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Dietary protein restriction in pregnant females reduces offspring birth weight and increases the risk of developing obesity, type 2 diabetes and cardiovascular disease. Despite these grave consequences, few studies have addressed the effects of preconceptional maternal malnutrition. Here we investigate how a preconceptional low-protein (LP) diet affects offspring body mass and insulin-regulated glucose metabolism. Ten-week-old female mice (C57BL/6JBom) received either an LP or isocaloric control diet (8% and 22% crude protein, respectively) for 10 weeks before conception, but were thereafter fed standard laboratory chow (22.5% crude protein) during pregnancy, lactation and offspring growth. When the offspring were 10 weeks old, they were subjected to an intraperitoneal glucose tolerance test (GTT), and sacrificed after a 5-day recovery period to determine visceral organ mass. Body mass of LP male offspring was significantly lower at weaning compared with controls. A similar, nonsignificant, tendency was observed for LP female offspring. These differences in body mass disappeared within 1 week after weaning, a consequence of catch-up growth in LP offspring. GTTs of 10-week-old offspring revealed enhanced insulin sensitivity in LP offspring of both sexes. No differences were found in body mass, food intake or absolute size of visceral organs of adult offspring. Our results indicate that maternal protein restriction imposed before pregnancy produces effects similar to postconceptional malnutrition, namely, low birth weight, catch-up growth and enhanced insulin sensitivity at young adulthood. This could imply an increased risk of offspring developing lifestyle-acquired diseases during adulthood.
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Journal of Developmental Origins of Health and Disease, Page 1 of 11.
&Cambridge University Press and the International Society for Developmental Origins of Health and Disease 2012
doi:10.1017/S2040174412000347
ORIGINAL ARTICLE
Maternal protein restriction before pregnancy reduces
offspring early body mass and affects glucose
metabolism in C57BL/6JBom mice
A. Dudele
1
*, S. Lund
2
, N. Jessen
2
, G. Wegener
3
, G. Winther
3
, J. Elnif
4
, S. Frische
5
, T. Wang
1
and
D. Mayntz
6,7
1
Department of Bioscience, Zoophysiology, University of Aarhus, Aarhus C, Denmark
2
Medical Research Laboratory and Medical Department M (Endocrinology and Diabetes), Aarhus University Hospital, Aarhus C,
Denmark
3
Center for Basic Psychiatric Research, Aarhus University Hospital, Risskov, Denmark
4
Animal Nutrition, Department of Basic Animal and Veterinary Sciences, University of Copenhagen, Copenhagen, Denmark
5
The Water and Salt Research Centre, Institute of Anatomy, University of Aarhus, Aarhus C, Denmark
6
Department of Bioscience, Ecology and Genetics, University of Aarhus, Aarhus C, Denmark
7
Department of Genetics and Biotechnology, University of Aarhus, Research Centre Foulum, Tjele, Denmark
Dietary protein restriction in pregnant females reduces offspring birth weight and increases the risk of developing obesity, type 2 diabetes
and cardiovascular disease. Despite these grave consequences, few studies have addressed the effects of preconceptional maternal malnutrition.
Here we investigate how a preconceptional low-protein (LP) diet affects offspring body mass and insulin-regulated glucose metabolism.
Ten-week-old female mice (C57BL/6JBom) received either an LP or isocaloric control diet (8% and 22% crude protein, respectively) for
10 weeks before conception, but were thereafter fed standard laboratory chow (22.5% crude protein) during pregnancy, lactation and offspring
growth. When the offspring were 10 weeks old, they were subjected to an intraperitoneal glucose tolerance test (GTT), and sacrificed after a
5-day recovery period to determine visceral organ mass. Body mass of LP male offspring was significantly lower at weaning compared with
controls. A similar, nonsignificant, tendency was observed for LP female offspring. These differences in body mass disappeared within 1 week
after weaning, a consequence of catch-up growth in LP offspring. GTTs of 10-week-old offspring revealed enhanced insulin sensitivity in LP
offspring of both sexes. No differences were found in body mass, food intake or absolute size of visceral organs of adult offspring. Our results
indicate that maternal protein restriction imposed before pregnancy produces effects similar to postconceptional malnutrition, namely, low
birth weight, catch-up growth and enhanced insulin sensitivity at young adulthood. This could imply an increased risk of offspring developing
lifestyle-acquired diseases during adulthood.
Received 8 February 2012; Revised 5 April 2012; Accepted 27 April 2012
Key words: glucose tolerance, low-protein diet, preconception nutrition
Introduction
Non-communicable diseases, such as cardiovascular disease
(CVD), type II diabetes mellitus (T2DM), cancer and chronic
respiratory diseases, are responsible for a large proportion of
early deaths worldwide.
1
Although certain lifestyle choices in
combination with genetic predisposition are recognized to
markedly increase the risk of developing these diseases,
2
the
physiological and nutritional state of the mother during
gestation and lactation also have a significant role in deter-
mining future health of the offspring.
3,4
It was highly controversial when Barker proposed the
concept of ‘Fetal origins of adult disease’ in 1990,
5
but epi-
demiological studies revealed a clear relationship between
maternal undernutrition during pregnancy and postnatal
exposure to nutrient-rich environment, with an increased
prevalence of CVD and T2DM in adult male offspring.
6,7
These observations lead to the formation of thrifty phenotype
hypothesis
8
that has been widely accepted in the present time.
In addition to epidemiological observations, animal models
have repeatedly shown that intrauterine growth restriction
imposed by maternal protein or caloric restriction during
pregnancy leads to low birth weight and development of
CVD, T2DM and obesity in the offspring.
9,10
Although the evidence appears strong and is consistent
when it comes to effects caused by maternal nutrition during
pregnancy, the effects of maternal nutrition before pregnancy
on the future offspring are far less studied. Although indistinct,
epidemiological evidence indicates an association of precon-
ceptional consumption of protein and vegetables with reduced
risk for low birth weight
11,12
and consecutively development
of T2DM and CVD.
13,14
Animal studies supplement these
*Address for correspondence: A. Dudele, M.Sc., Department of Bioscience,
Zoophysiology, University of Aarhus, C.F. Møllers alle
´,Building1131,
DK-8000, Aarhus C, Denmark.
(Email a.dudele@gmail.com)
findings by demonstrating that maternal caloric and protein
restriction exclusively before pregnancy leads to accelerated
postnatal growth in ewes and affects offspring vascular function
in ewes and mice,
15,16
as well as increases male offspring
adiposity at young adulthood in mice.
17
Here we study how reduced maternal protein intake
before conception affects offspring early body mass and their
insulin-mediated glucose metabolism. As the offspring are not
directly exposed to protein restriction during development,
we expect the effects to operate through maternal body
composition and/or physiological state, and we therefore
examine maternal food intake, body mass, organ size and
adiposity before conception to investigate whether any of
these factors account for changes induced in the offspring.
Materials and Methods
Animals and housing
All experiments were performed in accordance with the
principles and guidelines of Danish legislation on animal
welfare, permit number 2006/561–1257. Danish Inspecto-
rate for Animal Experiments specifically approved this study.
Eight-week-old virgin C57BL/6JBom mice (53 females and 36
males) were obtained from Taconic (Lille Skensved, Denmark)
and caged individually in type 2 Macrolon cages (length: 23 cm;
width: 17 cm; height: 14 cm) at 12:12 h light:dark regime, 238C
and 60% humidity. Food and water was available ad libitum at
all times, except for the overnight starvation before Glucose
Tolerance Test (GTT), when food was removed for 14 h.
Experimental diets
Four dry pellet diets were used in the experiment (composition
is described in Table 1). NIH #31 M rodent diet was purchased
from Taconic (Denmark) and two custom-made isocaloric
experimental diets – Low protein (LP) and Standard (ST) –
were purchased from Brogaarden/Altromin (Denmark). Both
diets were similar in composition apart from the crude protein
and carbohydrate content (Table 1). Laboratory chow diet
(Altromin 1310) for rats and mice (Brogaarden/Altromin,
Denmark) was used as a standard laboratory chow diet.
Experimental protocol
Before the start of the experiment, all 53 female mice were
allowed to acclimate to the facilities for 2 weeks; during this
period, they were fed the same diet as they received at the
breeding facilities (NIH #31 M rodent diet). Subsequently,
they were weighed and divided into two groups that received
either an LP (n527) or an ST (n526) diet for the next
9–10 weeks. Following that, nine mice from each treatment
were subjected to a GTT, allowed to recover from this test for
5 days and sacrificed by cervical dislocation. The remaining
mice (n535, n
ST
517; n
LP
518) were placed in cages, with
an 8–9-week-old male for every female, for 48 h. From this
point onwards, all animals received laboratory chow diet
(Altromin 1310). After mating, the male was removed, but
the female remained in the individual cage until offspring
were born and weaned onto laboratory chow diet (Altromin
1310) at the age of 23 days and caged individually. Labora-
tory chow diet (Altromin 1310) was used after mating and
thereafter to ensure that both groups of female mice were
exposed to a change in the diet close to the time of concep-
tion. In order to minimize handling stress upon the animals,
neither maternal nor offspring body masses were measured at
birth. Owing to the low litter numbers, litter sizes were not
adjusted. At the age of 10 weeks, the offspring were subjected
to GTT, allowed to recover from it for 5 days and sacrificed
by cervical dislocation (Fig. 1).
Food intake measurements
Maternal food intake was measured weekly using a spillage
collecting system, for example,
18
on individually caged animals.
Animals received a preweighed portion of food, large enough to
ensure that they had access to food at all times. After 1 week,
the food remaining in the feeding tray and spillage collector was
collected and stored at 2258C for later measurements. Before
the measurements, the collected food was defrosted and dried at
608C for 24 h, and weighed to the nearest 0.001 g. A standard
curve for conversion of wet mass to dry mass was created for
each feed separately.
Offspring food intake was measured on individually housed
animals once a week after weaning. As all offspring received
Table 1. Composition of the diets used before and during the experiment
Diet NIH #31 M rodent diet LP ST Laboratory chow diet (Altromin 1310)
Crude protein 18 8.4 21.5 22.5
Carbohydrates 53 60 47.2 50.5
Crude fat 5.3 7.2 7.2 5.0
Crude fiber 4.5 5.0 5.1 4.5
Energy (kcal/g) 3.4 3.3 3.3 2.99
LP, low protein; ST, standard diet.
Values presented as g per 100 g dry mass.
2A. Dudele et al.
laboratory chow diet (Altromin 1310), spillage of food was
expected to be similar between groups, and therefore only
the difference of given and leftover food in the feeding tray
was measured.
GTTs
GTTs were performed after a 14-h overnight fast. Upon
measuring fasting blood glucose, 10% glucose solution was
injected intraperitoneally (2 g/kg), and blood was collected
from an incision at the tip of the tail for determination
of glucose concentration at 15, 30, 60 and 120 min. All
blood glucose concentration measurements were performed
using Bayer Contour
R
glucometer and Bayer Contour
R
test strips.
Insulin levels
Blood samples for insulin measurements were collected after
the 14 h starvation preceding the GTT and 2 h after glucose
injection during GTT. Blood was collected from an incision
at the tip of the tail into a heparinized hematoctit tube. Trunk
blood was collected after sacrifice to determine non-fasting
insulin concentration. All collected blood samples were
heparinized and spun, and plasma was collected and frozen at
2808C for later analysis. Plasma insulin concentration was
measured using Ultra Sensitive Mouse Insulin ELISA kit
according to manufacturer’s instructions.
Organ size measurements
Five days after GTT, animals were weighed to a precision of
0.1 g and sacrificed by cervical dislocation and decapitation.
Kidneys, liver and heart were dissected, blotted on a filter paper
in a standardized manner and weighed. The gastrointestinal
tract was dissected free to determine the length from the pyloric
sphincter to the rectum and the length of the small intestine
with precision to 1 mm. Stomach, cecum and colon were
separated and flushed with 0.9% saline, dried at 608Cfor72h
and weighed. The small intestine was blotted on filter paper
and wet mass taken. The mucosal layer was separated from a
4–6-cm-long piece of the small intestine (as described by Jensen
et al.
19
). The proportion of mucosal layer to the layer of
muscular tissue of the small intestine was determined after
drying both layers separately for 72h at 608C. The rest of the
small intestine was used for another experiment, and therefore
wet mass to dry mass relationship of the 4–6-cm-long piece was
used to calculate the dry mass of the whole small intestine. All
weight measurements were recorded with precision to 0.1 mg.
Data analysis
Data are presented as means (6S.E.) or as least square means
(6S.E.), and the differences between treatment groups (ST v.
LP) were tested within the same sex by Student’s t-test. For
comparison of GTT results, the area under the GTT curve
[Area Under the Curve (AUC)] was calculated. The fasting
Fig. 1. Experimental protocol used during the study.
Effects of preconception nutrition on offspring 3
blood glucose concentration was compared between ST and
LP groups within the same gender by Student’s t-test, and if
the values were not significantly different – which was always
the case baseline was calculated (fasting glucose value 3
120 min) and subtracted from the total AUC.
Analysis of Covariance (ANCOVA) was used to test the
effects of body mass and dietary treatment on organ size of
females and the effects of maternal body mass and diet before
conception on offspring early body mass. Maternal diet and
offspring body mass were used as covariates when offspring
organ size was measured. ANCOVA was used to test the
effect of maternal body mass before conception on mean
body mass of male or female offspring per litter at weaning.
Maternal diet and litter size were included as covariates
initially, but were removed from the model if not significant.
Litter size was always included in the covariate model initially,
but was removed if nonsignificant, which was always the case.
Offspring growth and food intake were compared using
repeated measures Multivariate Analysis of Variance (MAN-
OVA) in both genders separately.
Homogeneity of variances was tested using Levene’s test
(a.0.05), and if the variances proved to be unequal data
were compared using Welch Analysis of Variance (ANOVA).
All data analysis was conducted using JMP 8 and Sigma
Plot 11.0.
Results
Food intake and body mass changes of mothers
After 9 weeks of feeding on experimental diets, the total food
consumption of females averaged 149 62g (dry mass) per
animal in both groups, and there was no significant difference
between LP and ST treatments (t-test, DF 551, t50.08,
P50.94). On average, after 9 weeks of feeding dams had
consumed a total of 493 65 kcal per animal and this result was
not affected by the dietary treatment (t-test, DF 551, t50.08,
P50.94). However, owing to differences in the protein
content of experimental diets, over 9 weeks of feeding females
on the LP diet ingested a total of 12.5 60.4 g crude protein,
whereas females on the ST diet consumed 2.6 times more
protein (32.1 60.4 g; t-test, DF 551, t539.64, P,0.0001).
At the end of the feeding period, we observed significant dif-
ferences in female body mass. Dams exposed to the LP diet had
lower body mass and weighed 21.7 60.3 g, whereas ST females
weighed 22.5 60.3 g (t-test, DF 551, t52.1, P50.04).
We found no significant differences in the body mass of
dams exposed to experimental diets and used for GTT and
organ size measurements (Table 2).
GTT and non-fasting plasma insulin concentration of
females fed on experimental diets
We found no significant differences in GTT results (Fig. 2;
t-test, DF 515, t52
0.71, P50.49) between females fed
the LP diet and those fed the ST diet for 9 weeks. However,
we observed a nearly significant reduction in the non-fasting
plasma insulin concentration of females fed the LP diet
(0.33 60.07 mg/l; n58) compared with the non-fasting
insulin levels of females fed the ST diet (0.66 60.14 mg/l;
n58; t-test, DF 514, t52.06, P50.06).
Organ sizes of females fed experimental diets
Females fed the ST diet had significantly larger kidneys than
females on the LP diet (Table 2). The difference remained
Table 2. Organ sizes of female mice that had been fed the ST or the LP diet for a period of 9 weeks
ST LP Student’s t-test (P-value)
Body mass (g) 22.7 60.5 22.1 60.5 0.41
Heart WM (mg) 119.7 65.0 111.4 65.0 0.26
Liver WM (mg) 940 640 937 640 0.95
Kidneys WM (mg) 257 67 22666 0.004
Ovary fat WM (mg) 464 621 521 621 0.067
Kidney fat WM (mg) 93.5 67.8 112.2 67.8 0.11
Kidney fat 1ovary fat WM (mg) 558 625 633 625 0.046
Gut length (cm) 39.7 60.7 39.6 60.7 0.90
Small intestine length (cm) 32.4 60.5 33.4 60.5 0.17
Small intestine WM (mg) 872 662 991 672 0.23
Small intestine DM (mg) 142.4 614.9 166.3 617.2 0.31
Stomach DM (mg) 36.4 60.8 37.3 60.7 0.36
Colon 1cecum DM (mg) 62.6 63.8 71.5 63.8 0.13
Total gut DM (mg) (intestine 1cecum 1colon) 206 618 234 620 0.31
Mucosa proportion (% of total dry mass) 71.8 62.3 74.9 62.3 0.36
ST, standard diet; LP, low-protein diet; WM, wet mass; DM, dry mass.
All values presented as mean 6S.E.(n59 in each group) and compared by Student’s t-test.
4A. Dudele et al.
significant when corrected for body mass (least square
means 6S.E., ST 5260 65, LP 5230 65, ANCOVA, body
mass F
1
535.46, P50.0001, diet: F
1
522.09, P50.0003).
Fat deposits were significantly larger in females fed the LP
diet (Table 2), even after correcting for total body mass
(least square means 6S.E., ST 5551 623, LP 5640 623,
ANCOVA, body mass F
1
54.4, P50.053, diet: F
1
57.63,
P50.01). Visceral organ masses varied with body mass, but
neither the absolute (Table 2) nor the body mass-corrected
organ masses proved to be significantly different between the
two treatments.
Mating success
From the 35 females that were mated (LP: n518, ST:
n517), 10 females delivered offspring (LP: n56, ST:
n54). The mean litter size in the LP group was 6 67 (range
3–8 pups) and in the ST group 5.5 60.9 (range 4–7 pups).
The mean body mass of LP females who delivered offspring
was 18.8 60.5 g before and 21.4 60.6 g after 9 weeks of
feeding on the LP diet (n56). The mean body mass of ST
diet females who delivered offspring was 20.0 60.6 g before
and 22.0 60.8 after the dietary treatment (n54). In total,
51 pups were born: LP females delivered 31 pups (females:
n523, males: n58) and ST females delivered 20 pups
(females: n513, males: n57).
Offspring growth and food intake
Male offspring born to mothers exposed to the LP diet before
conception had significantly lower body mass at weaning
(8.64 60.4 g) than male offspring from the ST group
(10.16 60.4 g; t-test, DF 513, t52.59, P50.02). A simi-
lar tendency was observed for female offspring, but it was
only marginally significant. LP female offspring weighed
8.88 60.2 g and ST female offspring had a body mass of
9.59 60.6 g (t-test, DF 534, t51.91, P50.065).
A positive significant relationship was observed between
the average male offspring body mass per litter at weaning
and maternal body mass before conception (ANCOVA,
maternal body mass before conception: F
1
56.25, P50.04).
A similar but nonsignificant tendency was observed for female
offspring (ANCOVA, maternal body mass before conception:
F
1
54.56, P50.06). These differences in offspring body
mass disappeared within 1 week after weaning (Fig. 3). The
differences in body mass at weaning were not affected by
litter size for either males (ANCOVA, litter size: F
1
50.007,
P50.94) or females (ANCOVA, litter size: F
1
50.16,
P50.69).
Over the entire female offspring growth period, maternal diet
and offspring age had significant effects on body mass; however,
there was no interaction between the two (Fig. 3a; repeated
measures MANOVA, maternal diet: F
1, 33
54.44, P50.04;
offspring age: F
7, 27
5254.6, P,0.0001;maternaldiet3time:
F
7, 27
50.95, P50.48). Thus, growth curves did not have a
significantly different pattern. Only offspring age had a sig-
nificant effect on male offspring body mass, indicating that
growth in body mass did occur during the experiment (Fig. 3b;
repeated measures MANOVA, maternal diet: F
1, 13
53.23,
P50.1; offspring age: F
7, 7
50.72, P,0.0001; maternal
diet 3offspring age: F
7, 7
50.72, P50.66).
After the 6-week-long post-weaning period, female offspring
total food consumption averaged at 197.1 64 g per individual
in each group and there was no significant difference between
ST and LP treatments (t-test, DF 534, t50.117, P50.91).
For the same period of time, male offspring total food intake
averaged at 193.7 65 g per individual in both ST and LP
groups (t-test, DF 514, t50.21, P50.84; Fig. 4).
Offspring GTT and insulin
GTT performed on adult offspring revealed that LP male
offspring were more glucose tolerant than ST male offspring
(Fig.5dand5e,DF511, t-test, t52.2, P50.047), but
this was not observed for female offspring (Fig. 5a and 5b,
t-test, DF 533, t520.06, P50.948). However, LP female
offspring had a significantly lower blood insulin concentration
Fig. 2. Intraperitoneal glucose tolerance test (GTT) results. (a) Plasma glucose concentrations during GTT and (b) calculated Area Under
the Curve (AUC). GTT was performed on female mice fed either the standard (ST; n59) or the low-protein (LP; n59) diet for a
period of 9 weeks (mean 6S.E.). AUC values were compared using Student’s t-test.
Effects of preconception nutrition on offspring 5
than ST female offspring 2 h after glucose challenge (Fig. 5c,
t-test, DF 515, t52.4, P50.03). Such differences were not
observed for male offspring (Fig. 5f, t-test, DF 57, t51.7,
P50.13). No differences were observed in male and female
offspring fasting (Fig. 5c and 5f, male offspring, t-test, DF 57,
t520.93, P50.38; female offspring, t-test, DF 526,
t50.94, P50.35) and non-fasting insulin concentrations
(male offspring, DF 512, t-test, t520.04, P50.97; female
offspring, t-test, DF 530, t51.02, P50.32).
Offspring organ size
We found no differences in offspring absolute and body
mass-corrected visceral organ size (Tables 3 and 4). Stomach
dry mass was significantly lower in LP female group (Table 3),
but this difference disappeared when stomach dry mass was
corrected for wet body mass (Table 4). Litter size had no
significant effect on any of the measured values.
Discussion
Our study shows that protein restriction in the diet before
conception may lower body mass at weaning, followed by
catch-up growth and increased insulin sensitivity at early
adulthood in mice. These effects are similar to those pre-
viously described for protein restriction during pregnancy and
shown to be an indicator for increased risk of developing
T2DM, CVD and obesity in humans and animals.
6,7,9,20–27
It is therefore possible that LP diet before pregnancy imposes
similar types of risks for the development of lifestyle diseases
in the offspring as those described for malnutrition during
pregnancy.
Offspring subjected to intrauterine growth restriction are
often observed to display rapid postnatal growth if exposed to
a nutrient-rich environment after birth.
21,28
It is not clear
whether catch-up growth is a risk factor that acts in addition
to protein restriction when it comes to development of
T2DM, CVD and obesity. However, in rodents, it appears to
reduce offspring lifespan.
28–30
In our study, male offspring
Fig. 4. Offspring food intake after weaning. Female (a) and
male (b) offspring born to mothers fed the standard (ST) or the
low-protein (LP) diet for 10 weeks before conception. Litter
number, ST: n54, LP: n56.
Fig. 3. Offspring body mass after weaning. Female (a)andmale(b)
offspring born to mothers fed the standard (ST) or the low-protein
(LP) diet for 10 weeks before conception. Litter number, ST: n54,
LP: n56. Body masses were compared by Student’s t-test within
the same gender for separate time points. *Denotes a significant
difference between the maternal dietary treatments, P,0.05.
6A. Dudele et al.
Fig. 5. Offspring glucose tolerance test (GTT) results and plasma insulin concentration. Blood glucose concentrations and Area Under the
Curve (AUC) during GTT in female (a,b, respectively) and male (d,e, respectively) offspring born to mothers fed either the standard
(ST) or the low-protein (LP) diet for 10 weeks before conception. Female (c) and male ( f) offspring plasma insulin concentration after
fasting and 2 h after glucose injection (2g/kg) during GTT. In (c) and ( f), numbers in brackets indicate number of samples tested. Litter
number, ST: n54, LP: n56. AUC and plasma insulin concentrations were compared by Student’s t-test within the same gender.
*Denotes a significant difference between the maternal dietary treatments, P,0.05.
Table 3. Organ size of female and male offspring born to mothers fed either the ST or the LP diet for 10 weeks before conception
Female offspring Male offspring
Maternal diet Maternal diet
ST LP PST LP P
Body mass at 11 weeks of age (g) 19.7 60.3 19.2 60.2 0.17 23.5 60.6 22.8 60.6 0.40
Heart WM (mg) 111.6 63.4 108.8 62.5 0.51 136.5 64.4 126.0 64.1 0.10
Liver WM (mg) 1026 630 1028 622 0.95 1263 639 1266 637 0.96
Kidney WM (mg) 241 64 244 63 0.56 296 66 288 66 0.34
Kidney fat (mg) 55.6 64.8 55.8 63.6 0.98 69.7 64.9 66.5 64.2 0.63
Gut length (cm) 41.4 60.7 41.8 60.5 0.67 42.5 61.1 41.3 61.0 0.46
Small intestine length (cm) 32.2 60.5 32.3 60.4 0.91 34.3 61.0 32.9 60.8 0.28
Small intestine WM (mg) 1074 637 1017 629 0.23 1106 645 1071 639 0.56
Small intestine DM (mg) 193.2 67.6 187.7 65.8 0.57 201.7 68.9 192.2 67.7 0.44
Stomach DM (mg) 32.5 60.8 29.8 60.6 0.011 31.6 61.1 32.9 61.1 0.45
Colon 1cecum DM (mg) 80.6 62.1 79.4 61.6 0.65 83.7 63.1 79.8 62.9 0.37
Total gut DM (mg) 274 69 267 67 0.55 287 611 272 69 0.32
Mucosa proportion (% of total dry mass) 74.7 62.1 77.1 61.6 0.35 73.9 63.2 80.0 63.0 0.20
ST, standard diet; LP, low-protein diet; WM, wet mass; DM, dry mass.
All values presented as mean 6S.E. (female offspring, ST: n513, LP: n523; male offspring, ST: n57, LP: n58; litter number, ST: n54,
LP: n56) and compared by Student’s t-test.
Effects of preconception nutrition on offspring 7
from the LP group had lower body mass at weaning than
those from the ST group, but these differences disappeared
within a few weeks after weaning (Fig. 3). This implies that
the offspring have gone through a period of fast catch-up
growth. Such catch-up growth has been shown in humans
and several different animal species,
24,31,32
including mice,
33
and is associated with enhanced insulin sensitivity.
34
This
pattern corresponds well with our observed results of low
male offspring early body mass, catch-up growth and
enhanced insulin sensitivity in both sexes (Fig. 5) and indi-
cates the presence of an event cascade similar to that induced
by maternal gestational malnutrition.
In other animal models, similar offspring responses have
been found. For example, rat offspring from mothers exposed
to the LP diet during pregnancy also have increased insulin
sensitivity at early adulthood (6–10 weeks), but this effect
disappears with age (15 months), when offspring develop
insulin resistance and glucose intolerance.
22–24
As our find-
ings from manipulating preconception nutrition resemble the
cascade of events caused by malnutrition during pregnancy, it
is possible that offspring born from mothers that received
the LP diet before pregnancy are at the same increased risk
of developing insulin resistance later in life. In a previous
study with the same mouse strain and the same experimental
protocol as in the current experiment, it was found that LP
male offspring have increased adiposity as early as 46 days
of age.
17
The two studies in combination indicate that
maternal protein restriction before pregnancy affects offspring
body mass, body composition and glucose metabolism and
can therefore modify offspring risk of developing lifestyle-
acquired diseases.
Although animal models using the LP diet after conception
are rather well studied in terms of produced offspring pheno-
types, the mechanisms underlying these effects are less clear.
35
Some of the effects of LP diet exposure during pregnancy have
been attributed to insufficient amount of amino acid supply to
the embryo during development;
9
however, our study and
previous experiments
17
have showed that similar effects can be
induced by preconceptional protein restriction. This indicates
that factors other than maternal diet during pregnancy can
be involved in fetal programming. Unfortunately, our study
design does not allow us to fully elucidate whether or not the
risks are imposed through maternal body composition or
physiological state, rather than by direct effects of amino acid
insufficiency during development. Although studies exposing
animals to protein restriction during pregnancy mostly ascribe
the effects obtained to reduced amino acid supply,
9
there is
evidence that maternal body mass index, body composition,
hypertension and diabetes among other factors can all have an
impact on offspring health.
36,37
Studies in human population show that maternal body
mass index before pregnancy and weight gain during preg-
nancy are good predictors of offspring birth weight.
12,38
Although we did not measure maternal weight gain during
pregnancy to avoid stress to the pregnant females,
39
we
observed a significant relationship between maternal body
mass at conception and male offspring body mass at
weaning and a similar, nearly significant tendency for female
offspring. It is likely that low body mass at weaning among
LP offspring can be explained, at least partly, by low maternal
body mass at conception, but these effects remain to be
investigated.
Table 4. Body mass-corrected organ size of female and male offspring born to mothers who were fed either the ST or the LP diet for 10 weeks before
conception
Female offspring Male offspring
Least square means 6S.E. Effect tests (P-value) Least square means 6S.E. Effect tests (P-value)
ST LP Body mass Maternal diet ST LP Body mass Maternal diet
Heart WM (mg) 109.3 62.9 110.0 62.2 0.0002 0.85 134.7 63.6 127.5 63.3 0.015 0.17
Liver WM (mg) 998 616 1037 612 ,0.0001 0.07 1247 631 1281 629 0.01 0.45
Kidney WM (mg) 238 64 246 63 0.0007 0.10 293 63 291 63,0.0001 0.68
Kidney fat (mg) 54.9 64.9 55.2 63.7 0.42 0.95 54.9 64.9 55.2 63.7 0.42 0.96
Intestine WM (mg) 1055 635 1029 627 0.01 0.57 1099 648 1076 641 0.62 0.73
Intestine DM (mg) 189.8 67.3 190.8 65.7 0.03 0.92 200.1 69.5 193.5 68.1 0.54 0.62
Stomach DM (mg) 31.1 60.9 33.3 60.8 0.006 0.10 31.1 60.9 33.3 60.8 0.006 0.097
Colon 1cecum DM (mg) 81.8 62.1 79.8 61.5 0.46 0.44 82.6 62.7 80.7 62.5 0.035 0.63
Total gut DM (mg) 270 68 271 67 0.037 0.96 283 611 275 69 0.29 0.57
ST, standard diet; LP, low-protein diet; WM, wet mass; DM, dry mass.
All values presented as least square mean 6S.E. (female offspring, ST: n513, LP: n523; male offspring, ST: n57, LP: n58; litter
number, ST: n54, LP: n56) of measures adjusted for wet body mass. In all analysis, litter size was used as a covariate first, but as it did not
have a significant effect it was removed from the final model.
8A. Dudele et al.
Studies on rodents pertaining to maternal obesity induced
by a high-fat diet show that obesity at conception programs
offspring obesity and insulin resistance at adulthood but do
not necessarily affect birth weight.
40
Our results showed that
females fed the LP diet had significantly larger fat deposits at
the time of conception than did females fed the ST diet
(Table 2). It is difficult to discriminate whether increased
adiposity in mothers fed the LP diet had an effect on off-
spring early body mass, but as suggested by previous studies it
could have influenced their glucose metabolism.
41
Another aspect of offspring programming is maternal
insulin-mediated glucose metabolism. Elevated maternal
blood glucose differentially affects birth weight: whereas mild
elevation above normoglycemia produces macrosomic off-
spring, highly elevated glucose levels induce fetal growth
restriction.
42
GTT on females at the end of preconceptional
feeding period showed no differences in fasting glucose values
or glucose tolerance (Fig. 2). However, non-fasting blood
insulin concentration was nearly significantly lower in LP-fed
dams, a known result of LP feeding.
43,44
As exposure of
preimplantation murine embryos to insulin increases fetal
growth rate in vitro,
45
it is possible that lowered maternal
insulin levels in the LP group at the time of conception have
restricted offspring growth.
Whereas our study mainly addresses the physiological
changes of the mother that can affect offspring development
and phenotype, other studies have shown that similar effects in
offspring can be induced without apparent changes in maternal
phenotype. Thus, female mice of the MF-1 strain fed an
LP diet for a very short period of time close to conception
(63.5 days, which includes the period of oocyte maturation
and preimplantation) produced offspring that at 21 and
52 weeks of age had elevated systolic blood pressure
16,46
and
attenuated endothelium-dependent vasodilation in mesenteric
arteries at the age of 22 weeks
16
compared with control
offspring whose mothers were supplied with sufficient protein.
As such short feeding periods may be expected to have
only minor effects on the body composition of the mothers,
compared with the 10-week period of feeding in our experi-
ment, the measured alterations in the MF-1 offspring could
very well have been caused by epigenetic changes in the mothers
eggs or in the young zygote. It is therefore of high importance
to investigate such epigenetic effects in the future studies on the
presented animal model of preconceptional nutrition.
Other studies have found that maternal LP nutrition
during pregnancy can program offspring food intake and
preferences,
47
which can potentially explain differences in
offspring growth, development of obesity, etc. In our study,
we measured offspring food intake throughout the experi-
ment and found no differences. Thus, we find no evidence
that differences observed in offspring phenotypes were caused
by differences in their feeding behavior after weaning.
Fertility rates observed in the study were rather low. Such
low conception rates may be explained by the use of
suboptimal mating protocol. Estrous cycle frequency in mice
is 4–5 days, but the females were only caged with the males
for 2 days, which may explain the low rate of conception. For
the future studies, females and males should be housed
together for a longer period (4–5 days). The study would have
had greater statistical power if the litter numbers had
been higher; however, the data presented were tested using
appropriate statistical tests, and the effects of maternal pre-
conceptional treatment are evident. It is, however, possible
that because of low litter numbers some of the effects could
not have not been detected, and therefore future studies need
to be carried out, where another mating protocol is used and
higher litter numbers are obtained.
As suggested by previous epidemiological studies, maternal
preconceptional nutrition and physiological state can have an
impact on the future health of the offspring in humans.
11,12
Our studies using an animal model of preconceptional
nutrition
17
confirm the importance of these effects. It is
therefore important to investigate the effects of maternal
preconceptional state further, as, once established, they can
provide effective grounds for intervention and prevention
of disease development in future generations of human
populations.
Overall, our findings show that maternal protein restriction
before pregnancy induces gender-specific effects in the off-
spring, namely, reduced early body mass, male offspring
catch-up growth and enhanced insulin sensitivity at early
adulthood in male and female offspring. These effects closely
resemble the effects of maternal protein restriction during
pregnancy on offspring physiology and may therefore imply
an increased risk for the offspring to develop obesity, type II
diabetes and CVD in later life.
The physiological mechanisms inducing these changes are
less clear and remain to be investigated.
Acknowledgments
D.M. and T.W. were funded by the grants from Danish
Research Council. G.W. was funded by Danish Medical
Research Council, grant no. 271-08-0768. We thank Heidi
Meldgaard Jensen and Rasmus Buchanan for their assistance
during experiments.
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Effects of preconception nutrition on offspring 11
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