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Hypoglycemia in the fetal alcohol syndrome in rat
Hypoglycemia In the Fetal Alcohol Syndrome In Rat Harumi Tanaka, MD, Nobuyuki Suzuki, MD and Masataka Arima, MD
As a treatable cause of central nervous system dysfunctions in the fetal alcohol syndrome, ethanolinduced hypoglycemia was studied in experimental rat models. Female Wistar rats were divided into ethanol and control groups. Before mating and during pregnancy, the ethanol group received 30% ethanol (E), or E with 20% sucrose (S) or 20% glucose (G), and the control group received water (W), or W with S or G. Pregnancies were terminated on gestational day (gd) 15, 18, and 21 by cesarean section or by spontaneous delivery. Dams and offspring were weighed and examined for several biochemical factors. Maternal blood glucose levels were higher on gd 15, but significantly lower on gd 18 and 21 in the ethanol group than in the control group. The fetal blood glucose levels were correlated with maternal blood glucose levels on gd 21. Maternal serum insulin levels were lower on gd 15 and 18 in the ethanol group than in the control group. The body and cerebral weights were significantly lower on gd 15, 18, 21 and postnatal day 1 in the ethanol offspring than in the controls. Administration of S or G with E during pregnancy resulted in no better effects on fetal biochemical development and blood glucose levels than administration of E alone. In this work we demonstrated hypoglycemia only in the late gestational and perinatal periods in experimental rat models, which may cause the high perinatal mortality and growth retardation in the fetal alcohol syndrome. Tanaka H, Suzuki N, Arima M. Hypoglycemia in the fetal alcohol syndrome in rat. Brain Dev 1982;4:97-103
The fetal alcohol syndrome (F AS) with mental retardation and midfacial anomalies has been recognized in many countries including Japan [1] . On the other hand, alcohol drinking is known to induce hypoglycemia in man and rat especially in males [2, 3]. It has been pointed out that facial malformations with anophthalmia and/or microphthalmia occur in rats borne by insulin-induced hypoglycemic mothers [4]. On the basis of the hypothesis that alcoholinduced hypoglycemia is one of the treatable From the Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for publication : December 17, 1981. Accepted for publication : January 29, 1982.
Key words: Maternal alcoholism, ethanol, hypogly· cemia, fetal development, fetal alcohol syndrome. Correspondence address: Harumi Tanaka, Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Ogawa-higashi-machi, Kodaira, Tokyo 187, Japan.
causes of the central nervous system dysfunctions of F AS, the relationship between hypoglycemia and several brain developmental factors in offspring of rat models was investigated. At the same time, the effect of additional sucrose or glucose administration to mothers together with ethanol on fetal development was studied. Materials and Methods Rat models of F AS were produced essentially as described previously [5] . Eighty-six virgin female and 23 male albino rats of the Wi star strain at 6 to 10 weeks of age were divided into ethanol and control groups. According to the treatment, two experiments were performed (Table 1). During the premating period for 91-137 days, females of the ethanol group received 30% ethanol diluted with drinking water (vol/vol) (E), and females of the control group and males received water (W). All rats of both
Table I Materials and methods Premating period
Mating period
Gest. period
Postnatal period
1. Sucrose treatment IS, 18, 21
0
:+S ~ W Sa Sr
Ea-1
~W'1
5d
tEa~ I
1
t ~ 1 tE~:~ t E+ Sa
Wa
Wa-"11 Sa
-f
Sr
-f
2. Glucose treatment
:+G W G
r-
o
Ea
L
--1
Wa..J ,-,
21d
Wa
Wa-"1 Ga--l
E: 30% ethanol, W: water, S: 20% sucrose, G: 20% glucose. Solid diet [a: ad libitum, r: restricted to 10 g/dayJ.
groups were placed on a solid diet (F-2, Funabashi Corp., Chiba, 4.41 kcalJg) ad lib. During mating male and female rats of both groups were given the solid diet with W ad lib. After successful matings confirmed by the presence of spermatozoa in vaginal smears, pregnant rats were kept on their respective diets during pregnancy as shown in Table 1. The ethanol group was divided into three groups; E and solid diet ad lib, E with 20% sucrose (S) and solid diet ad lib, and E with 20% glucose (G) and solid diet ad lib. The control group was divided into four groups; Wand solid diet ad lib, S and solid diet ad lib (Sa), or S and solid diet restricted to 10 gJday which was the same dose as that for the ethanol group (Sr), and G and solid diet ad lib . The day when vaginal spermatozoa were detected was regarded as gestational day (gd) O. Pregnancies were terminated on gd 15 (at 8 : 00 am), 18 (at 8:00 am) and 21 (at 10:00 am) by cesarean section after chloroform inhalation for 60 sec. The remaining pregnancies were terminated by spontaneous delivery. After delivery dams of both groups were given water ad lib . Pups were examined at birth and on postnatal day (pd) 5. All dams and offspring were examined for body weight, cerebral weight given by weighing brain excluding cerebellum and lower brainstem, litter size and gross abnormalities. Blood
Table 2 Effects of maternal ethanol ingestion on blood glucose, serum insulin levels, and body and cerebral weights in maternal, fetal or developing rats. gd 15
Blood glucose (mg/dl)
Serum insulin (}£U/ml) Body weight (g)
Cerebral weight (mg)
82±26(6) 98± 16(5)
gd 18 83 ±6(S) 71 ±9(4)**
gd 21
pd 1
pd5
55±16[8J 46±13[7J
101 ±15 [5J 96±30[6J
6S±13( 9) 51±13(12)**
D
W E
0
W E
D
W E
0
4.64±0.26( 9) 6.64 ±1.19 (5) 12.60±0.67(4) W 0.262 ±0 0. 23 (6) 1.47 ±0.06 (5) E 0.237±0.019(5)* 1.31 ±0.06(4)****3.38±0.91 (12)**** 5.08±1.02(7)** 12.71±1.06(3)
0
W ~ 142±11(6) E 121 ± 11 (5)***
6S ± 25 [22J 31 ± 15 [lSJ****
54 ±23 (6) 21 ± 10(5)***
32±21 (S) 21 ± 9(4)
t 77±4(5)
68±4(4)****
137±10( 9) 113 ±10(12)****
187±21(5) 147±24(5)**
369± 22(4) 436 ±36 (3)* *
W: water, E: 30% ethanol, gd: gestational day, pd: postnatal day, D: dam, 0: offspring. Data expressed as mean ± SD. Numbers in parentheses represent (the number of dams) or [the number of offspringJ used in each experiment. Significant from W; ****p < 0.01, ***p < 0.02, **p < 0.05, *p < 0.10. for g d 15 shows head weight.
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98 Brain & Development, Vol 4, No 2,1982
glucose, blood ethanol, serum insulin, DNA, RNA, protein in cerebrum, 14C-Ieucine incorporation into cerebral protein in vitro and 14C orotic acid incorporation into cerebral RNA in vivo were also measured. Maternal blood samples were collected by cardiac puncture. Fetal blood for glucose measurement was obtained from cervical vessels. Blood glucose levels were determined by Dextrostix using an Eyetone Mark 2 analyzer. Blood ethanol levels were determined with alcohol dehydrogenase and NAD [6]. Serum insulin levels were measured by a double-antibody radioimmunoassay. Extraction of DNA and RNA was based on the methods of Schmidt-ThannhauserSchneider [7, 8] and determination was done by the diphenylamine reaction and orcinal reaction or reading at wavelengths of 260 mJl and 275 mJl and calculation, respectively. Protein was determined by the method of Lowry et al [9]. 14C-leucine incorporation into cerebral protein in vitro (5 Jl Ci/lOO mg wet weight) was measured by the method of Haglid et al [10]. 14C orotic acid incorporation into cerebral RNA (1 Jl Cijfetus) was carried out by the method of Castles et al [11], 1 or 4 hrs after injection into the lateral ventricle through the right orbita. Radioactivity from 14C-leucine was expressed as total counts in protein per cerebrum, and the percent radioactivity in RNA removed from whole homogenate for 14C orotic acid. Mean significance was calculated according to the "student's" t test. Results Results for the blood glucose and serum insulin levels compared to body and cerebral weights from gd 15 to pd 5 are given in Table 2. The maternal blood glucose levels were higher on gd 15, but significantly lower on gd 18 and 21 in the ethanol group than in the control group. Higher blood glucose levels were observed in ethanol drinking female rats without fetuses after successful matings; mean values of 147, 137 and 99 mgjdl on gd 15, 18 and 21, respectively. Fetal blood glucose levels detected only on gd 21 were significantly lower in the ethanol group than in the control group. On the other hand, maternal serum insulin levels were lower on gd 15 and 18 in the ethanol group than in the control group. The body and cerebral weights were significantly lower on gd
15, 18, 21 and pd 1 in the ethanol offspring than in the controls. Blood ethanol levels at 8 : 00 am in 8 dams on gd 15 or 18 were 0 to 105 mgjdl with mean value of38 mgjdl. Table 3 summarizes the effects of additional sucrose or glucose on several factors indicating fetal development on gd 2l. Administration of S with E during pregnancy resulted in no better effect on fetal body and cerebral weights, stillbirth rate, cerebral DNA and RNA, and blood glucose levels in the living rats at 30 min after cesarean section than administration of E alone . During pregnancy ethanol contributed about 60% of the calories ingested in the ethanol group. The total calorie intake of the ethanol group was approximately 130% of that of the control group. For the Sr group, which received the same dose of solid diet as E group and the same calorie intake as the W group, good fetal development was observed compared to the E or E + S group. Administration of G with or without E during pregnancy resulted in no better effect on fetal biochemical factors such as protein, RNA and 14C orotic acid incorporation into cerebral RNA for 4 hrs and a rather worse effect on blood glucose levels, body and cerebral weights and stillbirth rate than administration of E alone or W alone. The ratio of total calories to ethanol derived calories was almost the same between sucrose and glucose treatments. In addition to the mean values, several factors for each dam and offspring are shown in Figs 1 to 3 for observing the variability in litters. In Fig 1 is shown the relationship between maternal and mean fetal blood glucose levels on gd 21 for the sucrose or glucose treatment. Fetal blood glucose levels were correlated with maternal blood glucose levels on gd 21 at least in the ethanol-treated groups. In Fig 2 are shown the fetal blood glucose levels and cerebral weights on gd 21 in the sucrose treatment experiment.. Both of the ethanol groups (E, E + S) showed lower blood glucose levels and cerebral weights than control groups and analysis of variance indicates that this difference is significant by X2 test with p < 0.005. In Fig 3 are shown the fetal total RNA in the cerebrum and 14C orotic acid incorporation into cerebral RNA on gd 21 in the glucose treatment experiment. Both of the ethanol groups (E, E + G) showed lower total RNA levels and slightly lower orotic acid incorporation ratios Tanaka et al: Hypoglycemia in FAS 99
......
Cl Cl
~
s·
Table 3 Development of fetuses of dams receiving dietary 20% sucrose or 20% glucose together with 30% ethanol for 21 days.
1. Sucrose trea tmen t
Ro b
"
B'
'l::l
Stillbirths (%)
~
;:s
......
~
.~
~
.w
...... '0
~
Blood glucose (mg/dl)
Body weight (g)
Cerebral weight (mg)
jJ.gDNA/ Cerebrum
Cerebrum 395 ± 38 (10)
~RNA/
E
22
28 ± 13 (10)
3.02 ± 1.19 (58)
115 ± 14 (44)
759 ± 56 (10)
E+S
35
25
2.18 ± 0.78 (37)
115 ± 12 (23)
744 ± 75 ( 6)
361 ± 42 ( 6)
815 ± 50 (10)**
458 ± 39 (10)****
±
18 ( 6)
W
0
72 ± 22 (10)****
4.59 ± 0.39 (64)****
141.± 14 (46)****
Sa
0
59 ± 4 ( 6)****
5.06 ± 0.75 (31)****
144 ± 13 (30)****
868 ± 47 ( 6)****
489 ± 52 ( 6)**** 487 ± 16 ( 6)****
Sr
0
61 ± 18 ( 6)****
4.83 ± 0.38 (33)****
148 ± 14 (33)****
869 ± 35 ( 6)****
Blood glucose (mg/dl)
Body weight (g)
Cerebral weight (mg)
mg protein/ Cerebrum
Cerebrum
Leucine in corp. (dpm x J04/cerebrum) 132 ± 29 (3) 259 ± 46 (3)***
2. Glucose treatment Stillbirths (%) E E+G W
G
~RNA/
Orotic acid incorp. (% of whole homog.) 11.89 ± 1.35 (10)
19 ± 16 (15)
3.55 ± 0.65 (68)
113 ± 10 (26)
7.73 ± 0.88 (21)
490 ± 48 (21)
18 ± 11 ( 6)
2.84 ± 0.45 (32)***
106 ± 8 (11)*
7.24 ± 1.06 ( 9)
464 ± 46 ( 9)
0
55 ± 29 (13)****
4 .72 ± 0.44 (56)****
131 ± 7 (29)****
8.82 ± 1.29 (18)****
541 ± 48 (18)****
12.01 ± 1.03 ( 9)
3
35 ± 18 ( 8)**
4.23 ± 0.77 (34)****
116 ± 14 (14)
8.97 ± 0.67 (13)****
535 ± 35 (13)****
12.08 ± 2.65 ( 4)
18 38
Data expressed as mean ± SD. Numbers in parentheses represent the number of fetuses . Significant from E; ****p < 0.01, ***p < 0.02, **p < 0.05, *p < 0.10. See text for details.
9.69 ± 1.38 ( 5)***
than the control groups. Discussion In our rat models, several anomalies were observed as described previously [12], and postnatal catch-up growth may be explained by the different development between man and rat. Therefore, this model can be considered as
mgldl
FAS . The evidence for the presence of hypoglycemia in F AS in man or experimental models has not been reported until now. Hypoglycemia in early pregnancy inteIferes with organogenesis in rat, which may explain the rrQldl
100
o
o
0
•
0
•• •• ••
• ~o
0
• •
#
DO
" "
•
0
•
u
0
.aen
0
J
"
o
J:I
"• • • •
°0 U--~~~·~------------~1~50~------~tw=· cerebral weight
i
-
;; 0
mg
Fig 2 Fetal blood glucose level and cerebral weight ongd 21. Key: .E, .E+S, 0 W, 0 Sa, 6 Sr. The details of groups are given in Table 1.
0
0
0 [l
[l
,.
0'1::i
•
0 ••• 0
••
mg/dl
,g
«
"
DO
CiI
DO
Fig 1 Relationship between maternal and fetal blood glucose levels on gd 21. Fetal blood glucose level is expressed as mean values for litters. Key: • E, • E + S or E + G, 0 W, 0 Sa or G, 6 Sr. The details of groups are given in Table 1.
ot •
0
0"
• • •at.~ • tIf." • • • • .~
°O~--------~OO~----------'~OO fetus
o ~o
•
310
0
o
o
o
0
0
De
0
o
100
e
• •• •• • •
0
0
0
•0 ~ • .0 • • .~ • • • •• • 0
080
[l
0
•
O~~------------~5~-----------'~O~----------~'5·h 1 hr 4hr I·e - orotic acid incorporation
Fig 3 Fetal total RNA content in cerebrum and "C· orotic acid incorporation into cerebral RNA on gd 21. Orotic acid incorpora· tion is expressed as percent radioactivity in RNA removed from whole homogenate for 1 hr and 4 hrs. Key : • E, • E + G, 0 W, 0 G. The details of groups are given in Tabll? 1.
Tanaka et al: Hypoglycemia in FAS 101
typical facial malformations. In the present study, however, we demonstrated hypoglycemia only during late pregnancy and the perinatal period in experimental raJ models, which may cause growth deficiency and high perinatal mortality in F AS. The inhibition of hepatic gluconeogenesis by ethanol is well known [13, 14]. In female rats after chronic ethanol ingestion, the liver glycogen level was decreased but the glucose concentration was not, which may suggest that peripheral glucose utilization was inhibited in the female [3] . The finding that in healthy women, moderate dose of alcohol significantly impaired glucose tolerance due to impaired glucose uptake by the tissues was also reported [15]. Our results in this study appear to indicate that the hypoglycemic status in late pregnancy through to the neonatal period is one of the causes of central nervous system dysfunctions but other manifestations in F AS were not attributed to the hypoglycemic status because of its absence in early pregnancy. Abell et a1 [16] reported that maternal hypoglycemia had a highly significant association with fetal growth retardation and that perinatal mortality was significantly increased in the presence of either hypoglycemia or hyperglycemia. The possible relationship between ethanol-induced impairment of glucose tolerance and symptoms of FAS should be studied further. In our data at 8: 00 am, there are no clear relationships between maternal ethanol levels and glucose levels or maternal body weight. Based on these results, we consider that central nervous system dysfunctions in FAS may be much closely related to ethano11eve1s than glucose levels. In order to prevent the central nervous system dysfunctions in FAS, sucrose or glucose addition was carried out. The reasons for the lack of success in this experiment may be the inhibition of glucose transport in the intestine by ethanol [17], and the continuous addition of a high concentration of sucrose or glucose throughout the gestational period. Dich et a1 [14] reported that simultaneous addition of glucose diminished the inhibition of protein synthesis by ethanol. Jones et a1 [18] demonstrated that the retarded growth of fetuses exposed to alcohol in utero is not due to impaired transfer of analogs of glucose from the maternal circulation to the fetus on gd 20. Further precise studies are needed for preven102 Brain & Development, Vol 4, No 2,1982
tion and treatment of FAS. The specific mechanism of FAS is unknown. Of those considered, impairment of protein synthesis at present seems the best documented [19]. Recently West et a1 [20] reported that prenatal exposure to ethanol alters the organization of hippocampal mossy fibers in rats, which may playa role in the mental retardation in FAS. On the other hand, the zinc concentration in the hippocampus, especially in mossy fibers, is higher than in the rest of the rat brain [21]. Flynn et a1 [22] also reported that manifestation of fetal dysmorphogenesis in alcoholic women appears to be related to the low zinc status. We also demonstrated the low zinc levels in fetal cerebrum in rat models [23] , which may lead to decreased protein synthesis and cause general growth retardation. Therefore, attention should be paid to the apparent beneficial effect of supplementary zinc treatment on fetal growth in F AS. Acknowledgments This study was supported by Grant No 81-01-07 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan.
References 1. Tanaka H, Arima M, Suzuki N. The fetal alcohol syndrome in Japan. Brain Dev (Tokyo) 1981;3: 305-11. 2. Freinkel N, Singer DL, Arky RA, Bleicher SJ, Anderson JB, Silbert CK. Alcohol hypoglycemia. 1. Carbohydrate metabolism of patients with clinical alcohol hypoglycemia and the experimental reproduction of the syndrome with pure ethanol. J Clin Invest 1963;42:1112-33. 3. Winston GW, Reitz RC. Effects of chronic ethanol ingestion on glucose homeostasis in males and females. Life Sci 1980;26:201-9. 4. Miyamoto M, Sakaguchi T, Midorikawa O. Teratogenicity of regular insulin and ultralente insulin in rats. Cong Anom (Nagoya) 1979;19 : 291-7. 5. Tanaka H, Suzuki N, Arima M. Experimental studies on the influence of male alcoholism on fetal development. Brain Dev (Tokyo) 1982;4: 7-12. 6. Lundquist F. The determination of ethyl alcohol in blood and tissues. Methods Biochem Anal 1959;7:217-51. 7. Schmidt G, Thannhauser SJ. A method for the determination of deoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J BioI Chem 1945;161:83-9.
8. Schneider WC. Phosphorus compounds in animal tissues. 3. A comparison of methods for the estimation of nucleic acids. J Bioi Chern 1946;164: 747-51. 9. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Bioi Chern 1951;193:265-75. 10. Haglid KG, Hamberger A. Cellular and subcellular distribution of protein-bound radioactivity in the tat brain during maturation after incorporation of [3H]leucine in vitro. Brain Res 1973;52:27787. 11. Castles TR, Campbell S, Gouge R, Lee CC. Nucleic acid synthesis in brains from rats tolerant to morphine analgesia. J Pharmacol Exp Ther 1972; 181:399-406. 12. Tanaka H, Suzuki N, Arirna M. The fetal alcohol syndrome in rat (in Japanese). Igaku No Ayumi (Tokyo) 1980;115 :929-932. 13. Krebs HA, Freedland RA, Hems R, Stubbs M. Inhibition of hepatic gluconeogenesis by ethanol. Biochem J 1969; 112 :117-24. 14. Dich J, Tc,6nnesen IC. Effects of ethanol, nutritional status, and composition of the incubation medium on protein synthesis in isolated rat liver parenchymal cells. Arch Biochem Biophys 1980; 204 :640-7. 15. Dornhost A, Ouyang A. Effect of alcohol on glucose tolerance. Lancet 1971;2:957-9 . 16. Abell DA. The significance of abnormal glucose
17.
18.
19.
20.
21. 22.
23.
tolerance (hyperglycemia and hypoglycemia) in pregnancy. Br J Obstet Gynaecol 1979;86 :21421. DeCastellarnau C, Moret6 M, Bolufer J. Effect of ethanol on glucose and tyrosine transport in the rat small intestine. Rev Esp FisioI1979;35:3216. Jones PJH, Leichter J, Lee M. Uptake of zinc, folate and analogs of glucose and amino acid by the rat fetus exposed to alcohol in utero. Nutr Rep Int 1981 ;24:75-83. Henderson GI, Patwardhan RV, Hoyumpa AM, Schenker S. Fetal alcohol syndrome: Overview of pathogenesis. Neurobehav Toxicol Teratol 1981 ;3:73-80. West JR, Hodges CA, Black AC. Prenatal exposure to ethanol alters the organization of hippocampal mossy fibers in rats. Science 1981; 211 :957-9. Fjerdingstad E, Danscher G, Fjerdingstad EJ. Zinc content in hippocampus and whole brain of normal rats. Brain Res 1974;79:338-42. Flynn A, Miller SI, Martier SS, Golden LN, Sokol RJ, DelVillano BC. Zinc status of pregnant alcoholic women: a determinant of fetal alcohol outcome. Lancet 1981;1:572-4. Tanaka H, Arima M, Suzuki N. The fetal alcohol syndrome. In : Proceedings of the symposium on developmental disabilities. Amsterdam: Excerpta Medica, 1982 (in press).
Tanaka et al: Hypoglycemia in F AS 103
As a treatable cause of central nervous system dysfunctions in the fetal alcohol syndrome, ethanolinduced hypoglycemia was studied in experimental rat models. Female Wistar rats were divided into ethanol and control groups. Before mating and during pregnancy, the ethanol group received 30% ethanol (E), or E with 20% sucrose (S) or 20% glucose (G), and the control group received water (W), or W with S or G. Pregnancies were terminated on gestational day (gd) 15, 18, and 21 by cesarean section or by spontaneous delivery. Dams and offspring were weighed and examined for several biochemical factors. Maternal blood glucose levels were higher on gd 15, but significantly lower on gd 18 and 21 in the ethanol group than in the control group. The fetal blood glucose levels were correlated with maternal blood glucose levels on gd 21. Maternal serum insulin levels were lower on gd 15 and 18 in the ethanol group than in the control group. The body and cerebral weights were significantly lower on gd 15, 18, 21 and postnatal day 1 in the ethanol offspring than in the controls. Administration of S or G with E during pregnancy resulted in no better effects on fetal biochemical development and blood glucose levels than administration of E alone. In this work we demonstrated hypoglycemia only in the late gestational and perinatal periods in experimental rat models, which may cause the high perinatal mortality and growth retardation in the fetal alcohol syndrome. Tanaka H, Suzuki N, Arima M. Hypoglycemia in the fetal alcohol syndrome in rat. Brain Dev 1982;4:97-103
The fetal alcohol syndrome (F AS) with mental retardation and midfacial anomalies has been recognized in many countries including Japan [1] . On the other hand, alcohol drinking is known to induce hypoglycemia in man and rat especially in males [2, 3]. It has been pointed out that facial malformations with anophthalmia and/or microphthalmia occur in rats borne by insulin-induced hypoglycemic mothers [4]. On the basis of the hypothesis that alcoholinduced hypoglycemia is one of the treatable From the Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for publication : December 17, 1981. Accepted for publication : January 29, 1982.
Key words: Maternal alcoholism, ethanol, hypogly· cemia, fetal development, fetal alcohol syndrome. Correspondence address: Harumi Tanaka, Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Ogawa-higashi-machi, Kodaira, Tokyo 187, Japan.
causes of the central nervous system dysfunctions of F AS, the relationship between hypoglycemia and several brain developmental factors in offspring of rat models was investigated. At the same time, the effect of additional sucrose or glucose administration to mothers together with ethanol on fetal development was studied. Materials and Methods Rat models of F AS were produced essentially as described previously [5] . Eighty-six virgin female and 23 male albino rats of the Wi star strain at 6 to 10 weeks of age were divided into ethanol and control groups. According to the treatment, two experiments were performed (Table 1). During the premating period for 91-137 days, females of the ethanol group received 30% ethanol diluted with drinking water (vol/vol) (E), and females of the control group and males received water (W). All rats of both
Table I Materials and methods Premating period
Mating period
Gest. period
Postnatal period
1. Sucrose treatment IS, 18, 21
0
:+S ~ W Sa Sr
Ea-1
~W'1
5d
tEa~ I
1
t ~ 1 tE~:~ t E+ Sa
Wa
Wa-"11 Sa
-f
Sr
-f
2. Glucose treatment
:+G W G
r-
o
Ea
L
--1
Wa..J ,-,
21d
Wa
Wa-"1 Ga--l
E: 30% ethanol, W: water, S: 20% sucrose, G: 20% glucose. Solid diet [a: ad libitum, r: restricted to 10 g/dayJ.
groups were placed on a solid diet (F-2, Funabashi Corp., Chiba, 4.41 kcalJg) ad lib. During mating male and female rats of both groups were given the solid diet with W ad lib. After successful matings confirmed by the presence of spermatozoa in vaginal smears, pregnant rats were kept on their respective diets during pregnancy as shown in Table 1. The ethanol group was divided into three groups; E and solid diet ad lib, E with 20% sucrose (S) and solid diet ad lib, and E with 20% glucose (G) and solid diet ad lib. The control group was divided into four groups; Wand solid diet ad lib, S and solid diet ad lib (Sa), or S and solid diet restricted to 10 gJday which was the same dose as that for the ethanol group (Sr), and G and solid diet ad lib . The day when vaginal spermatozoa were detected was regarded as gestational day (gd) O. Pregnancies were terminated on gd 15 (at 8 : 00 am), 18 (at 8:00 am) and 21 (at 10:00 am) by cesarean section after chloroform inhalation for 60 sec. The remaining pregnancies were terminated by spontaneous delivery. After delivery dams of both groups were given water ad lib . Pups were examined at birth and on postnatal day (pd) 5. All dams and offspring were examined for body weight, cerebral weight given by weighing brain excluding cerebellum and lower brainstem, litter size and gross abnormalities. Blood
Table 2 Effects of maternal ethanol ingestion on blood glucose, serum insulin levels, and body and cerebral weights in maternal, fetal or developing rats. gd 15
Blood glucose (mg/dl)
Serum insulin (}£U/ml) Body weight (g)
Cerebral weight (mg)
82±26(6) 98± 16(5)
gd 18 83 ±6(S) 71 ±9(4)**
gd 21
pd 1
pd5
55±16[8J 46±13[7J
101 ±15 [5J 96±30[6J
6S±13( 9) 51±13(12)**
D
W E
0
W E
D
W E
0
4.64±0.26( 9) 6.64 ±1.19 (5) 12.60±0.67(4) W 0.262 ±0 0. 23 (6) 1.47 ±0.06 (5) E 0.237±0.019(5)* 1.31 ±0.06(4)****3.38±0.91 (12)**** 5.08±1.02(7)** 12.71±1.06(3)
0
W ~ 142±11(6) E 121 ± 11 (5)***
6S ± 25 [22J 31 ± 15 [lSJ****
54 ±23 (6) 21 ± 10(5)***
32±21 (S) 21 ± 9(4)
t 77±4(5)
68±4(4)****
137±10( 9) 113 ±10(12)****
187±21(5) 147±24(5)**
369± 22(4) 436 ±36 (3)* *
W: water, E: 30% ethanol, gd: gestational day, pd: postnatal day, D: dam, 0: offspring. Data expressed as mean ± SD. Numbers in parentheses represent (the number of dams) or [the number of offspringJ used in each experiment. Significant from W; ****p < 0.01, ***p < 0.02, **p < 0.05, *p < 0.10. for g d 15 shows head weight.
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I
98 Brain & Development, Vol 4, No 2,1982
glucose, blood ethanol, serum insulin, DNA, RNA, protein in cerebrum, 14C-Ieucine incorporation into cerebral protein in vitro and 14C orotic acid incorporation into cerebral RNA in vivo were also measured. Maternal blood samples were collected by cardiac puncture. Fetal blood for glucose measurement was obtained from cervical vessels. Blood glucose levels were determined by Dextrostix using an Eyetone Mark 2 analyzer. Blood ethanol levels were determined with alcohol dehydrogenase and NAD [6]. Serum insulin levels were measured by a double-antibody radioimmunoassay. Extraction of DNA and RNA was based on the methods of Schmidt-ThannhauserSchneider [7, 8] and determination was done by the diphenylamine reaction and orcinal reaction or reading at wavelengths of 260 mJl and 275 mJl and calculation, respectively. Protein was determined by the method of Lowry et al [9]. 14C-leucine incorporation into cerebral protein in vitro (5 Jl Ci/lOO mg wet weight) was measured by the method of Haglid et al [10]. 14C orotic acid incorporation into cerebral RNA (1 Jl Cijfetus) was carried out by the method of Castles et al [11], 1 or 4 hrs after injection into the lateral ventricle through the right orbita. Radioactivity from 14C-leucine was expressed as total counts in protein per cerebrum, and the percent radioactivity in RNA removed from whole homogenate for 14C orotic acid. Mean significance was calculated according to the "student's" t test. Results Results for the blood glucose and serum insulin levels compared to body and cerebral weights from gd 15 to pd 5 are given in Table 2. The maternal blood glucose levels were higher on gd 15, but significantly lower on gd 18 and 21 in the ethanol group than in the control group. Higher blood glucose levels were observed in ethanol drinking female rats without fetuses after successful matings; mean values of 147, 137 and 99 mgjdl on gd 15, 18 and 21, respectively. Fetal blood glucose levels detected only on gd 21 were significantly lower in the ethanol group than in the control group. On the other hand, maternal serum insulin levels were lower on gd 15 and 18 in the ethanol group than in the control group. The body and cerebral weights were significantly lower on gd
15, 18, 21 and pd 1 in the ethanol offspring than in the controls. Blood ethanol levels at 8 : 00 am in 8 dams on gd 15 or 18 were 0 to 105 mgjdl with mean value of38 mgjdl. Table 3 summarizes the effects of additional sucrose or glucose on several factors indicating fetal development on gd 2l. Administration of S with E during pregnancy resulted in no better effect on fetal body and cerebral weights, stillbirth rate, cerebral DNA and RNA, and blood glucose levels in the living rats at 30 min after cesarean section than administration of E alone . During pregnancy ethanol contributed about 60% of the calories ingested in the ethanol group. The total calorie intake of the ethanol group was approximately 130% of that of the control group. For the Sr group, which received the same dose of solid diet as E group and the same calorie intake as the W group, good fetal development was observed compared to the E or E + S group. Administration of G with or without E during pregnancy resulted in no better effect on fetal biochemical factors such as protein, RNA and 14C orotic acid incorporation into cerebral RNA for 4 hrs and a rather worse effect on blood glucose levels, body and cerebral weights and stillbirth rate than administration of E alone or W alone. The ratio of total calories to ethanol derived calories was almost the same between sucrose and glucose treatments. In addition to the mean values, several factors for each dam and offspring are shown in Figs 1 to 3 for observing the variability in litters. In Fig 1 is shown the relationship between maternal and mean fetal blood glucose levels on gd 21 for the sucrose or glucose treatment. Fetal blood glucose levels were correlated with maternal blood glucose levels on gd 21 at least in the ethanol-treated groups. In Fig 2 are shown the fetal blood glucose levels and cerebral weights on gd 21 in the sucrose treatment experiment.. Both of the ethanol groups (E, E + S) showed lower blood glucose levels and cerebral weights than control groups and analysis of variance indicates that this difference is significant by X2 test with p < 0.005. In Fig 3 are shown the fetal total RNA in the cerebrum and 14C orotic acid incorporation into cerebral RNA on gd 21 in the glucose treatment experiment. Both of the ethanol groups (E, E + G) showed lower total RNA levels and slightly lower orotic acid incorporation ratios Tanaka et al: Hypoglycemia in FAS 99
......
Cl Cl
~
s·
Table 3 Development of fetuses of dams receiving dietary 20% sucrose or 20% glucose together with 30% ethanol for 21 days.
1. Sucrose trea tmen t
Ro b
"
B'
'l::l
Stillbirths (%)
~
;:s
......
~
.~
~
.w
...... '0
~
Blood glucose (mg/dl)
Body weight (g)
Cerebral weight (mg)
jJ.gDNA/ Cerebrum
Cerebrum 395 ± 38 (10)
~RNA/
E
22
28 ± 13 (10)
3.02 ± 1.19 (58)
115 ± 14 (44)
759 ± 56 (10)
E+S
35
25
2.18 ± 0.78 (37)
115 ± 12 (23)
744 ± 75 ( 6)
361 ± 42 ( 6)
815 ± 50 (10)**
458 ± 39 (10)****
±
18 ( 6)
W
0
72 ± 22 (10)****
4.59 ± 0.39 (64)****
141.± 14 (46)****
Sa
0
59 ± 4 ( 6)****
5.06 ± 0.75 (31)****
144 ± 13 (30)****
868 ± 47 ( 6)****
489 ± 52 ( 6)**** 487 ± 16 ( 6)****
Sr
0
61 ± 18 ( 6)****
4.83 ± 0.38 (33)****
148 ± 14 (33)****
869 ± 35 ( 6)****
Blood glucose (mg/dl)
Body weight (g)
Cerebral weight (mg)
mg protein/ Cerebrum
Cerebrum
Leucine in corp. (dpm x J04/cerebrum) 132 ± 29 (3) 259 ± 46 (3)***
2. Glucose treatment Stillbirths (%) E E+G W
G
~RNA/
Orotic acid incorp. (% of whole homog.) 11.89 ± 1.35 (10)
19 ± 16 (15)
3.55 ± 0.65 (68)
113 ± 10 (26)
7.73 ± 0.88 (21)
490 ± 48 (21)
18 ± 11 ( 6)
2.84 ± 0.45 (32)***
106 ± 8 (11)*
7.24 ± 1.06 ( 9)
464 ± 46 ( 9)
0
55 ± 29 (13)****
4 .72 ± 0.44 (56)****
131 ± 7 (29)****
8.82 ± 1.29 (18)****
541 ± 48 (18)****
12.01 ± 1.03 ( 9)
3
35 ± 18 ( 8)**
4.23 ± 0.77 (34)****
116 ± 14 (14)
8.97 ± 0.67 (13)****
535 ± 35 (13)****
12.08 ± 2.65 ( 4)
18 38
Data expressed as mean ± SD. Numbers in parentheses represent the number of fetuses . Significant from E; ****p < 0.01, ***p < 0.02, **p < 0.05, *p < 0.10. See text for details.
9.69 ± 1.38 ( 5)***
than the control groups. Discussion In our rat models, several anomalies were observed as described previously [12], and postnatal catch-up growth may be explained by the different development between man and rat. Therefore, this model can be considered as
mgldl
FAS . The evidence for the presence of hypoglycemia in F AS in man or experimental models has not been reported until now. Hypoglycemia in early pregnancy inteIferes with organogenesis in rat, which may explain the rrQldl
100
o
o
0
•
0
•• •• ••
• ~o
0
• •
#
DO
" "
•
0
•
u
0
.aen
0
J
"
o
J:I
"• • • •
°0 U--~~~·~------------~1~50~------~tw=· cerebral weight
i
-
;; 0
mg
Fig 2 Fetal blood glucose level and cerebral weight ongd 21. Key: .E, .E+S, 0 W, 0 Sa, 6 Sr. The details of groups are given in Table 1.
0
0
0 [l
[l
,.
0'1::i
•
0 ••• 0
••
mg/dl
,g
«
"
DO
CiI
DO
Fig 1 Relationship between maternal and fetal blood glucose levels on gd 21. Fetal blood glucose level is expressed as mean values for litters. Key: • E, • E + S or E + G, 0 W, 0 Sa or G, 6 Sr. The details of groups are given in Table 1.
ot •
0
0"
• • •at.~ • tIf." • • • • .~
°O~--------~OO~----------'~OO fetus
o ~o
•
310
0
o
o
o
0
0
De
0
o
100
e
• •• •• • •
0
0
0
•0 ~ • .0 • • .~ • • • •• • 0
080
[l
0
•
O~~------------~5~-----------'~O~----------~'5·h 1 hr 4hr I·e - orotic acid incorporation
Fig 3 Fetal total RNA content in cerebrum and "C· orotic acid incorporation into cerebral RNA on gd 21. Orotic acid incorpora· tion is expressed as percent radioactivity in RNA removed from whole homogenate for 1 hr and 4 hrs. Key : • E, • E + G, 0 W, 0 G. The details of groups are given in Tabll? 1.
Tanaka et al: Hypoglycemia in FAS 101
typical facial malformations. In the present study, however, we demonstrated hypoglycemia only during late pregnancy and the perinatal period in experimental raJ models, which may cause growth deficiency and high perinatal mortality in F AS. The inhibition of hepatic gluconeogenesis by ethanol is well known [13, 14]. In female rats after chronic ethanol ingestion, the liver glycogen level was decreased but the glucose concentration was not, which may suggest that peripheral glucose utilization was inhibited in the female [3] . The finding that in healthy women, moderate dose of alcohol significantly impaired glucose tolerance due to impaired glucose uptake by the tissues was also reported [15]. Our results in this study appear to indicate that the hypoglycemic status in late pregnancy through to the neonatal period is one of the causes of central nervous system dysfunctions but other manifestations in F AS were not attributed to the hypoglycemic status because of its absence in early pregnancy. Abell et a1 [16] reported that maternal hypoglycemia had a highly significant association with fetal growth retardation and that perinatal mortality was significantly increased in the presence of either hypoglycemia or hyperglycemia. The possible relationship between ethanol-induced impairment of glucose tolerance and symptoms of FAS should be studied further. In our data at 8: 00 am, there are no clear relationships between maternal ethanol levels and glucose levels or maternal body weight. Based on these results, we consider that central nervous system dysfunctions in FAS may be much closely related to ethano11eve1s than glucose levels. In order to prevent the central nervous system dysfunctions in FAS, sucrose or glucose addition was carried out. The reasons for the lack of success in this experiment may be the inhibition of glucose transport in the intestine by ethanol [17], and the continuous addition of a high concentration of sucrose or glucose throughout the gestational period. Dich et a1 [14] reported that simultaneous addition of glucose diminished the inhibition of protein synthesis by ethanol. Jones et a1 [18] demonstrated that the retarded growth of fetuses exposed to alcohol in utero is not due to impaired transfer of analogs of glucose from the maternal circulation to the fetus on gd 20. Further precise studies are needed for preven102 Brain & Development, Vol 4, No 2,1982
tion and treatment of FAS. The specific mechanism of FAS is unknown. Of those considered, impairment of protein synthesis at present seems the best documented [19]. Recently West et a1 [20] reported that prenatal exposure to ethanol alters the organization of hippocampal mossy fibers in rats, which may playa role in the mental retardation in FAS. On the other hand, the zinc concentration in the hippocampus, especially in mossy fibers, is higher than in the rest of the rat brain [21]. Flynn et a1 [22] also reported that manifestation of fetal dysmorphogenesis in alcoholic women appears to be related to the low zinc status. We also demonstrated the low zinc levels in fetal cerebrum in rat models [23] , which may lead to decreased protein synthesis and cause general growth retardation. Therefore, attention should be paid to the apparent beneficial effect of supplementary zinc treatment on fetal growth in F AS. Acknowledgments This study was supported by Grant No 81-01-07 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan.
References 1. Tanaka H, Arima M, Suzuki N. The fetal alcohol syndrome in Japan. Brain Dev (Tokyo) 1981;3: 305-11. 2. Freinkel N, Singer DL, Arky RA, Bleicher SJ, Anderson JB, Silbert CK. Alcohol hypoglycemia. 1. Carbohydrate metabolism of patients with clinical alcohol hypoglycemia and the experimental reproduction of the syndrome with pure ethanol. J Clin Invest 1963;42:1112-33. 3. Winston GW, Reitz RC. Effects of chronic ethanol ingestion on glucose homeostasis in males and females. Life Sci 1980;26:201-9. 4. Miyamoto M, Sakaguchi T, Midorikawa O. Teratogenicity of regular insulin and ultralente insulin in rats. Cong Anom (Nagoya) 1979;19 : 291-7. 5. Tanaka H, Suzuki N, Arima M. Experimental studies on the influence of male alcoholism on fetal development. Brain Dev (Tokyo) 1982;4: 7-12. 6. Lundquist F. The determination of ethyl alcohol in blood and tissues. Methods Biochem Anal 1959;7:217-51. 7. Schmidt G, Thannhauser SJ. A method for the determination of deoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J BioI Chem 1945;161:83-9.
8. Schneider WC. Phosphorus compounds in animal tissues. 3. A comparison of methods for the estimation of nucleic acids. J Bioi Chern 1946;164: 747-51. 9. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Bioi Chern 1951;193:265-75. 10. Haglid KG, Hamberger A. Cellular and subcellular distribution of protein-bound radioactivity in the tat brain during maturation after incorporation of [3H]leucine in vitro. Brain Res 1973;52:27787. 11. Castles TR, Campbell S, Gouge R, Lee CC. Nucleic acid synthesis in brains from rats tolerant to morphine analgesia. J Pharmacol Exp Ther 1972; 181:399-406. 12. Tanaka H, Suzuki N, Arirna M. The fetal alcohol syndrome in rat (in Japanese). Igaku No Ayumi (Tokyo) 1980;115 :929-932. 13. Krebs HA, Freedland RA, Hems R, Stubbs M. Inhibition of hepatic gluconeogenesis by ethanol. Biochem J 1969; 112 :117-24. 14. Dich J, Tc,6nnesen IC. Effects of ethanol, nutritional status, and composition of the incubation medium on protein synthesis in isolated rat liver parenchymal cells. Arch Biochem Biophys 1980; 204 :640-7. 15. Dornhost A, Ouyang A. Effect of alcohol on glucose tolerance. Lancet 1971;2:957-9 . 16. Abell DA. The significance of abnormal glucose
17.
18.
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20.
21. 22.
23.
tolerance (hyperglycemia and hypoglycemia) in pregnancy. Br J Obstet Gynaecol 1979;86 :21421. DeCastellarnau C, Moret6 M, Bolufer J. Effect of ethanol on glucose and tyrosine transport in the rat small intestine. Rev Esp FisioI1979;35:3216. Jones PJH, Leichter J, Lee M. Uptake of zinc, folate and analogs of glucose and amino acid by the rat fetus exposed to alcohol in utero. Nutr Rep Int 1981 ;24:75-83. Henderson GI, Patwardhan RV, Hoyumpa AM, Schenker S. Fetal alcohol syndrome: Overview of pathogenesis. Neurobehav Toxicol Teratol 1981 ;3:73-80. West JR, Hodges CA, Black AC. Prenatal exposure to ethanol alters the organization of hippocampal mossy fibers in rats. Science 1981; 211 :957-9. Fjerdingstad E, Danscher G, Fjerdingstad EJ. Zinc content in hippocampus and whole brain of normal rats. Brain Res 1974;79:338-42. Flynn A, Miller SI, Martier SS, Golden LN, Sokol RJ, DelVillano BC. Zinc status of pregnant alcoholic women: a determinant of fetal alcohol outcome. Lancet 1981;1:572-4. Tanaka H, Arima M, Suzuki N. The fetal alcohol syndrome. In : Proceedings of the symposium on developmental disabilities. Amsterdam: Excerpta Medica, 1982 (in press).
Tanaka et al: Hypoglycemia in F AS 103