Proteasomal modulation of cellular SNAT2 (SLC38A2) abundance and function by unsaturated fatty acid availability.

Expression and activity of the System A/SNAT2 (SLC38A2) amino acid transporter is up-regulated by amino acid starvation and hypertonicity by a mechanism dependent on both ATF4-mediated transcription of the SLC38A2 gene and enhanced stabilization of SNAT2 itself, which forms part of an integrated cellular stress response to nutrient deprivation and osmotic stress. Here we demonstrate that this adaptive increase in System A function is restrained in cells subjected to prior incubation with linoleic acid (LOA, an unsaturated C18:2 fatty acid) for 24 h. While fatty acid treatment had no detectable effect upon stress-induced SNAT2 or ATF4 gene transcription, the associated increase in SNAT2 protein/membrane transport activity were strongly suppressed in L6 myotubes or HeLa cells preincubated with LOA. Cellular ubiquitination of many proteins was increased by LOA and although the fatty acid-induced loss of SNAT2 could be attenuated by proteasomal inhibition, the functional increase in System A transport activity associated with amino acid starvation/hypertonicity that depends upon processing/maturation and delivery of SNAT2 to the cell surface could not be rescued. LOA up-regulated cellular expression of Nedd4.2, an E3-ligase implicated in SNAT2 ubiquitination, but shRNA-directed Nedd4.2 gene silencing could not curb fatty acid-induced loss of SNAT2 adaptation. However, expression of SNAT2 in which seven putative lysyl-ubiquitination sites in the cytoplasmic N-terminal domain were mutated to alanine protected SNAT2 against LOA-induced proteasomal degradation. Collectively, our findings indicate that increased availability of unsaturated fatty acids can compromise the stress-induced induction/adaptation in SNAT2 expression and function by promoting its degradation via the ubiquitin-proteasome system.

A Randomised Controlled Trial of Treatment for Post-Stroke Homonymous Hemianopia: Screening and Recruitment.

The authors report the screening process and recruitment figures for the VISION (Visual Impairment in Stroke; Intervention Or Not) trial. This is a prospective, randomised, single-blinded, three-arm controlled trial in 14 UK acute hospital stroke units. Stroke teams identified stroke survivors suspected as having homonymous hemianopia. Interventions included Fresnel prisms versus visual search training versus standard care (information only). Primary outcome was change in visual field assessment from baseline to 26 weeks. Secondary measures included change in quality-of-life questionnaires. Recruitment opened in May 2011. A total of 1171 patients were screened by the local principal investigators. Of 1171 patients, 178 (15.2%) were eligible for recruitment: 87 patients (7.4%) provided consent and were recruited; 91 patients (7.8%) did not provide consent, and 993 of 1171 patients (84.8%) failed to meet the eligibility criteria. Almost half were excluded due to complete/partial recovery of hemianopia (43.6%; n = 511). The most common ineligibility reason was recovery of hemianopia. When designing future trials in this area, changes in eligibility criteria/outcome selection to allow more patients to be recruited should be considered, e.g., less stringent levels of visual acuity/refractive error. Alternative outcomes measurable in the home environment, rather than requiring hospital attendance for follow-up, could facilitate increased recruitment.

CDK7 is a component of the integrated stress response regulating SNAT2 (SLC38A2)/System A adaptation in response to cellular amino acid deprivation.

Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/ß have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency.

Effects of Sodium and Amino Acid Substrate Availability upon the Expression and Stability of the SNAT2 (SLC38A2) Amino Acid Transporter.

The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AAs) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis, and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that while AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (N-methylaminoisobutyric acid and glutamine), but not non-substrates. This stabilization and substrate-induced repression were critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteasome system), because "grafting" this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.

Study protocol for a randomised controlled trial of insulin delivery by continuous subcutaneous infusion compared to multiple daily injections.

BACKGROUND: Intensive insulin therapy with continuous subcutaneous insulin infusion (CSII) devices or multiple daily injections (MDI) reduces the risk of long-term vascular complications of type I diabetes (TID). Both treatments are used routinely, but there is little evidence to demonstrate superiority of either treatment. If CSII treatment reduces the risk of long-term complications or is associated with an improved quality of life (QoL), the additional cost of this therapy may be compensated for by a reduction in long-term health expenditure. If there is no demonstrable difference between treatments, health-care resources may be better invested elsewhere. This study aims to address this gap in knowledge. METHODS/DESIGN: This is a pragmatic, randomised controlled trial (RCT). Fifteen centres, selected to represent a population with a broad demographic, will recruit 316 patients, newly diagnosed with TID, aged between 7 months and 15 years. Exclusion criteria include additional pathologies or treatments likely to affect glycaemic control and a first-degree relative with TID. Randomisation to CSII or MDI is stratified for age, gender and recruiting centre. The randomised treatment starts within 15 days of diagnosis. Patients will be trained to adjust their insulin dose according to carbohydrate intake and blood glucose level. Study visits coincide with routine clinic appointments at 3, 6, 9 and 12 months when data relating to routine clinical assessments, adverse events and concomitant medications are collected. Health utilities questionnaires are completed at each visit and a diabetes-specific QoL questionnaire (PedsQL) at diagnosis, 6 and 12 months. The primary outcome is glycaemic control (HbA1c) at 12 months. Secondary outcome measures include QoL, insulin use, growth and weight gain, adverse events and a health economics appraisal. DISCUSSION: This is the first adequately powered RCT comparing CSII and MDI in a non-selected population, treated according to standard practice guidelines. It will produce data that are meaningful to individual patients and local and national policymakers. TRIAL REGISTRATION: The study was registered with the European Clinical Trials Database on 4 November 2010, reference 2010-023792-25.

SNAT2 transceptor signalling via mTOR: a role in cell growth and proliferation?

We have investigated the effect of chronic competitive inhibition of SNAT2 (System A) amino acid (AA) transport, induced by incubation with a saturating dose of a non-metabolisable System A amino acid analogue (Me-AIB), on growth and proliferation of MCF-7 human breast cancer cells in complete culture medium. These cells express Na+- and pH-dependent SNAT2 AA transport and a saturating concentration of Me-AIB (10 mM) competitively inhibits (>90%) AA uptake via SNAT2. Incubation with Me-AIB for up to 5 days progressively reduced cell proliferation (~2-fold) and depleted intracellular concentrations of not only SNAT2 AA substrates but of essential branched chain AAs (e.g. leucine). Surprisingly, total cellular protein was maintained and cells subjected to chronic Me-AIB incubation exhibited a detectable increase in cell size. Analysis of mTOR signalling revealed that, despite a substantial reduction in size of the intracellular AA pool, Me-AIB elevated mTOR-dependent p70S6K1 phosphorylation. Proteomic analysis of TAP-tag purified SNAT2 fusion proteins identified two novel SNAT2-interacting proteins that may potentially function in conjunction with the SNAT2 transceptor to regulate signalling pathways influencing protein turnover and cell growth.

Distinct sensor pathways in the hierarchical control of SNAT2, a putative amino acid transceptor, by amino acid availability.

Mammalian nutrient sensors are novel targets for therapeutic intervention in disease states such as insulin resistance and muscle wasting; however, the proteins responsible for this important task are largely uncharacterized. To address this issue we have dissected an amino acid (AA) sensor/effector regulon that controls the expression of the System A amino acid transporter SNAT2 in mammalian cells, a paradigm nutrient-responsive process, and found evidence for the convergence of at least two sensor/effector pathways. During AA withdrawal, JNK is activated and induces the expression of SNAT2 in L6 myotubes by stimulating an intronic nutrient-sensitive domain. A sensor for large neutral AA (e.g. Tyr, Gln) inhibits JNK activation and SNAT2 up-regulation. Additionally, shRNA and transporter chimeras demonstrate that SNAT2 provides a repressive signal for gene transcription during AA sufficiency, thus echoing AA sensing by transceptor (transporter-receptor) orthologues in yeast (Gap1/Ssy1) and Drosophila (PATH). Furthermore, the SNAT2 protein is stabilized during AA withdrawal.