Investigation into the ion channels and plasma membrane properties of white adipocytes

Ca2+ is a ubiquitous intracellular signalling molecule that is involved in the regulation of numerous cellular functions. To date Ca2+ influx pathways present in white fat adipocytes have not been characterised. Additionally impaired [Ca2+]i management is implicated in the induction of the insulin resistant state in adipocytes. As adipocytes have a prominent role in the management of energy homeostasis, the presence of Ca2+ influx pathways was examined. Initial [Ca2+]i measurements confirmed the presence of functional Ca2+ influx and efflux pathways in adipocytes. Further [Ca2+]i measurements identified the Cav1.3 Voltage-gated Ca2+ channel (VGCC). The presence of the α1 subunit of Cav1.3 channel protein in adipocytes was confirmed by Western blotting, the expression of which was reduced in adipocyte samples sourced from Zucker obese rats. Initial [Ca2+]i imaging experiments utilising conditions of elevated extracellular K+ (50mM) did not stimulate Ca2+ influx. The plasma membrane potential (Vm) regulates many physiological processes, including cellular Ca2+ influx by VGCCs, with dysregulations in Vm underlying functional pathologies. K+ is widely believed to be the predominant ion that controls Vm for many cell types, however, whether K+ regulates adipocyte Vm is also unknown, prompting, investigation into the ionic species involved in the regulation of Vm in primary and differentiated 3T3-L1 adipocytes. As insulin and β-adrenoceptors regulate adipocyte function, their effect on Vm was also explored. The Vm of primary and 3T3-L1 adipocytes were -34.14mV (n=68) and -28.5mV (n=88) respectively. Elevation of extracellular K+ from 5.6mM to 50mM had no significant effect on the Vm of either type of adipocyte. The role of Cl- on adipocyte Vm was then investigated. Reduction of extracellular Cl- from 138 to 5mM, by equimolar substitution with Gluconate significantly depolarised the Vm of both primary and 3T3-L1 adipocytes. Patch clamp investigations also revealed a role of Na+ in adipocyte Vm. Neither insulin (100nM) or the β-adrenocpetor agonist isoprenaline (10µM) significantly changed adipocyte Vm. The role of Cl- in adipocyte Vm is indicative of the presence of Cl- channels, however electrophysiological studies failed to characterise the Cl- currents underlying adipocyte Vm. Overall, further investigations are required to characterise not only the Ca2+ influx pathways in adipocytes, and the roles thereof, but also the means by which they are regulated

Investigation into the ion channels and plasma membrane properties of white adipocytes

Ca2+ is a ubiquitous intracellular signalling molecule that is involved in the regulation of numerous cellular functions. To date Ca2+ influx pathways present in white fat adipocytes have not been characterised. Additionally impaired [Ca2+]i management is implicated in the induction of the insulin resistant state in adipocytes. As adipocytes have a prominent role in the management of energy homeostasis, the presence of Ca2+ influx pathways was examined. Initial [Ca2+]i measurements confirmed the presence of functional Ca2+ influx and efflux pathways in adipocytes. Further [Ca2+]i measurements identified the Cav1.3 Voltage-gated Ca2+ channel (VGCC). The presence of the α1 subunit of Cav1.3 channel protein in adipocytes was confirmed by Western blotting, the expression of which was reduced in adipocyte samples sourced from Zucker obese rats. Initial [Ca2+]i imaging experiments utilising conditions of elevated extracellular K+ (50mM) did not stimulate Ca2+ influx. The plasma membrane potential (Vm) regulates many physiological processes, including cellular Ca2+ influx by VGCCs, with dysregulations in Vm underlying functional pathologies. K+ is widely believed to be the predominant ion that controls Vm for many cell types, however, whether K+ regulates adipocyte Vm is also unknown, prompting, investigation into the ionic species involved in the regulation of Vm in primary and differentiated 3T3-L1 adipocytes. As insulin and β-adrenoceptors regulate adipocyte function, their effect on Vm was also explored. The Vm of primary and 3T3-L1 adipocytes were -34.14mV (n=68) and -28.5mV (n=88) respectively. Elevation of extracellular K+ from 5.6mM to 50mM had no significant effect on the Vm of either type of adipocyte. The role of Cl- on adipocyte Vm was then investigated. Reduction of extracellular Cl- from 138 to 5mM, by equimolar substitution with Gluconate significantly depolarised the Vm of both primary and 3T3-L1 adipocytes. Patch clamp investigations also revealed a role of Na+ in adipocyte Vm. Neither insulin (100nM) or the β-adrenocpetor agonist isoprenaline (10µM) significantly changed adipocyte Vm. The role of Cl- in adipocyte Vm is indicative of the presence of Cl- channels, however electrophysiological studies failed to characterise the Cl- currents underlying adipocyte Vm. Overall, further investigations are required to characterise not only the Ca2+ influx pathways in adipocytes, and the roles thereof, but also the means by which they are regulated

Etiology of the membrane potential of rat white fat adipocytes

The plasma membrane potential (Vm) is key to many physiological processes, however its ionic aetiology in white fat adipocytes is poorly characterised. To address this question, we have employed the perforated patch current-clamp and cell-attached patch-clamp methods in isolated primary white fat adipocytes and their cellular model: 3T3-L1. The resting Vm of primary and 3T3-L1 adipocytes were -32.1±1.2mV (n=95) and -28.8±1.2mV (n=87), respectively. Vm was independent of cell size and fat content. Elevation of extracellular [K+] to 50mM by equimolar substitution of bath Na+ did not affect Vm, whereas substitution of bath Na+ with the membrane impermeant cation N-methyl-D-glucamine+ hyperpolarized Vm by 16mV, data indicative of a non-selective cation permeability. Substitution of 133mM extracellular Cl- with gluconate, depolarised Vm to +5.5, whereas Cl- substitution with I- caused a -9mV hyperpolarization. Isoprenaline (10µM) but not insulin (100nM) significantly depolarized Vm. Single-channel ion activity was voltage independent; currents were indicative for Cl- with an inward slope conductance of 16±1.3pS (n=11) and a reversal potential close to the Cl- equilibrium potential: -29±1.6mV. Reduction of extracellular Cl- elevated the intracellular Ca2+ of adipocytes. In conclusion, the Vm of white fat adipocyte is well described by the Goldman-Hodgkin-Katz equation with a predominant permeability to Cl-. Consequently, changes in serum Cl- homeostasis or the adipocyte’s permeability to this anion via drugs will affect its Vm, intracellular Ca2+ and ultimately its function and its role in metabolic control

CaV1.2 and CaV1.3 voltage-gated L-type Ca2+ channels in rat white fat adipocytes

L-type channel antagonists are of therapeutic benefit in the treatment of hyperlipidaemia and insulin resistance. Our aim was to identify L-type voltage-gated Ca2+ channels in white fat adipocytes, and determine if they affect intracellular Ca2+, lipolysis and lipogenesis. We used a multidisciplinary approach of molecular biology, confocal microscopy, Ca2+ imaging and metabolic assays to explore this problem using adipocytes isolated from adult rat epididymal fat pads. CaV1.2, CaV1.3 and CaV1.1 alpha1, beta and alpha2delta subunits were detected at the gene expression level. The CaV1.2 and CaV1.3 alpha1 subunits were identified in the plasma membrane at the protein level. Confocal microscopy with fluorescent antibodies labelled CaV1.2 in the plasma membrane. Ca2+ imaging revealed that the intracellular Ca2+ concentration, [Ca2 +]i was reversibly decreased by removal of extracellular Ca2+, an effect mimicked by verapamil, nifedipine and Co2+, all blockers of L-type channels, whereas the Ca2+ channel agonist BAY-K8644 increased [Ca2+]i. The finding that the magnitude of these effects correlated with basal [Ca2+]i suggests that adipocyte [Ca2+]i is controlled by L-type Ca2+ channels that are constitutively active at the adipocyte depolarized membrane potential. Pharmacological manipulation of L-type channel activity modulated both basal and catecholamine-stimulated lipolysis but not insulin-induced glucose uptake or lipogenesis. We conclude that white adipocytes have constitutively active L-type Ca2+ channels which explains their sensitivity of lipolysis to Ca2+ channel modulators. Our data suggest CaV1.2 as a potential novel therapeutic target in the treatment of obesity

Characteristics of frequent emergency department presenters to an Australian emergency medicine network

<p>Abstract</p> <p>Background</p> <p>To describe the characteristics of emergency department (ED) patients defined as frequent presenters (FP) presenting to an Australian emergency department network and compare these with a cohort of non-frequent presenters (NFP).</p> <p>Method</p> <p>A retrospective chart review utilising an electronic emergency medicine patient medical record database was performed on patients presenting to Southern Health EDs from March 2009 to March 2010. Non-frequent presenters were defined as patients presenting less than 5 times and frequent presenters as presenting 8 or more times in the study period. Characteristics of both groups were described and compared.</p> <p>Results</p> <p>During the 12-month study period there were 540 FP patients with 4549 admissions and 73,089 NFP patients with 100,943 admissions. FP patients were slightly older with a significant increase in frequency of patients between the ages of 70 to 79 years and they were more likely to be divorced or separated than NFP patients. Frequent presenters to the emergency department were more likely to utilise the ambulance service to arrive at the hospital, or in the custody of police than NFP patients. FPs were more likely to be admitted to hospital, more likely to have an admission to a mental health bed than NFP patients and more likely to self-discharge from the emergency department while waiting for care.</p> <p>Conclusions</p> <p>There are major implications for the utilisation of limited ED resources by frequent presenters. By further understanding the characteristics of FP we may be able to address the specific health care needs of this population in more efficient and cost effective ways. Further research analysing the effectiveness of targeted multidisciplinary interventions aiming to reduce the frequency of ED attendances may be warranted.</p

Multi-ancestry study of blood lipid levels identifies four loci interacting with physical activity.

Many genetic loci affect circulating lipid levels, but it remains unknown whether lifestyle factors, such as physical activity, modify these genetic effects. To identify lipid loci interacting with physical activity, we performed genome-wide analyses of circulating HDL cholesterol, LDL cholesterol, and triglyceride levels in up to 120,979 individuals of European, African, Asian, Hispanic, and Brazilian ancestry, with follow-up of suggestive associations in an additional 131,012 individuals. We find four loci, in/near CLASP1, LHX1, SNTA1, and CNTNAP2, that are associated with circulating lipid levels through interaction with physical activity; higher levels of physical activity enhance the HDL cholesterol-increasing effects of the CLASP1, LHX1, and SNTA1 loci and attenuate the LDL cholesterol-increasing effect of the CNTNAP2 locus. The CLASP1, LHX1, and SNTA1 regions harbor genes linked to muscle function and lipid metabolism. Our results elucidate the role of physical activity interactions in the genetic contribution to blood lipid levels

Asymmetric division coordinates collective cell migration in angiogenesis

The asymmetric division of stem or progenitor cells generates daughters with distinct fates and regulates cell diversity during tissue morphogenesis. However, roles for asymmetric division in other more dynamic morphogenetic processes, such as cell migration, have not previously been described. Here we combine zebrafish in vivo experimental and computational approaches to reveal that heterogeneity introduced by asymmetric division generates multicellular polarity that drives coordinated collective cell migration in angiogenesis. We find that asymmetric positioning of the mitotic spindle during endothelial tip cell division generates daughters of distinct size with discrete ‘tip’ or ‘stalk’ thresholds of pro-migratory Vegfr signalling. Consequently, post-mitotic Vegfr asymmetry drives Dll4/Notch-independent self-organization of daughters into leading tip or trailing stalk cells, and disruption of asymmetry randomizes daughter tip/stalk selection. Thus, asymmetric division seamlessly integrates cell proliferation with collective migration, and, as such, may facilitate growth of other collectively migrating tissues during development, regeneration and cancer invasion

Trans-ethnic Meta-analysis and Functional Annotation Illuminates the Genetic Architecture of Fasting Glucose and Insulin

Knowledge of the genetic basis of the type 2 diabetes (T2D)-related quantitative traits fasting glucose (FG) and insulin (FI) in African ancestry (AA) individuals has been limited. In non-diabetic subjects of AA (n = 20,209) and European ancestry (EA; n = 57,292), we performed trans-ethnic (AA+EA) fine-mapping of 54 established EA FG or FI loci with detailed functional annotation, assessed their relevance in AA individuals, and sought previously undescribed loci through trans-ethnic (AA+EA) meta-analysis. We narrowed credible sets of variants driving association signals for 22/54 EA-associated loci; 18/22 credible sets overlapped with active islet-specific enhancers or transcription factor (TF) binding sites, and 21/22 contained at least one TF motif. Of the 54 EA-associated loci, 23 were shared between EA and AA. Replication with an additional 10,096 AA individuals identified two previously undescribed FI loci, chrX FAM133A (rs213676) and chr5 PELO (rs6450057). Trans-ethnic analyses with regulatory annotation illuminate the genetic architecture of glycemic traits and suggest gene regulation as a target to advance precision medicine for T2D. Our approach to utilize state-of-the-art functional annotation and implement trans-ethnic association analysis for discovery and fine-mapping offers a framework for further follow-up and characterization of GWAS signals of complex trait loc

Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses

Multi-ancestry sleep-by-SNP interaction analysis in 126,926 individuals reveals lipid loci stratified by sleep duration.

Both short and long sleep are associated with an adverse lipid profile, likely through different biological pathways. To elucidate the biology of sleep-associated adverse lipid profile, we conduct multi-ancestry genome-wide sleep-SNP interaction analyses on three lipid traits (HDL-c, LDL-c and triglycerides). In the total study sample (discovery + replication) of 126,926 individuals from 5 different ancestry groups, when considering either long or short total sleep time interactions in joint analyses, we identify 49 previously unreported lipid loci, and 10 additional previously unreported lipid loci in a restricted sample of European-ancestry cohorts. In addition, we identify new gene-sleep interactions for known lipid loci such as LPL and PCSK9. The previously unreported lipid loci have a modest explained variance in lipid levels: most notable, gene-short-sleep interactions explain 4.25% of the variance in triglyceride level. Collectively, these findings contribute to our understanding of the biological mechanisms involved in sleep-associated adverse lipid profiles