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S U M M A R Y 1. A progressive exercise test was performed on forty-four male and twenty-nine female healthy Europeans, aged between 20 and 64 years. Values for cardiac frequency (f H) and ventilation (V) were interpolated to standard (submaximal) oxygen uptakes (Vo,) of 0.75 l/min and 1.0 l/min. The tidal volume ( VT) at a ventilation of 20 and 30 1 BTPS/min was also determined (VT 20 and VT 30 respectively).2. The slope of the linear relationship between cardiac frequency or ventilation and oxygen uptake (SfH and SV respectively) can be used as a measure of the fitness of an individual, as it indicates the increase in f H or fr that is obligatory for an increase in energy expenditure equivalent to an additional oxygen uptake of 1.0 l/min (about the increase necessary for walking on the level at a normal speed). By analogy with the responses of an athlete, a 'fit' subject is one in whom responses are economically low,i.e. SfH and SV are lower than in sedentary individuals. Measures of SfH and SV can also be used to indicate the demands of everyday activities on f H and fr. 3. When SfH and SV are related to the individual's capacity to adapt f H and V from resting to predicted maximum values ('adaptation capacity' ACfH and ACT respectively), the resulting index (SfH x 100/ACfH or Sfr x 100/ACQ expresses the percentage of the adaptation capacity used for an additional energy expenditure equivalent to a To, of 1.0 l/min, and can be considered a measure of the 'physiological strain' of exercise. The effects on exercise responses of differences in body muscle can be allowed for by multiplying this index by lean body mass (LBM). The lower the (size-adjusted) physiological strain index, the fitter the individual subject.The maximum oxygen uptake (Vo,,,,,,.> has been widely used as the defining index of cardiorespiratory fitness (Robinson

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) have the potential to transform regenerative cardiac medicine and the modelling of cardiac disease. This is of particular importance in the context of diabetic cardiomyopathy where diabetic individuals exhibit reduced cardiac diastolic contractile performance in the absence of vascular disease, significantly contributing towards high cardiovascular morbidity. In this study, the capacity of iPSC-CM to act as a novel cellular model of cardiomyocytes was assessed. The diabetic phenotype is characterised by insulin resistance, therefore there was a specific focus upon metabolic parameters. Despite expressing crucial insulin signalling intermediates and relevant trafficking proteins, it was identified that iPSC-CM do not exhibit insulin-stimulated glucose uptake. iPSC-CM are spontaneously contractile however contraction mediated uptake was not found to mask any insulin response. The fundamental limitation identified in these cells was a critical lack of expression of the insulin sensitive glucose transporter GLUT4. Using comparative immunoblot analysis and the GLUT-selective inhibitor BAY-876 to quantify expression of these transporters, we show that iPSC-CM express high levels of GLUT1 and low levels of GLUT4 compared to primary cardiomyocytes and cultured adipocytes. Interventions to overcome this limitation were unsuccessful. We suggest that the utility of iPSC-CMs to study cardiac metabolic disorders may be limited by their apparent foetal-like phenotype.

SNARE proteins are integral to intracellular vesicular trafficking, which in turn is the process underlying the regulated expression of substrate transporters such as the glucose transporter GLUT4 at the cell surface of insulin target tissues. Impaired insulin stimulated GLUT4 trafficking is associated with reduced cardiac function in many disease states, most notably diabetes. Despite this, our understanding of the expression and regulation of SNARE proteins in cardiac tissue and how these may change in diabetes is limited. Here we characterize the array of SNARE proteins expressed in cardiac tissue, and quantify the levels of expression of VAMP2, SNAP23, and Syntaxin4-key proteins involved in insulin-stimulated GLUT4 translocation. We examined SNARE protein levels in cardiac tissue from two rodent models of insulin resistance, db/db mice and high-fat fed mice, and show alterations in patterns of expression are evident. Such changes may have implications for cardiac function.

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.