2.30
0.468 1.310 2.567 4.991 0.410 0.999 1.690 3.071
.420-0.543 .262-1.371 .476-2.726 .756-5.399 .358-0.454 ‘~8 O-1.122 .4 9 3-1.874 .198-3.758
0.27 0.48 0.78 0.68 0.29 0.34 0.56 0.85
i + * f i i f f
,009 .Ol .05 .04 ,007 .Ol .03 .08
0.22-0.43 0.43-0.67 0.67-1.04 0.48-0.87 0.184.38 0.284.44 0.424.69 0.50-1.13
0.363 1.106 2.122 4.416 0.340 0.894 1.533 2.512
1.324-0.412 .045-1.172 .802-2.521 ,.678-4.974 1.277-0.418 1.805-1.061 .116-2.021 1.005-3.048
0.75 0.79 0.85 0.75 0.20 0.19 0.09 0.62
f * f & f rt f i
.0x .07 .lO .07 .06 .OQ .04 .07
0.62-0.91 0.59-1.28 0.58-1.36 0.61-0.96 0.00-0.53 0.00-0.70 0.004.29 0.44-0.77
0.424 1.174 2.463 4.909 0.419 1.042 1.715 2.975
1.339-0.466 .lll-1.239 1.085-2.694 .509-5.191 1.370-0.462 1.953-1.145 .415-1.929 1.153-3.631
Glycogen Control
Insulin
44 14 ii 44 16 9 8
II
II
I I I
“Bound carbohydrate” (calculated) Control
Insulin
0.41 0.53 1.23 1.38 0.42 0.31 0.76 0.44
difference in regard to free sugar. All of the other components are decreased significantly on days 8 and 10. On the 12th day the “bound carbohydrate,” as in the yolk-sac membranes, was relatively more diminished than at the earlier stages. On this day the glycogen in the treated embryos is actually greater than in the controls. This increase
CARBOHYDRATE
237
METABOLISM
is just barely significant when the sbandard error of the differences is considered (gd = .087, difference of means = .172). When these data are considered in terms of what proportion each fraction is of the total carbohydrate present (Table IV) several significant differences may be noted between the control and experimental animals. The free sugar is diminished and the glycogen is increased at all stages after insulin treatment. The tendency, in the controls, for free sugar to decline and (Lbound carbohydrate” to become augmented is no longer noted in the treated embryos. In the latter the free sugar remains fairly constant except for day 10. (Even the elimination of those cases in which free sugar could not be detected would not alter these percentages very much. This treatment for day 10 would give us TABLE Carbohydrate
IV
Content of Embryos
(Per cent of total carbohydrate) co1nponent
6 days %
8 $YS
10 days 70
12 days
/a
%
Control Glycogen Free sugar “Bound carbohydrate”
18.9 52.4 28.6
26.6 43.9 29.4
27.2 29.7 43.0
24.2 26.9 49.1
Insulin Glycogen Free sugar “Bound carbohydrate”
31.8 22.0 46.1
40.4 22.6 36.9
39.7 6.4 53.9
44.5 32.4 23.0
10% free sugar and 51% “bound carbohydrate”.) “Bound carbohydrate” fluctuates but remains high at all but the la-day stage. Glycogen is, as in the controls, relatively constant after day 6. The differential in glycogen content between the control and experimental embryos remains fairly uniform at all stages. At the time that the investigation of blood sugar levels (6) was under way we made some measurements for free sugar in the fluid yolk of normal and injected embryos. These data have not been presented previously but may be pertinent to the present discussion. Assays were performed on either O.Ol-ml. or O.l-ml. samples which were obtained from the living intact embryo by means of a cannula which pierced the yolk-sac membrane at a point adjacent to the umbilical stalk (the Folin-Malmros technique was used). In the course of these studies it
238
EDGAR
ZWILLING
TABLE
V
Free Sugar in Fluid
Yolk
Age
days
No. of citses
Av. value in mg.-6X glucose (with S.E. of mean)
6 8 10 12
13 11 11 5
168 139 112 112
f f f f
6 8 10 12
14 10 4 4
146 126 126 113
fi 11.65 f 9.7 f 16.6 f 8.3
Range
Controls 11.1 12.9 10.4 7.6
86-242 60-197 70-163 100-143
Insulin 63-198 74-164 67-149 94-127
became evident that the fluid (digested) yolk was by no means homogeneous (note the large standard errors). Samples taken at different points on the same yolk-sac membrane gave widely divergent values. A more precise method would involve removing all of the yolk, centrifuging it, and assaying the supernatant fluid. Since we were interested in having our embryos survive this was not done. However, the values which we present here (Table V) do indicate that insulin has relatively little effect on the free sugar content of the digested yolk. Other data for days 7, 9, and 13 and some which we have for yolk from embryos treated with 2 units of insulin indicate the same thing. DISCUSSION
The most striking fact to emerge from this study is that the glycogen content of the yolk-sac membrane is greatly increased by the action of insulin. When this is considered together with the fact that the blood in the vitelline veins is hypoglycemic and that all of the carbohydrates in the embryo are diminished it is very unlikely that any reaction of the embryo itself causes the lowered blood sugar. It is much more likely that the reduced embryonic blood sugar results from the failure of the yolk-sac membrane to release carbohydrates in normal quantities. Such a reaction of the yolk-sac membrane to insulin is compatible with the observations that insulin inhibits glycogenolysis (especially in the presence of glucose) in mammalian liver (15) and that the liver may gain glycogen after insulin treatment when the proper levels
CARBOHYDRATE
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of blood sugar are maintained (16). The unaltered free sugar in the fluid yolk in our experiments provides the high sugar concentration requisite for these effects. It is likely, therefore, that the insulin, in our experiments, inhibits glycogenolysis in the yolk-sac membrane. Adrenal cortical extract produces a proportionate dwarfing of chick embryos. Together with insulin this extract exaggerates the teratogenic effects of the latter (3). The cortical extract alone produces a hyperglycemia and with insulin exaggerates the hypoglycemia (6). The effect of the hormone by itself (i.e., dwarfing and raised blood sugar) indicates that it interferes with embryonic carbohydrate utilization. It is likely that in the presence of the insulin effect a superimposed interference with the utilization of the smaller amounts of carbohydrate reaching the embryo may increase the inhibition of glycogenolysis in the yolk-sac membrane. If the cortical extract has a direct antiglycogenolytic effect on the yolk-sac membrane as found in liver (17), then it is marked only in the presence of insulin. Evidently nicotinamide and cu-ketoglutaric acid interfere with the insulin’s inhibition of glycogenolysis, since they tend to alleviate the hypoglycemia (7). Thus far only two hormones have been reported in the yolk of early chick embryos: an estrogen (18) and an insulin-like substance (19). It is unlikely that the former exerts any control over carbohydrate metabolism and doubtful whether the latter exists. E. H. Lang, at the Wellcome Research Laboratories, could not confirm Shikinami’s work (unpublished tests made in 1947). In the absence of the hormones usually associated with carbohydrate control it is likely that carbohydrate levels in the embryo are maintained by a relatively simple balance between sugar levels in the yolk and in the yolk-sac membrane until the embryonic hormones begin to function. This material provides experimental confirmation of Claude Bernard’s now classical concept of the embryonic “transitory liver”. According to this idea the placenta in mammals and the yolk-sac membrane in birds serve as glycogen storage structures until the fetal liver can assume this function. Unlike that of the yolk-sac membrane of the chick embryo the glycogen in mammalian placentas is thought to be quite refractory to the action of insulin (20,21). In general mammalian embryos are refractory to insulin (20-24). Some individual experiments in these reports indicate a fall in one 01 another of the carbohydrate components, and Corey (28) has demon-
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&rated that insulin may cross the placenta in rates and cause hypoglycemia. The failure of response in many of these experiments may be due to exchange with the maternal blood, since sugar can cross the placenta in dogs (25), but may also be due to the fact that most of the experiments were of short duration and involved late stages, in which the fetal hormones could affect the results. Certainly chick embryos, at the stages treated, do respond to insulin.4 All of the carbohydrate components are diminished. This may reflect, wholly or in part, the decreased amount of carbohydrate reaching the embryo. The return of free sugar and glycogen to normal levels by day 12 (Table III) probably indicates that the embryonic hormones are beginning t,o function. Whether insulin has a more direct effect on the embryo cannot’ be determined from these data. In an attempt to decide this point me injected insulin into the allantoic sacs of a group of G-day embryos (younger ones fail to survive this treatment). These did indeed become hypoglycemic and did show the same morphological effects as injections into the yolk. However, the yolk-sac membranes had an increased amount of glycogen after this t’reatment too, and we are unable to make any decision in this regard. It is interesting to speculate in view of the demonstrations by Soskin and his group (26) that insulin enhances sugar penetration (and possibly utilization) and that it is most effective at low sugar levels, that it is the insulin itself which al10ms the embryo to survive at hypoglycemic levels. We should like to emphasize that the chick embryo may be a very valuable object for fut’ure work on problems of carbohydrat’e metabolism. In effect, during the early stages, it is an eviscerated and glandless animal which gets all of its nutrients via the yolk-sac membrane and this membrane responds to insulin and other substances. 4 Mme. Guelin-Schedrina has reported (27) that the early embryo is refractory to insulin since there was no precocious appearance of glycogen in livers of embryos subjected to insulin. Her experiments consisted of injecting lo-20 mg. insulin (220-440 units!) into the blood stream of 5- and B-day embryos and fixing these 6-24 hr. afterwards for histochemical examination of glycogen. In other experiments she grafted 3-day embryos to the chorioallantoic membrane of ‘J-day hosts, in an effort to influence the early embryo by “embryonic ” insulin. These showed no difference (other than a loss in cardiac glycogen) after as long as 6 days on the host membrane. These experiments are certainly not conclusive since on the one hand an extremely toxic dose of insulin was employed and on the other the proper host stage for t)esting the action of embryonic insulin was not used,
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I should like, to acknowledge nay indebtedness to the follobving for their helpful of Connecticut; Dr. suggestions and criticisms: Dr. Walter Landauer, University Heinrich Wselsrh, K. T. State Psychiatric Institute; ant1 Dr. Salomc CluccksohnWaelsch, Columbia IJniversity.
1. Free sugar, glycogen, and total carbohydrate have been assayed in insulin-treated and control chick embryos and yolk-sac membranes at 6, 8, IO, and 12 days (5 units of insulin injected into the yolk sac at 5 days). “Hound carbohydrate” n-as calculated. 2. The glycogen content and tot,al carbohydrate of the yolk-sac membranes increased after insulin treatment, and the other carbohydrate components were diminished. All carbohydrate fract,ions decreased in the embryo proper. 3. Free sugar in the fluid yolk was not altered by the insulin. 4. It is concluded from these data that insulin which is injected into the yolk sac at 5 days produces its hypoglycemic effect’ by inhibiting glycogenolysis in the yolk-sac membrane. 5. It is not certain that the lowered carbohydrat’e content of the embryo results solely from the hypoglycemia or from an additional direct effect of the insulin. 6. These data are discussed with reference to the phenomenon of the “transitory liver” of Claude Bernard and to the action of insulin in the apparent absence of other hormones. REFEREKCES 1. LANDALTER,u'., J. Exptl. Zaol. 98, 65 (1945). 2. LANDAUER, W., J. Exptl. 2001. 105, 145 (1947). 3. LANDAUER, W., Endocrinology 41, 489 (1947). 4. LANDAUER, W., J. Exptl. 2001. 109, 283 (1948). 5. LANDAUER, Growth 12, 171 (1948). 6. ZWILLING, E., J. Exptl. Zool. 109, 197 (1948). 7. ZWILLING, E., hoc. Sot. Exptl. Biol. Med. 71, 609 (1949). 8. ZWVILLING,E., AND DERELL, J. T., J. Exptl. Zool. 115, 59 (1950). 9. RAHN, H., J. Morphol. 64, 483 (1939). 10. LILLIE, F’. R., The Development of the Chick. Henry Holt & Co., 19lY. Cambridge Univ. Press, 1931. Il. NEEDHAM, J., Chemical Embryology. 12. SOILIOGYI,hf., J. Biol. Chem. 160, 61 (1945). 13. SOrvfOGYI,hl., J. Biol. Chem. 160, 69 (1945). 14. GOOD,C. A., KRARIER, H., AND SORIOGYI,N., J. Biol. Chem. 100, 485 (1933).
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15. TAWENHAUS, M., LEVINE, R., AND SOSKIN, S., Proc. Sot. Exptl. Biol. Med. 42, 693 (1939). 16. BOUCKAERT, J. P., AND DE DUYE, CHR., Physiol. Revs. 27, 39 (1947). 17. COREY, E. L., AND BRITTON, S. W., Am. J. Physiol. 131, 783 (1941). 18. RIBOULLEAU, J., Compt. rend. sot. biol. 129, 914 (1938). 19. SHIKINAMI, Y., TGhoku J. Exptl. Med. 10, 1 (1928). 20. HUGGETT, A. ST. G., J. Physiol. (London) 67, 360 (1929). 21. COREY, E. L., Am. J. Physiol. 113, 450 (1935). 22. BRITTON, S. W., Am. J. Physiol. 95, 178 (1930). 23. SCHLOSSMANN, H., J. Physiol. (London) 92, 219 (1938). 24. PASSMORE, R., AND SCHLOSSMANN, H., J. Physiol. (London) 92, 459 (1938). 25. SCHLOSSMANN, H., Arch. exptl. Path. Pharmakol. 159, 213 (1931). 26. SOSKIN, S., AND LEVINE, R., Carbohydrate Metabolism. University of Chicago Press, 1946. 27. GUELIN-SCHEDRINA, A., Compt. rend. sot. biol. 121, 144 (1936). 28. COREY, E. L., Physiol. Zool. 5, 36 (1932).