Maternal obesity affects gene expression and cellular development in ...

Original research paper

Maternal obesity affects gene expression and cellular development in fetal brains Ewa K Stachowiak1, Saji Oommen 2, Vihas T Vasu 2, Malathi Srinivasan 3, Michal Stachowiak1, Kishorchandra Gohil 2, Mulchand S Patel 3 1

Department of Pathology and Anatomic Sciences, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA, 2Center for Comparative Respiratory Biology and Medicine, School of Medicine, University of California at Davis, Davis, CA, USA, 3Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA Objectives: Female rat neonates reared on a high carbohydrate (HC) milk formula developed chronic hyperinsulinemia and adult-onset obesity (HC phenotype). Furthermore, we have shown that fetal development in the HC intrauterine environment (maternal obesity complicated with hyperinsulinemia, hyperleptinemia, and increased levels of proinflammatory markers) resulted in increased levels of serum insulin and leptin in term HC fetuses and the spontaneous transfer of the HC phenotype to the adult offspring. The objectives of this study are to identify changes in global gene expression pattern and cellular development in term HC fetal brains in response to growth in the adverse intrauterine environment of the obese HC female rat. Methods: GeneChip analysis was performed on total RNA obtained from fetal brains for global gene expression studies and immunohistochemical analysis was performed on fetal brain slices for investigation of cellular development in term HC fetal brains. Results: Gene expression profiling identified changes in several clusters of genes that could contribute to the transfer of the maternal phenotype (chronic hyperinsulinemia and adult-onset obesity) to the HC offspring. Immunohistochemical analysis indicated diminished proliferation and neuronal maturation of stem-like cells lining the third ventricle, hypothalamic region, and the cerebral cortex in HC fetal brains. Discussion: These results suggest that maternal obesity during pregnancy could alter the developmental program of specific fetal brain cell-networks. These defects could underlie pathologies such as metabolic syndrome and possibly some neurological disorders in the offspring at a later age. Keywords: Fetal hyperinsulinemia and hyperleptinemia, Global gene expression, Maternal obesity, Neurogenesis, Neuronal maturation

Introduction The increasing incidence of overweight/obesity in the adult population worldwide is a major health concern. Obesity significantly increases the risk for the metabolic syndrome. In the United States, approximately 68% of the adult population is considered overweight and one half of this group is obese.1 A disturbing correlate of this observation is the marked increase in the incidence of overweight/obesity amongst women of child-bearing age. In the United States, this number exceeds 50%.1 An obese pregnancy has been shown to negatively impact on the health outcome of the adult offspring. Correspondence to: Mulchand S Patel, Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, 140 Farber Hall, 3435 Main Street, Buffalo, NY 14214, USA. Email: mspatel@ buffalo.edu

© W. S. Maney & Son Ltd 2013 DOI 10.1179/1476830512Y.0000000035

In addition to genes and poor life style choices, there is an increasing recognition of an early developmental component in the etiology of the development of obesity. The pioneering observations of Barker on the long-term effects of a malnourished pregnancy resulted in the recognition that a nutritional stimulus or insult experienced during early periods in life can impact on adult health outcome.2 The underlying principle for this hypothesis is the concept of metabolic programming which refers to the adjustments at various facets that occur in the organism in response to altered nutritional environments during critical early periods of development. Although beneficial for survival, these early responses result in permanent resetting of several homeostatic processes which predispose the organism for the development of metabolic disorders in later life.3,4

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Effect of maternal obesity on fetal brain cell-networks

In addition to pregnancy, organ development continues in the immediate postnatal period. The proliferation of neurons in the hypothalamus occurs during mid-gestation but the development of projection from these neurons to their downstream target sites is initiated postnatally.5 For example, the axonal projections from the neuropeptide Y/agouti-related polypeptide (NPY/AgRP) neurons in the arcuate nucleus (ARH) to the paraventricular nucleus occur around postnatal day 8–10.5 Studies with rodents have suggested that during early periods in life, both insulin and leptin function as trophic factors regulating the development of hypothalamic circuits that control energy.6 Therefore, altered levels of these hormones during the fetal and/or suckling periods could result in an altered development of hypothalamic structures involved in energy homeostasis that could underlie predisposition to metabolic disorders in later life. A rat model for adult-onset obesity has been developed in our laboratory by artificially rearing rat pups on a high carbohydrate (HC) milk formula in their immediate postnatal period.7 Although the HC female rats consumed a standard laboratory chow from the time of weaning, their body weight gains were markedly higher in the post-weaning period. During pregnancy, HC female rats were obese, hyperinsulinemic, and hyperleptinemic and had an increased proinflammatory response.7,8 Fetal development in the HC intrauterine environment resulted in hyperinsulinemia and hyperleptinemia in term HC fetuses.7,8 The offspring of HC female rats demonstrated increased body weight gain and chronic hyperinsulinemia in their post-weaning period.7 Results from animal models for an obese pregnancy have shown that maternal obesity increases the risk for obesity and associated metabolic disorders in the offspring.9 Additionally, offspring of obese dams show abnormalities in the development of neuronal circuits such as alterations in the hypothalamic leptin sensitivity and in expression of the orexigenic neuropeptides.10 Since both insulin and leptin are critically important for neuronal development during early periods of life, we hypothesized that the altered hormonal environment (hyperinsulinemia and hyperleptinemia) encountered by term HC fetuses may affect development of the HC fetal brain circuitry including hypothalamus which controls feeding behavior as well as other brain regions involved in diverse behavioral functions. In the present study we tested this hypothesis by examining global gene expression and cellular development in the term HC fetal brain.

by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The Institutional Animal Care and Use Committee at the University at Buffalo approved all animal procedures ( protocol # BCH 06064N). Intragastric cannulas were introduced into 4-day-old female rat pups under mild anesthesia (isoflurane inhalation). Buprenorphine (0.02 mg/kg body weight) was administrated as an analgesic. The pups were artificially reared away from their natural dams on an HC milk formula (carbohydrate 56%, fat 20%, and protein 24%) from postnatal day 4 to 24.7 The pups were housed individually in Styrofoam cups floating in a 37°C water bath and were fed at the rate of 0.45 kcal/g body weight/day. On postnatal day 24, the cannulas were cut close to the skin and the pups were weaned on to a standard rodent laboratory chow (Harlan Teklad, Madison, WI, USA) ad libitum on postnatal day 24.7 The control group consisted of female rat pups nursed by their natural dams (mother-fed; MF; caloric composition of rat milk: carbohydrate 8%, fat 68%, and protein 24%) and weaned onto a rodent chow on postnatal day 24.

Methods Generation of HC female rats

GeneChip analysis

The University at Buffalo’s Animal Care Program and the Laboratory Animal Facilities are fully accredited

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Generation of HC offspring HC and MF female rats (∼ 60-day-old) were bred with normal MF adult male rats. Pregnant rats continued to consume standard laboratory rodent lab chow during gestation and lactation. Pregnant rats were anesthetized (ketamine/xyalazine; 75 mg and 10 mg/kg body weight, respectively) and killed on gestational day 21 and the fetal brains (one fetus from each of six pregnant rats; no sex preference for fetal studies) were dissected out and frozen in liquid nitrogen. For immunohistochemical analysis term fetuses (two fetuses from each of four pregnant rats) were individually perfused with para-formaldehyde. Pregnant rats were anesthetized (ketamine/xyalazine) and the fetuses were rapidly removed from the anesthetized dam and further anesthetized (ketamine/xylazine) immediately before being perfused. The thorax was opened to fully expose the beating heart. A blunt needle was inserted into the left ventricle and a hole made in the right atrium. Ice-cold saline ( pH 7.35, 20 ml) was infused followed by ice cold 4% paraformaldehyde ( pH 7.35, 50 ml). The brain was then removed and placed in 4% paraformaldehyde overnight and subsequently transferred to 25% sucrose till the brain dropped to the bottom of the vial. The brains were slow frozen in isopentane and then stored at −80°C and processed as described below.

RNA was extracted from each of the whole fetal brains (n = 6) with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Twenty micrograms of RNA samples were

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processed for GeneChip analysis using Affymetrix GeneChips (Rat 230 2.0, containing probe sets for ∼28 000 genes). The data were analyzed with GeneChip Operating Software (GCOS) version 1.4, essentially as described by the manufacturer. The list of differentially expressed genes was edited to exclude all changes that were less than 2-fold and P value of change was >0.05 and subjected to DAVID analysis to group them into functional clusters.11

Immunocytochemistry Representative coronal sections (five sections/animal /3–4 animals per group) were immunostained and analyzed as described previously.12 Free floating sections were incubated in 10% normal goat serum (Immunogen; Grand Island, NY, USA), followed by overnight incubation with primary antibodies: rabbit anti-NPY (1:500 dilution; ABCAM, Cambridge, MA, USA), mouse anti-insulin receptor (IR) antibody (1:800 dilution; ABCAM), rabbit anti-α-MSH (1:20 000 dilution; ABCAM), rat anti-Nestin antibody (1:500 dilution; Pharmigen, Franklin Lakes, NJ, USA), mouse anti-βIII Tubulin monoclonal antibody (1:1000 dilution; Sigma-Aldrich, St Louis, MO, USA), goat anti DCX-(C-18; 1:250 dilution; Santa Cruz Laboratories, Santa Cruz, CA, USA); rabbit anti-Ki67 (1:1200 dilution; Vector, Burlingame, CA, USA), and TOPRO reagent (Invitrogen, Grand Island, NY, USA) for labeling double strand DNA. After multiple rinses in phosphate-buffered saline, Table 1 List of gene clusters altered in term fetal HC brains

Biological function of gene cluster

Number of annotations

Gene clusters up-regulated in term-fetal HC brains Cell homeostasis including feeding and 96 drinking behavior Neurotransmission/voltage gated 24 channels inter-intracellular communication/integral Membrane protein 21 Lipid metabolism 21 Protein kinase 18 Response to hormones 9 Cell death 8 Cell adhesion 7 G-protein signaling 7 Ion binding/transport 7 Neuronal/axonal 5 Purine metabolism 5 Protein metabolism 4 Immunoglobulin 4 Gene clusters down-regulated in term-fetal HC brains Muscle proteins 23 Transcription 21 Purine nucleotide 11 Cation-regulated 9 Circulation 6 Development/differentiation 6 Hormone 5 Cell migration 3 Cell death 3


sections were incubated for 2–3 hours with a dilution of the appropriate secondary antibodies. The following antibodies were used: Alexa Fluor 488 goat antimouse and Alexa Fluor 647 chicken anti-mouse (1:2000 and 1:200 dilution, respectively; Molecular Probes, Carlsbad, CA, USA); Cy3-conjugated goat anti-rabbit (1:2000 dilution; Jackson Immunoresearch, West Grove, PA, USA). Immunofluorescence staining was analyzed using Zeiss Axioimager Z1 microscope. Representative sections are shown.

Statistical analysis The significance of the difference of the means between the HC and MF groups of rats were determined by Students’ t test. P < 0.05 was considered significant.

Results Metabolic characteristics of the HC pregnancy In an earlier study it was shown that during pregnancy the HC female rat gained more body weight and had significantly increased levels of insulin, leptin, and Table 2 Selected list of genes whose expression is deregulated in HC term fetal brains Gene symbol

Gene

Fold change

Genes associated with myelination Myelin-associated glycoprotein Mag 2.5 Myelin oligodendrocyte glycoprotein Mog 6.5 Contactin associated protein 1 Cntnap 1 9.8 Myelin and lymphocyte protein Mal 10.6 Myelin basic protein Mbp 16 Proteolipid protein Plp 16 Myelin-associated oligodendrocytic Mobp 45.3 basic protein Glycerol-3-phosphate Gpd1 2.8 dehydrogenase Genes associated with metabolic syndrome Vasoactive intestinal poly peptide Vip 2.5 Hypocretin (orexin A) Hcrt 2.6 Angiotensinogen Agt 4.9 Arginine vasopressin Avp 5.3 Pro-melanin concentrating hormone Pmch 5.3 Oxytocin Oxt 5.7 Neurotensin receptor 2 Ntsr2 9.2 Developmental, signal transduction, and transcription factor genes Nuclear receptor subfamily 4 Nr4a3 −2.6 Jun D proto-oncogene JunD −2.5 Tubby homolog (mouse) Tub −2.5 Eomesodermin (T-box transcription LOC316 −2.1 factor) Kruppel-like factor 5 Klf 5 −2.1 Transcription factor E2a Tcfe2a −2.1 Nuclear receptor co-activator 4 Ncoa4 −2.0 F-box only protein 2 Fbxo2 2.1 Pleckstrin homology domain Plekhb1 2.8 containing family B (evictins) member 1 Myosin light polypeptide Myl2 −21.1 Myosin heavy chain Myh8 7.5 Creatin kinase Ckm −9.2 Troponin T3 Tnnt3 −8 Troponin C2 Tnnc2 −7


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pro-inflammatory markers compared to the MF pregnant rats.8 There were no significant differences in litter size or body weight between the HC and MF term fetuses.8

Gene array analysis In order to determine genome-wide alterations of the term HC fetal brain in response to development in the adverse HC intrauterine environment, gene array analysis was utilized. The expression of housekeeping genes such as β-actin and glyceraldehyde-3-phosphate dehydrogenase was very similar for the two groups (data not shown) indicating reproducibility and reliability of the multiple analytical steps in sample processing. Analysis of the gene expression data indicated that 236 genes were induced and 87 genes were repressed in the HC fetal brains compared with MF controls. Biological function clustering of the genes generated 14 clusters for up-regulated genes and 9 clusters for down-regulated genes in the HC fetal brain compared with the term MF fetal brain (Table 1). A detailed analysis of the identities of deregulated genes in term HC fetal brains revealed increased expression of genes related to the metabolic syndrome including neurotensin receptor 2 (9.2-fold), arginine vasopressin (5.3-fold), angiotensinogen (4.9-fold), and genes encoding orexigenic peptides such as hypocretin, pro-melanin concentrating hormone, and oxytocin (ranging from 2.6- to 5.7-fold; Table 2). Expression of genes involved in myelination also showed robust increases in the term HC fetal brains, including myelin-associated oligodendrocytic basic proteins (45.3-fold), myelin basic protein (16-fold), and proteolipid protein (16-fold). Table 2 also shows that the expression of genes associated with neural development, signal transduction, and transcription were markedly affected in term HC fetal brain. The expression of genes such as tubby homolog and nuclear receptor co-activator 4 were suppressed while factors F-box only protein 2 and pleckstrin homology domain containing family B member 1 was increased.

Immunohistochemical analysis To determine whether the deregulated expression of genes involved in neural development, signal transduction, and transcription is associated with an abnormal brain development, we analyzed cellular features of the term HC and MF fetal brains. The initial examination focused on the germinal layer of the third ventricle which contains the neural stem/progenitor cells that produce neurons and glia which eventually populate the entire brain. Brain sections encompassing diencephalon and the third ventricle were stained with TOPRO which marks DNA and reveals the stratification of cells in the tissue (Fig. 1A). In fetal MF brains, nuclei of the cells surrounding third ventricle formed

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3–4 tightly organized layers of cells in the ventral (Fig. 1A, left bottom panel) and in the medial regions of ventricle (Fig. 1A, right top panel). In contrast, in the HC fetal brains, the nuclear layers were fewer and the nuclei were more dispersed than in the MF fetal brains (Fig. 1A). Since term HC fetuses are hyperinsulinemic,8 we examined if the elevated insulin levels are accompanied by changes in the levels of brain IR. Immunostaining revealed strong IR immunoreactivity in the diencephalon parenchyma and walls of the third ventricle (inserts) in the brains of term MF fetuses. The IR immunoreactivity was greatly diminished in term HC fetuses (Fig. 1B) consistent with the reduced levels of IR mRNA shown in our earlier study using reverse transcriptase-polymerase chain reaction.8 The differences were observed in multiple brain sections, obtained from three fetuses per group. These results indicated that in term HC fetal brains the expression of IR is markedly reduced in the germinal zone of the third ventricle and its surrounding. Given the role of IR signaling in the stem cell biology, we assessed the proliferative activity of cells in the third ventricle and in other brain regions by staining brain sections with anti-Ki67 antibody. Ki67 protein is expressed by cells which maintain proliferative cycle, but is turned off in cells which enter the Go phase. As illustrated in Fig. 1C, in MF fetal brains many cells in the third ventricle walls were Ki67 positive, consistent with their well-established proliferative activity. Also, some of the cells that migrated out into the brain parenchyma maintained the expression of Ki67 indicating their continuous proliferation. Such cells were found in periventricular regions as well as in adjacent telencephalic structures, caudate nucleus, and in brain cortex. In contrast, in the term HC fetal brains the Ki67+ cells were rarely detected in the third ventricle and in the brain cortex and were also less frequent in the caudate nucleus. The sections shown in (Fig. 1C) represent typical differences between the MF and HC fetal brains. This finding indicated that the proliferative activity of the developing third ventricle and telencephalic cells is diminished in the HC fetuses. The brain progenitor cells and their immature progeny express nestin which is turned off and replaced with proteins typical to the neuronal or glial lineages as cells progress toward the differentiated state. Developing neuroblasts express doublecortin later replaced by βIIITubulin. The striking change found in the HC fetal brains was an increased appearance of nestin expressing cells in the diencephalon (Fig. 1D top). This observation indicated that more cells remained in an immature state, which was supported by reduced staining for β-III Tubulin suggesting a diminished neuronal differentiation (Fig. 1D bottom).

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Figure 1 Histological analysis of term fetal MF and HC rat brains. Brain sections were prepared and stained as described in Methods. (A) T3065TOPRO cell nuclei in the vicinity of the third ventricle. (B) Immunostaining of IR in the diencephalic region. Insets illustrate IR immunoreactivity in third ventricle walls. (C) Immunolabeling of proliferating cells using Ki67. Counting of Ki-67 cells on representative sections shows a decrease in the density of Ki67 cells in HC brains compared with MF controls. In the top and bottom regions of the third ventricle the density of Ki67 cells was reduced by 47 and 79%, respectively, by 57% in brain cortex and by 60% in caudate nucleus. (D) Immunostaining of Nestin-expressing neural progenitor cells and β-III Tubulinexpressing immature neurons in the third ventricular region. (E) Immunostaining of NPY- and MSH-expressing neurons in the hypothalamus. (F) Immunostaining of Nestin-expressing neural progenitor cells in the cortex. (G) Immunostaining of β-III Tubulin expressing immature neurons in the cortex. Bar size: A, B – 50 μm; C, D, E, F, G – 100 μm.

Consistent with the reduced numbers of the βIII-Tubulin expressing young neurons we observed a weaker staining for NPY in the term fetal brain

tissue surrounding the third ventricle of the HC rats (Fig. 1E). Similarly, the number of neurons that differentiated to express melanocyte-stimulating hormone


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(MSH) also appeared reduced in the vicinity of the third ventricle in HC fetal brains (Fig. 1E). Both changes indicated impaired differentiation of hypothalamic neurons in HC fetal brains. The results of Ki67 staining (Fig. 1C) indicated that changes in cell development are not limited to the hypothalamic and thalamic regions but may also include the telencephalic structures. Indeed, the appearance of nestin expressing immature cells was markedly increased in the hypothalamus, as well as in brain cortex of the HC fetal rats (Fig. 1F) confirming a broader spectrum of developmental changes. In the term HC fetal brains nestin+ cells were present throughout the layers of the dorsal and lateral cortices in contrast to the term fetal MF brains in which immature nestin+ cells were found only in the deeper layer. Differences were also observed in distribution of newly differentiated, βIII-Tubulin expressing neurons (Fig. 1G). In term MF fetal brains such neurons were found in all cortical layers. In contrast, in the term HC fetal brains the βIII-Tubulin expressing neurons were present in most outer layers of the cortex proximal to pial surface but appeared diminished in deeper layers of the brain cortex. The opposite changes in the stratification of nestin+ and βIII-Tubulin expressing cells suggest that neuronal maturation is delayed in term HC fetal brain cortex.

Discussion The results from this study add another dimension to the fetal programming effects in the HC fetus. The observations from the present investigation show that metabolic programming of female rats during postnatal development by HC diet has an unexpected impact on the brain development of their fetal progeny. In an earlier study it was observed that all HC fetuses (irrespective of gender differences) demonstrated increased body weights gains in the postweaning period and adult-onset obesity.13 The observed alterations in global gene expression and cellular development not only in the hypothalamic region but also in extra-hypothalamic regions of the term HC fetal brain suggest that an obese pregnancy could predispose the offspring not only for obesity but possibly also for neurological disorders. The results from this study underscore the importance of body weight control in females during pregnancy in the context of transmission of the predisposition to detrimental outcomes in the offspring. GeneChip analysis of the term HC fetal brain revealed deregulation of genes belonging to several functional clusters in brains of term HC fetuses (Table 2). We found a simultaneous and robust up-regulation of genes encoding myelin basic protein and proteolipid protein both of which are expressed

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by oligodendrocytes during myelination.14 The gene encoding glycerol-3-phosphate dehydrogenase is also included in this list because it participates in de novo synthesis of myelin lipids.14 Similarly, contactin associated protein 1 is essential for the formation of nodes of Ranvier during myelination of axons.15 The increased induction of this gene cluster in term HC fetal brain suggests changes in the biology of myelin-producing oligodendrocytes and potentially myelin formation. Other deregulated genes belonging to clusters such as ion channels, synaptic proteins, and neurotransmitters may affect normal development and activity of axons and synapses. Expression of genes encoding neuropeptides such as hypocretin (orexin A) and pro-melaninconcentrating hormone, which are involved in appetite regulation16 was markedly increased in term HC fetal brains. Also, expression of neurotensin receptor 2 implicated in the regulation of body temperature and feeding behavior17 and members of the nuclear receptor co-activator family regulating the expression of genes involved in lipid and glucose metabolism and development of obesity18 was significantly increased in term HC fetal brains. Similarly, genes encoding pleckstrin homology domain containing proteins, known to participate in insulin, leptin, and other growth factor signaling19 showed changes in their expression suggesting predisposition to developmental and metabolic deregulation in the HC offspring. Mutation in the tubby gene results in adult-onset obesity20 and its altered expression in the term HC fetal brain implicates a role for this gene in the development of obesity in the adult HC offspring. Taken together, the observed changes in the expression of the genes in term fetal HC brains indicate defects that could underlie the development of obesity and other metabolic disorders in the adult HC offspring. Further, brain development engages multiple signaling pathways and transcription factors. In this broad category of genes involved in brain development we found profound reduction in gene expression of troponins,21 eomesodermin,22 and creatine23 (Table 2). Consistent with these transcription data, are the results of our cytological analyses which indicated diminished proliferation and neuronal maturation of brain third ventricle lining stem-like cells. These changes were not limited to the third ventricle and hypothalamic region but were evident in the brain cortex. During development of the brain the walls of the third ventricle and lateral ventricles contain cells that exhibit self-renewal ability typical of stem cells. The slowly proliferating stem cells generate actively proliferating precursors which can migrate into brain parenchyma and differentiate into astrocytes, oligodendroglia, or various types of neurons. The

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diminished cell proliferation in term HC fetal brains was indicated by the reduced number of Ki67+ cells in the vicinity of the third ventricle in the brain as well as in diencephalic and telencephalic parenchyma. In addition, dissemination of immature nestinexpressing cells from their typical locales into the cortical and other brain regions indicates that neuronal differentiation is impaired in term HC fetuses. Typically, developing progenitor cells turn off the expression of nestin and acquire expression of neural markers like DCX or β-III Tubulin. In the hypothalamus some of these cells differentiate further to express NPY and MSH. Consistent with the increased numbers of Nestin+ cells throughout the hypothalamus, we observed a reduced number of cells expressing NPY, suggesting an impaired differentiation of neurons. A similar delay may occur in the multiple cortical brain areas and striatum where we observed an abnormally frequent occurrence of Nestin+ cells and reduced expression of β-III Tubulin. Our findings add to the existing reports that maternal obesity affects the cellular development in regions besides the endocrine-related areas in the brains of the offspring. Diet-induced maternal obesity in mice has been shown to decrease neurogenesis and brain-derived neurotropic factor concentrations in the hippocampus in the postnatal offspring.24 High-fat-diet-induced maternal obesity resulted in altered fetal hippocampal development as indicated by alterations in proliferation of neural precursors, decreased apoptosis and by decreased neuronal differentiation within the dentate gyrus.25 Fetuses of rats fed a high-fat diet during gestation had increased neural progenitor proliferation cells within the hypothalamus.9 Although the molecular mechanisms which initiate the abnormalities in gene expression and cell development in the HC offspring have not been directly examined, our findings indicate that these changes may be related in part to the maternal and fetal hyperinsulinemia and reduction of IR-β in the brains of term HC fetuses. Insulin and insulin-like growth factors comprise a family of neurotropic factors involved in early neural development that has been suggested to strongly affect long-term potentiation, learning, and memory processes. The IR signaling reportedly plays diverse roles in the central nervous system including neuronal development, synaptic plasticity, neuronal survival, lifespan learning and memory, and in neurological disorders.26 Hence, IR may play diverse and versatile functions in the central nervous system regulated by peripheral insulin crossing the brain–blood barrier as well as by intrinsic brain ligands. The reduced IR signaling has been implied in a variety of neurodegenerative disorders such as Alzheimer.27 Genetic disorders caused mainly by mutations in IR


are characterized by abnormalities in multiple organ development including neurological developmental delay.28 Studies in different models like Drosophila29 also demonstrate that insulin signaling is essential for the development of embryonic nervous system and that IR is required for both neural differentiation and expansion. Furthermore in vitro studies using mammalian hypothalamic neurospheres from embryonic day 20 indicated that insulin stimulated neuronal proliferation and differentiation into neurons and astrocytes.30 Abnormal levels of insulin in the fetal or early postnatal periods have been shown to induce alterations in the hypothalamic appetite-regulating mechanisms resulting in adult-onset obesity.31 Thus, in addition to affecting neurogenesis, as indicated by our studies, the hyperinsulinemia and depletion of IR may negatively impact on formation and functioning of the neuronal circuitry. Whether such additional defects occur in the HC offspring needs to be addressed in the future. Similar to insulin, leptin has also been shown to function as trophic factor during early periods of life. Alterations in leptin sensitivity and appetite-regulating networks have been reported in offspring of obese mothers.9 In addition to its function as a satiety factor in adults, leptin has been shown to regulate brain development in the prenatal and postnatal periods.32 For example, the lack of leptin in the Lep ob/Lep ob has been postulated to cause disruption of neural projection from arcuate nucleus in these mice.33 Lep ob/Lep ob mice demonstrate reduced locomotor activity and impairment of cognitive function and this could be due to abnormal development of the brain cortex.32 Serum leptin levels are significantly higher in the pregnant HC rat as well as in the term HC fetuses.8 Whether hyperleptinemia induces leptin resistance in term HC fetal brains and induces cellular alterations as observed in the Lep ob/Lep ob mice remains to be determined. Our studies further underscore the importance of weight regulation during pregnancy in the context of health outcomes in the offspring. The adverse intrauterine environment in pregnant HC rats is the net effect of increased body weight gains and associated hormonal alterations in the pre-pregnancy period. These effects are exaggerated during pregnancy resulting in further increases in body weight and in the serum levels of insulin and leptin.8 In addition, during pregnancy the levels of pro-inflammatory markers and markers of oxidative stress were markedly higher in the HC female.8 As indicated earlier approximately 50% of the women in the child-bearing age are overweight or obese even prior to pregnancy.1 In this connection, the HC rat model is appropriate for studies on the effects of overweight/obesity in


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women on their offspring. Alterations in the global gene expression and cellular development in brains from term HC fetuses revealed by the present investigation indicate that overweight/obesity during pregnancy may lead not only to metabolic disorders but also to neural-based diseases in the offspring. In this context it is of interest to note a recent study wherein it has been suggested that maternal metabolic conditions such as diabetes and obesity could predispose the offspring for the development of autism and related neurological disorders.34 Yet, another study reported35 that maternal obesity was linked to anxiety, depression, and attention deficit hyperactivity disorder in the offspring.

Acknowledgement

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References 1 Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity among US adults 1999–2008. JAMA 2010;303: 235–41. 2 Barker DJ. Intrauterine programming of adult disease. Mol Med Today 1995;1:418–23. 3 Fernandez-Twinn DS, Ozanne SE. Early life nutrition and metabolic programming. Ann New York Acad Sci 2010;1212: 78–96. 4 Li M, Sloboda DM, Vickers MH. Maternal obesity and developmental programming of metabolic disorders in offspring: evidence from animal models. Exp Diabetes Res 2011;2011: 592408. 5 Grove KL, Allen S, Grayson BE, Smith MS. Postnatal development of the hypothalamic neuropeptide Y system. Neuroscience 2003;116:393–406. 6 Bouret SG. Early life origins of obesity: role of hypothalamic programming. J Pediatr Gastroenterol Nutr 2009;48(Suppl. 1): S31–8. 7 Patel MS, Srinivasan M. Metabolic programming in the immediate postnatal life. Ann Nutr Metab 2011;58(Suppl. 2):18–28. 8 Srinivasan M, Dodds C, Ghanim H, Gao T, Ross PJ, Browne RW, et al. Maternal obesity and fetal programming: effects of a high-carbohydrate nutritional modification in the immediate postnatal life of female rats. Am J Physiol Endocrinol Metab 2008;295:E895–903. 9 Chang GQ, Gaysinskaya V, Karatayev O, Leibowitz SF. Maternal high-fat diet and fetal programming: increased proliferation of hypothalamic peptide-producing neurons that increase risk for overeating and obesity. J Neurosci 2008;28: 12107–19. 10 Tozuka Y, Wada E, Wada K. ‘Bio-communication’ between mother and offspring: lessons from animals and new perspectives for brain science. J Pharmacol Sci 2009;110:127–32. 11 Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009;4:44–57. 12 Stachowiak EK, Roy I, Lee YW, Capacchietti M, Aletta JM, Prasad PN, et al. Targeting novel integrative nuclear FGFR1 signaling by nanoparticle-mediated gene transfer stimulates neurogenesis in the adult brain. Integr Biol: Quant Biosci from Nano to Macro. 2009;1:394–403. 13 Vadlamudi S, Kalhan SC, Patel MS. Persistence of metabolic consequences in the progeny of rats fed a HC formula in their


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-61518 (MSP). We thank Dr Luidmila Pliss for performing the perfusion of the fetuses.

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