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Aims: To describe the process and outputs of a workshop convened to identify key priorities for future research in the area of diabetes and physical activity and provide recommendations to researchers and research funders on how best to address them. Methods: A 1‐day research workshop was conducted, bringing together researchers, people living with diabetes, healthcare professionals, and members of staff from Diabetes UK to identify and prioritise recommendations for future research into physical activity and diabetes. Results: Workshop attendees prioritised four key themes for further research: (i) better understanding of the physiology of exercise in all groups of people: in particular, what patient metabolic characteristics influence or predict the physiological response to physical activity, and the potential role of physical activity in beta cell preservation; (ii) designing physical activity interventions for maximum impact; (iii) promoting sustained physical activity across the life course; (iv) designing physical activity studies for groups with multiple long‐term conditions. Conclusions: This paper outlines recommendations to address the current gaps in knowledge related to diabetes and physical activity and calls on the research community to develop applications in these areas and funders to consider how to stimulate research in these areas. [ABSTRACT FROM AUTHOR]

Although evidence demonstrates the fundamental role of shear stress in vascular health, predominantly through the release of nitric oxide (NO), the mechanisms by which endothelial cells (EC)s sense and transduce shear are poorly understood. In cultured ECs tyrosine phosphorylation of PECAM-1 has been shown to activate eNOS in response to shear stress. However, in the human skeletal muscle microcirculation PECAM-1 was not activated in response to exercise or passive leg movement. Given this contradiction, this study aimed to assess the effect of exercise on conduit artery PECAM-1 and eNOS activation in humans. Eleven males were randomised to two groups; 30 min of handgrip exercise (n = 6), or a time-control group (n = 5). Protein content of eNOS and PECAM-1, alongside eNOS Ser1177 and PECAM-1 Tyr713 phosphorylation were assessed in ECs obtained from the radial artery pre- and post-intervention. Handgrip exercise resulted in a 5-fold increase in mean shear rate in the exercise group, with no change in the control group (group*time, P < 0.001). There was a 54% increase in eNOS Ser1177 phosphorylation in the exercise group, when compared to control group (group*time, P = 0.016), but no change was reported in PECAM-1 Tyr713 phosphorylation in either group (group*time, P > 0.05). eNOS and PECAM-1 protein content were unchanged (group*time, P > 0.05). Our data show that exercise-induced elevations in conduit artery shear rate increase eNOS Ser1177 phosphorylation but not PECAM-1 Tyr713 phosphorylation. This suggests PECAM-1 phosphorylation may not be involved in the vascular response to acute but prolonged elevations in exercise-induced shear rate in conduit arteries of healthy, active men. [ABSTRACT FROM AUTHOR]

We investigated whether 20 candidate single nucleotide polymorphisms (SNPs) were associated with in vivo exercise‐induced muscle damage (EIMD), and with an in vitro skeletal muscle stem cell wound healing assay. Sixty‐five young, untrained Caucasian adults performed 120 maximal eccentric knee‐extensions on an isokinetic dynamometer to induce EIMD. Maximal voluntary isometric/isokinetic knee‐extensor torque, knee joint range of motion (ROM), muscle soreness, serum creatine kinase activity and interleukin‐6 concentration were assessed before, directly after and 48 h after EIMD. Muscle stem cells were cultured from vastus lateralis biopsies from a separate cohort (n = 12), and markers of repair were measured in vitro. Participants were genotyped for all 20 SNPs using real‐time PCR. Seven SNPs were associated with the response to EIMD, and these were used to calculate a total genotype score, which enabled participants to be segregated into three polygenic groups: 'preferential' (more 'protective' alleles), 'moderate', and 'non‐preferential'. The non‐preferential group was consistently weaker than the preferential group (1.93 ± 0.81 vs. 2.73 ± 0.59 N ∙ m/kg; P = 9.51 × 10−4) and demonstrated more muscle soreness (p = 0.011) and a larger decrease in knee joint ROM (p = 0.006) following EIMD. Two TTN‐AS1 SNPs in linkage disequilibrium were associated with in vivo EIMD (rs3731749, p ≤ 0.005) and accelerated muscle stem cell migration into the artificial wound in vitro (rs1001238, p ≤ 0.006). Thus, we have identified a polygenic profile, linked with both muscle weakness and poorer recovery following EIMD. Moreover, we provide evidence for a novel TTN gene‐cell‐skeletal muscle mechanism that may help explain some of the interindividual variability in the response to EIMD. [ABSTRACT FROM AUTHOR]

Key points: Muscle glycogen and intramuscular triglycerides (IMTG, stored in lipid droplets) are important energy substrates during prolonged exercise.Exercise‐induced changes in lipid droplet (LD) morphology (i.e. LD size and number) have not yet been studied under nutritional conditions typically adopted by elite endurance athletes, that is, after carbohydrate (CHO) loading and CHO feeding during exercise.We report for the first time that exercise reduces IMTG content in both central and peripheral regions of type I and IIa fibres, reflective of decreased LD number in both fibre types whereas reductions in LD size were exclusive to type I fibres.Additionally, CHO feeding does not alter subcellular IMTG utilisation, LD morphology or muscle glycogen utilisation in type I or IIa/II fibres.In the absence of alterations to muscle fuel selection, CHO feeding does not attenuate cell signalling pathways with regulatory roles in mitochondrial biogenesis. We examined the effects of carbohydrate (CHO) feeding on lipid droplet (LD) morphology, muscle glycogen utilisation and exercise‐induced skeletal muscle cell signalling. After a 36 h CHO loading protocol and pre‐exercise meal (12 and 2 g kg–1, respectively), eight trained males ingested 0, 45 or 90 g CHO h–1 during 180 min cycling at lactate threshold followed by an exercise capacity test (150% lactate threshold). Muscle biopsies were obtained pre‐ and post‐completion of submaximal exercise. Exercise decreased (P < 0.01) glycogen concentration to comparable levels (∼700 to 250 mmol kg–1 DW), though utilisation was greater in type I (∼40%) versus type II fibres (∼10%) (P < 0.01). LD content decreased in type I (∼50%) and type IIa fibres (∼30%) (P < 0.01), with greater utilisation in type I fibres (P < 0.01). CHO feeding did not affect glycogen or IMTG utilisation in type I or II fibres (all P > 0.05). Exercise decreased LD number within central and peripheral regions of both type I and IIa fibres, though reduced LD size was exclusive to type I fibres. Exercise induced (all P < 0.05) comparable AMPKThr172 (∼4‐fold), p53Ser15 (∼2‐fold) and CaMKIIThr268 phosphorylation (∼2‐fold) with no effects of CHO feeding (all P > 0.05). CHO increased exercise capacity where 90 g h–1 (233 ± 133 s) > 45 g h–1 (156 ± 66 s; P = 0.06) > 0 g h–1 (108 ± 54 s; P = 0.03). In conditions of high pre‐exercise CHO availability, we conclude CHO feeding does not influence exercise‐induced changes in LD morphology, glycogen utilisation or cell signalling pathways with regulatory roles in mitochondrial biogenesis. Key points: Muscle glycogen and intramuscular triglycerides (IMTG, stored in lipid droplets) are important energy substrates during prolonged exercise.Exercise‐induced changes in lipid droplet (LD) morphology (i.e. LD size and number) have not yet been studied under nutritional conditions typically adopted by elite endurance athletes, that is, after carbohydrate (CHO) loading and CHO feeding during exercise.We report for the first time that exercise reduces IMTG content in both central and peripheral regions of type I and IIa fibres, reflective of decreased LD number in both fibre types whereas reductions in LD size were exclusive to type I fibres.Additionally, CHO feeding does not alter subcellular IMTG utilisation, LD morphology or muscle glycogen utilisation in type I or IIa/II fibres.In the absence of alterations to muscle fuel selection, CHO feeding does not attenuate cell signalling pathways with regulatory roles in mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]

Key points: Obesity and sedentary behaviour are associated with capillary rarefaction and impaired muscle microvascular vasoreactivity, due to reduced nitric oxide bioavailability.Low‐volume high‐intensity interval training (HIT) is a time‐efficient alternative to traditional moderate‐intensity continuous training (MICT), but its effect on the muscle microvasculature has not been studied.The applicability of current laboratory‐ and gym‐based HIT protocols for obese individuals with low fitness and mobility has been disputed by public health experts, who cite the strenuous nature and complex protocols as major barriers. Therefore, we developed a virtually supervised HIT protocol targeting this group that can be performed at home without equipment (Home‐HIT).This study is the first to show that 12 weeks of virtually supervised Home‐HIT in obese individuals with elevated cardiovascular disease risk leads to similar increases in capillarisation and eNOS/NAD(P)Hoxidase protein ratio within the muscle microvascular endothelium as virtually supervised home‐based MICT and laboratory‐based HIT, while reducing many of the major barriers to exercise. This study investigated the effect of a novel virtually supervised home‐based high‐intensity interval training (HIT) (Home‐HIT) intervention in obese individuals with elevated cardiovascular disease (CVD) risk on capillarisation and muscle microvascular eNOS/NAD(P)Hoxidase ratio. Thirty‐two adults with elevated CVD risk (age 36 ± 10 years; body mass index 34.3 ± 5 kg m−2; V̇O2 peak 24.6 ± 5.7 ml kg min−1), completed one of three 12‐week training programmes: Home‐HIT (n = 9), laboratory‐based supervised HIT (Lab‐HIT; n = 10) or virtually supervised home‐based moderate‐intensity continuous training (Home‐MICT; n = 13). Muscle biopsies were taken before and after training to assess changes in vascular enzymes, capillarisation, mitochondrial density, intramuscular triglyceride content and GLUT4 protein expression using quantitative immunofluorescence microscopy. Training increased V̇O2 peak (P < 0.001), whole‐body insulin sensitivity (P = 0.033) and flow‐mediated dilatation (P < 0.001), while aortic pulse wave velocity decreased (P < 0.001) in all three groups. Immunofluorescence microscopy revealed comparable increases in total eNOS content in terminal arterioles and capillaries (P < 0.001) in the three conditions. There was no change in eNOS ser1177 phosphorylation (arterioles P = 0.802; capillaries P = 0.311), but eNOS ser1177/eNOS content ratio decreased significantly following training in arterioles and capillaries (P < 0.001). Training decreased NOX2 content (arterioles P < 0.001; capillaries P < 0.001), but there was no change in p47phox content (arterioles P = 0.101; capillaries P = 0.345). All measures of capillarisation increased (P < 0.05). There were no between‐group differences. Despite having no direct supervision during exercise, virtually supervised Home‐HIT resulted in comparable structural and endothelial enzymatic changes in the skeletal muscle microvessels to the traditional training methods. We provide strong evidence that Home‐HIT is an effective novel strategy to remove barriers to exercise and improve health in an obese population at risk of CVD. Key points: Obesity and sedentary behaviour are associated with capillary rarefaction and impaired muscle microvascular vasoreactivity, due to reduced nitric oxide bioavailability.Low‐volume high‐intensity interval training (HIT) is a time‐efficient alternative to traditional moderate‐intensity continuous training (MICT), but its effect on the muscle microvasculature has not been studied.The applicability of current laboratory‐ and gym‐based HIT protocols for obese individuals with low fitness and mobility has been disputed by public health experts, who cite the strenuous nature and complex protocols as major barriers. Therefore, we developed a virtually supervised HIT protocol targeting this group that can be performed at home without equipment (Home‐HIT).This study is the first to show that 12 weeks of virtually supervised Home‐HIT in obese individuals with elevated cardiovascular disease risk leads to similar increases in capillarisation and eNOS/NAD(P)Hoxidase protein ratio within the muscle microvascular endothelium as virtually supervised home‐based MICT and laboratory‐based HIT, while reducing many of the major barriers to exercise. [ABSTRACT FROM AUTHOR]

Key points Skeletal muscle capillary density and vasoreactivity are reduced in obesity, due to reduced nitric oxide bioavailability., Sprint interval training (SIT) has been proposed as a time efficient alternative to moderate-intensity continuous training (MICT), but its effect on the skeletal muscle microvasculature has not been studied in obese individuals., We observed that SIT and MICT led to equal increases in capillarisation and endothelial eNOS content, while reducing endothelial NOX2 content in microvessels of young obese men., We conclude that SIT is equally effective at improving skeletal muscle capillarisation and endothelial enzyme balance, while being a time efficient alternative to traditional MICT., Abstract Sprint interval training (SIT) has been proposed as a time efficient alternative to moderate-intensity continuous training (MICT), leading to similar improvements in skeletal muscle capillary density and microvascular function in young healthy humans. In this study we made the first comparisons of the muscle microvascular response to SIT and MICT in an obese population. Sixteen young obese men (age 25 ± 1 years, BMI 34.8 ± 0.9 kg m−2) were randomly assigned to 4 weeks of MICT (40-60 min cycling at ∼65% [ABSTRACT FROM AUTHOR]

It is becoming increasingly apparent that a high vasodilator response of the skeletal muscle microvasculature to insulin and exercise is of critical importance for adequate muscle perfusion and long-term microvascular and muscle metabolic health. Previous research has shown that a sedentary lifestyle, obesity and ageing lead to impairments in the vasodilator response, while a physically active lifestyle keeps both microvascular density and vasodilator response high. To investigate the molecular mechanisms behind these impairments and the benefits of exercise training interventions, our laboratory has recently developed quantitative immunofluorescence microscopy methods to measure protein content of eNOS and NAD(P)Hoxidase specifically in the endothelial layer of capillaries and arterioles of human skeletal muscle. As eNOS produces nitric oxide (NO) and NAD(P)Hoxidase produces superoxide anions (O2−, quenching NO) we propose that the eNOS/NAD(P)Hoxidase protein ratio is a marker of vasodilator capacity. The novel methods show that endurance training (ET) and high intensity interval training (HIT), generally regarded as a time-efficient alternative to ET, increase eNOS protein content and the eNOS/NADP(H)oxidase protein ratio in previously sedentary lean and obese young men. Resistance exercise training had smaller but qualitatively similar effects. Western blot data of other laboratories suggest that endurance exercise training leads to similar changes in sedentary elderly men. Future research will be required to investigate the relative importance of other sources and tissues in the balance between NO and O2− production seen by the vascular smooth muscle layer of terminal arterioles. [ABSTRACT FROM AUTHOR]

Objective: The effects of RT on muscle mass, strength, and insulin sensitivity are well established, but the underlying mechanisms are only partially understood. The main aim of this study was to investigate whether RT induces changes in endothelial enzymes of the muscle microvasculature, which would increase NO bioavailability and could contribute to improved insulin sensitivity. Methods: Eight previously sedentary males (age 20 ± 0.4 years, BMI 24.5 ± 0.9 kg/m²) completed six weeks of RT 3x/week. Muscle biopsies were taken from the m. vastus lateralis and microvascular density; and endothelial-specific eNOS content, eNOS Ser1177 phosphorylation, and NOX2 content were assessed pre- and post-RT using quantitative immunofluorescence microscopy. Whole-body insulin sensitivity (measured as Matsuda Index), microvascular Kf (functional measure of the total available endothelial surface area), and arterial stiffness (AIx, central, and pPWV) were also measured. Results: Measures of microvascular density, microvascular Kf, microvascular eNOS content, basal eNOS phosphorylation, and endothelial NOX2 content did not change from pre-RT to post-RT. RT increased insulin sensitivity (p < 0.05) and reduced resting blood pressure and AIx (p < 0.05), but did not change central or pPWV. Conclusions: RT did not change any measure of muscle microvascular structure or function. [ABSTRACT FROM AUTHOR]

This article discusses the question of how urban shrinkage gets onto the agenda of public-policy agencies. It is based on a comparison of the agenda-setting histories of four European cities, Liverpool ( UK), Leipzig ( Germany), Genoa ( Italy) and Bytom ( Poland), which have all experienced severe population losses but show very different histories with respect to how local governments reacted to them. We use the political-science concepts of 'systemic vs. institutional agendas' and 'policy windows' as a conceptual frame to compare these experiences. The article demonstrates that shrinkage is hardly ever responded to in a comprehensive manner but rather that policies are only implemented in a piecemeal way in selected fields. Moreover, it is argued that variations in institutional contexts and political dynamics lead to considerable differences with regard to the chances of making shrinkage a matter of public intervention. Against this background, the article takes issue with the idea that urban shrinkage only needs to be 'accepted' by policymakers who would need to overcome their growth-oriented cultural perceptions, as has been suggested in a number of recent writings, and calls for a more differentiated, context-sensitive view. [ABSTRACT FROM AUTHOR]

Increases in insulin-mediated glucose uptake following endurance training ( ET) and sprint interval training ( SIT) have in part been attributed to concomitant increases in glucose transporter 4 ( GLUT4) protein content in skeletal muscle. This study used an immunofluorescence microscopy method to investigate changes in subcellular GLUT4 distribution and content following ET and SIT. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of 16 sedentary males in the overnight fasted state before and after 6 weeks of ET and SIT. An antibody was fully validated and used to show large (> 1 μm) and smaller (<1 μm) GLUT4-containing clusters. The large clusters likely represent trans-Golgi network stores and the smaller clusters endosomal stores and GLUT4 storage vesicles ( GSVs). Density of GLUT4 clusters was higher at the fibre periphery especially in perinuclear regions. A less dense punctate distribution was seen in the rest of the muscle fibre. Total GLUT4 fluorescence intensity increased in type I and type II fibres following both ET and SIT. Large GLUT4 clusters increased in number and size in both type I and type II fibres, while the smaller clusters increased in size. The greatest increases in GLUT4 fluorescence intensity occurred within the 1 μm layer immediately adjacent to the PM. The increase in peripheral localisation and protein content of GLUT4 following ET and SIT is likely to contribute to the improvements in glucose homeostasis observed after both training modes. [ABSTRACT FROM AUTHOR]

Key point Optimal vascular function is critical for health, and endurance training (ET) has previously been shown to be an effective method of improving this., Sprint interval training (SIT) has been proposed as a time efficient alternative to ET but its effect on skeletal muscle microvasculature has not been studied and no direct comparison with ET has been made., ET and SIT in this study were equally effective at decreasing arterial stiffness and increasing skeletal muscle capillarisation and eNOS content., The main results suggest that both training modes improve skeletal muscle microvascular and macrovascular function, with SIT being a time efficient alternative., Abstract Sprint interval training (SIT) has been proposed as a time efficient alternative to endurance training (ET) for increasing skeletal muscle oxidative capacity and improving certain cardiovascular functions. In this study we sought to make the first comparisons of the structural and endothelial enzymatic changes in skeletal muscle microvessels in response to ET and SIT. Sixteen young sedentary males (age 21 ± SEM 0.7 years, BMI 23.8 ± SEM 0.7 kg m−2) were randomly assigned to 6 weeks of ET (40-60 min cycling at ∼65%, 5 times per week) or SIT (4-6 Wingate tests, 3 times per week). Muscle biopsies were taken from the m. vastus lateralis before and following 60 min cycling at 65% to measure muscle microvascular endothelial eNOS content, eNOS serine1177 phosphorylation, NOX2 content and capillarisation using quantitative immunofluorescence microscopy. Whole body insulin sensitivity, arterial stiffness and blood pressure were also assessed. ET and SIT increased skeletal muscle microvascular eNOS content (ET 14%; P < 0.05, SIT 36%; P < 0.05), with a significantly greater increase observed following SIT ( P < 0.05). Sixty minutes of moderate intensity exercise increased eNOS ser1177 phosphorylation in all instances ( P < 0.05), but basal and post-exercise eNOS ser1177 phosphorylation was lower following both training modes. All microscopy measures of skeletal muscle capillarisation ( P < 0.05) were increased with SIT or ET, while neither endothelial nor sarcolemmal NOX2 was changed. Both training modes reduced aortic stiffness and increased whole body insulin sensitivity ( P < 0.05). In conclusion, in sedentary males SIT and ET are effective in improving muscle microvascular density and eNOS protein content. [ABSTRACT FROM AUTHOR]

We assessed the effects of post‐exercise cold‐water immersion (CWI) in modulating PGC‐1α mRNA expression in response to exercise commenced with low muscle glycogen availability. In a randomized repeated‐measures design, nine recreationally active males completed an acute two‐legged high‐intensity cycling protocol (8 × 5 min at 82.5% peak power output) followed by 10 min of two‐legged post‐exercise CWI (8°C) or control conditions (CON). During each trial, one limb commenced exercise with low (LOW: <300 mmol·kg−1 dw) or very low (VLOW: <150 mmol·kg−1 dw) pre‐exercise glycogen concentration, achieved via completion of a one‐legged glycogen depletion protocol undertaken the evening prior. Exercise increased (P < 0.05) PGC‐1α mRNA at 3 h post‐exercise. Very low muscle glycogen attenuated the increase in PGC‐1α mRNA expression compared with the LOW limbs in both the control (CON VLOW ~3.6‐fold vs. CON LOW ~5.6‐fold: P = 0.023, ES 1.22 Large) and CWI conditions (CWI VLOW ~2.4‐fold vs. CWI LOW ~8.0 fold: P = 0.019, ES 1.43 Large). Furthermore, PGC‐1α mRNA expression in the CWI‐LOW trial was not significantly different to the CON LOW limb (P = 0.281, ES 0.67 Moderate). Data demonstrate that the previously reported effects of post‐exercise CWI on PGC‐1α mRNA expression (as regulated systemically via β‐adrenergic mediated cell signaling) are offset in those conditions in which local stressors (i.e., high‐intensity exercise and low muscle glycogen availability) have already sufficiently activated the AMPK‐PGC‐1α signaling axis. Additionally, data suggest that commencing exercise with very low muscle glycogen availability attenuates PGC‐1α signaling. [ABSTRACT FROM AUTHOR]

Please cite this paper as: Cocks M, Shepherd SO, Shaw CS, Achten J, Costa ML, Wagenmakers AJM. Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle. Microcirculation 19: 642-651, 2012. Abstract Objective: The net production of NO by the muscle microvascular endothelium is a key regulator of muscle microvascular blood flow. Here, we describe the development of a method to quantify the protein content and phosphorylation of endothelial NO synthase (eNOS content and eNOS ser1177 phosphorylation) and NAD(P)H oxidase expression. Methods: Human muscle cryosections were stained using antibodies targeting eNOS, p-eNOS ser1177 and NOX2 in combination with markers of the endothelium and the sarcolemma. Quantitation was achieved by analyzing fluorescence intensity within the area stained positive for the microvascular endothelium. Analysis was performed in duplicate and repeated five times to investigate CV. In addition, eight healthy males (age 21 ± 1 year, BMI 24.4 ± 1.0 kg/m2) completed one hour of cycling exercise at ∼65% VO2max. Muscle biopsies were taken from the m. vastus lateralis before and immediately after exercise and analyzed using the new methods. Results: The CV of all methods was between 6.5 and 9.5%. Acute exercise increased eNOS serine1177 phosphorylation (fold change 1.29 ± 0.05, p < 0.05). Conclusions: These novel methodologies will allow direct investigations of the molecular mechanisms underpinning the microvascular responses to insulin and exercise, the impairments that occur in sedentary, obese and elderly individuals and the effect of lifestyle interventions. [ABSTRACT FROM AUTHOR]