Postnatal development in rat offspring delivered of dams with gestational hyperglycemia

Postnatal development in rat offspring delivered of dams with gestational hyperglycemia Shaobin Zhong, PhD: Joseph C. Dunbar, PhD,b and K-L. Catherine Jen, PhD" Detroit, Michigan OBJECTIVES: Our purpose was to test the hypotheses that (1) offspring delivered of dams with gestational hyperglycemia will show metabolic abnormalities and (2) dams with repeated pregnancy but without lactation experience will demonstrate abnormal glucose metabolism long after the delivery of the third litter. STUDY DESIGN: Female rats went through three cycles of gestation-lactation, gestation-nonlactation, or no mating at all. The offspring were reared to 3 months of age, when half of each group were mated. Intravenous glucose tolerance testing was conducted at different times in dams and adult offspring. RESULTS: Nonlactation dams showed gestational hyperglycemia, insulin resistance, and hyperlipidemia during the third pregnancy. Impaired intravenous glucose tolerance testing was also apparent 1 week and 3 months after weaning in dams. Adult offspring nursed by nonlactation dams were glucose intolerant and had higher hepatic gluconeogenic enzyme activities and higher lipid levels in the pregnant state. CONCLUSION: Gestational hyperglycemia produced by repeated gestation without lactation could have a long-lasting effect on adult offspring. (AM J OBSTET GVNECOL 1994; 171 :753-63.)

Key words: Pregnancy, nonlactation, insulin resistance, gluconeogenesis, gestational hyperglycemia

The physiologic changes during gestation tend to produce a state of insulin resistance. 1 The metabolic changes during lactation effectively promote the recovery from the pregnant state to the prepregnant state. If gestation is not followed by lactation, the recovery process may be slowed. Moore and Brasel" and Steingrimsdottir et aI.' found that in Osborne-Mendel rats pregnancy not followed by lactation resulted in increased fat cell numbers in retroperitoneal and parametrial pads. In comparison, lactating rats of the same strain had reduced subcutaneous fat because of a decrease in the size of adipocytes.' Also, Burnol et aI.' reported that in nonlacting rats blood glucose and insulin levels were 20% and 35% higher, respectively, compared with those of lactating rats. The studies of pregnancy with or without lactation conducted by other investigators only consisted of one reproduction cycle. Jen and Lin,5 Jen et aI.,6 Zhong et aI.,7 and Zhong and From the Department of Nutrition and Food Science" and the Department of Physiology, School ofMedicine/ Wayne State University. Supported by National Institutes of Health grant No. DK-40046 (K-L.CJ.). Received for publication January 24, 1994; revised April 20, 1994; accepted April 27, 1994. Reprint requests: K-L. Catherine Jen, PhD, Department of Nutrition and Food Science, 3009 Science Hall, Wayne State University, Detroit, MI 48202. Copyright © 1994 by Mosby-Year Book, Inc. 0002-9378194 $3.00 + 0 6/1157033

Jen S have conducted studies that included three cycles of repeated pregnancy with or without lactation and observed that repeated pregnancies without lactation in rats resulted in significantly increased fasting blood gkuose levels during the third gestational period. Rats with repeated pregnancy but without lacation produced some characteristics similar to those of human gestational diabetes, such as hyperglycemia in later pregnancy and an elevated spontaneous abortion rate. 6 - B To examine the postnatal development of offspring delivered of dams without previous lactation experience and the glycemic control of dams of an older age, we conducted the current study in obesity-prone Wistar rats.9 Offspring were obtained from previous lactating dams and dams not allowed to nurse in the previous two pregnancies but allowed to nurse the third litter. The hypotheses to be tested in this study were (1) offpsring delivered of dams with gestational hyperglycemia related to previous nonlactation experience will show metabolic abnormalities, especially during the period when metabolic demand is high, and (2) dams with repeated pregnancy but without lactation experience will demonstrate abnormal glucose metabolism long after the delivery of the third litter.

Material and methods Animals and diet. Sixty-seven Wistar female rats (Harlan-Sprague Dawley, Indianapolis, Ind.), with start-

753

754

Zhong, Dunbar, and Jan

ing weights of 225 to 250 gm, and part of their female offpsring (n = 34), were used in this study. Male rats of the same strains were used for mating. The female rats were housed individually in plastic maternal cages with wood shaving bedding in a colony room with 12-hour light/12-hour dark cycles. Purina Rodent Diet (No. 5001, Ralston Purina, St. Louis) and water were given to the rats ad libitum. The research protocol was approved by the Animal Investigation Committee of Wayne State University. Procedure of the animal treatment Dams. Female rats were divided randomly into three groups: lactation after pregnancy (n = 28), nonlactation after pregnancy (n = 28), and age-matched controls, no mating (n = 11). Body weight and food intake were measured three times per week. Food intake was determined by measuring the difference between the weight of food put in the food hopper and the weight of the leftover food after 2 days. The lactation and nonlactation rats were mated with male rats in stainless steel hanging cages. The day of conception was determined when vaginal plugs were found; this was designated as day 1 of pregnancy. The gestation period was typiGally 22 to 23 days. Each of the lactation dams nursed six healthy pups for 21 days. Nonlactation dams had their pups removed immediately after delivery and rested for 21 days without lactation. The above process was designated as reproduction cycle 1. The same routine was repeated in cycles 2 and 3. The interval between the cycles, from weaning (or 21 days after delivery for nonlacting rats) to next mating, was 7 to 10 days. In cycle 3 the rats were sub grouped randomly as follows: (1) a group oflactation (n = 10) and nonlactation (n = 10) rats were killed on day 19 of pregnancy without being given an intravenous glucose tolerance test (GTT) and (2) another group of lactation (n = 10) and nonlactation (n = 10) rats were given one intravenous GTT on pregnancy day 19 and another 7 to 10 days after weaning the third litter. They were then killed 2 days later; the remaining lactation (n = 8) and nonlactation (n = 8) rats were given an intravenous GTT 3 months after weaning and killed 2 days later. The intravenous GTT was performed on 11 control rats and they were killed as described above. For the intravenous GTT procedure each rat was fasted overnight and anesthetized with ketamine (60 mg/kg). A silicone rubber cannula (Silastic, Dow Coming, Midland, Mich.) was surgically implanted into the external jugular vein. A glucose load of 1 gmJkg of body weight was infused through this cannula to the rat after a baseline blood sample was obtained. Mter the glucose load blood samples were withdrawn at 1, 3, 5, 10, 15, 20, and 30 minutes. For each blood sampling dead space in the

September 1994 Am J Obstet Gynecol

cannula was withdrawn first and pure blood was then taken as sample. The cannula was kept patent by slow infusion of heparinized saline solution. During each pregnancy spontaneous abortions, defined as a positively identified pregnancy and rapid weight gain followed by a sudden weight drop to the prep regnant level, were recorded for lactation and nonlactation groups. Mter a 7- to 10-day rest those rats were mated again. Offspring. In cycle 3 part of the offpsring from lactation dams were raised to youngsters (n = 17). Also, another group of rats had two previous cycles of pregnancy and nonlactation, but in cycle 3 they were allowed to nurse six female pups per litter for 21 days. Some of these pups were randomly selected and raised to youngsters (n = 17). These dams were not used for any determinations because of their mixed nonlactation and lactation histories. When the youngsters were 3 months old 18 were mated with male rats of the same strain (pregnant lactation youngsters, n = 9; pregnant nonlactation youngsters, n = 9). They received an intravenous GTT on day 17 or 18 of pregnancy and were then killed on day 19 or 20 of pregnancy. The other 16 female youngsters served as controls (control lactation youngsters, n = 8; control nonlactation youngsters, n = 8) and were not mated, but they had an intravenous GTT and were killed 2 days later. At death rats were decapitated after being briefly exposed to carbon dioxide. Blood was collected into a heparin-ethylenediaminetetraacetic acid-saline solutionfilled centrifuge tube. The plasma was collected and stored for glucose, insulin, triglycerides, and total cholesterol assays. Other blood samples were collected into a ethylenediaminetetraacetic acid-glutathione-saline solution-filled tube. This plasma sample was stored in a -70 0 C freezer for catecholamine determinations. Liver was dissected out and immediately frozen with liquid nitrogen and stored in a -70 C freezer for hepatic enzyme determination. Biochemical assays. Adipose cell size and number from retroperitoneal and subcutaneous regions were measured by the method of Hirsch and Gallian.1O The total fat cell numbers in retroperitoneal pads were calculated by dividing total pad weight by the average cell size in each pad. The enzymatic determination of triglyceride was performed by the method of Bucolo and David" (Sigma, St. Louis). The total cholesterol was measured by the oxidase-peroxidase method of Allain et al. 12 (Sigma). The concentration of plasma glucose was assayed by the oxidase-peroxidase method of Trinder. IS Plasma insulin levels were determined with iodine 125~labeled insulin in a double-antibody radioimmunoassay, as de0

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Volume 171, Number 3 Am J Obstet Gynecol

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WEEKS Fig. 1. Body weight changes during three reproduction cycles and 7 to 10 days after weaning for three groups of rats. Points are group means. C, Control rats (n = 11) were never treated; N, rats (n = 28) had three cycles of pregnancy (preg) and nonlactation: L, rats (n = 28) had three cycles of pregnancy and lactation (lact). In each pregnancy period lactation and nonlactation rats were heavier than control rats. In each lactation period lactation rats were heavier than nonlactation and control rats. At end of lactation period in cycle 3 order of weights was as follows: lactation > nonlactation > control (p < 0.05); 7 to 10 days after weaning, nonlactation > control = lactation (p < 0.05).

scribed in Herbert et al. 14 The blood norepineprhine and epinephrine levels were measured by a modified method according to Hegstrand and Eichelman. 15 High-performance liquid chromatography with electrochemical detection was used. Liver homogenate was used for glucose-6-phosphate dehydrogenase, malic enzyme, fructose-I,6-bisphophatase, and phosphoenolpyruvate carboxykinase activity determinations. The assay procedures for glucose-6phosphate dehydrogenase have been described by Glock and McLean, 16 for malic enzyme by Ochoa,17 and for fructose-I, 6-biphosphatase and phosphoenolpyruvate carboxykinase by Opie and Newsholme. 18 Statistical analysis. Means and SEM were calculated. Analysis of variance with repeated measures or analysis of variance were performed to analyze data. If a significant result was obtained in analysis of variance within each period, Fisher's least significant difference test was then used to compare the difference between each pair of groups. Two-way analysis of variance was used to differentiate the effect of inheritance (from lactating or previously nonlactating dams) from the effect of pregnancy. A p value < 0.05 was considered significant. Z test was used to compare the spontaneous abortion rate in cycle 3 between lactation and nonlactation rats.

Results

Body weight changes and food intake. Fig. 1 shows the body weight changes during three reproduction cycles (n = 67). Lactation and nonlactation rats were heavier than control rats during pregnancy; the lactation group weighed more than the other two groups during lactation. The weights of nonlactation rats returned to control levels after weaning in cycles 1 and 2 but not in cycle 3. At the end of three cycles the order of body weight was lactation > nonlactation > control (control, 372 ± 10 gm; lactation, 407 ± 6 gm; nonlactation, 394 ± 9 gm); the difference between lactation and control rats was significant at p < 0.05. However, the nonlactation group surpassed the lactation group within 1 week after weaning in cycle 3 (control, 376 ± 9 gm; lactation, 374 ± 6 gm; nonlactation, 405 ± 9 gm), with nonlactation rats significantly higher than control and lactation rats at p < 0.05. Three months later there was no difference in body weight between lactation and nonlactation rats (lactation, n = 7, 397 ± 13 gm; nonlactation, n = 7, 395 ± 10 gm). The offspring of the lactating dams and from the previously nonla :tating dams had the same birth weights (6.65 ± () .16 gm vs 6.95 ± 0.10 gm, respectively, not significant). The average birth weight showed

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September 1994 Am J Obstet GynecoJ

Table I. Retroperitoneal and subcutaneous adipose cellularities at peak pregnancy in cycle 3 Control rats (n = 8) Subcutaneous cell size (fLg lipid/cell) Retroperitoneal fat pad weight (gm) Retroperitoneal cell size (fLg lipid/cell) Retroperitoneal cell number (10 6 )

0.78 26.7 0.96 4.61

± ± ± ±

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b

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± ± ± ±

0.07 1.6a . b 0.14b 0.48 a

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0.11 2.5 b 0.07 a , 0.52"

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P<

b

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an 11 % increase from cycle 1 to cycle 3 for nonlactation dams (6.25 ± 0.54 gm vs 6.95 ± 0.1 gm), but this increase was not statistically significant. The increase in birth weight from cycle 1 to cycle 3 for lactation dams was 5%. There was also no difference in body weight during the I2-week study period. Pregnant lactation youngsters and pregnant nonlactation youngsters rats had the same weights as well (control lactation youngsters 310 ± II gm, control nonlactation youngsters 316 ± 14 gm, not significant; pregnant lactation youngsters 354 ± 19 gm, pregnant nonlactation youngsters 350 ± 10 gm, not significant). During pregnancies, lactation and nonlactation rats consumed more food than control rats (jJ < 0.01). During lactation, lactation rats had significantly more food intake than control and nonlactation rats, whereas the latter two groups had similar intakes. Adipocyte cellularities. The retroperitoneal and subcutaneous adipose cellularities at late pregnancy in cycle 3 are shown in Table I. There was no difference in subcutaneous fat cells size among the groups. Nonlactation rats had significantly heavier retroperitoneal fat pads than did control rats. The lactation group had increased retroperitoneal cell size compared with the control group, and the nonlactation group had a higher retroperitoneal cell number. Blood glucose levels in intravenous GTT In late pregnancy of cycle 3. The glucose levels of the intravenous GTT on day 19 of the third pregnancy are shown in Fig. 2 (upper panel). The nonlactation group had a higher basal fasting glucose level compared with the lactation group. The glucose responses in the nonlactation group were higher at 1, 5, 10, 15, and 30 minutes after the glucose loading compared with the other two groups. After the difference in baseline glucose levels was adjusted for, the nonlactation group still had significantly elevated glucose responses compared with the lactation group. Postpartum after three cycles. Seven to ten days after weaning the basal glucose level in the nonlactation group was higher than in the other two groups. The nonlactation group also had higher glucose levels after glucose loading (Fig. 3, upper panel). After the

3-month recovery period, although the basal glucose level in the nonlactation group was not different from that in control and lactation rats, nonlactation rats still had significantly impaired glucose tolerance on intravenous GTT (Fig. 3, lower panel). Glucose levels in intravenous GTT in offspring. At 3 months of age no effects of pregnancy (day 16 to 17) on basal glucose levels and glucose responses in intravenous GTT in the youngsters from either the lactating dams or previously nonlactating dams was observed. However, the inheritance effect was apparent. Although the basal glucose levels in lactation and nonlactation youngsters were the same, nonlactation youngsters had significantly higher glucose responses than did lactation youngsters. The overall glucose levels of lactation (control + pregnant) and nonlactation (control + pregnant) youngsters in intravenous GTT were analyzed as shown in Fig. 4 (upper panel) to indicate the inheritance effect. Blood insulin levels in intravenous GTT. The insulin responses of each group of rats in intravenous GTT on pregnancy day 19 of cycle 3 are shown in Fig. 2 (lower panel). The nonlactation group showed higher insulin responses at all time points compared with the other two groups of rats. The peak insulin response was delayed in the nonlactation group. For youngsters the inheritance of nonlactation had an effect at 5 minutes. The overall insulin responses of lactation (control + pregnant) and nonlactation (control + pregnant) youngsters in intravenous GTT are shown in Fig. 4 (lower panel). Plasma triglyceride, total cholesterol, and catecholamine levels. The plasma triglyceride and total cholesterol levels in fasting and nonfasting conditions are displayed in Table II. The nonlactation rats had a higher blood triglyceride level than did the control rats in both the fasting and nonfasting states, whereas the lactation rats were different from the control rats in the nonfasting state. The blood total cholesterol level in the nonlactation group was significantly higher than that in the control group in both fasting and nonfasting states and higher than the lactation group in the nonfasting state.

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One week after weaning in cycle 3 nonlactation rats still had a significantly higher fasting blood triglyceride level than did control and lactation rats, but the differences in nonfasting triglyceride and fasting and nonfasting total cholesterol among the three groups were not significant. Three months after weaning the fasting blood total cholesterol level was higher in nonlactation rats. The blood triglyceride and total cholesterol levels in female offspring from lactating and previously nonlactating dams are shown in Fig. :). The order of the

fasting blood triglyceride levels was pregnant nonlactation youngsters > pregnant lactation youngsters = control nonlactation youngsters > control lactation youngsters, with pregnant nonlactation youngsters significantly higher than control lactation youngsters. The order of the nonfasting triglyceride levels was also pregnant nonlactation youngsters> pregnant lactation youngsters> control l1onlactation youngsters> control lactation youngsters, but this was only because of a pregnancy effect. The only significant diflC:.'!"cnce found

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September 1994 Am J Obstet Gynecol

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Fig. 3. Glucose responses during intravenous GIT for three groups of rats 7 to 10 days (upper panel) and 3 months (lower panel) after weaning after three cycles. Data are presented as mean ± SEM. C, Control rats, n = 11; L, pregnancy-lactation, n = 10; N, pregnancy-nonlactation, n = 10. Three months after weaning-lactation, n = 7; non lactation, n = 7. Asterisk, N onlactation value is significantly different from control at p < 0.05 or 0.0 I; nurnber sign with asterisk, non lactation significantly different from lactation but not control; two asterisks, nonlactation significantly difIerent from both control and lactation at p < 0.05 or 0.01.

total cholesterol levels was between control nonlactation youngsters and pregnant lactation youngsters (control nonlactation youngsters> pregnant lactation youngsters) in the fasting state. On pregnancy day 19 in cycle 3 the blood epinephIn

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nne levels of control, lactation, and nonlactation rats were undetectable. The pregnant rats (lactation and nonlactation) had significantly higher norepinephrine levels than did the control rats, whereas the lactation group had significantly higher norepinephrine levels than did the nonlactation group (control, 2.56 ± 0.06 f.LmoI!L; lactation, 3.43 ± 0.07 f.LmoI/L; nonlactation, 3.2 ± 0.07 f.LmoI/L, p < 0.05).

Volume 171. Number 3 Am J Obstet Gynecol

Zhong. Dunbar. and Jen

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Table II. Blood triglyceride and total cholesterol levels for three groups of rats Total cholesterol (mmol/ L)

Triglycerides (mmol/ L) Control

Pregnancy Day 19 (fasting) At death (nonfasting) After weaning 7-10 days (fasting) At death (nonfasting) 3 mo after weaning Fasting Nonfasting

I

Lactation

INonlactation ISignificance

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I

INonlactation ISignificance

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0 .90 ± 0.21" 2.71 ± 0.55 b

4.3 1 ± 0.80 b

P<

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0.42 ± 0 .05' 0. 37 ± 0.07'

0.67 ± O.Olb p < 0 0. 5

0.90 ± 0.20

0.87 ± 0.09

0.78 ± 0.08

0.40 ± 0.07 0.95 ± 0.10

0.46 ± 0.06 0.97 ± 0.07

1.31 ± 0.16 b

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1.40 ± 0.14' 1.47 ± 0.12'

1.91 ± 0.17 b

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1.24 ± 0.10

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1.66 ± 0.1 3

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1.24 ± 0.23 1.39 ± 0.23

b

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Hepatic enzyme activities. The phosphoenolpyruvate carboxykinase activities in different stages of cycle 3 are shown in Table III. On day 19 to 20 of the pregnancy in cycle 3, the phosphoenolpyruvate carboxy kinase activity was higher in nonlactation rats than in the other two groups. One week or 3 months after weaning non lactation rats continued to exhibit higher phosphoenolpyruvate carboxy kinase activity than lactation rats (p < 0.05). Both inheritance (from lactating or

previously nonlactating dams) and pregnancy had significant effects on phosphoenolpyruvate carboxykinase activity in the offspring, whereas the interactive effect was not significant. The order of the four groups of the youngsters was pregnant nonlactation youngsters > control nonlactation youngsters > pregnant lactation youngsters > control lactation youngsters, with control lactation youngsters significantly lower than all the other groups and pregnant lactation youngsters signifi-

760

Zhong, Dunbar, and Jen

September 1994 Am J Obstet Gynecol

Table III. Hepatic gluconeogenic and lipogenic enzyme activities in three groups of rats Lactation

Day 19 of pregnancy Liver weight (gm) Phosphoenolpyruvate carboxykinase (j.Lmol a-nicotinamide adenine dinucleotide/gm wet liver/min) Malic enzyme (j.Lmol j3-nicotinamide adenine dinucleotide phosphate, reduced form/gm wet liver/min) Glucose-6-phosphate dehydrogenase (j.Lmol j3-nicotinamide adenine dinucleotide phosphate, reduced form/gm wet liver/min) 7 -10 days after weaning Liver weight (gm) Phosphoenolpyruvate carboxykinase (j.Lmol a-nicotinamide adenine dinucleotide/gm wet liver/min) Malic enzyme (j.Lmol j3-nicotinamide adenine dinucleotide phosphate, reduced form/gm wet liver/min) Glucose-6-phosphate dehydrogenase (j.Lmol j3-nicotinamide adenine dinucleotide phosphate, reduced form/gm wet liver/min) 3 mo after weaning Liver weight (gm) Phosphoenolpyruvate carboxykinase (j.Lmol a-nicotinamide adenine dinucleotide/gm wet liver/min) Malic enzyme (j.Lmol j3-nicotinamide adenine dinucleotide phosphate, reduced form/gm wet liver/min) Glucose-6-phosphate dehydrogenase (j.Lmol j3-nicotinamide adenine dinucleotide phosphate, reduced form/gm wet liver/min)

(n = 10)

Nonlactation (n = 10)

12.0 ± 0.6 a 0.99 ± 0.07"

16.1 ± 0.6 h 1.12 ± 0.06"

16.0 ± 0.6 h 1.39 ± 0.10 b

p < 0.01

0.53 ± 0.08"

0.76 ± 0.08 h

0.92 ± 0.07 b

P < 0.05

0.77 ± 0.05 a

1.12 ± 0.09 h

1.23 ±

O.llh

P < 0.05

(n = 7) 12.7 ± 0.4 0.80 ± 0.04a

(n = 7) 12.2 ± 0.3 0.94 ± 0.05 b

P < 0.05

0.65 ± 0.10

0.60 ± 0.07

NS

0.77 ± 0.09

0.90 ± 0.10

NS

(n = 7) 12.5 ± 0.6 0.72 ± 0.06"

(n = 7) 11.3 ± 0.5 0.96 ± 0.06 b

0.48 ± 0.06

0.58 ± 0.11

NS

0.66 ± 0.07

0.79 ± 0.11

NS

Significance

P<

0.05

NS

NS 0.05

P<

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cantly lower than pregnant nonlactation youngsters (Fig. 6). The hepatic glucose-6-phosphate dehydrogenase and malic enzyme activities on day 19 to 20 of pregnancy of cycle 3, 1 week after weaning, and 3 months after weaning are shown in Table III. On pregnancy day 19 to 20 the two pregnant groups (lactation and nonlactation) had significantly higher glucose-6-phosphate dehydrogenase and malic enzyme activities than did control rats, but the difference between lactation and nonlactation rats in either glucose-6-phosphate dehydrogenase or malic enzyme was not significant. One week after weaning and 3 months later the three groups were not different. In female offspring a significant inheritance effect and an interaction effect on glucose-6-phosphate dehydrogenase activity was noted. As a result, pregnant rats from previously nonlactating dams had the highest activity. Only pregnancy had a significant effect on malic enzyme activity; inheritance and the combination of both had no effect. The glucose-6-phosphate dehydrogenase and malic enzyme activities of the four groups are shown in Fig. 6. Spontaneous abortion rates. In cycle 3 no cases of spontaneous abortion occun-ed in lactation rats. Five cases of spontaneous abortion occun-ed in nonlactation rats. The difference between lactation and nonlactation was statistically significant (z = 2.27, P < 0.05).

Comment

Hyperglycemia, hypertriglyceridemia, and glucose intolerance during late pregnancy. Pregnancy leads to a state of insulin resistance and an increase in body fat deposition. I. 19. 20 Lactation, on the other hand, assists the metabolic changes and body composition return to the prepregnant state. If pregnancy is not followed by lactation, the metabolic alterations may not return to the basal state as efficiently as that observed after lactation. We have previously reported that repeated pregnancy without lactation induced higher spontaneous abortion rates, elevated maternal body fat, and impaired glucose tolerance in later pregnancies. In this study hyperglycemia and an impaired intravenous GTT in later pregnancy were evident in previously nonlactating Wistar rats. The reason for the hyperglycemia in pregnancy is controversial. Stress plays an important role in the occun-ence of hyperglycemia,"1 and the stress intensity increases during pregnancy."" In the current study blood norepinephrine levels were significantly higher in the pregnant groups (lactation and nonlactation) than in the control group during the gestation period in cycle 3, which might at least partially explain why the pregnant rats had relatively higher blood glucose and insulin levels in the intravenous GTT than did the control rats. However, stress itself cannot explain why the previously nonlactating rats had higher basal glu-

Volume 171, Number 3 Am J Obsle l Gynecol

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Fig. 6. Hepatic phosphoenolpyruvate carboxykinase, glucose-6-phosphate dehydrogenase (G-6Data are lactation nonlactaraised by dams with two cycles of pregnancy-nonlactation but nursed pups in cycle 3. Control and control nonlactation youngsters are control rats, not mated. Pregnant lactation youngsters and pregnant nonlactation youngsters were killed at pregnancy day 20. Superscripts a, b, and c above bars represent values significantly different from each other at p < 0.05. NADPH, I3-Nicotinamide adenine dinucleotide phosphate, reduced form. PDH), and malic enzyme (ME) activities of female offspring rats at 3 months of age. presented as mean ± SEM. Control lactation youngsters (YLC) (n = 8) and pregnant youngsters (YLP) (n = 9) were from dams who had repeated pregnancy-lactation; control tion youngsters (YNC) (n = 8) and pregnant nonlactation youngsters (YNP) (n = 9) were

cose levels than did lactation rats. The previously nonlactating rats also exhibited an impaired intravenous GTT in both glucose and insulin levels but the lactating rats did not. Steingrimsdottir et al. 3 suggested that hypertrophy of

fat cells during pregnancy was the cause of insulin insensitivity. The results of the current study do not support this suggestion. The control, lactation, and non lactation groups had the same average subcutaneous fat cell size. However, nonlactation rats had heavier

762 Zhong, Dunbar, and Jen

retroperitoneal fat pad weight and more retroperitoneal fat cells. Because retroperitoneal fat weight correlated with total body fat mass,2' the higher body fat mass may have caused this insulin insensitivity and hyperglycemia. The binding affinity or postbinding defect must play more crucial roles in causing these, but further investigation is needed. During pregnancy the gluconeogenic activity in both humans and animals is usually elevated. 24. 25 Phosphoenolpyruvate carboxy kinase is one of the rate-limiting hepatic enzymes in the gluconeogenic process. In the current study the phosphoenolpyruvate carboxykinase activity was higher in pregnant rats than in control rats, which agreed with the previous findings by Jones et al." However, in the current study, the nonlactation group also had a significantly higher phosphoenolpyruvate carboxykinase activity than did lactation rats; this could at least partially contribute to the hyperglycemia in those nonlactation rats. The mechanism of this elevation is not clear. Phosphoenolpyruvate carboxykinase gene expression is inhibited by insulin. 26. 27 Because nonlactation rats also had hyperinsulinemia, this elevated phosphoenolpyruvate carboxy kinase activity with hyperinsulinemia may thus be an indicator of hepatic insulin insensitivity. During pregnancy blood lipid levels are usually increased in humans" and in rats (this study). We found that the nonlactating rats showed an even higher blood triglyceride level than did the lactating rats. Hypertriglyceridemia also indicates a state of insulin resistance. 28 In addition, this increased availability of blood triglyceride levels may spare blood glucose as fuel, thus further enhancing the maintenance of hyperglycemia. Therefore the insulin resistance in nonlactating rats was more serious than in normal pregnant rats. Glucose-6phosphate dehydrogenase and malic enzyme are two key hepatic lipogenic enzymes, and their activities were higher in pregnant rats (both lactation and nonlactation, nonlactation > lactation). This increased hepatic lipogenic activity may be closely associated with the increased blood lipid levels in the pregnant rats. The mechanism of this elevation needs to be further examined. Postweaning glycemic control and insulin sensitivity. A markedly increased hepatic uptake of gluconeogenic substrates in the late stage of gestation and the middle stage of lactation in rats was reported previously by Casado et al. 24 Phosphoenolpyruvate carboxykinase activity in guinea pigs" and gene expression (increased phosphoenolpyruvate carboxy kinase messenger ribonucleic acid) in rats 29 were also elevated during lactation. In this study 7 to 10 days after weaning, nonlactation rats showed a significantly higher phosphoenolpyruvate carboxykinase activity and did lactation rats. For lactating rats the increased gluconeogenic

September 1994 Am J Obstet Gynecol

activity could enhance lactose production in mammary glands. 25 However, the increased gluconeogenesis in nonlactating rats does not have any clear physiologic role. The increased phosphoenolpyruvate carboxykinase activity may at least partially explain the higher basal blood glucose and glcuose response to the intravenous GTT in nonlactation rats. In human gestational diabetes, although the hyperglycemia is restricted only to the gestational period, patients are more likely to have glucose intolerance later in life.'o In nonlactation rats the high phosphoenolpyruvate carboxy kinase activities and the significantly impaired intravenous GTT results were also maintained after a 3-month postweaning recovery period. Therefore repeated pregnancy without lactation may have a long-term effect on hyperglycemia and glucose intolerance, a phenomenon resembling human gestational diabetes mellitus. The health impact of gestational hyperglycemia on adult offspring. In our previous study with OsbomeMendel rats 7. 31 and the current study with Wistar rats significantly higher spontaneous abortion rates were observed in the previously nonlactating rats during the third pregnancy. Although the mechanism is not clear, this phenomenon is commonly observed in diabetic humans. A report from the Joslin Clinic (American Diabetes Association Professional Section Reports, 1989) suggested that frequent spontaneous abortions could be related to the elevated glycosylated hemoglobin levels. Further investigation is required to confirm this suggestion. Repeated pregnancy without lactation did not have any effect on parturition outcome (litter size, total weight, average pup weight, and sex distribution). The youngsters born to the previously nonlactating dams did not exhibit any abnormalities in birth weight and adult body weight. However, when they reached adulthood the glucose responses to the intravenous GTT were significantly higher in youngsters from the previously nonlactating dams than those from lactating dams, although the insulin responses were impaired to a lesser degree. Not coincidentally, Oh et al. 32 observed hyperinsulinemia in the offspring from mildly hyperglycemic dams induced by administering streptozotocin on day 5 of gestation. Gauguier et al." found that diabetes in early pregnancy affected glucose homeostasis in offspring. Jen et al. 34 had a similar finding that pups delivered of dams with hyperglycemia induced by fructose feeding were also hyperglycemic at birth. In the study of Grill et al." it was found that infusing glucose into the dams during the last week of gestation caused glucose intolerance in female adult offspring during pregnancy. All those results suggest that a relationship exists between dams and their offspring in glycemic control. The fetal metabolic environment may have great influence on the carbohydrate metabolism and the manifestation of diabetes in the

Volume 171. Number 3 Am J Obstet Gynecol

offspring. Furthermore, the youngsters born to the previously nonlactating dams had significantly higher hepatic gluconeogenic enzyme activities. These offspring also showed higher blood lipid levels and higher hepatic lipogenic enzyme activities. Because the lipid levels and hepatic gluconeogenic and lipogenic enzyme activities were also higher in those nonlactating dams during their pregnancy, there must be some mechanism linking the dams and pups prenatally. Insulin resistance apparently is the common underlying cause of these abnormalities in dams and offspring. Whether this insulin resistance occurs at the receptor or postreceptor level needs to be further addressed. It should be noted that the abnormalities shown in the offspring were manifested without any elevation of body weight at any time during the first 3 months of postnatal life. Therefore these abnormalities cannot be attributed to the difference in body weight, and maternal body weight does not affect the pup's birth weight. REFERENCES 1. Freinkel N. Of pregnancy and progeny. Diabetes 1980;29: 1023-35. 2. Moore B], Brasel]A. One cycle of reproduction consisting of pregnancy. lactation or no lactation. and recovery: effects on fat pad cellularity in ad libitum fed and foodrestricted rats. J Nutr 1984;114:1560-65. 3. Steingrimsdottir L, Brasel ]A, Greenwood MRC. Diet, pregnancy, and lactation: effects on adipose tissue, lipoprotein lipase, and fat cell size. Metabolism 1980;29:83741. 4. Burnol AF, Leturque A, Ferre p. Kande ]. Girard]. Increased insulin sensitivity and responsiveness during lactation in rats. Am] Physiol 1986;251:E537-41. 5. len KLC, Lin PKH. Hyperglycemia in pregnancy induced by repeated pregnancy without lactation in rats. Int ] Obesity 1987; 11 :423A. 6. len KLC, ]uuhl N, Lin PKH. Repeated pregnancy without lactation: effects on carcass composition and adipose tissue cellularity in rats.] Nutr 1988;1l8:93-8. 7. Zhong S, Almario R, Dubrinsky M, et al. Repeated pregnancy without lactation: effects on maternal glycemic control, pregnancy outcome, carcass composition, and fat distribution in rats. Metabolism 1990;39:1127-32. 8. Zhong S.] en KLC. Repeated pregnancy without lactation: effects on glucose homeostasis and in vitro lipogenesis in rats. Diabetes 1989;38(suppl 2):240A. 9. Schemel R, Mickelsen 0, Gill]L. Dietary obesity in rats: body weight and body fat accretion in seven strains of rats. J Nutr 1970;100:1041-8. 10. Hirsch J, Gallian MRC. Methods for the determination of adipose cell size in man and animals. ] Lipid Res 1968;9: 1l0-9. 11. Bucolo G, David H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chern 1973;19: 476-82. 12. Allain CA, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chern 1974;20:470-5. 13. Trinder P. Determination of blood glucose using an oxidase-peroxidase system with noncarcinogenic chromogen. ] Clin Pathol 1969;22: 158-61. 14. Herbert V. Lau K, Gottlieb CW, et al. Coated charcoal immunoassay of insulin. J Clin Endocrinol Metab 1965; 25:1375-84.

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