Effect of maternal diabetes and dietary copper on fetal development in rats

Reproductive Toxicology,Vol. 7, pp. 589-598, 1993 Printedin the U.S.A. All fightsreserved.

0890-6238/93$6.00 + .00 Copyright© 1993PergamonPress Ltd.

• Original Contribution

EFFECT OF MATERNAL DIABETES AND DIETARY COPPER FETAL DEVELOPMENT IN RATS

ON

MARGARET A. JANKOWSKI, JANET Y. URIU-HARE, ROBERT B. RUCKER, a n d CARL L. KEEN D e p a r t m e n t o f N u t r i t i o n , U n i v e r s i t y o f C a l i f o r n i a - D a v i s , D a v i s , California

Abstract m To test whether diabetes associated alterations in copper metabolism contribute to diabetesinduced teratogenicity in rats, pregnancy outcome was compared between diabetic and nondiabetic rats fed either a copper adequate (12/zg/g diet) or low copper diet (1/zg/g diet). The dietary regimen was begun two weeks prior to mating and continued throughout pregnancy. To facilitate the reduction of maternal copper stores in the low copper groups, the low copper diet was supplemented with a copper chelator, triethylenetetraamine, at 1% for one week; the chelator was removed from the diet one week prior to mating. Pregnancy was terminated on gestation day 20. Maternal and fetal tissues were assessed for copper concentrations, the activities of the cuproenzymes copper, zinc superoxide dismutase and cernloplasmin, and the copper binding protein metallothionein. Dams fed the low copper diet had low tissue copper concentrations, and low plasma ceruloplasrain and erythrocyte superoxide dismutase activities compared to copper-adequate dams. Fetuses in the low copper groups were characterized by low fiver copper concentrations. Gross structural and skeletal anomalies were only observed in the diabetic groups; maternal copper intake did not influence the frequency of these anomalies. However, fetuses in the low-copper nondiabetic group, and both diabetic groups, were characterized by low liver copper, zinc superoxide dismutase activity suggesting that fetal copper metabolism was influenced by both copper intake and diabetes. Key Words: copper; diabetes; development; pregnancy; superoxide dismutase; metallothionein; rats; zinc.

INTRODUCTION

pregnancy (3). For this reason, there has been interest in the idea that in addition to carbohydrate intolerance, other diabetic-related factors may be involved, such as alterations in the metabolism of essential nutrients (4-6). It is well recognized that magnesium (Mg), zinc (Zn), and copper (Cu) metabolism can be altered with diabetes (7-9), and it has been suggested that these alterations may contribute to diabetes-associated teratogenicity (10,11). While there have been reports concerning relationships between diabetesinduced alterations in Zn and Mg metabolism and developmental defects (11-13), there has been a paucity of work on associations between pregnancy outcome and diabetes-induced alterations in Cu metabolism. Streptozotocin-induced pregnant diabetic rat dams are characterized by high liver and kidney Cu concentrations (12,13), and fetuses from these dams can have low tissue Cu concentrations (12). Low Cu concentrations also occur in fetuses from Cudeficient dams (14). It is known that maternal Cu

An impressive reduction in diabetes associated pregnancy complications has occurred as a consequence of insulin therapy during the last two decades. However, the incidence of congenital malformations in infants from diabetic women continues at 8 to 10%, a value 2 to 3 times that of the nondiabetic population (1,2). This high risk for fetal abnormalities occurs in diabetic women even when they are characterized by relatively good blood glucose control throughout

This work was supported in part by National Institutes of Health (NIH) grants HD-26777 (CLK/RBR), NIH Individual National Research Service Award HD-07241 (JYUH), and a USDA Food and Agricultural Sciences National Needs Graduate Fellowship (MAJ). Address correspondence to Carl L. Keen, Nutrition Department, University of California-Davis, Davis, California, 956168669. Received 23 March 1993; Revision received 18 June 1993; Accepted 24 June 1993. 589

590

Reproductive Toxicology

deficiency can be teratogenic in many species including humans, cattle, sheep, and rats depending on the length and severity of the deficiency (15,16). In addition, several types of developmental abnormalities associated with maternal diabetes can occur as a consequence of maternal Cu deficiency (16). Consequently, the suggestion that marginal Cu deficiency may be a potential problem in some human populations (17), led us to ask if a synergism exists between maternal Cu deficiency and diabetes with respect to the induction of fetal anomalies. Data obtained in the current study show that under the conditions used, maternal diabetes had a more deleterious effect on fetal development than dietary Cu restriction. The data also show that the regulation of fetal cuproenzymes can be influenced by maternal diabetes.

MATERIALS A N D METHODS

Volume 7, Number 6, 1993 Table 1. Diet Components Spray dried egg white Corn starch Cerelose Corn oil Mineral mixa Cellulose Vitamin mixb

Amount g/kg diet 210 200 395 80 60 40 15

aLow Cu diets did not contain CuSO4 - 5H20. Mineral mix supplied the following(mmol/kgdiet): CaCO3 143.2, K2HPO4110.6, NaC144.1, MgSO432.9, CaHPO426.5, FeSO4 • H20 2.2, KI 0.07, ZnCO3 0.46, CuSO4 • 5H20 0.19, CrK(SO4)2 • 12H204.8 × 102, Mn(SO4h • 5H20 0.55, Na2SeO36.0 x 10-4. bVitamin mix supplied the following(mg/kg diet): Ca-pantothenate 37.5, thiamin-HCl22.5, pyridoxine-HC122.5, nicotinicacid 22.5, menadione 18.75, riboflavin7.5, p-aminobenzoicacid 7.5, folic acid 0.45, biotin 3.9, all-rac-a-tocopheryl acetate (Rovimix E-50) 178.5,retinylpalmitate (RovimixA-650)40.95, cholecalciferol (RovimixAD3 A650/D325) 3.45, vitamin B-12 (Merck 12 + mannitol) 22.5, choline chloride (70% solution) 1072.5, inositol 375, ascorbic acid as a preservative for the vitamin mixture 75. Rovimix was purchased from Hoffman-LaRoche(Nutley, NJ); reagents were purchased from Merck (Rahway, NJ).

Animals Virgin Sprague Dawley rats, 190 to 220 g, (Charles River, Wilmington, MA) were housed individually in suspended stainless steel cages maintained in a room at constant temperature (22 °C) and light (12/12 h light/dark cycle). After five days adaptation to the purified 12/xg Cu/g (0.19/.~mol/g) control diet, animals were assigned to a diabetic or nondiabetic group based on body weight. Diabetes was induced by two or three subcutaneous injections of increasing concentrations of streptozotocin (STZ; Sigma, St. Louis, MO; 38 to 45 mg/kg body weight) in 0.1 M citrate buffer, pH 4.5. Control animals were injected with an equal volume of citrate buffer (10). Animals with 6 h fasting blood glucose concentrations >- 13 mM five days after injection(s) were considered diabetic. Glucose concentration was measured (Beckman Glucose Analyzer II, Fullerton, CA) using plasma obtained from tail vein blood samples after being centrifuged at 1,500 × g, 4 °C, for 15 min. Diabetic and nondiabetic animals were assigned to the Cu adequate or a low Cu (I.0 /zg Cu/g; 0.016 /xmol/g), diet based on body weight. The level o f dietary Cu in the Cu adequate diet is consistent with the National Research Council's recommendation for this nutrient for pregnant rats (18). Animals in the low Cu group were initially fed the low Cu diet supplemented with 1% (wt/wt) triethylenetetraamine (TETA; Aldrich Chemical Company, Milwaukee, WI) for 5 days; for the remainder of the study they were fed the low Cu diet without TETA. This dietary treatment has been shown to result in suboptimal Cu status in rats as evidenced

by low liver and plasma Cu concentrations (19). The control groups were fed the Cu adequate diet throughout the study. The detailed composition of the diet is given in Table 1. Fourteen days after diabetic diagnosis, or blood glucose analysis in the nondiabetic group, rats were mated overnight with males of the same strain. Males were fed a stock commercial rodent chow (Ralston Purina #577) with the exception of the night they were mated when they had access to the purified diet. The presence of copulatory plugs the following morning was considered evidence of mating and designated gestation day 0. Maternal food intake and weight gain were recorded daily throughout gestation. On gestation day 20, dams were anesthetized with methoxyflurane (Metafane TM,Pitman-Moore, Inc., Washington Crossing, NJ), and killed by exsanguination following cardiac puncture. Maternal liver was perfused with 0.15 M saline, organs were removed, frozen in liquid nitrogen, and stored at - 7 0 °C until analyzed. Following maternal hematocrit analysis, blood was centrifuged at 1,500 × g, 4 °C for 15 min. Plasma and packed erythrocytes were collected and frozen separately at - 7 0 °C until analyzed.

Insulin analysis Plasma insulin concentrations were determined according to the methods of Yalow and Berson as modified by Desbuquois and Aurbach (20,21).

Diabetes and Cu metabolism • M. A. JANKOWSKIET AL.

Teratologic analysis Following laparotomy, the uterus was removed, the implantation sites counted, and the numbers of resorptions and live fetuses were recorded. Live fetuses were removed, examined for external malformations, sexed, measured for crown-rump length, and weighed. Placentas were removed, cleaned, and weighed. Every other fetus was fixed in 10% formalin for subsequent examination of internal tissues by a modified version of Wilson's cross section technique (22). The remaining fetuses were eviscerated and stored in 95% ethanol for subsequent staining with alizarin red S and alcian blue for skeletal analysis (23). Each skeleton was independently examined by two investigators who were unaware of the treatment group assignment at the time of analysis. Eviscerated fetal tissues were frozen in liquid nitrogen and stored at - 7 0 °C for further analysis.

591

tion at room temperature, the cells were centrifuged at 600 x g for 15 min at 4 °C. Lysate was removed and stored at - 7 0 °C until analyzed.

Superoxide dismutase (SOD) CuZnSOD activity was measured as a functional assessment of the tissue Cu concentrations; CuZnSOD activity is typically reduced by Cu deficiency and unaffected by Zn deficiency (25,26). Total SOD activity in maternal red blood cell hemoglobin-free lysate (27) and fetal liver was determined by the method of Marklund and Marklund (28). Cyanide sensitive SOD activity (designated MnSOD) was determined in fetal liver. CuZnSOD activity is defined as total SOD activity minus MnSOD activity. One unit of SOD activity was defined as the amount of sample needed to inhibit the auto-oxidation of pyrogallol by 50%. Tissue SOD activity is expressed as Units/mg protein. Red blood cell SOD (RBC SOD) activity is expressed as Units/mg hemoglobin.

Trace element analysis Aliquots of maternal liver and kidney, and one to two fetal livers per litter were wet ashed with concentrated ultrapure HNO3 (Fisher Scientific, Fair Lawn, NJ) as described by Clegg et al. (24). The Cu, Zn, manganese (Mn), and iron (Fe) concentrations in the ashed samples were determined using flame atomic absorption spectrophotometry, (AAS; Model 551, Wilmington, MA). Preparations of National Bureau of Standards bovine liver standard, (SRM bovine liver 1577a, Gaithersburg, MD) and appropriate blanks were processed simultaneously with each batch of samples to ensure accuracy of analysis. Zn, Mn, and Fe concentrations were determined because altered concentrations of these elements were observed in previous studies using this animal model. Mineral concentrations are shown as /zmol/g tissue wet weight.

Tissue preparation Maternal liver and kidney, and pooled fetal livers were homogenized with a Teflon tissue homogenizer in 0.25 M sucrose, 10 mM Tris buffer, pH 7.4. For the metallothionein assay, whole homogenate was centrifuged at 18,000 x g for 20 min at 4 °C. The supernate was removed and stored at - 7 0 °C until analyzed. For superoxide dismutase activity assays, whole homogenate was sonicated in an ice bath followed by centrifugation at 10,000 x g for 30 min at 4 °C. The supernate was removed and stored at - 7 0 °C until analyzed.

Erythrocyte lysate preparation An equal volume of deionized distilled H20 was added to red blood cells. Following a 15 min incuba-

Ceruloplasmin (Cp) Plasma Cp activity was measured based on the disappearance of o-dianisidine dihydrochloride (29). Plasma Cp is expressed as Units/L plasma.

Metallothionein (MT) The concentrations of the Cu and Zn binding protein, MT, were determined in maternal liver and kidney, and fetal liver based on the cadmium-heme saturation method using cadmium (Cd) in the form of CdCI2 (30). Cd concentration was determined using flame atomic absorption spectrophotometry, (AAS; Model 551 Instrumentation Laboratory, Wilmington, MA). MT concentration was calculated assuming 6 moles of Cd binds to 1 mole of metallothionein.

Protein quantification Protein concentrations were determined using a Bio Rad protein assay kit based on the Bradford method (31).

Hemoglobin (Hb) determination Erythrocyte lysate Hb concentrations were determined using a Sigma No. 525 Hb concentration kit (Sigma, St. Louis, MO).

Statistical analysis Analysis of variance was determined by one and two way ANOVA. Significant differences between groups were determined by Duncan's multiple range test. Statistical analyses were performed using SAS software (32). A P-value of - 0.05 was considered significant. Data are shown as mean --- SEM unless otherwise specified.

Reproductive Toxicology

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Volume 7, Number 6, 1993

Table 2. Influence of maternal diabetes and dietary Cu intake on maternal blood parameters Treatment Nondiabetic Adequate Cu Low Cu (7) (8) Plasma glucose (mM) -> 14 days pregestation gestation day 0 gestation day 20 Plasma insulin (pM) gestation day 20 Hematocrit (%)

7.7 - 0.1 a 8.2 ± 0.3a 6.9 --- 0.3a

7.2 8.6 ± 7.0 ±

284 ± 53a 34.6 ± 0.9a

Diabetic Adequate Cu (10)

Low Cu (10)

0.P 0.2 a 0.1 a

22.4 --- 0.9b 33.7 +-- 1.7b 31.8 ± 2.9b

22.3 ± 1.0b 34.6 ± 1.7b 34.9 ± 1.4b

420 --+ 114a 34.2 ± 0.6a

43 ± 6b 37.2 ± 0.6b

40 --- 5b 36.1 ± 0.6ab

Values are expressed as mean ± SEM. Values in a row with different superscript are different at P -< 0.05.

RESULTS

Maternal o u t c o m e B l o o d g l u c o s e m e a s u r e m e n t s t a k e n p r i o r to pregnancy, and on gestation day 0 and gestation day 20 c o n f i r m e d t h a t t h e S T Z t r e a t e d a n i m a l s w e r e d i a b e t i c ( T a b l e 2). I n a d d i t i o n , d i a b e t i c d a m s h a d 5 to 10 f o l d l o w e r p l a s m a i n s u l i n c o n c e n t r a t i o n s o n g e s t a t i o n d a y 20 t h a n n o n d i a b e t i c a n i m a l s . D i e t a r y C u d i d n o t a f f e c t b l o o d g l u c o s e o r p l a s m a insulin.

H e m a t o c r i t v a l u e s w e r e h i g h e r in d i a b e t i c - C u a d e q u a t e d a m s c o m p a r e d to n o n d i a b e t i c d a m s . D a i l y f o o d i n t a k e in t h e d i a b e t i c g r o u p s w a s n e a r l y t w i c e t h a t in t h e n o n d i a b e t i c g r o u p s ( F i g u r e 1). F o o d c o n s u m p t i o n w a s i n d e p e n d e n t o f d i e t a r y C u in n o n d i a b e t i c a n i m a l s , b u t in t h e d i a b e t i c g r o u p s o n g e s t a t i o n d a y s 1, 5, a n d 6, d i a b e t i c - l o w C u d a m s consumed less food than diabetic-Cu adequate d a m s . D e s p i t e h y p e r p h a g i a in t h e d i a b e t i c g r o u p , c u m u l a t i v e w e i g h t g a i n w a s l e s s in t h e d i a b e t i c ani-

60 A

4)

50

r" m

"0

40

0 0 IJ. m

eo r.

30

J,_.

4)

:E

20

10 0

5

10

15

20

Days of Gestation Fig. 1. Maternal daily food intake throughout gestation. • nondiabetic-Cu adequate, • nondiabetic-low Cu, [] diab e t i c - C u adequate, A diabetic-low Cu.

Diabetes and Cu metabolism • M. A. JAN~owsI~II~TAL.

593

~D O ~D >v A

m

m._

E =E

100,]

r,.,) ~ C

:Z o,

o

5

10

15

20

Days of Gestation Fig. 2. Maternal daily mean cumulative weight gain throughout gestation, t nondiabetic-Cu adequate, & nondiabetic-low Cu, 1~ diabetic-Cu adequate, A diabetic-low Cu.

mals than in the nondiabetic dams after gestation day 4 (Figure 2). Dietary Cu did not affect weight gain in the diabetic groups. In the nondiabetic animals low dietary Cu was associated with increased weight gain from gestation days 5 though 10. Pregnancy outcome Overall, diabetes had a detrimental affect on pregnancy outcome; diabetic dams had fewer implantantion sites and live births, and smaller fetuses with larger placentas than nondiabetic dams (Table 3). Diabetic dams had more resorptions than nondiabetic-low Cu dams, but had a similar incidence of resorptions as the Cu adequate dams. Gross external malformations were only noted in the diabetic groups; one case each of ectopic cordis, missing tail, and micrognathia were observed in the diabetic-low Cu group fetuses and one case of exencephaly was noted in the diabetic-Cu adequate group. Diabetes was associated with less fetal skeletal ossification and a higher incidence of skeletal variation (Tables 3 and 4). Maternal dietary Cu intake did not affect these parameters. Maternal tissue trace elements/metalloproteins Low Cu dams had lower liver and kidney Cu concentrations than their respective dietary controls (Table 5). Diabetic-Cu adequate dams had higher liver and kidney Cu concentrations than the nondia-

betic-Cu adequate animals, whereas similar liver and kidney Cu values were observed in the low Cu groups. Diabetic dams had higher liver and kidney Zn concentrations than nondiabetic animals. Regardless of dietary Cu intake, liver Mn concentrations in the diabetic dams were higher than in nondiabetic dams; kidney Mn concentrations were similar among the groups. Diabetes resulted in higher liver and kidney Fe concentrations in Cu adequate animals compared to nondiabetic-Cu adequate animals. There was no effect of dietary Cu on liver Fe concentrations. Liver and kidney MT concentrations were higher in the diabetic dams than in the nondiabetic dams regardless of dietary Cu (Table 5). Within the diabetic groups low Cu dams had lower liver MT concentrations than Cu adequate dams. Liver MT concentrations were not significantly affected by dietary Cu in the nondiabetic animals. Diabetic kidney MT levels were not influenced by dietary Cu, but kidney MT concentrations were higher in nondiabetic-Cu adequate dams than nondiabetic-low Cu dams. Both diabetes and dietary Cu affected plasma trace element concentrations (Table 6). Low Cu dams had low plasma Cu concentrations; diabetic-Cu adequate dams had lower plasma Cu concentrations than nondiabetic-Cu adequate dams. Plasma Fe levels were higher in diabetic-Cu ade-

Reproductive Toxicology

594 T a b l e 3. I n f l u e n c e

of maternal

diabetes

V o l u m e 7, N u m b e r 6, 1993

and dietary Cu intake on reproductive

outcome

Treatment Nondiabetic Adequate Cu L i t t e r s (no.) I m p l a n t a t i o n s i t e s (no.) L i v e p u p s (no.) F e t a l w e i g h t (g) Fetal crown-rump

17.57 15.86 3.61 3.84

l e n g t h (cm) P l a c e n t a w e i g h t (g) R e s o r p t i o n s (no.) Total resorption(%) T o t a l r e s o r p t i o n s / t o t a l i m p l a n t a t i o n sites* Total gross malformations/live fetuses Fetal skeletal ossification sites P u p s e x a m i n e d (no.) R i b s (no.) S t e r n u m (no.) C a u d a l v e r t e b r a e (no.) M e t a c a r p a l s (no.) A n t e r i o r p h a l a n g e s (no.)

7 -4- 0.92 a --- 0.88 a -+ 0.12 a --- 0.03 a

8 -+ -+ +-+

17.50 17.00 3.65 3.90

0.44 + 0.01 a 1.71 -+ 0.64 ab 9.4 -+ 3.3 ab 12/123 a 0/111

13.00 4.61 5.53 3.30 0.05

Diabetic Low Cu

0.65 a 0.78 a 0.07 a 0.03 a

0.40 + 0.01 a 0.50 -+ 0.27 t 3.0 + - 1 . 6 b 4/140 t 0/136

86 -+ 0.00 -+ 0.25 a -+ 0.24 a -+ 0.08 a -+ 0.04 a

13.00 4.12 5.48 3.18 0.00

102 -- 0.00 -+ 0.21 a -----0.10 a -+ 0.05 a --- 0.00 t

Adequate Cu

13.80 10.90 2.63 3.43

10 -+ 4++-

Low Cu

0.94 b 0.861 0.07 t 0.05 c

14.10 11.80 2.87 3.58

0.55 --+ 0.03 t 2.90 -+ 0.64 a 20.5 -+ 3.9 a 29/138c 1/109

13.00 1.58 3.88 2.93 0.00

10 - 0.62 t - 0.55 t - 0.09 t --+-0.04 t

0.61 --+ 0.04 b 2.30 -+ 0.47 a 16.0 -+ 3.0 a 23/141ac 3/118

81 -+ 0.00 -+ 0.20 b --+ 0.40 b -+ 0.07 b -+ 0.00 b

12.99 1.77 4.57 2.96 0.00

85 -+ 0.01 -+ 0.19 t --+ 0.26 b -+ 0.05 b -+ 0.00 b

V a l u e s are e x p r e s s e d as m e a n +- S E M u s i n g l i t t e r as t h e u n i t o f c o m p a r i s o n . V a l u e s in a r o w w i t h d i f f e r e n t s u p e r s c r i p t a r e d i f f e r e n t a t P -< 0.05. *Differences based on bionomial coefficient analysis.

quate dams than in the nondiabetic-low Cu dams. Plasma Zn concentrations were similar among the groups. Plasma Cp activity was lower in diabetic-Cu adequate dams than in nondiabetic-Cu adequate dams (Table 6). Low Cu dams had lower plasma Cp activities than Cu adequate dams regardless of diabetes. RBC SOD activity was lower in the low Cu groups (P = 0.055) compared to their respective dietary controls.

Fetal liver trace elements/metalloproteins Low maternal dietary Cu was associated with low fetal liver Cu concentrations compared to Cu

adequate groups (Table 7). Fetuses in the Cu adequate groups had similar liver Cu levels. Fetuses in the nondiabetic-low Cu group had lower liver Cu concentrations than all other groups. Overall, fetuses from the diabetic dams had lower liver Zn concentrations (P -< 0.0007), and higher liver Fe concentrations (P -< 0.0001) as assessed by a significant group effect by two way ANOVA, compared to fetuses from nondiabetic dams. Liver MT concentrations were lower in fetuses from the diabetic groups compared to the nondiabetic groups (Table 8). Fetuses from the nondiabetic-Cu adequate group had the highest CuZnSOD activities. Liver MnSOD activity was similar among the groups.

Table 4. Influence of maternal diabetes and dietary Cu intake on fetal skeletal variation Treatment Diabetic

Nondiabetic

F e t u s e s e x a m i n e d (no.) T o t a l no. f e t u s e s a f f e c t e d Unossified centra Hemi-vertebrae Supernumerary ribs R u d i m e n t a r y ribs 2 or more variations/fetus Total variations*

Adequate Cu

Low Cu

Adequate Cu

Low Cu

86 1a 1a 0a 0a 0a 0a Ia

102 4a 1a 0a 1a 3a 1a 5a

81 44 b 9b 13 t 4t

85 41 b 12 b 17 b

31 b

2 0 at

V a l u e s in a r o w w i t h d i f f e r e n t s u p e r s c r i p t are d i f f e r e n t at P -< 0.05. *A f e t u s m a y h a v e m o r e t h a n o n e v a r i a t i o n .

1ab

8b

81

57 b

50 t

Diabetes and Cu metabolism• M. A. JANKOWSKIET AL.

595

Table 5. Influence of maternal diabetes and dietary Cu intake on maternal liver and kidney Cu, Zn, Mn, and Fe (ftmol/g tissue) and metallothionein (MT) (nmoFg tissue) concentrations T r e a t m e n t (N) Nondiabetic Tissue Cu Zn Mn Fe MT

Liver Kidney Liver Kidney Liver Kidney Liver Kidney Liver Kidney

A d e q u a t e Cu 0.064 0.157 0.36 0.34 0.031 0.016 2.0 0.9 0.97 17.74

Diabetic L o w Cu

-+ 0.001a(6) ± 0.029a(6) --- 0.01a(6) -+ 0.02ab(6) -+ 0.002a(5) + 0.001(6) ± 0.3a(6) -+ 0.1a(6) -+0.16a(5) ±0.16a(7)

0.024 0.050 0.33 0.31 0.035 0.016 2.7 0.8 1.46 11.69

A d e q u a t e Cu

± 0.002b(6) -+ 0.00P(7) ± 0.0P(6) ± 0.0P(7) ± 0.005a(6) - 0.001(7) - 0.3ab(6) ~ - 0.&(7) ±0.20~(7) ± 1.02b(7)

0.122 0.881 0.50 0.41 0.060 0.016 3.3 1.4 19.82 31.70

L o w Cu

--+ 0.019e(5) + 0.075b(6) -+ 0.02b(5) --- 0.04b¢(7) --+ 0.004b(5) --- 0.001(7) ± 0.6b(5) + 0.2b(7) ± 1.83b(10) ± 1.47¢(5)

0.003 0.066 0.42 0.44 0.051 0.015 2.5 1.0 9.93 28.95

_ 0.002ab(7) -+ 0.004a(6) ± 0.02c(7) ----.0.03c(6) --- 0.004b(7) - 0.001(6) + 0.3ab(7) + 0.1a(6) --+ 1.35¢(10) ±3.05¢(7)

Values are e x p r e s s e d as m e a n --+ SEM. Values in a row with different suPerscripts are different at P -< 0.05.

DISCUSSION

trations, and 2) the low Cu diets fed were effective in producing signs of maternal Cu deficiency, (lower plasma Cp and RBC SOD activities (P = 0.055)); both observations suggesting that Cu was less available to the fetus. The influence of each of the above insults and their interactive effects are discussed below. Independent of diabetes, the period of low maternal dietary Cu restriction used inthe current study did not significantly increase the incidence of fetal anomalies. Diabetic pregnancies were associated with fewer implantation sites, more resorptions, and fewer viable fetuses compared to nondiabetic pregnancies. In addition, live fetuses from the diabetic dams were small and had large placentas compared to control fetuses. Aside from the lower number of implantation sites observed in diabetic dams, the above observations are consistent with previous reports (I0,12,13). Maternal dietary Cu intake did not affect the above findings with the exception that fetuses from diabetic dams fed the low Cu diet were characterized by being slightly longer than fetuses from the other groups. Similar to previous studies,

Both in human and in experimental rat models, diabetic pregnancy is characterized by a high incidence of malformations. Uriu-Hare and coworkers reported higher maternal liver Cu concentrations and lower fetal liver Cu conce~rations in fetuses from diabetic dams fed Cu adequate diets (12). Given that fetuses from Cu deficient dams also have lower liver Cu concentrations and can have soft tissue and skeletal anomalies similar to the ones occurring in fetuses from diabetic dams (16), we hypothesized that diabetes-induced changes in Cu metabolism may contribute to the poor pregnancy outcome associated with diabetes. We postulated that diabetes and maternal dietary Cu deficiency would be synergistic with respect to their reproductive risks. However in the current study, maternal dietary Cu intake did not significantly affect the frequency or severity of gross malformations observed in fetuses from the diabetic dams. The above is surprising given the findings that I) the diabetic condition was associated with marked elevations in maternal liver Cu concen-

T a b l e 6. I n f l u e n c e o f m a t e r n a l d i a b e t e s a n d d i e t a r y C u i n t a k e o n m a t e r n a l b l o o d p a r a m e t e r s Treatment Nondiabetic A d e q u a t e Cu (7) P l a s m a trace elements (/xM) Cu Zn Fe Plasma ceruloplasmin(U/L) R e d blood cell S O D (U/mg Hb)

29.4 13.9 36,8 199.7 3.28

-+ 1.3 a --1.0 ± 7.0 ab _+ 8.5 a ± 0.24

Diabetic L o w Cu (8)

1.7 14.6 17A 3.7 2.77

±0.2 c -+ 1.2 _ 1.7b ± 0.1 c ± 0.64

A d e q u a t e Cu (10) 21.3 12.2 49.5 141.0 2.83

+± -+ -+

IA b 1.2 10.8 a 10.9 b 0.17

Values are e x p r e s s e d as m e a n _+ SEM. Values in a row with different superscript are different at P -< 0.05.

L o w Cu (10) 4.2 15.3 37.3 10.2 2.38

_+ 0.9 c -+ 0.6 - 4.8 ab - 3.4 c ± 0.39

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Volume7, Number 6, 1993

Table 7. Influence of maternal diabetes and dietary Cu intake on fetal liver Cu, Zn, and Fe concentrations (/xmol/g tissue) Treatment Nondiabetic

Cu Zn Fe

Diabetic

Adequate Cu (7)

L o w Cu (8)

Adequate Cu (10)

L o w Cu (10)

0.223 ± 0.012 a 0.79 --- 0.03 ab 3.2 + 0.1 a

0.015 -+ 0.002 b 0.82 ± 0.02 a 3.0 ± 0.1 a

0.242 - 0.016 a 0.71 - 0.04 bc 4.1 ± 0.2 b

0.072 ± 0.013 c 0.67 ± 0.02 c 4.0 - 0.2 b

Values are expressed as mean - SEM using litter as the unit of comparison. Values in a row with different superscript are different at P - 0.05.

maternal diabetes was associated with delayed skeletal development as evidenced by fewer ossified sternebrae, caudal vertebrae, and metacarpals (12,33). Poor bone ossification has been observed in Cu deficient human infants and experimental animals (34). In the current study, maternal dietary Cu intake did not influence the degree of ossification in fetuses from diabetic or nondiabetic dams presumably due to the relatively short period of maternal Cu deficiency. While a longer period of maternal Cu deficiency (prior to mating) would have resulted in the subsequent induction of fetal anomalies, such a model would have less relevance to the human condition. Skeletal anomalies observed in fetuses from diabetic dams included unossified centra, hemivertebrae, supernumerary ribs, and rudimentary ribs. Maternal dietary Cu intake did not influence the above with the exception that the frequency of supernumerary ribs was lower in the diabetic-low Cu group compared to the diabetic-Cu adequate group. Gross malformations were only observed in fetuses from diabetic dams. One case of exencephaly occurred in the diabetic-Cu adequate group, and one case each of micrognathia, ectopic cordis, and missing tail occurred in the diabetic-low Cu group.

Similar to these observations, others have reported micrognathia and missing tail to be associated with maternal diabetes (13). In addition to compromised development, similar to previous observations, diabetes also affected maternal tissue mineral concentrations and metabolism (10,12). In the dam, diabetes resulted in high liver Cu, Zn, and Mn concentrations and high kidney Cu and Zn concentrations. Both diabetic and nondiabetic dams responded to the low Cu diet by lowering liver and kidney Cu concentrations, but the magnitude of the response was larger in diabetic dams compared to control dams. The higher liver and kidney Cu in diabetic animals is significantly associated with MT, a putative storage protein (8,35). Liver MT increased 10 to 20 fold after STZ treatment; assuming 2 to 3 moles of Cu binds to each mole of MT, this is enough to theoretically account for 33 to 49% of the liver Cu. It has been suggested that during pregnancy Cu is mobilized from MT in the liver and/or kidney for transport to the embryo/fetus (36). It is unknown if the diabetes-induced kidney and liver MT levels will affect Cu transport to the embryo/fetus. In the current study, liver and kidney metallothionein concentrations were higher in dia-

Table 8. Influence of maternal diabetes and dietary Cu intake on fetal liver metalloproteins Treatment Nondiabetic

MT (nmol/g tissue) CuZnSOD (U/mg protein) MnSOD (U/mg protein)

Diabetic

Adequate Cu

L o w Cu

Adequate Cu

L o w Cu

102.51 ± 8.00a(7)

87.05 + 5.43a(7)

64.10 ± 7.72b(7)

64.03 - 2.89b(7)

1.44 ± 0.16a(6)

0.98 -+ 0.22b(4)

0.97 ± 0.13b(5)

0.82 ± 0.09b(8)

1.23 --- 0.09(6)

0.97 ± 0.11(5)

1.10 ± 0.11(6)

1.15 -+ 0.07(8)

Values are expressed as mean -+ SEM. Values in a row with different superscript are different at P <- 0.05.

Diabetes and Cu metabolism • M. A. JANKOWSKIET AL.

betic dams compared to nondiabetic dams. Typically in nondiabetic rats, liver MT concentrations are relatively constant over a wide range of dietary Zn and Cu intakes (37). Consistent with this, in the present study, for nondiabetic rats the level of dietary Cu did not influence maternal liver MT concentrations. However, diabetic dams responded to the low Cu diet by lowering MT concentrations in the liver. In contrast to liver, kidney MT concentrations in the diabetic dams were not influenced by dietary Cu intake while they were in nondiabetic dams. Consequently, these data suggest differential regulation of MT in liver and kidney, and differential regulation between the diabetic and nondiabetic group. The impact of these alterations in liver and kidney MT metabolism on Cu delivery to the fetus has yet to be determined. While maternal dietary Cu intake did not influence the frequency of gross fetal defects from the diabetic dams, low maternal dietary Cu intake did result in fetuses with low liver Cu concentrations compared to controls. Fetuses from the diabetic dams fed the low Cu diet had Cu concentrations that were 30% those of fetuses from diabetic dams fed Cu adequate diets. In contrast, fetuses from nondiabetic dams fed low dietary Cu had liver Cu concentrations that were 10% of their respective controls. While fetuses from both groups of dams fed the Cu adequate diet had similar liver Cu concentrations, fetuses from diabetic dams had 33% lower liver CuZnSOD activity than fetuses from the Cu adequate-nondiabetic dams. Fetal liver CuZnSOD activities were similar in the diabetic and nondiabetic-low Cu groups. The observations of lower CuZnSOD activity, coupled with similar and higher Cu concentrations in the fetuses from the diabetic dams fed the Cu adequate and low Cu diet, respectively, suggest that maternal diabetes is altering fetal Cu metabolism. The observed lower liver CuZnSOD activity in fetuses from diabetic dams may result from 1) less Cu available for activation, 2) degradation of the CuZnSOD protein following oxidative modification (38,39), and/or 3) Cu activation of the cuproenzyme may be normal, but the CuZnSOD may be inactivated by glycosylation in the diabetic group (40). Fetuses from diabetic dams may be exposed to a pro-oxidant environment due to the observed alterations in Zn, Cu, and Fe concentrations and potentially high glucose concentrations. Fe can generate highly reactive hydroxy radicals through the Fenton reaction. Due to the low tissue concentrations of Zn in fetuses from diabetic dams, Fe may bind to vacant Zn binding sites on cellular mem-

597

branes resulting in the potential to generate hydroxy radicals (41). It is interesting that the response of fetal hepatic MT, a protein with putative antioxidant properties, to maternal diabetes and maternal Cu deficiency paralleled that of CuZnSOD. As a group, fetuses from diabetic dams had lower liver Zn and MT than fetuses from nondiabetic dams as has previously been reported (12). The low CuZnSOD activity, low Zn and MT concentrations, and high Fe concentrations observed in fetuses from diabetic dams may result in a compromised antioxidant system. Furthermore, high glucose concentrations have been suggested to accelerate the production of reactive oxygen species via trace element catalyzed glucose auto-oxidation and nonenzymatic glycation (42,43). Based on the above, it is reasonable to suggest that plasma and intracellular membranes, DNA, and select protein in fetuses from the diabetic dam may be particularly susceptible to free radical attack. Consistent with the above is the observation that in a rat embryo culture system, the addition of SOD lowered the incidence of malformations induced by high glucose concentrations (44). In summary, we did not see a synergistic effect of maternal diabetes and low maternal dietary Cu intake on pregnancy outcome as evaluated by gross teratologic analysis. However, both diabetes and low maternal dietary Cu intake were associated with altered fetal Cu and Zn metabolism. Whether these changes in mineral metabolism in the diabetic dam and perhaps compromised fetal antioxidant defense system increase the susceptibility of the fetus to defects needs to be addressed. REFERENCES 1. Simpson JL, Elias S, Martin AO, Palmer MS, Ogata ES, Radvany RA. Diabetes in pregnancy, Northwestern University series (1977-1981). Am J Obstet Gynecol. 1983;146: 263-270. 2. Small M, Cassidy L, Leiper JM, Paterson KR, Lunan CB, MacCuish AC. Outcome of pregnancy in insulin-dependent (type 1) diabetic women between 1971 and 1984. Q J Med. 1986;61:1159-1169. 3. Mills JL, Knopp RH, Simpson JL, et al. Lack of relation of increased malformation rates in infants of diabetic mothers to glycemic control during organogenesis. N Engl J Med. 1988;318:671-676. 4. Erikkson UJ, Karlsson M, Styrud J. Mechanisms of congenital malformations in diabetic pregnancy. Biol Neonate. 1987;51:113-118. 5. Freinkel N. Diabetic embryopathy and fuel-mediated organ teratogenesis: lessons from animal models. Horm Metabol Res. 1988;20:463-475. 6. Sadler TW, Hunter ES, Wynn RE, Phillips LS. Evidence for multifactorial origin of diabetes-induced embryopathies. Diabetes. 1989;38:70-74. 7. Wibell L, Gebre-Medhin M, Lindmark G. Magnesium and zinc in diabetic pregnancy. Acta Paediatr Scand. 1985; Suppl: 100-106.

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Volume 7, Number 6, 1993 27. Schacter LP, DelVillano BC, Gordon EM, Klein BL. Red cell superoxide dismutase and sickle cell anemia symptom severity. Am J Hematol. 1985;19:137-144. 28. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47:469-479. 29. Schosinsky KH, Lehmann HP, Beeler MF. Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin Chem. 1974;20:15561563. 30. Onosaka S, Cherian MG. Comparison of metallothionein determination by polarographic and cadmium-saturation methods. Toxicol Appl Pharmacol. 1982;63:270-274. 31. Spector T. Refinement of the coomassie blue method of protein quantitation. Anal Biochem. 1978;86:142-146. 32. SAS version 6.04. SAS Institute Inc. Copyright 1987 Cary, NC 27512-8000, U.S.A. 33. Erikkson UJ, Dahlstrom E, Hellerstrom C. Skeletal malformations in the offspring of diabetic rats after intermittent withdrawal of insulin in early gestation. Diabetes. 1983 ;32:1141-1145. 34. Mimouni F, Steichen JJ, Tsang RC, Hertzberg V, Miodovnik M. Decreased bone mineral content in infants of diabetic mothers. J. Perinatol. 1988;5:339-343. 35. Uriu-Hare JY, Walter RM, Keen CL. 65Zinc metabolism is altered during diabetic pregnancy in rats. J Nutr. 1992; 122:1988-1998. 36. Suzuki KT, Tamagawa H, Takahashi K, Shimojo N. Pregnancy-induced mobilization of copper and zinc bound to renal metallothionein in cadmium-loaded rats. Toxicology. 1990;60:199-210. 37. Bremner I. Nutritional and physiological significance of metallothionein. In: Riordan JF, Vallee BL, eds. Methods in enzymology; Volume 205, New York: Academic Press; 1991:25-35. 38. Goldberg A, Boches FS. Oxidized proteins in erythrocytes are rapidly degraded by the adenosine triphosphate-dependent proteolytic system. Science. 1982;215:1107-1109. 39. Salo DC, Pacifici RE, Lin SW, Giulivi C, Davies KJA. Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. J Biol Chem. 1990;265:11919-11927. 40. Arai K, Iizuka S, Tada Y, Oikawa K, Taniguchi N. Increase in the glucosylated form of erythrocyte Cu-Zn-superoxide dismutase in diabetes and close association of the nonenzymatic glucosylation with the enzyme activity. Biochim Biophys Acta. 1987;924:292-296. 41. Willson RL. Zinc and iron in free radical pathology and cellular control. In: Mills CF, ed. Zinc in human biology. New York: Springer-Verlag; 1989:147-172. 42. Wolff SP, Dean RT. Glucose autoxidation and protein modification. Biochem J. 1987;245:243-250. 43. Hunt JV, Wolff SP. Oxidative glycation and free radical production: a causal mechanism of diabetic complication. Free Rad Res Comm. 1991;12-13:115-123. 44. Erikkson U J, Borg LA. Protection by free oxygen radical scavenging enzymes against glucose-induced embryonic malformations in vitro. Diabetologia. 1991;34:325-331.