Diabetes Volume 64, February 2015 485

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 Signaling: Impact of Obesity and Type 2 Diabetes

Diabetes 2015;64:485–497 | DOI: 10.2337/db14-0590

Skeletal muscle is a heterogeneous tissue composed resistance is a common phenotype of obesity and type 2 of different fiber types. Studies suggest that insulin- diabetes (T2D) (2). Skeletal muscle is a heterogeneous tis- mediated glucose metabolism is different between fi sue composed of different ber types, which can be divided TRANSDUCTION SIGNAL muscle fiber types. We hypothesized that differences according to myosin heavy chain (MHC) isoform expres- are due to fiber type–specific expression/regulation of sion. Studies in rodents show that insulin-stimulated glu- insulin signaling elements and/or metabolic enzymes. cose uptake in the oxidative type I fiber–dominant muscles fi Pools of type I and II bers were prepared from biopsies is higher than in muscles with a high degree of glycolytic of the vastus lateralis muscles from lean, obese, and type 2 type II fibers (3–6). Whether this phenomenon is due to diabetic subjects before and after a hyperinsulinemic- differences in locomotor activity of individual muscles or euglycemic clamp. Type I fibers compared with type II a direct consequence of the fiber-type composition is largely fibers have higher levels of the insulin receptor, unknown. In incubated rat muscle, insulin-induced glucose GLUT4, hexokinase II, glycogen synthase (GS), and pyru- a a uptake was higher (;100%) in type IIa (oxidative/glycolytic) vate dehydrogenase-E1 (PDH-E1 ) and a lower protein fi content of Akt2, TBC1 domain family member 4 (TBC1D4), compared with IIx and IIb (glycolytic) bers (7,8), suggest- and TBC1D1. In type I fibers compared with type II ing that insulin-mediated glucose uptake is related to the fi fibers, the phosphorylation response to insulin was sim- oxidative capacity of the muscle ber. In humans, a posi- fi ilar (TBC1D4, TBC1D1, and GS) or decreased (Akt and tive correlation between proportions of type I bers PDH-E1a). Phosphorylation responses to insulin ad- in muscle and whole-body insulin sensitivity has been justed for protein level were not different between fiber demonstrated (9–11). Furthermore, insulin-stimulated types. Independently of fiber type, insulin signaling was glucose transport in human muscle strips was associated similar (TBC1D1, GS, and PDH-E1a) or decreased (Akt with the relative type I fiber content (12). Thus, it is likely and TBC1D4) in muscle from patients with type 2 diabe- that human type I fibers are more important than type II tes compared with lean and obese subjects. We con- fibers for maintaining glucose homeostasis in response to clude that human type I muscle fibers compared with insulin. Indeed, a decreased proportion of type I fibers has type II fibers have a higher glucose-handling capacity been found in various insulin resistant states such as the but a similar sensitivity for phosphoregulation by insulin. metabolic syndrome (9), obesity (13,14), T2D in some (10,13,14) but not all (12,15) studies and following bed- Skeletal muscle is important for whole-body insulin- rest (16), as well as in tetraplegic patients (17), and sub- stimulated glucose disposal (1), and skeletal muscle insulin jects with an insulin receptor mutation (18).

1Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Received 10 April 2014 and accepted 26 August 2014. August Krogh Centre, University of Copenhagen, Copenhagen, Denmark This article contains Supplementary Data online at http://diabetes 2 Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0590/-/DC1. 3Department of Endocrinology, Diabetes Research Center, Odense University © 2015 by the American Diabetes Association. Readers may use this article as Hospital, Odense, Denmark long as the work is properly cited, the use is educational and not for profit, and 4Section of Biology, Department of Oral Function and Molecular Biology, School of the work is not altered. Dentistry, Ohu University, Koriyama, Japan Corresponding author: Jørgen F.P. Wojtaszewski, [email protected]. 486 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

Mechanisms for a fiber type–dependent regulation of studies conducted at Odense University Hospital (Odense, glucose uptake could involve altered abundance/regulation Denmark). One fraction (eight lean, seven obese, and six of insulin-signaling elements and/or metabolic enzymes. In T2D) was from an already published study (31), while the rats, insulin receptor content and Akt and GLUT4 protein remaining subjects were from an unpublished study, in abundance are higher in type I compared with type II which subjects were investigated with an identical exper- fiber-dominated muscles (4,5,19–21). Furthermore, in imental protocol as previously described (31). Both stud- rats, Akt phosphorylation under insulin stimulation is ies were approved by the regional ethics committee and highest in type I compared with type II fiber-dominant carried out in accordance with the Declaration of Helsinki muscles (20). In humans, GLUT4 protein levels are higher II. Subject medication is detailed in the Supplementary in type I compared with type IIa and IIx muscle fibers Material. fi (14,22). Overall, these ndings suggest that insulin sig- Experimental Protocol naling to and effect on glucose transport is highest in type A detailed explanation of the in vivo study protocol has I fibers. Thus, a shift toward reduced type I and hence been published elsewhere (31). In short, all subjects were higher type II fiber content in obesity and T2D (10,13,14) instructed to refrain from strenuous physical activity 48 h could negatively influence muscle insulin action on glu- before the experimental day. After an overnight fast, sub- cose metabolism. Insulin resistance in obesity and T2D is jects underwent a 2-h basal tracer equilibration period characterized by a decreased ability of insulin to induce followed by a 4-h hyperinsulinemic-euglycemic clamp signaling proposed to mediate GLUT4 transloca- (Actrapid; Novo Nordisk) at an insulin infusion rate of tion by, for example, phosphorylation/activation of Akt 22 21 40 mU $ m $ min combined with tracer glucose and in- (23–25) and/or TBC1 domain family member 4 (TBC1D4) 3 direct calorimetry. A primed-constant [3- H]glucose infusion (23,25). Whether this relates to differences in the re- 3 was used throughout the 6-h study, and [3- H]glucose sponse to insulin between fiber types is unknown. was added to the glucose infusates to maintain plasma- Intracellular glucose metabolism could also be different specific activity constant at baseline levels during the 4-h between muscle fiber types. Glucose entering the muscle clamp period as described in detail previously (32). Vastus cell is initially phosphorylated by hexokinase (HK) and lateralis muscle biopsies were obtained before and after predominantly stored as glycogen or oxidized in the the clamp under local anesthesia (1% lidocaine) using a mitochondria through processes regulated by glycogen modified Bergström needle with suction. Muscle biopsies synthase (GS) and the pyruvate dehydrogenase complex, were immediately frozen in liquid nitrogen and stored respectively. HKII content is higher in human soleus below 280°C. muscle (;70% type I fibers) compared with gastrocnemius and vastus lateralis muscle (;50% type I fibers) (26). Also, Dissection of Individual Muscle Fibers the content of the pyruvate dehydrogenase (PDH) complex Muscle fibers were prepared as previously described (33) subunit E1a (PDH-E1a) is decreased in muscle of proliferator- but with minor modifications. A total of 20–60 mg of activated receptor g-coactivator-1a knockout mice (27), muscle tissue was freeze-dried for 48 h before dissection concomitant with a switch toward reduced type I fiber of individual muscle fibers in a climate-controlled room abundance (28). Furthermore, mitochondrial density is (20°C, ,35% humidity) using a dissection microscope (in higher in human type I compared with type II fibers (29). total, n = 5,384 fibers from 64 biopsies). The length of In contrast, no fiber type–specific expression pattern of GS each fiber was estimated under the microscope (1.5 6 0.4 has been shown (30). Altogether, these observations sug- mm [means 6 SD]) before being carefully placed in a PCR gest that glucose phosphorylation and oxidation but not tube and stored on dry ice. On the day of dissection, 5 mL storage rate capacity are enhanced in type I compared of ice-cooled Laemmli sample buffer (125 mmol/L Tris- with type II fibers. Whether HKII and PDH-E1a abundance HCl [pH 6.8], 10% glycerol, 125 mmol/L SDS, 200 mmol/L as well as GS and PDH-E1a regulation by insulin is different dithiothreitol, and 0.004% bromophenol blue) was added between human muscle fiber types is unknown. to each tube. During method optimization, addition of pro- We investigated whether proteins involved in glucose tease and phosphatase inhibitors was found to be unneces- metabolism were expressed and/or regulated by insulin in sary for preservation of either protein content or protein a fiber type–specific manner in human skeletal muscle. phosphorylation for this type of sample preparation (data This was achieved by creating pools of single fibers notshown).Afterthoroughmixingat4°C,eachtubewas expressing either MHC I (type I) or II (type II). These inspected under a microscope to confirm that the fiber was fibers were dissected from vastus lateralis muscle biopsies properly dissolved (if not, the tube was discarded). Each obtained from lean and obese normal glucose-tolerant sample was then heated for 10 min at 70°C and stored subjects as well as T2D patients. at 280°C. Preparation of Pooled Muscle Fiber Samples RESEARCH DESIGN AND METHODS A small fraction (1/5) of the solubilized fiber was used for Subjects identification of MHC expression using Western blotting and A total of 10 lean healthy, 11 obese nondiabetic, and 11 specific antibodies against MHC I or II (see IMMUNOBLOTTING). obese T2D subjects were randomly chosen from two Hybrid fibers (;5%) expressing more than one MHC diabetes.diabetesjournals.org Albers and Associates 487 isoform were discarded. Pools of type I and II fibers from on one gel, a standard curve of muscle homogenate was each biopsy were prepared (128 pools in total). The aver- loaded to ensure that quantification of each protein probed age number of type I and II fibers per muscle biopsy in- for was within the linear range. Following separation, cluded in each pool was 20 (range 9–36) and 42 (range proteins were transferred (semidry) from multiple gels to 22–147), respectively. a single polyvinylidene difluoride membrane that was Estimation of Protein Content and Test of Purity incubated with blocking agent (0.05% Tween 20 and 2% skimmed milk in Tris-buffered saline) for 45 min at room Protein content of the fiber-specific samples was esti- temperature, followed by incubation in primary antibody mated using 4–20% Mini-PROTEAN TGX stain-free gels (Bio-Rad), which allowed for gel-protein imaging follow- solution overnight at 4°C (for antibody details, see Supple- mentary Table 1). Membranes were incubated with ap- ing ultraviolet activation on a ChemiDoc MP Imaging Sys- propriate secondary antibodies (Jackson ImmunoResearch tem (Bio-Rad). The intensity of visualized protein bands Laboratories) that were conjugated to either horseradish (from 37–260 kDa) was compared with a standard curve peroxidase or biotin for 1 h at room temperature. Mem- from three different pools of human muscle homogenates branes incubated with biotin-conjugated antibody were with a known protein concentration (Supplementary Fig. 1). further treated with horseradish peroxidase–conjugated After gel imaging, the purity of each pooled sample was streptavidin. Protein bands were visualized using a ChemiDoc re-evaluated using Western blotting and MHC I– and II– MP imaging system (Bio-Rad) and enhanced chemi- specific antibodies (see IMMUNOBLOTTING). All fiber-specific luminescence (SuperSignal West Femto; Pierce). Band samples were diluted with Laemmli sample buffer to a pro- densitometry was performed using Image Laboratory (ver- tein concentration of 0.2 mg/mL. sion 4.0). Membranes were reprobed with an alternate Glycogen Determination in Muscle Fiber Pools antibody according to the scheme given in Supplementary Glycogen content in the fiber-specific pools was measured Table 2. by dot blotting using a specific antibody against glycogen Statistical Analyses (34,35). Briefly, 150 ng of protein was spotted onto a poly- vinylidene difluoride membrane. After air drying, the Subject characteristics and blood parameters were evalu- ated by a one-way ANOVA. To compare fiber type, insulin, membrane was reactivated in ethanol before blocking, in- and group effects, a three-way ANOVA with repeated cubation in primary and secondary antibody, and visual- measures for fiber type and insulin was used. If no triple ization as described in IMMUNOBLOTTING. The intensity of interaction was present, a two-way ANOVA on the in- each dot was compared with a standard curve (Supple- crement with insulin (Dinsulin-basal values) was performed mentary Fig. 2) obtained from a muscle homogenate for fiber type and group effects with repeated measures for with a glycogen content predetermined biochemically as fiber type. Main effects of group and significant interac- previously described (31) and expressed accordingly. tions were evaluated by Tukey post hoc testing. Statistical MHC Determination analyses were performed in SigmaPlot (version 12.5, Systat ForMHCdeterminationinmuscle biopsies, lysates were Software; one- and two-way ANOVA) and in SAS statistical prepared, and protein content was measured as previously software (version 9.2, SAS Institute; three-way ANOVA). described (31). Muscle lysates were diluted 1:3 with 100% Unless otherwise stated, n equals number of subjects as glycerol/Laemmli sample buffer (50/50) and run on 8% self- indicated in Table 1. Differences were considered signifi- cast stain-free gels containing 0.5% 2,2,2-trichloroethanol cant at P , 0.05. (36). A total of 3 mg of lysate protein was separated for ;16 h at 140 V as previously described (37). Protein RESULTS bands were visualized by ultraviolet activation of the stain- Clinical and Metabolic Characteristics free gel on a ChemiDoc MP Imaging System (Bio-Rad) and BMI and fat mass were higher in the obese and T2D groups fi quanti ed as stated below. Coomassie staining of the gel compared with the lean group (Table 1). Patients with T2D and the use of muscle homogenates provided similar re- compared with lean and obese subjects had elevated HbA1c sults as stain-free gel imaging and muscle lysates, respec- levels, increased fasting plasma glucose, insulin, and triglyc- tively (data not shown). eride (vs. lean only) concentrations (Tables 1 and 2). Dur- ing the hyperinsulinemic-euglycemic clamp, the glucose Immunoblotting disposal rate (GDR) was decreased in T2D versus lean For MHC determination of single muscle fibers and and obese subjects (Table 2). The decrease in GDR resulted evaluation of total and phosphorylated levels of relevant from both lower glucose oxidation rates and reduced non- proteins, equal amounts of sample volume (for MHC oxidative glucose metabolism (Table 2). determination) or protein amount were separated using either precast (Bio-Rad) or self-cast 7.5% gels. On each gel, Fiber Type Composition an internal control (muscle lysate) was loaded two times In muscle biopsies from lean and obese subjects, MHC I, per gel in order to minimize assay variation. Muscle fiber IIa, and IIx constituted 45, 46, and 9% (total 55% MHC pool values were divided by the average of the internal II), respectively (Fig. 1A). This fiber type composition control sample from the corresponding gel. Furthermore, is in accordance with previous observations using 488 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

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 2566 2556 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 1346 1516 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.

(immuno)histochemistry (9–11,13–15,26) and biochemical Fig. 3). Higher protein levels of insulin receptor b (16%), methods (18,22). In the T2D group, MHC I, IIa, and IIx HKII (470%), GLUT4 (29%), and electron transport constituted 35, 45, and 20% (total 65% MHC II), respec- chain complex II (35%) was found in type I versus II tively. In the T2D group compared with the lean and obese fibers (Fig. 1C–F). No differences between groups were group,therelativenumberoftypeImusclefibers was lower, observed except for a reduced (224%) insulin receptor and the relative number of type IIx muscle fibers was higher. b level in the T2D compared with the lean and obese MHC IIa expression was similar among all three groups. groups (Fig. 1C–F). Insulin Receptor, HKII, GLUT4, and Complex II Akt, Mammalian Target of Rapamycin, and N-myc As represented in Fig. 1B,allfiber pools contained one MHC Downstream-Regulated Gene 1 isoform only. Actin was used as a reference protein, and actin Akt2 protein content was lower (227%) in type I versus II abundance was equal between fiber pools (Supplementary fibers (Fig. 2C). In the three groups, the average increases

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 3446 5866 15***,†

Serum insulinclamp (pmol/L) 408 6 23 399 6 12 422 6 17 2 GDRbasal (mg/m /min) 76 6 3776 2806 4 2 GDRclamp (mg/m /min) 388 6 28 334 6 20 161 6 24***,††† 2 Glucose oxidationbasal (mg/m /min) 50 6 8476 4466 7 2 Glucose oxidationclamp (mg/m /min) 141 6 14 126 6 10 77 6 7***,†† 2 NOGMbasal (mg/m /min) 26 6 8306 4346 9 2 NOGMclamp (mg/m /min) 247 6 22 208 6 23 84 6 22***,†† 2 Lipid oxidationbasal (mg/m /min) 28 6 2306 2346 3 2 Lipid oxidationclamp (mg/m /min) 21 6 546 3196 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 means 6 SEM. NOGM, nonoxidative glucose metabolism; RER, respiratory exchange ratio. **P , 0.01, ***P , 0.001 vs. lean group; †P , 0.05, ††P , 0.01, †††P , 0.001 vs. obese group. diabetes.diabetesjournals.org Albers and Associates 489

Figure 1—MHC composition and muscle fiber type–specific protein abundance in lean, obese, and T2D subjects. A: MHC composition measured in whole muscle biopsies from lean, obese, and T2D subjects. B: The purity of each muscle fiber pool was checked by Western blotting of MHC I and II. Representative blots of MHC I and II muscle fiber pools from three subjects are shown. In muscle fiber pools, the protein 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 to 100. 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 fiber pools (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 testing was 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 effect of group compared with lean; (§)P = 0.06, §P < 0.05, §§P < 0.01 main effect of group compared with obese. AU, arbitrary units.

under insulin stimulation of phosphorylated (p-)AktThr308 type dependent, although the relative response to insulin and p-AktSer473 were 5.8- and 3.5-fold in type I fibers and was similar between fiber types (Supplementary Fig. 4A 6.1- and 3.7-fold in type II fibers, respectively (Fig. 2A and and B). In type I fibers, a higher protein level of mamma- B). In lean and obese groups, levels of insulin-stimulated lian target of rapamycin (mTOR) (20%) and its down- p-AktThr308 were lower (225%) in type I versus II fibers. stream target N-myc downstream-regulated gene (NDRG) In the T2D group, the insulin-stimulated p-AktThr308 and 1 (68%) compared with type II fibers was evident (Fig. 3B p-AktSer473 were lower in both fiber types compared with and D). Insulin had no effect on p-mTOR2481 but increased lean and obese groups. In response to insulin, phosphor- p-NDRG1Thr346 only in type I fibers from obese (86%) ylation of AktSer473/Akt2 but not AktThr308/Akt2 was fiber and T2D (100%) groups (Fig. 3A and C). No fiber-type 490 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

differences were evident when p-NDRG1Thr346 was ad- justed for NDRG1 protein abundance (Supplementary Fig. 4C). TBC1D1 and TBC1D4 TBC1D1 and TBC1D4 protein levels were lower (245% and 216%) in type I versus II fibers, respectively (Fig. 4B and G). Irrespective of fiber type, insulin stimulation in- creased p-TBC1D1Thr596 (36%) and p-TBC1D4 at all sites investigated (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 of phosphorylation of both TBC1D1 and TBC1D4. More spe- cifically, p-TBC1D1Thr596 (262%), p-TBC1D4Ser318 (221%), p-TBC1D4Ser588 (221%), p-TBC1D4Thr642 (224%), and p-TBC1D4Ser704 (224%) were lower in type I compared with type II fibers. No significant group differences in pro- tein abundance or protein phosphorylation of TBC1D1 and TBC1D4 were evident, although the response to insulin of p-TBC1D4Ser588 tended (P = 0.07) to be group dependent. Glycogen Content, GS Kinase 3, and GS In the basal state, glycogen content was lower (229%) in type 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 either of the fiber types. The protein levels of GS kinase (GSK) 3b were 14% less in type I versus type II, whereas GS protein was 53% higher in type I compared with type II fibers (Fig. 5C and F). In all three groups and in both fiber types, insulin induced a similar change in phosphorylation of GSK3bSer9 (62%), GS2+2a (236%) and GS3a+b (238%) (Fig. 5B, D,andE). Phosphorylation of GSK3bSer9 was lower (231%), whereas phosphorylation of GSsite2+2a and GSsite3a+b was, respectively, 68 and 51% higher in type I versus II fibers. No significant differences were evident between individual groups in protein abundance and protein phosphorylation of GSK3b and GS. PDH PDH-E1a protein content was 34% higher in type I versus II fibers (Fig. 6C). Basal levels of PDH-E1a site 1 phos- phorylation were similar between fiber types in all three groups (Fig. 6A). After insulin, the degree of phosphory- Figure 2—Akt in muscle fiber pools from lean, obese, and T2D fi fi subjects. Muscle fiber type–specific regulation of Akt phosphoryla- lation was signi cantly lower in type II versus I bers in tion on site Thr308 (A) and Ser473 (B) and protein content of Akt2 the obese and T2D groups only, indicating dephosphory- (C) was evaluated by Western blotting. Two bands are apparent lation by insulin in type II but not in type I fibers. In line, for human Akt2 when insulin stimulated [both being Akt2 (31)]. PDH-E1a site 2 phosphorylation was decreased by insu- Quantified values of each protein are related to the content of actin fi protein, and the basal type I fiber value in the lean group is set to lin, and this effect was dependent on ber type toward 100. Representative blots are shown above bars for each protein a greater effect of insulin in type II versus I fibers (Fig. 6B). probed for. White bars represent type I and black bars type II mus- Fiber-type differences were not evident when p-PDHsite1 cle fiber pools. Data are means 6 SEM. Post hoc testing was only site2 P < and p-PDH was adjusted for PDH-E1a content (Sup- performed when an interaction was evident. *** 0.001 vs. basal D E conditions; ††P < 0.01 vs. type I muscle fibers; ‡P < 0.05, ‡‡P < plementary Fig. 4 and ). 0.01, ‡‡‡P < 0.001 vs. lean group; §P < 0.05, §§P < 0.01, §§§P < 0.001 vs. obese group. AU, arbitrary units. DISCUSSION The current study is the first to evaluate changes in signaling events in response to insulin in fiber type–specific pools from human muscle. Based on our findings, we pro- pose a model in which human type I fibers have a greater diabetes.diabetesjournals.org Albers and Associates 491

Figure 3—mTOR and NDRG1 in muscle fiber pools from lean, obese, and T2D subjects. Muscle fiber type–specific regulation of mTOR phosphorylation 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 of each 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 blots are shown above bars for each protein probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6 SEM. 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.

abundance of proteins to transport (29% GLUT4), phos- accompanied by lower insulin receptor content and phorylate (470% HKII), and oxidize (35% electron trans- altered response to insulin of p-Akt308, p-Akt473, port chain complex II and 34% PDH) glucose and to p-TBC1D4Ser588 (P = 0.07), and p-NDRG1Thr346 in the synthesize glycogen (35% GS) compared with type II muscle fiber–specific pools from the T2D compared with fibers. These observations are supported by significant the lean and obese groups. In cells, NDRG1 phosphoryla- positive correlations between the MHC I content in whole tion has been suggested to be a readout of mTOR complex muscle lysates and insulin-stimulated GDR (r = 0.53; P = (mTORC) 2 activities (38). mTORC2 is also a widely ac- 0.002), glucose oxidation rate (r = 0.52; P = 0.003), and cepted upstream kinase for AktSer473 (39). Since the re- nonoxidative glucose metabolism (r = 0.44; P = 0.01) sponse to insulin of p-NDRG1Thr346/NDRG1 was similar (Supplementary Fig. 5). Interestingly, even though insulin between groups, these data could imply a specific dysfunc- receptor content was higher (16%) in type I fibers, phos- tional link between mTORC2 and p-AktSer473, as the latter phoregulation of TBC1D1, TBC1D4, and GS by insulin was decreased in response to insulin in both type I and II was similar between fiber types (all normalized to actin). fibers in T2D compared with the lean and obese groups. The apparent fiber-type differences in insulin-stimulated In rat muscle, abundance and insulin-stimulated phos- phosphorylation of Akt, NDRG1, and PDH-E1a (when phorylation of Akt were higher (660 and 160–180%, re- related to actin) were eliminated when adjusted for Akt2, spectively) in soleus muscle primarily containing type I NDRG1, and PDH-E1a protein abundance. These findings fibers, as opposed to epitrochlearis and extensor digito- suggest a similar sensitivity of type I and II muscle fibers rum longus muscles primarily consisting of type II fibers for regulation by insulin of the proteins investigated. (20). In contrast, in human muscle, we report a decreased Insulin-stimulated GDR, glucose oxidation rates, and Akt phosphorylation after insulin in type I versus II fibers, nonoxidative glucose metabolism were decreased in T2D due to higher Akt2 levels in type II fibers. Thus, findings compared with the lean and obese groups. This was in rat muscles with a diverse fiber-type composition could 492 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

Figure 4—TBC1D1 and TBC1D4 in muscle fiber pools from lean, obese, and T2D subjects. Muscle fiber–specific regulation of TBC1D1 phosphorylation at site Thr596 (A) and TBC1D4 phosphorylation on site Ser318 (C), Ser588 (D), Thr642 (E), and Ser704 (F) as well as protein content 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, and the basal type I fiber value in the lean group is set to 100. Representative blots are shown above bars for each protein probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6 SEM. AU, arbitrary units. diabetes.diabetesjournals.org Albers and Associates 493

Figure 5—Glycogen content, GSK3b, and GS in muscle fiber pools from lean, obese, and T2D subjects. A: Muscle fiber–specific glycogen content 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 each protein (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 blots are shown above bars for each protein probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6 SEM. 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.

simply result from differences in locomotor activity, al- the type I fiber–abundant soleus compared with the though species-related differences cannot be excluded. For type II fiber–abundant extensor digitorum longus muscle instance, TBC1D4 and TBC1D1 protein abundance in the (40). In rats, no significant correlations between MHC iso- current study are only modestly lower (216% and 245%) form abundance in various muscles and either TBC1D1 or in human type I versus II fibers. In mice, a high (.10-fold) TBC1D4 protein content were found (21). These findings TBC1D4 and a low (,20%) TBC1D1 content are evident in indicate that fiber-type differences in TBC1D4 and 494 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

TBC1D1 protein levels are highly dependent on the spe- cies investigated. In the current study, no differences in the response to insulin were observed between fiber types in phosphor- ylation of TBC1D4 and TBC1D1. We previously reported a decreased response to insulin of p-TBC1D4Ser318 and p-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 insulin was 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 in T2D versus control subjects (23). Thus, in the current study, the lack of significant defects in TBC1D4 regulation by insulin in the T2D group compared with control groups could be due to the physical fitness level of the groups studied. We found that p-TBC1D1Thr596 was increased by insulin in agreement with another study (41) and that the relative 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, while the absolute amount of phosphorylated protein is lower in type I versus II fibers. Whether a higher total amount of phosphorylated protein is important for the regula- tion of glucose uptake is unknown. To investigate the impact of the present findings on glucose uptake in dif- ferent human muscle fiber types, future studies need to examine the membrane-bound fraction of GLUT4 in dif- ferent fiber types or even measure single muscle fiber glucose transport as performed in rat muscle (7). Interestingly, Gaster et al. (14) previously reported that GLUT4 abundance was significantly lower in type I fibers only in muscle from T2D patients compared with lean and obese control subjects. This was not evident in the current study. However, we found a nonsignificantly lower GLUT4 content of the same magnitude (10–20%) as previously reported (14) in both type I and II fibers from the T2D compared with the lean and obese groups. Also, GLUT4 levels were generally higher in type I versus II fibers. Thus, fewer type I fibers in the T2D compared Figure 6—PDH-E1a in muscle fiber pools from lean, obese, and with the lean and obese groups possibly lowers the glu- T2D subjects. Muscle fiber type–specific regulation of PDH-E1a cose uptake capacity in diabetic skeletal muscle. In sup- phosphorylation on site 1 (A) and site 2 (B) as well as PDH-E1a protein content (C) were evaluated by Western blotting. Phospho- port, HKII content was higher in type I compared with specific PDH-E1a antibodies were directed against the phosphor- type II fibers. The influence of HKII protein levels on ylation of sites Ser293 (site 1) and Ser300 (site 2) on the human glucose uptake is controversial and has recently been es- PDH-E1a isoform. Due to sample limitations, protein levels of timated to control ;10% of human skeletal muscle glu- PDH-E1a were evaluated in a subset of fiber pools, with the number of samples indicated in each bar. Quantified values of each protein cose metabolism during insulin-stimulated conditions are related to the content of actin protein, and the basal type I fiber (42). In the current study, fiber type–specific HKII levels value in the lean group is set to 100. Representative blots are were not different between groups investigated. Thus, it shown above bars for each protein probed for. White bars represent is likely that decreased HKII levels reported in muscles type I and black bars type II muscle fiber pools. Data are means 6 fl SEM. Post hoc testing was only performed when an interaction was from T2D subjects (43) are at least partly in uenced by evident. †††P < 0.001 vs. type I muscle fibers. AU, arbitrary units. alowernumberoftypeIfibers in T2D versus control subjectsasalsoshowninthecurrentstudy.Interest- ingly, in contrast to HKII, HKI protein abundance was lower (219%) among the three groups in type I versus II diabetes.diabetesjournals.org Albers and Associates 495

fibers (Supplementary Fig. 6). This observation could in- fibers. No measure of physical activity was performed. It dicate a different role of HK isoforms in type I and II has been shown that training-induced increases in GLUT4 muscle fibers. content mainly occur in type I fibers (22). Thus, training A close correlation between the insulin-stimulated status of the subjects in the current study could poten- increase in nonoxidative glucose metabolism and GS tially influence differences between muscle fibers and/or activity has been reported (44). In the current study, groups. All measures were performed in muscle fibers from insulin-stimulated nonoxidative glucose metabolism the vastus lateralis muscle. Whether fiber type–specific was decreased in the T2D compared with the lean and differences in protein expression can be extended to other obese groups as shown by others (23,31,41,45). Thus, muscles is unknown, but has been challenged by one we investigated the fiber type–specific regulation of GS study (30), in which GLUT4 expression was higher in by insulin. We were unable to detect any differences in type I versus IIa and IIx fibers from vastus lateralis the response to insulin between fiber types, although the muscles but similar between fiber types in soleus and tri- absolute amount of phosphorylated GS was highest in ceps brachii muscles. The current study design did not type I fibers. Increased phosphorylation of GS in type I allow exploration of this further. To evaluate the biolog- fibers could be accounted for by a higher GS protein level ical impact of fiber-specific signaling events further, the in type I versus II fibers. Previously, a similar GS content methods used in the current study could be combined in type I, IIa, and IIx fiber pools was reported in muscle with ex vivo incubation of human muscle strips (12) from young (23 years) subjects (30). Thus, the present and the recently described method of single-fiber glucose findings of a higher GS content in type I versus II fibers uptake measurements (7). Such design demands open in muscle from middle-aged (;55 years) subjects indi- surgical biopsies and was therefore not applicable to the cates an age-dependent fiber type–specific regulation of cohort of the current study. GS abundance. The functional consequence of a differen- In conclusion, based on protein level measures, the tiated GS content between fiber types is unknown, since enzymatic capacities for glucose uptake, phosphorylation, we were unable to detect any differences in basal and and oxidation as well as for glycogen synthesis are higher insulin-stimulated glycogen content in both fiber types. in human type I compared with type II muscle fibers. In This is likely due to the relatively small (,6%) increase in response to insulin, most differences in phosphorylation glycogen content during a clamp procedure (46). If glyco- between fiber types were due to differences in protein gen levels were solely dependent on GS, the activity of levels. Thus, sensitivity for phosphoregulation by insulin this enzyme would be expected to be lower in type I of these proteins is similar between fiber types. Even versus II fibers. However, our data cannot support this though insulin-induced GDR was decreased in patients because the higher expression and phosphorylation of GS with type 2 diabetes compared with lean and obese subjects, indicates that total GS activity is in fact higher in type I few group differences in the muscle fiber–specific measure- versus II fibers. Thus, other factors than GS activity per se ments were observed. However, our observations favor the determines glycogen levels. idea that fewer type I fibers and a higher number of type IIx In a recent study, Nellemann et al. (47) did not find fibers in muscles from T2D patients contributes to the re- any changes in phosphorylation of PDH-E1a in human duced GDR under insulin-stimulated conditions compared skeletal muscle in response to insulin. Interestingly, with lean and obese subjects. in the current study, PDH-E1a phosphorylation was de- creased by insulin in type II fibers only. Thus, results by Acknowledgments. The authors thank M. Kleinert (University of Copen- Nellemann et al. (47) could have been influenced by fi – hagen, Denmark) for sharing know-how on the mTOR/NDRG1 analyses. The amuscle ber type dependent regulation not detected authors also thank the following for the donation of material essential for this in their whole-muscle biopsy preparation. An inverse re- work: L.J. Goodyear (Joslin Diabetes Center and Harvard Medical School, lationship between PDH-E1a phosphorylation and PDHa Boston, MA), O.B. Pedersen (University of Copenhagen, Denmark), and J. Hastie activity has been shown in human skeletal muscle during and D.G. Hardie (University of Dundee, U.K.). The monoclonal antibodies against exercise (48). Thus, findings in the current study suggest MHC I and II isoforms (A4.840 and A4.74) were developed by H.M. Blau, and an increased PDHa activity in response to insulin in type II antibody directed against MHC IIx (6H1) was developed by C. Lucas. All MHC fibers only. antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Study Limitations Human Development and maintained by The University of Iowa, Department All fiber pools were prepared from vastus lateralis muscle, of Biology, Iowa City, IA. Funding. This work was carried out as a part of the research programs which expresses relatively small (,10%) amounts of type “Physical activity and nutrition for improvement of health” funded by the Univer- IIx fibers (26). No significant differences in the MHC IIx fi sity of Copenhagen Excellence Program for Interdisciplinary Research and expression were observed between type II ber pools the UNIK project Food, Fitness & Pharma for Health and Disease (see www among the three groups (Supplementary Fig. 7). Thus, .foodfitnesspharma.ku.dk) supported by the Danish Ministry of Science, Tech- differences between type I and II fiber pools observed in nology and Innovation. This study was funded by the Danish Council for Inde- the current study are likely not influenced by differences pendent Research Medical Sciences, the Novo Nordisk Foundation, and a Clinical in protein abundance/regulation between type IIa and IIx Research Grant from the European Foundation for the Study of Diabetes. 496 Muscle Fiber Types and Insulin Signaling Diabetes Volume 64, February 2015

Duality of Interest. P.H.A. and J.N. are employees at Novo Nordisk A/S 15. He J, Watkins S, Kelley DE. Skeletal muscle lipid content and oxidative and own stocks in Novo Nordisk A/S. No other potential conflicts of interest enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity. relevant to this article were reported. Diabetes 2001;50:817–823 Author Contributions. P.H.A. was responsible for conception and design 16. Gallagher P, Trappe S, Harber M, et al. Effects of 84-days of bedrest and of research, performed analysis, interpreted results, drafted the manuscript, resistance training on single muscle fibre myosin heavy chain distribution in edited and revised the manuscript, and approved the final version. A.J.T.P. human vastus lateralis and soleus muscles. Acta Physiol Scand 2005;185: performed in vivo experiments and analysis, edited and revised the manuscript, 61–69 and approved the final version. J.B.B. performed analysis, interpreted results, 17. 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