- Email: [email protected]
Alterations in the offsprings' response to an oral glucose challenge associated with acute maternal malnutrition during late pregnancy
Nutrition Research, Vol. 19, No. 4, pp. 579-588, 1999 Copyright 0 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0271-5317/99/&w front matter
ELSEVIER
PI1 s0271-5317(9!900023-8
ALTERATIONS IN THE OFFSPRINGS’ RESPONSE TO AN ORAL GLUCOSE CHALLENGE ASSOCIATED WITH ACUTE MATERNAL MALNUTRITION DURING PREGNANCY
LATE
Sam N. Pennington, Ph.D.lz3; F. Melinda Carver, M.S.‘; Ivan A. Shibley, Jr., Ph.D.‘*4; Neil E. Jeansonne, Ph.D.‘; Debbie F. Miles, M.S.‘; Steve A. Lynch, B.S.‘; Benjamin R. King, Ph.D. ’and J. Sue Pennington, MS.*; Departments of ‘Biochemistry, ‘Comparative Medicine and 3Pediatrics, School of Medicine, East Carolina University, Greenville, NC 27858
ABSTRACT
An acutely malnourished pregnant rat model was used to test the effect of intrauterine growth retardation on the subsequent response of the offspring to a loading dose of glucose. Timed-pregnant Harlan-Sprague Dawley rats were given access to only tap water from day 17 through day 19 of their pregnancies (72 hours total). Control mothers had access to lab chow and water throughout their pregnancies. The litters were randomly culled to 4 males and 4 females, whenever possible, and the offspring were given oral glucose tolerance tests at 60, 180 and 360 days of age. At 60 and 180 days of age, malnourished offspring were glucose intolerant (e.g., at 60 days, control males 60 minute serum [glucose] = 9.3+O.lmM, [malnourished] = 10.1&0.2; [control females] = 9.6M.2 versus [malnourished] = 10.5M.3; (overall ~~0.05 for treatment). Plasma insulin levels for the malnourished offspring in response to the glucose load were not significantly different at 0 and 30 minutes after the glucose dose. However, at 60 and 120 minutes after the dose, serum insulin levels were significant lower in the malnourished animals ([control males] = 478f47 pMoles/liter versus 351ti4; [control females]=436f54 versus 300f33; ~~0.05 for treatment). Thus, the insulin to glucose ratio (pMoles/mM) were markedly decreased at the latter time points (control male ratio=49ti, malnourished=34+3; control female=44*4 versus 29&l; p< 0.05 for treatment). At 360 days of age, both male and female malnourished offspring had blood glucose levels comparable to controls in response to the oral glucose challenge. However, while the insulin levels of the malnourished males were comparable to control values, malnourished female insulin levels were lower at all time points. B 1999 Elsena Science Inc. Key Words: Malnutrition, Growth suppression, Insulin, Glucose uptake, Insulin synthesis Correapondii Author: Sam N. Pennington, Department of Biochemistry, School of Medicine, East Carolina University, Greenville, NC, USA 27858. Phone: 252-816-2076; Fax: 252-816-3260; E-mail: [email protected] 4Current Address: Box 7009 Reading,
Ivan A. Shibley, Jr., Department PA 19610 USA
579
of Chemistry,
Penn State Berks Campus
580
S.N. PENNINGTON et al.
INTRODUCTION Undernutrition during gestation leads to a number of untoward outcomes for the pregnancy. Data from studies in several different countries as well as comparisons of different ethnic groups living in the same country have shown that the incidence of obstetrical complications, premature labor and intrauterine growth retardation (IUGR) closely follows poor dietary habits (1). Both human and animal studies have shown that intrauterine growth retardation is associated with behavioral, metabolic and physiological abnormalities that may not fully appear until adult life (2-5). Maternal malnutrition leading to fetal IUGR has been reported to correlate with the development of glucose intolerance/insulin resistance in adulthood. Retrospective studies in humans (6-9) as well as prospective studies in animal models (10) have shown that a significant correlation exists between IUGR and the emergence of insulin resistance in the adult. In fact, many of these studies have suggested that the insulin resistance associated with IUGR could be related to the development of hypertension, hyperlipidemia, abnormal glucose homeostasis, and vascular disease, i.e. Syndrome X (11). Of the non-primate models of pregnancy, the rat model is widely used for developmental studies and to examine the effects of nutrition on fetal development. While the rat model does have differences relative to the human, the similar aspects of this model are recognized and the model is widely used. Studies using the food-deprived pregnant rat have also shown that maternal caloric deprivation results in a decrease in fetal insulin and insulin-like growth factor-I (IGF-I) levels but IGF-II was little changed (12-14). In the current study, acute maternal malnutrition during late pregnancy (72 hour fast on days 17- 19) was used to induce fetal IUGR and the offspring were subsequently examined at 60, 180 and 360 days of age to determine their response to an oral loading dose of glucose. Given the reported effects of maternal malnutrition on intrauterine growth and glucose metabolism in the offspring, the current experiments were conducted to determine if a very simple experimental model of maternal malnutrition could evoke outcomes similar to studies using more complex protein/calorie reduction paradigms. The simplicity of the maternal manipulation involved in the current paradigm suggested that the results should be highly reproducible within an individual laboratory and between laboratories. The dependent variables determined included maternal weight on days 17 and 20 of the pregnancy and at parturition, offspring weights for days 0.5-l 75, offspring blood glucose and insulin levels in response to an oral glucose challenge (2.0 g&g of body weight), and pancreatic weights.
MATERIALS
AND METHODS
Treatment Groups- Timed pregnant Sprague Dawley rats were obtained from Harlan Sprague Dawley, Indianapolis, IN. The rats were housed on beta-chip bedding throughout the study and they received tap water ad lib. and standard rat chow except that malnourished mothers were given access to only tap water from 0800 hours of day 17 through 0800 hours of day 20 of their pregnancies (72 hours total). Chow was returned to the fasted rats and their pups were born between days 21 and 22. All litters were culled to 8 pups per litter (4 females and 4 males, whenever possible). Pups were weaned on day 21 and individually housed thereafter. Pups were weighed at birth, weekly until age 60 days and then monthly thereafter. Oral Glucose Tolerance Test (GTT)- Rats from the malnourished (n= 26) and control (n= 24) litters received an oral GTT at 60, 180 and 360 days of age. Food and water were removed from the animals at 0200 hours and returned at 0800 hours. The six hours of food derivation was sufficient to produce a stable blood glucose level based upon a compilation of all “0” time values for the various treatment groups. The animals were weighed and gavaged with glucose (2 g/kg) dissolved in sterile water. Blood samples (0.4ml) were withdrawn from the tail vein of non-anesthetized animals at 0, 30, 60 and 120 minutes after glucose administration using 25 gauge-3/8 inch butterfly needles. Blood was collected in heparinized microcentri-
RESPONSE
TO A GLUCOSE
CHALLENGE
581
fuge tubes and immediately centrifuged at 10,000 x g for 20 seconds. Plasma was harvested and divided into 2 aliquots: one sample was placed on wet ice for glucose determination and one sample was snap frozen and stored at -80°C for insulin assay. Glucose levels were determined that day on a GemStar II Analyzer. Insulin Assay- Plasma insulin assays were performed in duplicate by a competitive binding method using a rat insulin RIA kit obtained from Linco Inc., St. Louis, MO. Briefly: plasma was thawed, combined with 12’I-tagged insulin and anti-rat insulin antibody and incubated at 4’C overnight. Precipitating antibody was added, the sample incubated for 20 minutes and then centrifuged at 3,000 x g for 40 minutes. Supernatants were discarded, the samples drained and the resulting pellet counted in a Beckman Gamma 7000 counter. The percent of maximum binding was calculated for both standards and the unknowns. A reference curve was generated from the standards and the insulin concentration was determined by interpolation to the reference curve. The results were recorded and converted to picomoles/liter. Quality control “low range” and “high range” standards were furnished by the kit manufacturer and these samples were included in every assay. The within and between variance for the assays were 7.6% and 9.90%, respectively. Statistical Analyses of Data- Group means and standard errors as well as post hoc testing of significant differences between the means for the experimental groups were calculated using the general linear model procedure of the SAS/PC statistical program. Statistically significant differences between group means for control and malnourished animals were determined by analysis of variance for repeated measures with pcO.05 accepted as significant. Significant differences between individual groups were determined by the use of the Least Mean Square (LMS) post hoc test. RESULTS In ongoing studies, we have found that maternal fasting for 72 hours on days 17-19 of the pregnancy resulted in a significant weight deficiency in both the mothers and their offspring (Figures 1 and 2). The malnourished offsprings’ weight deficits relative to controls were sustained into adulthood (Figure 2B).
-
-CONTROL
-
MALN.
*
200
, 17
I
, 18
I
I 19
I
, 20
I
, 21
I
,
= peO.05 vs CONTROL
I
22
DAY OF PREGNANCY FIG. 1 Effect of a 72 hour maternal fast on maternal weights on days 17, 20 and at parturition. Animals were weighed on the mornings of day 17 (start of fast) and day 20 (morning of the day when fast ended). They were also weighted on the morning after parturition (shown in the figure as day 21.5, which is a mean value) * = ~~0.05 for the malnourished maternal weights relative to control maternal weights on the same day. ’
S.N. PENNINGTON
582
et al.
18 17
E 9 ?
0
Control
m
Maln.
*
I-l
14 :: I
g (3 i
10.5
*
B
8.5 J
?--l0.5
7.0
PUP AGE (days) FIG 2-A- Effect of a 72 hour maternal fast on the weights of the offspring as neonates. Pups were weighed on the morning of each day indicated in the figure. While additional studies have shown that there is also a sex-related difference, the data in this figure are collapsed across sexes. * = ~~0.05 for the mean weight of pups from malnourished litters relative to the mean weight of pups from control litters compared on the same day. Data are means f S.E., n L 20 for each treatment group.
XXI
1 J
E _ b 400-
is-3oo-
5
200-
-Controtfemales -
Maln t&males
FIG 2-E Effect of a 72 hour maternal fast on the weights of the offspring. the mornings of the days indicated. * = ~~0.05 for the mean weight of pups tive to the mean weight of pups from control litters compared on the same weights at the early time points are masked by the “I”’ axis scale necessary figure. Data are means f SE., n > 6 offspring for each treatment group.
The animals were weighted on from malnourished litters reladay. Significant differences in to place all of the data in one
583
RESPONSE TO A GLUCOSE CHALLENGE
In response to an oral glucose challenge at sixty days of age (Figure 3, upper panels) the malnourished animals were hyperglycemic relative to controls. The initial insulin response (O-30 min.) to the oral glucose challenge was not different between the malnourished and control animals but the second phase of insulin release (30-120 min.) was significantly lower in the malnourished animals relative to control. (Figure 3, lower panels). This difference in insulin response resulted in a greatly diminished insulin to glucose ratio during the later phase of the glucose challenge/insulin release. Almost identical results, with rr+ spect to glucose levels and insulin response, were obtained when these animals were challenged with the same dose of glucose at 180 days of age (data not shown).
I
,,,,
0
r
25
50
75
’
I
lal
I
125
1
150
19'
TMIA=lER-
-b-w
150
i.
, 0
.
I
a
I
,
50
.
I
.
75
I
im
1
I
125
.
I
150
TM3uTmGl.m6Ew6E(~)
FIG 3 Comparison of the response of control and malnourished offspring to an oral glucose challenge at 60 days of age. Upper left panel= male blood glucose levels, upper right= female blood glucose; lower left panel= male insulin levels, lower right panel = female insulin levels. Data are means f S.E., n 2 6 pups for each time point. * = ~~0.05 relative to the control value at the same time point. In these same animals, the gross pancreatic weights were not different between malnourished and control animals (Figure 4).
S.N. PENNINGTON
584
et al.
FIG. 4 Effect of maternal malnutrition of the pancreatic weight of the offspring. Left panel= males, right panel=females lo-
101
6’ :.I,,.,.,.,,, 0
25
50
75
100
125
150
150
Y
-ED
I
I
0
25
’
I
50
’
I
75
I
100
’
1
125
’
I
150
TlwAFlERGLucosEDosE m-m
FIG. 5 Response to a glucose challenge in 360 da old offsRAng from control and malnourished litters. While the lasma lucose levels were not dt2 erent in etther the males or females, insulin levels revealed a mar Eed dt2 erence by which the animals achieved normal male blood glucose levels, upper right=female blood glucose; lower le lower ri t panel = female insulin levels. Data are means + S.E., n 2 6 *=p
RESPONSE TO A GLUCOSE CHALLENGE
585
Thus, the results of these initial experiments suggested that the marked caloric restriction experienced by these animals during the latter portion of their in utero development had altered their pancreatic function in some manner. It is known that the fetal pancreas undergoes significant growth during the period at which the caloric restriction occurred in these studies (15) but the pancreatic sizes showed little difference between treatment groups. Microscopic and functional studies of these tissues are currently ongoing and initial observations suggest that, contrary to reduced protein/calorie malnourished animals( 16), the in utero acutely food deprived offspring have comparable numbers and sizes pancreatic islets compared to controls(data not shown) Interestingly, when given a glucose challenge at one year of age, both the male and female malnourished animals had blood glucose levels comparable to the control offspring (Figure 5, upper panels). However, the insulin response of the malnourished males was very different from that of the malnourished females (Figure 5, lower panels) DISCUSSION The effect of early malnutrition on the subsequent response of the animal to the administration of glucose or insulin has been widely studied {see for example references (9,17,18)}. Swenne and coworkers have published a significant series of studies using the protein-energy deficient rat to examine the effect of early, postnatal malnutrition on the subsequent response to glucose and insulin administration as well as doing studies of the effect of aging on pancreatic function( 19). The paradigm used by Swenne and numerous other investigators is to maintain the experimental animals on a low protein (58%) or normal/high protein (18-25%) diet from three through six weeks of age and to examine these animals or their offspring for metabolic/hormonal changes. The results of the studies reported here using animals acutely malnourished in utero are comparable to those of Swenne and others in several ways. It is important to note that the outcome of the postnatal studies that malnourished the animals from three through six weeks of age are dependent on the particular tissue(s) studies (20) and also appear to give quite difference results depending on the timing of the protein deficiency (21). Neither of these parameters were examined in the current study. In the present study, the loss of tissue mass caused by the maternal fast was shared by both mother and fetuses inasmuch as immediately following parturition, weights of the malnourished mothers were significantly smaller than the weights of the chow-fed controls and the fetal/neonatal weights of the malnourished pups were also significantly lower. Brain weights were not different between the progeny (data not shown). Thus, the current model is comparable with respect to its effect on maternal/fetal weight to other malnourished models which reduce calories throughout the pregnancy. However, acute maternal fasting caused a very different response to a glucose challenge in the offspring. In studies of sustained caloric reduction models, retrospective population studies in humans (6-8) as well as prospective studies in animal models (10) have shown that a significant correlation exists between fetal growth suppression and the later emergence of insulin resistance in the adult. In the current study, short-term maternal fasting caused significant fetal growth suppression that was associated with hyperglycemia in response to a glucose load at 60 and 180 days of age but the offspring were not insulin resistant but rather were hypoinsulinemic during the 30- 120 minute period after the glucose dose. The timing of the decreased insulin release suggested that the second phase response in which de nova synthesized insulin is released may be defective (15). The response of the offspring from malnourished litters to a loading dose of glucose at 360 days of age was also distinct from the response of sustained protein-energy reduction models. While at 360 days of age acutely malnourished males and females both had blood glucose levels comparable to controls in response to a glucose challenge, the mechanisms by which the normal glycemia was achieved appeared to be quite different. The malnourished male animals had a normal glycemic response and a normal insulin re-
586
S.N. PENNINGTON
et al.
quite different. The malnourished male animals had a normal glycemic response and a normal insulin response suggesting that these animals had regained their ability to synthesize/secrete de now synthesized insulin while the female animals were normal glycemic in the presence of significantly less insulin at all time points (i.e., O-120 min. compared to the control females). Thus, by 360 days of age, the control female offspring appeared to have developed a hyperinsulinemic response to the glucose challenge while the malnourished females’ insulin levels were essentially “normal” based on the insulin levels observed in control animals at 60 and 180 days of age. Furthermore, at 360 days of age, the malnourished females remained smaller than their controls while the malnourished males were comparable in weight to the control males at this age. The known relationship between body mass index and insulin sensitivity could be related to the marked differences in the responses of the control females but this point remains conjecture at the present time. Obviously, these results require duplication and if confirmed, a study will be undertaken of the mechanism of the altered insulin response.
CONCLUSIONS I. 2. 3. 4. 5. 6.
7.
8.
Acute maternal malnutrition resulted in a significant weight deficiency in both the mothers and their offspring. The weight deficit of the malnourished offspring is sustained relative to non-malnourished control offspring. Gross pancreatic weights were not different between the two groups of offspring. At 60 and I80 days of age, the malnourished offspring were hyperglycemic in response to an oral GTT compared to non-malnourished controls. At 60 and 180 days of age, the initial phase (O-30 min.) insulin response to the oral GTT was not different between the malnourished and control offspring. At 60 and 180 days of age, the second phase (30-120 min.) insulin response of the malnourished animals was significantly depressed relative to control animals. This lack of insulin synthesis/secretion resulted in a greatly diminished insulin to glucose ratio during second phase insulin release. At 360 days of age, both the malnourished male and female animals were normal glycemic in response to the oral GTT. The mechanism by which the response had been normalized appeared quite different between the malnourished male and female animals. Both the control and malnourished male animals showed a normal insulin response at this age while the control female animals were hyperinsulinemic relative to the malnourished females through out the glucose challenge Coincidental with the changes in the insulin response of the malnourished male and female offspring at 360 days of age, the weight difference between the male treatment groups was lost while the malnourished females remained significantly smaller than the control females.
ACKNOWLEDGMENTS This work was supported, in part, by grants from the University of North Carolina Nutrition Institute and the Children’s Miracle Network to SNP. REFERENCES
1. Crawford MA. A think tank on nutrition in the primary prevention of low birthweight, cerebral palsy and related handicaps. Nut and Health I992;8:45-55.
587
RESPONSE TO A GLUCOSE CHALLENGE
2.
Lueder FL, Ogata ES. Uterine artery ligation in the maternal rat alters fetal tissue glucose utilization. Pediatr Res 1990:28:464-8.
3.
Barker DJP, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993:34 1:938-4 1.
4.
Woodside B, Cohen LR, Jans JE. Effects of food restriction during concurrent lactation and pregnancy in the rat. Physiol Behav 1987;40:613-5.
5.
Crouse JR. Ischemic heart disease. Lancet 1993;341:93 1-2.
6.
Parker DJP. Fetal growth and adult disease. Brit J Ob/Gyn 1992;99:275-6.
7.
Hales CN. Fetal nutrition and adult diabetes. Sci & Med 1994;1:54-63.
8.
Nestler JE. Assessment of insulin resistance. Sci & Med 1994; 1:58-67.
9.
Thompson CH, Sanderson AL, Sandeman D, Stein C, Brothwick A, Radda GK, Phillips DIW. Fetal growth and insulin resistance in adult life: Role of skeletal muscle morphology. Clinical Sciences 1997;92(3):291-6.
10. Simmons RA, Flozak AS, Ogata ES. The effect of insulin and insulin-like growth factor-l on glucose transport in normal and small for gestation age rats. Endocrinol 1993;133:1361-8. 11. Barker DJP, Hales CN, Fall CHD, Osmond C, Phillips K, Clark PMS. Type 2 (non-insulin dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relationship to reduced fetal growth. Diabetologia 1993;36:62-7. 12. Davenport ML, D’Ercole AJ, Underwood LE. Effect of maternal fasting on fetal growth, serum insulinlike growth factors (IGFs), and tissue IGF messenger ribonucleic acids. Endocrinol 1990;126:2062-7. 13. Straus DS, Ooi GT, Orlowski CC, Rechler MM. Expression of genes for insulin-like growth factor-I, IGF-II and IGF binding proteins- 1 and -2 in the fetal rat under conditions of intrauterine growth retardation caused by maternal fasting. Endocrinol 199 1; 128:5 18-25. 14. Bernstein IM, DeSouza MM, Copeland KC. Insulin-like growth factor I in substratedeprived, retarded fetal rats. Pediatr Res 1991:30:154-7.
growth-
15. Kroc M. ; Laing V, Go W, et al. editors.The Pancreas: Biology, Pathology and Disease. second ed. New York, NY: Raven Press, 1993; 38, Normal function of the endocrine pancreas. p. 75 l-8. 16. Eriksson UK, Swenne I. Diabetes of pregnancy: fetal macrosomia, hyperinsulinism and islet hyperplasia in the offspring of rats subject to temporary protein-energy malnutrition early in life. Pediatr Res 1993;34(6):791-5. 17. Grace cj, Swenne I, Milner RD. Long-term effects on glucose tolerance and insulin secretory response to glucose following a limited period of severe protein or energy malnutrition in young rats. Upsala J of Medicial Sciences 1991;96(3):177-83. 18. Holness MJ, Sugden MC. Suboptimal protein nutrition in early life later influences insulin action in pregnant rats. Diabetologia 1996;39( 1):12-2 1.
S.N. PENNINGTON et al.
588
19. Borg LA, Dahl N, Swenne I. Age-dependent differences in insulin secretion and intracellular handling of insulin in isolated pancreatic islets of the rat. Diabete et Metabolisme 1995;2 1(6):408-14. 20. Shepherd PR, Prins J, Smith CD. Poor fetal nutrition causes long-term changes in expression of insulin signaling components in adipocytes. Am J Physiology 1997;273( I ):E46-5 1. 2 1. Escriva F, Kergoat M, Bailbe D, Pascual-Leone AM, Portha B. Increased insulin action in the rat after protein malnutrition early in life. Diabetologia 1991;34(8):559-64. Accepted
for
publication
September
20,
1998.
ELSEVIER
PI1 s0271-5317(9!900023-8
ALTERATIONS IN THE OFFSPRINGS’ RESPONSE TO AN ORAL GLUCOSE CHALLENGE ASSOCIATED WITH ACUTE MATERNAL MALNUTRITION DURING PREGNANCY
LATE
Sam N. Pennington, Ph.D.lz3; F. Melinda Carver, M.S.‘; Ivan A. Shibley, Jr., Ph.D.‘*4; Neil E. Jeansonne, Ph.D.‘; Debbie F. Miles, M.S.‘; Steve A. Lynch, B.S.‘; Benjamin R. King, Ph.D. ’and J. Sue Pennington, MS.*; Departments of ‘Biochemistry, ‘Comparative Medicine and 3Pediatrics, School of Medicine, East Carolina University, Greenville, NC 27858
ABSTRACT
An acutely malnourished pregnant rat model was used to test the effect of intrauterine growth retardation on the subsequent response of the offspring to a loading dose of glucose. Timed-pregnant Harlan-Sprague Dawley rats were given access to only tap water from day 17 through day 19 of their pregnancies (72 hours total). Control mothers had access to lab chow and water throughout their pregnancies. The litters were randomly culled to 4 males and 4 females, whenever possible, and the offspring were given oral glucose tolerance tests at 60, 180 and 360 days of age. At 60 and 180 days of age, malnourished offspring were glucose intolerant (e.g., at 60 days, control males 60 minute serum [glucose] = 9.3+O.lmM, [malnourished] = 10.1&0.2; [control females] = 9.6M.2 versus [malnourished] = 10.5M.3; (overall ~~0.05 for treatment). Plasma insulin levels for the malnourished offspring in response to the glucose load were not significantly different at 0 and 30 minutes after the glucose dose. However, at 60 and 120 minutes after the dose, serum insulin levels were significant lower in the malnourished animals ([control males] = 478f47 pMoles/liter versus 351ti4; [control females]=436f54 versus 300f33; ~~0.05 for treatment). Thus, the insulin to glucose ratio (pMoles/mM) were markedly decreased at the latter time points (control male ratio=49ti, malnourished=34+3; control female=44*4 versus 29&l; p< 0.05 for treatment). At 360 days of age, both male and female malnourished offspring had blood glucose levels comparable to controls in response to the oral glucose challenge. However, while the insulin levels of the malnourished males were comparable to control values, malnourished female insulin levels were lower at all time points. B 1999 Elsena Science Inc. Key Words: Malnutrition, Growth suppression, Insulin, Glucose uptake, Insulin synthesis Correapondii Author: Sam N. Pennington, Department of Biochemistry, School of Medicine, East Carolina University, Greenville, NC, USA 27858. Phone: 252-816-2076; Fax: 252-816-3260; E-mail: [email protected] 4Current Address: Box 7009 Reading,
Ivan A. Shibley, Jr., Department PA 19610 USA
579
of Chemistry,
Penn State Berks Campus
580
S.N. PENNINGTON et al.
INTRODUCTION Undernutrition during gestation leads to a number of untoward outcomes for the pregnancy. Data from studies in several different countries as well as comparisons of different ethnic groups living in the same country have shown that the incidence of obstetrical complications, premature labor and intrauterine growth retardation (IUGR) closely follows poor dietary habits (1). Both human and animal studies have shown that intrauterine growth retardation is associated with behavioral, metabolic and physiological abnormalities that may not fully appear until adult life (2-5). Maternal malnutrition leading to fetal IUGR has been reported to correlate with the development of glucose intolerance/insulin resistance in adulthood. Retrospective studies in humans (6-9) as well as prospective studies in animal models (10) have shown that a significant correlation exists between IUGR and the emergence of insulin resistance in the adult. In fact, many of these studies have suggested that the insulin resistance associated with IUGR could be related to the development of hypertension, hyperlipidemia, abnormal glucose homeostasis, and vascular disease, i.e. Syndrome X (11). Of the non-primate models of pregnancy, the rat model is widely used for developmental studies and to examine the effects of nutrition on fetal development. While the rat model does have differences relative to the human, the similar aspects of this model are recognized and the model is widely used. Studies using the food-deprived pregnant rat have also shown that maternal caloric deprivation results in a decrease in fetal insulin and insulin-like growth factor-I (IGF-I) levels but IGF-II was little changed (12-14). In the current study, acute maternal malnutrition during late pregnancy (72 hour fast on days 17- 19) was used to induce fetal IUGR and the offspring were subsequently examined at 60, 180 and 360 days of age to determine their response to an oral loading dose of glucose. Given the reported effects of maternal malnutrition on intrauterine growth and glucose metabolism in the offspring, the current experiments were conducted to determine if a very simple experimental model of maternal malnutrition could evoke outcomes similar to studies using more complex protein/calorie reduction paradigms. The simplicity of the maternal manipulation involved in the current paradigm suggested that the results should be highly reproducible within an individual laboratory and between laboratories. The dependent variables determined included maternal weight on days 17 and 20 of the pregnancy and at parturition, offspring weights for days 0.5-l 75, offspring blood glucose and insulin levels in response to an oral glucose challenge (2.0 g&g of body weight), and pancreatic weights.
MATERIALS
AND METHODS
Treatment Groups- Timed pregnant Sprague Dawley rats were obtained from Harlan Sprague Dawley, Indianapolis, IN. The rats were housed on beta-chip bedding throughout the study and they received tap water ad lib. and standard rat chow except that malnourished mothers were given access to only tap water from 0800 hours of day 17 through 0800 hours of day 20 of their pregnancies (72 hours total). Chow was returned to the fasted rats and their pups were born between days 21 and 22. All litters were culled to 8 pups per litter (4 females and 4 males, whenever possible). Pups were weaned on day 21 and individually housed thereafter. Pups were weighed at birth, weekly until age 60 days and then monthly thereafter. Oral Glucose Tolerance Test (GTT)- Rats from the malnourished (n= 26) and control (n= 24) litters received an oral GTT at 60, 180 and 360 days of age. Food and water were removed from the animals at 0200 hours and returned at 0800 hours. The six hours of food derivation was sufficient to produce a stable blood glucose level based upon a compilation of all “0” time values for the various treatment groups. The animals were weighed and gavaged with glucose (2 g/kg) dissolved in sterile water. Blood samples (0.4ml) were withdrawn from the tail vein of non-anesthetized animals at 0, 30, 60 and 120 minutes after glucose administration using 25 gauge-3/8 inch butterfly needles. Blood was collected in heparinized microcentri-
RESPONSE
TO A GLUCOSE
CHALLENGE
581
fuge tubes and immediately centrifuged at 10,000 x g for 20 seconds. Plasma was harvested and divided into 2 aliquots: one sample was placed on wet ice for glucose determination and one sample was snap frozen and stored at -80°C for insulin assay. Glucose levels were determined that day on a GemStar II Analyzer. Insulin Assay- Plasma insulin assays were performed in duplicate by a competitive binding method using a rat insulin RIA kit obtained from Linco Inc., St. Louis, MO. Briefly: plasma was thawed, combined with 12’I-tagged insulin and anti-rat insulin antibody and incubated at 4’C overnight. Precipitating antibody was added, the sample incubated for 20 minutes and then centrifuged at 3,000 x g for 40 minutes. Supernatants were discarded, the samples drained and the resulting pellet counted in a Beckman Gamma 7000 counter. The percent of maximum binding was calculated for both standards and the unknowns. A reference curve was generated from the standards and the insulin concentration was determined by interpolation to the reference curve. The results were recorded and converted to picomoles/liter. Quality control “low range” and “high range” standards were furnished by the kit manufacturer and these samples were included in every assay. The within and between variance for the assays were 7.6% and 9.90%, respectively. Statistical Analyses of Data- Group means and standard errors as well as post hoc testing of significant differences between the means for the experimental groups were calculated using the general linear model procedure of the SAS/PC statistical program. Statistically significant differences between group means for control and malnourished animals were determined by analysis of variance for repeated measures with pcO.05 accepted as significant. Significant differences between individual groups were determined by the use of the Least Mean Square (LMS) post hoc test. RESULTS In ongoing studies, we have found that maternal fasting for 72 hours on days 17-19 of the pregnancy resulted in a significant weight deficiency in both the mothers and their offspring (Figures 1 and 2). The malnourished offsprings’ weight deficits relative to controls were sustained into adulthood (Figure 2B).
-
-CONTROL
-
MALN.
*
200
, 17
I
, 18
I
I 19
I
, 20
I
, 21
I
,
= peO.05 vs CONTROL
I
22
DAY OF PREGNANCY FIG. 1 Effect of a 72 hour maternal fast on maternal weights on days 17, 20 and at parturition. Animals were weighed on the mornings of day 17 (start of fast) and day 20 (morning of the day when fast ended). They were also weighted on the morning after parturition (shown in the figure as day 21.5, which is a mean value) * = ~~0.05 for the malnourished maternal weights relative to control maternal weights on the same day. ’
S.N. PENNINGTON
582
et al.
18 17
E 9 ?
0
Control
m
Maln.
*
I-l
14 :: I
g (3 i
10.5
*
B
8.5 J
?--l0.5
7.0
PUP AGE (days) FIG 2-A- Effect of a 72 hour maternal fast on the weights of the offspring as neonates. Pups were weighed on the morning of each day indicated in the figure. While additional studies have shown that there is also a sex-related difference, the data in this figure are collapsed across sexes. * = ~~0.05 for the mean weight of pups from malnourished litters relative to the mean weight of pups from control litters compared on the same day. Data are means f S.E., n L 20 for each treatment group.
XXI
1 J
E _ b 400-
is-3oo-
5
200-
-Controtfemales -
Maln t&males
FIG 2-E Effect of a 72 hour maternal fast on the weights of the offspring. the mornings of the days indicated. * = ~~0.05 for the mean weight of pups tive to the mean weight of pups from control litters compared on the same weights at the early time points are masked by the “I”’ axis scale necessary figure. Data are means f SE., n > 6 offspring for each treatment group.
The animals were weighted on from malnourished litters reladay. Significant differences in to place all of the data in one
583
RESPONSE TO A GLUCOSE CHALLENGE
In response to an oral glucose challenge at sixty days of age (Figure 3, upper panels) the malnourished animals were hyperglycemic relative to controls. The initial insulin response (O-30 min.) to the oral glucose challenge was not different between the malnourished and control animals but the second phase of insulin release (30-120 min.) was significantly lower in the malnourished animals relative to control. (Figure 3, lower panels). This difference in insulin response resulted in a greatly diminished insulin to glucose ratio during the later phase of the glucose challenge/insulin release. Almost identical results, with rr+ spect to glucose levels and insulin response, were obtained when these animals were challenged with the same dose of glucose at 180 days of age (data not shown).
I
,,,,
0
r
25
50
75
’
I
lal
I
125
1
150
19'
TMIA=lER-
-b-w
150
i.
, 0
.
I
a
I
,
50
.
I
.
75
I
im
1
I
125
.
I
150
TM3uTmGl.m6Ew6E(~)
FIG 3 Comparison of the response of control and malnourished offspring to an oral glucose challenge at 60 days of age. Upper left panel= male blood glucose levels, upper right= female blood glucose; lower left panel= male insulin levels, lower right panel = female insulin levels. Data are means f S.E., n 2 6 pups for each time point. * = ~~0.05 relative to the control value at the same time point. In these same animals, the gross pancreatic weights were not different between malnourished and control animals (Figure 4).
S.N. PENNINGTON
584
et al.
FIG. 4 Effect of maternal malnutrition of the pancreatic weight of the offspring. Left panel= males, right panel=females lo-
101
6’ :.I,,.,.,.,,, 0
25
50
75
100
125
150
150
Y
-ED
I
I
0
25
’
I
50
’
I
75
I
100
’
1
125
’
I
150
TlwAFlERGLucosEDosE m-m
FIG. 5 Response to a glucose challenge in 360 da old offsRAng from control and malnourished litters. While the lasma lucose levels were not dt2 erent in etther the males or females, insulin levels revealed a mar Eed dt2 erence by which the animals achieved normal male blood glucose levels, upper right=female blood glucose; lower le lower ri t panel = female insulin levels. Data are means + S.E., n 2 6 *=p
RESPONSE TO A GLUCOSE CHALLENGE
585
Thus, the results of these initial experiments suggested that the marked caloric restriction experienced by these animals during the latter portion of their in utero development had altered their pancreatic function in some manner. It is known that the fetal pancreas undergoes significant growth during the period at which the caloric restriction occurred in these studies (15) but the pancreatic sizes showed little difference between treatment groups. Microscopic and functional studies of these tissues are currently ongoing and initial observations suggest that, contrary to reduced protein/calorie malnourished animals( 16), the in utero acutely food deprived offspring have comparable numbers and sizes pancreatic islets compared to controls(data not shown) Interestingly, when given a glucose challenge at one year of age, both the male and female malnourished animals had blood glucose levels comparable to the control offspring (Figure 5, upper panels). However, the insulin response of the malnourished males was very different from that of the malnourished females (Figure 5, lower panels) DISCUSSION The effect of early malnutrition on the subsequent response of the animal to the administration of glucose or insulin has been widely studied {see for example references (9,17,18)}. Swenne and coworkers have published a significant series of studies using the protein-energy deficient rat to examine the effect of early, postnatal malnutrition on the subsequent response to glucose and insulin administration as well as doing studies of the effect of aging on pancreatic function( 19). The paradigm used by Swenne and numerous other investigators is to maintain the experimental animals on a low protein (58%) or normal/high protein (18-25%) diet from three through six weeks of age and to examine these animals or their offspring for metabolic/hormonal changes. The results of the studies reported here using animals acutely malnourished in utero are comparable to those of Swenne and others in several ways. It is important to note that the outcome of the postnatal studies that malnourished the animals from three through six weeks of age are dependent on the particular tissue(s) studies (20) and also appear to give quite difference results depending on the timing of the protein deficiency (21). Neither of these parameters were examined in the current study. In the present study, the loss of tissue mass caused by the maternal fast was shared by both mother and fetuses inasmuch as immediately following parturition, weights of the malnourished mothers were significantly smaller than the weights of the chow-fed controls and the fetal/neonatal weights of the malnourished pups were also significantly lower. Brain weights were not different between the progeny (data not shown). Thus, the current model is comparable with respect to its effect on maternal/fetal weight to other malnourished models which reduce calories throughout the pregnancy. However, acute maternal fasting caused a very different response to a glucose challenge in the offspring. In studies of sustained caloric reduction models, retrospective population studies in humans (6-8) as well as prospective studies in animal models (10) have shown that a significant correlation exists between fetal growth suppression and the later emergence of insulin resistance in the adult. In the current study, short-term maternal fasting caused significant fetal growth suppression that was associated with hyperglycemia in response to a glucose load at 60 and 180 days of age but the offspring were not insulin resistant but rather were hypoinsulinemic during the 30- 120 minute period after the glucose dose. The timing of the decreased insulin release suggested that the second phase response in which de nova synthesized insulin is released may be defective (15). The response of the offspring from malnourished litters to a loading dose of glucose at 360 days of age was also distinct from the response of sustained protein-energy reduction models. While at 360 days of age acutely malnourished males and females both had blood glucose levels comparable to controls in response to a glucose challenge, the mechanisms by which the normal glycemia was achieved appeared to be quite different. The malnourished male animals had a normal glycemic response and a normal insulin re-
586
S.N. PENNINGTON
et al.
quite different. The malnourished male animals had a normal glycemic response and a normal insulin response suggesting that these animals had regained their ability to synthesize/secrete de now synthesized insulin while the female animals were normal glycemic in the presence of significantly less insulin at all time points (i.e., O-120 min. compared to the control females). Thus, by 360 days of age, the control female offspring appeared to have developed a hyperinsulinemic response to the glucose challenge while the malnourished females’ insulin levels were essentially “normal” based on the insulin levels observed in control animals at 60 and 180 days of age. Furthermore, at 360 days of age, the malnourished females remained smaller than their controls while the malnourished males were comparable in weight to the control males at this age. The known relationship between body mass index and insulin sensitivity could be related to the marked differences in the responses of the control females but this point remains conjecture at the present time. Obviously, these results require duplication and if confirmed, a study will be undertaken of the mechanism of the altered insulin response.
CONCLUSIONS I. 2. 3. 4. 5. 6.
7.
8.
Acute maternal malnutrition resulted in a significant weight deficiency in both the mothers and their offspring. The weight deficit of the malnourished offspring is sustained relative to non-malnourished control offspring. Gross pancreatic weights were not different between the two groups of offspring. At 60 and I80 days of age, the malnourished offspring were hyperglycemic in response to an oral GTT compared to non-malnourished controls. At 60 and 180 days of age, the initial phase (O-30 min.) insulin response to the oral GTT was not different between the malnourished and control offspring. At 60 and 180 days of age, the second phase (30-120 min.) insulin response of the malnourished animals was significantly depressed relative to control animals. This lack of insulin synthesis/secretion resulted in a greatly diminished insulin to glucose ratio during second phase insulin release. At 360 days of age, both the malnourished male and female animals were normal glycemic in response to the oral GTT. The mechanism by which the response had been normalized appeared quite different between the malnourished male and female animals. Both the control and malnourished male animals showed a normal insulin response at this age while the control female animals were hyperinsulinemic relative to the malnourished females through out the glucose challenge Coincidental with the changes in the insulin response of the malnourished male and female offspring at 360 days of age, the weight difference between the male treatment groups was lost while the malnourished females remained significantly smaller than the control females.
ACKNOWLEDGMENTS This work was supported, in part, by grants from the University of North Carolina Nutrition Institute and the Children’s Miracle Network to SNP. REFERENCES
1. Crawford MA. A think tank on nutrition in the primary prevention of low birthweight, cerebral palsy and related handicaps. Nut and Health I992;8:45-55.
587
RESPONSE TO A GLUCOSE CHALLENGE
2.
Lueder FL, Ogata ES. Uterine artery ligation in the maternal rat alters fetal tissue glucose utilization. Pediatr Res 1990:28:464-8.
3.
Barker DJP, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993:34 1:938-4 1.
4.
Woodside B, Cohen LR, Jans JE. Effects of food restriction during concurrent lactation and pregnancy in the rat. Physiol Behav 1987;40:613-5.
5.
Crouse JR. Ischemic heart disease. Lancet 1993;341:93 1-2.
6.
Parker DJP. Fetal growth and adult disease. Brit J Ob/Gyn 1992;99:275-6.
7.
Hales CN. Fetal nutrition and adult diabetes. Sci & Med 1994;1:54-63.
8.
Nestler JE. Assessment of insulin resistance. Sci & Med 1994; 1:58-67.
9.
Thompson CH, Sanderson AL, Sandeman D, Stein C, Brothwick A, Radda GK, Phillips DIW. Fetal growth and insulin resistance in adult life: Role of skeletal muscle morphology. Clinical Sciences 1997;92(3):291-6.
10. Simmons RA, Flozak AS, Ogata ES. The effect of insulin and insulin-like growth factor-l on glucose transport in normal and small for gestation age rats. Endocrinol 1993;133:1361-8. 11. Barker DJP, Hales CN, Fall CHD, Osmond C, Phillips K, Clark PMS. Type 2 (non-insulin dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relationship to reduced fetal growth. Diabetologia 1993;36:62-7. 12. Davenport ML, D’Ercole AJ, Underwood LE. Effect of maternal fasting on fetal growth, serum insulinlike growth factors (IGFs), and tissue IGF messenger ribonucleic acids. Endocrinol 1990;126:2062-7. 13. Straus DS, Ooi GT, Orlowski CC, Rechler MM. Expression of genes for insulin-like growth factor-I, IGF-II and IGF binding proteins- 1 and -2 in the fetal rat under conditions of intrauterine growth retardation caused by maternal fasting. Endocrinol 199 1; 128:5 18-25. 14. Bernstein IM, DeSouza MM, Copeland KC. Insulin-like growth factor I in substratedeprived, retarded fetal rats. Pediatr Res 1991:30:154-7.
growth-
15. Kroc M. ; Laing V, Go W, et al. editors.The Pancreas: Biology, Pathology and Disease. second ed. New York, NY: Raven Press, 1993; 38, Normal function of the endocrine pancreas. p. 75 l-8. 16. Eriksson UK, Swenne I. Diabetes of pregnancy: fetal macrosomia, hyperinsulinism and islet hyperplasia in the offspring of rats subject to temporary protein-energy malnutrition early in life. Pediatr Res 1993;34(6):791-5. 17. Grace cj, Swenne I, Milner RD. Long-term effects on glucose tolerance and insulin secretory response to glucose following a limited period of severe protein or energy malnutrition in young rats. Upsala J of Medicial Sciences 1991;96(3):177-83. 18. Holness MJ, Sugden MC. Suboptimal protein nutrition in early life later influences insulin action in pregnant rats. Diabetologia 1996;39( 1):12-2 1.
S.N. PENNINGTON et al.
588
19. Borg LA, Dahl N, Swenne I. Age-dependent differences in insulin secretion and intracellular handling of insulin in isolated pancreatic islets of the rat. Diabete et Metabolisme 1995;2 1(6):408-14. 20. Shepherd PR, Prins J, Smith CD. Poor fetal nutrition causes long-term changes in expression of insulin signaling components in adipocytes. Am J Physiology 1997;273( I ):E46-5 1. 2 1. Escriva F, Kergoat M, Bailbe D, Pascual-Leone AM, Portha B. Increased insulin action in the rat after protein malnutrition early in life. Diabetologia 1991;34(8):559-64. Accepted
for
publication
September
20,
1998.