Peter H. Albers,1,2 Andreas J.T. Pedersen,3 Jesper B. Birk,1 Dorte E. Kristensen,1 Birgitte F. Vind,3

Otto Baba,4 Jane Nøhr,2 Kurt Højlund,3 and Jørgen F.P. Wojtaszewski1

Human Muscle Fiber Type–Specific Insulin Signaling:Impact of Obesity and Type 2DiabetesDiabetes 2015;64:485–497 | DOI: 10.2337/db14-0590

Skeletal muscle is a heterogeneous tissue composedof different fiber types. Studies suggest that insulin-mediated glucose metabolism is different betweenmuscle fiber types. We hypothesized that differencesare due to fiber type–specific expression/regulation ofinsulin signaling elements and/or metabolic enzymes.Pools of type I and II fibers were prepared from biopsiesof the vastus lateralis muscles from lean, obese, and type 2diabetic subjects before and after a hyperinsulinemic-euglycemic clamp. Type I fibers compared with type IIfibers have higher protein levels of the insulin receptor,GLUT4, hexokinase II, glycogen synthase (GS), and pyru-vate dehydrogenase-E1a (PDH-E1a) and a lower proteincontent of Akt2, TBC1 domain family member 4 (TBC1D4),and TBC1D1. In type I fibers compared with type IIfibers, the phosphorylation response to insulin was sim-ilar (TBC1D4, TBC1D1, and GS) or decreased (Akt andPDH-E1a). Phosphorylation responses to insulin ad-justed for protein level were not different between fibertypes. Independently of fiber type, insulin signaling wassimilar (TBC1D1, GS, and PDH-E1a) or decreased (Aktand TBC1D4) in muscle from patients with type 2 diabe-tes compared with lean and obese subjects. We con-clude that human type I muscle fibers compared withtype II fibers have a higher glucose-handling capacitybut a similar sensitivity for phosphoregulation by insulin.

Skeletal muscle is important for whole-body insulin-stimulated glucose disposal (1), and skeletal muscle insulin

resistance is a common phenotype of obesity and type 2diabetes (T2D) (2). Skeletal muscle is a heterogeneous tis-sue composed of different fiber types, which can be dividedaccording to myosin heavy chain (MHC) isoform expres-sion. Studies in rodents show that insulin-stimulated glu-cose uptake in the oxidative type I fiber–dominant musclesis higher than in muscles with a high degree of glycolytictype II fibers (3–6). Whether this phenomenon is due todifferences in locomotor activity of individual muscles ora direct consequence of the fiber-type composition is largelyunknown. In incubated rat muscle, insulin-induced glucoseuptake was higher (;100%) in type IIa (oxidative/glycolytic)compared with IIx and IIb (glycolytic) fibers (7,8), suggest-ing that insulin-mediated glucose uptake is related to theoxidative capacity of the muscle fiber. In humans, a posi-tive correlation between proportions of type I fibersin muscle and whole-body insulin sensitivity has beendemonstrated (9–11). Furthermore, insulin-stimulatedglucose transport in human muscle strips was associatedwith the relative type I fiber content (12). Thus, it is likelythat human type I fibers are more important than type IIfibers for maintaining glucose homeostasis in response toinsulin. Indeed, a decreased proportion of type I fibers hasbeen found in various insulin resistant states such as themetabolic syndrome (9), obesity (13,14), T2D in some(10,13,14) but not all (12,15) studies and following bed-rest (16), as well as in tetraplegic patients (17), and sub-jects with an insulin receptor gene mutation (18).

1Section of Molecular Physiology, Department of Nutrition, Exercise and Sports,August Krogh Centre, University of Copenhagen, Copenhagen, Denmark2Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark3Department of Endocrinology, Diabetes Research Center, Odense UniversityHospital, Odense, Denmark4Section of Biology, Department of Oral Function and Molecular Biology, School ofDentistry, Ohu University, Koriyama, Japan

Corresponding author: Jørgen F.P. Wojtaszewski, jwojtaszewski@nexs.ku.dk.

Received 10 April 2014 and accepted 26 August 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0590/-/DC1.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 64, February 2015 485

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Mechanisms for a fiber type–dependent regulation ofglucose uptake could involve altered abundance/regulationof insulin-signaling elements and/or metabolic enzymes. Inrats, insulin receptor content and Akt and GLUT4 proteinabundance are higher in type I compared with type IIfiber-dominated muscles (4,5,19–21). Furthermore, inrats, Akt phosphorylation under insulin stimulation ishighest in type I compared with type II fiber-dominantmuscles (20). In humans, GLUT4 protein levels are higherin type I compared with type IIa and IIx muscle fibers(14,22). Overall, these findings suggest that insulin sig-naling to and effect on glucose transport is highest in typeI fibers. Thus, a shift toward reduced type I and hencehigher type II fiber content in obesity and T2D (10,13,14)could negatively influence muscle insulin action on glu-cose metabolism. Insulin resistance in obesity and T2D ischaracterized by a decreased ability of insulin to inducesignaling proteins proposed to mediate GLUT4 transloca-tion by, for example, phosphorylation/activation of Akt(23–25) and/or TBC1 domain family member 4 (TBC1D4)(23,25). Whether this relates to differences in the re-sponse to insulin between fiber types is unknown.

Intracellular glucose metabolism could also be differentbetween muscle fiber types. Glucose entering the musclecell is initially phosphorylated by hexokinase (HK) andpredominantly stored as glycogen or oxidized in themitochondria through processes regulated by glycogensynthase (GS) and the pyruvate dehydrogenase complex,respectively. HKII content is higher in human soleusmuscle (;70% type I fibers) compared with gastrocnemiusand vastus lateralis muscle (;50% type I fibers) (26). Also,the content of the pyruvate dehydrogenase (PDH) complexsubunit E1a (PDH-E1a) is decreased in muscle of proliferator-activated receptor g-coactivator-1a knockout mice (27),concomitant with a switch toward reduced type I fiberabundance (28). Furthermore, mitochondrial density ishigher in human type I compared with type II fibers (29).In contrast, no fiber type–specific expression pattern of GShas been shown (30). Altogether, these observations sug-gest that glucose phosphorylation and oxidation but notstorage rate capacity are enhanced in type I comparedwith type II fibers. Whether HKII and PDH-E1a abundanceas well as GS and PDH-E1a regulation by insulin is differentbetween human muscle fiber types is unknown.

We investigated whether proteins involved in glucosemetabolism were expressed and/or regulated by insulin ina fiber type–specific manner in human skeletal muscle.This was achieved by creating pools of single fibersexpressing either MHC I (type I) or II (type II). Thesefibers were dissected from vastus lateralis muscle biopsiesobtained from lean and obese normal glucose-tolerantsubjects as well as T2D patients.

RESEARCH DESIGN AND METHODS

SubjectsA total of 10 lean healthy, 11 obese nondiabetic, and 11obese T2D subjects were randomly chosen from two

studies conducted at Odense University Hospital (Odense,Denmark). One fraction (eight lean, seven obese, and sixT2D) was from an already published study (31), while theremaining subjects were from an unpublished study, inwhich subjects were investigated with an identical exper-imental protocol as previously described (31). Both stud-ies were approved by the regional ethics committee andcarried out in accordance with the Declaration of HelsinkiII. Subject medication is detailed in the SupplementaryMaterial.

Experimental ProtocolA detailed explanation of the in vivo study protocol hasbeen published elsewhere (31). In short, all subjects wereinstructed to refrain from strenuous physical activity 48 hbefore the experimental day. After an overnight fast, sub-jects underwent a 2-h basal tracer equilibration periodfollowed by a 4-h hyperinsulinemic-euglycemic clamp(Actrapid; Novo Nordisk) at an insulin infusion rate of40 mU $ m22 $ min21 combined with tracer glucose and in-direct calorimetry. A primed-constant [3-3H]glucose infusionwas used throughout the 6-h study, and [3-3H]glucosewas added to the glucose infusates to maintain plasma-specific activity constant at baseline levels during the 4-hclamp period as described in detail previously (32). Vastuslateralis muscle biopsies were obtained before and afterthe clamp under local anesthesia (1% lidocaine) using amodified Bergström needle with suction. Muscle biopsieswere immediately frozen in liquid nitrogen and storedbelow 280°C.

Dissection of Individual Muscle FibersMuscle fibers were prepared as previously described (33)but with minor modifications. A total of 20–60 mg ofmuscle tissue was freeze-dried for 48 h before dissectionof individual muscle fibers in a climate-controlled room(20°C, ,35% humidity) using a dissection microscope (intotal, n = 5,384 fibers from 64 biopsies). The length ofeach fiber was estimated under the microscope (1.5 6 0.4mm [means 6 SD]) before being carefully placed in a PCRtube and stored on dry ice. On the day of dissection, 5 mLof ice-cooled Laemmli sample buffer (125 mmol/L Tris-HCl [pH 6.8], 10% glycerol, 125 mmol/L SDS, 200 mmol/Ldithiothreitol, and 0.004% bromophenol blue) was addedto each tube. During method optimization, addition of pro-tease and phosphatase inhibitors was found to be unneces-sary for preservation of either protein content or proteinphosphorylation for this type of sample preparation (datanot shown). After thorough mixing at 4°C, each tube wasinspected under a microscope to confirm that the fiber wasproperly dissolved (if not, the tube was discarded). Eachsample was then heated for 10 min at 70°C and storedat 280°C.

Preparation of Pooled Muscle Fiber SamplesA small fraction (1/5) of the solubilized fiber was used foridentification of MHC expression using Western blotting andspecific antibodies against MHC I or II (see IMMUNOBLOTTING).Hybrid fibers (;5%) expressing more than one MHC

486 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

isoform were discarded. Pools of type I and II fibers fromeach biopsy were prepared (128 pools in total). The aver-age number of type I and II fibers per muscle biopsy in-cluded in each pool was 20 (range 9–36) and 42 (range22–147), respectively.

Estimation of Protein Content and Test of PurityProtein content of the fiber-specific samples was esti-mated using 4–20% Mini-PROTEAN TGX stain-free gels(Bio-Rad), which allowed for gel-protein imaging follow-ing ultraviolet activation on a ChemiDoc MP Imaging Sys-tem (Bio-Rad). The intensity of visualized protein bands(from 37–260 kDa) was compared with a standard curvefrom three different pools of human muscle homogenateswith a known protein concentration (Supplementary Fig. 1).After gel imaging, the purity of each pooled sample wasre-evaluated using Western blotting and MHC I– and II–specific antibodies (see IMMUNOBLOTTING). All fiber-specificsamples were diluted with Laemmli sample buffer to a pro-tein concentration of 0.2 mg/mL.

Glycogen Determination in Muscle Fiber PoolsGlycogen content in the fiber-specific pools was measuredby dot blotting using a specific antibody against glycogen(34,35). Briefly, 150 ng of protein was spotted onto a poly-vinylidene difluoride membrane. After air drying, themembrane was reactivated in ethanol before blocking, in-cubation in primary and secondary antibody, and visual-ization as described in IMMUNOBLOTTING. The intensity ofeach dot was compared with a standard curve (Supple-mentary Fig. 2) obtained from a muscle homogenatewith a glycogen content predetermined biochemically aspreviously described (31) and expressed accordingly.

MHC DeterminationFor MHC determination in muscle biopsies, lysates wereprepared, and protein content was measured as previouslydescribed (31). Muscle lysates were diluted 1:3 with 100%glycerol/Laemmli sample buffer (50/50) and run on 8% self-cast stain-free gels containing 0.5% 2,2,2-trichloroethanol(36). A total of 3 mg of lysate protein was separatedfor ;16 h at 140 V as previously described (37). Proteinbands were visualized by ultraviolet activation of the stain-free gel on a ChemiDoc MP Imaging System (Bio-Rad) andquantified as stated below. Coomassie staining of the geland the use of muscle homogenates provided similar re-sults as stain-free gel imaging and muscle lysates, respec-tively (data not shown).

ImmunoblottingFor MHC determination of single muscle fibers andevaluation of total and phosphorylated levels of relevantproteins, equal amounts of sample volume (for MHCdetermination) or protein amount were separated usingeither precast (Bio-Rad) or self-cast 7.5% gels. On each gel,an internal control (muscle lysate) was loaded two timesper gel in order to minimize assay variation. Muscle fiberpool values were divided by the average of the internalcontrol sample from the corresponding gel. Furthermore,

on one gel, a standard curve of muscle homogenate wasloaded to ensure that quantification of each protein probedfor was within the linear range. Following separation,proteins were transferred (semidry) from multiple gels toa single polyvinylidene difluoride membrane that wasincubated with blocking agent (0.05% Tween 20 and 2%skimmed milk in Tris-buffered saline) for 45 min at roomtemperature, followed by incubation in primary antibodysolution overnight at 4°C (for antibody details, see Supple-mentary Table 1). Membranes were incubated with ap-propriate secondary antibodies (Jackson ImmunoResearchLaboratories) that were conjugated to either horseradishperoxidase or biotin for 1 h at room temperature. Mem-branes incubated with biotin-conjugated antibody werefurther treated with horseradish peroxidase–conjugatedstreptavidin. Protein bands were visualized using a ChemiDocMP imaging system (Bio-Rad) and enhanced chemi-luminescence (SuperSignal West Femto; Pierce). Banddensitometry was performed using Image Laboratory (ver-sion 4.0). Membranes were reprobed with an alternateantibody according to the scheme given in SupplementaryTable 2.

Statistical AnalysesSubject characteristics and blood parameters were evalu-ated by a one-way ANOVA. To compare fiber type, insulin,and group effects, a three-way ANOVA with repeatedmeasures for fiber type and insulin was used. If no tripleinteraction was present, a two-way ANOVA on the in-crement with insulin (Dinsulin-basal values) was performedfor fiber type and group effects with repeated measures forfiber type. Main effects of group and significant interac-tions were evaluated by Tukey post hoc testing. Statisticalanalyses were performed in SigmaPlot (version 12.5, SystatSoftware; one- and two-way ANOVA) and in SAS statisticalsoftware (version 9.2, SAS Institute; three-way ANOVA).Unless otherwise stated, n equals number of subjects asindicated in Table 1. Differences were considered signifi-cant at P , 0.05.

RESULTS

Clinical and Metabolic CharacteristicsBMI and fat mass were higher in the obese and T2D groupscompared with the lean group (Table 1). Patients with T2Dcompared with lean and obese subjects had elevated HbA1clevels, increased fasting plasma glucose, insulin, and triglyc-eride (vs. lean only) concentrations (Tables 1 and 2). Dur-ing the hyperinsulinemic-euglycemic clamp, the glucosedisposal rate (GDR) was decreased in T2D versus leanand obese subjects (Table 2). The decrease in GDR resultedfrom both lower glucose oxidation rates and reduced non-oxidative glucose metabolism (Table 2).

Fiber Type CompositionIn muscle biopsies from lean and obese subjects, MHC I,IIa, and IIx constituted 45, 46, and 9% (total 55% MHCII), respectively (Fig. 1A). This fiber type compositionis in accordance with previous observations using

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(immuno)histochemistry (9–11,13–15,26) and biochemicalmethods (18,22). In the T2D group, MHC I, IIa, and IIxconstituted 35, 45, and 20% (total 65% MHC II), respec-tively. In the T2D group compared with the lean and obesegroup, the relative number of type I muscle fibers was lower,and the relative number of type IIx muscle fibers was higher.MHC IIa expression was similar among all three groups.

Insulin Receptor, HKII, GLUT4, and Complex IIAs represented in Fig. 1B, all fiber pools contained one MHCisoform only. Actin was used as a reference protein, and actinabundance was equal between fiber pools (Supplementary

Fig. 3). Higher protein levels of insulin receptor b (16%),HKII (470%), GLUT4 (29%), and electron transportchain complex II (35%) was found in type I versus IIfibers (Fig. 1C–F). No differences between groups wereobserved except for a reduced (224%) insulin receptorb level in the T2D compared with the lean and obesegroups (Fig. 1C–F).

Akt, Mammalian Target of Rapamycin, and N-mycDownstream-Regulated Gene 1Akt2 protein content was lower (227%) in type I versus IIfibers (Fig. 2C). In the three groups, the average increases

Table 1—Subject characteristics at study entry

Lean Obese T2D

n (female/male) 10 (2/8) 11 (2/9) 11 (2/9)

Age (years) 54 6 2 56 6 2 55 6 2

Height (m) 1.77 6 0.03 1.77 6 0.03 1.75 6 0.03

BMI (kg/m2) 23.9 6 0.4 30.5 6 0.6*** 30.8 6 1.0***

Fat-free mass (kg) 59.3 6 3.3 68.5 6 3.5 63.3 6 3.3

Fat mass (kg) 16.2 6 0.6 28.1 6 1.1*** 31.8 6 2.7***

HbA1c (%) 5.4 6 0.1 5.2 6 0.1 6.8 6 0.2***,†††

HbA1c (mmol/mol) 35 6 1 34 6 1 51 6 3***,†††

Plasma cholesterol (mmol/L) 5.5 6 0.3 5.6 6 0.2 5.0 6 0.2

Plasma LDL cholesterol (mmol/L) 3.6 6 0.2 3.7 6 0.2 2.9 6 0.2†

Plasma HDL cholesterol (mmol/L) 1.6 6 0.1 1.4 6 0.1 1.0 6 0.1**,†

Plasma triglycerides (mmol/L) 0.9 6 0.1 1.4 6 0.2 2.6 6 0.6*

Diabetes duration (years) — — 4.0 6 1.5

Values are means 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 vs. lean group; †P , 0.05, †††P , 0.001 vs. obese group.

Table 2—Metabolic characteristics during hyperinsulinemic-euglycemic clamp

Lean Obese T2D

Plasma glucosebasal (mmol/L) 5.6 6 0.2 5.9 6 0.1 9.0 6 0.6***,†††

Plasma glucoseclamp (mmol/L) 5.5 6 0.1 5.3 6 0.2 5.5 6 0.1

Serum insulinbasal (pmol/L) 27 6 3 44 6 5 86 6 15***,†

Serum insulinclamp (pmol/L) 408 6 23 399 6 12 422 6 17

GDRbasal (mg/m2/min) 76 6 3 77 6 2 80 6 4

GDRclamp (mg/m2/min) 388 6 28 334 6 20 161 6 24***,†††

Glucose oxidationbasal (mg/m2/min) 50 6 8 47 6 4 46 6 7

Glucose oxidationclamp (mg/m2/min) 141 6 14 126 6 10 77 6 7***,††

NOGMbasal (mg/m2/min) 26 6 8 30 6 4 34 6 9

NOGMclamp (mg/m2/min) 247 6 22 208 6 23 84 6 22***,††

Lipid oxidationbasal (mg/m2/min) 28 6 2 30 6 2 34 6 3

Lipid oxidationclamp (mg/m2/min) 21 6 5 4 6 3 19 6 4**,†

RERbasal 0.82 6 0.01 0.81 6 0.01 0.80 6 0.01

RERclamp 0.98 6 0.03 0.95 6 0.02 0.87 6 0.02**,†

Plasma lactatebasal (mmol/L) 0.78 6 0.09 0.80 6 0.07 1.06 6 0.11

Plasma lactateclamp (mmol/L) 1.36 6 0.08 1.18 6 0.08 0.93 6 0.06***

Values are means6 SEM. NOGM, nonoxidative glucose metabolism; RER, respiratory exchange ratio. **P , 0.01, ***P , 0.001 vs. leangroup; †P , 0.05, ††P , 0.01, †††P , 0.001 vs. obese group.

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under insulin stimulation of phosphorylated (p-)AktThr308

and p-AktSer473 were 5.8- and 3.5-fold in type I fibers and6.1- and 3.7-fold in type II fibers, respectively (Fig. 2A andB). In lean and obese groups, levels of insulin-stimulatedp-AktThr308 were lower (225%) in type I versus II fibers.In the T2D group, the insulin-stimulated p-AktThr308 andp-AktSer473 were lower in both fiber types compared withlean and obese groups. In response to insulin, phosphor-ylation of AktSer473/Akt2 but not AktThr308/Akt2 was fiber

type dependent, although the relative response to insulinwas similar between fiber types (Supplementary Fig. 4Aand B). In type I fibers, a higher protein level of mamma-lian target of rapamycin (mTOR) (20%) and its down-stream target N-myc downstream-regulated gene (NDRG)1 (68%) compared with type II fibers was evident (Fig. 3Band D). Insulin had no effect on p-mTOR2481 but increasedp-NDRG1Thr346 only in type I fibers from obese (86%)and T2D (100%) groups (Fig. 3A and C). No fiber-type

Figure 1—MHC composition and muscle fiber type–specific protein abundance in lean, obese, and T2D subjects. A: MHC compositionmeasured in whole muscle biopsies from lean, obese, and T2D subjects. B: The purity of each muscle fiber pool was checked by Westernblotting of MHC I and II. Representative blots of MHC I and II muscle fiber pools from three subjects are shown. In muscle fiber pools, theprotein content of the insulin receptor b (C), HKII (D), GLUT4 (E ), and electron transport complex II (F ) was evaluated by Western blotting.Quantified values of each protein (C–F ) are related to the content of actin protein, and the basal type I fiber value in the lean group is set to100. Representative blots from three individuals are shown above each bar in A and C–F. White bars represent type I fibers (A) or type I fiberpools (C–F ), black bars type IIa fibers (A) or type II fiber pools (C–F ), and gray bars IIx fibers (A). Data are means 6 SEM. Post hoc testingwas only performed when an interaction was evident. †P < 0.05; †††P < 0.001 vs. type I muscle fibers; ‡P < 0.05, ‡‡P < 0.01 main effectof group compared with lean; (§)P = 0.06, §P < 0.05, §§P < 0.01 main effect of group compared with obese. AU, arbitrary units.

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differences were evident when p-NDRG1Thr346 was ad-justed for NDRG1 protein abundance (SupplementaryFig. 4C).

TBC1D1 and TBC1D4TBC1D1 and TBC1D4 protein levels were lower (245%and 216%) in type I versus II fibers, respectively (Fig. 4Band G). Irrespective of fiber type, insulin stimulation in-creased p-TBC1D1Thr596 (36%) and p-TBC1D4 at all sitesinvestigated (Ser318 [122%], Ser588 [59%], Thr642 [103%],and Ser704 [113%]) (Fig. 4A and C–F). Statistically signif-icant main effects of fiber type were evident for the level ofphosphorylation of both TBC1D1 and TBC1D4. More spe-cifically, p-TBC1D1Thr596 (262%), p-TBC1D4Ser318 (221%),p-TBC1D4Ser588 (221%), p-TBC1D4Thr642 (224%), andp-TBC1D4Ser704 (224%) were lower in type I comparedwith type II fibers. No significant group differences in pro-tein abundance or protein phosphorylation of TBC1D1 andTBC1D4 were evident, although the response to insulin ofp-TBC1D4Ser588 tended (P = 0.07) to be group dependent.

Glycogen Content, GS Kinase 3, and GSIn the basal state, glycogen content was lower (229%) intype I versus II fibers in the lean (P , 0.001), obese (P =0.09), and T2D (P = 0.09) groups (Fig. 5A). Insulin in-duced no significant changes in glycogen content in eitherof the fiber types. The protein levels of GS kinase (GSK)3b were 14% less in type I versus type II, whereas GSprotein was 53% higher in type I compared with type IIfibers (Fig. 5C and F). In all three groups and in both fibertypes, insulin induced a similar change in phosphorylationof GSK3bSer9 (62%), GS2+2a (236%) and GS3a+b (238%)(Fig. 5B, D, and E). Phosphorylation of GSK3bSer9 waslower (231%), whereas phosphorylation of GSsite2+2a

and GSsite3a+b was, respectively, 68 and 51% higher intype I versus II fibers. No significant differences wereevident between individual groups in protein abundanceand protein phosphorylation of GSK3b and GS.

PDHPDH-E1a protein content was 34% higher in type I versusII fibers (Fig. 6C). Basal levels of PDH-E1a site 1 phos-phorylation were similar between fiber types in all threegroups (Fig. 6A). After insulin, the degree of phosphory-lation was significantly lower in type II versus I fibers inthe obese and T2D groups only, indicating dephosphory-lation by insulin in type II but not in type I fibers. In line,PDH-E1a site 2 phosphorylation was decreased by insu-lin, and this effect was dependent on fiber type towarda greater effect of insulin in type II versus I fibers (Fig. 6B).Fiber-type differences were not evident when p-PDHsite1

and p-PDHsite2 was adjusted for PDH-E1a content (Sup-plementary Fig. 4D and E).

DISCUSSION

The current study is the first to evaluate changes insignaling events in response to insulin in fiber type–specificpools from human muscle. Based on our findings, we pro-pose a model in which human type I fibers have a greater

Figure 2—Akt in muscle fiber pools from lean, obese, and T2Dsubjects. Muscle fiber type–specific regulation of Akt phosphoryla-tion on site Thr308 (A) and Ser473 (B) and protein content of Akt2(C) was evaluated by Western blotting. Two bands are apparentfor human Akt2 when insulin stimulated [both being Akt2 (31)].Quantified values of each protein are related to the content of actinprotein, and the basal type I fiber value in the lean group is set to100. Representative blots are shown above bars for each proteinprobed for. White bars represent type I and black bars type II mus-cle fiber pools. Data are means 6 SEM. Post hoc testing was onlyperformed when an interaction was evident. ***P < 0.001 vs. basalconditions; ††P < 0.01 vs. type I muscle fibers; ‡P < 0.05, ‡‡P <0.01, ‡‡‡P < 0.001 vs. lean group; §P < 0.05, §§P < 0.01, §§§P <0.001 vs. obese group. AU, arbitrary units.

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abundance of proteins to transport (29% GLUT4), phos-phorylate (470% HKII), and oxidize (35% electron trans-port chain complex II and 34% PDH) glucose and tosynthesize glycogen (35% GS) compared with type IIfibers. These observations are supported by significantpositive correlations between the MHC I content in wholemuscle lysates and insulin-stimulated GDR (r = 0.53; P =0.002), glucose oxidation rate (r = 0.52; P = 0.003), andnonoxidative glucose metabolism (r = 0.44; P = 0.01)(Supplementary Fig. 5). Interestingly, even though insulinreceptor content was higher (16%) in type I fibers, phos-phoregulation of TBC1D1, TBC1D4, and GS by insulinwas similar between fiber types (all normalized to actin).The apparent fiber-type differences in insulin-stimulatedphosphorylation of Akt, NDRG1, and PDH-E1a (whenrelated to actin) were eliminated when adjusted for Akt2,NDRG1, and PDH-E1a protein abundance. These findingssuggest a similar sensitivity of type I and II muscle fibersfor regulation by insulin of the proteins investigated.

Insulin-stimulated GDR, glucose oxidation rates, andnonoxidative glucose metabolism were decreased in T2Dcompared with the lean and obese groups. This was

accompanied by lower insulin receptor content andaltered response to insulin of p-Akt308, p-Akt473,p-TBC1D4Ser588 (P = 0.07), and p-NDRG1Thr346 in themuscle fiber–specific pools from the T2D compared withthe lean and obese groups. In cells, NDRG1 phosphoryla-tion has been suggested to be a readout of mTOR complex(mTORC) 2 activities (38). mTORC2 is also a widely ac-cepted upstream kinase for AktSer473 (39). Since the re-sponse to insulin of p-NDRG1Thr346/NDRG1 was similarbetween groups, these data could imply a specific dysfunc-tional link between mTORC2 and p-AktSer473, as the latterwas decreased in response to insulin in both type I and IIfibers in T2D compared with the lean and obese groups.In rat muscle, abundance and insulin-stimulated phos-phorylation of Akt were higher (660 and 160–180%, re-spectively) in soleus muscle primarily containing type Ifibers, as opposed to epitrochlearis and extensor digito-rum longus muscles primarily consisting of type II fibers(20). In contrast, in human muscle, we report a decreasedAkt phosphorylation after insulin in type I versus II fibers,due to higher Akt2 levels in type II fibers. Thus, findingsin rat muscles with a diverse fiber-type composition could

Figure 3—mTOR and NDRG1 in muscle fiber pools from lean, obese, and T2D subjects. Muscle fiber type–specific regulation of mTORphosphorylation on site Ser2481 (A) and NDRG1 phosphorylation on site Thr346 (C) as well as protein content of mTOR (B) and NDRG1(D) were evaluated by Western blotting. Two bands are apparent for both p-NDRG1Thr346 and NDRG1 (both quantified). Quantified values ofeach protein are related to the content of actin protein, and the basal type I fiber value in the lean group is set to 100. Representative blotsare shown above bars for each protein probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6SEM. Post hoc testing was only performed when an interaction was evident. *P< 0.05; ***P< 0.001 vs. basal conditions; †††P< 0.001 vs.type I muscle fibers. AU, arbitrary units.

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Figure 4—TBC1D1 and TBC1D4 in muscle fiber pools from lean, obese, and T2D subjects. Muscle fiber–specific regulation of TBC1D1phosphorylation at site Thr596 (A) and TBC1D4 phosphorylation on site Ser318 (C ), Ser588 (D), Thr642 (E), and Ser704 (F ) as well as proteincontent of TBC1D1 (B) and TBC1D4 (G) were evaluated by Western blotting. Two bands are apparent for p-TBC1D1Thr596 and TBC1D1[long and medium/short isoform of TBC1D1 protein (49)]. Quantified values of each protein are related to the content of actin protein, andthe basal type I fiber value in the lean group is set to 100. Representative blots are shown above bars for each protein probed for. Whitebars represent type I and black bars type II muscle fiber pools. Data are means 6 SEM. AU, arbitrary units.

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simply result from differences in locomotor activity, al-though species-related differences cannot be excluded. Forinstance, TBC1D4 and TBC1D1 protein abundance in thecurrent study are only modestly lower (216% and 245%)in human type I versus II fibers. In mice, a high (.10-fold)TBC1D4 and a low (,20%) TBC1D1 content are evident in

the type I fiber–abundant soleus compared with thetype II fiber–abundant extensor digitorum longus muscle(40). In rats, no significant correlations between MHC iso-form abundance in various muscles and either TBC1D1 orTBC1D4 protein content were found (21). These findingsindicate that fiber-type differences in TBC1D4 and

Figure 5—Glycogen content, GSK3b, and GS in muscle fiber pools from lean, obese, and T2D subjects. A: Muscle fiber–specific glycogencontent measured by dot blotting. Muscle fiber–specific phosphorylation of GSK3b on site Ser9 (B) and GS phosphorylation on site 2+2a(D) and 3a+b (E) as well as protein abundance of GSK3b (C ) and GS (F ) were evaluated by Western blotting. Quantified values of eachprotein (B–F ) are related to the content of actin protein, and the basal type I fiber value in the lean group is set to 100. Representative blotsare shown above bars for each protein probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6SEM. Post hoc testing was only performed when an interaction was evident. (†)P = 0.09, †††P < 0.001 vs. type I muscle fibers. AU,arbitrary units.

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TBC1D1 protein levels are highly dependent on the spe-cies investigated.

In the current study, no differences in the response toinsulin were observed between fiber types in phosphor-ylation of TBC1D4 and TBC1D1. We previously reporteda decreased response to insulin of p-TBC1D4Ser318 andp-TBC1D4Ser588 in skeletal muscle from obese T2D sub-jects compared with weight-matched control subjects(23). In the current study, insulin-induced (delta values[insulin minus basal]) p-TBC1D4Ser588 was borderline (P =0.07) group dependent. The average response to insulinwas 62, 96, and 19% in the lean, obese, and T2D groups,respectively. It has been shown that exercise training nor-malizes defects in insulin action on TBC1D4 regulation inT2D versus control subjects (23). Thus, in the currentstudy, the lack of significant defects in TBC1D4 regulationby insulin in the T2D group compared with control groupscould be due to the physical fitness level of the groupsstudied. We found that p-TBC1D1Thr596 was increased byinsulin in agreement with another study (41) and that therelative increase was irrespective of fiber type and group.We conclude that the relative response to insulin of Akt,TBC1D4, and TBC1D1 is independent of fiber type, whilethe absolute amount of phosphorylated protein is lowerin type I versus II fibers. Whether a higher total amountof phosphorylated protein is important for the regula-tion of glucose uptake is unknown. To investigate theimpact of the present findings on glucose uptake in dif-ferent human muscle fiber types, future studies need toexamine the membrane-bound fraction of GLUT4 in dif-ferent fiber types or even measure single muscle fiberglucose transport as performed in rat muscle (7).

Interestingly, Gaster et al. (14) previously reportedthat GLUT4 abundance was significantly lower in type Ifibers only in muscle from T2D patients compared withlean and obese control subjects. This was not evident inthe current study. However, we found a nonsignificantlylower GLUT4 content of the same magnitude (10–20%) aspreviously reported (14) in both type I and II fibers fromthe T2D compared with the lean and obese groups. Also,GLUT4 levels were generally higher in type I versus IIfibers. Thus, fewer type I fibers in the T2D comparedwith the lean and obese groups possibly lowers the glu-cose uptake capacity in diabetic skeletal muscle. In sup-port, HKII content was higher in type I compared withtype II fibers. The influence of HKII protein levels onglucose uptake is controversial and has recently been es-timated to control ;10% of human skeletal muscle glu-cose metabolism during insulin-stimulated conditions(42). In the current study, fiber type–specific HKII levelswere not different between groups investigated. Thus, itis likely that decreased HKII levels reported in musclesfrom T2D subjects (43) are at least partly influenced bya lower number of type I fibers in T2D versus controlsubjects as also shown in the current study. Interest-ingly, in contrast to HKII, HKI protein abundance waslower (219%) among the three groups in type I versus II

Figure 6—PDH-E1a in muscle fiber pools from lean, obese, andT2D subjects. Muscle fiber type–specific regulation of PDH-E1aphosphorylation on site 1 (A) and site 2 (B) as well as PDH-E1aprotein content (C) were evaluated by Western blotting. Phospho-specific PDH-E1a antibodies were directed against the phosphor-ylation of sites Ser293 (site 1) and Ser300 (site 2) on the humanPDH-E1a isoform. Due to sample limitations, protein levels ofPDH-E1a were evaluated in a subset of fiber pools, with the numberof samples indicated in each bar. Quantified values of each proteinare related to the content of actin protein, and the basal type I fibervalue in the lean group is set to 100. Representative blots areshown above bars for each protein probed for. White bars representtype I and black bars type II muscle fiber pools. Data are means 6SEM. Post hoc testing was only performed when an interaction wasevident. †††P < 0.001 vs. type I muscle fibers. AU, arbitrary units.

494 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

fibers (Supplementary Fig. 6). This observation could in-dicate a different role of HK isoforms in type I and IImuscle fibers.

A close correlation between the insulin-stimulatedincrease in nonoxidative glucose metabolism and GSactivity has been reported (44). In the current study,insulin-stimulated nonoxidative glucose metabolismwas decreased in the T2D compared with the lean andobese groups as shown by others (23,31,41,45). Thus,we investigated the fiber type–specific regulation of GSby insulin. We were unable to detect any differences inthe response to insulin between fiber types, although theabsolute amount of phosphorylated GS was highest intype I fibers. Increased phosphorylation of GS in type Ifibers could be accounted for by a higher GS protein levelin type I versus II fibers. Previously, a similar GS contentin type I, IIa, and IIx fiber pools was reported in musclefrom young (23 years) subjects (30). Thus, the presentfindings of a higher GS content in type I versus II fibersin muscle from middle-aged (;55 years) subjects indi-cates an age-dependent fiber type–specific regulation ofGS abundance. The functional consequence of a differen-tiated GS content between fiber types is unknown, sincewe were unable to detect any differences in basal andinsulin-stimulated glycogen content in both fiber types.This is likely due to the relatively small (,6%) increase inglycogen content during a clamp procedure (46). If glyco-gen levels were solely dependent on GS, the activity ofthis enzyme would be expected to be lower in type Iversus II fibers. However, our data cannot support thisbecause the higher expression and phosphorylation of GSindicates that total GS activity is in fact higher in type Iversus II fibers. Thus, other factors than GS activity per sedetermines glycogen levels.

In a recent study, Nellemann et al. (47) did not findany changes in phosphorylation of PDH-E1a in humanskeletal muscle in response to insulin. Interestingly,in the current study, PDH-E1a phosphorylation was de-creased by insulin in type II fibers only. Thus, results byNellemann et al. (47) could have been influenced bya muscle fiber type–dependent regulation not detectedin their whole-muscle biopsy preparation. An inverse re-lationship between PDH-E1a phosphorylation and PDHaactivity has been shown in human skeletal muscle duringexercise (48). Thus, findings in the current study suggestan increased PDHa activity in response to insulin in type IIfibers only.

Study LimitationsAll fiber pools were prepared from vastus lateralis muscle,which expresses relatively small (,10%) amounts of typeIIx fibers (26). No significant differences in the MHC IIxexpression were observed between type II fiber poolsamong the three groups (Supplementary Fig. 7). Thus,differences between type I and II fiber pools observed inthe current study are likely not influenced by differencesin protein abundance/regulation between type IIa and IIx

fibers. No measure of physical activity was performed. Ithas been shown that training-induced increases in GLUT4content mainly occur in type I fibers (22). Thus, trainingstatus of the subjects in the current study could poten-tially influence differences between muscle fibers and/orgroups. All measures were performed in muscle fibers fromthe vastus lateralis muscle. Whether fiber type–specificdifferences in protein expression can be extended to othermuscles is unknown, but has been challenged by onestudy (30), in which GLUT4 expression was higher intype I versus IIa and IIx fibers from vastus lateralismuscles but similar between fiber types in soleus and tri-ceps brachii muscles. The current study design did notallow exploration of this further. To evaluate the biolog-ical impact of fiber-specific signaling events further, themethods used in the current study could be combinedwith ex vivo incubation of human muscle strips (12)and the recently described method of single-fiber glucoseuptake measurements (7). Such design demands opensurgical biopsies and was therefore not applicable to thecohort of the current study.

In conclusion, based on protein level measures, theenzymatic capacities for glucose uptake, phosphorylation,and oxidation as well as for glycogen synthesis are higherin human type I compared with type II muscle fibers. Inresponse to insulin, most differences in phosphorylationbetween fiber types were due to differences in proteinlevels. Thus, sensitivity for phosphoregulation by insulinof these proteins is similar between fiber types. Eventhough insulin-induced GDR was decreased in patientswith type 2 diabetes compared with lean and obese subjects,few group differences in the muscle fiber–specific measure-ments were observed. However, our observations favor theidea that fewer type I fibers and a higher number of type IIxfibers in muscles from T2D patients contributes to the re-duced GDR under insulin-stimulated conditions comparedwith lean and obese subjects.

Acknowledgments. The authors thank M. Kleinert (University of Copen-hagen, Denmark) for sharing know-how on the mTOR/NDRG1 analyses. Theauthors also thank the following for the donation of material essential for thiswork: L.J. Goodyear (Joslin Diabetes Center and Harvard Medical School,Boston, MA), O.B. Pedersen (University of Copenhagen, Denmark), and J. Hastieand D.G. Hardie (University of Dundee, U.K.). The monoclonal antibodies againstMHC I and II isoforms (A4.840 and A4.74) were developed by H.M. Blau, andantibody directed against MHC IIx (6H1) was developed by C. Lucas. All MHCantibodies were obtained from the Developmental Studies Hybridoma Bankdeveloped under the auspices of the National Institute of Child Health andHuman Development and maintained by The University of Iowa, Departmentof Biology, Iowa City, IA.Funding. This work was carried out as a part of the research programs“Physical activity and nutrition for improvement of health” funded by the Univer-sity of Copenhagen Excellence Program for Interdisciplinary Research andthe UNIK project Food, Fitness & Pharma for Health and Disease (see www.foodfitnesspharma.ku.dk) supported by the Danish Ministry of Science, Tech-nology and Innovation. This study was funded by the Danish Council for Inde-pendent Research Medical Sciences, the Novo Nordisk Foundation, and a ClinicalResearch Grant from the European Foundation for the Study of Diabetes.

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Duality of Interest. P.H.A. and J.N. are employees at Novo Nordisk A/Sand own stocks in Novo Nordisk A/S. No other potential conflicts of interestrelevant to this article were reported.Author Contributions. P.H.A. was responsible for conception and designof research, performed analysis, interpreted results, drafted the manuscript,edited and revised the manuscript, and approved the final version. A.J.T.P.performed in vivo experiments and analysis, edited and revised the manuscript,and approved the final version. J.B.B. performed analysis, interpreted results,edited and revised the manuscript, and approved the final version. D.E.K. per-formed analysis, edited and revised the manuscript, and approved the finalversion. B.F.V. performed in vivo experiments and analysis, edited and revisedthe manuscript, and approved the final version. O.B. and J.N. edited and revisedthe manuscript and approved the final version. K.H. interpreted results, editedand revised the manuscript, and approved the final version. J.F.P.W. was re-sponsible for conception and design of research, interpreted results, drafted themanuscript, edited and revised the manuscript, and approved the final version.J.F.P.W. is the guarantor of this work and, as such, had full access to all the datain the study and takes responsibility for the integrity of the data and the accuracyof the data analysis.

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