From palmitate oxidation, plasma-derived fatty acid oxidation was then calculated by dividing palmitate oxidation rate by the fractional contribution of palmitate to the total FFA concentration. Differences in measured variables before and after training were tested using paired t tests.

Repeated measures one-way ANOVA were used to detect differences in variables in time. For testing differences in blood parameters between treatments, areas under the concentration versus time curve where calculated for 0— min at rest and — during exercise.

On average, subjects completed a total of 31 ± 1. Therefore, the average exercise duration per week was 2. The week training program had no influence on percentage body fat or V o 2max Table 1.

At rest, total fat oxidation was not significantly influenced by the week training program ± 18 vs. Similarly, plasma-derived fatty acid oxidation was not significantly influenced by the week training program ± 24 vs. Plasma-derived fatty acid oxidation during exercise was not significantly influenced by the training program ± 88 vs.

Rate of appearance of FFA was not influenced by the training program, neither at rest ± 41 vs. The percentage of R a that was oxidized was also not influenced by the training program, neither at rest 40 ± 4 vs. At rest, carbohydrate oxidation was not significantly affected by the training program ± 9 vs.

Carbohydrate oxidation during exercise tended to be lower after training 1, ± vs. Energy expenditure, both at rest 4.

Acetate recovery, both at rest Plasma triglyceride concentrations Fig. Both at rest and during exercise, the average concentrations for plasma glucose at rest: 4. The week training program had no effect on two genes involved in the transport and oxidation of blood glucose: hexokinase II 2.

However, the expression of two genes encoding for key enzymes in fatty acid metabolism were affected by the training program: skeletal muscle ACC2 was significantly lower after training ± 24 vs. The expression of UCP3 The effect of endurance training on the contribution of different fat sources to total fat oxidation after endurance training is under debate.

Part of this controversy could be explained by the methodological difficulties in using [ 13 C]- and [ 14 C]-fatty acid tracers to estimate the oxidation of plasma fatty acids, especially in the resting state However, Sidossis et al.

We showed that this acetate recovery is reproducible 25 but has a high interindividual variation and is influenced by infusion period, metabolic rate, respiratory quotient, and body composition 21 and therefore needs to be determined in every individual under similar conditions and at similar time points as the measurement of plasma-derived fatty acid oxidation.

In the present study, we therefore measured the acetate recovery factor at all time points in each individual both before and after the training program at least 7 days separated from the last training session to exclude the influence of the last exercise bout on the measurements and were therefore able to correct plasma-derived fatty acid oxidation rate for loss of label in the TCA cycle.

With the available stable isotope tracer methodology, we cannot distinguish between IMTG- or VLDL-derived fatty acid oxidation. Using electron microscopy, it has previously been shown that endurance-trained athletes have increased IMTG concentrations 36 , and because endurance athletes have an increased fat oxidation capacity, it seems logical that this increased IMTG storage after endurance training is an adaptation mechanism to allow IMTG oxidation during exercise.

The localization of the IMTG near the mitochondria would make these triglyceride pools an efficient source of substrate, especially during exercise. However, biochemical analysis of IMTGs is problematic, and therefore the use of IMTG remains controversial.

On the other hand, the contribution of VLDL-derived fatty acids to fat oxidation during exercise is also still under debate 18 , The increased expression of LPL mRNA after training, as observed in our study, which is in accordance with previous studies showing increased LPL activity after endurance training in rodents 38 , 39 , and the reduced plasma triglyceride levels after the training program suggest that VLDL-derived fatty acids contribute significantly to total fat oxidation.

Alternatively, an increase in LPL after training might serve to provide fatty acids for the replenishment of IMTGs that have been oxidized during exercise Certainly, further studies are needed to clarify the contribution of IMTG- and VLDL-derived fatty acid oxidation to total fat oxidation.

Another important aspect of the present study is that we have examined the effect of a low-intensity training program for only 2 h per week. Because endurance training has been shown to increase the capacity to oxidize fatty acids, it has been proposed to be beneficial in overcoming the disturbances in fat oxidation often observed in obesity and diabetes 9.

To investigate the mechanisms behind the changes in substrate oxidation after the endurance-training program, we measured mRNA levels of several genes involved in glucose and fatty acid metabolism. A muscle biopsy was taken 6—7 days before the training program and 6—7 days after the last training session to exclude the influence of acute exercise on mRNA expression.

The expression of two genes involved in regulatory steps of glucose metabolism, i. As mentioned above, mRNA expression of LPL, which hydrolyzes plasma triglycerides and directs the released FFAs into the tissue 22 , tended to increase after training, suggesting that the capacity of skeletal muscle to hydrolyze VLDL triglycerides may be improved by the training program.

Inside the muscle cell, ACC2 activity has recently been suggested to control the rate of fatty acid oxidation and triglyceride storage ACC2 catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate that inhibits the activity of CPT1. CPT1 catalyzes the rate-limiting step in the transfer of fatty acyl-CoA into mitochondria, where they undergo oxidation.

Although we were not able to measure ACC2 enzyme activity, it is tempting to speculate that a decrease in ACC2 activity after training was responsible for the observed training-induced increase in fat oxidation.

Because high levels of malonyl-CoA have been associated with insulin resistance 42 , the reduction of ACC2 with endurance training could possibly be beneficial in the treatment of type 2 diabetes. Finally, we determined the expression of the human UCP3, which has recently also been implicated in the transport of fatty acids across the inner mitochondrial membrane In a cross-sectional study, we have previously found that UCP3 mRNA was lower in trained than in untrained subjects In the present study, we did not find a significant effect of the training program on UCP3 mRNA expression, suggesting that the training program was not severe enough to result in changes in UCP3 mRNA.

Remarkably, we recently found that, in the same study, UCP3 protein content was significantly decreased after training in all subjects The reason for the discrepancy between the effect of training on UCP3 mRNA expression and protein cannot be deduced from the present study but might involve posttranslational regulation, although the number of subjects is too limited to make such a conclusion.

The mechanism behind this adaptation seems to involve a chronic upregulation of LPL mRNA expression and a chronic downregulation of ACC2, potentially leading to lower malonyl-CoA concentration and less inhibition of CPT1.

In contrast to moderate- to high-intensity endurance training, the mild training protocol did not increase hexokinase II and GLUT4 expression, indicating that specifically fat oxidation was improved. This study was supported by a grant from the Netherlands Organization for Scientific Research NWO to P.

and a grant from the Netherlands Heart Foundation to D. The laboratories are members of the Concerted Action FATLINK FAIR-CT , supported by the European Commission.

The authors thank Paulette Vallier for help in mRNA analysis and Dr. Diraison for making and validating the ACC2 competitor. Address correspondence and reprint requests to Dr. Schrauwen, Department of Human Biology, Maastricht University, P. Box , MD Maastricht, the Netherlands.

E-mail: p. schrauwen hb. Sign In or Create an Account. Search Dropdown Menu. header search search input Search input auto suggest.

filter your search All Content All Journals Diabetes. Advanced Search. User Tools Dropdown. Sign In. Skip Nav Destination Close navigation menu Article navigation. Volume 51, Issue 7. Previous Article Next Article. RESEARCH DESIGN AND METHODS.

Article Information. Article Navigation. Pathophysiology July 01 The Effect of a 3-Month Low-Intensity Endurance Training Program on Fat Oxidation and Acetyl-CoA Carboxylase-2 Expression Patrick Schrauwen ; Patrick Schrauwen.

This Site. Google Scholar. Dorien P. van Aggel-Leijssen ; Dorien P. van Aggel-Leijssen. Gabby Hul ; Gabby Hul. Anton J. Wagenmakers ; Anton J.

Hubert Vidal ; Hubert Vidal. Wim H. Saris ; Wim H. Marleen A. van Baak Marleen A. van Baak. Diabetes ;51 7 — Get Permissions. toolbar search Search Dropdown Menu.

toolbar search search input Search input auto suggest. View large Download slide. TABLE 1 Subject characteristics. Age years View Large. TABLE 2 Palmitate and breath CO 2 enrichment before and after training. Time min. Breath 13 CO 2 enrichment TTR × 1, Physical Activity and Health: A Report of the Surgeon General.

Schrauwen P, Westerterp KR: The role of high-fat diets and physical activity in the regulation of body weight. Br J Nutr. Zurlo F, Larson K, Bogardus C, Ravussin E: Skeletal muscle metabolism is a major determinant of resting energy expenditure.

J Clin Invest. Blaak EE, van Aggel-Leijssen DP, Wagenmakers AJ, Saris WH, van Baak MA: Impaired oxidation of plasma-derived fatty acids in type 2 diabetic subjects during moderate-intensity exercise.

Colberg SR, Simoneau J-A, Thaete FL, Kelley DE: Skeletal muscle utilization of free fatty acids in women with visceral obesity. He J, Watkins S, Kelley DE: Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity.

Pan DA, Lillioja S, Kriketos AD, Milner MR, Baur LA, Bogardus C, Jenkins AB, Storlien LH: Skeletal muscle triglyceride levels are inversely related to insulin action.

Dobbins RL, Szczepaniak LS, Bentley B, Esser V, Myhill J, McGarry JD: Prolonged inhibition of muscle carnitine palmitoyltransferase-1 promotes intramyocellular lipid accumulation and insulin resistance in rats. Holloszy J, Coyle EF: Adaptations of skeletal muscle to endurance exercise and their metabolic consequences.

J Appl Physiol. Turcotte LP, Richter EA, Kiens B: Increased plasma FFA uptake and oxidation during prolonged exercise in trained vs. untrained humans. Am J Physiol Endocrinol Metab. van Loon LJ, Jeukendrup AE, Saris WH, Wagenmakers AJ: Effect of training status on fuel selection during submaximal exercise with glucose ingestion.

Klein S, Coyle EF, Wolfe RR: Fat metabolism during low-intensity exercise in endurance-trained and untrained men. Horowitz JF, Leone TC, Feng W, Kelly DP, Klein S: Effect of endurance training on lipid metabolism in women: a potential role for PPARalpha in the metabolic response to training.

Hurley BF, Nemeth PM, Martin WHI, Hagberg JM, Dalsky GP, Holloszy JO: Muscle triglyceride utilization during exercise: effect of training. Martin WH III, Dalsky GP, Hurley BF, Matthews DE, Bier DM, Hagberg JM, Rogers MA, King DS, Holloszy JO: Effect of endurance training on plasma free fatty acid turnover and oxidation during exercise.

Phillips SM, Green HJ, Tarnopolsky MA, Heigenhauser GF, Hill RE, Grant SM: Effects of training duration on substrate turnover and oxidation during exercise.

Kiens B, Richter EA: Utilization of skeletal muscle triacylglycerol during postexercise recovery in humans. Kiens B, Essen-Gustavsson B, Christensen NJ, Saltin B: Skeletal muscle substrate utilization during submaximal exercise in man: effect of endurance training.

J Physiol Lond. Sidossis LS, Coggan AR, Gastaldelli A, Wolfe RR: A new correction factor for use in tracer estimations of plasma fatty acid oxidation.

Schrauwen P, Wagenmakers AJM, Marken Lichtenbelt WDv, Saris WHM, Westerterp KR: Increase in fat oxidation on a high-fat diet is accompanied by an increase in triglyceride-derived fatty acid oxidation. Schrauwen P, Blaak EE, Van Aggel-Leijssen DP, Borghouts LB, Wagenmakers AJ: Determinants of the acetate recovery factor: implications for estimation of [13C]substrate oxidation.

Clin Sci Colch. Levak-Frank S, Radner H, Walsh A, Stollberger R, Knipping G, Hoefler G, Sattler W, Weinstock PH, Breslow JL, Zechner R: Muscle-specific overexpression of lipoprotein lipase causes a severe myopathy characterized by proliferation of mitochondria and peroxisomes in transgenic mice.

Abu-Elheiga L, Matzuk MM, Abo-Hashema KA, Wakil SJ: Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Schrauwen P, Saris WH, Hesselink MK: An alternative function for human uncoupling protein 3: protection of mitochondria against accumulation of nonesterified fatty acids inside the mitochondrial matrix.

FASEB J. Schrauwen P, Aggel-Leijssen DPCv, Marken Lichtenbelt WDv, Baak MAv, Gijsen AP, Wagenmakers AJM: Validation of the [1,2- 13 C] -acetate recovery factor for correction of [U- 13 C] -palmitate oxidation rates in humans.

J Physiol. On the other hand, it has also been observed that the contribution of IMCL to total fat oxidation was higher in trained athletes vs individuals with type 2 diabetes when matched for plasma NEFA levels [ 1 ]. This suggests that liberation of fatty acid from myocellular lipid droplets in individuals with type 2 diabetes is compromised relative to trained athletes Fig.

Skeletal muscle lipid metabolism: acute exercise and endurance training effects. Contrarily, in people who are metabolically compromised i. obese and type 2 diabetic individuals , the same amount of IMCL is stored in fewer, but larger lipid droplets b.

Triacylglycerols are shown within the lipid droplets. Lipid droplet—mitochondria interaction is higher in athletes vs metabolically compromised individuals.

Upon endurance-exercise intervention training depicted by the calendar , lipid droplet morphology and lipid droplet—mitochondria interactions changes towards the athlete-like phenotype in individuals who are metabolically compromised c.

d , e During an acute endurance exercise bout, fatty acids originating from lipid droplets, as well as from the circulation are used as an energy source. Endurance-trained athletes rely more heavily on IMCL to fuel exercise and have a higher lipid-droplet turnover i.

storage of circulation-derived fatty acids in lipid droplets and release of fatty acids originating from lipid droplets for fatty acid oxidation than those who are metabolically compromised.

This reduces the number of lipid droplets, as depicted by a smaller stack of lipid droplets in d vs e. The interaction between lipid droplets and mitochondria is higher in endurance-trained athletes. This may facilitate fatty acid oxidation during exercise. Changes that occur upon exercise training in metabolically compromised individuals are shown in b and c , i.

an increased lipid droplet—mitochondrial interaction, and smaller and more lipid droplets. The hypothesised changes upon an acute exercise bout after metabolically compromised individuals have followed an endurance training intervention are represented in e and f : lipid droplet—mitochondrial interaction is anticipated to increase during exercise, and lipid turnover and IMCL utilisation starts to mimic the events in athletes.

Hypothetical changes are depicted using transparent illustrations. This figure is available as part of a downloadable slideset. Myocellular lipid droplets are viewed as dynamic organelles that store and release fatty acids upon changes in energy demand and supply [ 10 ].

Lipid droplet characteristics, such as number, size, location and protein decoration, are determinants of insulin resistance [ 5 , 11 ] and are remarkably different between athletes and individuals with type 2 diabetes.

Unlike athletes, those with type 2 diabetes store more lipid droplets in the subsarcolemmal region [ 5 , 8 , 11 ] in glycolytic type II muscle fibres [ 5 ].

Lipid droplet coating proteins of the perilipin PLIN family play a role in lipid-droplet turnover by interacting with lipases, such as adipose triglyceride lipase ATGL and hormone sensitive lipase HSL , and their co-activators.

PLIN2, PLIN3 and PLIN5 are the main PLINs present in human skeletal muscle [ 10 ]. PLIN2 negatively regulates ATGL-mediated lipid droplet lipolysis by hindering access of ATGL to the lipid droplet surface [ 12 ]. PLIN3 coats nascent lipid droplets and associates with fat oxidation rates [ 13 ].

PLIN5 regulates lipolytic rate in an energy demand-dependent fashion to match fatty acid release from lipid droplets with mitochondrial fatty acid oxidation [ 10 ]. While acute exercise does not affect total PLIN5 or ATGL content [ 1 ], redistribution of PLIN5 and ATGL upon exercise to match the acute changes in energy demand may occur.

Examination of the subcellular redistribution of proteins involved in myocellular lipid droplet lipolysis upon exercise has recently become possible at the level of individual lipid droplets via advanced imaging [ 10 ]. Thus, it has been shown that healthy lean participants preferentially use lipid droplets coated with PLIN2 [ 14 , 15 ] and PLIN5 [ 14 ] during endurance exercise.

Interestingly, the number of PLIN5-coated lipid droplets in endurance-trained athletes is higher than in individuals type 2 diabetes [ 6 ]. In addition, we observed that people with type 2 diabetes have a higher myocellular PLIN2 protein content than endurance-trained athletes [ 5 ].

Although it is commonly accepted that PLIN2 that is not bound to the lipid droplet surface is ubiquitinated and targeted for degradation, it has not yet been proven that the higher PLIN2 content in the muscle of type 2 diabetic individuals indeed implies increased decoration of the lipid droplet surface with PLIN2.

Taken together, this indicates that the muscle of endurance-trained athletes is equipped for a higher exercise-mediated lipid-droplet turnover than that of individuals with type 2 diabetes. 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 droplet-derived 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.

These studies provide novel and important insights on lipid droplet utilisation in relation to their location and protein decoration and give a better understanding of how lipid-droplet turnover is regulated during exercise in healthy individuals.

This type of data, however, is lacking in individuals with type 2 diabetes. For full comprehension of why lipid droplet utilisation is compromised during endurance exercise in individuals with type 2 diabetes, a tracer study to make the distinction between whether plasma or lipid droplet-derived fatty acids are used for oxidation, along with lipid droplet-specific analysis of lipid droplet coat proteins and analysis of lipid droplet location, should be performed pre- and post-endurance exercise in individuals with type 2 diabetes.

The more pronounced utilisation of intramyofibrillar lipid droplets during exercise may well be related to the observation that, in skeletal muscle, most lipid droplets predominantly in the trained state, in the intramyofibrillar area are in close proximity to mitochondria [ 17 , 18 , 19 ].

At the interaction sites of mitochondria and lipid droplets, there is an abundance of PLIN5 [ 18 ]. In line with the role of PLIN5 in matching lipolytic rate to fatty acid oxidation rate, PLIN5 may play a role in shuttling or chaperoning lipid droplet-released fatty acids to mitochondria for oxidation [ 18 ].

Recent studies have suggested that, when interacting with lipid droplets, mitochondria have different cellular functions than non-lipid-droplet-interacting mitochondria [ 19 , 20 ]. For skeletal muscle, it has been suggested that mitochondria that are in contact with lipid droplets have a greater capacity for ATP production than non-lipid-droplet-interacting mitochondria [ 19 ].

Thus, lipid droplet—mitochondrial tethering may facilitate high fat oxidation by liberating fatty acids in the direct vicinity of mitochondria with a high capacity to oxidise fatty acids, thereby contributing to ATP maintenance during exercise.

At present, experimental proof in humans for these functional processes is lacking. It should be noted, though, that trained individuals possess higher PLIN5 levels, have more PLIN5-coated lipid droplets [ 6 ] and may, thus, have more lipid droplet—mitochondrial interaction sites than individuals with type 2 diabetes.

Lipid droplet—mitochondria interactions are not different between healthy lean and healthy obese participants [ 21 , 22 ], but these data are lacking for individuals with type 2 diabetes in comparison with endurance-trained athletes. Data on changes in lipid droplet—mitochondria tethering during exercise are only available for endurance-trained athletes.

In male elite cross-country skiers, lipid droplet—mitochondria interactions increase upon an acute exercise bout despite unaltered IMCL content [ 16 ].

In endurance-trained 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 droplet—mitochondria 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.

The underlying mechanism for increased mitochondria—lipid droplet tethering during exercise and whether PLIN5 is important for the capacity to increase lipid droplet—mitochondrial tethering are so far unknown. Furthermore, it is not clear whether lipid droplet—mitochondrial tethering is disturbed in individuals with type 2 diabetes.

The literature indicates that PLIN5 is important for lipid droplet—mitochondrial tethering [ 18 , 20 ] in oxidative tissues. PLIN5 protein quantification in individual lipid droplets should be performed concomitantly with lipid droplet—mitochondrial interaction analyses in athletes and in those with type 2 diabetes upon an acute exercise bout to gain a better understanding of how lipid droplet—mitochondrial tethering works and if the capacity to tether additional mitochondria to lipid droplets upon exercise is compromised in individuals with type 2 diabetes Fig.

Compromised mitochondrial respiratory capacity is frequently reported in type 2 diabetes [ 25 , 26 , 27 ] and obesity [ 26 ], albeit not always confirmed [ 28 ]. A potent way to increase mitochondrial respiratory capacity and a concomitant increase in fat oxidation is endurance training.

Several studies have shown that mitochondrial respiratory capacity and fat oxidation increases upon endurance exercise training, even in type 2 diabetic [ 25 , 29 ] and obese [ 25 , 30 ] participants. As well as increasing mitochondrial capacity, endurance training also is an effective intervention to improve fat oxidation and modulate fat storage in the skeletal muscle of lean sedentary participants [ 31 ].

Several studies have shown that endurance training 4—16 weeks may affect lipid droplet characteristics without major changes in total IMCL content in type 2 diabetic [ 5 , 11 , 25 , 29 , 32 ], obese [ 21 , 25 , 33 ], and healthy lean, sedentary [ 21 , 34 , 35 ] participants.

In most of these studies, however, insulin sensitivity improved. To understand this seemingly paradoxical observation, we need to focus on what happens at the lipid droplet level, rather than at the total IMCL content level.

Upon exercise training, lipid droplet size [ 5 , 22 , 32 ] and subsarcolemmal lipid droplet content [ 11 , 21 , 22 ] reduces, while intramyofibrillar lipid droplet content increases [ 22 ]. These exercise-mediated changes, in previously untrained insulin-resistant individuals, resembles the IMCL storage pattern observed in insulin-sensitive endurance-trained athletes.

In contrast, in individuals with type 2 diabetes, fewer but larger lipid droplets are observed, with a higher fraction of lipid droplets in the subsarcolemmal region of type II muscle fibres [ 5 ].

Lipid droplet—mitochondrial tethering increases upon endurance training in obese participants [ 21 , 22 ], while no such effect was observed in individuals with type 2 diabetes [ 36 ]. All of these athlete-like changes were observed in training programmes that were carried out for more than 10 weeks Fig.

Short-term training 4 weeks in obese participants did not change lipid droplet size and number, but lipid droplet—mitochondrial interaction was increased [ 33 ]. This indicates that an athlete-like shift in lipid droplet phenotype permits storage of IMCL without impeding insulin sensitivity.

A training-induced improvement in lipid droplet—mitochondrial tethering appears to be an early adaptation of endurance training that is crucial for remodelling of the IMCL storage pattern.

Training studies in healthy lean participants show that endurance training for 6 weeks promotes IMCL utilisation during exercise [ 14 , 35 , 37 ]. While in the untrained state PLIN2- and PLIN5-coated lipid droplets are preferentially used during exercise, 6 weeks of endurance training resulted in preferred utilisation of PLIN5-coated lipid droplets during exercise [ 14 ].

While the effect of exercise training on proteins involved in lipid-droplet turnover, such as PLIN2, PLIN5 and ATGL, has been measured, data on the effect of endurance training on IMCL utilisation and lipid-droplet turnover during an exercise bout in obese participants and individuals with type 2 diabetes is lacking Fig.

PLIN5 gene expression and protein content upon an endurance training intervention increases in obese participants and individuals with type 2 diabetes [ 5 , 33 , 38 , 39 ]. For PLIN2 [ 5 , 33 , 38 , 39 , 40 ], PLIN3 [ 5 , 33 , 38 ] and ATGL [ 5 , 38 ] the training effects are less consistent, either showing an increase or no change in the general population.

Increased PLIN5 protein content upon endurance training indicates that IMCL use during exercise is facilitated and that lipolysis rates of lipid droplets are better matched to mitochondrial fatty acid oxidation rates in individuals with type 2 diabetes vs baseline. To test these mechanisms in a human setting, acute exercise studies in participants with type 2 diabetes are needed and should include fatty acid tracers and muscle biopsies to study IMCL utilisation during exercise, and changes in PLIN5 protein content at the lipid droplet surface before and after training.

Additionally, in vitro studies in human primary myotubes obtained from endurance-trained athletes and individuals with type 2 diabetes, in combination with imaging of fatty acid tracers with live-cell imaging, can give important insights into turnover of individual lipid droplets upon exposure to different stimuli resembling exercise.

Moreover, to study the direct role of PLIN5 in lipid-droplet turnover, these in vitro studies should be combined with overexpression of fluorescently tagged PLIN5 to test whether PLIN5-coated lipid droplets indeed have a higher lipid-droplet turnover.

In most of the studies discussed above, the timing of meal intake relative to the training sessions was not monitored strictly or intentionally timed so that participants trained fasted.

Interestingly, training in the overnight fasted state has gained popularity to promote fat oxidative capacity. Upon fasting, adipose tissue lipolysis and plasma NEFA levels increase.

The increase in NEFA drives myocellular uptake of fatty acids and, thus, can promote IMCL storage and oxidation of fatty acids. Indeed, fat oxidation rates during acute exercise in the fasted state are higher than in the fed state [ 41 , 42 ].

Also, the sustained increase in NEFA levels upon exercise in the fasted state can hypothetically provide ligands for peroxisome proliferator-activated receptor PPAR -mediated gene expression and, thereby, promote an adaptive response in regard to fat metabolism. Interestingly, endurance training in the fasted state improves glucose tolerance to a greater extent than training in the fed state [ 43 ].

Data on functional adaptations like increased fat oxidative capacity following training in the fasted state are inconsistent [ 35 , 37 , 44 , 45 ]. Acute exercise studies measuring IMCL utilisation with fatty acid tracers and in muscle biopsies have been performed in the fasted state and show IMCL utilisation during exercise [ 1 , 14 , 15 ].

Compared with exercise in the fed state, exercising in the fasted state results in higher NEFA levels, higher fat oxidation rates and a drop in IMCL content [ 42 ]. We previously observed that, over a wide range of interventions, elevated plasma fatty acids promote IMCL storage.

Whether this also occurs during exercise in the fasted state and translates into a higher flux of fatty acids in lipid droplets during exercise remains to be studied. Upon 6 weeks of endurance training, IMCL content drops during a single exercise bout in the fasted state.

This drop in IMCL content upon acute exercise was similar if the training was performed in the carbohydrate-fed state vs that fasted state [ 35 , 37 ]. Currently, most training interventions under fasted conditions have only been performed in healthy lean participants and translation towards the type 2 diabetes population should be done carefully.

Based on the results in healthy lean individuals, training while fasted may induce more IMCL remodelling due to a higher stimulus for lipid-droplet turnover in individuals with type 2 diabetes.

Before drawing these conclusions, training interventions in the fasted vs fed state should be performed in individuals with type 2 diabetes. Intrahepatic lipid IHL storage is associated with type 2 diabetes and cardiovascular diseases.

The poor accessibility of the liver in healthy individuals means that most studies towards the effect of acute exercise and exercise training on IHLs and lipid metabolism in humans are based upon non-invasive techniques, such as MRI and tracer studies.

Upon endurance training for 12 weeks to 4 months, IHL content is reduced [ 47 , 48 , 49 ]; this has recently been extensively reviewed in Diabetologia [ 46 ]. While a drop in IHL levels after endurance training generally occurs in the absence of changes in body weight, we observed that the training-mediated drop in IHL correlated with a drop in body fat mass [ 46 , 47 ].

Increased IHL storage is, in general, not associated with disturbed VLDL-triacylglycerol secretion rates [ 46 ], and data on VLDL -triacylglycerol secretion rates upon endurance training is contradictory, either showing no change [ 49 ] or a decrease [ 50 ] Table 1.

It is tempting to speculate that exercise-mediated improvements in whole-body insulin sensitivity include reduced de novo lipogenesis in the liver, thereby contributing to a lower IHL content. While we are not aware of any studies underpinning this notion, it is interesting to note that a short-term 7 day training programme resulted in altered composition but not content of IHL.

After training, IHL contained more polyunsaturated fatty acids [ 51 ]; this is in line with lower de novo lipogenesis, which gives rise to saturated fat Fig. Liver lipid metabolism: acute exercise and endurance training effects. IHL content is lower in healthy lean individuals than in those who are metabolically compromised.

This may be a consequence of lower plasma NEFA levels and lower rates of de novo lipogenesis in lean vs metabolically compromised individuals. a Upon acute endurance exercise, especially in the fasted state, IHL content rises, most likely due to increased plasma NEFA levels. Furthermore, VLDL-triacylglycerol secretion rates drop during acute exercise, and de novo lipogenesis is blunted due to higher postprandial glycogen synthesis by the muscle, thereby reducing glucose availability for lipid synthesis by the liver.

b The underlying mechanisms that are hypothetically involved during endurance training in metabolically compromised individuals are shown exercise training depicted by the calendar ; these include reduced de novo lipogenesis, and improved postprandial glucose and NEFA uptake by the muscle and, thus, lower availability of glucose and NEFA for the liver to synthesise lipids.

In addition, VLDL-triacylglycerol secretion rate upon endurance training in metabolically compromised individuals drops or is unchanged. As exercise training reduces IHL content [ 47 , 48 ], one could suggest that IHL also drops upon acute exercise. We observed that, upon 2 h of endurance exercise, IHL content was unaffected, irrespective of participants being in the fed or fasted stated.

After exercise and upon recovery in the fasted state, however, we observed an increase in IHL [ 41 ]. Additionally, IHL increases upon an exercise bout in active lean participants who consumed a light meal before the start of the exercise [ 52 ].

Interestingly, in both studies [ 41 , 52 ], increased IHL content after exercise occurred in the presence of elevated plasma NEFA levels. If this rise in plasma NEFAs is prevented by providing a glucose drink every half hour during and after exercise, IHL does not increase. This indicates that the rise in plasma NEFA levels upon exercise drives the increased IHL content after an exercise bout.

IHL can be used during exercise, upon secretion of VLDL-triacylglycerols into the bloodstream. VLDL-triacylglycerol kinetic analyses during an acute exercise bout in the fasted state show that VLDL-triacylglycerol secretion rates drop during exercise and that the contribution of these particles to total energy expenditure is decreased [ 53 ].

Thus, besides the increase in NEFA influx, the lower VLDL-triacylglycerol secretion rates during exercise may also contribute to the increase in IHL content after acute exercise in the fasted state Fig. In lean, normoglycaemic but insulin-resistant individuals, postprandial IHL synthesis and de novo lipogenesis is lower after a single bout of exercise compared with rest [ 54 ].

Overall, IHL may increase upon acute exercise, but is lower after training, possibly due to lower postprandial de novo lipogenesis during recovery. It is also lower in endurance-trained individuals. It is currently unknown how the apparent increase in IHL after acute exercise turns into reduced IHL content after endurance training.

We cannot exclude that training, per se, is not the major determinant of IHL but that the dietary habits of trained individuals may also make an important contribution. IMCL and IHL content are increased, and fat oxidative capacity decreased in metabolically compromised individuals, such as obese individuals and those with type 2 diabetes.

While endurance exercise training reduces total intracellular fat content in the liver, the effects in muscle indicate remodelling rather than lowering of the myocellular lipid droplet pool.

In fact, in most populations and under most conditions, endurance exercise training augments IMCL content. Thus, the ability of exercise to modulate lipid droplet dynamics in the liver and muscle contributes to differences in fat oxidative metabolism.

Endurance training in individuals with type 2 diabetes remodels IMCL content towards an athlete-like phenotype, while IHL content is reduced. While many training intervention studies have been performed in metabolically compromised individuals, the effects of acute exercise have not been extensively studied, particularly not in participants with type 2 diabetes.

Thus, it is unclear why IMCL utilisation during exercise is lower in individuals with type 2 diabetes and whether the observed IMCL remodelling towards the athlete-like phenotype in these individuals also translates into the anticipated increase in IMCL utilisation during exercise.

Study findings on the effects of sex differences and exercise intensity on IMCL use during exercise or lipid droplet remodelling upon training are either contradictory or lacking. Compared with skeletal muscle, the underlying mechanisms of the effects of exercise and training on IHL are even more poorly understood.

The reduction in IHL content upon training that is observed in metabolically compromised individuals may partly originate from reduced postprandial de novo lipogenesis. Since diurnal rhythms are present in lipid metabolism, future studies should also focus on the effect of timing of exercise on the parameters discussed in this review in order to elucidate the optimal conditions for exercise-induced improvements in insulin sensitivity in individuals with type 2 diabetes.

Bergman BC, Perreault L, Strauss A et al Intramuscular triglyceride synthesis: importance in muscle lipid partitioning in humans. Am J Physiol Endocrinol Metab 2 :E—E Article CAS PubMed Google Scholar.

Kiens B Skeletal muscle lipid metabolism in exercise and insulin resistance. Physiol Rev 86 1 — van Loon LJ, Greenhaff PL, Constantin-Teodosiu D, Saris WH, Wagenmakers AJ The effects of increasing exercise intensity on muscle fuel utilisation in humans. J Physiol 1 — Article PubMed PubMed Central Google Scholar.

Goodpaster BH, He J, Watkins S, Kelley DE Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes.

J Clin Endocrinol Metab 86 12 — Mol Metab — Article CAS PubMed PubMed Central Google Scholar. Gemmink A, Daemen S, Brouwers B et al Dissociation of intramyocellular lipid storage and insulin resistance in trained athletes and type 2 diabetes patients; involvement of perilipin 5?

J Physiol 5 — Boon H, Blaak EE, Saris WH, Keizer HA, Wagenmakers AJ, van Loon LJ Substrate source utilisation in long-term diagnosed type 2 diabetes patients at rest, and during exercise and subsequent recovery. Diabetologia 50 1 — Chee C, Shannon CE, Burns A et al Relative contribution of intramyocellular lipid to whole-body fat oxidation is reduced with age but subsarcolemmal lipid accumulation and insulin resistance are only associated with overweight individuals.

Diabetes 65 4 — van Loon LJ, Manders RJ, Koopman R et al Inhibition of adipose tissue lipolysis increases intramuscular lipid use in type 2 diabetic patients. Diabetologia 48 10 — Gemmink A, Goodpaster BH, Schrauwen P, Hesselink MKC Intramyocellular lipid droplets and insulin sensitivity, the human perspective.

Biochim Biophys Acta Mol Cell Biol Lipids 10 Pt B — Nielsen J, Mogensen M, Vind BF et al Increased subsarcolemmal lipids in type 2 diabetes: effect of training on localization of lipids, mitochondria, and glycogen in sedentary human skeletal muscle.