Maternal obesity causes fetal hypothalamic insulin resistance and disrupts development of hypothalamic feeding pathways.

OBJECTIVE: Perinatal exposure to maternal obesity results in predisposition of offspring to develop obesity later in life. Increased weight gain in offspring exposed to maternal obesity is usually associated with hyperphagia, implicating altered central regulation of food intake as a cause. We aimed to define how maternal obesity impacts early development of the hypothalamus to program lasting dysfunction in feeding regulatory pathways. METHODS: Mice offspring of diet-induced obese mothers were compared to the offspring of lean control mothers. We analysed gene expression in the fetal hypothalamus, alongside neurosphere assays to investigate the effects of maternal obesity on neural progenitor cell proliferation in vitro. Western blotting was used to investigate the insulin signalling pathway in the fetal hypothalamus. Characterisation of cell type and neuropeptide profile in adulthood was linked with analyses of feeding behaviour. RESULTS: There was a reduction in the expression of proliferative genes in the fetal hypothalamus of offspring exposed to maternal obesity. This reduction in proliferation was maintained in vitro when hypothalamic neural progenitor cells were grown as neurospheres. Hypothalamic fetal gene expression and neurosphere growth correlated with maternal body weight and insulin levels. Foetuses of obese mothers showed hypothalamic insulin resistance, which may be causative of reduced proliferation. Furthermore, maternal obesity activated the Notch signalling pathway in neonatal offspring hypothalamus, resulting in decreased neurogenesis. Adult offspring of obese mothers displayed an altered ratio of anorexigenic and orexigenic signals in the arcuate nucleus, associated with an inability to maintain energy homeostasis when metabolically challenged. CONCLUSIONS: These findings show that maternal obesity alters the molecular signature in the developing hypothalamus, which is associated with disrupted growth and development of hypothalamic precursor cells and defective feeding regulation in adulthood. This is the first report of fetal hypothalamic insulin resistance in an obese pregnancy and suggests a mechanism by which maternal obesity causes permanent changes to hypothalamic structure and function.

Catch-up growth following intra-uterine growth-restriction programmes an insulin-resistant phenotype in adipose tissue.

BACKGROUND: It is now widely accepted that the early-life nutritional environment is important in determining susceptibility to metabolic diseases. In particular, intra-uterine growth restriction followed by accelerated postnatal growth is associated with an increased risk of obesity, type-2 diabetes and other features of the metabolic syndrome. The mechanisms underlying these observations are not fully understood. AIM: Using a well-established maternal protein-restriction rodent model, our aim was to determine if exposure to mismatched nutrition in early-life programmes adipose tissue structure and function, and expression of key components of the insulin-signalling pathway. METHODS: Offspring of dams fed a low-protein (8%) diet during pregnancy were suckled by control (20%)-fed dams to drive catch-up growth. This 'recuperated' group was compared with offspring of dams fed a 20% protein diet during pregnancy and lactation (control group). Epididymal adipose tissue from 22-day and 3-month-old control and recuperated male rats was studied using histological analysis. Expression and phosphorylation of insulin-signalling proteins and gene expression were assessed by western blotting and reverse-transcriptase PCR, respectively. RESULTS: Recuperated offspring at both ages had larger adipocytes (P<0.001). Fasting serum glucose, insulin and leptin levels were comparable between groups but increased with age. Recuperated offspring had reduced expression of IRS-1 (P<0.01) and PI3K p110ß (P<0.001) in adipose tissue. In adult recuperated rats, Akt phosphorylation (P<0.01) and protein levels of Akt-2 (P<0.01) were also reduced. Messenger RNA expression levels of these proteins were not different, indicating a post-transcriptional effect. CONCLUSION: Early-life nutrition programmes alterations in adipocyte cell size and impairs the protein expression of several insulin-signalling proteins through post-transcriptional mechanisms. These indices may represent early markers of insulin resistance and metabolic disease risk.

Programming of adipose tissue miR-483-3p and GDF-3 expression by maternal diet in type 2 diabetes.

Nutrition during early mammalian development permanently influences health of the adult, including increasing the risk of type 2 diabetes and coronary heart disease. However, the molecular mechanisms underlying such programming are poorly defined. Here we demonstrate that programmed changes in miRNA expression link early-life nutrition to long-term health. Specifically, we show that miR-483-3p is upregulated in adipose tissue from low-birth-weight adult humans and prediabetic adult rats exposed to suboptimal nutrition in early life. We demonstrate that manipulation of miR-483-3p levels in vitro substantially modulates the capacity of adipocytes to differentiate and store lipids. We show that some of these effects are mediated by translational repression of growth/differentiation factor-3, a target of miR-483-3p. We propose that increased miR-483-3p expression in vivo, programmed by early-life nutrition, limits storage of lipids in adipose tissue, causing lipotoxicity and insulin resistance and thus increasing susceptibility to metabolic disease.

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.

Altered hepatic insulin signalling in male offspring of obese mice.

Individuals exposed in utero to maternal obesity have increased risk of developing type 2 diabetes mellitus and obesity in adulthood. The molecular mechanisms underlying this association are unknown. We have therefore used a murine model of maternal obesity, in which the offspring of obese dams develop hyperinsulinaemia by 3 months of age indicative of insulin resistance. Here, we investigate the effects of maternal diet-induced obesity on the expression/phosphorylation of key hepatic insulin signalling proteins and the expression of anti-oxidant enzymes in male offspring. At 3 months of age, offspring of obese dams had decreased levels of insulin receptor substrate (IRS) 1 (P < 0.01), whereas the ratio of phosphorylated IRS1 Ser307 to total IRS1 was significantly increased (P < 0.001), suggesting that it was less active. Protein expression of the PI3K p85α subunit was decreased (P < 0.01) and there was a tendency for phosphorylation of Akt at Ser473 to be reduced (P = 0.08) in the offspring of obese dams. protein kinase Cζ (P < 0.001) and glycogen synthase kinase 3ß (P < 0.05) levels were increased in these animals in comparison with controls. Maternal obesity also resulted in increased phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182 (P < 0.01) and raised c-Jun N-terminal kinase 1 expression (P < 0.5) in the offspring. The expression of antioxidant enzymes was also affected by maternal obesity with CuZnSOD (P < 0.001) and glutathione reductase (P < 0.05) being increased, whereas glutathione peroxidase 1 was reduced (P < 0.05) in the offspring. We conclude that maternal obesity leads to alterations in hepatic insulin signalling protein expression and phosphorylation. These molecular changes may contribute to the development of insulin resistance.

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.

Mechanisms by which poor early growth programs type-2 diabetes, obesity and the metabolic syndrome.

Fetal programming is gaining momentum as a highly documented phenomenon which links poor early growth to adult disease. It is backed up by large cohorts in epidemiological studies worldwide and has been tested in various animal models. The root causes of programming link closely with maternal condition during pregnancy, and therefore the fetal environment. Suboptimal fetal environments due to poor or inadequate nutrition, infection, anemia, hypertension, inflammation, gestational diabetes or hypoxia in the mother expose the fetus to hormonal, growth factor, cytokine or adipokine cues. These in turn act to alter metabolic, immune system, vascular, hemodynamics, renal, growth and mitochondrial parameters respectively and most evidently in the later stages of life where they impact on the individual as poor glucose homeostasis, insulin resistance, type 2 diabetes, hypertension, cardiovascular disease, obesity and heart disease. These events are compounded by over-nutrition or lifestyle choices which are in conflict with the programming of the fetus. We and others have utilised various species to test the early life programming hypothesis and to identify key molecular mechanisms. With parallel studies of human cohorts, these molecular markers can be validated as realistic 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.