Figure 1
Transmission electron microscopy and point-counting of subcellular lipid droplets A, acquisition of electron micrographs was systematically randomized to cover the subsarcolemmal (SS), superficial myofibrillar (SMF) and central myofibrillar regions (CMF) of each longitudinally oriented fibre. The arrow indicates the sarcolemma. (original magnification ×600, scale bar: 20 µm). B, subcellular localisations of lipid droplets. The arrow indicates the sarcolemma. C, close-up view of point-counting of subsarcolemmal lipid droplet volume fraction with grid (size 135 nm) overlay. The open circles indicate intersections touching a lipid droplet. * , a subsarcolemmal lipid droplet; * * , an intermyofibrillar lipid droplet; Mi, mitochondria; Z, Z-line. (Original magnification ×10,000, scale bar: 1 µm.)
Source publication
Key points:
Although lipid droplets in skeletal muscle are an important energy source during endurance exercise, our understanding of lipid metabolism in this context remains incomplete. Using transmission electron microscopy, two distinct subcellular pools of lipid droplets can be observed in skeletal muscle - one beneath the sarcolemma and the o...
Context in source publication
Context 1
... make robust and unbiased volume estimates of lipid droplets and mitochondria (for fibre-typing, see below), we used point-counting techniques, with reference to Cavalieri's principle and the Delesse principle (Weibel, 1979), to estimate areas of mitochondria and lipid droplets in the electron micrographs (Fig. 1). Identification of mitochondria in micrographs was based on previously reported work on mammalian skeletal muscles (Hoppeler et al. 1973). Our criteria for identifying lipid droplets included having a circular white-greyish appearance with a fuzzy border (absence of distinct membrane) and a minimum diameter of 200 ...
Citations
... Effects of acute exercise bout on IMCL content is not conclusive. Consistent with our results, several previous studies reported no change, whereas a decrease in IMCL has also been reported (Schrauwen-Hinderling et al. 2003a;Koh et al. 2017). Exercise induces lipolysis at a rate that exceeds the oxidation of FFA, resulting in elevated plasma FFA levels, mainly from the adipose tissues (Schrauwen-Hinderling et al. 2003b). ...
... There is very little information available on effects of acute endurance exercise on lipid droplet morphology. Koopman et al. (2006) reported a decrease in lipid droplet size after resistance exercise and Koh et al. (2017) reported no change in the lipid droplet size after 52-63 minutes of skiing in athletes. Smaller lipid droplets have been associated with better insulin sensitivity, improved aerobic fitness, increased mitochondrial size, increased oxidative enzyme activity and whole-body lipid oxidation (Covington et al. 2017;He et al. 2004). ...
... This study was limited to only males, as with the previous similar studies on lipid droplet morphological effect of acute exercise (Koopman et al. 2006;Koh et al. 2017;Strauss et al. 2020). Since there is sex based differences in lipid deposition and substrate utilization (Devries 2016), future studies should be expanded to both genders. ...
Purpose
Smaller lipid droplet morphology and GLUT 4 protein expression have been associated with greater muscle oxidative capacity and glucose uptake, respectively. The main purpose of this study was to determine the effect of an acute long-duration exercise bout on skeletal muscle lipid droplet morphology, GLUT4, perilipin 3, and perilipin 5 expressions.
Methods
Twenty healthy men (age 24.0 ± 1.0 years, BMI 23.6 ± 0.4 kg/m²) were recruited for the study. The participants were subjected to an acute bout of exercise on a cycle ergometer at 50% VO2max until they reached a total energy expenditure of 650 kcal. The study was conducted after an overnight fast. Vastus lateralis muscle biopsies were obtained before and immediately after exercise for immunohistochemical analysis to determine lipid, perilipin 3, perilipin 5, and GLUT4 protein contents while GLUT 4 mRNA was quantified using RT-qPCR.
Results
Lipid droplet size decreased whereas total intramyocellular lipid content tended to reduce (p = 0.07) after an acute bout of endurance exercise. The density of smaller lipid droplets in the peripheral sarcoplasmic region significantly increased (0.584 ± 0.04 to 0.638 ± 0.08 AU; p = 0.01) while larger lipid droplets significantly decreased (p < 0.05). GLUT4 mRNA tended to increase (p = 0.05). There were no significant changes in GLUT 4, perilipin 3, and perilipin 5 protein levels.
Conclusion
The study demonstrates that exercise may impact metabolism by enhancing the quantity of smaller lipid droplets over larger lipid droplets.
... Human type II fibres are hence enriched with creatine phosphate (CrP) and glycogen energy depots 75 and contain higher levels of adenylate kinase 21 , glycogenolysis and glycolysis metabolic machinery 14,21 . Conversely, type I fibres are more abundant in peroxisomes 14 , mitochondria 14,21,76,77 and intramyocellular lipids (IMCLs) [76][77][78] , consistent with their slower ATP turnover 74 (Supplementary Fig. 1b). ...
... Glycogen granules are nonuniformly distributed between intramyofibrillar, intermyofibrillar and subsarcolemmal pools 35,267,268 . Alternatively, intramyocellular lipids are stored in lipid droplets (LDs) found predominantly at central (intermyofibrillar) but also peripheral (subsarcolemmal) regions within healthy muscle fibres 76,78 . During submaximal 54 and longer-duration high-intensity interval 55 exercise most ATP in muscle is regenerated by mitochondrial oxidative phosphorylation (OXPHOS) (see the section 'Acute exercise muscle metabolism') ( Fig. 2). ...
... Intramyocellular lipids (IMCLs) are stored in the hydrophobic core of lipid droplet ellipsoids 77 at peripheral (subsarcolemmal, SS LD ) and central (intermyofibrillar, IMF LD ) regions within fibres [76][77][78]268 . Women may have ~43% more individual lipid droplets in muscle, contributing to a greater (~84%) density of total IMCLs than in men 334 . ...
Viewing metabolism through the lens of exercise biology has proven an accessible and practical strategy to gain new insights into local and systemic metabolic regulation. Recent methodological developments have advanced understanding of the central role of skeletal muscle in many exercise-associated health benefits and have uncovered the molecular underpinnings driving adaptive responses to training regimens. In this Review, we provide a contemporary view of the metabolic flexibility and functional plasticity of skeletal muscle in response to exercise. First, we provide background on the macrostructure and ultrastructure of skeletal muscle fibres, highlighting the current understanding of sarcomeric networks and mitochondrial subpopulations. Next, we discuss acute exercise skeletal muscle metabolism and the signalling, transcriptional and epigenetic regulation of adaptations to exercise training. We address knowledge gaps throughout and propose future directions for the field. This Review contextualizes recent research of skeletal muscle exercise metabolism, framing further advances and translation into practice.
... Additionally, there are a number of metabolic differences between these upper and lower body muscle groups, particularly related to fat utilization. Specifically, compared to legs, arm muscles have been reported to display lower fat oxidation capacity [51], lower 3-hydroxy-acyl-CoAdehydrogenase (HAD) activity (necessary for fatty acid oxidation) [39], lower intramyocellular lipid (IMCL) content [52], and higher exercise-induced lactate release [53]. While these factors are not directly related to fat-free mass, they highlight some considerable differences in upper and lower body skeletal muscle metabolism, which could have implications for the effects of vitamin D status on muscle size in the upper and lower extremities. ...
Vitamin D insufficiency is a global health concern and low vitamin D status is regularly associated with reduced muscle mass and sarcopenia in observational research. Recent research using Mendelian randomization (MR) has highlighted the potentially causal positive effect of serum vitamin D (25(OH)D) on total, trunk and upper body appendicular fat-free mass (FFM). However, no such effect was found in lower body FFM, a result that mirrors the outcomes of some vitamin D intervention studies. Here we review the current literature on vitamin D, muscle mass and strength and discuss some potential mechanisms for the differing effects of vitamin D on upper and lower body FFM. In particular, differences in distribution of the vitamin D receptor as well as androgen receptors, in the upper and lower body musculature, will be discussed.
... It has recently been reported that~35% of isolated rat cortical astrocytes 24 h after the seeding (~6 × 10 4 cells/coverslip) contain multiple LDs with an average diameter of~450 nm (ranging between 0.2 and 1 µm; [37]), comparable with the measurements performed on hepatocytes [22,43], skeletal muscle [44,45], and tumor cell lines [46]. In contrast, adipocytes normally contain a single LD, occupying most of the cytoplasm and ranging up to 100 µm in diameter [47]. ...
In recent years, increasing evidence regarding the functional importance of lipid droplets (LDs), cytoplasmic storage organelles in the central nervous system (CNS), has emerged. Although not abundantly present in the CNS under normal conditions in adulthood, LDs accumulate in the CNS during development and aging, as well as in some neurologic disorders. LDs are actively involved in cellular lipid turnover and stress response. By regulating the storage of excess fatty acids, cholesterol, and ceramides in addition to their subsequent release in response to cell needs and/or environmental stressors, LDs are involved in energy production, in the synthesis of membranes and signaling molecules, and in the protection of cells against lipotoxicity and free radicals. Accumulation of LDs in the CNS appears predominantly in neuroglia (astrocytes, microglia, oligodendrocytes, ependymal cells), which provide trophic, metabolic, and immune support to neuronal networks. Here we review the most recent findings on the characteristics and functions of LDs in neuroglia, focusing on astrocytes, the key homeostasis-providing cells in the CNS. We discuss the molecular mechanisms affecting LD turnover in neuroglia under stress and how this may protect neural cell function. We also highlight the role (and potential contribution) of neuroglial LDs in aging and in neurologic disorders.
... There is growing interest in elucidating how contracting muscles use lipid droplets (LD) in order to sustain exercise metabolism and/or for unknown purposes. For instance, during high-volume, high-intensity exercise (i.e., 57 min and 11 mmol/L blood lactate), LD within myofibrils but not those LD located close to the sarcolemma are used [58]. This is surprising as this kind of exercise mostly relies on oxidative metabolism mainly through fatty acid and glucose oxidation [59]. ...
... Furthermore, during innate immune response, LD increase their size [62]. This may also reveal different effects of LDs within skeletal muscle fibers, for instance, type 2 fibers have lower LD density, but they seems to have higher size than those LD form type 1 fibers in the leg skeletal muscle [58]. Although the current evidence is scarce, it would be interesting to test whether exercise impacts innate immune function by altering LD-mitochondria contact. ...
The incidence and severity of metabolic diseases can be reduced by introducing healthy lifestyle habits including moderate exercise. A common observation in age-related metabolic diseases is an increment in systemic inflammation (the so-called inflammaging) where mitochondrial reactive oxygen species (ROS) production may have a key role. Exercise prevents these metabolic pathologies, at least in part, due to its ability to alter immunometabolism, e.g., reducing systemic inflammation and by improving immune cell metabolism. Here, we review how exercise regulates immunometabolism within contracting muscles. In fact, we discuss how circulating and resident macrophages alter their function due to mitochondrial signaling, and we propose how these effects can be triggered within skeletal muscle in response to exercise. Finally, we also describe how exercise-induced mitochondrial adaptations can help to fight against virus infection. Moreover, the fact that moderate exercise increases circulating immune cells must be taken into account by public health agencies, as it may help prevent virus spread. This is of interest in order to face not only acute respiratory-related coronavirus (SARS-CoV) responsible for the COVID-19 pandemic but also for future virus infection challenges.
... First, we investigated fibre type and region (peripheral and central)-specific differences in IMTG content detected using BODIPY and ORO in muscle biopsies obtained after an overnight fast (study 1). In this context, subcellular distribution is an important consideration because IMTG use during exercise occurs primarily in the central (intermyofibrillar) region of type I fibres of the cell (Jevons et al. 2020;Koh et al. 2017), and IMTG deposited in the peripheral (subsarcolemmal) region is inversely related to insulin sensitivity Nielsen et al. 2010). We also critically reviewed several steps in the immunohistochemical protocol and analytical procedure that can affect the visualisation and quantification of IMTG content. ...
... White bar = 30 µm be considered to more accurately reflect subcellular differences in IMTG content. In this regard, studies employing transmission electron microscopy to investigate subcellular lipid content support this assertion, because they also demonstrate IMTG content to be greater in the central (or intermyofibrillar) region (Koh et al. 2017). Thus, it appears that the choice of lipid dye will have implications for the conclusions that are drawn from studies investigating regional differences in IMTG content and LD morphology in human skeletal muscle in relation to health and disease. ...
... While IMTG utilisation was of a similar magnitude in the central and peripheral regions when using BODIPY, utilisation of IMTG was specific to the peripheral region when examined using ORO. This latter finding is in direct contrast with a recent study employing transmission electron microscopy which reported that IMTG utilisation during high-intensity exercise occurred exclusively in the intermyofibrillar region (Koh et al. 2017). Whilst we acknowledge that using BODIPY may also not entirely replicate the observations made using high-resolution electron microscopy (because we observed exercise-induced decreases in IMTG content in both subcellular regions), our data suggest subcellularspecific IMTG utilisation during exercise should not be investigated using ORO. ...
Despite over 50 years of research, a comprehensive understanding of how intramuscular triglyceride (IMTG) is stored in skeletal muscle and its contribution as a fuel during exercise is lacking. Immunohistochemical techniques provide information on IMTG content and lipid droplet (LD) morphology on a fibre type and subcellular-specific basis, and the lipid dye Oil Red O (ORO) is commonly used to achieve this. BODIPY 493/503 (BODIPY) is an alternative lipid dye with lower background staining and narrower emission spectra. Here we provide the first quantitative comparison of BODIPY and ORO for investigating exercise-induced changes in IMTG content and LD morphology on a fibre type and subcellular-specific basis. Estimates of IMTG content were greater when using BODIPY, which was predominantly due to BODIPY detecting a larger number of LDs, compared to ORO. The subcellular distribution of intramuscular lipid was also dependent on the lipid dye used; ORO detects a greater proportion of IMTG in the periphery (5 μm below cell membrane) of the fibre, whereas IMTG content was higher in the central region using BODIPY. In response to 60 min moderate-intensity cycling exercise, IMTG content was reduced in both the peripheral (- 24%) and central region (- 29%) of type I fibres (P < 0.05) using BODIPY, whereas using ORO, IMTG content was only reduced in the peripheral region of type I fibres (- 31%; P < 0.05). As well as highlighting some methodological considerations herein, our investigation demonstrates that important differences exist between BODIPY and ORO for detecting and quantifying IMTG on a fibre type and subcellular-specific basis.
... To the best of our knowledge, a relationship between PFO or Fat max and performance has not been elucidated in endurance sports dominated by upper body exercise. Interestingly, there are limb-specific differences in fiber-type composition, glycolytic and oxidative capacity, oxygen extraction, and storage of intramuscular triacylglycerol in highly trained cross-country (CC) skiers with equally trained upper and lower bodies, [13][14][15][16] which implies that the relationship between fat oxidation and performance may be different in upper body compared with lower body exercise. Furthermore, even at similar exercise intensities different fat oxidation rates have been reported between whole-body exercise modalities, [17][18][19] but it remains unknown whether this affects the potential relationship between peak fat oxidation and prolonged endurance performance. ...
... The reliance on intramuscular triacylglycerol (IMTG) is higher in trained compared with untrained individuals, 47,48 and in a study on elite CC-skiers, a 53% reduction in IMTG volume fraction in m. triceps brachii together with a solid positive correlation between IMTG degradation during a 20-km CC-skiing time trial and baseline IMTG volume fraction was reported. 16 This implies that the higher peak fat oxidation in ES compared with RS might derive from superior IMTG utilization. ...
The peak fat oxidation rate (PFO) and the exercise intensity that elicits PFO (Fatmax) is associated with endurance performance during exercise primarily involving lower body musculature, but it remains elusive whether these associations are present during predominant upper body exercise. The aim was to investigate the relationship between PFO and Fatmax determined during a graded exercise test on a ski‐ergometer using double poling (GET‐DP) and performance in the long‐distance cross‐country skiing race, Vasaloppet.
Forty‐three healthy men completed GET‐DP and Vasaloppet and were divided into two subgroups; recreational (RS, n=35) and elite skiers (ES, n=8). Additionally, RS completed a cycle‐ergometer GET (GET‐Cycling) to elucidate whether the potential relationships were specific to exercise modality.
PFO (r²=0.10, p=0.044) and Fatmax (r²=0.26, p<0.001) were correlated with performance, however, V̇O2peak was the only independent predictor of performance (adj. R²=0.36) across all participants. In ES, Fatmax was the only variable associated with performance (r²=0.54, p=0.038). Within RS, DP V̇O2peak (r²=0.11, p=0.047) and ski specific training background (r²=0.30, p=0.001) were associated with performance. Between the two GETs Fatmax (r²=0.20, p=0.006) but not PFO (r²=0.07, p=0.135) was correlated. Independent of exercise mode, neither PFO nor Fatmax were associated with performance in RS (p>0.05).
These findings suggest that prolonged endurance performance is related to PFO and Fatmax but foremost to V̇O2peak during predominant upper body exercise. Interestingly, Fatmax may be an important determinant of performance among ES. Among RS, DP V̇O2peak and skiing experience appeared as performance predictors. Additionally, whole‐body fat oxidation seemed specifically coupled to exercise modality.
... In addition, the site of lipid storage, with athletes having more lipid droplets in the intramyofibrillar area than individuals with type 2 diabetes, spatially and functionally matches a high lipid dropletderived fat oxidative capacity. Indeed, reduction in lipid droplet number and content in the intramyofibrillar area upon acute exercise is observed [8,16], suggesting a preferential utilisation of intramyofibrillar lipid droplets during exercise. ...
... Data on changes in lipid droplet-mitochondria tethering during exercise are only available for endurance-trained athletes. In male elite cross-country skiers, lipid dropletmitochondria interactions increase upon an acute exercise bout despite unaltered IMCL content [16]. In endurancetrained women, lipid droplet-mitochondria tethering increases during exercise, with a concomitant reduction in IMCL content [23]. ...
... The latter study suggests that lipid droplet-mitochondrial interaction upon exercise promotes fatty acid oxidation. The seemingly contradictory finding that an exercise-mediated increase in lipid dropletmitochondria interaction is paralleled by reduced IMCL content in women [23] but not in men [16] might originate from sex differences, as reviewed recently [24]. A lack of a reduction in IMCL upon exercise (as observed in the male elite cross-country skiers) may also be reflective of a high IMCL turnover (IMCL utilisation during exercise matches fatty acid incorporation into lipid droplets). ...
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
... Muscle contains two distinct types of mitochondria, intermyofibular and subsarcolemmal, each with a corresponding pool of LDs. In highly trained skiers, LDs positioned in the intermyofibrillar region, but not LDs positioned in the subsarcolemmal region, were consumed during 1 hour of exhaustive exercise (Koh et al., 2017). Another study obtained similar results, showing that trained individuals store the bulk of their LDs in the intermyofibrillar region while individuals with type 2 diabetes had a higher portion in the subsarcolemmal region. ...
Lipid droplets (LDs) are fat storage organelles integral to energy homeostasis and a wide range of cellular processes. LDs physically and functionally interact with many partner organelles, including the endoplasmic reticulum, mitochondria, lysosomes, and peroxisomes. Recent findings suggest that the dynamics of LD interorganelle contacts is in part controlled by LD intracellular motility. LDs can be transported directly by motor proteins along either actin filaments or microtubules, via Kinesin-1, Cytoplasmic Dynein, and type V Myosins. LDs can also be propelled indirectly, by hitchhiking on other organelles, cytoplasmic flows, and potentially actin polymerization. Although the anchors that attach motors to LDs remain elusive, other regulators of LD motility have been identified, ranging from modification of the tracks to motor cofactors to members of the perilipin family of LD proteins. Manipulating these regulatory pathways provides a tool to probe whether altered motility affects organelle contacts and has revealed that LD motility can promote interactions with numerous partners, with profound consequences for metabolism. LD motility can cause dramatic redistribution of LDs between a clustered and a dispersed state, resulting in altered organelle contacts and LD turnover. We propose that LD motility can thus promote switches in the metabolic state of a cell. Finally, LD motility is also important for LD allocation during cell division. In a number of animal embryos, uneven allocation results in a large difference in LD content in distinct daughter cells, suggesting cell-type specific LD needs.
... Interestingly, lower fat oxidation rates were reported during double poling (predominantly upper body exercise) compared with leg skiing in highly trained CC skiers. 12 Lower fat oxidation rates during double poling in highly trained CC skiers may be a consequence of limb-specific differences in the capacities for free fatty acid deliverance and uptake in the skeletal muscle 13 and the utilization of intramuscular triacylglycerol, 14 which could be attributed to limb differences in fiber type composition, glycolytic and oxidative activity, oxygen extraction, and intramuscular triacylglycerol storage. [14][15][16] These findings indicate differences in substrate utilization between limbs, which may reflect a difference in the relationship between fat oxidation rates and performance during prolonged upper body and lower body exercise (≥4 hours). ...
... 12 Lower fat oxidation rates during double poling in highly trained CC skiers may be a consequence of limb-specific differences in the capacities for free fatty acid deliverance and uptake in the skeletal muscle 13 and the utilization of intramuscular triacylglycerol, 14 which could be attributed to limb differences in fiber type composition, glycolytic and oxidative activity, oxygen extraction, and intramuscular triacylglycerol storage. [14][15][16] These findings indicate differences in substrate utilization between limbs, which may reflect a difference in the relationship between fat oxidation rates and performance during prolonged upper body and lower body exercise (≥4 hours). In order to further elucidate potential differences in fat oxidation rates between limbs, a GE protocol assessing upper body PFO and Fat max should be suitable to CC skiers. ...
Peak fat oxidation rate (PFO) and the intensity that elicits PFO (Fatmax) are commonly determined by a validated graded exercise test (GE) on a cycling‐ergometer with indirect calorimetry. However, for upper body exercise fat oxidation rates are not well elucidated and no protocol has been validated. Thus, our aim was to test validity and inter‐method reliability for determination of PFO and Fatmax in trained men using a GE protocol applying double poling on a ski‐ergometer. PFO and Fatmax was assessed during two identical GE tests (GE1 and GE2) and validated against separated short continuous exercise bouts (SCE) at 35%, 50% and 65% of V̇O2peak on the ski‐ergometer in 10 trained men (V̇O2peak: 65.1±1.0 ml·min−1·kg−1, mean±SEM). Between GE tests no differences were found in PFO (GE1: 0.42±0.03; GE2: 0.45±0.03 g·min−1, p=0.256) or Fatmax (GE1: 41±2%; GE2: 43±3% of V̇O2peak, p=0.457) and the intra‐individual coefficient of variation (CV) was 8±2% and 11±2% for PFO and Fatmax, respectively. Between GE and SCE tests PFO (GEavg: 0.44±0.03; SCE; 0.47±0.06 g·min−1, p=0.510) was not different, whereas a difference in Fatmax (GEavg: 42±2%; SCE: 52±4% of V̇O2peak, p=0.030) was observed with a CV of 17±4% and 15±4% for PFO and Fatmax, respectively. In conclusion, GE has a high day‐to‐day reliability in determination of PFO and Fatmax in trained men, whereas it is unclear if PFO and Fatmax determined by GE reflect continuous exercise in general. This article is protected by copyright. All rights reserved.
