Poor early growth and excessive adult calorie intake independently and additively affect mitogenic signaling and increase mammary tumor susceptibility.

We previously showed that offspring of rat dams receiving a protein-restricted (low protein) diet throughout pregnancy and lactation develop mammary tumors more quickly. Rapid post-weaning mammary growth and mammary tissue overexpression of insulin receptor, insulin-like growth factor-1 receptor (IGF-1R), estrogen receptor isoform alpha and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), correlated with this risk. The objectives of this study were therefore (i) to identify underlying mechanisms of increased risk through candidate and global approaches; (ii) to determine if excessive calorie intake further increased risk and if so, (iii) to identify the molecular mechanisms mediating this. We provide evidence for transcriptional upregulation of IGF-1R by Sp1 in LP mammary tissue (P < 0.01). Cell cycle control and DNA damage repair gene cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21waf1) was also upregulated (P < 0.05) as was transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cell (P < 0.05) and adhesion gene CDH1 (P < 0.05). Invasion and metastasis markers matrix metalloproteinase 9 and serpin peptidase inhibitor, clade E, member 1 (SERPIN1) were upregulated (both P < 0.05), whereas metastasis suppressor gene NME1 was downregulated (P < 0.01). Feeding a highly palatable diet (HPD) to increase calorie intake from puberty, additively and independently increased early mammary tumor risk, which correlated with increased serum insulin and triglyceride concentrations (P < 0.05). PTEN gene expression was reduced both by early protein restriction (P < 0.05) and HPD (P < 0.01), which may induce Akt in cell survival pathways. Progesterone receptor and ERBB2 (both P < 0.05) expression increased as an effect of an interaction between maternal diet and adult nutrition, with subsequent downstream activation of the mitogen-activated protein kinase pathway. We conclude that poor early growth and excessive calorie intake exert independent and additive effects on mitogenic growth factor signaling to influence mammary tumor susceptibility.

Compensatory mammary growth following protein restriction during pregnancy and lactation increases early-onset mammary tumor incidence in rats.

Breast cancer incidence is increased in women with both high and low birth weight. The latter is also associated with hyperglycaemia, insulin resistance and type-2 diabetes, each of which independently increases breast cancer risk. We showed previously in our model of poor early-growth that pregnancy estradiol levels were raised while offspring developed type-2 diabetes. We hypothesized that nutritionally-induced poor early-growth influences breast cancer risk and investigated this in our model. Wistar rat dams were given either a control diet (20% casein) or an isocaloric low-protein (LP) diet (8% casein) throughout pregnancy and lactation. Offspring postnatal mammary gland development was assessed by morphometry. To identify potential growth mechanisms, we measured protein expression of receptors involved in insulin and hormone signaling, both in cleared mammary gland lysates and isolated epithelial cells. Mammary tumor incidence and latency (n=96) was monitored after three weekly intraperitoneal nitrosomethylurea injections (50 mg/kg body wt). LP offspring displayed reduced postnatal ductal branching and epithelial invasion at 3 weeks, followed by compensatory mammary growth 1 week later coinciding with increased protein expression of receptors to insulin, IGF-1 and estrogen. Significantly, early-mammary tumor incidence (0-16 weeks post-treatment) was doubled in LP offspring [RR, 2.13 (1.02, 4.45); P=0.046]. The data suggest that poor early nutrition has an important influence on the mammary primordium, and increases future susceptibility to breast cancer. Up-regulated growth factor and hormone signaling during compensatory mammary growth may mediate this increased susceptibility and present potential targets for intervention.

Maternal protein restriction leads to hyperinsulinemia and reduced insulin-signaling protein expression in 21-mo-old female rat offspring.

Human adult diseases such as cardiovascular disease, hypertension, and type 2 diabetes have been epidemiologically linked to poor fetal growth and development. Male offspring of rat dams fed a low-protein (LP) diet during pregnancy and lactation develop diabetes with concomitant alterations in their insulin-signaling mechanisms. Such associations have not been studied in female offspring. The aim of this study was to determine whether female LP offspring develop diabetes in later life. Control and LP female offspring groups were obtained from rat dams fed a control (20% protein) or an isocaloric (8% protein) diet, respectively, throughout pregnancy and lactation. Both groups were weaned and maintained on 20% normal laboratory chow until 21 mo of age when they underwent intravenous glucose tolerance testing (IVGTT). Fasting glucose was comparable between the two groups; however, LP fasting insulin was approximately twofold that of controls (P < 0.02). Glucose tolerance during IVGTT was comparable between the two groups; however, LP peak plasma insulin at 4 min was approximately threefold higher than in controls (P < 0.001). LP plasma insulin area under the curve was 1.9-fold higher than controls (P < 0.02). In Western blots, both muscle protein kinase C-zeta expression and p110beta-associated p85alpha in abdominal fat were reduced (P < 0.05) in LPs. Hyperinsulinemia in response to glucose challenge coupled with attenuation of certain insulin-signaling molecules imply the development of insulin resistance in LP muscle and fat. These observations suggest that intrauterine protein restriction leads to insulin resistance in females in old age and, hence, an increased risk of type 2 diabetes.

The maternal endocrine environment in the low-protein model of intra-uterine growth restriction.

Many adult diseases, including type 2 diabetes, hypertension and cardiovascular disease, are related to low birth weight. The mechanistic basis of this relationship is not known. To investigate the role of fetal undernutrition, we used a rat model of maternal protein restriction in which dams were fed a diet containing 80 g protein/kg (v. 200 g/kg in the control group) throughout gestation and lactation. Offspring were born smaller than controls and in adulthood developed diabetes, hyperinsulinaemia and tissue insulin resistance. To determine possible mechanisms of fetal programming, circulating levels of several hormones were measured in maternal plasma at gestational days 14, 17 and 21 and fetal plasma at gestational day 21. Several differences were noted at day 14, when glucose concentrations in maternal and feto-placental blood were raised significantly (P=0.04 and P=0.0001 respectively); insulin levels in the low-protein (LP) dams were raised (P=0.04), prolactin levels were raised (P=0.047) and progesterone levels were reduced (P=0.02). Circulating 17beta-oestradiol in the LP dams was raised by 35 % over those of the controls from day 17 to day 21 (P=0.008). A significant decrease in maternal leptin levels (P=0.004) was observed at gestation on day 21. Neither oestradiol nor leptin levels were altered in the fetal circulation at day 21. Maternal and fetal corticosterone levels were comparable with control levels, suggesting that they do not initiate the programming effects in this model. Our present results suggest that maternal protein restriction imposes changes in maternal levels of glucose, insulin, prolactin, progesterone, oestradiol and leptin; these changes could influence the programming of eventual adult disease in the developing fetus.