Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function

Adiponectin (Ad) is linked to various disease states and mediates antidiabetic and anti-inflammatory effects. While it was originally thought that Ad expression was limited to adipocytes, we demonstrate here that Ad is expressed in mouse skeletal muscles and within differentiated L6 myotubes, as assessed by RT-PCR, Western blot, and immunohistochemical analyses. Serial muscle sections stained for fiber type, lipid content, and Ad revealed that muscle fibers with elevated intramyocellular Ad expression were consistently type IIA and IID fibers with detectably higher intramyocellular lipid (IMCL) content. To determine the effect of Ad on muscle phenotype and function, we used an Ad-null [knockout (KO)] mouse model. Body mass increased significantly in 24-wk-old KO mice [+5.5 +/- 3% relative to wild-type mice (WT)], with no change in muscle mass observed. IMCL content was significantly increased (+75.1 +/- 25%), whereas epididymal fat mass, although elevated, was not different in the KO mice compared with WT (+35.1 +/- 23%; P = 0.16). Fiber-type composition was unaltered, although type IIB fiber area was increased in KO mice (+25.5 +/- 6%). In situ muscle stimulation revealed lower peak tetanic forces in KO mice relative to WT (-47.5 +/- 6%), with no change in low-frequency fatigue rates. These data demonstrate that the absence of Ad expression causes contractile dysfunction and phenotypical changes in skeletal muscle. Furthermore, we demonstrate that Ad is expressed in skeletal muscle and that its intramyocellular localization is associated with elevated IMCL, particularly in type IIA/D fibers.     

Exercise-induced alterations in intramyocellular lipids and insulin resistance: the athlete's paradox revisited

We previously reported an "athlete's paradox" in which endurance-trained athletes, who possess a high oxidative capacity and enhanced insulin sensitivity, also have higher intramyocellular lipid (IMCL) content. The purpose of this study was to determine whether moderate exercise training would increase IMCL, oxidative capacity of muscle, and insulin sensitivity in previously sedentary overweight to obese, insulin-resistant, older subjects. Twenty-five older (66.4 +/- 0.8 yr) obese (BMI = 30.3 +/- 0.7 kg/m2) men (n = 9) and women (n = 16) completed a 16-wk moderate but progressive exercise training program. Body weight and fat mass modestly but significantly (P < 0.01) decreased. Insulin sensitivity, measured using the euglycemic hyperinsulinemic clamp, was increased (21%, P = 0.02), with modest improvements (7%, P = 0.04) in aerobic fitness (Vo2peak). Histochemical analyses of IMCL (Oil Red O staining), oxidative capacity [succinate dehydrogenase activity (SDH)], glycogen content, capillary density, and fiber type were performed on skeletal muscle biopsies. Exercise training increased IMCL by 21%. In contrast, diacylglycerol and ceramide, measured by mass spectroscopy, were decreased (n = 13; -29% and -24%, respectively, P < 0.05) with exercise training. SDH (19%), glycogen content (15%), capillary density (7%), and the percentage of type I slow oxidative fibers (from 50.8 to 55.7%), all P < or = 0.05, were increased after exercise. In summary, these results extend the athlete's paradox by demonstrating that chronic exercise in overweight to obese older adults improves insulin sensitivity in conjunction with favorable alterations in lipid partitioning and an enhanced oxidative capacity within muscle. Therefore, several key deleterious effects of aging and/or obesity on the metabolic profile of skeletal muscle can be reversed with only moderate increases in physical activity.     

Muscle glycogen content in type 2 diabetes mellitus

Muscle contains the largest reservoir of glycogen (Glyc), a depot that is closely regulated and with influence on insulin sensitivity. The current study examines muscle Glyc in type 2 diabetes mellitus (T2DM) and obesity and with respect to muscle fiber type, intramyocellular lipid content (IMCL), and mitochondrial function (oxidative enzyme activity; OX-Enz). There is increasing interest in the relation of IMCL and mitochondrial dysfunction with insulin resistance (IR), yet the association with muscle Glyc has not been examined with regard to these parameters. Using a quantitative histological approach specific to muscle fiber types, we assessed muscle Glyc, IMCL, and OX-Enz in vastus lateralis obtained by percutaneous biopsy in lean nondiabetic (L; n = 16), obese nondiabetic (Ob; n = 15), and T2DM volunteers (n = 14). Insulin sensitivity was estimated using homeostasis model assessment (HOMA)-IR. Muscle Glyc was reduced in T2DM, a deficit evident for type IIa fibers, yet minor in types I and IIb fibers. Low Glyc in T2DM correlated with fasting hyperglycemia. Also, in T2DM and Ob, there was significantly higher IMCL and lower OX-Enz in all fiber types. The IMCL-to-OX-Enz ratio, especially for type I fibers, correlated strongly with IR. Similarly, a Glyc-to-OX-Enz ratio correlated with IR, particularly for type IIb fibers. This ratio tended to be higher in Ob and T2DM. In summary, there is decreased muscle Glyc in T2DM yet a disproportional Glyc-to-OX-Enz relationship that is related to IR, although not as robustly as the IMCL-to-OX-Enz ratio.     

Myofibrillar distribution of succinate dehydrogenase activity and lipid stores differs in skeletal muscle tissue of paraplegic subjects

Lack of physical activity has been related to an increased risk of developing insulin resistance. This study aimed to assess the impact of chronic muscle deconditioning on whole body insulin sensitivity, muscle oxidative capacity, and intramyocellular lipid (IMCL) content in subjects with paraplegia. Nine subjects with paraplegia and nine able-bodied, lean controls were recruited. An oral glucose tolerance test was performed to assess whole body insulin sensitivity. IMCL content was determined both in vivo and in vitro using (1)H-magnetic resonance spectroscopy and fluorescence microscopy, respectively. Muscle biopsy samples were stained for succinate dehydrogenase (SDH) activity to measure muscle fiber oxidative capacity. Subcellular distributions of IMCL and SDH activity were determined by defining subsarcolemmal and intermyofibrillar areas on histological samples. SDH activity was 57 ± 14% lower in muscle fibers derived from subjects with paraplegia when compared with controls (P < 0.05), but IMCL content and whole body insulin sensitivity did not differ between groups. In muscle fibers taken from controls, both SDH activity and IMCL content were higher in the subsarcolemmal region than in the intermyofibrillar area. This typical subcellular SDH and IMCL distribution pattern was lost in muscle fibers collected from subjects with paraplegia and had changed toward a more uniform distribution. In conclusion, the lower metabolic demand in deconditioned muscle of subjects with paraplegia results in a significant decline in muscle fiber oxidative capacity and is accompanied by changes in the subcellular distribution patterns of SDH activity and IMCL. However, loss of muscle activity due to paraplegia is not associated with substantial lipid accumulation in skeletal muscle tissue.     

Effect of weight loss on muscle lipid content in morbidly obese subjects

The purpose of this study was to test the hypothesis that weight loss results in a reduction in intramuscular lipid (IMCL) content that is concomitant with enhanced insulin action. Muscle biopsies were obtained from morbidly obese individuals [body mass index (BMI) 52.2 +/- 2.5 kg/m(2); n = 6] before and after gastric bypass surgery, an intervention that improves insulin action. With intervention, there was a 47% reduction (P < 0.01) in BMI and a 93% decrease in homeostasis model assessment, or HOMA (7.0 +/- 1.9 vs. 0.5 +/- 0.1). Histochemically determined IMCL content decreased (P < 0.05) by approximately 30%. In relation to fiber type, IMCL was significantly higher in type I vs. type II fibers. In both fiber types, there were reductions in IMCL and trends for muscle atrophy. Despite these two negating factors, the IMCL-to-fiber area ratio still decreased by approximately 44% with weight loss. In conclusion, despite differing initial levels and possible atrophy, weight loss appears to decrease IMCL deposition to a similar relative extent in type I and II muscle fibers. This reduction in intramuscular triglyceride may contribute to enhanced insulin action seen with weight loss.     

High-fat diet overrules the effects of training on fiber-specific intramyocellular lipid utilization during exercise

In this study, we compared the effects of endurance training in the fasted state (F) vs. the fed state [ample carbohydrate intake (CHO)] on exercise-induced intramyocellular lipid (IMCL) and glycogen utilization during a 6-wk period of a hypercaloric (～+30% kcal/day) fat-rich diet (HFD; 50% of kcal). Healthy male volunteers (18-25 yrs) received a HFD in conjunction with endurance training (four times, 60-90 min/wk) either in F (n = 10) or with CHO before and during exercise sessions (n = 10). The control group (n = 7) received a HFD without training and increased body weight by ～3 kg (P < 0.001). Before and after a HFD, the subjects performed a 2-h constant-load bicycle exercise test in F at ～70% maximal oxygen uptake rate. A HFD, both in the absence (F) or presence (CHO) of training, elevated basal IMCL content by ～50% in type I and by ～75% in type IIa fibers (P < 0.05). Independent of training in F or CHO, a HFD, as such, stimulated exercise-induced net IMCL breakdown by approximately twofold in type I and by approximately fourfold in type IIa fibers. Furthermore, exercise-induced net muscle glycogen breakdown was not significantly affected by a HFD. It is concluded that a HFD stimulates net IMCL degradation by increasing basal IMCL content during exercise in type I and especially IIa fibers. Furthermore, a hypercaloric HFD provides adequate amounts of carbohydrates to maintain high muscle glycogen content during training and does not impair exercise-induced muscle glycogen breakdown.     

Cross contamination of intramyocellular lipid signals through loss of bulk magnetic susceptibility effect differences in human muscle using (1)H-MRSI at 4 T.

Cross contamination of intramyocellular lipid (IMCL) signals through loss of bulk magnetic susceptibility (BMS) differences was detected in human muscles using proton magnetic resonance spectroscopic imaging ((1)H-MRSI) at 4 T by varying nominal voxel sizes on healthy subjects. In soleus muscle the IMCL content estimated in 1.00-ml-sized voxels was 15% and 30% higher than that in 0.25-ml voxels for nonobese (P < 0.05) and obese (P < 0.01) subjects, respectively, whereas no effect was observed on IMCL estimation in tibialis posterior (TP) and tibialis anterior (TA) regions for different voxel sizes. The unbiased 0.25-ml voxel size (1)H-MRSI method was applied to measure IMCL content in nonobese sedentary (NOB-Sed), moderately trained (Ath), sedentary obese (OB), and Type 2 diabetic mellitus (DM) subjects. IMCL content in soleus was greatest in OB, with decreasing content in DM, Ath, and NOB-Sed, respectively (12.6 +/- 1.6, 9.7 +/- 1.8, 7.4 +/- 1.0, 4.9 +/- 0.5 mmol/kg wet wt; P < 0.05 by ANOVA; P < 0.05 OB vs. NOB-Sed or Ath). In TA, IMCL was equivalently elevated in DM and OB, which was higher than in Ath or NOB-Sed, respectively (4.2 +/- 0.4 and 4.2 +/- 0.7 vs. 2.7 +/- 0.5 and 1.5 +/- 0.3 mmol/kg wet wt; ANOVA, P < 0.05; P < 0.05 DM or OB vs. NOB-Sed). We conclude that IMCL content is overestimated when voxel size exceeds 0.25 ml despite measurement by optimized high-resolution (1)H-MRSI at high field. When IMCL is measured unbiased by concomitant obesity, we find that it is strongly influenced by muscle type, training status, and the presence of obesity and Type 2 diabetes.     

Intramyocellular lipid content is increased after exercise in nonexercising human skeletal muscle.

Intramyocellular lipid (IMCL) content has been reported to decrease after prolonged submaximal exercise in active muscle and, therefore, seems to form an important local substrate source. Because exercise leads to a substantial increase in plasma free fatty acid (FFA) availability with a concomitant increase in FFA uptake by muscle tissue, we aimed to investigate potential differences in the net changes in IMCL content between contracting and noncontracting skeletal muscle after prolonged endurance exercise. IMCL content was quantified by magnetic resonance spectroscopy in eight trained cyclists before and after a 3-h cycling protocol (55% maximal energy output) in the exercising vastus lateralis and the nonexercising biceps brachii muscle. Blood samples were taken before and after exercise to determine plasma FFA, glycerol, and triglyceride concentrations, and substrate oxidation was measured with indirect calorimetry. Prolonged endurance exercise resulted in a 20.4 +/- 2.8% (P < 0.001) decrease in IMCL content in the vastus lateralis muscle. In contrast, we observed a substantial (37.9 +/- 9.7%; P < 0.01) increase in IMCL content in the less active biceps brachii muscle. Plasma FFA and glycerol concentrations were substantially increased after exercise (from 85 +/- 6 to 1450 +/- 55 and 57 +/- 11 to 474 +/- 54 microM, respectively; P < 0.001), whereas plasma triglyceride concentrations were decreased (from 1498 +/- 39 to 703 +/- 7 microM; P < 0.001). IMCL is an important substrate source during prolonged moderate-intensity exercise and is substantially decreased in the active vastus lateralis muscle. However, prolonged endurance exercise with its concomitant increase in plasma FFA concentration results in a net increase in IMCL content in less active muscle.     

Overexpression of PLIN5 in skeletal muscle promotes oxidative gene expression and intramyocellular lipid content without compromising insulin sensitivity

Aims/hypothesis: While lipid deposition in the skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like the skeletal muscle. Here we investigated the effects of PLIN5 overexpression – in comparison with the effects of PLIN2 – on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. Methods: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in the skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. Results: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. Conclusions/interpretation: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in the skeletal muscle promoted expression of a cluster of genes under control of PPARα and PGC1α involved in FA catabolism and mitochondrial oxidation.     

Postexercise fat intake repletes intramyocellular lipids but no faster in trained than in sedentary subjects

The hypotheses that postexercise replenishment of intramyocellular lipids (IMCL) is enhanced by endurance training and that it depends on fat intake were tested. Trained and untrained subjects exercised on a treadmill for 2 h at 50% peak oxygen consumption, reducing IMCL by 26-22%. During recovery, they were fed 55% (high fat) or 15% (low fat) lipid energy diets. Muscle substrate stores were estimated by (1)H (IMCL)- and (13)C (glycogen)-magnetic resonance spectroscopy in tibialis anterior muscle before and after exercise. Resting IMCL content was 71% higher in trained than untrained subjects and correlated significantly with glycogen content. Both correlated positively with indexes of insulin sensitivity. After 30 h on the high-fat diet, IMCL concentration was 30-45% higher than preexercise, whereas it remained 5-17% lower on the low-fat diet. Training status had no significant influence on IMCL replenishment. Glycogen was restored within a day with both diets. We conclude that fat intake postexercise strongly promotes IMCL repletion independently of training status. Furthermore, replenishment of IMCL can be completed within a day when fat intake is sufficient.     

Relationship between visceral adiposity and intramyocellular lipid content in two rat models of insulin resistance

High visceral adiposity and intramyocellular lipid levels (IMCL) are both associated with the development of type 2 diabetes. The relationship between visceral adiposity and IMCL levels was explored in diet- and glucocorticoid-induced models of insulin resistance. In the diet-induced model, lean and fa/fa Zucker rats were fed either normal or high-fat (HF) chow over 4 wk. Fat distribution, IMCL content in the tibialis anterior (TA) muscle (IMCL(TA)), and whole body insulin resistance were measured before and after the 4-wk period. The HF diet-induced increase in IMCL(TA) was strongly correlated with visceral fat accumulation and greater glucose intolerance in both groups. The increase in IMCL(TA) to visceral fat accumulation was threefold greater for fa/fa rats. In the glucocorticoid-induced model, insulin sensitivity was impaired with dexamethasone. In vivo adiposity and IMCL(TA) content measurements were combined with ex vivo analysis of plasma and muscle tissue. Dexamethasone treatment had minimal effects on visceral fat accumulation while increasing IMCL(TA) levels approximately 30% (P < 0.05) compared with controls. Dexamethasone increased plasma glucose by twofold and increased the saturated fatty acid content of plasma lipids [fatty acid (CH2)n/omegaCH3 ratio +15%, P < 0.05]. The lipid composition of the TA muscle was unchanged by dexamethasone treatment, indicating that the relative increase in IMCL(TA) observed in vivo resulted from a decrease in lipid oxidation. Visceral adiposity may influence IMCL accumulation in the context of dietary manipulations; however, a "causal" relationship still remains to be determined. Dexamethasone-induced insulin resistance likely operates under a different mechanism, i.e., independently of visceral adiposity.     

Elevated intramyocellular lipid concentration in obese subjects is not reduced after diet and exercise training

To determine the effects of weight loss on intramyocellular energy substrates, vastus lateralis muscle biopsies were taken from six obese subjects (body mass index 34 +/- 5 kg/m(2)) before, after 15 wk of energy restriction (ER; -700 kcal/day), and after a further average 20.7 +/- 1.6 wk of endurance training plus low-fat diet (ET-LFD). Body weight fell from 100 +/- 6 to 89 +/- 6 kg during ER and to 84 +/- 4 kg after ET-LFD. Lipids and glycogen were histochemically measured in type I, IIA, and IIB fibers. Total muscle glycogen content (MGC; per 100 fibers) decreased after ER [from 72 +/- 13 to 55 +/- 8 arbitrary units (AU)]. A similar but not significant decrease was seen in total muscle lipid content (MLC; 14 +/- 5 to 9 +/- 1 AU). After ET-LFD, MGC returned to initial values (74 +/- 8 AU), and MLC approached near-initial values (12 +/- 3 AU). Individual fiber lipid concentration did not change throughout the protocol in all fiber types, whereas glycogen concentration increased after ET-LFD. The training effects of ET-LFD were measured as increasing activities of key mitochondrial enzymes. Although total muscle energy reserves can be reduced after weight loss, their concentration within individual myofibers remains elevated. Weight loss does not appear sufficient to correct the potential detrimental effects of high intracellular lipid concentrations.     

Single muscle fiber contractile properties of young competitive distance runners.

The purpose of this investigation was to characterize the contractile properties of individual slow- and fast-twitch myofibers from highly trained distance runners. Muscle biopsies were obtained from the gastrocnemius of eight competitive runners (Run) and eight recreationally active individuals (Rec). Slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) myofibers were isolated and analyzed for diameter (microm), peak force (Po; mN), unloaded contraction velocity (Vo; fiber lengths/s), and power. Maximum oxygen uptake was higher (P<0.05) in Run (71+/-1 vs. 47+/-2 ml.kg(-1).min(-1)). Diameter of MHC I and MHC IIa fibers from Run subjects was approximately 20% greater (P<0.05) than Rec. Peak force of the MHC IIa fibers was 31% higher (P<0.05) in Run, whereas Po of MHC I fibers was not different between groups. No differences for specific tension (Po/cross-sectional area) were present between groups for either fiber type. Vo was higher (P<0.05) in MHC I (+70%) and MHC IIa (+18%) fibers from Run subjects. In vitro peak absolute power (microN.s(-1)) of both fiber types was greater (P<0.05) in Run (131 and 85% for MHC I and MHC IIa, respectively). Additionally, normalized power (W/l) of the MHC I fibers was 64% higher in Run, whereas no differences were noted for normalized power of MHC IIa fibers. These data indicate that highly trained endurance runners have elevated contraction velocity in both slow- and fast-twitch myofibers. These characteristics of the fast-twitch muscle fibers have not been previously reported in competitive endurance athletes and may contribute to the high level of running performance in these athletes.     

Intramyocellular lipid content in human skeletal muscle

Fat can be stored not only in adipose tissue but also in other tissues such as skeletal muscle. Fat droplets accumulated in skeletal muscle [intramyocellular lipids (IMCLs)] can be quantified by different methods, all with advantages and drawbacks. Here, we briefly review IMCL quantification methods that use biopsy specimens (biochemical quantification, electron microscopy, and histochemistry) and non-invasive alternatives (magnetic resonance spectroscopy, magnetic resonance imaging, and computed tomography). Regarding the physiological role, it has been suggested that IMCL serves as an intracellular source of energy during exercise. Indeed, IMCL content decreases during prolonged submaximal exercise, and analogously to glycogen, IMCL content is increased in the trained state. In addition, IMCL content is highest in oxidative, type 1 muscle fibers. Together, this, indeed, suggests that the IMCL content is increased in the trained state to optimally match fat oxidative capacity and that it serves as readily available fuel. However, elevation of plasma fatty acid levels or dietary fat content also increases IMCL content, suggesting that skeletal muscle also stores fat simply if the availability of fatty acids is high. Under these conditions, the uptake into skeletal muscle may have negative consequences on insulin sensitivity. Besides the evaluation of the various methods to quantify IMCLs, this perspective describes IMCLs as valuable energy stores during prolonged exercise, which, however, in the absence of regular physical activity and with overconsumption of fat, can have detrimental effects on muscular insulin sensitivity.     

Insulin resistance,intramyocellular lipid content,and plasma adiponectin in patients with type 1 diabetes

Insulin resistance is a key pathogenic factor of type 2 diabetes (T2DM); in contrast, in type 1 diabetes (T1DM) it is considered a secondary alteration. Increased intramyocellular lipid (IMCL) content accumulation and reduced plasma adiponectin were suggested to be pathogenic events of insulin resistance in T2DM. This study was designed to assess whether IMCL content and plasma adiponectin were also associated with the severity of insulin resistance in T1DM. We studied 18 patients with T1DM, 7 older and overweight/obese patients with T2DM, and 15 nondiabetic, insulin-resistant offspring of T2DM parents (OFF) and 15 healthy individuals (NOR) as appropriate control groups matched for anthropometric features with T1DM patients by means of the euglycemic hyperinsulinemic clamp combined with the infusion of [6,6-2H2]glucose and 1H magnetic resonance spectroscopy of the calf muscles. T1DM and T2DM patients showed reduced insulin-stimulated glucose metabolic clearance rate (MCR: 5.1 +/- 0.6 and 3.2 +/- 0.8 ml x kg(-1) min(-1)) similar to OFF (5.3 +/- 0.4 ml x kg(-1) x min(-1)) compared with NOR (8.5 +/- 0.5 ml x kg(-1) min(-1), P < 0.001). Soleus IMCL content was increased in T1DM (112 +/- 15 AU), T2DM (108 +/- 10 AU) and OFF (82 +/- 13 AU) compared with NOR (52 +/- 7 AU, P < 0.05) and the result was inversely proportional to the MCR (R2 = 0.27, P < 0.001); an association between IMCL content and Hb A1c was found only in T1DM (R2 = 0.57, P < 0.001). Fasting plasma adiponectin was reduced in T2DM (7 +/- 1 microg/ml, P = 0.01) and OFF (11 +/- 1 microg/ml, P = 0.03) but not in T1DM (25 +/- 6 microg/ml), whose plasma level was increased with respect to both OFF (P = 0.03) and NOR (16 +/- 2 microg/ml, P = 0.05). In conclusion, in T1DM, T2DM, and OFF, IMCL content was associated with insulin resistance, demonstrating that IMCL accretion is a marker of insulin resistance common to both primary genetically determined and secondary metabolic (chronic hyperglycemia) alterations. The increased adiponectin levels in insulin-resistant patients with T1DM, in contrast to the reduced levels found in patients with T2DM and in OFF, demonstrated that the relationship of adiponectin to insulin resistance in humans is still unclear.     

Intramyocellular triacylglycerol in prolonged cycling with high- and low-carbohydrate availability.

Vastus lateralis intramyocellular lipid (IMCL) content was assessed by (1)H-magnetic resonance spectroscopy before and after prolonged time trial cycling bouts of approximately 3-h duration. Six highly trained male cyclists completed a double-blind, randomized, crossover design of two experimental trials after a strenuous exercise bout and 48 h of high (HC) (9.32 +/- 0.08 g. kg(-1). day(-1)) and low (LC) (0.59 +/- 0.21 g. kg(-1). day(-1)) dietary carbohydrate. Resting IMCL content was significantly higher after LC vs. HC (P < 0.01) and was reduced during exercise by 64 and 57%, respectively. IMCL was not different between conditions after exercise (P > 0.05). The approximately twofold increase in IMCL degradation in LC compared with HC suggests that higher rates of whole body lipid metabolism in LC were in part attributable to a greater IMCL utilization. Four subjects experienced reductions of IMCL in excess of 70% during exercise. To our knowledge, this is the first study to report near depletion of IMCL during prolonged cycling, indicating that IMCL, presumably the triacylglycerol component, may be exhausted by prolonged strenuous exercise.     

Regional differences in intramyocellular lipids in humans observed by in vivo 1H-MR spectroscopic imaging.

Regional differences in the content of intramyocellular lipids (IMCL), extramyocellular lipids, and total creatine (TCr) were quantified in soleus (S), tibialis posterior (TP), and tibialis anterior (TA) muscles in humans using in vivo 1H proton spectroscopic imaging at 4 T. Improved spatial resolution (0.25-ml nominal voxel resolution) made it feasible to measure IMCL in S, TP, and TA simultaneously in vivo. The most significant regional difference was found in the content of IMCL compared with extramyocellular lipids or TCr. The concentrations of TCr were found to be 29-32 mmol/kg, with little regional variation. IMCL content was measured to be 4.8 +/- 1.6 mmol/kg tissue wt in S, 2.8 +/- 1.3 mmol/kg tissue wt in TP, and 1.6 +/- 0.9 mmol/kg tissue wt in TA in the order of S > TP > TA (P < 0.05). It is likely that these IMCL values are consistent with the known fiber types of these muscles, with S having the greatest fraction of type I (slow-twitch, oxidative) fibers and TA having a large fraction of type IIb (fast-twitch, glycolytic) fibers.     

Scanning electron microscopic analysis of intramyocellular lipid droplets in an animal model of type 2 diabetes

Objective: To evaluate the accumulation pattern of intramyocellular lipids (IMCLs) in striated muscle during the development and progression of diabetes, using a novel scanning electron microscopic method. Methods and Procedures: Hyperglycemia was induced by feeding diabetes‐prone (DP) Psammomys obesus a high‐energy (HE) diet. Lipid accumulation within gastrocnemius muscle fibers was assessed in formalin‐fixed muscle samples during the development of hyperglycemia using high resolution imaging in a scanning electron microscope. We evaluated the temporal relationship between changes in IMCL quantity and morphology and the altered glucose metabolism and assessed the effect of reversal of hyperglycemia on IMCL level and morphology. Diabetes‐resistant (DR) P. obesus served as controls. Results: Lipid accumulation in the muscle fibers of DP animals was increased with the development of hyperglycemia. This was characterized by increased lipid density as well as by an abundance of large lipid droplets. Reversal of the phenotype resulted in the disappearance of large lipid droplets. The IMCL level and the distribution of lipid droplet size were similar in muscles of both the normoglycemic DR and DP animals, with an abundance of small lipid droplets. This profile was changed following a HE diet only in the DP animals. Discussion: Lipid accumulation in the muscle of P. obesus during the development of hyperglycemia is characterized by increased quantity and accumulation of large lipid droplets. These changes were reversible upon normalization of blood glucose. The evaluated methodology is a useful tool for the study of the dynamics of lipid accumulation in different metabolic conditions.     

Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity

Impaired mitochondrial function and structure and intramyocellular lipid (IMCL) accumulation have been associated with obesity and Type 2 diabetes. We examined whether endurance exercise training and sex influenced IMCL and mitochondrial morphology using electron microscopy, whole-body substrate use, and mitochondrial enzyme activity. Untrained men (n = 5) and women (n = 7) were tested before and after 7 wk of endurance exercise training. Testing included 90 min of cycle ergometry at 60% Vo(2 peak) with preexercise muscle biopsies analyzed for IMCL and mitochondrial size/area using electron microscopy and short-chain beta-hydroxyacyl-CoA dehydrogenase (SCHAD) and citrate synthase (CS) enzyme activity. Training increased the mean lipid area density (P = 0.090), the number of IMCL droplets (P = 0.055), the number of IMCL droplets in contact with mitochondria (P = 0.010), the total mitochondrial area (P < 0.001), and the size of individual mitochondrial fragments (P = 0.006). Women had higher mean lipid area density (P = 0.030) and number of IMCL droplets (P = 0.002) before and after training, but higher individual IMCL area only before training (P = 0.013), compared with men. Women oxidized more fat (P = 0.027) and less carbohydrate (P = 0.032) throughout the study. Training increased Vo(2 peak) (P < 0.001), %fat oxidation (P = 0.018), SCHAD activity (P = 0.003), and CS activity (P = 0.042). In summary, endurance exercise training increased IMCL area density due to an increase in the number of lipid droplets, whereas the increase in total mitochondrial area was due to an increase in the size of individual mitochondrial fragments. In addition, women have higher IMCL content compared with men due mainly to a greater number of individual droplets. Finally, endurance exercise training increased the proportion of IMCL in physical contact with mitochondria.     

Effects of Weight Loss and Physical Activity on Muscle Lipid Content and Droplet Size

Objectives : To address the potential effects of weight loss and physical activity (WL + Ex) on intramyocellular lipid (IMCL) and lipid droplet size in overweight and obese previously sedentary individuals. Research Methods and Procedures : IMCL and lipid droplet size was determined in vastus lateralis, obtained by percutaneous biopsy, from 21 obese volunteers (9 men/12 women), using Oil Red O staining, along with succinate dehydrogenase histochemistry and mitochondrial immunohistochemistry as measures of skeletal muscle oxidative capacity. Insulin sensitivity (IS) was determined by glucose clamp. Results : A 4‐month WL + Ex intervention resulted in ～10% WL and ～15% increase in maximal oxygen uptake, leading to a 46% increase in IS (all p < 0.01). IMCL did not significantly change (p = 0.36). However, the size of lipid droplets decreased after WL + Ex (p < 0.01), and this decrease in lipid droplet size correlated with increased IS (p < 0.01) and the amount of physical activity (p < 0.05). Succinate dehydrogenase activity and mitochondrial labeling increased significantly (p < 0.01), without a significant shift in fiber type distribution. Discussion : In summary, IMCL does not decrease in response to WL + Ex in obese, previously sedentary individuals, yet the lipid within muscle is dispersed into smaller droplets. This change in the size of lipid droplets, likely coupled with a concomitant increase in oxidative enzyme capacity, is correlated to improved IS.