ObjectiveIntramyocellular lipid (IMCL) storage negatively associates with insulin resistance, albeit not in endurance-trained athletes. We investigated the putative contribution of lipid droplet (LD) morphology and subcellular localization to the so-called athlete's paradox.MethodsWe performed quantitative immunofluorescent confocal imaging of muscle biopsy sections from endurance Trained, Lean sedentary, Obese, and Type 2 diabetes (T2DM) participants (n = 8/group). T2DM patients and Trained individuals were matched for IMCL content. Furthermore we performed this analysis in biopsies of T2DM patients before and after a 12-week exercise program (n = 8).ResultsWe found marked differences in lipid storage morphology between trained subjects and T2DM: the latter group mainly store lipid in larger LDs in the subsarcolemmal (SS) region of type II fibers, whereas Trained store lipid in a higher number of LDs in the intramyofibrillar (IMF) region of type I fibers. In addition, a twelve-week combined endurance and strength exercise program resulted in a LD phenotype shift in T2DM patients partly towards an ‘athlete-like’ phenotype, accompanied by improved insulin sensitivity. Proteins involved in LD turnover were also more abundant in Trained than in T2DM and partly changed in an ‘athlete-like’ fashion in T2DM patients upon exercise training.ConclusionsOur findings provide a physiological explanation for the athlete's paradox and reveal LD morphology and distribution as a major determinant of skeletal muscle insulin sensitivity.

Exercise training reduces intrahepatic lipid (IHL) content in people with elevated liver fat content. It is unclear, however, whether exercise training reduces IHL content in people with normal liver fat content. Here, we measured the effect of exercise training on IHL content in people with and people without nonalcohol fatty liver. We further measured changes in insulin sensitivity and hepatic energy metabolism. Eleven males with nonalcoholic fatty liver (NAFL) and 11 body mass index-matched individuals without nonalcoholic fatty liver (CON) completed a 12-wk supervised exercise training program. IHL content (proton magnetic resonance spectroscopy), maximal oxidative capacity (V̇o, spiroergometry), total muscle strength, body composition, insulin sensitivity (hyperinsulinemic-euglycemic clamp), hepatic ATP-to-total phosphorus ratio, and the hepatic phosphomonoester-to-phosphodiester (PME/PDE) ratio (phosphorus magnetic resonance spectroscopy) were determined. IHL content reduced with exercise training ( P = 0.014) in the whole study population. The relative reduction in IHL content was comparable in NAFL (-34.5 ± 54.0%) and CON (-28.3 ± 60.1%) individuals ( P = 0.800). V̇o ( P < 0.001), total muscle strength ( P < 0.001), and skeletal muscle insulin sensitivity ( P = 0.004) increased, whereas adipose tissue ( P = 0.246) and hepatic ( P = 0.086) insulin sensitivity did not increase significantly. Hepatic ATP-to-total phosphorus ratio ( P = 0.987) and PME/PDE ratio ( P = 0.792) did not change. Changes in IHL content correlated with changes in body weight ( r = 0.451, P = 0.035) and changes in hepatic PME/PDE ratio ( r = 0.569, P = 0.019). In conclusion, exercise training reduced intrahepatic lipid content in people with nonalcoholic fatty liver and in people with normal intrahepatic lipid content, and the percent reduction in intrahepatic lipid content was similar in both groups.

Aims/hypothesisIn contrast to insulin-resistant individuals, insulin-sensitive athletes possess high intramyocellular lipid content (IMCL), good mitochondrial function and high perilipin 5 (PLIN5) levels, suggesting a role for PLIN5 in benign IMCL storage. We hypothesised a role for PLIN5 in modulating fasting-mediated insulin resistance.MethodsTwelve men were fasted for 60 h, before and after which muscle biopsies were taken and stained for lipid droplets (LDs), PLIN5 and laminin. Confocal microscopy images were analysed for LD size, number, PLIN5 association and subcellular distribution.ResultsFasting elevated IMCL content 2.8-fold and reduced insulin sensitivity (by 55%). Individuals with the most prominent increase in IMCL showed the least reduction in insulin sensitivity (r = 0.657; p = 0.028) and mitochondrial function (r = 0.896; p = 0.006). During fasting, PLIN5 gene expression or PLIN5 protein content in muscle homogenates was unaffected, microscopy analyses revealed that the fraction of PLIN5 associated with LDs (PLIN5+) increased significantly (+26%) upon fasting, suggesting PLIN5 redistribution. The significant increase in LD number (+23%) and size (+23%) upon fasting was entirely accounted for by PLIN5+ LDs, not by LDs devoid of PLIN5. Also the association between IMCL storage capacity and insulin resistance and mitochondrial dysfunction was only apparent for PLIN5+ LDs.Conclusions/interpretationFasting results in subcellular redistribution of PLIN5 and promotes the capacity to store excess fat in larger and more numerous PLIN5-decorated LDs. This associates with blunting of fasting-induced insulin resistance and mitochondrial dysfunction, suggesting a role for PLIN5 in the modulation of fasting-mediated lipotoxicity.Trial registration:trialregister.nl NTR 2042Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-3865-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Aims/hypothesis Human brown adipose tissue (BAT) has recently emerged as a potential target in the treatment of type 2 diabetes, owing to its capacity to actively clear glucose from the circulation-at least upon cold exposure. The effects of insulin resistance on the capacity of human BAT to take up glucose are unknown. Prolonged fasting is known to induce insulin resistance in peripheral tissues in order to spare glucose for the brain. Methods We studied the effect of fasting-induced insulin resistance on the capacity of BAT to take up glucose during cold exposure as well as on cold-stimulated thermogenesis. BAT glucose uptake was assessed by means of cold-stimulated dynamic 2-deoxy-2-[ 18 F]fluoro-D-glucose positron emission tomography/computed tomography ([ 18 F]FDG-PET/CT) imaging.Results We show that a 54 h fasting period markedly decreases both cold-induced BAT glucose uptake and nonshivering thermogenesis (NST) during cold stimulation. In vivo molecular imaging and modelling revealed that the reduction of glucose uptake in BAT was due to impaired cellular glucose uptake and not due to decreased supply. Interestingly, decreased BAT glucose uptake upon fasting was related to a decrease in core temperature during cold exposure, pointing towards a role for BAT in maintaining normothermia in humans. Conclusions/interpretation Cold-stimulated glucose uptake in BAT is strongly reduced upon prolonged fasting. When cold-stimulated glucose uptake in BAT is also reduced under other insulin-resistant states, such as diabetes, cold-induced activation of BAT may not be a valid way to improve glucose clearance by BAT under such conditions. Trial registration: www.trialregister.nl NTR3523 Funding: This work was supported by the EU FP7 project DIABAT (HEALTH-F2-2011-278373

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Skeletal muscle can store excess fat as subcellular lipid droplets (LDs). While originally viewed as uninteresting static balls of triacylglycerol, it is now clear that myocellular LDs play an active role in myocellular (patho)physiology. In this review we aim to discuss the role of LDs in muscle cell insulin sensitivity and identify parameters which appear to affect this relationship. Moreover, we discuss the application of novel tools permitting detailed examination of these parameters. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.

Intramyocellular lipid (IMCL) hampers insulin sensitivity, albeit not in endurance-trained athletes (Trained). Compared to type 2 diabetes mellitus (T2DM) patients, Trained subjects have high levels of perilipin 5 (PLIN5). In the present study, we tested whether the fraction of PLIN5-coated lipid droplets (LDs) is a determinant of skeletal muscle insulin sensitivity and contributes to the athlete's paradox. Muscle biopsies were taken from eight Trained, Lean sedentary, Obese and T2DM subjects. Trained, Obese and T2DM subjects were matched for total IMCL content. Confocal images were analysed for lipid area fraction, LD size and number and PLIN5+ and PLIN5- LDs were measured. A stepwise linear regression was performed to identify factors explaining observed variance in glucose infusion rate (GIR). Trained and T2DM subjects stored IMCL differently; Trained subjects had a higher number of LDs compared to T2DM subjects (0.037 ± 0.004 μm vs. 0.023 ± 0.003 μm , P = 0.024) that were non-significantly smaller (0.27 ± 0.01 μm vs. 0.32 ± 0.02 μm , P = 0.197, Trained vs. T2DM). Even though total PLIN5 protein content was almost double in Trained vs. T2DM subjects (1.65 ± 0.21 AU vs. 0.89 ± 0.09 AU, P = 0.004), PLIN5 coating did not affect LD number or size significantly. Of the observed variance in GIR, the largest fraction by far (70.2%) was explained by maximal oxygen uptake. Adding PLIN5 protein content or PLIN5+ LDs increased the explained variance in GIR (74.7% and 80.7% for PLIN5 protein content and PLIN5+ LDs, respectively). Thus, the putative relationship between PLIN5 and insulin sensitivity is at best indirect and is apparent only in conjunction with maximal oxygen uptake. Hence, PLIN5 abundance cannot be causally linked to the athlete's paradox.