Perinatal Outcomes in Early and Late Gestational Diabetes Mellitus After Treatment From 24-28 Weeks' Gestation: A TOBOGM Secondary Analysis.

OBJECTIVE: In most gestational diabetes mellitus (GDM) studies, cohorts have included women combined into study populations without regard to whether hyperglycemia was present earlier in pregnancy. In this study we sought to compare perinatal outcomes between groups: women with early GDM (EGDM group: diagnosis before 20 weeks but no treatment until 24-28 weeks if GDM still present), with late GDM (LGDM group: present only at 24-28 weeks), and with normoglycemia at 24-28 weeks (control subjects). RESEARCH DESIGN AND METHODS: This is a secondary analysis of a randomized controlled treatment trial where we studied, among women with risk factors, early (<20 weeks' gestation) GDM defined according to World Health Organization 2013 criteria. Those receiving early treatment for GDM treatment were excluded. GDM was treated if present at 24-28 weeks. The primary outcome was a composite of birth before 37 weeks' gestation, birth weight ≥4,500 g, birth trauma, neonatal respiratory distress, phototherapy, stillbirth/neonatal death, and shoulder dystocia. Comparisons included adjustment for age, ethnicity, BMI, site, smoking, primigravity, and education. RESULTS: Women with EGDM (n = 254) and LGDM (n = 467) had shorter pregnancy duration than control subjects (n = 2,339). BMI was lowest with LGDM. The composite was increased with EGDM (odds ratio [OR] 1.59, 95% CI 1.18-2.12)) but not LGDM (OR 1.19, 95% CI 0.94-1.50). Induction of labor was higher in both GDM groups. In comparisons with control subjects there were higher birth centile, higher preterm birth rate, and higher rate of neonatal jaundice for the EGDM group (but not the LGDM group). The greatest need for insulin and/or metformin was with EGDM. CONCLUSIONS: Adverse perinatal outcomes were increased with EGDM despite treatment from 24-28 weeks' gestation, suggesting the need to initiate treatment early, and more aggressively, to reduce the effects of exposure to the more severe maternal hyperglycemia from early pregnancy.

Somatostatin receptor expression and clinical outcome of multilineage pituitary tumours expressing PIT1 and SF1.

The application of transcription factor immunohistochemistry to pituitary neuroendocrine tumour (PitNET) assessment has allowed identification of tumours that do not conform to a single lineage. Multilineage pituitary transcription factor 1 (PIT1) and steroidogenic factor 1 (SF1) PitNETs are a rare and relatively newly described tumour subtype. These tumours express both transcription factors and may also express combinations of hormones corresponding to both lineages. Histological and clinical characteristics can vary, and overall clinical behaviour and prognosis is not known. We describe the clinical outcomes and somatostatin receptor status (SSTR) of a series of nine cases identified from our cohort of pituitary tumours at Westmead Hospital. Eight PitNETs (88.9%) expressed growth hormone and caused acromegaly at presentation. Of the seven macrotumours that caused acromegaly, one had cavernous sinus invasion. The Ki-67 labeling index score ranged from 0.6% to 3.6%. About 88% of tumours that secreted excess growth hormone exhibited strong immunostaining for SSTR 2 and all tumours displayed weak immunoreactivity for SSTR5. In 62.5% of patients with acromegaly, cure was achieved after surgical resection. Somatostatin receptor ligands resulted in clinical remission in cases where medical treatment was initiated. There was no new tumour recurrence or regrowth over an overall mean follow-up period of 62.5 months.

Revealing KRas4b topology on the membrane surface.

KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

Aneurysmal subarachnoid haemorrhage in the pituitary fossa of a patient with acromegaly - a rare phenomenon.

Summary: The anatomy of the pituitary fossa is complex. The wall of the fossa can vary, resulting in inconsistencies in the nature and integrity of the sella barrier. Cerebrospinal fluid is generally confined to the subarachnoid space and does not circulate freely in the pituitary fossa. Spontaneous haemorrhage in the fossa typically occurs in the context of pre-existing intrasellar pathology such as a pituitary adenoma. Extravasation of blood into the subarachnoid space can rarely be observed following pituitary apoplexy. We describe the unique occurrence of subarachnoid haemorrhage in a largely empty pituitary fossa after the rupture of a cerebral aneurysm. Learning points: Pituitary apoplexy and subarachnoid haemorrhage (SAH) are both high in the differential diagnosis of sudden onset severe headaches. Haemorrhagic pituitary apoplexy may result in extravasation into the subarachnoid space, resulting in typical SAH symptoms and signs. This is the first reported case of primary SAH resulting in blood pooling in an empty sella arising from previous surgical resection of a large macroadenoma.

Insulin-like growth factor role in determining the anti-cancer effect of metformin: RCT in prostate cancer patients.

Objective: Androgen deprivation therapy (ADT), a principal therapy in patients with prostate cancer, is associated with the development of obesity, insulin resistance, and hyperinsulinemia. Recent evidence indicates that metformin may slow cancer progression and improves survival in prostate cancer patients, but the mechanism is not well understood. Circulating insulin-like growth factors (IGFs) are bound to high-affinity binding proteins, which not only modulate the bioavailability and signalling of IGFs but also have independent actions on cell growth and survival. The aim of this study was to investigate whether metformin modulates IGFs, IGF-binding proteins (IGFBPs), and the pregnancy-associated plasma protein A (PAPP-A) - stanniocalcin 2 (STC2) axis. Design and methods: In a blinded, randomised, cross-over design, 15 patients with prostate cancer on stable ADT received metformin and placebo treatment for 6 weeks each. Glucose metabolism along with circulating IGFs and IGFBPs was assessed. Results: Metformin significantly reduced the homeostasis model assessment as an index of insulin resistance (HOMA IR) and hepatic insulin resistance. Metformin also reduced circulating IGF-2 (P < 0.05) and IGFBP-3 (P < 0.01) but increased IGF bioactivity (P < 0.05). At baseline, IGF-2 correlated significantly with the hepatic insulin resistance (r2= 0.28, P < 0.05). PAPP-A remained unchanged but STC2 declined significantly (P < 0.05) following metformin administration. During metformin treatment, change in HOMA IR correlated with the change in STC2 (r2= 0.35, P < 0.05). Conclusion: Metformin administration alters many components of the circulating IGF system, either directly or indirectly via improved insulin sensitivity. Reduction in IGF-2 and STC2 may provide a novel mechanism for a potential metformin-induced antineoplastic effect.

Ectopic insulin secretion by a large-cell neuroendocrine carcinoma of the cervix.

In patients presenting with hyperinsulinemic hypoglycemia with a nonpancreatic neuroendocrine tumor, the diagnosis of an ectopic insulin-secreting tumor should be considered, and investigated further with confirmatory insulin staining.

Androgen deprivation in prostate cancer: benefits of home-based resistance training.

INTRODUCTION: Androgen deprivation therapy (ADT) has detrimental effects on body composition, metabolic health, physical functioning, bone mineral density (BMD) and health-related quality of life (HRQOL) in men with prostate cancer. We investigated whether a 12-month home-based progressive resistance training (PRT) programme, instituted at the start of ADT, could prevent these adverse effects. METHODS: Twenty-five patients scheduled to receive at least 12 months of ADT were randomly assigned to either usual care (UC) (n = 12) or PRT (n = 13) starting immediately after their first ADT injection. Body composition, body cell mass (BCM; a functional component of lean body mass), BMD, physical function, insulin sensitivity and HRQOL were measured at 6 weeks and 6 and 12 months. Data were analysed by a linear mixed model. RESULTS: ADT had a negative impact on body composition, BMD, physical function, glucose metabolism and HRQOL. At 12 months, the PRT group had greater reductions in BCM by - 1.9 ± 0.8 % (p = 0.02) and higher gains in fat mass by 3.1 ± 1.0 % (p = 0.002), compared to the UC group. HRQOL domains were maintained or improved in the PRT versus UC group at 6 weeks (general health, p = 0.04), 6 months (vitality, p = 0.02; social functioning, p = 0.03) and 12 months (mental health, p = 0.01; vitality, p = 0.02). A significant increase in the Matsuda Index in the PRT versus UC group was noted at 6 weeks (p = 0.009) but this difference was not maintained at subsequent timepoints. Between-group differences favouring the PRT group were also noted for physical activity levels (step count) (p = 0.02). No differences in measures of BMD or physical function were detected at any time point. CONCLUSION: A home-based PRT programme instituted at the start of ADT may counteract detrimental changes in body composition, improve physical activity and mental health over 12 months. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry, ACTRN12616001311448.

The Adverse Effects of Androgen Deprivation Therapy in Prostate Cancer and the Benefits and Potential Anti-oncogenic Mechanisms of Progressive Resistance Training.

Prostate cancer has the second highest incidence of all cancers amongst men worldwide. Androgen deprivation therapy (ADT) remains a common form of treatment. However, in reducing serum testosterone to castrate levels and rendering men hypogonadal, ADT contributes to a myriad of adverse effects which can affect prostate cancer prognosis. Physical activity is currently recommended as synergistic medicine in prostate cancer patients to alleviate the adverse effects of treatment. Progressive resistance training (PRT) is an anabolic exercise modality which may be of benefit in prostate cancer patients given its potency in maintaining and positively adapting skeletal muscle. However, currently, there is a scarcity of RCTs which have evaluated the use of isolated PRT in counteracting the adverse effects of prostate cancer treatment. Moreover, although physical activity in general has been found to reduce relapse rates and improve survival in prostate cancer, the precise anti-oncogenic effects of specific exercise modalities, including PRT, have not been fully established. Thus, the overall objective of this article is to provide a rationale for the in-depth investigation of PRT and its biological effects in men with prostate cancer on ADT. This will be achieved by (1) summarising the metabolic effects of ADT in patients with prostate cancer and its effect on prostate cancer progression and prognosis, (2) reviewing the existing evidence regarding the metabolic benefits of PRT in this cohort, (3) exploring the possible oncological pathways by which PRT can affect prostate cancer prognosis and progression and (4) outlining avenues for future research.

PIP2 Influences the Conformational Dynamics of Membrane-Bound KRAS4b.

KRAS4b is a small GTPase involved in cellular signaling through receptor tyrosine kinases. The activation of KRAS4b occurs only after recruitment of the regulatory proteins to the plasma membrane, thus making the role of the phospholipid bilayer an integral part of the signaling mechanism. Phospholipids, primarily with anionic headgroups, interact with both the membrane-anchoring hypervariable (HVR) region and the G-domain (catalytic domain) and influence the orientation of KRAS4b on the membrane surface, potentially playing a key role in the regulation of activation. Although there has been significant research focused on the role of the anionic phosphatidylserine, less effort has been spent on the role of the important signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Using instrumentation to measure the fluorescence anisotropy decay of site specifically labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) Nanodiscs over a wide frequency range, we quantitate the binding of KRAS4b to Nanodiscs containing either 30% phosphatidylserine (PS) or 10% l-α-phosphatidylinositol 4,5-bisphosphate by measuring the rotational correlation time of the Nanodisc-KRAS4b complex. We find that KRAS4b binds significantly tighter to Nanodiscs containing PIP2 but that at any level of binding saturation of KRAS4b, both 30% PS and 10% PIP2 containing Nanodiscs display similar rotational correlation times. This shows that the overall hydrodynamic radii of the KRAS4b-Nanodisc complexes are similar regardless of the incorporated anionic lipid. Atomic force microscopy is used to visualize KRAS4b when bound to individual Nanodiscs. Clean images are observed with the PIP2-doped Nanodiscs, but significantly blurred images are obtained when the anionic lipid is PS. This suggests that KRAS4b is not only more tightly bound overall with PIP2 as the anionic lipid but also less mobile on the bilayer surface. Microsecond molecular dynamics simulations of KRAS4b on PS- and PIP2-containing membranes show that the dynamics of the G-domain at the bilayer surface are significantly altered in the presence of PIP2, due to the formation of long-lived salt bridges with basic residues on the G-domain. The orientation and dynamics of KRAS4b on the membrane are critical to understanding the mechanisms of oncoprotein signaling, and our results with the GDP-bound form show subtle differences from that published for GTP-KRAS4b.

A potent liver-mediated mechanism for loss of muscle mass during androgen deprivation therapy.

CONTEXT: Androgen deprivation therapy (ADT) in prostate cancer results in muscular atrophy, due to loss of the anabolic actions of testosterone. Recently, we discovered that testosterone acts on the hepatic urea cycle to reduce amino acid nitrogen elimination. We now hypothesize that ADT enhances protein oxidative losses by increasing hepatic urea production, resulting in muscle catabolism. We also investigated whether progressive resistance training (PRT) can offset ADT-induced changes in protein metabolism. OBJECTIVE: To investigate the effect of ADT on whole-body protein metabolism and hepatic urea production with and without a home-based PRT program. DESIGN: A randomized controlled trial. PATIENTS AND INTERVENTION: Twenty-four prostate cancer patients were studied before and after 6 weeks of ADT. Patients were randomized into either usual care (UC) (n = 11) or PRT (n = 13) starting immediately after ADT. MAIN OUTCOME MEASURES: The rate of hepatic urea production was measured by the urea turnover technique using 15N2-urea. Whole-body leucine turnover was measured, and leucine rate of appearance (LRa), an index of protein breakdown and leucine oxidation (Lox), a measure of irreversible protein loss, was calculated. RESULTS: ADT resulted in a significant mean increase in hepatic urea production (from 427.6 ± 18.8 to 486.5 ± 21.3; P < 0.01) regardless of the exercise intervention. Net protein loss, as measured by Lox/Lra, increased by 12.6 ± 4.9% (P < 0.05). PRT preserved lean body mass without affecting hepatic urea production. CONCLUSION: As early as 6 weeks after initiation of ADT, the suppression of testosterone increases protein loss through elevated hepatic urea production. Short-term PRT was unable to offset changes in protein metabolism during a state of profound testosterone deficiency.

Disparate Effect of Aromatization on the Central Regulation of GH Secretion by Estrogens in Men and Postmenopausal Women.

CONTEXT: Estrogen receptor antagonism by tamoxifen inhibits GH secretion in both men and postmenopausal women, suggesting that estrogen, albeit at low concentration, stimulates GH secretion. However, systemic estrogen replacement in postmenopausal women does not enhance GH secretion. To clarify the role of estrogen in mediating GH secretion, we investigated the effect of estrogen deprivation by using aromatase inhibitors. AIM: To determine whether estrogens mediate GH secretion in men and postmenopausal women. DESIGN: The effects of letrozole, an aromatase inhibitor, and tamoxifen were compared in an open-label crossover study. Eight men and 14 women received tamoxifen (20 mg/d) and letrozole (2.5 mg/d) for 2 weeks each. The primary endpoints were GH response to arginine stimulation and gonadal steroid levels. RESULTS: In men, letrozole significantly (P < 0.05) reduced the peak GH response to arginine (mean ± SEM; Δ -49.4% ± 18.1%). Tamoxifen also reduced the mean peak GH, but this did not reach statistical significance. In postmenopausal women, letrozole did not affect peak GH, whereas tamoxifen significantly (P < 0.05) reduced peak GH (Δ -47.3% ± 10%). In men, letrozole reduced circulating estradiol (from 43.1 ± 2.8 to 12.7 ± 1.3 pmol/L; P < 0.001), whereas in women estradiol was undetectable (<11 pmol/L) at baseline and throughout letrozole therapy. CONCLUSION: Because estrogen deprivation reduced circulating GH, we conclude that estrogens regulate GH secretion in men. In postmenopausal women, the neutral effect of aromatase inhibition is likely explained by pre-existing estrogen deficiency. The inhibition of GH secretion by tamoxifen in menopause suggests a non-estrogen receptor-mediated mechanism of action. In contrast to men, estrogen is unlikely to mediate GH secretion in postmenopausal women.

Nanodiscs: A Controlled Bilayer Surface for the Study of Membrane Proteins.

The study of membrane proteins and receptors presents many challenges to researchers wishing to perform biophysical measurements to determine the structure, function, and mechanism of action of such components. In most cases, to be fully functional, proteins and receptors require the presence of a native phospholipid bilayer. In addition, many complex multiprotein assemblies involved in cellular communication require an integral membrane protein as well as a membrane surface for assembly and information transfer to soluble partners in a signaling cascade. Incorporation of membrane proteins into Nanodiscs renders the target soluble and provides a native bilayer environment with precisely controlled composition of lipids, cholesterol, and other components. Likewise, Nanodiscs provide a surface of defined area useful in revealing lipid specificity and affinities for the assembly of signaling complexes. In this review, we highlight several biophysical techniques made possible through the use of Nanodiscs.

The Association of Falling Insulin Requirements With Maternal Biomarkers and Placental Dysfunction: A Prospective Study of Women With Preexisting Diabetes in Pregnancy.

OBJECTIVE: To investigate the association of falling insulin requirements (FIR) among women with preexisting diabetes with adverse obstetric outcomes and maternal biomarkers longitudinally in pregnancy. RESEARCH DESIGN AND METHODS: A multicenter prospective cohort study of 158 women (41 with type 1 diabetes and 117 with type 2 diabetes) was conducted. Women with FIR of ≥15% from the peak total daily dose after 20 weeks' gestation were considered case subjects (n = 32). The primary outcome was a composite of clinical markers of placental dysfunction (preeclampsia, small for gestational age [≤5th centile], stillbirth, premature delivery [<30 weeks], and placental abruption). Maternal circulating angiogenic markers (placental growth factor [PlGF] and soluble fms-like tyrosine kinase 1 [sFlt-1]), placental hormones (human placental lactogen, progesterone, and tumor necrosis factor-α), HbA1c, and creatinine were studied serially during pregnancy. RESULTS: FIR ≥15% were associated with an increased risk of the composite primary outcome (odds ratio [OR] 4.38 [95% CI 1.9-10.3]; P < 0.001), preeclampsia (OR 6.76 [95% CI 2.7-16.7]; P < 0.001), and was more common among women with type 1 diabetes (36.6 vs. 14.5%; P = 0.002). Creatinine was modestly elevated among women with FIR ≥15%; however, there was no difference in HbA1c. The ratio of sFlt-1 to PlGF was significantly higher among women with FIR at 25, 30, and 36 weeks, with differences maintained in the subgroup that developed preeclampsia. There was no difference in placental hormones between the groups. CONCLUSIONS: This is the first prospective study to associate FIR with altered expression of placental antiangiogenic factors and preeclampsia. FIR are an important clinical sign, among women with preexisting diabetes, that should alert the clinician to investigate underlying placental dysfunction.

Interaction of KRas4b with anionic membranes: A special role for PIP2.

KRas4b is a small G-protein whose constitutively active oncogenic mutants are present in 90% of pancreatic cancers. Using fully post-translationally modified KRAS4b, we investigated the role of lipid identity in the recruitment of KRas4b to a membrane surface of defined composition. Application of a newly developed single frequency fluorescence anisotropy decay experiment to this system revealed that KRas4b has a significant binding preference for Nanodisc bilayers containing PIP2. We conducted molecular dynamics simulations to look for an origin of this specificity. In the case of membranes containing PIP2 the protein formed long-lived salt bridges with PIP2 head groups but not the monovalent DMPS, explaining the experimentally observed lipid specificity. Additionally, we report that PIP2 forms key contacts with Helix-4 on the catalytic domain of KRas4b that orient the protein in a manner expected to facilitate association with upstream and downstream signaling partners.

Ovulatory disorders are an independent risk factor for pregnancy complications in women receiving assisted reproduction treatments.

BACKGROUND: Conception using assisted reproduction treatments (ART) has been associated with an increased risk of pregnancy complications. It is uncertain if this is caused by ART directly, or is an association of the underlying factors causing infertility. AIMS: We assessed the relationship between assisted conception (AC) and maternal or fetal complications in a large retrospective cohort study. In a nested cohort of women receiving infertility treatment, we determined if such risk rests predominantly with certain causes of infertility. MATERIALS AND METHODS: Retrospective database analysis of 50 381 women delivering a singleton pregnancy in four public hospital obstetric units in western Sydney, and a nested cohort of 508 women receiving ART at a single fertility centre, in whom the cause of infertility was known. RESULTS: A total of 1727 pregnancies followed AC; 48 654 were spontaneous conceptions. Adjusted for age, body mass index and smoking, AC was associated with increased risk of preterm delivery (OR 1.73, 95% CI 1.50-2.02), hypertension (OR 1.55, 95% CI 1.34-1.82) and diabetes (OR 1.51, 95% CI 1.30-1.75). In the nested cohort, ovulatory dysfunction was present in 145 women and 336 had infertility despite normal ovulatory function. Ovulatory dysfunction was associated with increased risk of diabetes (OR 2.94, 95% CI 1.72-5.02) and hypertension (OR 2.40, 95% CI 1.15-5.00) compared to women with normal ovulatory function. CONCLUSIONS: Assisted conception is associated with increased risk of pregnancy complications. This risk appears greatest for women whose underlying infertility involves ovulatory dysfunction. Such disorders probably predispose towards diabetes and hypertension, which is then exacerbated by pregnancy.

Predicting Weight Gain in Patients Treated With Clozapine: The Role of Sex, Body Mass Index, and Smoking.

OBJECTIVES: Weight gain on clozapine is highly variable and poorly predictable. Its mechanisms are not well understood. This study explores the factors that predict weight gain between 3 and 12 months of clozapine therapy in community-dwelling patients. METHODS: We conducted a retrospective audit of patients attending an outpatient clozapine clinic. Weight change from 3 to 12 months of therapy was recorded, expressed as a percentage of the 3-month weight. Univariate analyses compared percent weight change according to sex, smoking status, country of birth, and baseline body mass index. Correlations between weight gain, age, and clozapine dose were explored. A general linear model identified independent predictors of weight gain. RESULTS: The mean weight change from 3 to 12 months in 117 patients was +3.1% (range, -17% to +30%). Females gained more weight than males (+5.5% vs +1.3%, P = 0.01), smokers gained more than nonsmokers (+5.1% vs +1.2%, P = 0.02), and obese patients gained less than normal or overweight individuals (0.15% vs 4.6% and 5.2%, respectively, P = 0.01). Age and clozapine dose had no relation to weight change. On multivariate analysis, baseline BMI and smoking status remained independent predictors of percent weight change in females. These 2 predictors explained 25% of weight change in females in the first 3 to 12 months of therapy. These associations were not observed in males. CONCLUSIONS: We hypothesize that smoking affects weight change by promoting clozapine metabolism to norclozapine via cytochrome P450 enzymes. Verifying this hypothesis and exploring the mechanisms underpinning the sex dichotomy are areas for further research.

p38 MAPK regulates steroidogenesis through transcriptional repression of STAR gene.

STAR/StarD1, part of a protein complex, mediates the transport of cholesterol from the outer to inner mitochondrial membrane, which is the rate-limiting step for steroidogenesis, and where steroid hormone synthesis begins. Herein, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of STAR gene transcription, using model steroidogenic cell lines. Our data indicate that oxidant activation of p38 MAPK exhibits a negative regulatory role in the induction of functional expression of STAR, as evidenced by enhanced induction of STAR (mRNA/protein) expression and increased steroidogenesis during pharmacological inhibition of p38 MAPK or in cells with increased transient overexpression of a dominant-negative (dn) form of p38 MAPKα or p38 MAPKß. Studies with rat Star-promoter demonstrated that overexpression of p38 MAPKα-wt, -ß, or -γ significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKα-dn, -ß, or -γ enhanced the Star promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dn constructs and designer knock-out cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKα-mediated inhibition of Star promoter activity. In addition, our studies raised the possibility of CREB being a potential target of the p38 MAPK inhibitory effect on Star promoter activity. Collectively, these data provide novel mechanistic information about how oxidant-sensitive p38 MAPKs, particularly p38 MAPKα, contribute to the negative regulation of Star gene expression and inhibit steroidogenesis.

Beta-cell ARNT is required for normal glucose tolerance in murine pregnancy.

AIMS: Insulin secretion increases in normal pregnancy to meet increasing demands. Inability to increase beta-cell function results in gestational diabetes mellitus (GDM). We have previously shown that the expression of the transcription factor ARNT (Aryl-hydrocarbon Receptor Nuclear Translocator) is reduced in the islets of humans with type 2 diabetes. Mice with a beta-cell specific deletion of ARNT (ß-ARNT mice) have impaired glucose tolerance secondary to defective insulin secretion. We hypothesised that ARNT is required to increase beta-cell function during pregnancy, and that ß-ARNT mice would be unable to compensate for the beta-cell stress of pregnancy. The aims of this study were to investigate the mechanisms of ARNT regulation of beta-cell function and glucose tolerance in pregnancy. METHODS: ß-ARNT females were mated with floxed control (FC) males and FC females with ß-ARNT males. RESULTS: During pregnancy, ß-ARNT mice had a marked deterioration in glucose tolerance secondary to defective insulin secretion. There was impaired beta-cell proliferation in late pregnancy, associated with decreased protein and mRNA levels of the islet cell-cycle regulator cyclinD2. There was also reduced expression of Irs2 and G6PI. In contrast, in control mice, pregnancy was associated with a 2.1-fold increase in ARNT protein and a 1.6-fold increase in cyclinD2 protein, and with increased beta-cell proliferation. CONCLUSIONS: Islet ARNT increases in normal murine pregnancy and beta-cell ARNT is required for cyclinD2 induction and increased beta-cell proliferation in pregnancy.

Telomere shortening and DNA damage of embryonic stem cells induced by cigarette smoke.

Embryonic stem cells (ESCs) provide a valuable in vitro model for testing toxicity of chemicals and environmental contaminants including cigarette smoke. Mouse ESCs were acutely or chronically exposed to smoke components, cigarette smoke condensate (CSC), or cadmium, an abundant component of CSC, and then evaluated for their self-renewal, apoptosis, DNA damage and telomere function. Acute exposure of ESCs to high dose of CSC or cadmium increased DNA damage and apoptosis. Yet, ESCs exhibited a remarkable capacity to recover following absence of exposure. Chronic exposure of ESCs to low dose of CSC or cadmium resulted in shorter telomeres and DNA damage. Together, acute exposure of ESCs to CSC or cadmium causes immediate cell death and reduces pluripotency, while chronic exposure of ESCs to CSC or cadmium leads to DNA damage and telomere shortening. Notably, a sub-proportion of ESCs during passages is selected to resist to smoke-induced oxidative damage to telomeres.

Telomere susceptibility to cigarette smoke-induced oxidative damage and chromosomal instability of mouse embryos in vitro.

Cigarette smoke is associated with high risk of lung, cardiovascular, and degenerative diseases, reduced fertility, and possibly the health of newborns. Cigarette smoke contains many components and exerts its genotoxicity in part by generating reactive oxidative stress. Telomeres consist of repeated 'G' rich sequences and associated proteins located at the chromosomal ends that maintain chromosomal integrity. We tested the hypothesis that telomere shortening and dysfunction are implicated in smoke associated oxidative damage and chromosomal instability using early mouse embryos in vitro and short-telomere mouse model. Mouse embryos exposed to smoke components, cigarette smoke condensate (CSC) at the concentration of 0.02 mg/ml continuously or 0.1mg/ml for 20 h, or cadmium at 5-100 microM, exhibited increased oxidative stress and telomere shortening and loss, associated with chromosomal instability, apoptosis, and compromised embryo cleavage and development. Remarkably, reduction of oxidative stress by an antioxidant N-acetyl-L-cysteine (NAC) greatly reduced these toxicities. Notably, cadmium led to more severe oxidative damage and telomere dysfunction, which could be more effectively rescued by antioxidant treatment, than did CSC. Moreover, short telomeres predisposed embryos to smoke component-induced oxidative damage. These data further extend our understanding of mechanisms underlying smoke-induced oxidative damage to include telomere dysfunction and chromosomal instability.