EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events.

Adipocyte dysfunction is a crucial driver of insulin resistance and type 2 diabetes. We identified EH domain-containing protein 2 (EHD2) as one of the most highly upregulated genes at the early stage of adipose-tissue expansion. EHD2 is a dynamin-related ATPase influencing several cellular processes, including membrane recycling, caveolae dynamics, and lipid metabolism. Here, we investigated the role of EHD2 in adipocyte insulin signaling and glucose transport. Using C57BL6/N EHD2 knockout mice under short-term high-fat diet conditions and 3T3-L1 adipocytes we demonstrate that EHD2 deficiency is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation. Furthermore, we show that lack of EHD2 is linked with altered plasma membrane lipid and protein composition, reduced insulin receptor expression, and diminished insulin-dependent SNARE protein complex formation. In conclusion, these data highlight the importance of EHD2 for the integrity of the plasma membrane milieu, insulin receptor stability, and downstream insulin receptor signaling events, involved in glucose uptake and ultimately underscore its role in insulin resistance and obesity.

Cervical cerclage technique: what do experts actually achieve?

BACKGROUND: Cervical cerclage is a recognized intervention in the management of women at risk of preterm birth and midtrimester loss. The mechanism of action of cerclage is unclear, and the technique has been poorly researched. OBJECTIVE: This study aimed to evaluate cerclage technique among experienced obstetricians, using a previously developed and evaluated cerclage simulator. STUDY DESIGN: This prospective experimental simulation and observational study used identical simulators for 28 consultant obstetricians who were asked to perform their normal cerclage. Suture type, height, knot site, and free thread length were recorded. Using computed tomography, depth of bite and tension (by reduction in area of cervix) were calculated. RESULTS: A total of 52 cervical cerclages were completed (Mersilene tape, n=20; monofilament suture, n=32). Mean suture height was 33 mm (standard deviation, 7.7 mm), greater with monofilament suture than with Mersilene tape, and associated with smaller needle size. Mean depth of bite and mean reduction of starting area did not differ by suture type. Seven procedures showed ≥1 suture bite that had entered the cervical canal once or more. CONCLUSION: This study assessed cerclage technique of experienced obstetricians using simulators and computed tomography imaging, and demonstrated wide variation in technique; this may affect the efficacy of the procedure. Further work should establish optimal technique and consensus for training and clinical practice.

Cervical cerclage training: Development and assessment of a simulator.

BACKGROUND: Cervical cerclage is a mainstay intervention for the prevention of spontaneous preterm birth in high-risk women. Simulation training facilitates high-level skill transfer in a low-consequence environment, and is being integrated into obstetrics and gynecology training. OBJECTIVE: This study aimed to develop a simulator for cervical cerclage, determine its validity as a simulator, and identify parameters suitable as proxy markers for performance. STUDY DESIGN: The 3 aims of this study were achieved, namely: (1) simulator design by obstetricians and a commercial company; (2) survey of obstetricians and gynecologists across a variety of training stages to determine need for and opinion of the simulator; and (3) comparison of novice and expert groups across a variety of proxy markers for successful cerclage insertion. RESULTS: Obstetricians and gynecologists found the simulator to be similar to clinical scenarios and suitable for skill training. Novice participants stated that the use of the simulator improved their confidence (P=.016). In a comparison between 6 expert and 8 novice surgeons, there seemed to be variations across multiple measurements of cerclage placement. CONCLUSION: Simulation is an increasingly prominent training modality for surgical skills. The simulator described herein was considered suitable for training by obstetricians and gynecologists. Further work should focus on the validations of proxy markers of successful insertion, longitudinal assessment of trainees, and correlation of training outcomes with clinical outcomes.

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

Knockout of syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion.

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport that is underpinned by the insulin-stimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similar mechanism. We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells. This article has an associated First Person interview with Hannah L. Black and Rachel Livingstone, joint first authors of the paper.

Pulsatility of glucocorticoid hormones in pregnancy: Changes with gestation and obesity.

OBJECTIVE: Hypothalamic-pituitary-adrenal axis (HPA) activity is decreased in obese pregnancy and associates with increased foetal size. Pulsatile release of glucocorticoid hormones regulates their action in target tissues. Glucocorticoids are essential for normal foetal growth, but little is known about glucocorticoid pulsatility in pregnancy. We aimed to investigate the ultradian rhythm of glucocorticoid secretion during obese and lean pregnancy and nonpregnancy. DESIGN: Serum cortisol, cortisone, corticosterone and 11-dehydrocorticosterone were measured by LC-MS/MS from samples obtained at 10-minute intervals between 08.00-11.00 hours and 16.00-19.00 hours, from 8 lean (BMI <25 kg/m2 ) and 7 obese (BMI > 35 kg/m2 ) pregnant women between 16-24 weeks gestation and again at 30-36 weeks), and nonpregnant controls (lean n = 3, obese n = 4) during the luteal phase of their menstrual cycle. Interstitial fluid cortisol was measured by ELISA, from samples obtained using a portable microdialysis and automated collection device at 20-minute intervals over 24 hours. RESULTS: Serum cortisol AUC, highest peak and lowest trough increased significantly with gestation in lean and obese pregnant compared with nonpregnant subjects. Pulsatility of cortisol was detected in interstitial fluid. In pregnant subjects, interstitial fluid pulse frequency was significantly lower with advancing gestation in obese, but not in lean. CONCLUSIONS: We demonstrate cortisol pulsatility in interstitial fluid. Pulse frequency is altered with increased gestation and BMI. This may be a novel mechanism to explain decreased HPA activity in obese pregnancy.

Transfer and Metabolism of Cortisol by the Isolated Perfused Human Placenta.

Context: Fetal overexposure to glucocorticoids in utero is associated with fetal growth restriction and is postulated to be a key mechanism linking suboptimal fetal growth with cardiovascular disease in later life. Objective: To develop a model to predict maternal-fetal glucocorticoid transfer. We hypothesized placental 11-ß-hydroxysteroid dehydrogenase-type 2 (11ß-HSD2) would be the major rate-limiting step in maternal cortisol transfer to the fetus. Design: We used a deuterated cortisol tracer in the ex vivo placental perfusion model, in combination with computational modeling, to investigate the role of interconversion of cortisol and its inactive metabolite cortisone on transfer of cortisol from mother to fetus. Participants: Term placentas were collected from five women with uncomplicated pregnancies, at elective caesarean delivery. Intervention: Maternal artery of the isolated perfused placenta was perfused with D4-cortisol. Main Outcome Measures: D4-cortisol, D3-cortisone, and D3-cortisol were measured in maternal and fetal venous outflows. Results: D4-cortisol, D3-cortisone, and D3-cortisol were detected and increased in maternal and fetal veins as the concentration of D4-cortisol perfusion increased. D3-cortisone synthesis was inhibited when 11-ß-hydroxysteroid dehydrogenase (11ß-HSD) activity was inhibited. At the highest inlet concentration, only 3.0% of the maternal cortisol was transferred to the fetal circulation, whereas 26.5% was metabolized and 70.5% exited via the maternal vein. Inhibiting 11ß-HSD activity increased the transfer to the fetus to 7.3% of the maternal input, whereas 92.7% exited via the maternal vein. Conclusions: Our findings challenge the concept that maternal cortisol diffuses freely across the placenta and confirm that 11ß-HSD2 acts as a major "barrier" to cortisol transfer to the fetus.

Glucocorticoids are lower at delivery in maternal, but not cord blood of obese pregnancies.

Glucocorticoids are vital for lung maturation. We previously showed that cortisol is lower in obese pregnancy. Whether this is maintained at delivery is unknown but is clinically relevant as maternal and cord blood cortisol levels are correlated and offspring of obese are more likely to need neonatal respiratory support. We hypothesized that glucocorticoids are lower in maternal and cord blood at delivery in obese pregnancies. Glucocorticoids (cortisol and corticosterone) and their inactive versions (cortisone and 11-dehydrocorticosterone) were measured by LC-MS/MS in maternal and cord plasma from 259 Caucasian women at delivery (BMI 18-55 kg/m2). Analyses adjusted for labour status, delivery mode, offspring gender, birthweight and gestational age. Cortisol and corticosterone were significantly higher in maternal than cord blood. Inactive versions were significantly higher in cord than maternal blood. Increased maternal BMI associated with lower maternal cortisol, corticosterone and 11-dehydrocorticosterone. Despite significant positive correlations between maternal and cord blood glucocorticoid levels, increased maternal BMI was not associated with lower cord blood glucocorticoid levels. Conditions at delivery may overcome any potential negative effects of low maternal glucocorticoids on the fetus in the short-term. This may not preclude the longer-term effects of fetal exposure to lower glucocorticoid levels during obese pregnancy.

Does attendance at a specialist antenatal clinic improve clinical outcomes in women with class III obesity compared with standard care? A retrospective case-note analysis.

OBJECTIVES: To determine whether attendance at a specialised multidisciplinary antenatal clinic for women with class III obesity (BMI >40 kg/m2) is associated with improved clinical outcomes compared with standard antenatal care. DESIGN: Retrospective cohort study using routinely collected data from electronic patient record. SETTING: Community and hospital based antenatal care. PARTICIPANTS: Women with a singleton pregnancy with class III obesity booked for antenatal care and delivered in one of two hospitals in NHS Lothian, Scotland, UK between 2008 and 2014. Maternal and offspring outcomes were compared in women who attended a specialised obesity clinic (n=511) compared with standard antenatal care (n=502). MAIN OUTCOME MEASURES: Included stillbirth, low birth weight, gestational diabetes, induction of labour and caesarean section. RESULTS: Compared with standard care, women receiving specialist care were less likely to have a stillbirth (OR 0.12, 95% CI 0.06 to 0.97) and a low birthweight baby (OR 0.57, 95% CI 0.33 to 0.99) and more likely to be screened for (100% vs 73.6%; p<0.001) and diagnosed with (26.0% vs 12.5%; p<0.001) gestational diabetes, to require induction of labour (38.4% vs 29.9%; p=0.009), an elective (20.3% vs 17.7%; p<0.001) and emergency (23.9% vs 20.3%; p<0.001) caesarean section and attend antenatal triage one or more times during pregnancy (77.7% vs 53.1%; p<0.001). Women attending the specialist clinic had a higher BMI (44.5 kg/m2 (4.3) vs 43.2 kg/m2 (3.1); p<0.001) and were more likely to be nulliparous (46.0% vs 24.9%; p<0.001). There were no other differences in maternal demographic or maternal and offspring outcomes between groups. CONCLUSIONS: Attendance at a specialised antenatal clinic for obesity is associated with reduced rates of stillbirth and low birth weight and improved detection of gestational diabetes. The improvement in clinical outcomes is associated with an increase in healthcare attendance to obstetric triage and clinical interventions including induction of labour and caesarean section.

The effect of fetal growth and nutrient stresses on steroid pathways.

The early life environment is a crucial time for establishing the trajectory of future health. Low birthweight is considered a marker of an adverse in utero environment and predisposes to cardio-metabolic disease later in life. It has been proposed that this is mediated by glucocorticoids, with life-long activation of the HPA axis. Here we review the evidence to support this hypothesis, with particular emphasis on the effects of fetal growth and nutrient stresses in utero on steroid pathways of the HPA axis. A better understanding of the mechanisms underlying these processes could help to optimize in utero health, and identify individuals at greatest risk of future disease.

Decreased maternal hypothalamic-pituitary-adrenal axis activity in very severely obese pregnancy: Associations with birthweight and gestation at delivery.

BACKGROUND: The maternal hypothalamic-pituitary-adrenal-axis (HPAA) undergoes dramatic activation during pregnancy. Increased cortisol and corticotrophin-releasing-hormone (CRH) associate with low birthweight and preterm labor. In non-pregnant obesity, the HPAA is activated but circulating cortisol levels are normal or lower than in lean women. We hypothesized that maternal cortisol levels would be lower in obese pregnancy, and would associate with increased fetal size and length of gestation. METHOD: Fasting serum cortisol was measured at 16, 28 and 36 weeks gestation and at 3-6 months postpartum in 276 severely obese and 135 lean women. In a subset of obese (n=20) and lean (n=20) we measured CRH, hormones that regulate bioavailable cortisol (corticosteroid-binding-globulin, estradiol, estriol, and progesterone). Urinary glucocorticoid metabolites were measured in pregnant (obese n=6, lean n=5) and non-pregnant (obese n=7, lean n=7) subjects. RESULTS: Maternal cortisol and HPAA hormones were lower in obese pregnancy. Total urinary glucocorticoid metabolites increased significantly in lean pregnancy, but not in obese. Lower maternal cortisol in obese tended to be associated with increased birthweight (r=-0.13, p=0.066). In obese, CRH at 28 weeks correlated inversely with gestational length (r=-0.49, p=0.04), and independently predicted gestational length after adjustment for confounding factors (mean decrease in CRH of -0.25 pmol/L (95% CI -0.45 to -0.043 pmol/L) per/day increase in gestation). CONCLUSION: In obese pregnancy, lower maternal cortisol without an increase in urinary glucocorticoid clearance may indicate a lesser activation of the HPAA than in lean pregnancy. This may offer a novel mechanism underlying increased birthweight and longer gestation in obese pregnancy.

Screening and management of gestational diabetes mellitus in Scottish obstetric units: a national survey.

BACKGROUND AND AIMS: The last study of screening practices for gestational diabetes (GDM) in the UK concluded that a lack of consensus about screening was due to a lack of clinical guidelines. We aimed to determine current practices in Scotland since new guidelines recommended that diagnosis should be made at a lower level of hyperglycaemia. METHOD AND RESULTS: An online questionnaire designed to investigate the screening and management of GDM was distributed to all maternity units in Scotland managing women with GDM (n = 15) for completion by relevant clinical team members. The response rate was 100%. Considerable variation in clinical practice existed between units. Thirteen units (86.7%) had adopted the lower glucose tolerance values for diagnosis of GDM (fasting ≥5.1 mmol/L; 2-h ≥8.5 mmol/L) recommended by the Scottish Intercollegiate Guidelines Network in 2010. Available data from units using this guideline (n = 3) revealed a significant increase in the percentage of women diagnosed with GDM between 2010 and 2012 (2010: 1.28%, 2012: 2.54%; p < 0.0001). CONCLUSION: Despite provision of clinical guidelines, there are still inconsistencies in screening and management of GDM in Scotland. If a similar increase in the prevalence of GDM is experienced across Scotland, there will be major implications for health care provision and resource allocation.

Homotypic vacuole fusion in yeast requires organelle acidification and not the V-ATPase membrane domain.

Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have implicated the vacuolar H(+)-ATPase (V-ATPase). The V-ATPase is a multisubunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H(+)-translocation/vacuole acidification function, rather than the physical presence of the V-ATPase, that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole-fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion.

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells.

The insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in human fat and muscle cells and the nitrogen-regulated trafficking of the general amino acid permease Gap1 in the yeast Saccharomyces cerevisiae share several common features: Both Gap1 and GLUT4 are nutrient transporters that are mobilised to the cell surface from an intracellular store in response to an environmental cue; both are polytopic membrane proteins harbouring amino acid targeting motifs in their C-terminal tails that are required for their regulated trafficking; ubiquitylation of both Gap1 and GLUT4 plays an important role in their regulated trafficking, as do the ubiquitin-binding GGA (Golgi-localised, γ-ear-containing, ARF-binding) adaptor proteins. Here, we find that when expressed heterologously in yeast, human GLUT4 is subject to nitrogen-regulated trafficking in an ubiquitin-dependent manner similar to Gap1. In addition, by expressing a GLUT4/Gap1 chimeric protein in adipocytes we show that the carboxy-tail of Gap1 directs intracellular sequestration and insulin-regulated trafficking in adipocytes. These findings demonstrate that the trafficking signals and their cognate molecular regulatory machinery that mediate regulated exocytosis of membrane proteins are conserved across evolution.

Cell-specific inositol 1,4,5 trisphosphate 3-kinase mediates epithelial cell apoptosis in response to oxidative stress in Drosophila.

Organismal stress responses to oxidative stress are relevant to ageing and disease and involve key cell-/tissue-specific signal transduction mechanisms. Using Drosophila, an established in vivo model for stress studies, we show that cell-specific inositol phosphate signalling specifically via inositol 1,4,5 trisphosphate 3-kinase (InsP(3) 3-K, IP(3)K), negatively regulates organismal responses to oxidative stress. We demonstrate that the Drosophila Malpighian tubule (equivalent to vertebrate kidney and liver) is a key epithelial sensor for organismal oxidative stress responses: precise targeting of either gain-of-function constructs of Drosophila IP(3)Ks (IP(3)K-1 and IP(3)K-2), or loss-of-function (RNAi) constructs to only one cell type in tubule reversibly modulates survival of stress-challenged adult flies. In vivo, targeted IP(3)K-1 directly increases H(2)O(2) production, pro-apoptotic caspase-9 activity and mitochondrial membrane potential. The mitochondrial calcium load in tubule principal cells-assessed by luminescent and fluorescent genetically-encoded mitochondrial calcium reporters-is significantly increased by IP(3)K-1 under oxidative stress conditions, leading to apoptosis. The Drosophila orthologues of human apoptotic bcl-2 genes include debcl and buffy. Oxidative stress challenge does not modulate gene expression of either debcl or buffy in tubules; and altered debcl expression does not influence survival rates under oxidative stress challenge. Finally, targeted over-expression of either debcl or buffy to tubule principal cells does not impact on tubule caspase-9 activity. Thus, IP(3)K-1 modulates epithelial cell apoptosis without involvement of bcl-2-type proteins.