Clara M. Cheng

A physiologic role for testosterone in limiting estrogenic stimulation of the breast.

ObjectiveThe normal ovary produces abundant testosterone in addition to estradiol (E2) and progesterone, but usually only the latter two hormones are “replaced” in the treatment of ovarian failure and menopause. Some clinical and genetic evidence suggests, however, that endogenous androgens normally inhibit estrogen-induced mammary epithelial proliferation (MEP) and thereby may protect against breast cancer. DesignTo investigate the role of endogenous androgen in regulating mammary epithelial proliferation, normal-cycling rhesus monkeys were treated with flutamide, an androgen receptor antagonist. To evaluate the effect of physiological testosterone (T) supplementation of estrogen replacement therapy, ovariectomized monkeys were treated with E2, E2 plus progesterone, E2 plus T, or vehicle. ResultsWe show that androgen receptor blockade in normal female monkeys results in a more than twofold increase in MEP, indicating that endogenous androgens normally inhibit MEP. Moreover, we show that addition of a small, physiological dose of T to standard estrogen therapy almost completely attenuates estrogen-induced increases in MEP in the ovariectomized monkey, suggesting that the increased breast cancer risk associated with estrogen treatment could be reduced by T supplementation. Testosterone reduces mammary epithelial estrogen receptor (ER) &agr; and increases ER&bgr; expression, resulting in a marked reversal of the ER&agr;/&bgr; ratio found in the estrogen-treated monkey. Moreover, T treatment is associated with a significant reduction in mammary epithelial MYC expression, suggesting that Ts antiestrogenic effects at the mammary gland involve alterations in ER signaling to MYC. ConclusionsThese findings suggest that treatment with a balanced formulation including all ovarian hormones may prevent or reduce estrogenic cancer risk in the treatment of girls and women with ovarian failure.

Insulin-like growth factor 1 is essential for normal dendritic growth†

This study evaluated somatic and dendritic growth of neurons in the frontoparietal cortex of Igf1–/– brains. Pyramidal neuron density was increased by ∼25% (P = .005) and soma size reduced by ∼10% (P < .001). Golgi staining revealed that cortical layer II–III neurons exhibited a significant reduction in dendritic length and complexity in Igf1 null mice. Dendritic spine density and presumably synaptic contacts were reduced by 16% (P = .002). Similar findings were obtained for cortical layer V and piriform cortex pyramids. Supporting a reduction in synapses, synaptotagmin levels were reduced by 30% (P < .02) in the Igf1 null brain. Investigation of factors critically involved in dendritic growth and synaptogenesis showed an ∼50% reduction in cortical CDC42 protein expression (P < .001) and an ∼10% reduction in brain cholesterol levels (P < .01) in Igf1 null mice. Evidence is presented that Igf1 deletion causes disruptions in lipid and microtubule metabolism, leading to impaired neuronal somatic and dendritic growth. Published 2003 Wiley‐Liss, Inc.

Biochemical and Morphometric Analyses Show that Myelination in the Insulin-like Growth Factor 1 Null Brain Is Proportionate to Its Neuronal Composition

To elucidate the role of insulin-like growth factor 1 (IGF1) in the normal development of brain myelination, we used behavioral, biochemical, and histological analyses to compare the myelination of brains from Igf1−/− and wild-type (WT) littermate mice. The studies were conducted at postnatal day 40, at which time the Igf1−/− mice weighed ∼66% less than wild-type mice. However, theIgf1−/− brain weight was only reduced by ∼34%. Formal neurological testing showed no sign of central or peripheral myelinopathy inIgf1−/− mice. Myelin composition was not significantly different, and myelin concentration, normalized to brain weight or protein, was equal inIgf1−/− and WT mice. Likewise, concentrations of myelin-specific proteins (MBP, myelin proteolipid protein, MAG, and 2′,3′-cyclic nucleotide,3′-phosphodiesterase) were not significantly different inIgf1−/− and WT mice. The myelin-associated lipids galactocerebroside and sulfatide were modestly reduced in Igf1−/− brains. Regional oligodendrocyte populations and myelin staining patterns were comparable in Igf1−/− and WT brains, with the notable exception of the olfactory system. TheIgf1−/− olfactory bulb was profoundly reduced in size and was depleted of mitral neurons and oligodendrocytes, and its efferent tracts were depleted of myelin. In summary, this study shows that myelination of theIgf1−/− brain is proportionate to its neuronal composition. Where projection neurons are preserved despite the deletion of IGF1, as in the cerebellar system, oligodendrocytes and myelination are indistinguishable from wild type. Where projection neurons are depleted, as in the olfactory bulb, oligodendrocytes are also depleted, and myelination is reduced in proportion to the reduced projection neuron mass. These data make a strong case for the primacy of axonal factors, not including IGF1, in determining oligodendrocyte survival and myelination.

Endogenous IGF1 enhances cell survival in the postnatal dentate gyrus

The dentate gyrus is selectively reduced in size in the insulin‐like growth factor 1 (IGF1) null mouse brain. The purpose of this study was to determine whether this defect is due to reduced granule cell numbers, and if so, to determine whether altered cell proliferation, survival, or both contribute to attenuation of dentate gyrus size. At postnatal day 10 (P10), granule cell numbers were not significantly different in IGF1 null and littermate wildtype (WT) dentate gyri. The subgranular zone cell population, however, was relatively increased, and the granule cell layer population relatively decreased in the IGF1 null dentate gyrus. By P50, total dentate cell numbers were decreased by 20% (P = 0.01) in the IGF1 null mouse, although IGF1 null subgranular zone progenitor cells remained relatively increased compared with WT (38%, P < 0.05). IGF1 null dentate cell proliferation, assessed by thymidine analogue incorporation, was actually increased at P10 (33%, P < 0.05) and P50 (167%, P = 0.001). Dentate granule cell death, assessed by the appearance of pycnotic cells and DNA fragmentation, was also significantly increased in the IGF1 null dentate (61%, P < 0.05 and 101%, P = 0.03). These data suggest that endogenous IGF1 serves an important role in dentate granule cell survival during the course of postnatal brain development. In addition, this work suggests the potential of a compensatory mechanism promoting increased dentate cell proliferation in the face of impaired cell survival during postnatal neurogenesis. J. Neurosci. Res. 64:341–347, 2001. Published 2001 Wiley‐Liss, Inc.

Caloric restriction augments brain glutamic acid decarboxylase-65 and -67 expression

The ketogenic diet is a very low‐carbohydrate, high‐fat diet used to treat refractory epilepsy. We hypothesized that this diet may act by increasing expression of glutamic acid decarboxylase (GAD), the rate‐limiting enzyme in γ‐aminobutyric acid (GABA) synthesis. Thus, we evaluated brain GAD levels in a well‐established, seizure‐suppressing, rodent model of the ketogenic diet. Because the diet is most effective when administered with a modest (∼10%) calorie restriction, we studied three groups of animals: rats fed ad libitum standard rat chow (Ad lib‐Std); calorie‐restricted standard chow (CR‐Std); and an isocaloric, calorie‐restricted ketogenic diet (CR‐Ket). We found that GAD67 mRNA was significantly increased in the inferior and superior colliculi and cerebellar cortex in both CR diet groups compared with control (e.g., by 45% in the superior colliculus and by 71% in the cerebellar cortex; P < .001). GAD65 mRNA was selectively increased in the superior colliculus and temporal cortex in both CR‐Std and CR‐Ket diet groups compared with ad lib controls. The only apparent CR‐Ket‐specific effect was a 30% increase in GAD67 mRNA in the striatum (P = .03). Enhanced GAD immunoreactivity was detected in parallel with the mRNA changes. These data clearly show that calorie restriction increases brain GAD65 and ‐67 expression in several brain regions, independent of ketogenic effects. These observations may explain why caloric restriction improves the efficacy of the ketogenic diet in treating epilepsy and suggest that diet modification might be useful in treatment of a number of brain disorders characterized by impaired GAD or GABA activity.

X-Chromosome Gene Dosage and the Risk of Diabetes in Turner Syndrome

BACKGROUND Turner syndrome (TS) is caused by the absence or fragmentation of the second sex chromosome. An increased risk of diabetes mellitus (DM) has consistently been noted, but the specific phenotype and genetic etiology of this trait are unknown. METHODS In a prospective study, we examined the prevalence of DM in adult participants in an intramural National Institutes of Health (NIH) TS study. Results were analyzed with respect to karyotype, age, body mass index (BMI), and autoimmune indices. Insulin sensitivity and secretion were compared in age- and BMI-matched euglycemic women with TS and healthy female controls. We compared gene expression profiles in lymphocytes from differentially affected TS groups. RESULTS Type 2 DM was present in 56 of 224 (25%) of the women with TS; type 1 DM was found in only one woman (<0.5%). DM was more prevalent among women with an isoXq chromosome compared to X monosomy (40.0 vs. 17.3%; P = 0.004). Euglycemic women with TS (n = 72; age, 33 +/- 12 yr; BMI, 23 +/- 3 kg/m(2)) had significantly higher glycemic and lower insulin responses to OGTT, with insulin sensitivity similar to controls. Gene expression profiles comparing 46,X,i(X)q vs. 45,X groups showed a significant increase in Xq transcripts and in potentially diabetogenic autosomal transcripts in the isoXq group. CONCLUSION Type 2 DM associated with deficient insulin release is significantly increased among women with monosomy for the X-chromosome but is increased even more among women with monosomy for Xp coupled with trisomy for Xq. These data suggest that haploinsufficiency for unknown Xp genes increases risk for DM and that excess dosage of Xq genes compounds the risk.

Bicuspid aortic valve and aortic coarctation are linked to deletion of the X chromosome short arm in Turner syndrome

Background Congenital heart disease (CHD) is a cardinal feature of X chromosome monosomy, or Turner syndrome (TS). Haploinsufficiency for gene(s) located on Xp have been implicated in the short stature characteristic of the syndrome, but the chromosomal region related to the CHD phenotype has not been established. Design We used cardiac MRI to diagnose cardiovascular abnormalities in four non-mosaic karyotype groups based on 50-metaphase analyses: 45,X (n=152); 46,X,del(Xp) (n=15); 46,X,del(Xq) (n=4); and 46,X,i(Xq) (n=14) from peripheral blood cells. Results Bicuspid aortic valves (BAV) were found in 52/152 (34%) 45,X study subjects and aortic coarctation (COA) in 19/152 (12.5%). Isolated anomalous pulmonary veins (APV) were detected in 15/152 (10%) for the 45,X study group, and this defect was not correlated with the presence of BAV or COA. BAVs were present in 28.6% of subjects with Xp deletions and COA in 6.7%. APV were not found in subjects with Xp deletions. The most distal break associated with the BAV/COA trait was at cytologic band Xp11.4 and ChrX:41,500 000. One of 14 subjects (7%) with the 46,X,i(Xq) karyotype had a BAV and no cases of COA or APV were found in this group. No cardiovascular defects were found among four patients with Xq deletions. Conclusions The high prevalence of BAV and COA in subjects missing only the X chromosome short arm indicates that haploinsufficiency for Xp genes contributes to abnormal aortic valve and aortic arch development in TS.

Insulin-like growth factor-1 promotes neuronal glucose utilization during brain development and repair processes.

Abstract We have reviewed several lines of evidence suggesting that IGF-1 augments neuronal glucose utilization during brain development. To briefly recapitulate, brain glucose utilization parallels IGF-1 receptor expression during brain development. In normal murine brain development, IGF-1 is produced in greatest abundance by growing cerebellar and sensory projection neurons during the time of dendrite elaboration and synaptogenesis. Glucose utilization is significantly reduced in developing IGF1 -/-brain, particularly in those sites where IGF-1 expression is normally most abundant. The defect in glucose utilization in IGF1 -/-brains is demonstrable at the terminal level in vitro , and is reversed by IGF-1. It appears that IGF-1 promotes glucose allocation to growing neurons in an autocrine manner, enabling the extraordinary elaboration of processes characterizing these complex information-processing systems. These sensory processing centers continue to exhibit high-level glucose utilization in the mature brain, after IGF-1 expression has receded, reflecting the extraordinary dendritic complexity and synaptic density achieved by these structures. IGF-1 is not likely to play a major role in the rapid, neural activity-based glucose utilization in the mature brain, as discussed in Section IV. It will be interesting to determine whether IGF-1 is involved in activity-induced synaptic remodelling, as a number of studies suggest ( Torres-Aleman, 2000 ). IGF-1 is highly expressed by reactive (GFAP-positive) astrocytes in various injury models ( Komoly et al. , 1992 ; Lee et al. , 1992 ; Lee and Bondy, 1993 ; Gehrmann et al. , 1994 ; Yao et al. , 1995 ; Walter et al. , 1997 ; Beilharz et al. , 1998 ; Li et al. , 1998 ). We have found that astrocytic IGF-1 expression several days after MCAO is closely correlated with increased glucose utilization in the injury site, as shown in Fig. 7 ( Lee et al. , 1992 ). The significance of this local increase in glucose utilization is unclear, but it may be attributed to increased anabolic activity by astrocytes synthesizing and secreting collagen and other extracellular proteins involved in scar formation. PKB/Akt and GSK3β appear to be central players in IGF signaling to the brain (Fig. 13). PKB/Akt phosphorylation in IGF-1-expressing neurons is associated with increased GLUT4 expression and translocation from intracellular to the plasma membrane. IGF1s apparent link with this “insulin-sensitive” transporter may represent a specific anabolic pathway, distinct from the glucose transport pathways involving GLUTs 1 and 3. PKB/Akt phosphorylation also leads to the inhibition of GSK3β in IGF-1-expressing neurons (Figs. 11 and 13). This inhibition is expected to facilitate glycogen and protein synthesis, as GSK3β normally inhibits both glycogen synthase and eIF2B (Fig. 13). As a result, there is accumulation of glycogen in IGF-1-expressing neurons ( Cheng et al. , 2000 ), which may serve to create a relative “sink” for G-6-P, promoting further glucose transport into the neuron. GSK3β also phosphorylates the microtubule-associated protein tau. Tau is hyper-phosphorylated in the IGF1 null brain, as expected if IGF-1 normally inhibits this multifunctional enzyme. Tau hyperphosphorylation is the mechanisms governing normal cell- and developmental stage-specific regulation of IGF-1 expression, and to attempt to restore normal patterns of expression in conditions such as fetal alcohol exposure and malnutrition, and possibly in other forms of mental retardation.

Monosomy for the X chromosome

Dosage compensation serves to equalize X chromosome gene expression in mammalian males and females and involves extensive silencing of the 2nd X chromosome in females. If dosage compensation mechanisms completely suppressed the 2nd X chromosome, then actual physical loss of this “eXtra” chromosome should have few consequences. However, X monosomy has major effects upon normal development, fertility and longevity in humans and some other species. This article reviews observations and arguments attempting to explain the phenotypic effects of X monosomy in humans and other mammals in terms of X chromosome gene dosage.

High-level expression of Dok-1 in neurons of the primate prefrontal cortex and hippocampus†

The docking protein p62Dok‐1 (Dok‐1) has a central role in cell signaling mediated by a wide range of protein tyrosine kinases, including intrinsic membrane kinases, such as the insulin‐like growth factor‐1 (IGF‐1) receptor. To elucidate potential IGF signaling mechanisms, we used DNA array technology to investigate novel kinase targets expressed in the primate dorsolateral prefrontal cortex (DLPFC). Dok‐1 transcripts were among the most abundant found in this structure. Because Dok‐1 expression has not been characterized in brain, we evaluated its expression pattern using immunoblotting, in situ hybridization, and immunohistochemistry in the rhesus monkey prefrontal cortex and hippocampal formation. Dok‐1 antibodies identified a 62‐kDa band in lysates from the DLPFC, consistent with the known size for Dok‐1. In situ hybridization showed that Dok‐1 mRNA was expressed in all layers of the DLPFC and in all neuronal subregions of the hippocampal formation. Immunohistochemical analysis showed Dok‐1 immunoreactivity concentrated in pyramidal neurons of cortical layers IV–V and throughout Ammons horn and in granule neurons of the dentate gyrus. Dok‐1 expression was also identified in endothelial cells of cerebral blood vessels. These expression patterns are very similar to those of the IGF‐1 receptor and suggest that Dok‐1 could be among the downstream targets of IGF signaling in areas of the primate brain involved in learning and memory. Published 2003 Wiley‐Liss, Inc.