System-L amino acid transporters play a key role in pancreatic b-cell signalling and function

The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influences β-cell signalling and function. In this report we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L transporter LAT1 is abundantly expressed in islets and that knock-down of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the System-L transporter LAT1 is required for regulating β-cell signaling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type-2 diabetes

pVAC-Seq: A genome-guided in silico approach to identifying tumor neoantigens

Cancer immunotherapy has gained significant momentum from recent clinical successes of checkpoint blockade inhibition. Massively parallel sequence analysis suggests a connection between mutational load and response to this class of therapy. Methods to identify which tumor-specific mutant peptides (neoantigens) can elicit anti-tumor T cell immunity are needed to improve predictions of checkpoint therapy response and to identify targets for vaccines and adoptive T cell therapies. Here, we present a flexible, streamlined computational workflow for identification of personalized Variant Antigens by Cancer Sequencing (pVAC-Seq) that integrates tumor mutation and expression data (DNA- and RNA-Seq). pVAC-Seq is available a

Effect of the pharmacist-managed cardiovascular risk reduction services on diabetic retinopathy outcome measures

Background: Diabetic retinopathy (DR) is a progressive, sight-threatening long-term complication of diabetes. Diabetes disease management reduces the risk of developing or progression to a severe form of DR. However, there are no reports of the potential role of pharmacists in DR progression. Objective: For this study, we performed a retrospective data analysis of patients with diabetes seen at cardiovascular risk reduction services provided by pharmacists with an objective to determine the potential role of pharmacists in the DR progression. These services involve pharmacists working in collaborative drug therapy management (CDTM), using a collaborative practice agreement (CPA) with primary care physicians. Methods: Patient records and ophthalmological notes were collected for 317 individuals seen by the pharmacists (intervention group) and 320 individuals seen only by a physician (control). Results: Statistical analysis was performed on 148 individuals in an intervention group and 120 individuals in the control group for which complete records were available. Retinopathy progression remained stable in 89.6 % of individuals in the intervention group compared to 87.9% in the control group. Moreover, the relative risk of retinopathy progressing to a severe form was 1.17 for the control group compared the intervention group. Conclusions: Our studies provide a proof-of-concept that pharmacists-managed care possesses a potential role in protection from DR, and paves a way for future pharmacists managed care with an emphasis on reducing diabetic complications

Metformin reduces airway glucose permeability and hyperglycaemia-induced Staphylococcus aureus load independently of effects on blood glucose

Background Diabetes is a risk factor for respiratory infection, and hyperglycaemia is associated with increased glucose in airway surface liquid and risk of Staphylococcus aureus infection. Objectives To investigate whether elevation of basolateral/blood glucose concentration promotes airway Staphylococcus aureus growth and whether pretreatment with the antidiabetic drug metformin affects this relationship. Methods Human airway epithelial cells grown at air–liquid interface (±18 h pre-treatment, 30 μM–1 mM metformin) were inoculated with 5×105 colony-forming units (CFU)/cm2 S aureus 8325-4 or JE2 or Pseudomonas aeruginosa PA01 on the apical surface and incubated for 7 h. Wild-type C57BL/6 or db/db (leptin receptor-deficient) mice, 6–10 weeks old, were treated with intraperitoneal phosphate-buffered saline or 40 mg/kg metformin for 2 days before intranasal inoculation with 1×107 CFU S aureus. Mice were culled 24 h after infection and bronchoalveolar lavage fluid collected. Results Apical S aureus growth increased with basolateral glucose concentration in an in vitro airway epithelia–bacteria co-culture model. S aureus reduced transepithelial electrical resistance (RT) and increased paracellular glucose flux. Metformin inhibited the glucose-induced growth of S aureus, increased RT and decreased glucose flux. Diabetic (db/db) mice infected with S aureus exhibited a higher bacterial load in their airways than control mice after 2 days and metformin treatment reversed this effect. Metformin did not decrease blood glucose but reduced paracellular flux across ex vivo murine tracheas. Conclusions Hyperglycaemia promotes respiratory S aureus infection, and metformin modifies glucose flux across the airway epithelium to limit hyperglycaemia-induced bacterial growth. Metformin might, therefore, be of additional benefit in the prevention and treatment of respiratory infection

The catalytic subunit of the system L1 amino acid transporter (S<i>lc7a5</i>) facilitates nutrient signalling in mouse skeletal muscle

The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass

Thioredoxin Interacting Protein Is Required for a Chronic Energy-Rich Diet to Promote Intestinal Fructose Absorption

Increased consumption of fats and added sugars has been associated with an increase in metabolic syndromes. Here we show that mice chronically fed an energy-rich diet (ERD) with high fat and moderate sucrose have enhanced the absorption of a gastrointestinal fructose load, and this required expression of the arrestin domain protein Txnip in the intestinal epithelial cells. ERD feeding induced gene and protein expression of Glut5, and this required the expression of Txnip. Furthermore, Txnip interacted with Rab11a, a small GTPase that facilitates the apical localization of Glut5. We also demonstrate that ERD promoted Txnip/Glut5 complexes in the apical intestinal epithelial cell. Our findings demonstrate that ERD facilitates fructose absorption through a Txnip-dependent mechanism in the intestinal epithelial cell, suggesting that increased fructose absorption could potentially provide a mechanism for worsening of metabolic syndromes in the setting of a chronic ERD

Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A

International audienceAims/Hypothesis: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA), whereas mono and polyunsaturated fatty acids (MUFA and PUFA) not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A). This study investigated the effects of oleic acid (OA; a MUFA), linoleic acid (LOA; a PUFA) and palmitate (PA; a SFA) in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. Principal Findings: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt-and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine 307 phosphorylation-events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A.Conclusions/Interpretation: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A

Obesity-Related Upregulation of Monocyte Chemotactic Factors in Adipocytes : Involvement of Nuclear Factor-κB and c-Jun NH2-Terminal Kinase Pathways

OBJECTIVE—We sought to evaluate the entire picture of all monocyte chemotactic factors that potentially contribute to adipose tissue macrophage accumulation in obesity

Evaluation of the groundnut model PNUTGRO for crop response to water availability, sowing dates, and seasons

Field experiments were conducted during the 1987, 1991 and 1992 rainy seasons at Patancheru (latitude 17°32′N; longitude 78°16′E; elevation 545 m), Andhra Pradesh, India, to collect data to test and validate the hedgerow version of the groundnut model PNUTGRO for predicting phenological development, light interception, canopy growth, dry matter production, pod and seed yields of groundnut (Arachis hypogaea L.) as influenced by row spacing and plant population. The model was calibrated using the crop growth and phenology data of groundnut (cv. Robut 33-1) obtained from the 1987 and 1991 rainy season experiments. In these experiments groundnut was grown at plant populations ranging from 5 to 45 plants/m2 with and without irrigation. Changes were made in the cultivar-specific coefficients related to the light penetration into the crop canopy and dry matter production. The model was validated against independent data obtained from a 1992 rainy season experiment. In 1992, groundnut was grown at plant populations ranging from 10 to 40 plants/m2 and at row spacings of 20, 30 and 60 cm. The model predicted the occurrence of vegetative and reproductive stages, canopy development, total dry matter production and its partitioning to pods and seed accurately. Maximum leaf area index observed during the season was significantly correlated with simulated values (r2 = 0.95). In spite of some incidence of diseases and pests, the correlation between simulated and observed pod yield was significant (r2 = 0.61). It is concluded from this study that the hedgerow version of the groundnut model PNUTGRO can be used to quantify groundnut growth and yields as influenced by plant population and row spacing

Comparing mutation calls in fixed tumour samples between the Affymetrix OncoScan® Array and PCR based next-generation sequencing

Background: The importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® Array platform is designed to produce actionable mutation calls in archival material. Methods: We compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls. Results: A small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5–10%) than product claims indicated. Conclusions: This benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results