maternal overnutrition impacts offspring adiposity and brain appetite markers-modulation by...

ORIGINAL ARTICLE

Maternal Overnutrition Impacts Offspring Adiposity and Brain AppetiteMarkers-Modulation by Postweaning DietS. Rajia,* H. Chen*� and M. J. Morris*

*Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.

�Department of Medical and Molecular Bioscience, Faculty of Science, University of Technology, Broadway, NSW, Australia.

Obesity is a worldwide epidemic in both adults and children. In

many societies, maternal obesity is becoming the most common

perturbation in pregnancy (1). This increases the likelihood of a

cluster of disorders in offspring, including impaired glucose toler-

ance, insulin resistance, dyslipidaemia, hypertension and obesity (2).

Animal studies indicate that maternal nutrition exerts an important

influence on the development of hypothalamic neurons involved in

energy homeostasis and thus metabolic profile in offspring (3–5).

The process by which prenatal and early postnatal environmental

factors have long-term effects in adulthood is called programming.

This is now considered a potential mechanism underlying the

development of obesity, hyperphagia, cardiovascular disease and

metabolic disorders later in life whereby altered gene expression

occurs as a consequence of an adaptation to environmental

changes (6–8). The plasticity of the hypothalamus in late gestation

and early postnatal life (7) plays a key role in programming where

adipose tissue secreted cytokines such as leptin are considered to

act as key neurotrophic factors during brain development, which

may permanently influence appetite and metabolism (3, 4, 9). Both

maternal and postweaning HFD can impact on the expression of

hypothalamic gene that regulates food intake and metabolism (5,

10).

Journal ofNeuroendocrinology

Correspondence to:

Margaret J. Morris, Department of

Pharmacology, School of Medical

Sciences, The University of New South

Wales, Sydney, NSW 2052, Australia

(e-mail: [email protected]).

Maternal obesity has long-term consequences for the development of hypothalamic neurones

involved in energy homeostasis and the metabolic profile in offspring. In the present study, we

compared the effects of maternal obesity induced by longstanding high-fat diet (HFD) with

milder postnatal overfeeding during suckling induced by litter size reduction. Female Sprague–

Dawley rats consumed chow (C) or HFD. On postnatal day 1, litters from chow dams were

adjusted to three per dam (small litter, CS) versus 12 control (normal litter, CN). Litters from

HFD dams were adjusted to 12 per dam and fed HFD after weaning to induce obesity (HN).

Thus, two degrees of maternal overnutrition were produced (CS and HN). To test whether post-

weaning diet can amplify the effects of maternal obesity, male offspring weaned onto chow or

HFD were followed to 21 weeks. Maternal postnatal overnutrition (CS) and HFD-induced mater-

nal obesity (HN) increased body weight and fat mass in offspring compared to those from con-

trol dams (CN). Significant glucose intolerance was induced by both degrees of maternal

overnutrition, but only in offspring consuming HFD. HFD-induced maternal obesity (HN) was

linked to increased offspring leptin, insulin, lipids, insulin resistance and hyperphagia, and was

exaggerated by postweaning HFD. No effect of maternal postnatal overnutrition (CS) was seen

on these parameters. Hypothalamic signal transducer and activator of transcription-3 and sup-

pressor of cytokine signalling-3 mRNA were significantly elevated by maternal HFD (HN) in the

HFD-fed offspring. The data obtained suggest that even mild maternal overnutrition (CS) led to

increased adiposity, glucose intolerance and altered brain appetite regulators in offspring. A

greater impact of HFD-induced maternal obesity was evident. Marked additive effects were

observed when animals consumed a HFD postweaning.

Key words: maternal overnutrition, high-fat diet, glucose homeostasis, leptin, adiposity, appetite

regulation.

doi: 10.1111/j.1365-2826.2010.02005.x

Journal of Neuroendocrinology 22, 905–914

ª 2010 The Authors. Journal Compilation ª 2010 Blackwell Publishing Ltd

Journal of NeuroendocrinologyFrom Molecular to Translational Neurobiology

The circulating hormones insulin and leptin constitute important

homeostatic signals influencing hypothalamic fuel metabolism. The

adipocyte-derived hormone leptin regulates body weight and food

intake via leptin receptors (Ob-Rb) located predominantly in the

hypothalamus (11). Subsequent phosphorylation of signal transduc-

tion and activator of transcription-3 (STAT-3) inhibits the expression

of orexigenic neuropeptide Y (NPY) and agouti-related protein

(AgRP), whereas anorexigenic pro-opiomelanocortin (POMC) and

cocaine- and amphetamine-regulated transcript are stimulated to

inhibit food intake (12). However, the high leptin levels in obese

subjects, proportional to adipose tissue mass (13), fail to bring

about weight loss as a result of central leptin resistance (14). In

particular, suppressor of cytokine signalling-3 (SOCS-3) has been

suggested as a potential mediator of central leptin resistance in

obesity (15). Leptin has also been reported to increase sympathetic

nerve activity resulting in hypertension (16).

Many studies have explored the impact of maternal undernutri-

tion on programming of obesity in offspring (8, 17–19); more

recently, attention has focused on maternal overnutrition (6, 10,

20–22), which is a more prominent health problem worldwide.

However, the issue of the extent of maternal obesity is important,

and the effect of maternal overnutrition induced early in life, as

a result of litter size reduction, on offspring is not known. We

hypothesised that frank maternal obesity induced by the overcon-

sumption of a high-fat diet (HFD) would exert greater effects on

plasma hormones and brain appetite regulators in offspring than

mild maternal early postnatal overnutrition induced by litter size

reduction. Given the link between maternal obesity and offspring

cardiovascular disease, blood pressure was measured in offspring,

in addition to food intake, glucose tolerance, plasma leptin, insulin

and triglyceride levels. To investigate alterations in hypothalamic

appetite regulators, we also examined mRNA expression of NPY,

POMC, Ob-Rb and its downstream mediators STAT-3 and SOCS-3.

Materials and methods

Animals and diet

All animal experiments were approved by the Animal Care and Ethics Com-

mittee of the University of New South Wales. Female out bred Sprague–

Dawley rats (Animal Resource Centre, Murdoch, WA, Australia) aged 9 weeks

were housed at 20 � 2 �C and maintained under a 12 : 12 h light ⁄ dark

cycle. They were fed normal chow (11 kJ ⁄ g, 14% fat, 21% protein, 65% car-

bohydrate by energy; Gordon’s Specialty Stockfeeds, Yanderra, NSW, Austra-

lia) or palatable cafeteria-style (HFD) (15.33 kJ ⁄ g, 34% fat, 19% protein,

47% carbohydrate by energy) as described previously (5, 23) for 5 weeks to

produce two groups: Chow and HFD. At 14 weeks, they were mated with

chow fed male Sprague–Dawley rats from the same source. On postnatal

day 1, litters from chow fed dams were adjusted to 3 per dam (small litter,

CS) inducing postnatal overnutrition by increasing milk availability (24, 25)

versus 12 per dam (normal litter, CN) and both groups were fed chow after

weaning. Pups from HFD fed dams were maintained in normal litters (12

per dam, HN) and were fed HFD after weaning (Table 1).

Thus, three goups of mother were generated: control and two degrees of

overnutrition, one by early postnatal overnutrition after litter size reduction

(chow, small litter CS) and another rendered obese by HFD consumption

(HFD, normal litter HN; Table 1). They were mated at 14 weeks of age with

chow fed males. After delivery, the litters were adjusted to normal size and

at weaning (day 20) the males were divided into two groups of equal body

weight and provided chow (CNC, CSC, HNC) or HFD (CNH, CSH, HNH), produc-

ing six groups of rats (n = 12–17; Table 1).

Body weight and energy intake

Body weight of offspring was monitored every 3 days till weaning (20 days)

and weekly thereafter. Average 24-h food intake (g) was calculated weekly

from 5–15 weeks by carefully collecting and weighing the food remaining in

the cage, and then subtracting this from the known amount given. The total

energy (kJ) consumed by each group was calculated from the energy infor-

mation supplied on the packet.

Blood pressure measurement

Blood pressure was measured at 12 weeks of age in conscious animals

using tail-cuff pulse plethysmography (CODA-6; Kent Scientific Corporation,

Torrington, CT, USA). Rats were pre-warmed by placing at 37 �C for

5–10 min to facilitate tail blood flow before blood pressure was measured.

After five acclimation readings, the average of three to seven readings was

used for each rat.

Glucose tolerance test

At 16 weeks, an i.p. glucose tolerance test (IPGTT) was performed on con-

scious animals after 6 h of fasting. After baseline blood glucose was deter-

mined (Accu-Chek Advantage Glucometre; Roche Diagnostics, Castle Hill,

NSW, Australia), rats were administered 2 g glucose ⁄ kg body weight (30%

w ⁄ v) by i.p. injection. Further blood samples were taken at 15, 30, 45, 60,

90 and 120 min for glucose determination.

Table 1. Experimental Procedure.

Diet Maternal (intervention) Offspring (diet) Offspring ID

Chow Normal litter consuming chow (CN); n = 7 Chow CN-chow (CNC); n = 17

HFD CN-HFD (CNH); n = 14

Small litter consuming chow (CS); n = 6 Chow CS-chow (CSC); n = 12

HFD CS-HFD (CSH); n = 12

HFD Normal litter consuming HFD (HN); n = 7 Chow HN-chow (HNC); n = 17

HFD HN-HFD (HNH); n = 15

HFD, high-fat diet.

906 S. Rajia et al.

ª 2010 The Authors. Journal Compilation ª 2010 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 22, 905–914

Sample collection

Blood samples (0.3 ml) were obtained from the tail vein of warmed con-

scious rats using a heparinised syringe (Pfizer, New York, NY, USA;

1000 IU ⁄ ml saline) at 14 weeks of age for fasting glucose and insulin levels.

The insulin resistance index was estimated by homeostasis model

assessment (HOMA): plasma insulin (ng ⁄ ml) · fasting plasma glucose

(mM) ⁄ (22.5 · 0.0417): the greater the HOMA value, the greater the level of

insulin resistance (26). Although the correlation between HOMA and eugly-

caemic clamp data is stronger in the human than the rodent, HOMA was

significantly correlated with euglycaemic clamp outcome as an indicator of

insulin resistance in the rat and is more feasible in small animals (27).

At 21 weeks, nonfasted offspring were anaesthetised (xylazine ⁄ ketamine

20 ⁄ 80 mg ⁄ kg). After measurement of naso–anal length and girth, blood

was removed by cardiac puncture for plasma hormone analysis. After decap-

itation, the brain was placed ventral side up; coronal cuts were made at the

optic chiasm and the rostral border of the hypothalamus. The hypothalamus

(minus preoptic area) was separated by making incisions at the lateral edges

of the hypothalamic sulcus and above the third ventricle. The hypothalamus

was dissected into dorsal and ventral segments. The ventral hypothalamus

containing the arcuate nucleus was snap frozen. White adipose tissue (WAT:

right retroperitoneal (rRP), gonadal and mesenteric) and liver were collected

and weighed. Hypothalamus and rRP WAT were snap frozen in liquid nitro-

gen and stored at )80 �C for later measurement of mRNA expression. Tibia

length was recorded. Body mass index (BMI) was calculated as kg ⁄ m2.

Plasma biochemistry

Glucose was measured and blood was centrifuged at 4472 g for 8 min

(Eppendorf Minispin; Crown Scientific, Minto, NSW, Australia). Separated

plasma was stored at )20 �C. Plasma leptin and insulin concentrations were

analysed using commercially available radioimmunoassay kits according to

manufacturer’s instructions (Linco, St Charles, MO, USA). Plasma triglycerides

were analysed colorimetrically (490 nm; Bio-Rad 680XR, Bio-Rad,Gladesville,

NSW, Australia) using commercially available triglyceride reagent (Roche)

and standard (Sigma, Croydon, Victoria, Australia).

Haematoxylin and eosin staining of liver

A section of liver from each rat was placed in 4% formaldehyde overnight,

transferred to 70% ethanol embedded in paraffin wax and stained for

hematoxylin and eosin. Fatty change in liver was scored by an observer

blinded to the treatment by assessing the amount and size of white vacu-

oles present throughout the stained section (0 = no fat vacuoles; 1 = few

small lipid vacuoles; 2 = increased number of small fat vacuoles and some

big vacuoles; 3 = hepatocytes filled by big fat vacuoles). The average of the

grades within each treatment group was then calculated. Grading of liver

histology used a scoring method adapted from a previous publication (28).

Real-time polymerase chain reaction (PCR)

Quantitative real-time PCR was used to determine mRNA expression of NPY,

POMC, SOCS-3, STAT-3, and Ob-Rb in ventral hypothalamus and adipose tri-

glyceride lipase (ATGL) in rRP WAT. Total RNA was extracted by homogenis-

ing tissue with Tri reagent (Sigma, St Louis, MO, USA), separation by

chloroform, precipitated with isopropyl alcohol and then washed with 75%

ethanol. Spectrophotometric measurements were done using the Nanodrop

Spectrophotometre (Nanodrop Technologies, Wilmington, DE, USA) to deter-

mine RNA concentration and purity. The purified total RNA was used as a

template to generate single-strand cDNA using M-MLV Reverse Transcrip-

tase, RNase H Minus, Point Mutant Kit, (Promega Corporation, Madison, WI,

USA). Pre-optimised probe ⁄ primers (Applied Biosystems, Foster City, CA,

USA) were used for quantitative PCR (Realplex software, Eppendorf,

Hamburg, Germany). Ob-Rb, STAT-3, SOCS-3, NPY and POMC mRNA levels

in hypothalamus were compared to housekeeping gene ribosomal 18s RNA.

ATGL mRNA levels in rRP were compared to housekeeping gene b-actin. The

housekeeping gene was labelled by VIC and target gene by FAM. An individ-

ual sample from the control CN-chow group was then arbitrarily assigned

as a calibrator against which all other samples are expressed as fold differ-

ence.

Statistical analysis

Results are expressed as the mean � SEM. Maternal characteristics and pre-

weaning data were analysed by one-way ANOVA followed by least significant

difference (LSD) using SPSS, version 17 (SPSS Inc., Chicago, IL, USA). Post-

weaning weekly body weights and IPGTT glucose concentrations were analy-

sed by one-way ANOVA with repeated measures, followed by LSD. Food intake,

blood pressure and endpoint organ weights, body weight, fat masses,

plasma markers, mRNA levels were analysed by two-way ANOVA followed by

least significant difference test using SPSS. If two-way ANOVA showed signifi-

cant overall maternal effects, then it was followed by one-way ANOVA to

examine maternal effects separately in chow and HFD groups. Correlation

between the body weight and fat masses was assessed by Pearson’s correla-

tion.

Results

Maternal characteristics

HN mothers showed marked increases in body weight at 14 weeks

when mating began (Table 2). Mothers were killed at 22 weeks,

after offspring were weaned; at this time, they were 17% heavier

than CN females, with a marked increase in sampled WAT mass

(P < 0.001) and a doubling of plasma leptin and triglyceride con-

centrations (HN versus CN; P < 0.01 and 0.05, respectively; Table 2).

Postnatal overnutrition via litter size reduction resulted in increased

body weight compared to CN dams at 14 weeks (CS versus CN;

P < 0.05; Table 2). No overall effect of litter size reduction was

seen at 21 weeks on body weight, plasma leptin, insulin and tri-

glyceride concentration (CS versus CN; Table 2). Although total fat

mass was increased 24%, this failed to reach significance.

Offspring body weight

Preweaning

No difference was observed in offspring day 1 body weight across

maternal groups (Fig. 1A). During suckling, the offspring of obese

HN dams were significantly heavier (P < 0.01) by 4 days and those

from CS dams showed increased body weight (P < 0.01) from post-

natal day 7 versus those of lean CN dams (Fig. 1A).

Postweaning

HFD-induced maternal obesity (HN) and maternal postnatal

overnutrition (CS) as a result of litter size reduction both led to

significantly greater postweaning body weight gain in offspring.

Maternal adiposity influences offspring hormones and metabolic disorders 907


HFD-induced maternal obesity (HN) effects were significant from

5 weeks (P < 0.001; Fig. 1B) and those of maternal early postnatal

overnutrition (CS) were evident from 9–19 weeks (P < 0.05; Fig. 1B).

This was observed in both diet groups, with the greatest impact in

offspring of HN mothers consuming HFD. At the end of the study

(21 weeks), body weights of offspring of HN and CS dams were

16% and 11% greater than those of CN dams consuming chow

(HNC versus CNC, CSC versus CNC; P < 0.001 and 0.01, respectively;

Table 3). In HFD animals, no impact of maternal postnatal overnu-

trition via litter size reduction (CS) was evident; whereas offspring

from obese HN dams were 19% heavier than CN offspring (HNH

versus CNH; P < 0.01; Table 3). HFD-induced maternal obesity also

caused increase in BMI in both diet groups (HNC versus CNC, HNH

versus CNH; P < 0.001; Table 3) and increase in naso–anal length

in HFD fed animals (HNH versus CNH, P = 0.05; Table 3). No signifi-

cant effect of maternal postnatal overnutrition as a result of small

litter size was observed in body length, whereas BMI was signifi-

cantly increased by maternal litter size reduction across both diet

groups (CSC versus CNC; CSH versus CNH P < 0.001; Table 3). As

expected, postweaning HFD consumption led to significant

increases in body weight from 8 weeks across all maternal groups

(P < 0.001; Fig. 1B) and was maintained over time. At 21 weeks,

body length and BMI were also significantly greater in HFD animals

(Table 3).

Glucose homeostasis

Baseline glucose in offspring was not different by maternal effects

within diet groups (Fig. 2C). No effect of maternal status was

observed on IPGTT in rats consuming chow (Fig. 2A). However, a

greater glucose excursion was found at 30 min with both degrees

of maternal overnutrition versus control in HFD fed offspring

(Fig. 2B), and this was more pronounced in those from obese HN

dams, reflected by continued higher blood glucose levels at 90 min

(HNH versus CNH; P < 0.01; Fig. 2B). Postweaning HFD significantly

increased both baseline blood glucose (Fig. 2C) and glucose at each

time point during IPGTT experiment in all maternal groups

(P < 0.001 at 15 and 30 min; P < 0.05 from 60 to 120 min).

In rats consuming HFD, the area under the glucose tolerance

test AUC was increased by both degrees of maternal overnutrition

compared to offspring from lean dams (HNH, CSH versus CNH;

P < 0.05, Fig. 2D). AUC were also increased when offspring con-

sumed HFD postweaning across all maternal groups (P < 0.001,

Fig. 2D).

Table 2. Maternal Characteristics.

Mothers CN (n = 7) CS (n = 6) HN (n = 7)

BW at mating (14 weeks) 258.7 � 7.66 284.2 � 7.80* 363.0 � 5.43��

BW at kill (22 weeks) 327.2 � 14.5 345.6 � 16.0 382.1 � 8.0��

rRP (% BW) 0.24 � 0.05 0.29 � 0.05 1.93 � 0.56��

Gonadal (% BW) 0.48 � 0.08 0.65 � 0.06 1.96 � 0.19��

Mesenteric (% BW) 0.40 � 0.06 0.50 � 0.04 1.45 � 0.18��

Total WAT (% BW) 1.25 � 0.19 1.64 � 0.11 5.95 � 0.44��

Leptin (ng ⁄ ml) 1.59 � 0.24 1.52 � 0.11 3.23 � 0.16��

Insulin (ng ⁄ ml) 0.20 � 0.03 0.44 � 0.14 0.28 � 0.07

Triglycerides (mM) 0.16 � 0.03 0.17 � 0.02 0.28 � 0.04�

Values expressed as the mean � SEM. Data were analysed by one-way ANOVA followed by least significant difference. *P < 0.05 [small litter ⁄ consuming chow (CS)

versus normal litter ⁄ consuming chow (CN)]; �, ��, ��high-fat diet effect P < 0.05, 0.01, 0.001 [normal litter/ high-fat diet (HN) versus CN]. BW, body weight;

WAT, white adipose tissue.

40

50 800

600

400

200

0

30

20

10

00 3 6 9 12 15 18 21

CNCS

HN

CNC

CNHHNCCSC

CSH

HNH

Age (days)4 6 8 10 12 14 1816 20

Age (weeks)

Body

wei

ght

(g)

Body

wei

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†††####***

†††

†††#

†††#††

(A) (B)

Fig. 1. Offspring body weight (A) pre-weaning from day 1 to day 19 and (B) postweaning from 5 to 20 weeks from normal litter ⁄ consuming chow (CN, circle),

small litter ⁄ consuming chow (CS, triangle) and normal litter ⁄ high-fat diet (HFD) (HN, square) mothers. Results are expressed as the mean � SEM; n = 34–44

(A) or n = 10–11 (B). Data were analysed by one-way ANOVA with repeated measures, followed by least significant difference. (B) Clear and solid symbols refer

to offspring’s postweaning diet chow or HFD, respectively. ��, ��Maternal HFD effect: P < 0.01, 0.001 (HN versus CN offspring); #, ###Maternal small litter

size effect: P < 0.05, 0.001 (CS versus CN offspring); ***Overall postweaning HFD effect: P < 0.001 (HFD versus chow fed offspring across all mother groups).

908 S. Rajia et al.


At 14 weeks, insulin concentration (Fig. 2E) and HOMA (Fig. 2F)

were significantly higher in the offspring of obese HN dams relative

to those of lean CN dams in both diet groups (P < 0.001). No sig-

nificant effect of maternal postnatal overnutrition via litter size

reduction (CS) was observed on offspring plasma insulin and

HOMA. Postweaning HFD consumption in offspring caused signifi-

cant increases in insulin concentration and HOMA in offspring

(P < 0.001, Fig. 2E, F).

Adiposity

In line with the increased body weight, a significant effect of HFD-

induced maternal obesity (HN) was observed on offspring total WAT

expressed as either net weight or percent body weight in both diet

groups (P < 0.001, Fig. 3A; Table 3). A significant effect of maternal

postnatal overnutrition as a result of small litter size (CS) on WAT

was only observed in chow fed offspring (P < 0.01; Fig. 3A; Table 3).

When both diet groups were combined, there was a significant

positive correlation between body weight and percent total fat

across all maternal groups (CN offspring, r = 0.833, P < 0.001; CS

offspring r = 0.882, P < 0.001; HN offspring r = 0.870, P < 0.001).

Postweaning HFD consumption led to significant increases in fat

masses both when expressed as net or percent body weight across

all maternal groups (P < 0.001; Fig. 3A and Table 3).

Leptin was increased by HFD-induced maternal obesity (HN) in

both diet groups (P < 0.001; Fig. 3B). No significant effect of mater-

nal postnatal overnutrition as a result of small litter size was

observed. As expected, postweaning consumption of HFD led to

marked increases in leptin (P < 0.001; Fig. 3B). Leptin concentration

was correlated with percent total fat (r = 0.853, P < 0.001, n = 86)

when all six groups were combined.

Lipids

Significant effects of HFD-induced maternal obesity (HN) (P < 0.001

versus CN) and offspring postweaning HFD (P < 0.01) were

observed on plasma triglycerides (Fig. 3C). No effect of maternal

postnatal overnutrition was observed on plasma triglycerides (CS

versus CN offspring; Fig. 3C). Histological scoring of the liver sec-

tions revealed that postweaning HFD consumption led to signifi-

cantly greater accumulation of lipids vacuoles within hepatocytes

(P < 0.001, Fig. 4). In those consuming HFD, liver fatty changes

were increased by both degrees of maternal overnutrition (HNH,CSH

versus CNH; P < 0.01 Fig. 4).

In rRP WAT, no significant maternal effects was observed, but

mRNA expression of ATGL in offspring was significantly decreased

by postweaning HFD across all mother groups (P < 0.05, Fig. 3D).

Blood pressure

Obesity induced by postweaning HFD consumption was associated

with significant increases in systolic and diastolic pressures

(P < 0.01; Table 3). No significant maternal effect was observed on

blood pressure and heart rate (Table 3).

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15 30 45 60 75 90 105 120

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15 30 45 60 75 90 105 120

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15

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cose

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(A) (B)

(C) (D)

(E) (F)

Fig. 2. Blood glucose concentrations during i.p. glucose tolerance test (IPGTT) in offspring fed (A) chow or (B) high-fat diet (HFD) from normal litter ⁄ consum-

ing chow (CN, circle), small litter ⁄ consuming chow (CS, triangle) and normal litter ⁄ high-fat diet (HN, square) mothers; (C) Baseline glucose and (D) area under

the curve (AUC) of IPGTT at 16 weeks (E) Fasting insulin concentrations and (F) homeostasis model assessment (HOMA) at 14 weeks of CN-offspring fed chow

(CNC) and HFD (CNH), CS-offspring fed chow (CSC) and HFD (CSH) and HN-offspring fed chow (HNC) and HFD (HNH). Results are expressed as the mean � SEM

(n = 8–12). Data were analysed by one-way ANOVA with repeated measures, followed by least significant difference (LSD) in (A) and (B). Other data were analysed

by two-way ANOVA followed by LSD and one-way ANOVA was performed to examine maternal effects separately in chow and HFD groups. �, ��, ��Maternal

HFD effect: P < 0.05, 0.01, 0.001 (HNC versus CNC ⁄ HNH versus CNH); #, ##Maternal small litter size effect: P < 0.05, 0.01 (CSH versus CNH); **, ***Overall post-

weaning HFD effect: P < 0.05, 0.01, 0.001 (HFD versus chow fed offspring across all mother groups).

CNC CSC HNC CNH CSH HNH CNC CSC HNC CNH CSH HNH

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910 S. Rajia et al.


Food intake and central appetite regulators

The total energy consumed by the offspring of obese HN dams

was 11% higher than that of offspring of lean CN dams in both

chow and HFD groups (P < 0.05 and 0.01, respectively; Table 3).

A similar pattern was observed when the net weight (gram) of

food consumed was examined (data not shown). No effect of

maternal postnatal overnutrition was observed. Across all mater-

nal groups, consumption of postweaning HFD led to a doubling

of energy intake compared to chow fed offspring (P < 0.001;

Table 3).

Within the chow diet group, maternal postnatal overnutrition as

a result of small litter size was associated with decreased hypotha-

lamic Ob-Rb mRNA expression in offspring (P < 0.05; Fig. 5A). Sig-

nificant increases in Ob-Rb mRNA were observed in offspring of

obese HN mothers consuming HFD (P < 0.01; Fig. 5A). STAT-3

mRNA expression showed a similar pattern to that of Ob-Rb

(Fig. 5B). HFD-induced maternal obesity also caused a significant

increase in SOCS-3 mRNA expression in offspring consuming HFD

(HNH versus CNH; P < 0.05; Fig. 5C). Postweaning HFD consumption

also caused an overall increase in SOCS-3 expression (P < 0.01;

Fig. 5C). No significant differences were observed in hypothalamic

NPY and POMC mRNA expression (Fig. 5D, E).

Discussion

A number of animal studies indicate that obesity may have a ‘pro-

grammed origin’ (3, 6, 10, 22). The present study shows that even a

short period of postnatal overnutrition in mother very early in life,

as a result of litter size reduction, can increase adiposity and glu-

cose intolerance in offspring. Frank maternal obesity as a result of

HFD feeding prior to and during pregnancy exerted greater effects

on offspring adiposity, and glucose and lipid homeostasis. Postwea-

ning diet also had an important impact because the effects of

maternal obesity appeared to be amplified when offspring con-

sumed HFD.

Maternal HFD consumption during pregnancy and lactation is an

important contributing factor to the development of obesity both

early life and adulthood (5, 10, 20). Altered milk composition of

obese mothers (29), as well as an increased quantity of milk con-

sumed by pups of obese mothers (5), may have contributed to their

early-onset obesity. As a result, in the present study, offspring from

obese dams (HN) showed significant increases in body weight gain

during development and at the endpoint they were heavier with

elevated BMI in both diet groups. Early postnatal overnutrition

imposed by reduced competition for milk during suckling did not

result in marked changes in adiposity or body weight in mothers.

Previous work from this and other laboratories showed litter size

reduction resulted in time-dependent increases in body weight, fat

mass and fat cell number (24, 25, 30). This may be related to the

Fig. 3. (A) Total white adipose tissue (WAT) sampled expressed as percent of body weight, (B) plasma leptin and (C) triglycerides (D) mRNA expression of adi-

pose triglyceride lipase (ATGL) in retroperitoneal WAT (rRP) at 21 weeks in offspring of normal litter ⁄ consuming chow (CN) mother fed chow (CNC) and high-

fat diet (HFD) (CNH), small litter ⁄ consuming chow (CS) mother fed chow (CSC) and HFD (CSH) and normal litter ⁄ high-fat diet (HN) mother fed chow (HNC) and

HFD (HNH). Results are expressed as the mean � SEM (n = 12–17). Data were analysed by two-way ANOVA followed by least significant difference and one-way

ANOVA was performed to examine maternal effects separately in chow and HFD groups. ��Maternal HFD effect: P < 0.001 (HNC versus CNC ⁄ HNH versus CNH);

##Maternal small litter size effect: P < 0.01 (CSC versus CNC); *, **, ***Overall postweaning HFD effect: P < 0.05, 0.01, 0.001 (HFD versus chow fed offspring

across all mother groups).

Score 0

Score 2 Score 3

Score 1

2.5

(A)

(B)

2.0

1.5

***

# ††

1.0

0.5

0.0

Ave

rage

live

r sc

ore


Fig. 4. (A) Representative histological sections of liver, stained with hae-

motoxylin and eosin, from each of the four scoring grades (0 = No fat vac-

uoles; 1 = few fat vacuoles; 2 = increased number of small fat vacuoles

and some big vacuoles; 3 = hepatocytes filled by big fat vacuoles). (B) Aver-

age liver score at 21 weeks in offspring of normal litter ⁄ consuming chow

(CN) mother fed chow (CNC) and high-fat diet (HFD) (CNH), small litter ⁄ con-

suming chow (CS) mother fed chow (CSC) and HFD (CSH) and normal litter ⁄high-fat diet (HN) mother fed chow (HNC) and HFD (HNH). Results are

expressed as the mean � SEM (n = 8). Data were analysed by two-way ANO-

VA followed by least significant difference, and one-way ANOVA was performed

to look at maternal effects separately in chow and HFD groups. ��Maternal

HFD effect: P < 0.01 (HNH versus CNH); #Maternal small litter size effect:

P < 0.05 (CSH versus CNH); ***Overall postweaning HFD effect: P < 0.001

(HFD versus chow fed offspring across all mother groups).



time point examined because, previously, significant effects of litter

size adjustment were observed early in life (up to 10 weeks) and

abated over time (25). To our knowledge, this is the first study doc-

umenting an impact of maternal early postnatal overnutrition

induced by litter size reduction on offspring. A late onset effect of

maternal postnatal overnutrition (CS) on offspring body weight was

observed from 9 weeks of age in both diet groups. At 21 weeks,

this effect was observed in only chow fed offspring, with increased

fat mass. This suggests that maternal early postnatal overnutrition

plays an important role in determining the adiposity in offspring;

however, postweaning HFD consumption has a much stronger

impact, in that it could override the impact of early maternal over-

nutrition.

Maternal obesity induced by HFD caused hyperleptinaemia in off-

spring, in line with their increased fat content, in agreement with

other studies (5, 21). In the present study, litter size reduction in

mothers did not affect their plasma leptin concentration, so it is

perhaps not surprising that leptin was normal in the CS offspring.

Earlier reports on rats from small size litters showed an elevation in

plasma leptin at 8 weeks (25, 31), although these normalised by

16 weeks (25). The time points chosen may limit our ability to

observe any significant changes in plasma leptin concentration in

dams after postnatal overnutrition.

In agreement with other studies examining adult offspring,

maternal obesity induced by HFD led to hyperinsulinaemia with sig-

nificantly higher HOMA in offspring, suggesting that maternal obes-

ity is linked to insulin resistance in offspring (21, 32). Postweaning

consumption of HFD also resulted in a greater HOMA value. Mater-

nal postnatal overnutrition by litter size reduction did not have any

effect on HOMA; however, it impaired glucose uptake during IPGTT.

The offspring of CS dams showed delayed glucose clearance at

30 min, with more marked delay in HFD fed rats from obese dams.

Higher glucose levels resulting from lower glucose utilisation in

peripheral tissues is a typical feature in obesity. Increased area

under the IPGTT curve was seen in offspring from both CS and HN

mothers, although this was only unmasked when offspring con-

sumed HFD, suggesting an interaction between maternal phenotype

and current diet increased the risk of glucose intolerance.

Maternal obesity as a result of a fat rich diet during pregnancy

and lactation (10, 33), as well as a lifetime of HFD consumption


CNC

0.8

0.6

0.4

0.2

0.0

3

(A)

(B) (C)

(E)(D)

5

4

3

2

1

0

2

1

0

1.5

1.0

0.5

0.0

1.5

1.0

0.5

0.0

Ob-

Rb m

RNA

STA

T-3

mRN

AN

PY m

RNA

POM

C m

RNA

SOC

S-3

mRN

A

#

#

**

††

†† †

CSC HNC CNH CSH HNH


CNC CSC HNC CNH CSH HNH CNC CSC HNC CNH CSH HNH

Fig. 5. mRNA expression of (A) Ob-Rb receptor, (B) signal transducer and activator of transcription (STAT-3), (C) suppressor of cytokine signalling (SOCS-3),

(D) neuropeptide Y (NPY), (E) pro-opiomelanocortin (POMC) in ventral hypothalamus at 21 weeks in offspring of normal litter ⁄ consuming chow (CN) mother

fed chow (CNC) and high-fat diet (HFD) (CNH), small litter ⁄ consuming chow (CS) mother fed chow (CSC) and HFD (CSH) and normal litter ⁄ high-fat diet (HN)

mother fed chow (HNC) and HFD (HNH). Results are expressed as the mean � SEM (n = 8). Data were analysed by two-way ANOVA followed by least significant

difference and one-way ANOVA was performed to examine maternal effects separately in chow and HFD groups. �, ��Maternal HFD effect: P < 0.05, 0.01 (HNH

versus CNH); #Maternal small litter size effect: P < 0.05 (CSC versus CNC); **Overall postweaning HFD effect: P < 0.01 (HFD versus chow fed offspring across

all mother groups).

912 S. Rajia et al.


(34), might cause a ‘malprogramming’ of hypothalamic neuropept-

idergic systems in offspring, leading to permanently altered appe-

tite regulatory function. Maternal junk food during gestation and

lactation has been linked to an exacerbated preference for fatty,

sugary and salty foods (20). This is possibly related to defective

leptin signalling in the hypothalamus. In the present study, hypo-

thalamic Ob-Rb was up-regulated in HFD-fed offspring from obese

dams (HN). Changes in mRNA expression of its downstream medi-

ator STAT-3 mirrored those in the Ob-Rb receptor. However, the

simultaneous up-regulation of the suppressor SOCS-3 may

counteract increased STAT-3 expression to cause central leptin

resistance (15). Although cafeteria diet and leptin are known to

modulate the hypothalamic appetite regulators NPY and POMC (5,

11, 23), central leptin resistance as a result of up-regulated

SOCS-3 may help to explain why, in the present study, elevated

leptin in HFD-fed rats failed to change hypothalamic NPY and

POMC expression in offspring from HN dams. Hyperphagia would

be consistent with SOCS-3 overexpression by HFD-induced mater-

nal obesity and offspring of HN dams ate more than the other

groups, especially when consuming HFD. This suggests that the

hypothalamic neuronal changes as a result of maternal obesity

led to transmission of increased susceptibility to hyperphagia and

obesity in offspring. An overall up-regulation of SOCS-3 was also

found with offspring following prolonged postweaning HFD con-

sumption, as shown previously (35). This may contribute to central

leptin resistance and thus lead to hyperphagia. Early postnatal

overnutrition led to hyperleptinaemia and down-regulation of

hypothalamic Ob-Rb receptor in rats, also suggesting leptin

resistance (31). In the present study, an effect of maternal early

postnatal overnutrition was also observed in the 21-week-old

chow fed offspring, with reduced expression of hypothalamic

Ob-Rb, which may reduce leptin binding and weaken its signalling.

This could further contribute to the downregulated STAT-3

observed in chow fed offspring. Overall, these data suggest that

different degrees of maternal overnutrition can alter offspring

appetite control differently.

Postweaning HFD caused increased fat mass with elevated leptin

and down-regulation of ATGL in rRP WAT. Adipose ATGL mRNA

expression has been shown to have strong negative correlations

with obesity and insulin resistance (36). There is some adaptive

increase in ATGL mRNA expression in chow fed offspring from HN

mothers, which was not sufficient to reverse adiposity and lipide-

mia. ATGL was down-regulated in insulin resistant HFD-fed off-

spring across all maternal groups in the present study, suggesting

a slower rate of lipolysis in HFD rats, contributing to concomitant

increases in plasma triglycerides and body fat mass.

Studies in human (2, 37) and animals (21) showed increased

blood pressure in offspring of obese mothers. Maternal obesity is

also associated with fatty liver (38, 39), which has been linked to

increased risk of cardiovascular disease (40). In the present study,

both degrees of maternal overnutrition led to increased hepatic

lipid content, but not on blood pressure, possibly as a result of the

tail-cuff methodology employed. Postweaning HFD caused an eleva-

tion in systolic and diastolic blood pressure, as shown previously

(25). Hyperleptinaemia and hyperinsulinaemia have both been linked

to sympathetic activation associated with obesity related hyperten-

sion (41, 42). This may explain our finding of an overall HFD effect

on blood pressure with increased leptin and insulin across all

mother groups. The HFD fed rats were also hyperphagic and most

likely leptin resistant, related to SOCS-3 overexpression. Selective

leptin resistance may contribute to the inability of leptin to activate

downstream signalling pathways in the arcuate nucleus but preser-

vation of leptin action in other cardiovascular-related hypothalamic

areas (41). Further investigation is needed to examine the cardio-

vascular consequences of maternal overnutrition and postweaning

HFD.

To conclude, the most important finding of the present study is

that even mild overnutrition of the mother early in life, as induced

by litter size reduction, led to increased adiposity, glucose intoler-

ance and altered brain appetite regulators in their offspring at

adulthood. A far greater impact of HFD-induced frank maternal

obesity was observed on offspring, who were at the highest risk of

metabolic disorders. Thus, the different degrees of maternal overnu-

trition differentially affected offspring adiposity, hormones, glucose

homeostasis and appetite regulators. Given the rising incidence of

obesity in women of childbearing age, maternal nutrition and body

weight prior to pregnancy has important implications for the health

of the next generation.

Acknowledgements

This work was supported by a project grant of the National Health and

Medical Research Council of Australia to MJ Morris and an Australian Post-

graduate Award to S Rajia. The authors declare that there are no competing

financial interests in relation to this work.

Received: 29 January 2010,

revised 25 March 2010,

accepted 5 April 2010

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