Hexosamines Provoke Membrane Cholesterol Accrual, Filamentous Actin Loss, and GLUT4 Dysregulation in Adipocytes through Transcriptional Activation of Specificity Protein 1

poster abstractThe hexosamine biosynthesis pathway (HBP) serves as a sensor of excess nutrient bioavailability and has been implicated in the pathogenesis of type 2 diabetes. Previous study observed that hyperinsulinemic culturing conditions akin to those seen clinically activate the HBP provoking gains in plasma membrane (PM) cholesterol content in L6 myotubes and 3T3-L1 adipocytes. This, in turn, compromised the cortical filamentous actin (F-actin) structure necessary for the proper incorporation of the insulin sensitive glucose transporter GLUT4 into the membrane. The mechanism(s), however, by which HBP activation provokes PM cholesterol accrual, remains unclear. Here, the hypothesis that HBP engages a cholesterolgenic transcriptional response resulting in PM cholesterol accrual/toxicity was tested. In 3T3-L1 adipocytes, pathophysiologically relevant doses of hyperinsulinemia (0.25, 0.5, and 5 nM) resulted in a dose-dependent gain in PM cholesterol as well as mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Immunoprecipitation experiments demonstrated that hyperinsulinemia induced elevations in O-linked N-acetylglucosamine post-translational modification of the cholesterolgenic transcription factor specificity protein 1 (Sp1). This modification was prevented in cells in which the HBP was inhibited. Chromatin immunoprecipitation demonstrated that hyperinsulinemia induced a ~4 fold increase in the affinity of Sp1 to the promoter region of HMGR, which was lost with HBP inhibition. Luciferase assays confirmed that this altered binding resulted in a ~50% increase in promoter activity of this cholesterolgenic gene. Hyperinsulinemia also augmented Sp1 binding to the promoter of the sterol response element binding protein gene, resulting in increased total and nuclear content of this factor. To further delineate the role of Sp1 in this process, a specific inhibitor, mithramycin (MTR), of Sp1 binding to DNA was employed. This inhibitor prevented against hyperinsulinemia-induced gains in HMGR and PM cholesterol as well as F-actin loss. Importantly, this treatment corrected the impaired insulin-stimulated GLUT4 translocation and glucose transport induced by hyperinsulinemia. These data suggest hyperinsulinemia-induced HBP activity provokes cholesterol synthesis and PM cholesterol accrual/F-actin loss that compromises GLUT4/glucose transport regulation by insulin

Evidence that Hyperinsulinemia, known to Accelerate Diabetes Progression, may also Contribute to Dyslipidemia via Impairing ApoA1/ABCA1-Mediated Cholesterol Efflux

poster abstractLow levels of plasma high-density lipoprotein cholesterol (HDL-C) are associated with insulin resistance and type 2 diabetes (T2D). As it is well appreciated that hyperinsulinemia contributes to the progression/worsening of insulin resistance, we tested here if this key metabolic derangement impaired cellular mechanisms of HDL-C generation. An initial event in this process is the binding of apolipoprotein A1 (ApoA1) to the plasma membrane (PM)-localized ATPbinding cassette cholesterol transporter protein ABCA1. Subcellular fractionation analyses revealed that 3T3-L1 adipocytes exposed to chronic insulin (12h, 5nM) displayed a 25% decrease (P<0.05) in PM ABCA1 content and a reciprocal increase in endosomal ABCA1 content. These insulin-induced changes in cellular ABCA1 distribution occurred concomitantly with a decrease in ApoA1-mediated cellular cholesterol efflux. Consistent with endosomal/cytosolic cycling of the small molecular GTPase Rab8 playing a functional role in ABCA1 vesicle trafficking, we found a 50% increase (P<0.05) in endosomal Rab8 content and a 30% decrease (P<0.05) in cytosolic Rab8 content. New data shows that increased HBP activity increases cholesterol biosynthesis and increased endosomal cholesterol content inhibits the functional cycling of Rab proteins. In line with these observations, we found that cells treated with the cholesterol-lowering agent methyl-β-cyclodextrin were protected against insulininduced defects in ABCA1/Rab8 vesicle trafficking and PM cholesterol accrual. These data are consistent with the concept that the coexistence of low plasma HDL-C with insulin resistance and T2D may reflect a negative influence of hyperinsulinemia on Rab8-mediated trafficking of ABCA1 to the PM for ApoA1-mediated cholesterol efflux

Effect of Corncob bedding on feed conversion efficiency in a high-fat diet-induced prediabetic model in C57Bl/6J mice

Laboratory facilities use many varieties of contact bedding, including wood chips, paper products, and corncob, each with its own advantages and disadvantages. Corncob bedding, for example, is often used because of its high absorbency, ability to minimize detectable ammonia, and low cost. However, observations that mice eat the corncob lead to concerns that its use can interfere with dietary studies. We evaluated the effect of corncob bedding on feed conversion (change in body weight relative to the apparent number of kcal consumed over 7 d) in mice. Four groups of mice (6 to 12 per group) were housed in an individually ventilated caging system: (1) low-fat diet housed on recycled paper bedding, (2) low-fat diet housed on corncob bedding, (3) high-fat diet housed on recycled paper bedding, and (4) high-fat diet housed on corncob bedding. After 4 wk of the high-fat diet, feed conversion and percentage body weight change both were lower in corncob-bedded mice compared with paper-bedded mice. Low-fat-fed mice on corncob bedding versus paper bedding did not show statistically significant differences in feed conversion or change in percentage body weight. Average apparent daily feed consumption did not differ among the 4 groups. In conclusion, these data suggest that corncob bedding reduces the efficiency of feed conversion in mice fed a high-fat diet and that other bedding choices should be favored in these models

Chromium Enhances Insulin Responsiveness via AMPK

Trivalent chromium (Cr3+) is known to improve glucose homeostasis. Cr3+ has been shown to improve plasma membrane-based aspects of glucose transporter GLUT4 regulation and increase activity of the cellular energy sensor 5′ AMP-activated protein kinase (AMPK). However, the mechanism(s) by which Cr3+ improves insulin responsiveness and whether AMPK mediates this action is not known. In this study we tested if Cr3+ protected against physiological hyperinsulinemia-induced plasma membrane cholesterol accumulation, cortical filamentous actin (F-actin) loss and insulin resistance in L6 skeletal muscle myotubes. In addition, we performed mechanistic studies to test our hypothesis that AMPK mediates the effects of Cr3+ on GLUT4 and glucose transport regulation. Hyperinsulinemia-induced insulin-resistant L6 myotubes displayed excess membrane cholesterol and diminished cortical F-actin essential for effective glucose transport regulation. These membrane and cytoskeletal abnormalities were associated with defects in insulin-stimulated GLUT4 translocation and glucose transport. Supplementing the culture medium with pharmacologically relevant doses of Cr3+ in the picolinate form (CrPic) protected against membrane cholesterol accumulation, F-actin loss, GLUT4 dysregulation and glucose transport dysfunction. Insulin signaling was neither impaired by hyperinsulinemic conditions nor enhanced by CrPic, whereas CrPic increased AMPK signaling. Mechanistically, siRNA-mediated depletion of AMPK abolished the protective effects of CrPic against GLUT4 and glucose transport dysregulation. Together these findings suggest that the micronutrient Cr3+, via increasing AMPK activity, positively impacts skeletal muscle cell insulin sensitivity and glucose transport regulation

Exercise training prevents skeletal muscle plasma membrane cholesterol accumulation, cortical actin filament loss, and insulin resistance in C57BL/6J mice fed a western‐style high‐fat diet

Insulin action and glucose disposal are enhanced by exercise, yet the mechanisms involved remain imperfectly understood. While the causes of skeletal muscle insulin resistance also remain poorly understood, new evidence suggest excess plasma membrane (PM) cholesterol may contribute by damaging the cortical filamentous actin (F‐actin) structure essential for GLUT4 glucose transporter redistribution to the PM upon insulin stimulation. Here, we investigated whether PM cholesterol toxicity was mitigated by exercise. Male C57BL/6J mice were placed on low‐fat (LF, 10% kCal) or high‐fat (HF, 45% kCal) diets for a total of 8 weeks. During the last 3 weeks of this LF/HF diet intervention, all mice were familiarized with a treadmill for 1 week and then either sham‐exercised (0 m/min, 10% grade, 50 min) or exercised (13.5 m/min, 10% grade, 50 min) daily for 2 weeks. HF‐feeding induced a significant gain in body mass by 3 weeks. Sham or chronic exercise did not affect food consumption, water intake, or body mass gain. Prior to sham and chronic exercise, “pre‐intervention” glucose tolerance tests were performed on all animals and demonstrated that HF‐fed mice were glucose intolerant. While sham exercise did not affect glucose tolerance in the LF or HF mice, exercised mice showed an improvement in glucose tolerance. Muscle from sham‐exercised HF‐fed mice showed a significant increase in PM cholesterol, loss of cortical F‐actin, and decrease in insulin‐stimulated glucose transport compared to sham‐exercised LF‐fed mice. These HF‐fed skeletal muscle membrane/cytoskeletal abnormalities and insulin resistance were improved in exercised mice. These data reveal a new therapeutic aspect of exercise being regulation of skeletal muscle PM cholesterol homeostasis. Further studies on this mechanism of insulin resistance and the benefits of exercise on its prevention are needed

Evening and morning peroxiredoxin-2 redox/oligomeric state changes in obstructive sleep apnea red blood cells: Correlation with polysomnographic and metabolic parameters

We have examined the effects of Obstructive Sleep Apnea (OSA) on red blood cell (RBC) proteome variation at evening/morning day time to uncover new insights into OSA-induced RBC dysfunction that may lead to OSA manifestations. Dysregulated proteins mainly fall in the group of catalytic enzymes, stress response and redox regulators such as peroxiredoxin 2 (PRDX2). Validation assays confirmed that at morning the monomeric/dimeric forms of PRDX2 were more overoxidized in OSA RBC compared to evening samples. Six month of positive airway pressure (PAP) treatment decreased this overoxidation and generated multimeric overoxidized forms associated with chaperone/transduction signaling activity of PRDX2. Morning levels of overoxidized PRDX2 correlated with polysomnographic (PSG)-arousal index and metabolic parameters whereas the evening level of disulfide-linked dimer (associated with peroxidase activity of PRDX2) correlated with PSG parameters. After treatment, morning overoxidized multimer of PRDX2 negatively correlated with fasting glucose and dopamine levels. Overall, these data point toward severe oxidative stress and altered antioxidant homeostasis in OSA RBC occurring mainly at morning time but with consequences till evening. The beneficial effect of PAP involves modulation of the redox/oligomeric state of PRDX2, whose mechanism and associated chaperone/transduction signaling functions deserves further investigation. RBC PRDX2 is a promising candidate biomarker for OSA severity and treatment monitoring, warranting further investigation and validation.Project partially supported by Harvard Medical School-Portugal Program (HMSPICJ/0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013), FCT/Poly-Annual Funding Program and FEDER/Saúde XXI Program (Portugal) and postdoctoral fellowship (SFRH/BPD/43365/2008) of Fundação para a Ciência e a Tecnologia (FCT), Portugal.info:eu-repo/semantics/publishedVersio

Proteomics of Red Blood Cells from Patients with Obstructive Sleep Apnea

This work was approved by the Ethical Committee of INSA.I.P.-Lisboa, Centro Hospitalar Lisboa-Norte., Faculdade de Ciências Médicas da Universidade Nova de Lisboa and Comissão Nacional de Proteção de Dados, Portugal.DIGE images were obtained in ITQB.Obstructive sleep apnea (OSA) is a common public health concern causing metabolic and cardiovascular consequences. Although OSA is a systemic disease, the molecular mechanisms and specific genes/proteins associated with such processes remain poorly defined.To identify dysregulated proteins that could be useful as candidate biomarkers of diagnosis/prognosis of OSA., is the aim of this studyProject - Obstructive sleep apnea and associated metabolic/cardiovascular disorders: understanding mechanisms towards early diagnosis and prognosis prediction. Work partially supported by Harvard Medical School-Portugal Program (HMSP-ICJ/0022/2011), FCT/Poly-Annual Funding Program and FEDER/Saúde XXI Program (Portugal

Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia

ATP-binding cassette (ABC) transporters in normal and pathological lung

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (mal)function in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD) have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases