Understanding and scaffolding Danish schoolteachers' motivation for using classroom-based physical activity: study protocol for a mixed methods study.

INTRODUCTION: The benefits of physical activity for children's health, both mental and physical, and its positive effects on academic achievement are well established. Research also emphasises that schools could provide a natural setting for regular physical activity. There is, however, a limited amount of knowledge about teachers' views when it comes to integrating physical activity as part of teaching. The aim of this study is to understand teachers' motivation for integrating physical activity as part of teaching and to assess their need for guidance and support. METHODS AND ANALYSIS: The study uses an explanatory sequential mixed-methods design. Schools from across Denmark are included in the sample. The design comprises two separated phases-a quantitative and qualitative phase. The quantitative phase is guided by the self-determination theory where teachers' motivation will be measured using the Work Task Motivation Scale for Teachers. The theory of scaffolding guides the qualitative phase, which consists of in-depth interviews with participants selected from the quantitative phase based on levels of motivation and on demographic information. In accordance with the study aims, the analysis of data will identify teachers' internal and external levels of motivation. The purpose of the qualitative phase is to enhance understanding of teachers' motivation and of their need for support in the use of physical activity in teaching. ETHICS AND DISSEMINATION: All relevant ethics approvals have been acquired. All participants in this study will provide written informed consent prior to data collection. All data emerging from the quantitative and qualitative phase will be anonymised for analysis. Ethics approval was requested from the Regional Committee on Health Research Ethics for Southern Denmark approval ID S-20162000-40 and the Danish Data Protection Agency approval ID 16/15491). The study was deemed not notifiable by both authorities. TRIAL REGISTRATION NUMBER: NCT02894346; Pre-results.

Body position influences arterial occlusion pressure: implications for the standardization of pressure during blood flow restricted exercise.

PURPOSE: Arterial occlusion pressure (AOP) measured in a supine position is often used to set cuff pressures for blood flow restricted exercise (BFRE). However, supine AOP may not reflect seated or standing AOP, thus potentially influencing the degree of occlusion. The primary aim of the study was to investigate the effect of body position on AOP. A secondary aim was to investigate predictors of AOP using wide and narrow cuffs. METHODS: Twenty-four subjects underwent measurements of thigh circumference, skinfold and blood pressure, followed by assessments of thigh AOP in supine and seated positions with a wide and a narrow cuff, respectively, using Doppler ultrasound. RESULTS: In the supine position, AOP was 148 ± 19 and 348 ± 94 mmHg with the wide and narrow cuff, respectively. This increased to 177 ± 20 and 409 ± 101 mmHg in the seated position, with correlations between supine and seated AOP of R 2 = 0.81 and R 2 = 0.50 for the wide and narrow cuff, respectively. For both cuff widths, thigh circumference constituted the strongest predictor of AOP, with diastolic blood pressure explaining additional variance with the wide cuff. The predictive strength of these variables did not differ between body positions. CONCLUSION: Our results indicate that body position strongly influences lower limb AOP, especially with narrow cuffs, yielding very high AOP (≥ 500-600 mmHg) in some subjects. This should be taken into account in the standardization of cuff pressures used during BFRE to better control the physiological effects of BFRE.

Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues.

BACKGROUND: Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio. METHODOLOGY/PRINCIPAL FINDINGS: A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed. CONCLUSIONS/SIGNIFICANCE: Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.

Agonism and antagonism at the insulin receptor.

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B'29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1-10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints ((3)H-thymidine incorporation), and not on metabolic endpoints ((14)C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity ((3)H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B'29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.

Insight into the molecular basis for the kinetic differences between the two insulin receptor isoforms.

More than 20 years after the description of the two IR (insulin receptor) isoforms, designated IR-A (lacking exon 11) and IR-B (with exon 11), nearly every functional aspect of the alternative splicing both in vitro and in vivo remains controversial. In particular, there is no consensus on the precise ligand-binding properties of the isoforms. Increased affinity and dissociation kinetics have been reported for IR-A in comparison with IR-B, but the opposite results have also been reported. These are not trivial issues considering the reported possible increased mitogenic potency of IR-A, and the reported link between slower dissociation and increased mitogenesis. We have re-examined the ligand-binding properties of the two isoforms using a novel rigorous mathematical analysis based on the concept of a harmonic oscillator. We found that insulin has 1.5-fold higher apparent affinity towards IR-A and a 2-fold higher overall dissociation rate. Analysis based on the model showed increased association (3-fold) and dissociation (2-fold) rate constants for binding site 1 of IR in comparison with IR-B. We also provide a structural interpretation of these findings on the basis of the structure of the IR ectodomain and the proximity of the sequence encoded by exon 11 to the C-terminal peptide that is a critical trans-component of site 1.

Structural basis of allosteric ligand-receptor interactions in the insulin/relaxin peptide family: implications for other receptor tyrosine kinases and G-protein-coupled receptors.

The insulin/relaxin superfamily of peptide hormones comprises 10 members in humans. The three members of the insulin-related subgroup bind to receptor tyrosine kinases (RTKs), while four of the seven members of the relaxin-like subgroup are now known to bind to G-protein-coupled receptors (GPCRs), the so-called relaxin family peptide receptors (RXFPs). Both systems have a long evolutionary history and play a critical role in fundamental biological processes, such as metabolism, growth, survival and longevity, and reproduction. The structural biology and ligand-binding kinetics of the insulin and insulin-like growth factor I receptors have been studied in great detail, culminating in the recent crystal structure of the insulin receptor extracellular domain. Some of the fundamental properties of these receptors, including constitutive dimerization and negative cooperativity, have recently been shown to extend to other RTKs and GPCRs, including RXFPs, confirming kinetic observations made over 30 years ago.

Dimerization and negative cooperativity in the relaxin family peptide receptors.

Peptides of the relaxin family bind to the relaxin family peptide receptors or RXFPs, members of the G-protein-coupled receptor (GPCR) superfamily. For many years, ligand binding to GPCRs was thought to take place as monomeric complexes, ignoring early evidence of negative cooperativity. However, recent research has shown that most GPCRs form constitutive dimers or larger oligomers. The connection between dimerization and negative cooperativity has now been shown for several GPCRs, including the thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone receptors, which like RXFP1 and -2 belong to the leucine-rich repeat-containing subgroup of class A GPCRs. We recently demonstrated homodimerization and negative cooperativity for RXFP1 and RXFP2 as well as their heterodimerization. Another study showed that RXFP1 has to homodimerize in order to be transported from the endoplasmic reticulum to the cell membrane.