Trends and Insight

Binding Protein Plays Key Role in Stress Response and Memory

Stress can cause glucocorticoids to flood the brain and interfere with memory performance and other behaviors. Corticosteroid-binding globulin (CBG), a glycoprotein with high affinity for binding glucocorticoids in blood, is believed to play an important role in this process.

Studies have shown that mice with CBG defi- ciency react to stressful conditions with lower glucocorticoid signaling, so researchers have hypothesized that plasma CBG plays a crucial role in the amount of corticosterone that gains access to the brain. To test this hypothesis, a research team led by Marie-Pierre Moisan, Ph.D., of the University of Bordeaux, France, investigated the involvement of CBG levels in the stressinduced rise of corticosterone in the hippocampus.

The researchers compared the performance of CBG-deficient mice and controls in a memory retrieval task known to be affected by hippocampal glucocorticoid levels. When the control mice were subjected to acute stress before the test, they suffered from the impaired memory retrieval associated with an increase in corticosterone in the plasma and brain. Stress did not affect the memory retrieval of the CBG-deficient mice; the animals experienced a markedly reduced surge of corticosterone in plasma and no rise in corticosterone in the hippocampus.

To demonstrate that the reduced rise in corticosterone was responsible for the absence of the stress-induced memory change, the researchers infused corticosterone into the hippocampus to mimic a stress-induced increase. Control and CBG-deficient mice reacted with similar memory retrieval impairments. The researchers found that the adrenal glands of the CBG-deficient mice responded normally to stress in increasing corticosterone production, but the lack of CBG binding led to increased clearance, hence the absence of a rise in plasma corticosterone.

In an upcoming Endocrinology [endo. endojournals.org] article, the researchers state that this clear-cut evidence that CBG retains glucocorticoids in plasma, thereby promoting their access to the hippocampus, has important implications for understanding the role of CBG and glucocorticoids in stress-related psychiatric disorders.

MicroRNAs Can Be TSH Mediators

Researchers are continually tracing hormone actions back to their most basic levels, including interactions with genes. Although much is known about the pathways by which thyroid-stimulating hormone (TSH) regulates thyroid cell growth and secretion of thyroid hormone, little is known about its interactions with microRNAs (miRNAs), short segments of RNA that bind to target genes and suppress their expression.

To explore the role of miRNAs in TSH activity, Koichi Suzuki, Ph.D., of Japan’s National Institute of Infectious Diseases in Tokyo, led a team that performed an miRNA microarray analysis and demonstrated that TSH significantly decreased the expression of 47 miRNAs in thyroid cells. Using miRNA agonists, the researchers identified two miRNAs, miR-16 and miR-195, as mediators of TSH’s capacity to induce cell proliferation. High levels of miR-16 and miR-195 suppressed the cell-cycle progression and DNA synthesis that TSH induces.

The target genes suppressed by the two miRNAs were Mapk8, Ccne1, and Cdc6—genes known to be upregulated by TSH as part of cell-cycle regulation in the thyroid. The genes are involved in the phosphatidylinositol 3-kinase (PI3K) and cyclic adenosine monophosphate (cAMP) pathways—activation of these pathways induces a series of transcription factors and cell-cycle regulating proteins that lead to cell proliferation.

An inhibitor of PI3K not only reversed the effect of TSH, but also increased miR-16 and miR-195 expression, and an inhibitor of the cAMP pathway had similar effects. These results led the researchers to suggest that TSH activates the cAMP and P13K signaling cascades to decrease miR-16 and miR-95, which induces the Mapk8, Ccne1, and Cdc6 genes to activate cell proliferation. In a paper accepted for publication in Molecular Endocrinology [mend. endojournals.org] the authors write that these findings provide another step toward understanding the physiological regulation of thyroid cell growth and function.

Excess Androgen Linked with Fatty Liver in PCOS

Affecting as many as 20 percent of reproductive-age women, polycystic ovary syndrome (PCOS) goes hand in hand with infertility as well as liver disease and obesity. Though multiple studies have shown a higher incidence of nonalcoholic fatty liver disease (NAFLD) in women with PCOS, none has accounted for obesity’s possible confounding effects. Does PCOS itself increase risk of NAFLD or is NAFLD in this population purely caused by obesity? Furthermore, does androgen level, a defining trait of PCOS, have anything to do with this dynamic?

Scientists led by Daniel Cuthbertson, M.D., Ph.D., University of Liverpool, United Kingdom, used proton magnetic resonance spectroscopy to measure liver fat in 29 women with PCOS and 22 age-matched controls. In their paper, to be published soon in The Journal of Clinical Endocrinology & Metabolism [jcem.endojournals.org], the researchers report that the hyperandrogenic subgroup of women with PCOS had increased liver fat, a result that held up after adjusting for body mass index. The PCOS subgroup with normal androgen levels showed no significant difference from controls in hepatic steatosis.

The researchers conclude that NAFLD in PCOS is not entirely caused by obesity or insulin resistance, but is instead connected with the hyperandrogenic subtype of PCOS. Knowing that high levels of free androgens in PCOS is a risk factor for developing NAFLD may lead to more aggressive strategies, including lifestyle intervention, in these women.

Nursing from Birth Is Important for Cervical Development

Many experts recommend breast-feeding, especially the consumption of “first milk” or colostrum. Although nutritional and immunological benefits of nursing are well recognized, the role of colostrum as a conduit for delivery of milk-borne bioactive factors (MbFs), including hormones and growth factors, to neonates, termed lactocrine signaling, is less well defined. Scientists at Rutgers and Auburn Universities have been conducting research to determine the effect of lactocrine signaling on female reproductive tract (FRT) development. In an upcoming study in Endocrinology [endo. endojournals.org], a team of researchers led by Carol Bagnell and Frank Bartol, sought to determine whether nursing affects the development of neonatal cervical tissues.

As a component of the FRT, the cervix plays an important role in reproduction, serving as a conduit for sperm transport, a protective uterine barrier during gestation and as a dynamic component of the birth canal at parturition. “Earlier studies by our group established that nursing is required to support normal uterine development in the neonate,” wrote Carol Bagnell of Rutgers University. “By extension, given the wellestablished role of relaxin, a prototypical MbF, in cervical growth and remodeling, we hypothesized that aspects of cervical development in the neonate should be both relaxin and lactocrine sensitive.”

Using pigs as their model, the researchers found that nursing for two days from birth was required for neonatal expression of cervical proteins important for growth and remodeling including, estrogen receptor-α, vascular endothelial growth factor and matrix metalloproteinase 9. Treatment with relaxin altered expression of these cervical proteins only in pigs that nursed showing that cooperative factors absent in replacer-fed gilts are needed for the cervical response to relaxin.

Returning replacer-fed gilts to nursing two days after birth failed to rescue the lactocrine-null cervical phenotype by postnatal day 14, suggesting that a critical window for lactocrine signaling exists within the first two days after birth. Changes in milk composition and/or development of the gastrointestinal tract may make absorbing MbFs difficult after postnatal day two, the authors suggest.

The researchers conclude that delaying nursing for even two days from birth disrupts cervical development. “This work has significant implications for humans as it pertains to how breast-feeding may support optimal patterns of infant growth and development,” Bagnell wrote. “All mammals, including humans, evolved to nurse their young. Consequently, lactocrine signaling is likely to represent a conserved mechanism that extends biochemical communication from mother to offspring into the postnatal period.”

Hair Provides Cortisol Record for Cyclical Cushing’s Syndrome

Hair is being used increasingly as the body’s historical record of drug use, environmental exposure, and hormone production. The newest application could be cortisol measurement to identify patients with cyclical Cushing’s syndrome.

Cyclical Cushing’s is a rare disorder characterized by alternating periods of excess and normal cortisol secretion. The diagnosis is difficult because cycles can be regular or irregular, with normal periods ranging from days to years. The standard screening tests cover only a day, through 24-hour urine collections or midnight saliva tests.

Researchers led by Laura Manenschijn, M.D., of the Erasmus Medical Center in Rotterdam, The Netherlands, collected scalp hair from patients with confirmed Cushing’s syndrome, patients suspected of having cyclical Cushing’s, and a non-obese control group to test for cortisol.

The cortisol levels were significantly higher in Cushing’s patients than controls. The sensitivity and specificity for a Cushing’s diagnosis, based on the upper limit of the reference range of healthy individuals, were 86 percent and 98 percent, respectively. These diagnostic percentages compare favorably with those of the commonly used 24- hour urine collections and midnight saliva measurement—tests that had to be performed repeatedly on the patients suspected of having cyclical Cushing’s in order to catch them during an episode of excess secretion.

The new technique even offers the possibility of providing a timeline of the disease; the hair samples were measured in 1-cm segments, each corresponding to a period of about a month. The times of high cortisol exposure reconstructed in this way corresponded with the clinical course of the disease, in both patients with Cushing’s and cyclical Cushing’s. This allowed researchers to create retrospective timelines of cortisol exposure that correlated with symptomatic periods in patients suspected of cyclical Cushing’s.

In an article slated for The Journal of Clinical Endocrinology & Metabolism [jcem.endojournals.org], the researchers conclude that this new hair-based diagnostic tool could improve patient care through early recognition cyclic Cushing’s syndrome.

The “Pill” for Men?

A compound originally synthesized to block a gene that causes cancer may lead to a contraceptive pill for men, say scientists at the Dana-Farber Cancer Institute and the Baylor College of Medicine. Their research in mice suggests that the compound, called JQ1, interferes with the creation and maturation of sperm in the testes.

In a study led by Baylor’s Martin M. Matzuk, M.D., Ph.D., and DanaFarber’s James E. Bradner, M.D. that appeared in the August 17 issue of the journal Cell, the team compared mice injected with 50 mg/kg of JQ1 daily with untreated mice and found that sperm counts in the treated mice dropped 72 percent after three weeks. By the sixth week, sperm counts in the treated mice had dropped even further, a total of 89 percent. The treatment affected sperm motility, as well. After six weeks, only 5 percent of the few sperm from the treated group were fully motile. Although several treated mice became fathers after 6–10 weeks of treatment with the lower dose of the JQ1, raising the dose to 75 mg/kg or 100 mg/kg per day rendered them sterile by the end of the mating period.

The effects of JQ1 appear to be temporary and limited to fertility; treatment did not affect the animals’ testosterone levels and the treated mice continued to mate with female mice as often as before. The mice regained their fertility less than two months after stopping treatment, and offspring conceived afterward were healthy.

The research shows that JQ1 works by binding to a protein called BRDT, which plays a role in chromatin remodeling when the testes generate sperm. During this process, chromatin—the combination of DNA and proteins in the nucleus of a cell—is “remodeled” to give the proteins that regulate genes access to genetic material. BRDT is conserved in mice and men and is related to the cancercausing gene BRD4, for which the compound was originally developed.

Testing in humans is a long way off. Derivatives of JQ1 would have to be developed and optimized for delivery in a pill or implant. However, the compound is a breakthrough because it crosses the difficult barrier that separates blood and sperm.

Gene Variation Tied to Early Menarche

Divergences in the age at which a woman begins menstruating are associated with many disorders—an early age of menarche is associated with breast and endometrial cancers and a late age increases the risk of Alzheimer’s disease and osteoporosis.

Because genetic factors play an important but little-understood role in determining the age of menarche, researchers led by Yao-Zhong Liu, M.D., Ph.D., of Tulane University studied the role of a form of genetic variation called copy number variation (CNV).

In CNV, a DNA segment is repeated (copied) two or more times within a chromosome—and sometimes it is omitted. The repeated segments can range in size from thousands to millions of DNA bases. These gains and losses of large chunks of DNA are believed to account for three times as much genetic variation as is caused by single nucleotide polymorphisms, which have received much more attention. Extra copies of a segment can lead to higher levels of the gene’s product, disrupt coding sequences, and affect gene expression even outside the CNV region.

Dr. Liu’s team did a genome-wide association study of CNV and age of menarche in 1,654 Caucasian females. They identified a CNV, variation_38399, associated with age of menarche: Test subjects with only one copy of the variant had a mean age of menarche of 14.0 years, more than a year later than the subjects with two copies of the variant, at 12.9 years. The researchers then con- firmed the association in a cohort of 752 Chinese women.

The variant is located about 75 kilobases upstream of the diazepambinding inhibitor gene, a gene known to regulate estrogen levels, a key factor for menarche.

In a paper in The Journal of Clinical Endocrinology & Metabolism [jcem.endojournals.org], the researchers write that this first study of CNV and menarche supports the existence of a mechanism by which variation_38399 modulates the age of menarche by influencing the signaling pathway mediated by the diazepam-binding inhibitor gene.

Oxytocin Secretion Linked to Eating Disorder

Animal studies have shown that oxytocin is an appetite-regulating hormone. A new study looks at how oxytocin secretion dynamics may differ in anorexia nervosa.

Scientists led by Elizabeth A. Lawson, M.D., M.Sc., at the Harvard Medical School and Massachusetts General Hospital in Boston, studied 13 women with anorexia, 9 women recovered from anorexia, and 13 controls. Participants were given standardized high-protein, high-carbohydrate, low-fat breakfasts, after which serial blood samples were taken. Functional magnetic resonance imaging (fMRI) was also done to assess brain activity in areas involved in appetite, such as the insula, where food intake and emotion related to eating are processed.

In their paper, to be published soon in The Journal of Clinical Endocrinology & Metabolism [jcem.endojournals. org], the researchers report that, as expected, peripheral post-prandial oxytocin levels were highest in women with active disorder and lowest in recovered women and that higher oxytocin levels were associated with greater severity of disordered eating psychopathology. Postprandial oxytocin secretion was also associated with differences in fMRI activation of brain regions involved in appetite in women with anorexia nervosa, even after recovery.

The researchers conclude that oxytocin may be an independent appetite regulator, and its dysregulation may contribute to symptoms of disordered eating in anorexia nervosa, possibly by desensitizing the anorexic woman to her internal state. “Inherent abnormalities in oxytocin pathways may contribute to underlying deficits that increase susceptibility to developing and sustaining anorexia nervosa,” says Dr. Lawson. The next step in unmasking oxytocin’s therapeutic possibility is to study its effects when given to humans.

Leydig Cell Regenerators Appear to be Stem Cells

Stem cells appear to be the precursors to replacements for Leydig cells, the testosterone-producing cells of the adult testes, and researchers say that they have come up with a new way of studying the regenerative process.

Leydig cells rarely die or divide if left undisturbed but can regenerate if necessary. For example, if Leydig cells are depleted in rats by the injection of the alkylating agent, ethane dimethanesulfonate (EDS), a new generation forms. To better understand this process, Haolin Chen, Ph.D., of Johns Hopkins Bloomberg School of Medicine, led a team to investigate whether the new cells are regenerated by stem cells (which can grow into many forms) or quiescent progenitor cells (cells already in the Leydig cell lineage).

The researchers isolated cells that expressed platelet-derived growth factor α but not 3β-hydroxysteroid dehydrogenase (3β-HSD), a key enzyme in the steroid synthesis pathway. Depending on the culture conditions, these cells, called 3β-HSD-negative, could differentiate into forms that produce testosterone—this ability to differentiate is a characteristic of stem cells.

To determine the cells’ location, the researchers separated the seminiferous tubules from the testicular interstitium and cultured both. The seminiferous tubule cells developed 3β-HSD-positive cells capable of producing testosterone, but the interstitial cells did not, which suggested that the tubule surfaces contained stem cells. When the 3β-HSD-positive cells were removed from the seminiferous tubule surfaces with EDS and cultured again, the testosterone-producing cells reappeared—this regenerative ability provided further evidence of the presence of stem cells.

The researchers posit in an article scheduled for Endocrinology [endo. endojournals.org] that the precursors for the newly formed Leydig cells are stem cells, mainly on the surfaces of the seminiferous tubules, showing yet another role for stem cells in regenerative processes. They propose that their tubule culture system, which contains not only the stem cells but also their niche, could provide a valuable example for research into the components and properties of low-turnover adult stem cells, in general, in complex tissues of all mammals.

Pancreas Size Shrinks Before Diabetes Onset

Diabetes is presaged by several factors, some not apparent until after diagnosis. Shrinking of the pancreas is common in type 1 diabetes, reducing it by as much as 48 percent in adult patients 10 years after disease onset. Recently, other data show pancreas size shrinking by as much as 31 percent less than 6 months after diagnosis. Researchers led by Alistair Williams, B.Sc., of the University of Bristol have now extended these findings in a larger group of patients.

Twenty adult male diabetes patients and 24 healthy male controls were scanned by MRI. The age of the patients and controls was similar, being centered around 27 years, and each diabetes patient had scans from 1 month to 8 months after being diagnosed. The pancreas size of the patients had decreased by 26 percent compared to their control counterparts.

In the study’s findings, to be published in The Journal of Clinical Endocrinology and Metabolism [jcem.endojournals.org], age and disease-duration made no signifi- cant contribution to pancreas size. Neither did glucose levels nor the number of autoantibodies. Reduced pancreas size, in turn, did not produce exocrine deficiency, which is common in long-term diabetes.

The study suggests, however, that if pancreas size decreases by 48 percent 10 years after onset and 26 percent just months following diagnosis, then half of that reduction happens in the pre-diabetic period. Furthermore, this atrophy may begin many years before onset, allowing reduction in pancreas size to be useful as a marker of pancreatic β-cell loss before and after diagnosis.

Although the link between β-cell mass and pancreatic size is uncertain, it might provide a measure of disease progression. Coupled with the low risk of non-invasive MRI, this information may give diabetes patients a chance to seek treatment sooner, when effective preventive therapies become available.

Vitamin D’s Effects on Arterial Health

Like most of our body parts, arteries lose their elasticity. Evidence has suggested a link between vitamin D deficiency and arterial stiffening, which would support the notion that vitamin D supplementation reduces the risk for cardiovascular disease. A new study specifically examines the possible connection.

Francesco Giallauria, M.D., Ph.D., at the National Institute on Aging in Baltimore and the University of Naples in Italy, says, “The higher prevalence of hypervitaminosis D is due to several factors that may coexist—low levels of sunlight exposure, sedentary lifestyle, and inadequate consumption of fresh food.” His team used pulse-wave velocity (PWV) to test whether low serum levels of 25OHD contribute to carotid–femoral atherosclerosis in 1,288 participants in a multiethnic, cross-sectional analysis as part of the Baltimore Longitudinal Study of Aging.

In their paper, to be published soon in The Journal of Clinical Endocrinology & Metabolism [jcem. endojournal.org], the researchers report that the lowest serum 25OHD levels correlated with the highest PWV measurements, which indicates artery hardening, and that this inverse relationship held up even after adjusting for potential confounders, such as age, sex, ethnicity, and blood-draw timing. Additional adjustments were made for specific cardiovascular factors, such as weight, amount of exercise, and smoking as well as for conditions known to affect the heart such as diabetes

The researchers conclude that 25OHD level is an independent predictor of arterial stiffness. They are planning to undertake large-scale studies to focus on the underlying mechanism and to determine definitively whether vitamin D supplementation improves arterial elasticity.

Body Rhythms Reset Through Site-specific Receptors

Our circadian rhythms are synchronized between the suprachiasmatic nucleus (SCN) in the hypothalamus and peripheral oscillators, the body’s ancillary clocks. Glucocorticoids secreted by the adrenal gland, such as corticosterone, provide timing signals so the phases of these clocks act in harmony. Damage to the SCN disrupts the rhythm of these secretions and although peripheral tissues can maintain their oscillations, their phases no longer operate together. The majority of glucocorticoid receptors, however, are outside the SCN.

By removing the adrenal glands of rats, Michael Menaker, Ph.D., Pinar Pezuk, Ph.D., and fellow researchers at the University of Virginia were able to show how important glucocorticoids are to entraining the peripheral oscillators. Following surgery, phase shifts occurred in some tissues but not others. Lungs and the pituitary, pineal, and salivary glands continued normally, although the kidneys, liver, and cornea all lost normal phase relationships with their light-dark cycles.

Hydrocortisone treatments restored the phases of the kidney and cornea but caused dyssynchrony in the liver and the lungs, perhaps attributable to a high-glucocorticoid threshold as seen in responses to stress. Methamphetamine also caused phase shifts in the cornea as did restrictive feeding, which pushes peripheral oscillators to split from the SCN. Animals without adrenal signaling reset faster than intact animals suggesting that glucocorticoids block this uncoupling.

Although adrenalectomy did not cause phase shifts in all tissues, it did advance re-entrainment in almost all peripheral oscillators and the SCN when other phase shifts were induced. Corticosterone itself is slow to adjust to shifts in the light cycle. This may slow recovery of the peripheral tissues for intact animals. In its absence, resynchronization was more rapid.

Circadian rhythms change with our environment. The report of this study in an upcoming article in Endocrinology [endo.endojournals.org] provides a good view of how our organs reset apart from the main body clock through the versatility of glucocorticoids.

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