The Sections on Growth and Metabolism and Women’s Health integrate basic and clinical research to elucidate genetic and physiological mechanisms governing male and female development. We are particularly interested in identifying X-chromosome genes involved in sexually dimorphic development of the reproductive systems and brain and in distinguishing these effects from those of steroid hormones.

Growth Factors, Hormones, and Brain Development
Cheng, Chen, Wang, Smith, Bondy
The growing brain consumes about 50 percent of the total fuel available to the organism as a whole during early postnatal development, despite the fact that its mass is less than 10 percent of the whole. How the brain competes so successfully with peripheral tissues for energy and substrates has been unclear. Insulin preferentially enhances fuel use by peripheral tissues, but not by the brain. We previously demonstrated that endogenous brain IGF1 serves an insulin-like role in promoting neuronal glucose utilization and hence growth during postnatal development. We have shown that brain growth in Igf1 null mice falls behind that of normal littermates by almost 40 percent during the postnatal period, which is when brain IGF1 expression is normally most abundant. We have also demonstrated that brain glucose uptake and utilization is profoundly reduced in the Igf1 null brain during this period. Our studies have implicated IGF1-induced phosphorylation of Akt/PKB in translocation of glucose across the neuronal membrane and IGF1-induced phosphorylation of GSK3b in neuronal glycogenesis, suggesting that IGF1 augments neuronal glucose uptake and storage by familiar, insulin-like pathways. Over the past year, we have investigated the consequences of IGF1 deletion for neuronal survival and growth/morphogenesis.

While cell numbers were preserved throughout most brain structures in the Igf1 null brain, there was a significant reduction in dentate granule cell number in the hippocampal formation, a structure critical for memory. Cell proliferation was actually either normal or increased in the Igf1 null dentate germinal zone, but cell death increased even more, explaining the reduction in dentate size in the IGF1’s absence. Neuronal numbers were preserved, but morphometric analysis showed that pyramidal neuron soma size decreased by about 10 percent in the Igf1 null frontoparietal cortex. Golgi staining revealed that cortical neurons exhibited a significant reduction in dendritic length and complexity in Igf1 null mice. In addition, the density of dendritic spines and, presumably, synaptic contacts declined by 16 percent in the Igf1 null brain ( Fig. 2).

Figure 2

Camera lucida drawings of Golgi-stained layer 2–3 pyramids. A & B are wild type (WT) and C & D are knockout (KO) from Igf1 null brains.


Synaptotagmin levels decreased by 30 percent (P = 0.01) in the Igf1 null brain, yielding evidence in support of a reduction in synapses.

We have proposed that IGF1’s primary physiological role in normal brain development is to promote the growth of projection neurons. IGF1 expression is most abundant in neurons (e.g., Purkinje cells) destined to be the largest and most complex in the brain, implicating IGF1’s insulin-like, anabolic effects in this extraordinary growth. IGF1-expressing neurons grow large perikarya, long axons, and extraordinarily prolific dendritic arbors, which are severely hypoplastic in the IGF1 null brain. Homozygous IGF1 deletion results in mental retardation, showing that the effects of this anabolic peptide on neuronal metabolism and growth have important implications for cognitive function. Current work in our laboratory is aimed at discovering factors regulating neuronal IGF1 expression with a view to therapeutic manipulation to increase the potential for cognitive development and for successful responses to brain injury.

Estrogen Regulation of Gene Expression in the Primate Neocortex
Cheng, Wang, Smith, Bondy
Estrogen has well-established neurotrophic effects in vitro and is reputed to protect against Alzheimer’s disease in post-menopausal women. To elucidate the molecular basis for these neuroprotective effects, we have investigated the effects of estradiol (E2) versus placebo on brain gene expression in a primate model system: ovariectomized rhesus monkeys. We previously showed that E2 enhances the expression of glucose transporters and IGF1 in the monkey prefrontal cortex, a brain region involved in higher-level cognitive functioning, thus suggesting a role for IGF1 and augmented glucose transport in estrogen’s salutary effects on brain function. Using DNA array technology, studies over the past year have focused on profiling a larger spectrum of cortical genes regulated by estrogen. E2 treatment produced significant alterations in gene expression profiles in the prefrontal cortex. The levels of 61 transcripts increased while those of 77 decreased more than 1.5-fold in the E2-treated group, with greatest effects including an approximately four-fold increase in the ribosomal protein S6 kinase and a 4.3-fold decrease in malate dehydrogenase 1. About 15 percent of the altered transcripts encode metabolic enzymes, 12 percent transcription factors, 12 percent cytoskeletal proteins, and 10 percent protein kinases, with the remainder belonging to diverse functional categories.

Figure 3

Estrogen decreases the level of transcription factors TF2B and E2F1 and increases the expression of the synaptic protein VAMP in the primate prefrontal cortex. Data from immunoblots of protein extracted from estrogen and placebo-treated monkeys (n=4–5/group).

Fig. 3 shows confirmation of some of these changes at the protein level. The findings show robust and complex genomic effects by E2 on a brain structure that is of major importance for higher cognitive functions. Identification of cell types involved in these changes and investigation of potential functional interrelationships between the altered gene products should elucidate E2’s role in cognitive function and neuroprotection.

Effects of X-Chromosome Deletion on Bone Fragility and Intermediary Metabolism
Bakalov, Hanton, Luo, Zinn, Bondy
Turner Syndrome (TS) involves the loss of all or part of one sex chromosome and is observed in one out of 2,500 live female births. We have recently initiated genotype-phenotype studies aimed at identifying X-chromosome genes responsible for abnormalities in development and function of the brain, cardiovascular system, and ovary in TS. The new studies are also focusing on the clinical characterization and genetic tracking of metabolic disorders, including osteoporosis, glucose intolerance, hypertension, and dyslipidemia, which appear to affect greater-than-expected numbers of individuals with TS. The short stature typical of the disorder is thought to be due primarily to haplo-insufficiency for the SHOX gene product. Several studies have reported an increased prevalence of bone fractures in TS, which has been attributed either to SHOX deletion or to estrogen insufficiency owing to ovarian failure. We have shown that, when fracture history is carefully documented through personal interviews with both subjects and age-matched controls, there is no excess of fractures in TS. Moreover, we have demonstrated that bone mineral density, when measured by using volumetric methods not dependent on bone size, is virtually normal in TS women on conventional hormone replacement treatment. This analysis appears to exclude a role for the SHOX gene product in preventing osteoporosis or bone fragility.

Reports on diabetes mellitus (DM) in TS have varied widely, with some studies indicating prevalence up to 40 percent and others finding no increase over normal populations (about 5 percent). We have now shown that, while most girls and women with TS have normal fasting glucose and insulin, the response to a glucose challenge is markedly abnormal in at least 40 percent of lean young women after exclusion of obese subjects from the analysis. Multivariate analyses have also excluded significant contributions of family history and ovarian failure to this diabetic phenotype. Preliminary data suggest a primary insulin secretory defect in young lean women and girls with TS, without evidence of insulin resistance as seen in typical type 2 DM. Since about 40 percent of the group is affected and thus 40 percent gets its single normal X from the father, we hypothesize that a gene promoting pancreatic insulin synthesis/secretion is selectively active on the maternal X but imprinted or silent on the paternal X chromosome. Genotyping of parental chromosomes will allow testing of this hypothesis, and evaluation of subjects with partial X deletions will allow us to search for specific genes responsible for this metabolic phenotype.

Role of Ovarian Androgens in Protection of the Mammary Gland
Zhou, Dimitrakakis, Bondy
Androgens are normally quite abundant in healthy women; indeed, they are relatively more abundant than estrogens throughout the entire life cycle. Androgens’ normal physiological role in women is thought to involve augmentation of lean body mass, energy, and libido. We have proposed that another important role for endogenous androgen in women is to protect the mammary gland from “unopposed” estrogenic stimulation. We have shown that treatment of normally cycling rhesus monkeys with flutamide, an androgen receptor antagonist, enhances mammary epithelial proliferation by about 50 percent, indicating that androgen receptor activation normally suppresses mammary epithelial proliferation. To evaluate the efficacy of physiologic androgen supplementation in limiting estrogen replacement therapy–induced mammary epithelial proliferation, we employed an ovariectomized rhesus monkey model of menopause. Proliferation was increased about four-fold in the groups treated with estradiol and estradiol plus progesterone but was no different from vehicle-treated control in the group on estradiol plus testosterone. These observations suggest that endogenous androgens normally limit mammary epithelial proliferation and that androgen supplementation of estrogen therapy may reduce estrogen-induced proliferation and breast cancer risk. Potentially explaining at least partially the mechanism of testosterone’s protective effects, we have shown that estrogen receptor (ER) alpha is down-regulated and ER beta up-regulated by testosterone. Since the alpha isoform mediates proliferative effects and MYC expression, while the beta isoform does not, the dramatic alteration in ER alpha/beta ratio may be integral to testosterone’s inhibition of estrogen-induced proliferation. Indeed, we have also demonstrated a significant reduction in MYC expression in the estrogen/testosterone group (Fig. 4).

Figure 4

Estrogen (E) and estrogen plus progesterone (E/P) strongly induce MYC expression in the primate mammary epithelium while estrogen plus testosterone (E/T) do not; compared with control (Con).