myofibrillar distribution of succinate dehydrogenase ... and metabolism.pdfin skeletal muscle tissue...

doi:10.1152/ajpendo.00270.2011 302:E365-E373, 2012. First published 8 November 2011;Am J Physiol Endocrinol Metab

Josephine PrompersFeyter, Stephan F. E. Praet, Klaas Nicolay, Luc J. C. van Loon and Jeanine Richard A. M. Jonkers, Marlou L. Dirks, Christine I. H. C. Nabuurs, Henk M. Deof paraplegic subjects

tissueactivity and lipid stores differs in skeletal muscle Myofibrillar distribution of succinate dehydrogenase

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

Richard A. M. Jonkers,1 Marlou L. Dirks,2 Christine I. H. C. Nabuurs,1 Henk M. De Feyter,1

Stephan F. E. Praet,2 Klaas Nicolay,1 Luc J. C. van Loon,2 and Jeanine Josephine Prompers1

1Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven; and 2NUTRIMSchool for Nutrition, Toxicology, and Metabolism, Department of Human Movement Sciences, Maastricht University MedicalCentre�, Maastricht, The Netherlands

Submitted 1 June 2011; accepted in final form 2 November 2011

Jonkers RA, Dirks ML, Nabuurs CI, De Feyter HM, Praet SF,Nicolay K, van Loon LJ, Prompers JJ. Myofibrillar distribution ofsuccinate dehydrogenase activity and lipid stores differs in skeletalmuscle tissue of paraplegic subjects. Am J Physiol Endocrinol Metab302: E365–E373, 2012. First published November 8, 2011;doi:10.1152/ajpendo.00270.2011.—Lack of physical activity has beenrelated to an increased risk of developing insulin resistance. Thisstudy aimed to assess the impact of chronic muscle deconditioning onwhole body insulin sensitivity, muscle oxidative capacity, and in-tramyocellular lipid (IMCL) content in subjects with paraplegia. Ninesubjects with paraplegia and nine able-bodied, lean controls wererecruited. An oral glucose tolerance test was performed to assesswhole body insulin sensitivity. IMCL content was determined both invivo and in vitro using 1H-magnetic resonance spectroscopy andfluorescence microscopy, respectively. Muscle biopsy samples werestained for succinate dehydrogenase (SDH) activity to measure mus-cle fiber oxidative capacity. Subcellular distributions of IMCL andSDH activity were determined by defining subsarcolemmal and inter-myofibrillar areas on histological samples. SDH activity was 57 �14% lower in muscle fibers derived from subjects with paraplegiawhen compared with controls (P � 0.05), but IMCL content andwhole body insulin sensitivity did not differ between groups. Inmuscle fibers taken from controls, both SDH activity and IMCLcontent were higher in the subsarcolemmal region than in the inter-myofibrillar area. This typical subcellular SDH and IMCL distributionpattern was lost in muscle fibers collected from subjects with para-plegia and had changed toward a more uniform distribution. Inconclusion, the lower metabolic demand in deconditioned muscle ofsubjects with paraplegia results in a significant decline in muscle fiberoxidative capacity and is accompanied by changes in the subcellulardistribution patterns of SDH activity and IMCL. However, loss ofmuscle activity due to paraplegia is not associated with substantiallipid accumulation in skeletal muscle tissue.

intramyocellular lipids; mitochondrial oxidative capacity; decondi-tioned muscle; paraplegia; insulin resistance

INDIVIDUALS WHO ARE DEPENDENT largely on a wheelchair formobility have an increased risk of developing glucose intoler-ance and insulin resistance (4, 10, 34, 46). The latter is likelyattributed to reduced physical activity, loss of leg muscle mass,and altered body composition (24, 59, 67). Whole body insulinresistance is generally accompanied by ectopic lipid depositionin skeletal muscle tissue (39, 49), which has been associatedwith muscle mitochondrial dysfunction and a reduced capacityto oxidize fatty acids (41, 45). However, little is known about

lipid content and mitochondrial oxidative capacity in decondi-tioned skeletal muscle tissue in relation to whole body insulinsensitivity in subjects with paraplegia (PAR).

Using nuclear magnetic resonance, previous studies havereported increased intramuscular lipid content in deconditionedleg muscles of PAR individuals (21, 23, 55). In addition, aninitial reduction of muscle mitochondrial oxidative activity hasbeen observed directly after spinal cord injury, followed by arecovery to nearly normal levels after 6 mo (13). However, themyofibrillar distribution of mitochondria in deconditionedmuscle of PAR subjects has not been evaluated in previousstudies. It has been suggested that subsarcolemmal (SS) mito-chondria are of more metabolic relevance for fat oxidation,facilitating glucose transport, and the optimal functioning ofthe insulin-signaling pathway when compared with intermyo-fibrillar (IMF) mitochondria, which instead support contractileactivity (37, 53). In healthy, sedentary, and well-trained malesit has been shown that mitochondrial density is two- to three-fold higher in the SS area when compared with the IMF area(16). Furthermore, the volume density of intramyocellular lipid(IMCL) droplets in the SS layer is higher when compared withthe IMF region (31, 62). Recently, it was shown that, inpatients with type 2 diabetes, oxidative capacity (47) andelectron transport chain activity (53) of SS mitochondria arereduced, whereas IMCL content in the SS layer was threefoldhigher in diabetes patients when compared with healthy, nor-moglycemic controls (47). To date, however, it is unknownhow subcellular distribution patterns of IMCL and muscle fiberoxidative capacity change in response to muscle decondition-ing following paralysis.

The aim of our study was to compare whole body insulinsensitivity, muscle fiber oxidative capacity, and IMCL contentbetween PAR subjects and age-, sex-, and body mass index-matched able-bodied controls. An oral glucose tolerance testwas performed to assess glucose tolerance and estimate wholebody insulin sensitivity. Furthermore, total mixed-muscle lipidcontent was determined both in vivo and in vitro using 1H-magnetic resonance spectroscopy (1H-MRS) and fluorescencemicroscopy, respectively. In addition, muscle fiber type-spe-cific IMCL content and its subcellular distribution was as-sessed using immunofluorescence microscopy. Muscle sec-tions were stained for succinate dehydrogenase (SDH) activityas a measurement for muscle fiber oxidative capacity. Wehypothesized that PAR subjects have a reduced whole bodyinsulin sensitivity, which is accompanied by reduced SDHactivity and increased IMCL deposition in the deconditionedmuscle. The latter changes are expected to be visible particu-larly in the SS location of the muscle fibers.

Address for reprint requests and other correspondence: J. J. Prompers,Biomedical NMR, Dept. of Biomedical Engineering, Eindhoven University ofTechnology, P. O. Box 513, 5600 MB Eindhoven, The Netherlands (e-mail:[email protected]).

Am J Physiol Endocrinol Metab 302: E365–E373, 2012.First published November 8, 2011; doi:10.1152/ajpendo.00270.2011.

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MATERIALS AND METHODS

Ethical approval. Nine healthy subjects with PAR and nine able-bodied, lean, age- and sex-matched controls (CON) were recruited(Table 1). Daily energy intake was determined from a food intakequestionnaire, which was filled out by the subjects for 2 days prior tothe study. Sports participation and exercise frequency and duration inparticular were determined from a questionnaire. All subjects wereinformed about the nature and possible risks of the experimentalprocedures before their written informed consent was obtained, afterapproval by the Medical Ethical Committee of the Maastricht Uni-versity Medical Centre�, The Netherlands.

Diet and physical activity prior to testing. On the evening prior tothe measurement, subjects consumed a standardized meal [41.2 kJ/kgbody wt, providing 72 energy percent (En%) carbohydrate, 11 En%fat, and 17 En% protein] at 8 PM, after which they remained fasted for12 h. Subjects were instructed to refrain from any exhaustive physicallabor and exercise training for 48 h prior to testing.

Experimental trials. On the day of the experiment, subjects arrivedat the laboratory by car or public transport at 8 AM. A Teflon catheter

was inserted into an antecubital vein for blood sampling. After 30 minof supine rest, a basal blood sample was collected and a muscle biopsytaken from the m. vastus lateralis. Muscle biopsy samples wereobtained from the midbelly region of the vastus lateralis (15 cm abovethe patella) and �3 cm below entry through the fascia using thepercutaneous needle biopsy technique (6). Muscles samples weredissected, freed from any visible nonmuscle material, embedded inTissue-Tek (Sakura; Finetek, Zoeterwoude, The Netherlands), andrapidly frozen in liquid nitrogen-cooled isopentane.

Thereafter, a standard oral glucose tolerance test was performed,and blood samples were collected subsequently at t � 30, 60, 90, and120 min following ingestion of 75 g of glucose (dissolved in 250 mlof water) to monitor plasma glucose and insulin responses.

After completion of the oral glucose tolerance test, localized1H-MRS experiments were performed on a 1.5-T whole body scanner(Philips Gyroscan S15/ACS; Philips Medical Systems, Best, TheNetherlands) to measure lipid levels in the vastus lateralis of thecontralateral leg at approximately the same proximo-distal position asfor the muscle biopsy.

1H-MRS. IMCL content was measured in subjects in the supineposition. Five single-voxel measurements were performed in thevastus lateralis using the point-resolved spectroscopy sequence (rep-etition time/echo time � 1,500/35 ms), as described previously (17).Briefly, the voxels (10 � 10 � 15 mm3) were placed at differentlocations within the vastus lateralis, avoiding any subcutaneous fatand minimizing the amount of visible interstitial fat using standardT1-weighted images (Fig. 1). From each voxel, a spectrum without (32acquisitions) and with (128 acquisitions) chemical shift-selectivewater suppression was collected. Water, extramyocellular lipid(EMCL) CH2, and IMCL-CH2 peak areas were quantified from theunsuppressed and suppressed spectra, respectively, using a nonlinearleast-squares algorithm (advanced method for accurate, robust, andefficient spectral fitting) in the jMRUI software package (64), asdescribed previously (17). Both IMCL and EMCL content wereexpressed as a percentage of the water signal in the unsuppressedspectrum measured in the same voxel, and data from the differentvoxels of each subject were averaged.

Plasma analyses. Blood samples were collected in EDTA-contain-ing tubes and centrifuged at 1,000 g and 4°C for 10 min. Aliquots ofplasma were frozen immediately in liquid nitrogen and stored at�80°C. Samples were analyzed for plasma glucose and insulin toassess whole body insulin sensitivity using the oral glucose insulinsensitivity index (42). Insulin was analyzed by radioimmunoassay(Insulin RIA Kit; Linco Research, St. Charles, MO). Plasma glucose(ABX Pentra Glucose HK CP; Radiometer Nederland, Zoetermeer,The Netherlands), free fatty acids (NEFA-C kit; Wako Chemicals,Neuss, Germany), triglyceride (ABX Pentra Trig CP; Radiometer

Table 1. Subjects’ characteristics

CON PAR

Sex (males/females) 7/2 7/2Age, yr 31 � 3 34 � 4Height, m 1.80 � 0.02 1.81 � 0.01Body mass, kg 74.4 � 3.1 82.1 � 5.9BMI, kg/m 23.1 � 1.2 25.1 � 1.6Energy intake, kcal/day 2,066 � 131 1,456 � 141*Exercise frequency, wk�1 3.9 � 0.4 3.5 � 0.8Exercise duration, min 92 � 9 101 � 11Time postinjury, yr NA 10.0 � 2.6Whole body insulin sensitivity and

glycemic controlFasting glucose, mmol/l 5.1 � 0.2 5.7 � 0.3Fasting insulin, pmol/l 64 � 7 78 � 13OGIS120 index, ml �min�1 �m�2 432 � 22 361 � 29

Plasma lipid profileFree fatty acids, �mol/l 499 � 61 444 � 49Triacylglycerols, mmol/l 0.85 � 0.90 1.30 � 0.26HDL cholesterol, mmol/l 1.36 � 0.08 1.21 � 0.10LDL cholesterol, mmol/l 2.62 � 0.21 2.86 � 0.41LDL/HDL ratio 2.05 � 0.30 2.43 � 0.37Total cholesterol, mmol/l 4.37 � 0.19 4.67 � 0.44

Values are means � SE. CON, controls; PAR, subjects with paraplegia;BMI, body mass index; NA, not applicable; OGIS, oral glucose insulinsensitivity (42). *Significantly different from CON (P � 0.01).

Fig. 1. Typical examples of transversal T1-weigted images of the upper leg. Presentedare images of a healthy control (A: field ofview � 0.20 � 0.20 m2) and a subject withparaplegia (B: field of view � 0.18 � 0.18m2). The white rectangle outside the legcircumference originates from a phantomlocated in the center of the surface coil,which was used for proper positioning on them. vastus lateralis. A substantially higheramount of fat tissue (bright signal) can beobserved in the subject with paraplegia.

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Nederland), total cholesterol (ABX Pentra Cholesterol CP; Radiom-eter Nederland), high-density lipoprotein cholesterol (ABX PentraCholesterol; Radiometer Nederland), and low-density lipoprotein cho-lesterol concentrations were analyzed with a COBAS FARA semiau-tomatic analyzer.

Muscle biopsy analyses. Multiple serial cryostat sections (5 �m)from biopsy samples collected from a subject of the CON group werethaw-mounted together with biopsy samples collected from a subjectof the PAR group on uncoated, precleaned glass slides.

Slides were stained for muscle fiber composition, as describedpreviously (65). After fixation, cryosections were incubated withprimary antibodies directed against laminin (polyclonal rabbit anti-laminin, dilution 1:50; Sigma, Zwijndrecht, The Netherlands), slowtype 1 skeletal muscle fibers (A4.840, dilution 1:25; DevelopmentalStudies Hybridoma Bank, Iowa City, IA), and slow and fast type 2afibers (N2.261, dilution 1:25; Developmental Studies HybridomaBank) diluted in 0.05% Tween-phosphate-buffered saline (PBS).Slides were then washed and incubated with the conjugated secondaryantibodies goat anti-rabbit IgG Alexa Fluor 350, goat anti-mouse IgMAlexa Fluor 488, and goat anti-mouse IgG Alexa Fluor 555 (dilutionsof 1:130, 1:200, and 1:200, respectively; Molecular Probes, Invitro-gen, Breda, The Netherlands) diluted in 0.05% Tween-PBS. After afinal washing step, all slides were mounted with Mowiol (Calbio-chem, Amsterdam, The Netherlands) and cover glasses.

An Oil Red O (ORO) staining, combined with a staining forlaminin (primary antibody: polyclonal rabbit anti-laminin; secondaryantibody: goat anti-rabbit IgG Alexa Fluor 488), was performed asdescribed previously by Koopman et al. (36) and van Loon et al (63)as a measurement for IMCL content. To determine fiber type-specificIMCL content, slides stained for muscle fiber composition were usedas a reference.

Muscle fiber type-specific oxidative capacity was estimated bydetermining maximal SDH activity in the muscle cross-sections (25,51). To assess intramyocellular SDH activity, a modified SDH stain-ing was used as described previously (25, 56), allowing direct, fibertype-specific determination of SDH activity. The staining procedureswere as follows. Freshly prepared cryosections were incubated for 60min at 37°C in a phosphate buffer at pH 7.6 [13 ml of 0.2 M NaH2PO4

(Sigma) � 87 ml of 0.2 M Na2HPO4 (Sigma)] containing 0.1 Msodium succinate (Sigma) and 1.2 mM nitro-blue tetrazolium (Sigma).After incubation, the slides were rinsed (3 times in deionized water).The unbound nitro-blue tetrazolium was removed with three ex-changes in increasing solutions of acetone in deionized water (30, 60,and 90%, respectively). When the slides were dry, sections wereincubated for 60 min with the primary antibodies described above.Slides were then washed (3 � 5 min with PBS), after which theappropriate conjugated secondary antibodies were applied for 30 min.Incubation was followed by a final washing step (3 � 5 min withPBS), and the slides were mounted with Mowiol and coverslips.

After 24 h, glass slides were examined using a Nikon E800fluorescence microscope (Uvikon, Bunnik, The Netherlands) coupledto a Basler A113 C progressive-scan color charged-coupled devicecamera with a Bayer color filter. Epifluorescence signal was recordedusing a Texas-red excitation filter (540–580 nm) for ORO andA4.840, a fluorescein isothiocyanate excitation filter (465–495 nm)for N2.261 and laminin (for ORO staining), and a 4=,6-diamidino-2-phenylindole UV excitation filter (340–380 nm) for laminin (for fibertype staining). All image recordings and analyses were performed bya researcher blinded for subject coding. Digitally captured images(�240 magnification) were processed and analyzed using Lucia 4.81software (Nikon, Düsseldorf, Germany).

The signal derived from the antibody against laminin was used toselect single muscle fibers, and to differentiate between fiber typeswithin each image, serial cross-section-stained images for fiber typecomposition were used. The ORO-epifluorescence signal was re-corded for each muscle fiber at a �240 magnification. An intensitythreshold representing minimal intensity values corresponding to lipid

droplets was set manually and used uniformly for all images. Totalcross-sectional area, number of objects, and area emitting ORO-epifluorescence signal were measured. Identical settings were used forall muscle cross-sections, because the recorded ORO signal dependson staining efficiency as well as on the applied image acquisitionsettings (intensity threshold and integration time). Lipid droplet sizewas calculated by dividing the area emitting ORO-epifluorescencesignal by the number of objects. Fiber type-specific IMCL content isexpressed as the fraction of the cross-sectional area that was stainedwith ORO. Images were analyzed for subcellular localization ofIMCL in different consecutive 2-�m layers, as described previously(60, 62). The location of lipid droplets and resultant area fractionwithin successive bands of 2 �m in width were recorded from the SSregion toward the central area of each muscle fiber type-specificcross-section.

In bright-field light microscopy, sections stained for SDH activitywere captured at �120 magnification. A laminin, A4.840, and N2.261staining on the same tissue was recorded and processed as describedabove. The bright-field images of the SDH-activity staining wereconverted post hoc to 8-bit grayscale values. The mean optical densityof the SDH-generated signal per individual fiber was determined byaveraging the optical density (OD) measured in every pixel in the celland corrected for the mean OD of the background stain measured ina field of view containing no muscle fibers. The location of SDHactivity and resultant OD within successive bands of 2 �m in widthwas recorded from the SS toward the central region of each musclefiber type-specific cross-section.

Statistics. All data are expressed as means � SE. Subject, plasma,and mixed-muscle data were compared between groups by means ofan independent t-test. Skeletal muscle fiber type-specific variableswere analyzed with mixed-model repeated-measures analysis of vari-ance (RM-ANOVA), with fiber type (type 1, type 2a, and type 2x) asthe within-subjects factor and group (PAR compared with CON) asthe between-subjects factor. Relative differences in both IMCL con-tent and SDH activity within the skeletal muscle cell were calculatedand analyzed by ANOVA-RM, with location (SS and IMF) and fibertype (type 1, type 2a, and type 2x) as within-subjects factor and groupas between-subjects factor. In case of a significant interaction, inde-pendent t-tests were performed to determine group effects, pairedt-tests to determine location differences, and RM-ANOVA for fibertype-specific effects. Bonferroni corrections were applied when ap-propriate. To evaluate the relationships between variables, Pearsoncorrelation coefficients were calculated. Statistical analyses wereperformed using the SPSS 17.0 software package (SPSS, Chicago,IL). The level of significance was set at P � 0.05.

RESULTS

Subjects’ characteristics. The characteristics of the CONand PAR subjects are shown in Table 1. Both groups consistedof seven male and two female subjects. No differences in meanage, height, body mass, or body mass index were observedbetween groups. Energy intake was 31 � 3% lower in PARsubjects compared with CON subjects (P � 0.01). Sportsparticipation did not differ between PAR and CON subjects.The PAR subjects had been dependent on a wheelchair in dailylife for 10.0 � 2.6 yr due to incomplete or complete spinal cordinjury (n � 7; injury levels: C5–C6, C5–C7, C8, T4, T4, T6,T9–T10), spina bifida (n � 1; injury level: L4–L5), or paral-ysis due to another cause (n � 1). Mean fasting plasma glucoseand insulin concentrations were within the normal range forhealthy individuals in both groups (Table 1), although accord-ing to the guidelines of the American Diabetes Association (1),two individuals in the PAR group showed impaired glucosetolerance (2-h plasma glucose 8.31 and 8.09 mmol/l). In the

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PAR group, fasting plasma glucose concentrations tended to be12 � 2% higher (P � 0.06), and whole body insulin sensitivityas determined by the oral glucose insulin sensitivity indextended to be 20 � 3% lower (P � 0.07) when compared withthe CON group. Plasma lipid profiles did not differ betweengroups.

Skeletal muscle fiber type composition. The PAR group hada 43 � 2% lower percentage of type 1 fibers and a 22 � 10%higher percentage of type 2a fibers compared with the CONgroup (P � 0.05, Table 2). The percentage of type 2x fiberstended to be 21 � 9% higher in the PAR subjects whencompared with the CON subjects (P � 0.06). Cross-sectionalarea of myofibers from PAR subjects was 18 � 6% smallerwhen compared with CON subjects independent of fiber type(P � 0.05; Table 2).

Muscle lipid content and SDH activity. Examples of 1H-MRspectra from the vastus lateralis of a CON and a PAR subjectare shown in Fig. 2, and examples of muscle tissue cross-sections stained for IMCL and SDH activity are shown inFigs. 3 and 4, respectively. The results of the 1H-MRS analysesof mixed-muscle EMCL and IMCL content and the histologi-cal analyses of IMCL content and SDH activity are presentedin Table 3. EMCL content in vastus lateralis, measured bylocalized 1H-MRS, was higher in PAR compared with CONsubjects (4.58 � 0.72 vs. 0.99 � 0.13% of unsuppressed watersignal, P � 0.001) and tended to correlate negatively withwhole body insulin sensitivity (r � �0.416, P � 0.086). In

contrast, IMCL content, analyzed by both localized 1H-MRSand ORO staining, did not significantly differ between groups.The analyses of fiber type-specific IMCL content with OROstaining showed a tendency for a fiber type effect (P � 0.07),independent of group, with greater mean area fractions in type1 vs. type 2a vs. type 2x fibers. The lipid droplet size was50 � 12% larger in PAR individuals compared with CONsubjects (P � 0.05) independent of fiber type. Lipid dropletsize was inversely correlated with whole body insulin sensi-tivity (r � �0.815, P � 0.001).

SDH activity in PAR subjects was 57 � 14% lower com-pared with CON subjects (P � 0.05) independent of fiber type(Table 3). SDH activity was significantly greater in type 1fibers than in type 2 fibers (P � 0.05), and SDH activity in type2a fibers was greater than in type 2x fibers (P � 0.01), allindependent of group. SDH activity did not correlate withwhole body insulin sensitivity.

Myofibrillar distribution. Following the analyses of totalIMCL content and SDH activity, the spatial distribution ofIMCL and SDH activity inside skeletal muscle fibers is pre-sented in Figs. 5 and 6, respectively. Recordings of successive2-�m bands were merged into a SS layer (defined as the first4 �m from the sarcolemma) and an IMF region (defined as thearea from the SS layer to the center of the cell).

In the CON group, 62 � 1% of mixed-muscle IMCL (Fig.5A) and 60 � 1% of mixed-muscle SDH activity (Fig. 6A)were located in the SS region, whereas the remaining 38 � 1and 40 � 1% were located in the IMF region (P � 0.01 andP � 0.04, respectively). Similar spatial distributions for IMCLand SDH activity were observed in fiber type-specific analysesfor the CON group (Figs. 5, B–D, and 6, B–D). In contrast, inthe PAR group, no differences were observed between the SSand the IMF region for either IMCL or SDH activity. In the SSlayer, SDH activity was greater in the CON subjects whencompared with the PAR subjects (Fig. 6).

DISCUSSION

The present study aimed to elucidate the effects of chronicmuscle deconditioning on whole body insulin sensitivity, musclefiber oxidative capacity, and lipid accumulation. We hypothesizedthat PAR subjects have reduced whole body insulin sensitivity

Table 2. Muscle fiber type composition

CON PAR

Myofiber distribution by area, %Type 1 56 � 6 13 � 5*Type 2a 35 � 4 57 � 8*†Type 2x 9 � 3†‡ 30 � 10

Myofiber size, �m3

Type 1 6,392 � 359 5,513 � 871#Type 2a 6,569 � 359 5,044 � 445#Type 2x 6,684 � 224 5,447 � 661#

Values are means � SE. *Significantly different from CON (P � 0.05);†significantly different from type 1 (P � 0.005); ‡significantly different fromtype 2a (P � 0.005); #significantly different from CON, independent of fibertype (P � 0.05).

Fig. 2. 1H-MR spectra from the m. vastuslateralis of a control subject (CON; A) and asubject with paraplegia (PAR; B) The spec-tra were processed with 1-Hz line broaden-ing. Peak annotations: tCr CH2/CH3, CH2/CH3 protons of total creatine; TMA, CH3

protons of trimethylammonium groups;EMCL CH2/CH3, CH2/CH3 protons of ex-tramyocellular lipid; IMCL CH2/CH3, CH2/CH3 protons of intramyocellular lipid.





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accompanied by a lower muscle fiber oxidative capacity andgreater IMCL deposition. However, despite the loss of functionalability of the legs, young healthy PAR individuals did not show asubstantial decline in glucose tolerance and/or whole body insulinsensitivity. The loss of leg muscle recruitment was accompaniedby lower muscle fiber oxidative capacity as determined from SDHactivity, whereas mixed-muscle lipid deposition was unaffected.In healthy muscle tissue of controls, both SDH activity and IMCLcontent were greater in the SS region when compared with theIMF region. The latter myofibrillar distribution pattern was nolonger present in the deconditioned muscle of the PAR subjects.

Whole body insulin sensitivity. Insulin sensitivity is loweredfollowing a prolonged period of reduced habitual physicalactivity (40, 58). Individuals who are dependent largely on awheelchair are expected to have a reduced physical activitylevel and, therefore, to be less insulin sensitive. Althoughfasting plasma glucose and insulin levels in the PAR subjectswere within the normal range according to the guidelines of theAmerican Diabetes Association (1), two subjects were diag-nosed with impaired glucose tolerance. The tendency towardelevated fasting glucose levels concomitant with the tendencytoward reduced insulin sensitivity in the PAR group is inaccordance with previous observations of a higher prevalenceof glucose intolerance in individuals with spinal cord injurycompared with able-bodied subjects (2–4, 18). However, thePAR subjects in this study were young, healthy individualswith an active lifestyle and without any excessive weight gainor upper body disability. Sports participation did not differ

between PAR and CON subjects, and the PAR subjects wereusing manual wheelchairs. This might explain that insulinsensitivity of the PAR subjects was not substantially lowerwhen compared with the able-bodied controls.

In addition to the reduced physical activity, other factors,such as altered body composition and increased visceral adi-posity in particular, have been implicated in the developmentof insulin resistance in individuals with spinal cord injury (19,24). In this study, visceral adipose tissue mass was not as-sessed, but body mass and body mass index did not differsignificantly between PAR and CON subjects.

Muscle fiber oxidative capacity. It is known that with inac-tivity (14, 50, 52), immobilization (22, 48), and denervation(26, 43), muscle oxidative capacity is reduced. The latter has atleast partly been attributed to a change in muscle fiber typecomposition toward more type 2 fibers (54) and in particulartype 2x fibers (12, 13, 57). In accord, we observed a reducedproportion of type 1 fibers and an elevated proportion type 2muscle fibers in the deconditioned muscle compared with thehealthy muscle (Table 2). In addition, besides the substantialchanges in muscle fiber type composition, we observed that forall fiber types maximal SDH activity was lower in muscleobtained from the PAR vs. able-bodied subjects.

In addition to muscle fiber type-specific analyses, histologyalso allows determination of subcellular distribution of musclefiber oxidative capacity. In healthy muscle tissue of the controlgroup, oxidative capacity as determined from SDH activitywas 1.5 times higher in the SS region compared with the IMF

Fig. 3. M. vastus lateralis tissue cross-sec-tions (�240 magnification) of a CON (A)and a PAR subject (B) stained for IMCL,with the cell membranes in green. The imageof the CON subject consisted mainly of type1 fibers, whereas the image of the PARsubject consisted mainly of type 2a fibers.

Fig. 4. M. vastus lateralis tissue cross-sections(�240 magnification) of a CON (A and B) anda PAR subject (C and D) stained for succinatedehydrogenase (SDH) activity (A and C) andfiber type composition, with the cell membranein blue, type 1 fibers in red, and type 2a fibersin green (B and D). The darkest fibers wereanalyzed as type 2x fibers.





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region. This is in line with previous reports, which show agreater mitochondrial density and greater lipid oxidative ca-pacity in the SS layer when compared with the IMF area of themuscle fiber (16, 20, 37, 38). Furthermore, endurance-typeexercise training has been reported to particularly increase the

mitochondrial density in the SS area of the muscle fibers (9, 30,31). In contrast to the results in healthy muscle, oxidativecapacity in the SS region of deconditioned muscle tissue fromthe PAR subjects was similar to that in the IMF area. More-over, muscle fiber oxidative capacity in the SS region wassubstantially lower in PAR subjects when compared withable-bodied subjects. These results are in agreement with arecent study by Nielsen et al. (48), who showed that SSmitochondrial content decreased by 33% after 2 wk of muscleimmobilization by whole leg casting, whereas central IMFmitochondrial content remained unchanged. In contrast, Ferettiet al. (22) reported a 17% decrease in IMF mitochondria, withno significant decrease in SS mitochondrial content, in youngsubjects after 37 days of bed rest. The difference in mode ofdisuse and activity level could explain the observed discrep-ancies. In our study and the study by Nielsen et al. (48) themuscle disuse was restricted to the legs while the upperbody remained active, whereas the subjects in the bed reststudy (22) remained refrained from any physical activityduring the bed rest period. Our study confirms the hypoth-esis that SS mitochondria respond to a greater degree tomuscle use or disuse than IMF mitochondria (32, 33, 44, 47,48). In contrast, Chomentowski et al. (15) reported recentlythat in skeletal muscle tissue of obese, insulin-resistantsubjects with or without diabetes, IMF mitochondrial con-tent was reduced, whereas SS mitochondrial content wasunaffected compared with sedentary controls. Apparently,adaptive muscle fiber oxidative responses to muscle disuseare markedly different from those in insulin resistance,indicating that the relationship between muscle disuse andinsulin resistance is not unambiguous.

Table 3. Lipid content and SDH activity

CON PAR

Lipid content assessed with 1H-MRS,%unsuppressed water signal

EMCL 0.99 � 0.13 4.58 � 0.72**IMCL 1.14 � 0.10 1.36 � 0.07

IMCL content assessed with ORO,area fraction·10�3

Mixed 23.8 � 9.1 18.1 � 7.3Type 1 40.1 � 17.8 35.2 � 22.2Type 2a 15.7 � 7.1 20.6 � 7.6Type 2x 2.8 � 1.0 14.6 � 5.3

IMCL droplet size, �m2

Mixed 0.38 � 0.04 0.65 � 0.08*Type 1 0.43 � 0.10 0.66 � 0.19Type 2a 0.43 � 0.07 0.64 � 0.12Type 2x 0.27 � 0.05 0.64 � 0.11

SDH activity, ODMixed 44.0 � 11.8 18.8 � 2.5*Type 1 50.3 � 14.0 24.3 � 4.7Type 2a† 36.4 � 8.6 18.7 � 2.3Type 2x†‡ 29.1 � 6.6 14.2 � 1.8

Values are means � SE. 1H-MRS, 1H-magnetic resonance spectroscopy;EMCL, extramyocellular lipids; IMCL, intramyocellular lipids; ORO, Oil RedO; SDH, succinate dehydrogenase; OD, optical density. **Significantly dif-ferent from CON (P � 0.001); *significantly different from CON, independentof fiber type (P � 0.05); †significantly different from type 1 (P � 0.05);‡significantly different from type 2a (P � 0.05).

Fig. 5. IMCL content distribution in m. vas-tus lateralis of the CON and the PAR groups.Subcellular regions were divided in the sub-sarcolemmal (SS) layer [the 1st two 2-�mbands (4 �m)] and intermyofibrillar (IMF)area (from the SS layer toward the center ofthe cell). Data are displayed for mixed (A),type 1 (B), type 2a (C), and type 2x musclefibers (D) and are expressed as means � SE.#Significantly different from the SS region(P � 0.05); †P � 0.055 vs. the SS region;*significantly different from the CON group(P � 0.05). AF, area fraction.





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Muscle lipid content. Our study showed a 4.6-fold greaterEMCL content in vastus lateralis muscle of PAR subjectscompared with controls, which is in line with a previousobservation of increased EMCL content in m. soleus of spinalcord injury patients (21, 23, 55). EMCL content tended tocorrelate negatively with whole body insulin sensitivity, whichis in agreement with a study by Elder et al. (21) demonstratingthat EMCL is a good predictor of insulin sensitivity in indi-viduals with spinal cord injury.

We hypothesized that, as a result of reduced muscle fiberoxidative capacity and an associated reduced mitochondrialcapacity to oxidize fatty acids, IMCL content would bestrongly elevated in deconditioned muscle of PAR subjects.However, in contrast to the study of Shah and et al. (55), weobserved no significant differences in IMCL content betweenPAR subjects and able-bodied controls. Our findings are re-markable and suggest that, at least in healthy PAR subjects,negative feedback systems prevent excessive accumulation ofIMCL during an extensive period of muscle disuse. One factorthat might have contributed to the normal IMCL levels in thePAR group is the fiber type transformation toward faster, lessoxidative fibers, which tended to store less IMCL. In addition,it is known that chronic inactivity results in a loss of musclecapillaries (28), potentially limiting lipid deposition in decon-ditioned muscle of PAR subjects. Another factor that may haveplayed a role in keeping normal IMCL content is that energyintake was 31 � 3% lower in PAR subjects compared withCON subjects, whereas sports participation did not differsignificantly between PAR and CON subjects. A negativefeedback mechanism preventing excess intracellular lipid ac-cumulation also could have occurred at the molecular level. It

has been shown that both acute and chronic physical inactivitylead to downregulation of skeletal muscle lipoprotein lipaseactivity and a reduction of plasma triglyceride uptake intomuscle (7, 68). In addition, physical inactivity has been shownto decrease fatty acid translocase CD36 mRNA expression (5),and muscle denervation was shown to decrease long-chainfatty acid transport, which was associated with reductions inplasmalemmal CD36 and plasma membrane-associated fattyacid-binding protein (35). Unfortunately, because of the min-imal yield of the biopsy samples from PAR subjects, we werenot able to determine lipoprotein lipase activity or CD36 andplasma membrane-associated fatty acid-binding protein con-tent in muscle tissue from PAR subjects.

Although no significant differences in IMCL content be-tween PAR and CON groups have been observed in the presentstudy, IMCL droplet size appeared to be greater in decondi-tioned muscle tissue (Table 3). Droplet size was inverselycorrelated with whole body insulin sensitivity, which is inaccord with previously published data (27). A larger dropletsize has been postulated to contribute to an impairment in fattyacid mobilization from the IMCL pool in obese type 2 diabetespatients as a result of an impaired interaction of lipases withtriacylglycerols within the droplets due to a reduced surface-to-volume ratio (8, 11, 29, 61). This could eventually lead to anexcessive accumulation of IMCL in skeletal muscle and, assuch, exacerbate insulin resistance.

In lean muscle tissue, the majority of IMCL droplets are locatedclose to mitochondria (30, 66), and therefore, the distributionpattern of IMCL in lean myofibers is similar to the distributionpattern of oxidative capacity; i.e., IMCL shows an exponentialdecline from the periphery toward the central region of the

Fig. 6. SDH activity distribution in m. vastuslateralis of the CON and PAR groups. Sub-cellular regions were divided in the SS layer[the 1st two 2-�m bands (4 �m)] and IMFarea (from the SS layer toward the center ofthe cell). Data are displayed for mixed (A),type 1 (B), type 2a (C), and type 2x musclefibers (D) and are expressed as means � SE.*Significantly different from the CON group(P � 0.05); #significantly different from theSS region (P � 0.05); ‡P � 0.068 vs. theCON group. OD, optical density.





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myofiber. This typical IMCL distribution has been observed inboth lean sedentary and trained subjects (37, 60). Also in ourstudy, IMCL content in the SS region of muscle fibers of CONsubjects was higher than in the IMF area. In contrast, in thedeconditioned muscle tissue of PAR subjects, IMCL was homo-geneously distributed in the muscle cell in parallel with theuniform distribution of oxidative capacity. It has been suggestedthat SS mitochondria are of greater metabolic significance for fatoxidation, glucose transport, and the optimal functioning of theinsulin-signaling cascade (37, 53). Therefore, the parallel changesin subcellular distribution patterns of SDH activity and IMCLcontent in deconditioned skeletal muscle point toward an adaptiveresponse to a lower metabolic demand. This is in contrast withfindings in type 2 diabetes patients, in whom the decreased muscleoxidative capacity in the SS region was accompanied by anincreased IMCL content, indicating a mismatch between lipiddemand and supply (47).

Conclusions. In conclusion, deconditioned muscle tissue ofPAR subjects shows a large decline in muscle oxidative ca-pacity, which is accompanied by a change in the typicalmyofibrillar distribution of SDH activity and IMCL toward amore uniform distribution without a substantial rise in totalIMCL content. These structural changes in leg muscle tissueare not accompanied by a major decline in whole body insulinsensitivity. These findings suggest that in healthy subjectsnegative feedback systems prevent excess lipid deposition inmuscle tissue during an extensive period of muscle disuse,reflecting an adaptive response to a lower metabolic demand.

ACKNOWLEDGMENTS

We acknowledge all volunteers who participated in this research. We alsothank Larry de Graaf for technical support with the MR scanner, Hans Keizerand Milou Beelen for obtaining muscle biopsy samples, and René Koopmanand Ralph Manders for assistance with obtaining muscle biopsy samples.

Present address of C. H. I. C. Nabuurs: NUTRIM School for Nutrition,Toxicology, and Metabolism, Depts. of Human Movement Sciences andRadiology, Maastricht University Medical Centre�, P. O. Box 616, 6200 MOMaastricht, The Netherlands.

Present address of H. M. De Feyter: Department of Diagnostic Radiology,Magnetic Resonance Research Center, Yale University School of Medicine,300 Cedar St., New Haven, CT 06520-8043.

Present address of S. F. E. Praet: Department of Rehabilitation and PhysicalTherapy, Erasmus University Medical Centre, P. O. Box 2040, 3000 CARotterdam, The Netherlands.

DISCLOSURES

No conflicts of interest, financial or otherwise, are reported by the authors.

AUTHOR CONTRIBUTIONS

R.A.J., C.I.N., H.M.D.F., K.N., L.J.v.L., and J.J.P. did the conception anddesign of the research; R.A.J., M.L.D., C.I.N., H.M.D.F., S.F.P., L.J.v.L., andJ.J.P. performed the experiments; R.A.J., M.L.D., C.I.N., L.J.v.L., and J.J.P.analyzed the data; R.A.J., M.L.D., C.I.N., L.J.v.L., and J.J.P. interpreted theresults of the experiments; R.A.J. and J.J.P. prepared the figures; R.A.J. andJ.J.P. drafted the manuscript; R.A.J., L.J.v.L., and J.J.P. edited and revised themanuscript; R.A.J., M.L.D., C.I.N., H.M.D.F., S.F.P., K.N., L.J.v.L., and J.J.P.approved the final version of the manuscript.

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