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Oncolytic virotherapy is a promising cancer treatment using genetically modified viruses. Unfortunately, virus particles rapidly decay inside the body, significantly hindering their efficacy. In this article, treatment perturbations that could overcome obstacles to oncolytic virotherapy are investigated through the development of a Voronoi Cell-Based model (VCBM). The VCBM derived captures the interaction between an oncolytic virus and cancer cells in a 2-dimensional setting by using an agent-based model, where cell edges are designated by a Voronoi tessellation. Here, we investigate the sensitivity of treatment efficacy to the configuration of the treatment injections for different tumour shapes: circular, rectangular and irregular. The model predicts that multiple off-centre injections improve treatment efficacy irrespective of tumour shape. Additionally, we investigate delaying the infection of cancer cells by modifying viral particles with a substance such as alginate (a hydrogel polymer used in a range of cancer treatments). Simulations of the VCBM show that delaying the infection of cancer cells, and thus allowing more time for virus dissemination, can improve the efficacy of oncolytic virotherapy. The simulated treatment noticeably decreases the tumour size with no increase in toxicity. Improving oncolytic virotherapy in this way allows for a more effective treatment without changing its fundamental essence.

BACKGROUND: Metabolic complications are highly prevalent in cancer survivors treated with irradiation but the underlying mechanisms remain unknown. METHODS: Chow or high fat-fed C57Bl/6J mice were irradiated (6Gy) before investigating the impact on whole-body or skeletal muscle metabolism and profiling their lipidomic signature. Using a transgenic mouse model (Tg:Pax7-nGFP), we isolated muscle progenitor cells (satellite cells) and characterised their metabolic functions. We recruited childhood cancer survivors, grouped them based on the use of total body irradiation during their treatment and established their lipidomic profile. RESULTS: In mice, irradiation delayed body weight gain and impaired fat pads and muscle weights. These changes were associated with impaired whole-body fat oxidation in chow-fed mice and altered ex vivo skeletal muscle fatty acid oxidation, potentially due to a reduction in oxidative fibres and reduced mitochondrial enzyme activity. Irradiation led to fasting hyperglycaemia and impaired glucose uptake in isolated skeletal muscles. Cultured satellite cells from irradiated mice showed decreased fatty acid oxidation and reduced glucose uptake, recapitulating the host metabolic phenotype. Irradiation resulted in a remodelling of lipid species in skeletal muscles, with the extensor digitorum longus muscle being particularly affected. A large number of lipid species were reduced, with several of these species showing a positive correlation with mitochondrial enzymes activity. In cancer survivors exposed to irradiation, we found a similar decrease in systemic levels of most lipid species, and lipid species that increased were positively correlated with insulin resistance (HOMA-IR). CONCLUSION: Irradiation leads to long-term alterations in body composition, and lipid and carbohydrate metabolism in skeletal muscle, and affects muscle progenitor cells. Such changes result in persistent impairment of metabolic functions, providing a new mechanism for

Background: Low tissue concentrations of carotenoids have been suggested to contribute to insulin resistance in obesity. Objectives: The objectives of the study were to 1) evaluate the relations of adipose tissue and serum carotenoids with body fat, abdominal fat distribution, muscle, adipose tissue and liver insulin resistance, and dietary intake; 2) evaluate the relations and distributions of carotenoids detected in adipose tissue and serum; and 3) compare serum carotenoids and retinol concentrations in subjects with and without obesity. Methods: Post hoc analysis of serum and adipose tissue carotenoids in individuals [n = 80; 31 men, 49 women; age (mean ± SEM): 51.4 ± 1.1 y] who participated in 2 separate studies conducted at the Clinical Research Facility at the Garvan Institute of Medical Research (Sydney) between 2008 and 2013. Retinol, α-carotene, β-carotene, ζ-carotene, lutein, lycopene, phytoene, and phytofluene were measured using HPLC. Body composition was measured by dual-energy X-ray absorptiometry. Insulin resistance was measured by 2-step hyperinsulinemic-euglycemic clamps with deuterated glucose (n = 64), and subcutaneous and visceral abdominal volume and liver and pancreatic fat by MRI (n = 60). Periumbilical subcutaneous fat biopsy was performed and carotenoids and retinol measured in the tissue (n = 16). Results: We found that ζ-carotene, phytoene, and phytofluene were stored in considerable amounts in adipose tissue (25% of adipose tissue carotenoids). Carotenoid concentrations in adipose tissue and serum correlated significantly, but they followed different distributions: ζ-carotene was 3-fold higher in adipose tissue compared with serum, while lutein and lycopene made up 20% and 21% of serum carotenoids compared with 2% and 12% of adipose tissue carotenoids, respectively. Liver (P ≤ 0.028) and adipose tissue (P = 0.023), but not muscle (P ≥ 0.16), insulin resistance correlated inversely with many of the serum carotenoids. Conclusions: Multiple

Identifying the range of plants and/or animals processed by pounding and/or grinding stones has been a rapidly developing research area in world prehistory. In Australia, grinding and pounding stones are ubiquitous across the semi-arid and arid zones and the associated tasks have been mostly informed by ethnographic case studies. More recently, plant microfossil studies have provided important insights to the breadth of plants being exploited in a range of contexts and over long time periods. The preservation of starch and/or phytoliths on the used surfaces of these artefacts is well documented, though the factors determining the survival or destruction of use-related starch residues are still largely unknown. Some of these artefacts have also been used for grinding up small animals and these tasks can be identified by specific staining methods for organic remains such as collagen. In this study, 25 grinding and pounding stones identified during an archaeological project in arid South Australia, were examined for starch and collagen residues. The artefacts were from 3 locations in central South Australia, all located in exposed settings. Of these localities, Site 11 in the Western Valley near Woomera is an important Aboriginal landscape specifically associated with male ceremonial practice in the recent past. The remaining two sites, one in the adjacent Nurrungar Valley and the other near Andamooka 100 km distant, have unrestricted access and potentially a different suite of residues. The Kokatha Mula Nations, the Traditional Owners of Woomera, requested that this study be undertaken to explore the range of plants that may have been processed here. It provided an opportunity to investigate the preservation potential of starch and collagen on grinding stones; explore the range of taphonomic factors involved in the persistence of residues in extreme environmental conditions; and test the methodological developments in identifying specific plant origin of starch residu

Background: Large cohort longitudinal studies have almost unanimously concluded that metabolic health in obesity is a transient phenomenon, diminishing in older age. We aimed to assess the fate of insulin sensitivity per se over time in overweight and obese individuals. Methods: Individuals studied using the hyperinsulinaemic-euglycaemic clamp at the Garvan Institute of Medical Research from 2008 to 2010 (n = 99) were retrospectively grouped into Lean (body mass index (BMI) < 25 kg/m2) or overweight/obese (BMI ≥ 25 kg/m2), with the latter further divided into insulin-sensitive (ObSen) or insulin-resistant (ObRes), based on median clamp M-value (M/I, separate cut-offs for men and women). Fifty-seven individuals participated in a follow-up study after 5.4 ± 0.1 years. Hyperinsulinaemic-euglycaemic clamp, dual-energy X-ray absorptiometry and circulating cardiovascular markers were measured again at follow-up, using the same protocols used at baseline. Liver fat was measured using computed tomography at baseline and proton magnetic resonance spectroscopy at follow-up with established cut-offs applied for defining fatty liver. Results: In the whole cohort, M/I did not change over time (p = 0.40); it remained significantly higher at follow-up in ObSen compared with ObRes (p = 0.02), and was not different between ObSen and Lean (p = 0.41). While BMI did not change over time (p = 0.24), android and visceral fat increased significantly in this cohort (ptime ≤ 0.0013), driven by ObRes (p = 0.0087 and p = 0.0001, respectively). Similarly, systolic blood pressure increased significantly over time (ptime = 0.0003) driven by ObRes (p = 0.0039). The best correlate of follow-up M/I was baseline M/I (Spearman’s r = 0.76, p = 1.1 × 10-7). Conclusions: The similarity in insulin sensitivity between the ObSen and the Lean groups at baseline persisted over time. Insulin resistance in overweight and obese individuals predisposed to further metabolic deterioration over time.

Akt/PKB is an important crosstalk node at the junction between a number of major signalling pathways in the mammalian cell. As a significant nutrient sensor, Akt plays a central role in many cellular processes, including cell growth, cell survival and glucose metabolism. The dysregulation of Akt signalling is implicated in the development of many diseases, from diabetes to cancer. The translocation of Akt from cytosol to plasma membrane is a crucial step in Akt activation. Akt is initially synthesized on the endoplasmic reticulum, but translocates to the plasma membrane (PM) in response to insulin stimulation, where it may be activated. The Akt is then recycled to the cytoplasm. The activated Akt may propagate signals to downstream substrates both at the PM and in the cytosol, hence understanding the translocation dynamics is an important step in dissecting the signalling system. At the present time, however, knowledge concerning the translocation of either activated and unactivated Akt is scant. Here we present a simple, deterministic, three-compartment ordinary differential equation model of Akt translocation in vitro. This model can reproduce the salient features of Akt translocation in a manner consistent with the experimental data. Furthermore, we demonstrate that this system is equivalent to a damped harmonic oscillator, and analyse the steady state and transient behaviour of the model over the entire parameter space.

Oncolytic virotherapy is an experimental cancer treatment that uses genetically engineered viruses to target and kill cancer cells. One major limitation of this treatment is that virus particles are rapidly cleared by the immune system, preventing them from arriving at the tumour site. To improve virus survival and infectivity Kim et al. (Biomaterials 32(9):2314–2326, 2011) modified virus particles with the polymer polyethylene glycol (PEG) and the monoclonal antibody herceptin. Whilst PEG modification appeared to improve plasma retention and initial infectivity, it also increased the virus particle arrival time. We derive a mathematical model that describes the interaction between tumour cells and an oncolytic virus. We tune our model to represent the experimental data by Kim et al. (2011) and obtain optimised parameters. Our model provides a platform from which predictions may be made about the response of cancer growth to other treatment protocols beyond those in the experiments. Through model simulations, we find that the treatment protocol affects the outcome dramatically. We quantify the effects of dosage strategy as a function of tumour cell replication and tumour carrying capacity on the outcome of oncolytic virotherapy as a treatment. The relative significance of the modification of the virus and the crucial role it plays in optimising treatment efficacy are explored.

© 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Combined virotherapy and immunotherapy has been emerging as a promising and effective cancer treatment for some time. Intratumoural injections of an oncolytic virus instigate an immune reaction in the host, resulting in an influx of immune cells to the tumour site. Through combining an oncolytic viral vector with immunostimulatory cytokines an additional antitumour immune response can be initiated, whereby immune cells induce apoptosis in both uninfected and virus infected tumour cells. We develop a mathematical model to reproduce the experimental results for tumour growth under treatment with an oncolytic adenovirus co-expressing the immunostimulatory cytokines interleukin 12 (IL-12) and granulocyte-monocyte colony stimulating factor (GM-CSF). By exploring heterogeneity in the immune cell stimulation by the treatment, we find a subset of the parameter space for the immune cell induced apoptosis rate, in which the treatment will be less effective in a short time period. Therefore, we believe the bivariate nature of treatment outcome, whereby tumours are either completely eradicated or grow unbounded, can be explained by heterogeneity in this immune characteristic. Furthermore, the model highlights the apparent presence of negative feedback in the helper T cell and APC stimulation dynamics, when IL-12 and GM-CSF are co-expressed as opposed to individually expressed by the viral vector.

Skeletal muscle tropomyosin (Tpm1.1) is an elongated, rod-shaped, alpha-helical coiled-coil protein that forms continuous head-to-tail polymers along both sides of the actin filament. In this study we use single molecule fluorescence TIRF microscopy combined with a microfluidic device and fluorescently labelled proteins to measure Tpm1.1 association to and dissociation from single actin filaments. Our experimental setup allows us to clearly resolve Tpm1.1 interactions on both sides of the filaments. Here we provide a semi-automated method for the extraction and quantification of kymograph data for individual actin filaments bound at different Tpm1.1 concentrations. We determine boundaries on the kymograph on each side of the actin filament, based on intensity thresholding, performing fine manual editing of the boundaries (if needed) and extracting user defined kinetic properties of the system. Using our analytical tools we can determine (i) nucleation point(s) and rates, (ii) elongation rates of Tpm1.1, (iii) identify meeting points after the saturation of filament, and when dissociation occurs, (iv) initiation point(s), (v) the final dissociation point(s), as well as (vi) dissociation rates. All of these measurements can be extracted from both sides of the filament, allowing for the determination of possible differences in behaviour on the two sides of the filament, and across concentrations. The robust and repeatable nature of the method allows quantitative, semi-automated analyses to be made of large studies of acto-tropomyosin interactions, as well as for other actin binding proteins or filamentous structures, opening the way for dissection of the dynamics underlying these interactions.

The use of viruses as a cancer treatment is becoming increasingly more robust; however, there is still a long way to go before a completely successful treatment is formulated. One major challenge in the field is to select which virus, out of a burgeoning number of oncolytic viruses and engineered derivatives, can maximise both treatment spread and anticancer cytotoxicity. To assist in solving this problem, an in-depth understanding of the virus-tumour interaction is crucial. In this article, we present a novel integro-differential system with distributed delays embodying the dynamics of an oncolytic adenovirus with a fixed population of tumour cells in vitro, allowing for heterogeneity to exist in the virus and cell populations. The parameters of the model are optimised in a hierarchical manner, the purpose of which is not to obtain a perfect representation of the data. Instead, we place our parameter values in the correct region of the parameter space. Due to the sparse nature of the data it is not possible to obtain the parameter values with any certainty, but rather we demonstrate the suitability of the model. Using our model we quantify how modifications to the viral genome alter the viral characteristics, specifically how the attenuation of the E1B 19 and E1B 55 gene affect the system performance, and identify the dominant processes altered by the mutations. From our analysis, we conclude that the deletion of the E1B 55 gene significantly reduces the replication rate of the virus in comparison to the deletion of the E1B 19 gene. We also found that the deletion of both the E1B 19 and E1B 55 genes resulted in a long delay in the average replication start time of the virus. This leads us to propose the use of E1B 19 gene-attenuated adenovirus for cancer therapy, as opposed to E1B 55 gene-attenuated adenoviruses.

In recent years, interest in the capability of virus particles as a treatment for cancer has increased. In this work, we present a mathematical model embodying the interaction between tumour cells and virus particles engineered to infect and destroy cancerous tissue. To quantify the effectiveness of oncolytic virotherapy, we conduct a local stability analysis and bifurcation analysis of our model. In the absence of tumour growth or viral decay, the model predicts that oncolytic virotherapy will successfully eliminate the tumour cell population for a large proportion of initial conditions. In comparison, for growing tumours and decaying viral particles there are no stable equilibria in the model; however, oscillations emerge for certain regions in our parameter space. We investigate how the period and amplitude of oscillations depend on tumour growth and viral decay. We find that higher tumour replication rates result in longer periods between oscillations and lower amplitudes for uninfected tumour cells. From our analysis, we conclude that oncolytic viruses can reduce growing tumours into a stable oscillatory state, but are insufficient to completely eradicate them. We propose that it is only with the addition of other anti-cancer agents that tumour eradication may be achieved by oncolytic virus.

Different mouse strains exhibit variation in their inherent propensities to develop metabolic disease. We recently showed that C57BL6, 129X1, DBA/2 and FVB/N mice are all susceptible to high-fat diet-induced glucose intolerance, while BALB/c mice are relatively protected, despite changes in many factors linked with insulin resistance. One parameter strongly linked with insulin resistance is ectopic lipid accumulation, especially metabolically active ceramides and diacylglycerols (DAG). This study examined diet-induced changes in the skeletal muscle lipidome across these five mouse strains. High-fat feeding increased total muscle triacylglycerol (TAG) content, with elevations in similar triacylglycerol species observed for all strains. There were also generally consistent changes across strains in the abundance of different phospholipid (PL) classes and the fatty acid profile of phospholipid molecular species, with the exception being a strain-specific difference in phospholipid species containing two polyunsaturated fatty acyl chains in BALB/c mice (i.e. a diet-induced decrease in the other four strains, but no change in BALB/c mice). In contrast to TAG and PL, the high-fat diet had a minor influence on DAG and ceramide species across all strains. These results suggest that widespread alterations in muscle lipids are unlikely a major contributors to the favourable metabolic profile of BALB/c mice and rather there is a relatively conserved high-fat diet response in muscle of most mouse strains.

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.

The timing and nature of hunter-gather exploitation of tropical rainforests is a topic of ongoing debate. In contrast to most other tropical regions, permanent settlement in Australian rainforests developed much later, and in the absence of adjacent agricultural economies. Here we explore how the tropical rainforests of northern Queensland were exploited during the late Holocene through an ancient starch and phytolith record spanning the last 2000 years. Sequences at the two sites under study – Urumbal Pocket (a ‘Eucalyptus pocket’ surrounded by rainforest) and Goddard Creek (within the rainforest) – indicate a human presence since the early Holocene, coincident with the re-establishment of rainforest in the region. Toxic starchy nuts and the associated complex processing underpinned permanent settlement. Using a geometric morphometric approach to starch analysis, a range of economic starch producing plant species were identified including Endiandra palmerstonii, E. insignis, Lasjia whelani and Beilschmiedia bancroftii in the Urumbal pocket sequence. The phytolith record shows that Urumbal Pocket has been a ‘Eucalyptus pocket’ for at least the last 2000 years, the open nature of the vegetation maintained by regular burning. Goddard Creek, on the other hand has been closed forest, with a changing profile as fire was used more frequently over time. The starch and phytolith sequence provide a unique insight into the local history of these rainforest archaeological sites, with a record that can be viewed against the backdrop of regional sequences documenting climatic and environmental patterns during the late Holocene.

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-receptorrelated 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.

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.

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.

Background: AS160 regulates insulin-sensitive glucose transport in adipocytes. Results: Knockdown of the AS160 substrate Rab10 differentially affected Glut4 and LRP1 in adipocytes and fibroblasts. Conclusion: Glut4 and LRP1 are sorted from a Rab10-independent constitutive trafficking pathway into a highly regulated Rab10-dependent pathway in adipocytes. Significance: Rab10 is a marker for the specialized insulin-sensitive pathway in adipocytes and an AS160 substrate that limits exocytosis through this pathway.

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 fromthe cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites.

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.

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.

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.

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% (k en = 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 (k ex = 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 (k seq) 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.