Conceived and designed the experiments: AJW ESL TPF. Performed the experiments: AJW ESL AW FRAC. Analyzed the data: AJW ESL. Contributed reagents/materials/analysis tools: AJW ESL FRAC. Wrote the paper: AJW ESL AW FRAC.
The authors have declared that no competing interests exist.
Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (
Human and animal model data support an association between sub-optimal intrauterine environments, altered fetal growth and the predisposition to adult-onset disease
The majority of studies manipulating the maternal environment have examined the interaction between macro- or micronutrient intake during gestation on offspring development and health. In the rat, maternal food restriction (30–50% of
However, there is now increasing evidence that maternal nutrition during the periconceptional period, and even prior to conception, may contribute to offspring disease risk
Using our established mouse model, we have narrowed the window of sensitivity further, identifying the terminal stages of oocyte maturation, as well as the preimplantation period, as being critically sensitive to maternal nutrition. Maternal LPD administered exclusively during these stages elevated fetal and postnatal growth, induced hypertension and vascular dysfunction and altered behavioural profiles in the adult offspring
There are few animal or human studies investigating whether deficient maternal nutrition around the time of conception contributes to the development of metabolic syndrome in aging animals. Such studies would determine the transitory or permanent nature of periconceptional programming processes. We have therefore extended our previous mouse studies
All mice and experimental procedures were conducted using protocols approved by, and in accordance with, the UK Home Office Animal (Scientific Procedures) Act 1986 and local ethics committee at the University of Southampton under UK Home Office Project Licence PPL30/2467. Maternal dietary treatments and offspring postnatal analyses were conducted as previously described
Females were fed either normal protein diet (NPD) throughout oocyte maturation (3.5 days prior to mating; Egg-NPD), low protein diet (LPD) exclusively during oocyte maturation (Egg-LPD), NPD though out gestation, LPD throughout gestation (LPD) or LPD exclusively during preimplantation development (3.5 days following mating; Emb-LPD).
g/kg Inclusion | ||
NPD | LPD | |
Starch Maize | 425 | 485 |
Sucrose | 213 | 243 |
Casein | 180 | 90 |
Corn Oil | 100 | 100 |
Cellulose | 50 | 50 |
Mineral mix |
20 | 20 |
Vitamin mix |
5 | 5 |
DL-methionine | 5 | 5 |
Choline Chloride | 2 | 2 |
Gross Energy, MJ/kg | 18.39 | 18.27 |
Mineral mix (AIN-76): (Special Diet Services).
Vitamin mix (AIN-76): (Special Diet Services).
Egg-NPD | Egg-LPD | NPD | LPD | Emb-LPD | ||
|
|
16.1±0.9 | 15.8±0.7 | 13.0±0.5 | 13.7±0.5 | 13.2±0.5 |
|
46.7±0.7 | 49.5±0.7 | 47.2±1.7 | 48.5±2 | 47.3±1.6 | |
|
109.0±0.6 | 113.1±1.3 | 110.8±1.6 | 110.1±0.7 | 111.0±1.9 | |
|
|
15.9±0.8 | 15.8±0.5 | 13.0±0.6 | 13.1±0.4 | 12.7±0.4 |
|
40.1±0.8 | 41.6±0.7 | 37.3±1 | 38.5±1.2 | 41.9±1.6 |
|
|
105.8±1.5 | 110.3±2.0 | 110.7±1.7 | 113.6±1.4 | 111.9±2.0 |
Values are means ± SEM for 9–13, 11–12, 9–12, 8–10 and 9–11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males and females respectively;
*P = 0.023 when compared to NPD females.
Egg-NPD | Egg-LPD | NPD | LPD | Emb-LPD | ||||||
6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | |
|
16.2±0.9 | 16.1±0.9 | 14.9±0.7 | 15.8±0.7 | 13.3±0.3 | 13.0±0.5 | 13.5±0.5 | 13.7±0.5 | 13.6±0.3 | 13.2±0.5 |
|
47.1±0.7 | 46.7±0.7 | 46.3±0.7 | 49.5±0.7 | 48.1±1 | 47.2±1.7 | 47.1±0.8 | 48.5±2.0 | 48.9±0.6 | 47.3±1.6 |
|
103.1±0.6 | 109±0.6 |
111.1±1.3 | 113.1±1.3 | 105.6±1.1 | 110.8±1.6 |
111.1±0.7 | 110.1±0.7 | 109.6±0.9 | 111.0±1.9 |
Values are means ± SEM for 55–56, 42–52, 38–53, 46–61 and 47–57 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males respectively for the 6 month cohort and 8–13, 11–12, 12, 7–8 and 11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males respectively for the 1 year cohort;
*P<0.05 when compared to the same treatment group.
Egg-NPD | Egg-LPD | NPD | LPD | Emb-LPD | ||||||
6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | 6 Month Cohort | 1 Year Cohort | |
|
15.0±0.8 | 15.9±0.8 | 14.5±0.5 | 15.8±0.5 | 12.3±0.4 | 13.0±0.6 | 12.6±0.2 | 13.1±0.4 | 13.4±0.3 | 12.7±0.4 |
|
40.2±0.8 | 40.3±0.8 | 40.3±0.7 | 41.6±0.7 | 39.5±1.1 | 37.3±1.0 | 39.9±0.6 | 38.5±1.2 | 42.8±0.7 | 41.9±1.6 |
|
102.6±0.8 | 105.8±1.5 | 108.8±1.24 | 110.3±2.0 | 104.2±1 | 110.7±1.7 |
110.4±1 | 113.6±1.4 | 112.1±0.9 | 111.9±2.0 |
Values are means ± SEM for 51–54, 50–57, 48–50, 48–50 and 48–49 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD females respectively for the 6 month cohort and 10–12, 10–12, 12, 8–9 and 11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD females respectively for the 1 year cohort;
*P<0.05 when compared to the same treatment group.
At 52 weeks of age, offspring SBP was measured by tail-cuff plethysmography as previously described
At 52 weeks of age animals were deprived of food (
Serum glucose concentrations were measured using the Infinity™ Glucose Oxidase Liquid Stable Reagent kit (Thermo) according to manufacturer's instructions. All samples were analysed in duplicate against a standard curve (0–200 mg/dL) and a reference sample analysed in every assay to measure inter-assay variation (10.46%).
Serum insulin was measured using a mouse ultra-sensitive ELISA (DRG Instruments) according to manufacturer's instructions. All samples were analysed against a standard curve (ranging from 0–6.5 µg/L) and a reference sample analysed in every assay to measure inter-assay variation (1.77%).
RNA was extracted from IBAT and retroperitoneal fat using the RNeasy® Lipid Tissue Mini Kit (QIAGEN, UK) according to manufacturer's instructions. On-column DNase I digestion was performed to remove contaminating genomic DNA. RNA was reverse transcribed to cDNA using the ImProm™II kit (Promega, UK) and a random priming strategy, according to the manufacturer's instructions. Intron-spanning primers used in this study are detailed in
Gene Name | Gene Symbol | Accession Number | Primer Sequences | Amplicon Length | Primer efficiency | |
Forward Primer | Reverse Primer | |||||
Uncoupling protein 1 (mitochondrial, proton carrier) |
|
NM_009463.2 |
|
|
84 | 1.99 |
Insulin receptor |
|
NM_010568.1 |
|
|
102 | 1.93 |
Adrenergic receptor, beta 3 |
|
NM_013462.3 |
|
|
61 | 1.99 |
Insulin-like growth factor I receptor |
|
NM_010513.2 |
|
|
89 | 1.98 |
Succinate dehydrogenase complex, subunit A, flavoprotein |
|
NM_023281 |
|
|
66 | 1.99 |
Phosphoglycerate kinase 1 |
|
NM_008828 |
|
|
65 | 1.99 |
TATA box binding protein |
|
NM_013684.3 |
|
|
90 | 1.94 |
Peptidylprolyl isomerase B |
|
NM_011149 |
|
|
92 | 1.95 |
All data (postnatal weights, SBP, organ allometry, serum insulin and glucose levels and fat pad gene expression) were analysed using a multilevel random effects regression model (SPSS version 18) as described previously
No difference in postnatal body weight was observed between Egg-LPD and Egg-NPD males or females for up to 1 year of age (
Effect of maternal LPD given either (A) exclusively during oocyte maturation (Egg-LPD), or (B) either throughout gestation (LPD) or exclusively during preimplantation development (Emb-LPD) on offspring growth Z-score profiles for up to 52 weeks when compared with offspring fed maternal normal protein diet (Egg-NPD and NPD respectively). n = 9–13, 11–12, 9–12, 8–10 and 9–11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males and females respectively; *P<0.01.
Egg-LPD males and females displayed elevated SBP at 52 weeks of age when compared to Egg-NPD offspring (
Mean systolic blood pressure at 52 weeks of age from (A) offspring fed NPD or LPD exclusively during oocyte maturation (Egg-NPD and Egg-LPD respectively), or (B) fed NPD or LPD throughout gestation, or LPD exclusively during preimplantation development (Emb-LPD). Values are means for 9–13, 11–12, 9–12, 8–10 and 9–11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males and females respectively ± SEM; *P<0.01.
No differences were observed between Egg-NPD and Egg-LPD offspring, or between NPD, LPD and Emb-LPD offspring in mean serum glucose or insulin levels (
NPD | LPD | Emb-LPD | Egg-NPD | Egg-LPD | |||||||
|
|
|
|
|
|
|
|
|
|
||
|
Males | 2.400 | 0.190 | 2.160 | 0.161 | 2.546 | 0.153 | 1.990 | 0.088 | 2.202 | 0.115 |
Females | 2.004 | 0.155 | 1.842 | 0.056 | 1.972 | 0.135 | 1.991 | 0.122 | 1.882 | 0.051 | |
|
Males | 174.767 | 21.51 | 214.501 | 19.031 | 169.887 | 18.523 | 166.634 | 12.341 | 156.187 | 12.626 |
Females | 153.665 | 8.989 | 149.36 | 7.317 | 162.561 | 17.852 | 150.881 | 9.386 | 151.341 | 8.937 |
Values are means ± SEM for 9–13, 11–12, 9–12, 8–10 and 9–11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males and females respectively.
Egg-LPD males had a reduced lung weight ratio (when expressed as percentage of body weight) compared to Egg-NPD males (P<0.001) and an increased carcass weight (body weight minus combined weight of organs and fat pads; P = 0.04) (
No differences in individual organ weights were observed between NPD, LPD and Emb-LPD offspring (
Mean fat pad: body weight ratio from (A) male or (B) female offspring fed NPD or LPD exclusively during oocyte maturation (Egg-NPD and Egg-LPD respectively), or (C) male or (D) female offspring fed NPD or LPD throughout gestation, or LPD exclusively during preimplantation development (Emb-LPD). Values are means for 9–13, 11–12, 9–12, 8–10 and 9–11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males and females respectively ± SEM; *P<0.02.
In our previous analysis of offspring development at 6 months, we identified positive associations between perinatal weight (at 3 weeks of age) and adult health markers, specific to the timing and duration of maternal diet
Correlations with Weight at 52 weeks; r values | Weight at 3 Weeks | Total Fat Pad Weight | Serum Insulin | Serum Glucose |
NPD | 0.366 | 0.613 |
0.578 |
0.085 |
LPD | 0.162 | 0.774 |
0.631 |
0.589 |
Emb-LPD | 0.614 |
0.632 |
0.691 |
−0.513 |
Egg-NPD | −0.085 | 0.657 |
0.551 |
0.215 |
Egg-LPD | −0.088 | 0.468 |
0.456 |
0.180 |
Values are means for 9–13, 11–12, 9–12, 8–10 and 9–11 Egg-NPD, Egg-LPD, NPD, LPD and Emb-LPD males and females respectively;
*p<0.03.
Weight at 1 year was significantly positively correlated with total fat pad mass and with serum insulin levels in all treatment groups (
As significant differences in body and fat pad weights were observed between NPD, LPD and Emb-LPD offspring, the expression profiles of several key adipose regulating genes from IBAT and white adipose tissue (WAT; from the retroperitoneum) were determined by RTqPCR. Emb-LPD females had higher expression of
Relative real-time RT-PCR expression values from male and female (A) interscapular and (B) retroperitoneal fat tissues. Values are means for 9–12, 8–10 and 9–11 NPD, LPD and Emb-LPD males and females respectively ± SEM; *P≤0.05.
Positive correlations of adult (52 weeks of age) weight and IBAT
IBAT | WAT | |||
|
|
|
|
|
|
0.774 |
0.623 |
−0.657 |
−0.739 |
|
0.614 |
0.689 |
−0.652 |
−0.792 |
|
0.396 | 0.622 |
−0.44 | −0.608 |
Values are means for 9–12, 8–10 and 9–11 NPD, LPD and Emb-LPD males and females respectively;
*p<0.05.
IBAT | WAT | |||
|
|
|
|
|
|
0.092 | 0.255 | −0.062 | −0.204 |
|
0.338 | 0.516 |
−0.099 | −0.324 |
|
−0.066 | 0.051 | 0.109 | −0.029 |
Values are means for 9–12, 8–10 and 9–11 NPD, LPD and Emb-LPD males and females respectively;
*p<0.05.
IBAT | WAT | |||
|
|
|
|
|
|
0.414 | 0.244 | −0.43 | −0.455 |
|
0.549 |
0.691 |
−0.528 |
−0.634 |
|
0.388 | 0.595 |
−0.442 | −0.583 |
Values are means for 9–12, 8–10 and 9–11 NPD, LPD and Emb-LPD males and females respectively;
*p<0.05.
IBAT | WAT | |||
|
|
|
|
|
|
0.013 | −0.159 | −0.129 | 0.128 |
|
0.510 |
0.418 | −0.597 |
−0.598 |
|
0.060 | −0.095 | −0.105 | 0.089 |
Values are means for 9–12, 8–10 and 9–11 NPD, LPD and Emb-LPD males and females respectively;
*p<0.05.
Development of the metabolic syndrome has been identified as a significant risk factor for future adult health. Metabolic syndrome components (obesity, insulin and glucose insensitivity and hypertension) have been identified in offspring derived from a range of different maternal gestational nutritional challenges
This study aimed to determine whether altered adult phenotypes evident at 6 months, induced through maternal periconceptional diet
To gain further mechanistic insights into the changes in body weight observed, we assessed whole body adiposity and gene expression profiles in white (retroperitoneal; WAT) and brown adipose tissue (interscapular; IBAT). Whilst LPD females had significantly reduced inguinal and retroperitoneal fat pad weights, no significant changes were observed in Emb-LPD female adiposity when compared to NPD females. Analysis of gene expression patterns revealed Emb-LPD females had elevated
The DOHaD hypothesis proposes that offspring metabolic homeostatic levels are set during fetal development in direct response to maternal nutritional cues. However, subsequent mismatch between predicted and actual nutritional levels increases adult disease risk
Adipose tissue and the adipokines it secrets also has a role in the development of cardiovascular disease. Factors such as angiotensinogen, adiponectin, leptin, angiotensin-converting enzyme and plasminogen activator inhibitor-1 (PAI-1) are all secreted from adipose tissue and have been implicated in the development of cardiovascular disease in the offspring
Offspring glucose and insulin homeostasis appear equally sensitive to maternal gestational nutrition. Both over- and under-nutrition result in offspring hyperphagia, adiposity and insulin resistance
As these and our own data highlight, sex specific phenotypic responses are often observed following maternal dietary manipulation. Female offspring from mice fed high-fat diets prior to gestation become hypertensive and hypercholesterolemic and have reduced locomotor activity
In conclusion, our results are the first to demonstrate the effects of maternal periconceptional nutrition on adult body weight, cardiovascular physiology and adiposity regulation in aging animals. Our findings reveal subtle but significant differences in central regulatory processes influencing adult body weight and adipose tissue development and function dependent upon the duration and stage in development during which maternal nutrition is manipulated. Whilst the evidence from human and other animal model studies support the premise that early sub-optimal environmental conditions can alter adult physiology and disease risk, the mechanisms underlying these long-term alterations are not yet fully understood. Further research is therefore essential for the identification of fundamental factors important for prevention of metabolic diseases in adulthood.
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We thank the Biomedical Facility, University of Southampton, for their technical support.