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Abstract Professional advisory groups, with patient and public involvement (PPI) representatives, can be used for co-production within research projects. This paper aims to document the benefits and challenges of undertaking stakeholder and participant engagement for an implementation research project within NHS (National Health Service) Wales. A patient focused research project, initiated by clinicians, on the use of patient empowerment tools within standard patient care, used a professional advisory group to identify appropriate tools to use within the research proposal. The professional advisory group was made up of therapists, NHS stakeholders, academics and PPI representatives. A hybrid-meeting style was employed to optimise participation for all members of the group. Benefits of the professional advisory group included increased engagement and ownership of the study due to co-creation, and obtaining important contextual information and lived experience. However, challenges included keeping the discussion on topic due to pre-conceived agendas, pleasing everyone in the room due to varied backgrounds, and technological issues. Future professional advisory groups should consider how to facilitate the full involvement of PPI representatives within the discussion and having a variety of resources to present the topic of discussion. Furthermore, clearly communicating what the purpose and direction of the research project is and how it fits into the wider system, should be carefully considered. Overall, it was recognised that the professional advisory group was of significant value to shape the research proposal. Due to the situational challenges faced by healthcare professional within the NHS and preconceived ideas for solutions, it is hoped that by involving stakeholders early in the process there will be greater acceptance and usability of the research findings.

Self-management of long-term conditions requires health professionals to understand and develop capabilities that empower the population they serve. A rapid evidence review was undertaken to assess the current evidence based on the psychometric properties of patient empowerment tools. MEDLINE was searched, and data were extracted for each publication and scored using a modified Psychometric and Pragmatic Evidence Rating Scale (PAPERS) evidence rating scale. The results were grouped into the following domains: (a) health literacy; (b) patient activation; (c) long-term conditions; (d) self-management needs and behaviors. A full-text review of 65 publications led to the inclusion of 29 primary studies. The highest scoring tools were selected with respect to performance for each domain: (a) Newest Vital Sign and the Brief Health Literacy Screen; (b) Consumer Health Activation Index and PAM-13; (c) LTCQ and LTCQ8; and (d) SEMCD and Patient Enablement Instrument. PAPERS was a useful tool in determining the generalizability, validity, and reliability of these patient empowerment tools. However, further research is required to establish whether an individual's health literacy status influences patient empowerment tool outcomes.

Abstract The regulated translocation of the glucose transporter, GLUT4, to the surface of adipocytes and muscle is a key action of insulin. This is underpinned by the delivery and fusion of GLUT4-containing vesicles with the plasma membrane. Recent studies have revealed that a further action of insulin is to mediate the dispersal of GLUT4 molecules away from the site of GLUT4 vesicle fusion with the plasma membrane. Although shown in adipocytes, whether insulin-stimulated dispersal occurs in other cells and/or is exhibited by other proteins remains a matter of debate. Here we show that insulin stimulates GLUT4 dispersal in the plasma membrane of adipocytes, induced pluripotent stem cell-derived cardiomyocytes and HeLa cells, suggesting that this phenomenon is specific to GLUT4 expressed in all cell types. By contrast, insulin-stimulated dispersal of TfR was not observed in HeLa cells, suggesting that the mechanism may be unique to GLUT4. Consistent with dispersal being an important physiological mechanism, we observed that insulin-stimulated GLUT4 dispersal is reduced under conditions of insulin resistance. Adipocytes of different sizes have been shown to exhibit distinct metabolic properties: larger adipocytes exhibit reduced insulin-stimulated glucose transport compared to smaller cells. Here we show that both GLUT4 delivery to the plasma membrane and GLUT4 dispersal are reduced in larger adipocytes, supporting the hypothesis that larger adipocytes are refractory to insulin challenge compared to their smaller counterparts, even within a supposedly homogeneous population of cells.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the ‘insulin responsive vesicles’ are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro. Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.

A major consequence of insulin binding its receptor on fat and muscle cells is the stimulation of glucose transport into these tissues. This is achieved through an increase in the exocytic trafficking rate of the facilitative glucose transporter GLUT4 from intracellular stores to the cell surface. Delivery of GLUT4 to the cell surface requires the formation of functional SNARE complexes containing Syntaxin 4, SNAP23, and VAMP2. Insulin stimulates the formation of these complexes and concomitantly causes phosphorylation of Syntaxin 4. Here, we use a combination of biochemistry and cell biological approaches to provide a mechanistic link between these observations. We present data to support the hypothesis that Tyr-115 and Tyr-251 of Syntaxin 4 are direct substrates of activated insulin receptors, and that these residues modulate the protein’s conformation and thus regulate the rate at which Syntaxin 4 forms SNARE complexes that deliver GLUT4 to the cell surface. This report provides molecular details on how the cell regulates SNARE-mediated membrane traffic in response to an external stimulus.

Translational research should examine racism and bias and improve health equity. We designed and implemented a course for the Master of Science in Clinical Investigation program of the Northwestern University Clinical and Translational Sciences Institute. We describe curriculum development, content, outcomes, and revisions involving 36 students in 2 years of “Anti-Racist Strategies for Clinical and Translational Science.” Ninety-six percent of students reported they would recommend the course. Many reported changes in research approaches based on course content. A course designed to teach anti-racist research design is feasible and has a positive short-term impact on learners.

Adipocytes play multiple roles in the regulation of glucose metabolism which rely on the regulation of membrane traffic. These include secretion of adipokines and serving as an energy store. Central to their energy storing function is the ability to increase glucose uptake in response to insulin, mediated through translocation of the facilitative glucose transporter GLUT4 to the cell surface. The trans-Golgi reticulum localized SNARE protein syntaxin 16 (Sx16) has been identified as a key component of the secretory pathway required for insulin-regulated trafficking of GLUT4. We used CRISPR/Cas9 technology to generate 3T3-L1 adipocytes lacking Sx16 to understand the role of the secretory pathway on adipocyte function. GLUT4 mRNA and protein levels were reduced in Sx16 knockout adipocytes and insulin stimulated GLUT4 translocation to the cell surface was reduced. Strikingly, neither basal nor insulin-stimulated glucose transport were affected. By contrast, GLUT1 levels were upregulated in Sx16 knockout cells. Levels of sortilin and insulin regulated aminopeptidase were also increased in Sx16 knockout adipocytes which may indicate an upregulation of an alternative GLUT4 sorting pathway as a compensatory mechanism for the loss of Sx16. In response to chronic insulin stimulation, Sx16 knockout adipocytes exhibit elevated insulin-independent glucose transport and significant alterations in lactate metabolism. We further show that the adipokine secretory pathways are impaired in Sx16 knockout cells. Together this demonstrates a role for Sx16 in the control of glucose transport, the response to elevated insulin, cellular metabolic profiles and adipocytokine secretion.

Abstract Introduction The regulated delivery of the glucose transporter GLUT4 from intracellular stores to the plasma membrane underpins insulin‐stimulated glucose transport. Insulin‐stimulated glucose transport is impaired in skeletal muscle of patients with type‐2 diabetes, and this may arise because of impaired intracellular trafficking of GLUT4. However, molecular details of any such impairment have not been described. We hypothesized that GLUT4 and/or levels of proteins involved in intracellular GLUT4 trafficking may be impaired in skeletal muscle in type‐2 diabetes and tested this in obese individuals without and without type‐2 diabetes. Methods We recruited 12 participants with type‐2 diabetes and 12 control participants. All were overweight or obese with BMI of 25–45 kg/m2. Insulin sensitivity was measured using an insulin suppression test (IST), and vastus lateralis biopsies were taken in the fasted state. Cell extracts were immunoblotted to quantify levels of a range of proteins known to be involved in intracellular GLUT4 trafficking. Results Obese participants with type‐2 diabetes exhibited elevated fasting blood glucose and increased steady state glucose infusion rates in the IST compared with controls. Consistent with this, skeletal muscle from those with type‐2 diabetes expressed lower levels of GLUT4 (30%, p = .014). Levels of Syntaxin4, a key protein involved in GLUT4 vesicle fusion with the plasma membrane, were similar between groups. By contrast, we observed reductions in levels of Syntaxin16 (33.7%, p = 0.05), Sortilin (44%, p = .006) and Sorting Nexin‐1 (21.5%, p = .039) and −27 (60%, p = .001), key proteins involved in the intracellular sorting of GLUT4, in participants with type‐2 diabetes. Conclusions We report significant reductions of proteins involved in the endosomal trafficking of GLUT4 in skeletal muscle in obese people with type 2 diabetes compared with age‐ and weight‐matched controls. These abnormalities of intracellular GLUT4 trafficking may contribute to reduced whole body insulin sensitivity.

Summary: Fluorescent biosensors are powerful tools allowing the concentration of metabolites and small molecules, and other properties such as pH and molecular crowding to be measured inside live single cells. The technology has been hampered by lack of simple software to identify cells and quantify biosensor signals in single cells. We have developed a new software package, FRETzel, to address this gap and demonstrate its use by measuring insulin-stimulated glucose uptake in individual fat cells of varying sizes for the first time. Our results support the long-standing hypothesis that larger fat cells are less sensitive to insulin than smaller ones, a finding that has important implications for the battle against type 2 diabetes. FRETzel has been optimized using the messy and crowded environment of cultured adipocytes, demonstrating its utility for quantification of FRET biosensors in a wide range of other cell types, including fibroblasts and yeast via a simple user-friendly quantitative interface.

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.

With 2.3 million individuals incarcerated in the United States, incarceration remains a pressing social influence on health. While risk factors for incarceration are known, research has been slow to identify protective factors. Characterizing adolescent protective and risk factors for incarceration outcomes can inform interventions to prevent incarceration. Using survey data from the National Longitudinal Study of Adolescent to Adult Health, we tested the influence of adolescent risk and protective factors for incarceration of young people. We used ordinal logistic regressions, investigating the relationship between adolescent characteristics (wave I, grades 7–12) with two outcomes: incarceration onset (no incarceration, onset of incarceration as a juvenile, and onset of incarceration as an adult) and incarceration frequency (never incarcerated, incarcerated once, incarcerated more than once) during adolescence and into adulthood (N = 12,136, wave IV, ages 24–32). Adolescent protective factors against incarceration onset and higher incarceration frequency centered on education, including a higher grade point average and a higher likelihood of having future plans to attend college. Risk factors included disruptive behavior, physical or sexual abuse in childhood, parental incarceration, and living with a stepfather figure. Stratified analyses suggested that Latinos may have unique risk profiles compared to African American and White youth. The educational system may be a locus for protective interventions promoting academic achievement and educational aspirations. Understanding both incarceration risk and protective factors may allow interventions to be tailored appropriately.

Glucose transporter 4 (GLUT4) is sequestered inside muscle and fat and then released by vesicle traffic to the cell surface in response to postprandial insulin for blood glucose clearance. Here, we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively secreted GLUT1 transporter and localize CHC22 to the ER-to-Golgi intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4, and sortilin, and downregulation of either p115 or CHC22, but not GM130 or sortilin, abrogates insulin-responsive GLUT4 release. This indicates that CHC22 traffic initiates human GLUT4 sequestration from the ERGIC and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.

Self-management of long-term conditions requires health professionals to understand and develop capabilities that empower the population they serve. A rapid evidence review was undertaken to assess the current evidence based on the psychometric properties of patient empowerment tools. MEDLINE was searched, and data were extracted for each publication and scored using a modified Psychometric and Pragmatic Evidence Rating Scale (PAPERS) evidence rating scale. The results were grouped into the following domains: (a) health literacy; (b) patient activation; (c) long-term conditions; (d) self-management needs and behaviors. A full-text review of 65 publications led to the inclusion of 29 primary studies. The highest scoring tools were selected with respect to performance for each domain: (a) Newest Vital Sign and the Brief Health Literacy Screen; (b) Consumer Health Activation Index and PAM-13; (c) LTCQ and LTCQ8; and (d) SEMCD and Patient Enablement Instrument. PAPERS was a useful tool in determining the generalizability, validity, and reliability of these patient empowerment tools. However, further research is required to establish whether an individual's health literacy status influences patient empowerment tool outcomes. [ABSTRACT FROM AUTHOR]

Adipocytes are key to metabolic regulation, exhibiting insulinstimulated glucose transport that is underpinned by the insulinstimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasmamembranewhere they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similarmechanism.We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ~50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulinstimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells. [ABSTRACT FROM AUTHOR]