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Objectives The objective of the study was to validate PCr/ATP ratios as an in vivo marker for cardiac mitochondrial function.Background Cardiac energy status, measured as PCr/ATP ratio with 31P-MRS in vivo, was shown to be a prognostic factor in heart failure and is lowered in cardiometabolic disease. As mitochondrial function is also hampered in these diseases and oxidative phosphorylation is the major contributor to ATP synthesis, the PCr/ATP ratio might be a reflection of cardiac mitochondrial function.Methods Thirty-eight patients scheduled for open heart surgery were enrolled in this study. Before surgery, cardiac 31P-MRS was performed. During surgery, tissue specimens from the right atrial appendage were obtained for the ex vivo assessment of mitochondrial function using high-resolution respirometry.Results The patient population included was heterogenous resulting in wide ranges of PCr/ATP ratios and ADP-stimulated respiration rates (PCr/ATP ranging from 0.533 to 1.717; ADP-stimulated respiration rates ranging from 28.5 to 94.6 pmol/mg/s). Correlation analysis however showed no relationship between PCr/ATP and ADP-stimulated respiration rates fueled by various substrates (octanoylcarnitine R2 2 2 = 0.005, p = 0.71; pyruvate R2 = 0.040, p = 0.26).Conclusions Our results do not support the use of cardiac energy status (PCr/ATP) as a surrogate marker of mitochondrial function in the heart. The dissociation of the two parameters in the present study suggests that mitochondrial function is not the only determinant of cardiac energy status.Condensed abstractThis study does not support the use of in vivo cardiac energy status (PCr/ATP ratio) as a surrogate marker of ex vivo mitochondrial function (maximal oxidative respiration). Although both PCr/ATP and mitochondrial function are reduced in cardiovascular disease, the dissociation of the two parameters in the present study shows that PCr/ATP is determined by more factors than only mitochondrial function. Hence, PCr/ATP is not a good marker for mitochondrial function, but it can be a valuable marker for cardiometabolic health in cardiometabolic studies.Trial registration: https://clinicaltrials.gov (NCT03049228).

beta(2)-agonist treatment improves skeletal muscle glucose uptake and whole-body glucose homeostasis in rodents, likely via mTORC2-mediated signalling. However, human data on this topic is virtually absent. We here investigate the effects of two-weeks treatment with the beta(2)-agonist clenbuterol (40 mu g/day) on glucose control as well as energy- and substrate metabolism in healthy young men (age: 18-30 years, BMI: 20-25 kg/m(2)) in a randomised, placebo-controlled, double-blinded, cross-over study (ClinicalTrials.gov-identifier: NCT03800290). Randomisation occurred by controlled randomisation and the final allocation sequence was seven (period 1: clenbuterol, period 2: placebo) to four (period 1: placebo, period 2: clenbuterol). The primary and secondary outcome were peripheral insulin-stimulated glucose disposal and skeletal muscle GLUT4 translocation, respectively. Primary analyses were performed on eleven participants. No serious adverse events were reported. The study was performed at Maastricht University, Maastricht, The Netherlands, between August 2019 and April 2021. Clenbuterol treatment improved peripheral insulin-stimulated glucose disposal by 13% (46.6 +/- 3.5 versus 41.2 +/- 2.7 mu mol/kg/min, p = 0.032), whereas skeletal muscle GLUT4 translocation assessed in overnight fasted muscle biopsies remained unaffected. These results highlight the potential of beta(2)-agonist treatment in improving skeletal muscle glucose uptake and underscore the therapeutic value of this pathway for the treatment of type 2 diabetes. However, given the well-known (cardiovascular) side-effects of systemic beta(2)-agonist treatment, further exploration on the underlying mechanisms is needed to identify viable therapeutic targets.beta 2-agonist treatment improves skeletal muscle glucose uptake and whole-body glucose homeostasis in rodents. Here the authors report that two-weeks of treatment with the beta 2-agonist clenbuterol improves insulin-stimulated glucose disposal in healthy young men in a double-blinded, randomized cross-over trial.

Background Primary teachers' pedagogical practices (TPP) are strongly focused on supporting pupils' psychological needs, creating a safe learning climate, and encouraging pupils' developmental and learning processes. As a core motivation for teachers is the desire to interact constructively with children, pedagogical practices can be understood as central to teachers' work. A familiar problem in many international contexts is that TPP can come under pressure, typically as the result of interplay between personal and contextual factors. However, which contextual factors influence TPP, and how they do so, remains unclear. Purpose In this study, we aimed to better understand how context influences primary school teachers' pedagogical practices, within the setting of primary education in the Netherlands. We were particularly interested in which contextual factors were perceived as important and how they influenced TPP. Methods Data were collected through open questions in a survey among 215 primary school teachers in the northern part of the Netherlands. Focus group interviews were then held with 11 of the survey participants. The data were analysed qualitatively, using a framework approach with five stages of familiarisation: identifying themes, indexing, charting, and mapping and interpretation. Findings The analysis identified nine contextual factors that, according to participants, both positively and negatively affected TPP. The most frequently mentioned threats to TPP were educational accountability and standards, and organisation of work in the classroom, whilst school improvement and pupils' educational needs were regarded as the most important stimulating factors. We clustered the factors into four patterns which reflected the way that teachers appraised them: organisation of daily classroom practices, school culture, educational improvement, and contribution to pupils' development. Conclusions According to the teachers, the interplay between TPP and the context can cause pressure, frustration, and feelings of incompetence because there is insufficient time for achieving pedagogical goals. Our study draws attention to the complex challenges that teachers face in terms of balancing pedagogical practices and other educational tasks, and draws out implications for policy and practice.

Fatty acids are an important energy source during exercise. Training status and substrate availability are determinants of the relative and absolute contribution of fatty acids and glucose to total energy expenditure. Endurance-trained athletes have a high oxidative capacity, while, in insulin-resistant individuals, fat oxidation is compromised. Fatty acids that are oxidised during exercise originate from the circulation (white adipose tissue lipolysis), as well as from lipolysis of intramyocellular lipid droplets. Moreover, hepatic fat may contribute to fat oxidation during exercise. Nowadays, it is clear that myocellular lipid droplets are dynamic organelles and that number, size, subcellular distribution, lipid droplet coat proteins and mitochondrial tethering of lipid droplets are determinants of fat oxidation during exercise. This review summarises recent insights into exercise-mediated changes in lipid metabolism and insulin sensitivity in relation to lipid droplet characteristics in human liver and muscle. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00125-020-05170-z) contains a slideset of the figures for download, which is available to authorised users.

Aims/hypothesis Physical inactivity, low mitochondrial function, increased intramyocellular lipid (IMCL) deposition and reduced insulin sensitivity are common denominators of chronic metabolic disorders, like obesity and type 2 diabetes. Yet, whether low mitochondrial function predisposes to insulin resistance in humans is still unknown. Methods Here we investigated, in an intervention study, whether muscle with low mitochondrial oxidative capacity, induced by one-legged physical inactivity, would feature stronger signs of lipid-induced insulin resistance. To this end, ten male participants (age 22.4 ± 4.2 years, BMI 21.3 ± 2.0 kg/m2) underwent a 12 day unilateral lower-limb suspension with the contralateral leg serving as an active internal control. Results In vivo, mitochondrial oxidative capacity, assessed by phosphocreatine (PCr)-recovery half-time, was lower in the inactive vs active leg. Ex vivo, palmitate oxidation to 14CO2 was lower in the suspended leg vs the active leg; however, this did not result in significantly higher [14C]palmitate incorporation into triacylglycerol. The reduced mitochondrial function in the suspended leg was, however, paralleled by augmented IMCL content in both musculus tibialis anterior and musculus vastus lateralis, and by increased membrane bound protein kinase C (PKC) θ. Finally, upon lipid infusion, insulin signalling was lower in the suspended vs active leg. Conclusions/interpretation Together, these results demonstrate, in a unique human in vivo model, that a low mitochondrial oxidative capacity due to physical inactivity directly impacts IMCL accumulation and PKCθ translocation, resulting in impaired insulin signalling upon lipid infusion. This demonstrates the importance of mitochondrial oxidative capacity and muscle fat accumulation in the development of insulin resistance in humans. Trial registration ClinicalTrial.gov NCT01576250. Funding PS was supported by a ‘VICI’ Research Grant for innovative research from the Netherlands Organization for Scientific Research (Grant 918.96.618).

Falls among older adults are often attributed to declining muscle strength with ageing. Associations between muscle strength and balance control have been reported, but the evidence for, and key mechanisms of resistance exercise in fall prevention are unclear. No studies have directly examined the relationship between muscle fibre characteristics and reactive balance control. Here, we address whether or not Type II muscle fibre characteristics associate with reactive balance during walking in young and older adults with varying muscle fibre type composition. We analyse muscle biopsy-derived fibre characteristics and stability during a treadmill-based walking perturbation (trip-like) task of healthy young adults, healthy, normally active older adults, trained older adults and physically impaired older adults. We find no significant associations between Type II muscle fibre properties and reactive balance during walking, indicating that practitioners and researchers should consider more than just the muscle tissue properties when assessing and intervening on fall risk.

Objective Storage of triglycerides in lipid droplets is governed by a set of lipid droplet-associated proteins. One of these lipid droplet-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair lipid droplet breakdown in macrophages and cancer cells by inhibiting adipose triglyceride lipase. Here, we aimed to better characterize the role and mechanism of action of HILPDA in hepatocytes. Methods We performed studies in HILPDA-deficient and HILPDA-overexpressing liver cells, liver slices, and mice. The functional role and physical interactions of HILPDA were investigated using a variety of biochemical and microscopic techniques, including real-time fluorescence live-cell imaging and Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM). Results Levels of HILPDA were markedly induced by fatty acids in several hepatoma cell lines. Hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglycerides in mice with non-alcoholic steatohepatitis. Similarly, deficiency of HILPDA in mouse liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labeled fatty acids in lipid droplets, respectively, which was independent of adipose triglyceride lipase. Fluorescence microscopy showed that HILPDA partly colocalizes with lipid droplets and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging further revealed that HILPDA preferentially localizes to lipid droplets that are being remodeled. Overexpression of HILPDA in liver cells increased the activity of diacylglycerol acyltransferases (DGAT) and DGAT1 protein levels, concurrent with increased lipid storage. Confocal microscopy coupled to FRET-FLIM analysis demonstrated that HILPDA physically interacts with DGAT1 in living liver cells. The stimulatory effect of HILPDA on lipid storage via DGAT1 was corroborated in adipocytes. Conclusions Our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity. Our findings suggest a novel regulatory mechanism by which fatty acids promote triglyceride synthesis and storage., Graphical abstract Image 1, Highlights • HILPDA expression is induced by fatty acids in hepatoma cells. • HILPDA deficiency modestly decreases liver triglyceride storage in mice with NASH. • HILPDA preferentially associates with newly synthesized lipid droplets and active lipid droplets. • HILPDA promotes lipid storage at least in part independently of ATGL. • HILPDA physically interacts and induces DGAT1.

In many different cell types neutral lipids can be stored in lipid droplets (LDs). Nowadays, LDs are viewed as dynamic organelles, which store and release fatty acids depending on energy demand (LD dynamics). Proteins like perilipin 2 (PLIN2) and PLIN5 decorate the LD membrane and are determinants of LD lipolysis and fat oxidation, thus affecting LD dynamics. Trained athletes and type 2 diabetes (T2D) patients both have high levels of intramyocellular lipid (IMCL). While IMCL content scales negatively with insulin resistance, athletes are highly insulin sensitive in contrast to T2D patients, the so-called athlete's paradox. Differences in LD dynamics may be an underlying factor explaining the athlete's paradox. We aimed to quantify PLIN2 and PLIN5 content at individual LDs as a reflection of the ability to switch between fatty acid release and storage depending on energy demand. Thus, we developed a novel fluorescent super-resolution microscopy approach and found that PLIN2 protein abundance at the LD surface was higher in T2D patients than in athletes. Localization of adipocyte triglyceride lipase (ATGL) to the LD surface was lower in LDs abundantly decorated with PLIN2. While PLIN5 abundance at the LD surface was similar in athletes and T2D patients, we have observed previously that the number of PLIN5 decorated LDs was higher in athletes, indicating more LDs in close association with mitochondria. Thus, in athletes interaction of LDs with mitochondria was more pronounced and LDs have the protein machinery to be more dynamic, while in T2D patients the LD pool is more inert. This observation contributes to our understanding of the athlete's paradox.

Mild cold acclimation for 10 days has been previously shown to markedly improve insulin sensitivity in patients with type 2 diabetes. Here we show in a single-arm intervention study (Trialregister.nl ID: NL4469/NTR5711) in nine patients with type 2 diabetes that ten days of mild cold acclimation (16–17 °C) in which observable, overt shivering was prevented, does not result in improved insulin sensitivity, postprandial glucose and lipid metabolism or intrahepatic lipid content and only results in mild effects on overnight fasted fat oxidation, postprandial energy expenditure and aortic augmentation index. The lack of marked metabolic effects in this study is associated with a lack of self-reported shivering and a lack of upregulation of gene expression of muscle activation or muscle contraction pathways in skeletal muscle and suggests that some form of muscle contraction is needed for beneficial effects of mild cold acclimation., Cold acclimation has been shown to have beneficial metabolic effects, including improved insulin sensitivity in patients with type 2 diabetes. Here the authors show that a mild cold acclimation regiment during which overt shivering was prevented did not result in improved insulin sensitivity in a small group of patients with type 2 diabetes.

The aim of this study is to explore Dutch primary school teachers' perceptions regarding their pedagogical practices, and, specifically, whether they consider these practices under pressure. We conducted a survey among a sample of 261 primary school teachers from 115 schools, that included open and closed questions. The analyses showed that primary school teachers' perceptions of pressure varied: 75% of the teachers indicated that their pedagogical practices are under pressure and one-quarter of the teachers reported experiencing low or no pressure. Furthermore, the variety in experienced pressure is not influenced by teachers' experience or educational beliefs. The data show that a lack of coherence between teachers' personal vision and the school's vision influences the extent to which teachers experience pressure. This study builds on theoretical research into teachers' pedagogical practices by showing that pressure on teachers' pedagogical practices is a general problem in primary education.