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Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy. Unexpectedly, this marked elevation in protein synthesis was accompanied by enhanced protein stability and folding and not by markers of proteostasis stress such as protein carbonylation and aggregation. The improvement in proteostasis was attributed to a coordinate up-regulation of proteins in the global proteostasis network, including ribosomal, proteasomal, chaperone, and endoplasmic reticulum/mitochondrial unfolded protein response proteins. We conclude that defects associated with hyperactivation of the insulin signaling pathway are unlikely attributed to defective proteostasis because up-regulation of protein synthesis by insulin is accompanied by up-regulation of proteostatic machinery., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Reconstitution of actin filaments on surfaces for observation of filament-associated protein dynamics by fluorescence microscopy is currently an exciting field in biophysics. Here we examine the effects of attaching actin filaments to surfaces on the binding and dissociation kinetics of a fluorescence-labeled tropomyosin, a rod-shaped protein that forms continuous strands wrapping around the actin filament. Two attachment modalities of the actin to the surface are explored: where the actin filament is attached to the surface at multiple points along its length; and where the actin filament is attached at one end and aligned parallel to the surface by buffer flow. To facilitate analysis of actin-binding protein dynamics, we have developed a software tool for the viewing, tracing and analysis of filaments and co-localized species in noisy fluorescence timelapse images. Our analysis shows that the interaction of tropomyosin with actin filaments is similar for both attachment modalities. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

In a recent study, we showed that in response to high fat feeding C57BL/6, 129X1, DBA/2 and FVB/N mice all developed glucose intolerance, while BALB/c mice displayed minimal deterioration in glucose tolerance and insulin action. Lipidomic analysis of livers across these five strains has revealed marked strain-specific differences in ceramide (Cer) and sphingomyelin (SM) species with high-fat feeding; with increases in C16-C22 (long-chain) and reductions in C>22 (very long-chain) Cer and SM species observed in the four strains that developed HFD-induced glucose intolerance. Intriguingly, the opposite pattern was observed in sphingolipid species in BALB/c mice. These strain-specific changes in sphingolipid acylation closely correlated with ceramide synthase 2 (CerS2) protein content and activity, with reduced CerS2 levels/activity observed in glucose intolerant strains and increased content in BALB/c mice. Overexpression of CerS2 in primary mouse hepatocytes induced a specific elevation in very long-chain Cer, but despite the overall increase in ceramide abundance, there was a substantial improvement in insulin signal transduction, as well as decreased ER stress and gluconeogenic markers. Overall our findings suggest that very long-chain sphingolipid species exhibit a protective role against the development of glucose intolerance and hepatic insulin resistance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background & Aim: Western diets rich in animal protein and poor in fruit and vegetables increase the body acid load, a predictor of type 2 diabetes risk. The relationships between dietary acid load, mild metabolic acidosis and insulin resistance remain unclear. The objective of this study was to assess the association between dietary acid load, body acid/base markers and peripheral insulin resistance at baseline and following a short-term overfeeding intervention in healthy individuals., Methods: In a cross-sectional study of 104 men and women, insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp. Plasma lactate, a marker of metabolic acidosis, was assessed and acid load scores (potential renal acid load, PRAL and net endogenous acid production, NEAP) derived from diet diaries. The cohort was grouped into lean and overweight/obese and the latter further classified as insulin-sensitive (Obsen) and insulin-resistant (Obres) based on hyperinsulinemic-euglycemic clamp glucose infusion rate (GIR, top tertile vs. bottom 2 tertiles). A subset of 40 individuals participated in an overfeeding intervention (+1250 kcal/day) for 28 days and studies repeated., Results: Obsen and Obres were matched for adiposity (BMI and fat mass, both P = 1). Fasting plasma lactate was higher in Obres (0.78 [0.63-1.14] mmol/L) compared with both lean (0.71 [0.44-0.90] mmol/L, P = 0.02) and Obsen (0.67 [0.56-0.79] mmol/L, P = 0.04) and not different between lean and Obsen (P = 0.9). Overfeeding was characterized by an increase in dietary acid load scores PRAL (P = 0.003) and NEAP (P = 0.05), a reduction in GIR necessary to maintain euglycemia (P = 0.03) and an increase in fasting plasma lactate (P = 0.02). The change in lactate was inversely associated with the change in GIR (r = -0.36, P = 0.03)., Conclusions: Mild metabolic acidosis, measured by plasma lactate, aligns with insulin resistance independent of obesity and is induced by short-term increases in energy and dietary acid load in healthy humans. Further studies are required to determine whether buffering mild metabolic acidosis improves insulin resistance and reduces diabetes risk., (Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

Akt/PKB is a biochemical regulator that functions as an important cross-talk node between several signalling pathways in the mammalian cell. In particular, Akt is a key mediator of glucose transport in response to insulin. The phosphorylation (activation) of only a small percentage of the Akt pool of insulin-sensitive cells results in maximal translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This enables the diffusion of glucose into the cell. The dysregulation of Akt signalling is associated with the development of diabetes, cancer and cardiovascular disease. Akt is synthesised in the cytoplasm in the inactive state. Under the influence of insulin, it moves to the PM, where it is phosphorylated to form pAkt. Although phosphorylation occurs only at the PM, pAkt is found in many cellular locations, including the PM, the cytoplasm, and the nucleus. Indeed, the spatial distribution of pAkt within the cell appears to be an important determinant of downstream regulation. Here we present a simple, linear, four-compartment ordinary differential equation (ODE) model of Akt activation that tracks both the biochemical state and the physical location of Akt. This model embodies the main features of the activation of this important cross-talk node and is consistent with the experimental data. In particular, it allows different downstream signalling motifs without invoking separate feedback pathways. Moreover, the model is computationally tractable, readily analysed, and elucidates some of the apparent anomalies in insulin signalling via Akt., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Insulin increases glucose uptake by increasing the rate of exocytosis of the facilitative glucose transporter isoform 4 (Glut4) relative to its endocytosis. Insulin also releases Glut4 from highly insulin-regulated secretory compartments (GSVs or Glut4 storage vesicles) into constitutively cycling endosomes. Previously it was shown that both overexpression and knockdown of the small GTP-binding protein Rab14 decreased Glut4 translocation to the plasma membrane (PM). To determine the mechanism of this perturbation, we measured the effects of Rab14 knockdown on the trafficking kinetics of Glut4 relative to two proteins that partially co-localize with Glut4, the transferrin (Tf) receptor and low-density-lipoprotein-receptor-related protein 1 (LRP1). Our data support the hypothesis that Rab14 limits sorting of proteins from sorting (or 'early') endosomes into the specialized GSV pathway, possibly through regulation of endosomal maturation. This hypothesis is consistent with known Rab14 effectors. Interestingly, the insulin-sensitive Rab GTPase-activating protein Akt substrate of 160 kDa (AS160) affects both sorting into and exocytosis from GSVs. It has previously been shown that exocytosis of GSVs is rate-limited by Rab10, and both Rab10 and Rab14 are in vitro substrates of AS160. Regulation of both entry into and exit from GSVs by AS160 through sequential Rab substrates would provide a mechanism for the finely tuned 'quantal' increases in cycling Glut4 observed in response to increasing concentrations of insulin., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Objective: Alterations in lipids in muscle and plasma have been documented in insulin-resistant people with obesity. Whether these lipid alterations are a reflection of insulin resistance or obesity remains unclear., Methods: Nondiabetic sedentary individuals not treated with lipid-lowering medications were studied (n = 51). Subjects with body mass index (BMI) > 25 kg/m(2) (n = 28) were stratified based on median glucose infusion rate during a hyperinsulinemic-euglycemic clamp into insulin-sensitive and insulin-resistant groups (above and below median, obesity/insulin-sensitive and obesity/insulin-resistant, respectively). Lean individuals (n = 23) served as a reference group. Lipidomics was performed in muscle and plasma by liquid chromatography electrospray ionization-tandem mass spectrometry. Pathway analysis of gene array in muscle was performed in a subset (n = 35)., Results: In muscle, insulin resistance was characterized by higher levels of C18:0 sphingolipids, while in plasma, higher levels of diacylglycerol and cholesterol ester, and lower levels of lysophosphatidylcholine and lysoalkylphosphatidylcholine, indicated insulin resistance, irrespective of overweight/obesity. The sphingolipid metabolism gene pathway was upregulated in muscle in insulin resistance independent of obesity. An overweight/obesity lipidomic signature was only apparent in plasma, predominated by higher triacylglycerol and lower plasmalogen species., Conclusions: Muscle C18:0 sphingolipids may play a role in insulin resistance independent of excess adiposity., (© 2016 The Obesity Society.)

Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR-DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR-DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Insulin is a potent peptide hormone that regulates glucose levels in the blood. Insulin-sensitive cells respond to insulin stimulation with the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM), enabling the clearance of glucose from the blood. Defects in this process can give rise to insulin resistance and ultimately diabetes. One widely cited model of insulin signalling leading to glucose transport is that of Sedaghat et al. (2002) Am. J. Physiol. Endocrinol. Metab. 283, E1084-E1101. Consisting of 20 deterministic ordinary differential equations (ODEs), it is the most comprehensive model of insulin signalling to date. However, the model possesses some major limitations, including the non-conservation of key components. In the current work, we detail mathematical and sensitivity analyses of the Sedaghat model. Based on the results of these analyses, we propose a reduced state space model of the insulin receptor subsystem. This reduced model maintains the input-output relation of the original model but is computationally more efficient, analytically tractable and resolves some of the limitations of the Sedaghat model., (© The Authors 2015. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.)

Aside from glucose metabolism, insulin regulates a variety of pathways in peripheral tissues. Under insulin-resistant conditions, it is well known that insulin-stimulated glucose uptake is impaired, and many studies attribute this to a defect in Akt signaling. Here we make use of several insulin resistance models, including insulin-resistant 3T3-L1 adipocytes and fat explants prepared from high fat-fed C57BL/6J and ob/ob mice, to comprehensively distinguish defective from unaffected aspects of insulin signaling and its downstream consequences in adipocytes. Defective regulation of glucose uptake was observed in all models of insulin resistance, whereas other major actions of insulin such as protein synthesis and anti-lipolysis were normal. This defect corresponded to a reduction in the maximum response to insulin. The pattern of change observed for phosphorylation in the Akt pathway was inconsistent with a simple defect at the level of Akt. The only Akt substrate that showed consistently reduced phosphorylation was the RabGAP AS160 that regulates GLUT4 translocation. We conclude that insulin resistance in adipose tissue is highly selective for glucose metabolism and likely involves a defect in one of the components regulating GLUT4 translocation to the cell surface in response to insulin., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background and Aims: L-glutamine is an efficacious glucagon-like peptide (GLP)-1 secretagogue in vitro. When administered with a meal, glutamine increases GLP-1 and insulin excursions and reduces postprandial glycaemia in type 2 diabetes patients. The aim of the study was to assess the efficacy and safety of daily glutamine supplementation with or without the dipeptidyl peptidase (DPP)-4 inhibitor sitagliptin in well-controlled type 2 diabetes patients., Methods: Type 2 diabetes patients treated with metformin (n = 13, 9 men) with baseline glycated hemoglobin (HbA1c) 7.1±0.3% (54±4 mmol/mol) received glutamine (15 g bd)+ sitagliptin (100 mg/d) or glutamine (15 g bd) + placebo for 4 weeks in a randomized crossover study., Results: HbA1c (P = 0.007) and fructosamine (P = 0.02) decreased modestly, without significant time-treatment interactions (both P = 0.4). Blood urea increased (P<0.001) without a significant time-treatment interaction (P = 0.8), but creatinine and estimated glomerular filtration rate (eGFR) were unchanged (P≥0.5). Red blood cells, hemoglobin, hematocrit, and albumin modestly decreased (P≤0.02), without significant time-treatment interactions (P≥0.4). Body weight and plasma electrolytes remained unchanged (P≥0.2)., Conclusions: Daily oral supplementation of glutamine with or without sitagliptin for 4 weeks decreased glycaemia in well-controlled type 2 diabetes patients, but was also associated with mild plasma volume expansion., Trial Registration: ClincalTrials.gov NCT00673894.

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min(-1) versus 0.6 min(-1)). Differentiation decreases Glut4 ken 40% (ken = 0.12 min(-1)). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025-0.038 min(-1)), not through the fast Tf receptor pathway (kex = 0.2 min(-1)). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min(-1)). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objectives: While chronic obesity is associated with alterations in circulating glycerolipids, sphingolipids and plasmalogens, the effects of short-term overfeeding in humans are unclear., Design and Methods: Healthy individuals (n = 40) were overfed by 1,250 kcal day(-1) for 28 days. Insulin sensitivity (hyperinsulinemic-euglycemic clamp), abdominal fat distribution and serum lipidomics (mass spectrometry) were assessed., Results: Overfeeding increased liver fat, insulin resistance, serum C-reactive protein and urinary F2-isoprostanes. HDL increased (11% ± 2%, P < 0.001) while LDL, triglycerides and nonesterified fatty acids were unchanged. Three hundred and thirty three serum lipids were detected, of which 13% increased and 20% decreased with overfeeding. Total diacylglycerol and lysoalkylphosphatidylcholine (LPC(O)) concentrations decreased (P < 0.01), while total ceramide, Cer22:0 and Cer24:0 increased (P ≤ 0.01). The most notable increases were observed in the HDL-associated phosphatidylethanolamine-based plasmalogens and their precursors alkylhosphatidylethanolamine (18 ± 5% and 38 ± 8% respectively, P ≤ 0.01)., Conclusions: Overfeeding led to weight gain and changes in the serum lipid profile. Increases in ceramides were noted, which left unchecked may promote systemic insulin resistance. Uniform increases were observed in plasmalogens and their precursors. Because plasmalogens are powerful antioxidants, this may be an appropriate response against increased oxidative stress generated by over-nutrition. The metabolic consequences of changes in concentrations of many circulating lipid species with overfeeding require further study. Copyright © 2013 The Obesity Society., (Copyright © 2013 The Obesity Society.)

Aims/hypothesis: Muscle insulin resistance, one of the earliest defects associated with type 2 diabetes, involves changes in the phosphoinositide 3-kinase/Akt network. The relative contribution of obesity vs insulin resistance to perturbations in this pathway is poorly understood., Methods: We used phosphospecific antibodies against targets in the Akt signalling network to study insulin action in muscle from lean, overweight/obese and type 2 diabetic individuals before and during a hyperinsulinaemic-euglycaemic clamp., Results: Insulin-stimulated Akt phosphorylation at Thr309 and Ser474 was highly correlated with whole-body insulin sensitivity. In contrast, impaired phosphorylation of Akt substrate of 160 kDa (AS160; also known as TBC1D4) was associated with adiposity, but not insulin sensitivity. Neither insulin sensitivity nor obesity was associated with defective insulin-dependent phosphorylation of forkhead box O (FOXO) transcription factor. In view of the resultant basal hyperinsulinaemia, we predicted that this selective response within the Akt pathway might lead to hyperactivation of those processes that were spared. Indeed, the expression of genes targeted by FOXO was downregulated in insulin-resistant individuals., Conclusions/interpretation: These results highlight non-linearity in Akt signalling and suggest that: (1) the pathway from Akt to glucose transport is complex; and (2) pathways, particularly FOXO, that are not insulin-resistant, are likely to be hyperactivated in response to hyperinsulinaemia. This facet of Akt signalling may contribute to multiple features of the metabolic syndrome.

Insulin triggers glucose uptake into muscle and adipose tissue by stimulating the translocation of the glucose transporter glut4 from intracellular vesicles to the plasma membrane (pm). insulin leads to a rapid increase in glut4 at the pm from approximately 5% to 40-50%. this effect is time and dose-dependent, reaching a new steady state after 30 min of insulin stimulation. previous kinetic analyses in adipocytes has revealed that this is regulated by two mechanisms-increasing the amount of glut4 in the endosomal recycling system and increasing the exocytosis rate constant. fazakerley et al.1 focuses on GLUT4 kinetics in the L6 skeletal muscle cell line. Despite displaying a similar redistribution of GLUT4 to the cell surface with insulin to that seen in adipocytes, the mechanism for this effect in L6 cells was completely different. Insulin had a modest effect to increase the amount of GLUT4 in the recycling system with the dominant effect being on reduction of the endocytosis rate constant. Similar findings were observed with AMPK agonists. These studies indicate that different cell types are capable of achieving the same cell biological endpoint but using completely distinct mechanisms.

The phosphatidylinositol 3-kinase/Akt pathway regulates many biological processes, including insulin-regulated GLUT4 insertion into the plasma membrane. However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this, we examined insulin regulation of components within the signaling cascade in a quantitative kinetic and dose-response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the plasma membrane strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with Thr(P)-642 grouped with other Akt substrates. Further experiments suggested that atypical protein kinase Czeta phosphorylates AS160 at Ser-588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signaling components for biological specificity.

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of approximately 20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min(-1)) and was decreased during the steady-state responses to insulin (0.18 min(-1)), AICAR (0.16 min(-1)), and A-769662 (0.24 min(-1)). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.

Recycling via the plasma membrane is a key feature that is shared by many membrane proteins. Using a combination of indirect immunofluorescence labeling and fluorescence detection using a fluorescence multiwell plate reader, we exploited the possibilities of quantitatively measuring the trafficking kinetics of transmembrane proteins. Parameters that can be studied include dynamic appearance/presence at the cell surface, recycling via the cell surface, and internalization. For the insulin-responsive glucose transporter GLUT4 (glucose transporter number 4), details are presented on how to quantitatively measure insulin-induced GLUT4 translocation toward the plasma membrane (transition state) and to analyze cell surface recycling of GLUT4 in basal and insulin-stimulated cells (steady state).

The insulin-sensitive glucose transporter GLUT4 mediates the uptake of glucose into adipocytes and muscle cells. In this study we have used a novel 96-well plate fluorescence assay to study the kinetics of GLUT4 trafficking in 3T3-L1 adipocytes. We have found evidence for a graded release mechanism whereby GLUT4 is released into the plasma membrane recycling system in a nonkinetic manner as follows: the kinetics of appearance of GLUT4 at the plasma membrane is independent of the insulin concentration; a large proportion of GLUT4 molecules do not participate in plasma membrane recycling in the absence of insulin; and with increasing insulin there is an incremental increase in the total number of GLUT4 molecules participating in the recycling pathway rather than simply an increased rate of recycling. We propose a model whereby GLUT4 is stored in a compartment that is disengaged from the plasma membrane recycling system in the basal state. In response to insulin, GLUT4 is quantally released from this compartment in a pulsatile manner, leaving some sequestered from the recycling pathway even in conditions of excess insulin. Once disengaged from this location we suggest that in the continuous presence of insulin this quanta of GLUT4 continuously recycles to the plasma membrane, possibly via non-endosomal carriers that are formed at the perinuclear region.

A generic framework for the automated classification of human movements using an accelerometry monitoring system is introduced. The framework was structured around a binary decision tree in which movements were divided into classes and subclasses at different hierarchical levels. General distinctions between movements were applied in the top levels, and successively more detailed subclassifications were made in the lower levels of the tree. The structure was modular and flexible: parts of the tree could be reordered, pruned or extended, without the remainder of the tree being affected. This framework was used to develop a classifier to identify basic movements from the signals obtained from a single, waist-mounted triaxial accelerometer. The movements were first divided into activity and rest. The activities were classified as falls, walking, transition between postural orientations, or other movement. The postural orientations during rest were classified as sitting, standing or lying. In controlled laboratory studies in which 26 normal, healthy subjects carried out a set of basic movements, the sensitivity of every classification exceeded 87%, and the specificity exceeded 94%; the overall accuracy of the system, measured as the number of correct classifications across all levels of the hierarchy, was a sensitivity of 97.7% and a specificity of 98.7% over a data set of 1309 movements.

The insulin-responsive glucose transporter GLUT4 plays an essential role in glucose homeostasis. A novel assay was used to study GLUT4 trafficking in 3T3-L1 fibroblasts/preadipocytes and adipocytes. Whereas insulin stimulated GLUT4 translocation to the plasma membrane in both cell types, in nonstimulated fibroblasts GLUT4 readily cycled between endosomes and the plasma membrane, while this was not the case in adipocytes. This efficient retention in basal adipocytes was mediated in part by a C-terminal targeting motif in GLUT4. Insulin caused a sevenfold increase in the amount of GLUT4 molecules present in a trafficking cycle that included the plasma membrane. Strikingly, the magnitude of this increase correlated with the insulin dose, indicating that the insulin-induced appearance of GLUT4 at the plasma membrane cannot be explained solely by a kinetic change in the recycling of a fixed intracellular GLUT4 pool. These data are consistent with a model in which GLUT4 is present in a storage compartment, from where it is released in a graded or quantal manner upon insulin stimulation and in which released GLUT4 continuously cycles between intracellular compartments and the cell surface independently of the nonreleased pool.

Accelerometry offers a practical and low cost method of objectively monitoring human movements, and has particular applicability to the monitoring of free-living subjects. Accelerometers have been used to monitor a range of different movements, including gait, sit-to-stand transfers, postural sway and falls. They have also been used to measure physical activity levels and to identify and classify movements performed by subjects. This paper reviews the use of accelerometer-based systems in each of these areas. The scope and applicability of such systems in unsupervised monitoring of human movement are considered. The different systems and monitoring techniques can be integrated to provide a more comprehensive system that is suitable for measuring a range of different parameters in an unsupervised monitoring context with free-living subjects. An integrated approach is described in which a single, waist-mounted accelerometry system is used to monitor a range of different parameters of human movement in an unsupervised setting.

We assessed the feasibility of using a waist-mounted, wireless triaxial accelerometer (TA) to monitor human movements in an unsupervised home setting to detect changes in functional status. A pilot study was carried out with six healthy subjects aged 80-86 years. The subjects wore a TA unit every day for two to three months. Each morning they carried out a short routine of directed movements that included standing, sitting, lying and walking. Important movement variables were measured. During the rest of the day, subjects were monitored for falls, and variables such as metabolic energy expenditure were measured. All subjects remained healthy; there was no overall change in functional status and there were only slight fluctuations in health status. No longitudinal changes were detected in any of the variables measured during the directed routine. There was a moderate correlation between weekly self-reported health status and energy expenditure: subjects reported a lower health status for weeks in which they expended less energy. The TA system was found to be practical for long-term, unsupervised home monitoring. All subjects found the system simple to use and the TA unit unobtrusive and comfortable to wear. High compliance rates were achieved: the TA units were worn on 88% of the days in the study, for an average of 11.2 hours per day.

Triaxial accelerometers have been employed to monitor human movements in a variety of circumstances. The study considered the use of data from a single waist-mounted triaxial accelerometer to distinguish between activity states and rest A method using acceleration magnitude was applied to data collected from 26 normal subjects performing sit-to-stand and stand-to-sit transitions and walking. The effects of three parameters were investigated: the length n of a smoothing median filter, the width w of the averaging window used to process the signal and the value of the acceleration magnitude threshold th. These were found to be inter-related, and sets of parameters that resulted in accurate discrimination were determined by the relationship between th and the product of w and n, and by the relationship between n and w. The subjects were randomly divided into control (N = 13) and test (N = 13) groups. Optimum parameter sets were determined using the control group. Eleven sets of parameters yielded the same optimum results of a sensitivity of 1.0 and a specificity of 0.96 in the control group. Upon application to the test group, using these parameters, the system successfully distinguished between activity and rest, giving sensitivities greater than 0.98 and specificities between 0.88 and 0.94.

When a brief current pulse is incident on excitable cells in cardiac and other nervous tissue, a change in phase of the cell's response is usually observed. In cardiac tissue, the cells are exposed to external stimulation of mainly positive currents, which depolarize the cells. We performed a systematic study of the effect of depolarizing stimuli, covering timing, magnitude, and duration, and demonstrated that all of these parameters influence the phase response of the cell. The phase response of our model cell compares favorably with measurements on isolated sinoatrial node cells. We investigated the phase response to single depolarizing stimuli as a function of the stimulus parameters (phase response curves), and then studied cell responses to the combined effect of a pulse train (entrainment phenomena). The range of magnitudes and durations for the stimuli were 0.01-5 nA and 0.01-50 ms. Comparisons of the entrainment properties of the model with experimental results show good agreement with similar modes and different entrainment ratios occurring for similar basic cycle lengths (as functions of the unperturbed cell period). Our results demonstrate that any combination of parameters that provide the same charge transfer to the cell causes a similar phase response, independent of the specific magnitude and duration for the entire range of stimuli investigated.