human muscle fiber type specific insulin signaling – impact of...

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Human muscle fiber type specific insulin signaling – Impact of obesity and type 2 diabetes

Peter H. Albers1,2

, Andreas J.T. Pedersen3, Jesper B. Birk

1, Dorte E. Kristensen

1, Birgitte F. Vind

3,

Otto Baba4, Jane Nøhr

2, Kurt Højlund

3, Jørgen F.P. Wojtaszewski

1

1Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, August Krogh

Centre, University of Copenhagen, Denmark

2Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark

3Diabetes Research Center, Department of Endocrinology, Odense University Hospital, Odense,

Denmark

4Section of Biology, Department of Oral Function & Molecular Biology, School of Dentistry, Ohu

University, Koriyama, Japan

*Corresponding Author: Jørgen F.P. Wojtaszewski, PhD. Universitetsparken 13, DK-2100

Copenhagen Ø, Denmark. Phone no: (+45) 28751625, e-mail: [email protected]

Running title: Muscle fiber types and insulin signaling

Word count (abstract): 197

Word count (main text): 3979

References: 48

Number of tables+figures: 2+6

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Diabetes Publish Ahead of Print, published online September 3, 2014

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ABSTRACT

Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that

insulin-mediated glucose metabolism is different between muscle fiber types. We hypothesized that

differences are due to fiber-type specific expression/regulation of insulin signaling elements and/or

metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis

muscles from lean, obese and type 2 diabetic subjects before and after a hyperinsulinemic-

euglycemic clamp. Type I fibers compared to type II fibers have higher protein levels of the insulin

receptor, GLUT4, hexokinase II, glycogen synthase (GS), pyruvate dehydrogenase (PDH-E1α) and

a lower protein content of Akt2, TBC1D4 and TBC1D1. In type I fibers compared to type II fibers,

the phosphorylation-response to insulin was similar (TBC1D4, TBC1D1 and GS) or decreased (Akt

and PDH-E1α). Phosphorylation-responses to insulin adjusted for protein level were not different

between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS and

PDH-E1α) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared

to lean and obese subjects. We conclude that human type I muscle fibers compared to type II fibers

have a higher glucose handling capacity but a similar sensitivity for phosphor-regulation by insulin.

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Keywords

Skeletal muscle • Insulin sensitivity • Glucose disposal rate • Indirect calorimetric • Insulin signaling •

Myosin heavy chain composition • Glycogen • GLUT4 • Glycogen synthase • TBC1 domain family member

• Pyruvate dehydrogenase • Akt

Abbreviations

BMI Body mass index

EDL Extensor digitorum longus

GDR Glucose disposal rate

GS Glycogen synthase

GSK Glycogen synthase kinase

HK Hexokinase

HRP Horseradish peroxidase

mTOR mammalian target of rapamycin

mTORC mammalian target of rapamycin complex

MHC Myosin heavy chain

NDRG N-myc downstream-regulated gene

NOGM Non-oxidative glucose uptake

PDC Pyruvate dehydrogenase complex

PDH-E1α Pyruvate dehydrogenase-E1 alpha subunit

RT Room temperature

T2D Type 2 diabetic

TBC1 Tre-2/USP6, BUB2, cdc16

TBC1D TBC1 domain family member

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INTRODUCTION

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 2 diabetes (2). Skeletal muscle

is a heterogeneous tissue composed of different fiber types, which can be divided according to

myosin heavy chain (MHC) isoform expression. Studies in rodents show that insulin-stimulated

glucose uptake in the oxidative type I fiber-dominant muscles is higher than in muscles with a high

degree of glycolytic type II fibers (3-6). Whether this phenomenon is due to differences in

locomotor activity of individual muscles or a direct consequence of the fiber-type composition is

largely unknown. In incubated rat muscle, insulin-induced glucose uptake was higher (~100%) in

type IIa (oxidative/glycolytic) compared to IIx and IIb (glycolytic) fibers (7;8), suggesting that

insulin-mediated glucose uptake is related to the oxidative capacity of the muscle fiber. In humans,

a positive correlation between proportions of type I fibers in muscle and whole-body insulin

sensitivity has been demonstrated (9-11). Furthermore, insulin-stimulated glucose transport in

human muscle strips was associated with the relative type I fiber content (12). Thus, it is likely that

human type I fibers are more important than type II fibers for maintaining glucose homeostasis in

response to insulin. Indeed, a decreased proportion of type I fibers has been found in various insulin

resistant states such as the metabolic syndrome (9), obesity (13;14), type 2 diabetes in some

(10;13;14) but not all (12;15) studies and following bedrest (16) as well as in tetraplegic patients

(17) and subjects with an insulin receptor gene mutation (18).

Mechanisms for a fiber-type dependent regulation of glucose uptake could involve altered

abundance/regulation of insulin signaling elements and/or metabolic enzymes. In rats, insulin

receptor content and Akt and GLUT4 protein abundance are higher in type I compared to type II

fiber dominated muscles (4;5;19-21). Furthermore, in rats, Akt phosphorylation under insulin

stimulationare highest in type I compared to type II fiber dominant muscles (20). In humans,

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GLUT4 protein levels are higher in type I compared to type IIa and IIx muscle fibers (14;22).

Overall, these findings suggest that insulin signaling to and effect on glucose transport is highest in

type I fibers. Thus, a shift towards reduced type I and hence higher type II fiber content in obesity

and type 2 diabetes (10;13;14) could negatively influence muscle insulin action on glucose

metabolism. Insulin resistance in obesity and type 2 diabetes is characterized by a decreased ability

of insulin to induce signaling proteins proposed to mediate GLUT4 translocation by i.e.

phosphorylation/activation of Akt (23-25) and/or TBC1 domain family member (TBC1D) 4 (23;25).

Whether this relates to differences in the response to insulin between fiber types is unknown.

Intracellular glucose metabolism could also be different between muscle fiber types. Glucose

entering the muscle cell is initially phosphorylated by hexokinase (HK) and predominantly stored as

glycogen or oxidized in the mitochondria, through processes regulated by glycogen synthase (GS)

and the pyruvate dehydrogenase complex (PDC), respectively. HKII content is higher in human

soleus muscle (~70% type I fibers) compared to gastrocnemius and vastus lateralis muscle (~50%

type I fibers) (26). Also, the content of the PDC subunit PDH-E1α is decreased in muscle of

proliferator-activated receptor gamma-coactivator-1α knock-out mice (27), concomitant with a

switch towards reduced type I fiber abundance (28). Furthermore, mitochondrial density is higher in

human type I compared to type II fibers (29). In contrast, no fiber-type specific expression pattern

of GS has been shown (30). All together these observations suggest that glucose phosphorylation

and oxidation but not storage rate capacity are enhanced in type I compared to type II fibers.

Whether HKII and PDH-E1α abundance as well as GS and PDH-E1α regulation by insulin is

different between human muscle fiber types is unknown.

We investigated whether proteins involved in glucose metabolism were expressed and/or regulated

by insulin in a fiber type specific manner in human skeletal muscle. This was achieved by creating

pools of single fibers expressing either MHC I (type I) or II (type II). These fibers were dissected

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from vastus lateralis muscle biopsies obtained from lean and obese normal glucose tolerant subjects

as well as type 2 diabetic patients.

RESEARCH DESIGN AND METHODS

Subjects. 10 lean healthy, 11 obese non-diabetic and 11 obese type 2 diabetic (T2D) subjects were

randomly chosen from two studies conducted at Odense University Hospital, Odense, Denmark.

One fraction (8 lean, 7 obese, 6 T2D) were from an already published study (31), while the

remaining subjects were from an unpublished study, in which subjects were investigated with an

identical experimental protocol as previously described (31). Both studies were approved by the

regional ethics committee and carried out in accordance with the Declaration of Helsinki II. Subject

medication is detailed in supplemental materials.

Experimental protocol. Detailed explanation of the in vivo study protocol has been published

elsewhere (31). In short, all subjects were instructed to refrain from strenuous physical activity 48 h

before the experimental day. After an overnight fast, subjects underwent a 2 h basal tracer

equilibration period followed by a 4 h hyperinsulinemic-euglycemic clamp at an insulin (Actrapid,

Novo Nordisk, Denmark) infusion rate of 40 mU·m-2

·min-1

combined with tracer glucose and

indirect calorimetry. A primed-constant [3-3H]glucose infusion was used throughout the 6-h study,

and [3-3H]glucose was added to the glucose infusates to maintain plasma specific activity constant

at baseline levels during the 4-h clamp period as described in detail previously (32). Vastus lateralis

muscle biopsies were obtained before and after the clamp under local anesthesia (1% lidocaine)

using a modified Bergström needle with suction. Muscle biopsies were immediately frozen in liquid

nitrogen and stored below -80⁰C.

Dissection of individual muscle fibers. Muscle fibers were prepared as previously described (33)

but with minor modifications. 20-60 mg of muscle tissue were freeze-dried for 48 h before

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dissection of individual muscle fibers in a climate-controlled room (20⁰C, <35% humidity) using a

dissection microscope (in total n= 5384 fibers from 64 biopsies). The length of each fiber was

estimated under the microscope (1.5±0.4 mm, mean±SD) before being carefully placed in a PCR-

tube and stored on dry-ice. On the day of dissection 5 µl of ice-cooled Laemmli sample buffer (125

mM Tris-HCl, pH 6.8, 10% glycerol, 125 mM SDS, 200 mM DTT, 0.004% Bromophenol Blue)

was added to each tube. During method optimization addition of protease and phosphatase

inhibitors were found to be unnecessary for preservation of either protein content or protein

phosphorylation for this type of sample preparation (data not shown). After thorough mixing at 4⁰C,

each tube was inspected under a microscope to confirm that the fiber was properly dissolved (if not,

the tube was discarded). Each sample was then heated for 10 minutes at 70⁰C and stored at -80⁰C.

Preparation of pooled muscle fiber samples. A small fraction (1/5) of the solubilized fiber was

used for identification of MHC expression using Western Blotting and specific antibodies against

MHC I or II (see section on immunoblotting). Hybrid fibers (~5%) expressing more than one MHC

isoform were discarded. Pools of type I and II fibers from each biopsy were prepared (128 pools in

total). The average number of type I and II fibers per muscle biopsy included in each pool were 20

(range: 9-36) and 42 (range: 22-147), respectively.

Estimation of protein content and test of purity. Protein content of the fiber specific samples was

estimated using 4-20% Mini-PROTEAN TGX stain-free gels (BioRad, CA), which allowed for gel-

protein imaging following UV-activation on a ChemiDoc MP Imaging System (BioRad, CA). The

intensity of visualized protein bands (from 37-260 kDa) was compared to a standard curve from 3

different pools of human muscle homogenates with a known protein concentration (supplementary

figure 1). After gel imaging, the purity of each pooled sample was re-evaluated using Western

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Blotting and MHC I and II specific antibodies (see section on Immunoblotting). All fiber specific

samples were diluted with Laemmli sample buffer to a protein concentration of 0.2 mg/ml.

Glycogen determination in muscle fiber pools. Glycogen content in the fiber specific pools was

measured by dot-blotting using a specific antibody against glycogen (34;35). Briefly, 150 ng of

protein was spotted onto a PVDF-membrane. After air-drying, the membrane was re-activated in

ethanol before blocking, incubation in primary and secondary antibody and visualization as

described in the section on Immunoblotting. The intensity of each dot was compared to a standard

curve (supplementary figure 2) from a muscle homogenate with an glycogen content pre-

determined biochemically as previously described (31) and expressed accordingly.

MHC determination. For MHC determination in muscle biopsies, lysates were prepared and

protein content was measured as previously described (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). 3 µg of lysate protein was separated for ~16 h at 140 V as

previously described (37). Protein bands were visualized by UV-activation of the stain free gel on a

ChemiDoc MP Imaging System (Biorad, CA) and quantified as stated below. Coomassie staining of

the gel and the use of muscle homogenates provided similar results as stain free gel imaging and

muscle lysates, respectively (data not shown).

Immunoblotting. For MHC determination of single muscle fibers and evaluation of total and

phosphorylated levels of relevant proteins, equal amounts of sample volume (for MHC

determination) or protein amount were separated using either pre-cast (Biorad, CA) or self-cast 7.5%

gels. On each gel, an internal control (muscle lysate) was loaded two times per gel in order to

minimize assay variation. Muscle fiber pool values were divided by the average of the internal

control sample from the corresponding gel. Furthermore, on one gel a standard curve of muscle

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homogenate was loaded to ensure that quantification of each protein probed for was within the

linear range. Following separation, proteins were transferred (semidry) from multiple gels to a

single PVDF-membrane which was incubated with blocking agent (0.05% Tween 20 and 2%

skimmed milk in TBS) for 45 minutes at room temperature (RT), followed by incubation in primary

antibody solution overnight at 4⁰C (for antibody details, see supplementary table 1). Membranes

were incubated with appropriate secondary antibodies (Jackson ImmunoResearch, PA) which were

conjugated to either horseradish peroxidase (HRP) or biotin for 1 h at RT. Membranes incubated

with biotin-conjugated antibody were further treated with HRP-conjugated streptavidin. Protein

bands were visualized using a ChemiDoc MP imaging system (BioRad, CA) and enhanced

chemiluminescence (SuperSignal West Femto, Pierce, IL). Band densitometry was performed using

Image Lab (version 4.0). Membranes were re-probed with an alternate antibody according to the

scheme given in supplementary table 2.

Statistical analyses. Subject characteristics and blood parameters were evaluated by a one-way

ANOVA. To compare fiber type, insulin and group effects, a three-way ANOVA with repeated

measures for fiber type and insulin was used. If no triple-interaction was present, a two-way

ANOVA on the increment with insulin (∆insulin-basal values) was performed for fiber type and

group effects with repeated measures for fiber type. Main-effects of group and significant

interactions were evaluated by Tukey post hoc testing. Statistical analyses were performed in

SigmaPlot (version 12.5, Systat Software, IL; one- and two-way ANOVA) and in SAS statistical

software (version 9.2, SAS Institute, NC; three-way ANOVA). Unless otherwise stated n equals

number of subjects as indicated in table I. Differences were considered significant at p<0.05.

RESULTS

Clinical and metabolic characteristics. BMI and fat mass were higher in the obese and T2D

groups compared to the lean group (table 1). Patients with type 2 diabetes compared to lean and

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obese subjects had elevated HbA1c levels, increased fasting plasma glucose, insulin and triglyceride

(vs lean only) concentrations (table 2). During the hyperinsulinemic-euglycemic clamp the glucose

disposal rate (GDR) was decreased in type 2 diabetic vs lean and obese subjects (table 2). The

decrease in GDR resulted from both lower glucose oxidation rates and reduced non-oxidative

glucose metabolism.

Fiber type composition. In muscle biopsies from lean and obese subjects MHC I, IIa and IIx

constituted 45, 46 and 9% (totally 55% MHCII), respectively (Figure 1A). This fiber type

composition is in accordance with previous observations using (immuno) histochemistry (9-11;13-

15;26) and biochemically methods (18;22). In the T2D group MHC I, IIa and IIx constituted 35, 45

and 20% (totally 65% MHC II), respectively. In the T2D group compared to the lean and obese

group, 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 between all three groups.

Insulin receptor, hexokinase II, GLUT4 and complex II. As represented in figure 1B, all fiber

pools contained one MHC isoform only. Actin was used as reference protein and actin abundance

was equal between fiber pools (supplementary figure 3). Higher protein levels of insulin receptorβ

(+16%), HKII (+470%), GLUT4 (+29%) and electron transport chain complex II (+35%) was

found in type I vs II fibers (Figure 1C-F). No differences between groups were observed except for

a reduced (-24%) insulin receptorβ level in the T2D compared to the lean and obese groups (Figure

1C-F).

Akt, mTOR and NDRG1. Akt2 protein content was lower (-27%) in type I vs II fibers (Figure 2C).

In the three groups, the average increases under insulin stimulation of p-AktThr308

and p-AktSer473

were 5.8 and 3.5 fold in type I fibers and 6.1 and 3.7 fold in type II fibers, respectively (Figure

2A+B). In lean and obese groups levels of insulin-stimulated p-AktThr308

were lower (-25%) in type

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I vs II fibers. In the T2D group the insulin-stimulated p-AktThr308

and p-AktSer473

were lower in both

fiber types compared to lean and obese groups. In response to insulin, phosphorylation of

AktSer473

/Akt2 but not AktThr308

/Akt2 was fiber type dependent, although the relative response to

insulin was similar between fiber types (supplementary figure 4A+B). In type I fibers, a higher

protein level of mammalian target of rapamycin (mTOR) (+20%) and its downstream target N-myc

downstream-regulated gene (NDRG) 1 (+68%) compared to type II fibers was evident (Figure

3B+D). Insulin had no effect on p-mTOR2481

but increased p-NDRG1Thr346

only in type I fibers

from obese (+86%) and T2D (+100%) groups (Figure 3A+C). No fiber type differences were

evident when p-NDRG1Thr346

was adjusted for NDRG1 protein abundance (supplementary figure

4C).

TBC1D1 and TBC1D4. TBC1D1 and TBC1D4 protein levels were -45% and -16% in type I vs II

fibers, respectively (Figure 4B+G). Irrespective of fiber type, insulin stimulation increased p-

TBC1D1Thr596

(+36%) and p-TBC1D4 at all sites investigated (Ser318

(+122%), Ser588

(+59%),

Thr642

(+103%) and Ser704

(+113%)) (Figure 4A+C-F). Statistically significant main effects of fiber

type were evident for the level of phosphorylation of both TBC1D1 and TBC1D4. More

specifically p-TBC1D1Thr596

(-62%), p-TBC1D4Ser318

(-21%), p-TBC1D4Ser588

(-21%), p-

TBC1D4Thr642

(-24%) and p-TBC1D4Ser704

(-24%) were lower in type I compared to type II fibers.

No significant group differences in protein 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, GSK3 and glycogen synthase. In the basal state, glycogen content was lower (-

29%) in type I vs II fibers in the obese (p=0.09) and type 2 diabetic (p=0.09) group; Figure 5A).

Insulin induced no significant changes in glycogen content in either of the fiber types. The protein

levels of glycogen synthase kinase (GSK) 3β was 14% less in type I vs type II, whereas GS protein

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was 53% higher in type I compared to type II fibers (Figure 5C+F). In all 3 groups and in both fiber

types insulin induced a similar change in phosphorylation of GSK3βSer9

(+62%), GS2+2a

(-36%) and

GS3a+b

(-38%) (Figure 5B+D+E). Phosphorylation of GSK3βSer9

was lower (-31%), whereas

phosphorylation of GSsite2+2a

and GSsite3a+b

were respectively 68% and 51 % higher in type I vs II

fibers. No significant differences were evident between individual groups in protein abundance and

protein phosphorylation of GSK3β and GS.

Pyruvate dehydrogenase. PDH-E1α protein content was 34% higher in type I vs II fibers (Figure

6C). Basal levels of PDH-E1α site1 phosphorylation were similar between fiber types in all 3

groups (Figure 6A). After insulin the degree of phosphorylation was significantly lower in type II vs

I fibers in the obese and T2D groups only, indicating de-phosphorylation by insulin in type II but

not in type I fibers. In line, PDH-E1α site2 phosphorylation was decreased by insulin, and this

effect was dependent on fiber type towards a greater effect of insulin in type II vs I fibers (Figure

6B). Fiber type differences were not evident when p-PDHsite1

and p-PDHsite2

was adjusted for PDH-

E1α content (supplementary figure 4D+E).

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 propose a model in which

human type I fibers have a greater abundance of proteins to transport (+29% GLUT4),

phosphorylate (+470% HKII) and oxidize (+35% ETC complex II and +34% PDH) glucose and to

synthesize glycogen (+35% GS) compared to type II fibers. These observations are supported by

significant positive correlations between the MHC I content in whole muscle lysates and insulin-

stimulated glucose disposal rate (r=0.53, p=0.002), glucose oxidation rate (r=0.52, p=0.003) and

non-oxidative glucose metabolism (r=0.44, p=0.01) (supplementary figure 5). Interestingly, even

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though insulin receptor content was higher (+16%) in type I fibers, phosphor-regulation of TBC1D1,

TBC1D4 and GS by insulin was similar between fiber types (all normalized to actin). The apparent

fiber-type differences in insulin-stimulated phosphorylation of Akt, NDRG1 and PDH-E1α (when

related to actin) were eliminated when adjusted for Akt2, NDRG1 and PDH-E1α protein abundance.

These findings suggest a similar sensitivity of type I and II muscle fibers for regulation by insulin of

the proteins investigated.

Insulin-stimulated glucose disposal rate, glucose oxidation rates and non-oxidative glucose

metabolism were decreased in T2D compared to the lean and obese groups. This was accompanied

by lower insulin receptor content and altered response to insulin of p-Akt308

, p-Akt473

, p-

TBC1D4Ser588

(p=0.07) and p-NDRG1Thr346

in the muscle fiber-specific pools from the T2D

compared to the lean and obese groups. In cells, NDRG1 phosphorylation has been suggested to be

a read-out of mTOR complex (mTORC) 2 activities (38). mTORC2 is also a widely accepted

upstream kinase for AktSer473

(39). Since the response to insulin of p-NDRG1Thr346

/NDRG1 was

similar between groups, these data could imply a specific dysfunctional link between mTORC2 and

p-AktSer473

as the latter was decreased in response to insulin in both type I and II fibers in T2D

compared to the lean and obese groups. In rat muscle, abundance and insulin-stimulated

phosphorylation of Akt were higher (+660% and +160-180%, respectively) in soleus muscle

primarily containing type I fibers, as opposed to epitrochlearis and extensor digitorum longus (EDL)

muscles primarily consisting of type II fibers (20). In contrast, in human muscle, we report a

decreased Akt phosphorylation after insulin in type I vs II fibers, due to higher Akt2 levels in type

II fibers. Thus, findings in rat muscles with a diverse fiber-type composition could simply result

from differences in locomotor activity, although species-related differences cannot be excluded. For

instance, TBC1D4 and TBC1D1 protein abundance in the present study is only modestly lower (-16

and -45%) in human type I vs II fibers. In mice, a high (>10-fold) TBC1D4 and a low (<20%)

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TBC1D1 content are evident in the type I fiber abundant soleus compared to the type II fiber

abundant EDL muscle (40). In rats, no significant correlations between MHC-isoform abundance in

various muscles and either TBC1D1 or TBC1D4 protein content was found (21). These findings

indicate that fiber-type differences in TBC1D4 and TBC1D1 protein levels are highly dependent on

the species investigated.

In the present study, no differences in the response to insulin were observed between fiber types in

phosphorylation of TBC1D4 and TBC1D1. We previously reported a decreased response to insulin

of p-TBC1D4Ser318

and p-TBC1D4Ser588

in skeletal muscle from obese type 2 diabetic subjects

compared to weight-matched controls (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 normalizes defects in insulin action on TBC1D4 regulation in

type 2 diabetic vs control subjects (23). Thus, in the present study, the lack of significant defects in

TBC1D4 regulation by insulin in the T2D group compared to 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 vs

II fibers. Whether a higher amount of phosphorylated protein is important for the regulation of

glucose uptake is unknown. To investigate the impact of the present findings on glucose uptake in

different human muscle fiber types, future studies need to examine the membrane-bound fraction of

GLUT4 in different fiber types or even measure single muscle fiber glucose transport as performed

in rat muscle (7).

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Interestingly, Gaster et al. (14) previously reported that GLUT4 abundance was significantly lower

in type I fibers only, in muscle from type 2 diabetic patients compared to lean and obese controls.

This was not evident in the current study. However, we found a non-significantly lower GLUT4

content of the same magnitude (10-20%) as previously reported (14) in both type I and II fibers

from the T2D compared to the lean and obese groups. Also, GLUT4 levels were generally higher in

type I vs II fibers. Thus, fewer type I fibers in the T2D compared to the lean and obese groups

possibly lowers the glucose uptake capacity in diabetic skeletal muscle. In support, HKII content

was higher in type I compared to type II fibers. The influence of HKII protein levels on glucose

uptake is controversial and has recently been estimated to control ~10% of human skeletal muscle

glucose metabolism during insulin-stimulated conditions (42). In the current study, fiber type

specific HKII levels were not different between groups investigated. Thus, it is likely that decreased

HKII levels reported in muscles from T2D subjects (43) are at least partly influenced by a lower

number of type I fibers in T2D vs control subjects as also shown in the present study. Interestingly,

in contrast to HKII, HKI protein abundance was lower (-19%) among the three groups in type I vs

II fibers (supplementary figure 6). This observation could indicate a different role of HK isoforms

in type I and II muscle fibers.

A close correlation between the insulin-stimulated increase in non-oxidative glucose metabolism

and GS activity has been reported (44). In the present study, insulin-stimulated non-oxidative

glucose metabolism was decreased in the T2D compared to the lean and obese groups as shown by

others (23;31;41;45). Thus, we investigated the fiber-type specific regulation of GS by insulin. We

were unable to detect any differences in the response to insulin between fiber types, although the

absolute amount of phosphorylated GS was highest in type I fibers. Increased phosphorylation of

GS in type I fibers could be accounted for by a higher GS protein level in type I vs II fibers.

Previously, a similar GS content in type I, IIa and IIx fiber pools was reported in muscle from

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young (23 yrs) subjects (30). Thus, the present findings of a higher GS content in type I vs II fibers

in muscle from middle-aged (~55 yrs) subjects, indicates an age dependent fiber type specific

regulation of GS abundance. The functional consequence of a differentiated GS content between

fiber types is unknown, since we were unable to detect any differences in basal and insulin-

stimulated glycogen content in both fiber types. This is likely due to the relatively small (<6%)

increase in glycogen content during a clamp procedure (46). If glycogen levels were solely

dependent on glycogen synthase, the activity of this enzyme would be expected to be lower in type I

vs II fibers. However, our data cannot support this because the higher expression and

phosphorylation of GS indicates that total GS activity is infact higher in type I vs II fibers. Thus,

other factors than GS activity per se determines glycogen levels.

In a recent study, Nellemann et al. (47) did not find any changes in phosphorylation of PDH-E1α in

human skeletal muscle in response to insulin. Interestingly, in the present study, PDH-E1α

phosphorylation was decreased by insulin in type II fibers only. Thus, results by Nellemann et al.

could have been influenced by a muscle fiber-type dependent regulation not detected in their whole

muscle biopsy preparation. An inverse relationship between PDH-E1α phosphorylation and PDHa

activity has been shown in human skeletal muscle during exercise (48). Thus, findings in the

present study suggest an increased PDHa activity in response to insulin in type II fibers only.

Study limitations:

All fiber pools were prepared from vastus lateralis muscle, which expresses relatively small (<10%)

amounts of type IIx fibers (26). No significant differences in the MHC IIx expression were

observed between type II fiber pools among the three groups (supplementary figure 7). Thus,

differences between type I and II fiber pools observed in the present study are likely not influenced

by differences in protein abundance/regulation between type IIa and IIx fibers. No measure of

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physical activity was performed. It has been shown that training-induced increases in GLUT4

content mainly occur in type I fibers (22). Thus, training status of the subjects in the present study

could potentially influence differences between muscle fibers and/or groups. All measures were

performed in muscle fibers from the vastus lateralis muscle. Whether fiber-type specific differences

in protein expression can be extended to other muscles is unknown, but has been challenged by one

study (30), in which GLUT4 expression was higher in type I vs. IIa and IIx fibers from vastus

lateralis muscles but similar between fiber types in soleus and triceps brachii muscles. The present

study design did not allow exploration of this further. To evaluate the biological impact of fiber

specific signaling events further, the methods used in the present study could be combined with ex

vivo incubation of human muscle strips (12) and the recently described method of single fiber

glucose uptake measurements (7). Such design demands open surgical biopsies and was therefore

not applicable to the cohort of the present study.

In conclusion, based on protein level measures, the enzymatic capacities for glucose uptake,

phosphorylation and oxidation as well as for glycogen synthesis are higher in human type I

compared to type II muscle fibers. In response to insulin, most differences in phosphorylation

between fiber types were due to differences in protein levels. Thus, sensitivity for phosphor-

regulation by insulin of these proteins is similar between fiber types. Even though insulin-induced

glucose disposal rate was decreased in patients with type 2 diabetes compared to lean and obese

subjects, few group differences in the muscle fiber specific measurements were observed. However,

our observations favor the idea that fewer type I fibers and a higher number of type IIx fibers in

muscles from type 2 diabetic patients contributes to the reduced glucose disposal rate under insulin-

stimulated conditions compared to lean and obese subjects.

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ACKNOWLEDGMENTS

Author contributions: Conception and design of research: P.H.A., J.F.P.W. Performed in vivo

experiments: A.J.T.P., B.F.V. Performed analysis: P.H.A., A.J.T.P., J.B.B., D.E.K., B.F.V.

Interpreted results: P.H.A., J.B.B., K.H., J.F.P.W. Drafted manuscript: P.H.A., J.F.P.W. Edited and

revised manuscript: All. Approved final version: All. Jørgen F.P. Wojtaszewski is the guarantor of

this work and, as such, had full access to all the data in the study and takes responsibility for the

integrity of the data and the accuracy of the data analysis.

Assistance/donations: Maximilian Kleinert (University of Copenhagen, Denmark) is

acknowledged for sharing his knowhow on the mTOR/NDRG1 analyses. Also, we are grateful for

the kind donation of material essential for this work by the following scientists: LJ Goodyear (Joslin

Diabetes Center and Harvard medical school, Boston, MA), OB Pedersen (University of

Copenhagen, Denmark), J Hastie and DG Hardie (University of Dundee, UK). The monoclonal

antibodies against MHC I and II isoforms (A4.840 and A4.74) were developed by H.M. Blau and

antibody directed against MHC IIx (6H1) was developed by C. Lucas. All MHC antibodies were

obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the

NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

Funding/financial support: This work was carried out as a part of the research programs "Physical

activity and nutrition for improvement of health" funded by the University of Copenhagen (UCPH)

Excellence Program for Interdisciplinary Research; and the UNIK project: Food, Fitness & Pharma

for Health and Disease (see www.foodfitnesspharma.ku.dk) supported by the Danish Ministry of

Science, Technology and Innovation. This study was funded by the Danish Council for Independent

Research Medical Sciences (FSS), the Novo Nordisk Foundation and a Clinical Research Grant

from the European Foundation for the study of Diabetes (EFSD). Disclosure statement: P.H.A. is

financed as an industrial PhD student by the Danish Agency for Science, Technology and

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Innovation and Novo Nordisk A/S and owns stocks in Novo Nordisk A/S. J.N. is an employee at

Novo Nordisk A/S and owns stocks in Novo Nordisk A/S. A.J.T.P., J.B.B., D.E.K., B.F.V., O.B.,

K.H., J.F.P.W. have nothing to disclose.

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REFERENCES

1. DeFronzo,RA, Jacot,E, Jequier,E, Maeder,E, Wahren,J, Felber,JP: The effect of insulin on the

disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral

venous catheterization. Diabetes 30:1000-1007, 1981

2. Dohm,GL, Tapscott,EB, Pories,WJ, Dabbs,DJ, Flickinger,EG, Meelheim,D, Fushiki,T,

Atkinson,SM, Elton,CW, Caro,JF: An in vitro human muscle preparation suitable for

metabolic studies. Decreased insulin stimulation of glucose transport in muscle from morbidly

obese and diabetic subjects. J Clin Invest 82:486-494, 1988

3. James,DE, Jenkins,AB, Kraegen,EW: Heterogeneity of insulin action in individual muscles in

vivo: euglycemic clamp studies in rats. Am J Physiol 248:E567-E574, 1985

4. Marette,A, Richardson,JM, Ramlal,T, Balon,TW, Vranic,M, Pessin,JE, Klip,A: Abundance,

localization, and insulin-induced translocation of glucose transporters in red and white muscle.

Am J Physiol 263:C443-C452, 1992

5. Bonen,A, Tan,MH, Watson-Wright,WM: Insulin binding and glucose uptake differences in

rodent skeletal muscles. Diabetes 30:702-704, 1981

6. Ploug,T, Galbo,H, Vinten,J, Jorgensen,M, Richter,EA: Kinetics of glucose transport in rat

muscle: effects of insulin and contractions. Am J Physiol 253:E12-E20, 1987

7. Mackrell,JG, Cartee,GD: A novel method to measure glucose uptake and myosin heavy chain

isoform expression of single fibers from rat skeletal muscle. Diabetes 61:995-1003, 2012

8. Mackrell,JG, Arias,EB, Cartee,GD: Fiber type-specific differences in glucose uptake by single

fibers from skeletal muscles of 9- and 25-month-old rats. J Gerontol A Biol Sci Med Sci

67:1286-1294, 2012

9. Stuart,CA, McCurry,MP, Marino,A, South,MA, Howell,ME, Layne,AS, Ramsey,MW,

Stone,MH: Slow-Twitch Fiber Proportion in Skeletal Muscle Correlates with Insulin

Responsiveness. J Clin Endocrinol Metab 2013

10. Oberbach,A, Bossenz,Y, Lehmann,S, Niebauer,J, Adams,V, Paschke,R, Schon,MR, Bluher,M,

Punkt,K: Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity

in skeletal muscle of patients with type 2 diabetes. Diabetes Care 29:895-900, 2006

11. Coen,PM, Dube,JJ, Amati,F, Stefanovic-Racic,M, Ferrell,RE, Toledo,FG, Goodpaster,BH:

Insulin resistance is associated with higher intramyocellular triglycerides in type I but not type

II myocytes concomitant with higher ceramide content. Diabetes 59:80-88, 2010

12. Zierath,JR, He,L, Guma,A, Odegoard,WE, Klip,A, Wallberg-Henriksson,H: Insulin action on

glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with

NIDDM. Diabetologia 39:1180-1189, 1996

13. Marin,P, Andersson,B, Krotkiewski,M, Bjorntorp,P: Muscle fiber composition and capillary

density in women and men with NIDDM. Diabetes Care 17:382-386, 1994

of 44Diabetes

21

14. Gaster,M, Staehr,P, Beck-Nielsen,H, Schroder,HD, Handberg,A: GLUT4 is reduced in slow

muscle fibers of type 2 diabetic patients: is insulin resistance in type 2 diabetes a slow, type 1

fiber disease? Diabetes 50:1324-1329, 2001

15. He,J, Watkins,S, Kelley,DE: Skeletal muscle lipid content and oxidative enzyme activity in

relation to muscle fiber type in type 2 diabetes and obesity. Diabetes 50:817-823, 2001

16. Gallagher,P, Trappe,S, Harber,M, Creer,A, Mazzetti,S, Trappe,T, Alkner,B, Tesch,P: Effects

of 84-days of bedrest and resistance training on single muscle fibre myosin heavy chain

distribution in human vastus lateralis and soleus muscles. Acta Physiol Scand 185:61-69, 2005

17. Grimby,G, Broberg,C, Krotkiewska,I, Krotkiewski,M: Muscle fiber composition in patients

with traumatic cord lesion. Scand J Rehabil Med 8:37-42, 1976

18. Kristensen,JM, Skov,V, Petersson,SJ, Ortenblad,N, Wojtaszewski,JF, Beck-Nielsen,H,

Hojlund,K: A PGC-1alpha- and muscle fibre type-related decrease in markers of

mitochondrial oxidative metabolism in skeletal muscle of humans with inherited insulin

resistance. Diabetologia 2014

19. James,DE, Strube,M, Mueckler,M: Molecular cloning and characterization of an insulin-

regulatable glucose transporter. Nature 338:83-87, 1989

20. Song,XM, Ryder,JW, Kawano,Y, Chibalin,AV, Krook,A, Zierath,JR: Muscle fiber type

specificity in insulin signal transduction. Am J Physiol 277:R1690-R1696, 1999

21. Castorena,CM, Mackrell,JG, Bogan,JS, Kanzaki,M, Cartee,GD: Clustering of GLUT4, TUG,

and RUVBL2 protein levels correlate with myosin heavy chain isoform pattern in skeletal

muscles, but AS160 and TBC1D1 levels do not. J Appl Physiol 111:1106-1117, 2011

22. Daugaard,JR, Nielsen,JN, Kristiansen,S, Andersen,JL, Hargreaves,M, Richter,EA: Fiber type-

specific expression of GLUT4 in human skeletal muscle: influence of exercise training.

Diabetes 49:1092-1095, 2000

23. Vind,BF, Pehmoller,C, Treebak,JT, Birk,JB, Hey-Mogensen,M, Beck-Nielsen,H, Zierath,JR,

Wojtaszewski,JF, Hojlund,K: Impaired insulin-induced site-specific phosphorylation of TBC1

domain family, member 4 (TBC1D4) in skeletal muscle of type 2 diabetes patients is restored

by endurance exercise-training. Diabetologia 54:157-167, 2011

24. Hojlund,K, Birk,JB, Klein,DK, Levin,K, Rose,AJ, Hansen,BF, Nielsen,JN, Beck-Nielsen,H,

Wojtaszewski,JF: Dysregulation of glycogen synthase. J Clin Endocrinol Metab 94:4547-

4556, 2009

25. Karlsson,HK, Zierath,JR, Kane,S, Krook,A, Lienhard,GE, Wallberg-Henriksson,H: Insulin-

stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type

2 diabetic subjects. Diabetes 54:1692-1697, 2005

26. Jensen,TE, Leutert,R, Rasmussen,ST, Mouatt,JR, Christiansen,ML, Jensen,BR, Richter,EA:

EMG-normalised kinase activation during exercise is higher in human gastrocnemius

compared to soleus muscle. PLoS One 7:e31054, 2012

of 44 Diabetes

22

27. Kiilerich,K, Adser,H, Jakobsen,AH, Pedersen,PA, Hardie,DG, Wojtaszewski,JF, Pilegaard,H:

PGC-1alpha increases PDH content but does not change acute PDH regulation in mouse

skeletal muscle. Am J Physiol Regul Integr Comp Physiol 299:R1350-R1359, 2010

28. Handschin,C, Chin,S, Li,P, Liu,F, Maratos-Flier,E, LeBrasseur,NK, Yan,Z, Spiegelman,BM:

Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1alpha

muscle-specific knock-out animals. J Biol Chem 282:30014-30021, 2007

29. Howald,H, Hoppeler,H, Claassen,H, Mathieu,O, Straub,R: Influences of endurance training

on the ultrastructural composition of the different muscle fiber types in humans. Pflugers Arch

403:369-376, 1985

30. Daugaard,JR, Richter,EA: Muscle- and fibre type-specific expression of glucose transporter 4,

glycogen synthase and glycogen phosphorylase proteins in human skeletal muscle. Pflugers

Arch 447:452-456, 2004

31. Vind,BF, Birk,JB, Vienberg,SG, Andersen,B, Beck-Nielsen,H, Wojtaszewski,JF, Hojlund,K:

Hyperglycaemia normalises insulin action on glucose metabolism but not the impaired

activation of AKT and glycogen synthase in the skeletal muscle of patients with type 2

diabetes. Diabetologia 55:1435-1445, 2012

32. Hother-Nielsen,O, Henriksen,JE, Holst,JJ, Beck-Nielsen,H: Effects of insulin on glucose

turnover rates in vivo: isotope dilution versus constant specific activity technique. Metabolism

45:82-91, 1996

33. Murphy,RM: Enhanced technique to measure proteins in single segments of human skeletal

muscle fibers: fiber-type dependence of AMPK-alpha1 and -beta1. J Appl Physiol 110:820-

825, 2011

34. Baba,O: [Production of monoclonal antibody that recognizes glycogen and its application for

immunohistochemistry]. Kokubyo Gakkai Zasshi 60:264-287, 1993

35. Prats,C, Gomez-Cabello,A, Nordby,P, Andersen,JL, Helge,JW, Dela,F, Baba,O, Ploug,T: An

optimized histochemical method to assess skeletal muscle glycogen and lipid stores reveals

two metabolically distinct populations of type I muscle fibers. PLoS One 8:e77774, 2013

36. Ladner,CL, Yang,J, Turner,RJ, Edwards,RA: Visible fluorescent detection of proteins in

polyacrylamide gels without staining. Anal Biochem 326:13-20, 2004

37. Kohn,TA, Myburgh,KH: Electrophoretic separation of human skeletal muscle myosin heavy

chain isoforms: the importance of reducing agents. J Physiol Sci 56:355-360, 2006

38. Garcia-Martinez,JM, Alessi,DR: mTOR complex 2 (mTORC2) controls hydrophobic motif

phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1).

Biochem J 416:375-385, 2008

39. Sarbassov,DD, Guertin,DA, Ali,SM, Sabatini,DM: Phosphorylation and regulation of

Akt/PKB by the rictor-mTOR complex. Science 307:1098-1101, 2005

of 44Diabetes

23

40. Taylor,EB, An,D, Kramer,HF, Yu,H, Fujii,NL, Roeckl,KS, Bowles,N, Hirshman,MF, Xie,J,

Feener,EP, Goodyear,LJ: Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-

stimulated signaling nexus in mouse skeletal muscle. J Biol Chem 283:9787-9796, 2008

41. Vendelbo,MH, Clasen,BF, Treebak,JT, Moller,L, Krusenstjerna-Hafstrom,T, Madsen,M,

Nielsen,TS, Stodkilde-Jorgensen,H, Pedersen,SB, Jorgensen,JO, Goodyear,LJ,

Wojtaszewski,JF, Moller,N, Jessen,N: Insulin resistance after a 72-h fast is associated with

impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal

muscle. Am J Physiol Endocrinol Metab 302:E190-E200, 2012

42. Ng,JM, Bertoldo,A, Minhas,DS, Helbling,NL, Coen,PM, Price,JC, Cobelli,C, Kelley,DE,

Goodpaster,BH: Dynamic PET Imaging Reveals Heterogeneity of Skeletal Muscle Insulin

Resistance. J Clin Endocrinol Metab 2013

43. Pendergrass,M, Koval,J, Vogt,C, Yki-Jarvinen,H, Iozzo,P, Pipek,R, Ardehali,H, Printz,R,

Granner,D, DeFronzo,RA, Mandarino,LJ: Insulin-induced hexokinase II expression is reduced

in obesity and NIDDM. Diabetes 47:387-394, 1998

44. Poulsen,P, Wojtaszewski,JF, Petersen,I, Christensen,K, Richter,EA, Beck-Nielsen,H, Vaag,A:

Impact of genetic versus environmental factors on the control of muscle glycogen synthase

activation in twins. Diabetes 54:1289-1296, 2005

45. Hojlund,K, Staehr,P, Hansen,BF, Green,KA, Hardie,DG, Richter,EA, Beck-Nielsen,H,

Wojtaszewski,JF: Increased phosphorylation of skeletal muscle glycogen synthase at NH2-

terminal sites during physiological hyperinsulinemia in type 2 diabetes. Diabetes 52:1393-

1402, 2003

46. Wojtaszewski,JF, Hansen,BF, Gade, Kiens,B, Markuns,JF, Goodyear,LJ, Richter,EA: Insulin

signaling and insulin sensitivity after exercise in human skeletal muscle. Diabetes 49:325-331,

2000

47. Nellemann,B, Vendelbo,MH, Nielsen,TS, Bak,AM, Hogild,M, Pedersen,SB, Bienso,RS,

Pilegaard,H, Moller,N, Jessen,N, Jorgensen,JO: Growth hormone-induced insulin resistance

in human subjects involves reduced pyruvate dehydrogenase activity. Acta Physiol (Oxf) 2013

48. Pilegaard,H, Birk,JB, Sacchetti,M, Mourtzakis,M, Hardie,DG, Stewart,G, Neufer,PD, Saltin,B,

van,HG, Wojtaszewski,JF: PDH-E1alpha dephosphorylation and activation in human skeletal

muscle during exercise: effect of intralipid infusion. Diabetes 55:3020-3027, 2006

49. Jessen,N, An,D, Lihn,AS, Nygren,J, Hirshman,MF, Thorell,A, Goodyear,LJ: Exercise

increases TBC1D1 phosphorylation in human skeletal muscle. Am J Physiol Endocrinol

Metab 301:E164-E171, 2011

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Figure legends

Figure 1. Myosin heavy chain composition and muscle fiber-type specific protein abundance in

lean, obese and type 2 diabetic subjects. Myosin heavy chain composition measured in whole

muscle biopsies from lean, obese and type 2 diabetic (T2D) subjects (A). The purity of each muscle

fiber pool was checked by Western Blotting of myosin heavy chain (MHC) I and II (B).

Representative blots of type I (MHC I) and type II (MHC II) muscle fiber pools from three subjects

are shown (B). In muscle fiber pools, the protein content of the insulin receptorβ (C), hexokinase II

(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+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±SEM. Post hoc testing was only performed when an interaction was evident . AU, arbitrary

units; MHC, myosin heavy chain. †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.

Figure 2. Akt in muscle fiber pools from lean, obese and type 2 diabetic subjects. Muscle fiber-type

specific regulation of Akt phosphorylation on site Thr308

(A) and Ser473

(B) and protein content of

Akt2 (C) was evaluated by Western Blotting. Two bands are apparent for human Akt2 when insulin

stimulated (both being Akt2 (31)). 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±SEM. Post hoc testing was only performed when an interaction

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was evident. AU, arbitrary units. ***p<0.001 vs basal conditions; ††

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.

Figure 3. Mammalian target of rapamycin (mTOR) and N-myc downstream-regulated gene 1

(NDRG1) in muscle fiber pools from lean, obese and type 2 diabetic 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±SEM. Post hoc testing was only performed when an interaction was evident. AU, arbitrary

units. *p<0.05, ***p<0.001 vs basal conditions; †††

p<0.001 vs type I muscle fibers.

Figure 4. TBC1 domain family member 1 and 4 (TBC1D1 and TBC1D4) in muscle fiber pools from

lean, obese and type 2 diabetic 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) was 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±SEM. AU, arbitrary units.

Figure 5. Glycogen content, glycogen synthase kinase 3β (GSK3β), glycogen synthase (GS) in

muscle fiber pools from lean, obese and type 2 diabetic subjects. Muscle fiber specific glycogen

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content measured by dot-blotting (A). Muscle fiber specific phosphorylation of GSK3β on site Ser9

(B) and GS phosphorylation on site 2+2a (D) and 3a+b (E) as well as protein abundance of GSK3β

(C) and GS (F) was 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±SEM. Post hoc testing was only

performed when an interaction was evident. AU, arbitrary units. (†)

p=0.09, †††

p<0.001 vs type I

muscle fibers.

Figure 6. Pyruvate dehydrogenase (PDH)-E1α in muscle fiber pools from lean, obese and type 2

diabetic subjects. Muscle fiber-type specific regulation of PDH-E1α phosphorylation on site 1 (A)

and site 2 (B) as well as PDH-E1α protein content (C) were evaluated by Western Blotting.

Phospho-specific PDH-E1α antibodies were directed against the phosphorylation of sites Ser293

(site

1) and Ser300

(site 2) on the human PDH-E1α isoform. Due to sample limitations, protein levels of

PDH-E1α were evaluated in a subset of fiber pools, with the number of samples indicated in each

bar. 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±SEM. Post hoc testing was only performed when an interaction was evident. AU, arbitrary

units. †††

p<0.001 vs type I muscle fibers.

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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 ± 2 56 ± 2 55 ± 2

Height (m) 1.77 ± 0.03 1.77 ± 0.03 1.75 ± 0.03

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

Fat free mass (kg) 59.3 ± 3.3 68.5 ± 3.5 63.3 ± 3.3

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

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

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

Plasma cholesterol (mmol/l) 5.5 ± 0.3 5.6 ± 0.2 5.0 ± 0.2

Plasma LDL-cholesterol (mmol/l) 3.6 ± 0.2 3.7 ± 0.2 2.9 ± 0.2†

Plasma HDL-cholesterol (mmol/l) 1.6 ± 0.1 1.4 ± 0.1 1.0 ± 0.1**,†

Plasma triglycerides (mmol/l) 0.9 ± 0.1 1.4 ± 0.2 2.6 ± 0.6*

Diabetes duration (years) - - 4.0 ± 1.5

Values are means±SEM. *p<0.05, **p<0.01, ***p<0.001 vs lean group; †p<0.05,

†††p<0.001 vs

obese group.

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Table 2 Metabolic characteristics during hyperinsulinemic-euglycemic clamp

Lean Obese T2D

Plasma glucosebasal (mmol/l) 5.6 ± 0.2 5.9 ± 0.1 9.0 ± 0.6***,†††

Plasma glucoseclamp (mmol/l) 5.5 ± 0.1 5.3 ± 0.2 5.5 ± 0.1

Serum insulinbasal (pmol/l) 27 ± 3 44 ± 5 86 ± 15***,†

Serum insulinclamp (pmol/l) 408 ± 23 399 ± 12 422 ± 17

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

GDRclamp (mg/m2/min) 388 ± 28 334 ± 20 161 ± 24***

,†††

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

Glucose oxidationclamp (mg/m2/min) 141 ± 14 126 ± 10 77 ± 7***

,††

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

NOGMclamp (mg/m2/min) 247 ± 22 208 ± 23 84 ± 22***

,††

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

Lipid oxidationclamp (mg/m2/min) -1 ± 5 4 ± 3 19 ± 4**,†

RERbasal 0.82 ± 0.01 0.81 ± 0.01 0.80 ± 0.01

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

Plasma lactatebasal (mmol/l) 0.78 ± 0.09 0.80 ± 0.07 1.06 ± 0.11

Plasma lactateclamp (mmol/l) 1.36 ± 0.08 1.18 ± 0.08 0.93 ± 0.06***

Values are means±SEM. GDR, glucose disposal rate; NOGM, non-oxidative 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.

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Total protein expression

202x207mm (300 x 300 DPI)

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Akt phosphorylation and Akt 2 protein expression

200x376mm (300 x 300 DPI)

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Phosphorylation and Protein expression of mTor and NDRG1

138x96mm (300 x 300 DPI)

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Protein expression and phosphorylation of TBC1D1 and TBC1D4

266x360mm (300 x 300 DPI)

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Phosphorylation and protein expression of GSK3 beta and Glycogen synthase (GS) 202x206mm (300 x 300 DPI)

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Pyruvate dehydrogenase (PDH) expression and phosphorylation

201x380mm (300 x 300 DPI)

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Online supplemental materials

Subject medications. Patients with type 2 diabetes were treated either by diet alone (n=2) or diet in

combination with metformin (n=7), metformin and long-acting insulin (n=1) or rosiglitazone and

sulfonylurea (n=1). Oral anti-diabetics were withdrawn one week prior to the study together with

antihypertensive (n=5) and lipid lowering (n=7) drugs. In addition, one lean subject were treated with a

proton-pump inhibitor, 2 obese subjects were treated with angiotensin-converting-enzyme (ACE) inhibitor

whereas 4 patients with type 2 diabetes were treated with ACE-inhibitor (n=3) or AT2-blocker (n=1). Long-

acting insulin was withdrawn one day before the study. The patients with type 2 diabetes were GAD65

antibody negative and without any signs of diabetic retinopathy, nephropathy, neuropathy or macro vascular

complications. All woman included were post-menopausal. Lean and obese control subjects had no family

history of diabetes.

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Supplementary table 1. Primary antibodies used for Western Blotting and DOT-Blotting Antibody Manufacturer/donator Cat. #

MHC I Developmental Studies Hybridoma Bank (Australia) A4.840

MHC II Developmental Studies Hybridoma Bank (Australia) A4.74

MHC IIx Developmental Studies Hybridoma Bank (Australia) 6H1

Actin Sigma Aldrich (MO, USA) A2066

Insulin-Rβ Santa Cruz Biotechnology (CA, USA) SC-711

Hexokinase I Cell Signaling Technology (MA, USA) 2024

Hexokinase II Cell Signaling Technology (MA, USA) 2867

GLUT4 Thermo Scientific (Pierce, IL, USA) PA1-1065

Complex 2 Molecular Probes, Invitrogen (CA, USA) A11142

p-AktThr308

Cell Signaling Technology (MA, USA) 9275

p-AktSer473


Akt2 Cell Signaling Technology, MA, USA 3063

p-mTORSer2481

Cell Signaling Technology, MA, USA 2974

mTOR Cell Signaling Technology, MA, USA 2972

p-NDRG1Thr346


NDRG1 Cell Signaling Technology, MA, USA 9485

p-TBC1D1Thr596


TBC1D1 James Hastie and Grahame Hardie, University of Dundee, UK -

p-TBC1D4Ser318


p-TBC1D4Ser588


p-TBC1D4Thr642

Symansis (New Zealand) 3028-P1

p-TBC1D4Ser704

Laurie J Goodyear, Joslin Diabetes Center and Harvard medical

school (Boston, MA, USA)

-

TBC1D4 Upstate Biotechnology (Millipore) (MA, USA) 07-741

Glycogen Otto Baba, Tokyo Medical and Dental University, Tokyo, Japan -

p-GSK3βSer9


GSK3β BD Transduction Laboratories, NJ, USA 610202

p-GSsite2+2a Grahame Hardie, University of Dundee, UK -

p-GSsite3a+b

Grahame Hardie, University of Dundee, UK -

GS Oluf B. Pedersen, University of Copenhagen, Denmark -

p-PDHsite1


p-PDHsite2


List of primary antibodies used for Western Blotting analysis of skeletal muscle fiber type specific pools.

Commercial available antibodies are listed with company name and catalog number while non-commercial

antibodies were kindly donated by persons as stated in the table.

of 44Diabetes

3

Supplementary table 2. Protein analysis order for each Western Blotting performed

Overview of each Western Blotting analysis performed on muscle fiber specific pools from lean, obese and type 2 diabetic subjects. Two runs of

Western Blotting (Top: Western blot #1 and bottom: Western blot #2) were performed. Each gel was cut into 4-5 strips where after proteins from

corresponding gel strips were semi-dry transferred to a single PVDF-membrane, blocked and probed with primary antibodies as listed. On each

membrane, 3-7 different antibodies were used with either a sodium-azide treatment or a stripping procedure in between. The use of 0.02% sodium-azide

in the primary antibody solution irreversible inhibits the enzyme activity of horseradish peroxidase already present on the membrane. This procedure

allowed for reprobing of the membrane with an antibody recognizing a protein with a different molecular weight than the previously detected antibody.

The stripping procedure was performed by incubating the membrane for 1 h at 58⁰C in stripping buffer (63 mM Tris-HCl (pH 6.7), 2% SDS, 0.8% β-

mercaptoethanol). Following each stripping procedure, membranes were incubated in appropriate secondary antibody in order to verify, that the primary

antibody was lost during stripping. If not, membranes were stripped again. Use of the sodium-azide procedure and the stripping procedure is marked

with an asterisk (*) and a dagger (†) symbol, respectively, in front of the re-probed antibody. N/A, not applicable due to unsuccessful re-probing of the

membrane.

Western Blot #1 MW (kDa) Primary antibody

gel cut off 1. 2. 3. 4. 5. 6.

Membrane 1 250

p-TBC1D4Thr642 †p-TBC1D4Ser704 N/A N/A †TBC1D4 125

Membrane 2 125

p-GSsite2+2a

*HKII †p-GS

site3a+b

†Insulin-Rβ

75

Membrane 3 75

p-AKTSer473

N/A †p-AKT

Thr308

†Akt2 N/A

†Complex II

50

Membrane 4 50

Actin N/A N/A †GLUT4 †p-PDHsite1 †p-PDHsite2 37


gel cut off 1. 2. 3. 4. 5. 6. 7.

Membrane 1 -

p-mTORSer2481 N/A †mTOR 250

Membrane 2 250

p-TBC1D1Thr596

N/A †TBC1D1 N/A N/A

†p-TBC1D4

Ser588

†p-TBC1D4

Ser318

125

Membrane 3 125

N/A N/A †GS

75

Membrane 5 50

p-NDRG1Thr346 *Actin †NDRG1 †GSK3β †p-GSK3βSer9 37

of 44 Diabetes

4

Supplementary figure 1. In order to quantify the protein content of individual muscle fiber pools Mini-

Protean TGX Stain-free gels with an acryl-amide gradient of 4-20% was used. 3 µl of pooled muscle fiber

sample was loaded and compared against a standard curve. Individual gels were compared using the same

internal gel standard sample (termed Std. in (A)). The standard curve consisted of data obtained from three

different pools of human muscle homogenate samples. Each homogenate sample was loaded in triplicates in

the range from 0.25-2 µg of protein. Pooled muscle fiber samples were expected to be and in fact were

within this protein range. An example of a stain-free image from one of the standard homogenate samples,

loaded in triplicates is shown (A). Each protein lane from the stain-free image was quantified from ~30-250

kDa, due to a higher variation in the bands appearing between type I and II muscle fiber pools outside this

range (data not shown). The averaged variation coefficient of each triplicate measure was 2.9% (range: 0.5-

5.5%). In (B) the x-axis states the amount of muscle homogenate protein loaded on the gel, while the y-axis

states the quantified values of the stain free image in arbitrary units (AU). The value of each muscle

homogenate is shown as well as the standard curve from which signal intensity from the muscle fiber pools

were compared to. The coefficient of determination of the standard curve was R2=1.00.

of 44Diabetes

5

Supplementary figure 2. In order to quantify the glycogen content of individual muscle fiber pools, 4

standard curves (50-400 ng of protein) from 4 different muscle homogenate samples with a known

(biochemical determined) glycogen concentration was spotted onto the same PVDF-membrane as the pooled

fiber samples. The x-axis states how much glycogen was spotted. The y-axis states the quantified values of

the visualized dot-blot in arbitrary units (AU). Each single value of the different homogenate samples is

shown as well as the fitted linear curve (R2=0.90). All muscle fiber pools were compared against this

standard curve in order to estimate the glycogen content.

of 44 Diabetes

6

Supplementary figure 3. Actin content in muscle fiber pools from lean, obese and type 2 diabetic subjects.

Muscle fiber specific expression of actin was evaluated in the two runs of Western Blotting. No significant

differences were observed between muscle fibers, clamp conditions and between groups in western blot #1

(A) and #2 (B). Quantified values are raw data with the basal type I fiber value in the lean group being 100.

A representative blot is shown for each protein probed for. White bars represent type I fibers and black bars

type II muscle fiber pools. Primary antibody used is stated in supplementary table 1. Data are means±SEM.

AU, arbitrary units.

of 44Diabetes

7

Supplementary figure 4. Phosphorylation of Akt on sites Thr308 (A) and Ser473 (B) related to the total

protein expression of Akt2 as well as phosphorylation of NDRG1 on site Thr346

related to the total content of

NDRG1 (C). Phosphorylation of PDH-E1α on site 1 (Ser293

) (D) and site 2 (Ser300

) (E) was related to PDH-

E1α protein expression. Since PDH-E1α expression was determined in a subset of samples the total number

of samples is indicated in each bar (D+E). The basal type I fiber value in the lean group is set at 100. White

bars represent type I fibers and black bars type II muscle fiber pools. Data are means±SEM. AU, arbitrary

units. ***p<0.001 vs basal conditions.

of 44 Diabetes

8

Supplementary figure 5. Correlation between the MHC 1 content in whole muscle lysate preparation and

the glucose disposal rate (A; r=0.53, p=0.002), net glucose oxidation rates (B; r=0.52, p=0.003) and non-

oxidative glucose metabolism (C; r=0.44, p=0.01) during insulin stimulated conditions. Circles represent

lean controls, squares represent obese controls and triangles represent type 2 diabetic subjects. A Pearson

product-moment correlation was performed for all values combined using SigmaPlot (version 12.5, Systat

Software, IL).

of 44Diabetes

9

Supplementary figure 6. Hexokinase I protein expression was evaluated in a subgroup of type I and II

muscle fiber specific pools from lean, obese and type 2 diabetic subjects. The number of samples is indicated

in each bar. Quantified values are related to actin with the basal value in the lean group being 100. White

bars represent type I fibers and black bars type II muscle fiber pools. Data are means±SEM. AU, arbitrary

units.

of 44 Diabetes

10

Supplementary figure 7. MHC IIx protein expression in type II muscle fiber specific pools from lean, obese

and type 2 diabetic subjects. Quantified values are related to actin with the basal value in the lean group

being 100. No significant differences in the MHC IIx expression between type II muscle fiber pools were

observed. White bars represent basal and black bars insulin-stimulated conditions. Primary antibody used is

stated in supplementary table 1. Data are means±SEM. AU, arbitrary units.

of 44Diabetes

SUPPLEMENTARY DATA

©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0590/-/DC1

Subject medications. Patients with type 2 diabetes were treated either by diet alone (n=2) or diet in combination with metformin (n=7), metformin and long-acting insulin (n=1) or rosiglitazone and sulfonylurea (n=1). Oral anti-diabetics were withdrawn one week prior to the study together with antihypertensive (n=5) and lipid lowering (n=7) drugs. In addition, one lean subject were treated with a proton-pump inhibitor, 2 obese subjects were treated with angiotensin-converting-enzyme (ACE) inhibitor whereas 4 patients with type 2 diabetes were treated with ACE-inhibitor (n=3) or AT2-blocker (n=1). Long-acting insulin was withdrawn one day before the study. The patients with type 2 diabetes were GAD65 antibody negative and without any signs of diabetic retinopathy, nephropathy, neuropathy or macro vascular complications. All woman included were post-menopausal. Lean and obese control subjects had no family history of diabetes.

SUPPLEMENTARY DATA


Supplementary Table 1. Primary antibodies used for Western Blotting and DOT-Blotting

Antibody Manufacturer/donator Cat. # MHC I Developmental Studies Hybridoma Bank (Australia) A4.840 MHC II Developmental Studies Hybridoma Bank (Australia) A4.74 MHC IIx Developmental Studies Hybridoma Bank (Australia) 6H1 Actin Sigma Aldrich (MO, USA) A2066 Insulin-Rβ Santa Cruz Biotechnology (CA, USA) SC-711 Hexokinase I Cell Signaling Technology (MA, USA) 2024 Hexokinase II Cell Signaling Technology (MA, USA) 2867 GLUT4 Thermo Scientific (Pierce, IL, USA) PA1-1065 Complex 2 Molecular Probes, Invitrogen (CA, USA) A11142 p-AktThr308 Cell Signaling Technology (MA, USA) 9275 p-AktSer473 Cell Signaling Technology (MA, USA) 9271 Akt2 Cell Signaling Technology, MA, USA 3063 p-mTORSer2481 Cell Signaling Technology, MA, USA 2974 mTOR Cell Signaling Technology, MA, USA 2972 p-NDRG1Thr346 Cell Signaling Technology, MA, USA 3217 NDRG1 Cell Signaling Technology, MA, USA 9485 p-TBC1D1Thr596 Cell Signaling Technology (MA, USA) 6927 TBC1D1 James Hastie and Grahame Hardie, University of Dundee, UK - p-TBC1D4Ser318 Cell Signaling Technology (MA, USA) 8619 p-TBC1D4Ser588 Cell Signaling Technology (MA, USA) 8730 p-TBC1D4Thr642 Symansis (New Zealand) 3028-P1 p-TBC1D4Ser704 Laurie J Goodyear, Joslin Diabetes Center and Harvard medical

school (Boston, MA, USA) -

TBC1D4 Upstate Biotechnology (Millipore) (MA, USA) 07-741 Glycogen Otto Baba, Tokyo Medical and Dental University, Tokyo, Japan - p-GSK3βSer9 Cell Signaling Technology, MA, USA 9331 GSK3β BD Transduction Laboratories, NJ, USA 610202 p-GSsite2+2a Grahame Hardie, University of Dundee, UK - p-GSsite3a+b Grahame Hardie, University of Dundee, UK - GS Oluf B. Pedersen, University of Copenhagen, Denmark - p-PDHsite1 Grahame Hardie, University of Dundee, UK - p-PDHsite2 Grahame Hardie, University of Dundee, UK -

List of primary antibodies used for Western Blotting analysis of skeletal muscle fiber type specific pools. Commercial available antibodies are listed with company name and catalog number while non-commercial antibodies were kindly donated by persons as stated in the table.

SUPPLEMENTARY DATA


Supplementary Table 2. Protein analysis order for each Western Blotting performed

Overview of each Western Blotting analysis performed on muscle fiber specific pools from lean, obese and type 2 diabetic subjects. Two runs of Western Blotting (Top: Western blot #1 and bottom: Western blot #2) were performed. Each gel was cut into 4-5 strips where after proteins from corresponding gel strips were semi-dry transferred to a single PVDF-membrane, blocked and probed with primary antibodies as listed. On each membrane, 3-7 different antibodies were used with either a sodium-azide treatment or a stripping procedure in between. The use of 0.02% sodium-azide in the primary antibody solution irreversible inhibits the enzyme activity of horseradish peroxidase already present on the membrane. This procedure allowed for reprobing of the membrane with an antibody recognizing a protein with a different molecular weight than the previously detected antibody. The stripping procedure was performed by incubating the membrane for 1 h at 58⁰C in stripping buffer (63 mM Tris-HCl (pH 6.7), 2% SDS, 0.8% β-mercaptoethanol). Following each stripping procedure, membranes were incubated in appropriate secondary antibody in order to verify, that the primary antibody was lost during stripping. If not, membranes were stripped again. Use of the sodium-azide procedure and the stripping procedure is marked with an asterisk (*) and a dagger (†) symbol, respectively, in front of the re-probed antibody. N/A, not applicable due to unsuccessful re-probing of the membrane.

Western Blot #1 MW (kDa) Primary antibody gel cut off 1. 2. 3. 4. 5. 6.

Membrane 1 250 p-TBC1D4Thr642 †p-TBC1D4Ser704 N/A N/A †TBC1D4 125

Membrane 2 125 p-GSsite2+2a *HKII †p-GSsite3a+b †Insulin-Rβ 75

Membrane 3 75 p-AKTSer473 N/A †p-AKTThr308 †Akt2 N/A †Complex II 50

Membrane 4 50 Actin N/A N/A †GLUT4 †p-PDHsite1 †p-PDHsite2 37


gel cut off 1. 2. 3. 4. 5. 6. 7.

Membrane 1 - p-mTORSer2481 N/A †mTOR 250

Membrane 2 250 p-TBC1D1Thr596 N/A †TBC1D1 N/A N/A †p-TBC1D4Ser588 †p-TBC1D4Ser318 125

Membrane 3 125 N/A N/A †GS

75

Membrane 5 50

p-NDRG1Thr346 *Actin †NDRG1 †GSK3β †p-GSK3βSer9 37

SUPPLEMENTARY DATA


Supplementary Figure 1. In order to quantify the protein content of individual muscle fiber pools Mini-Protean TGX Stain-free gels with an acryl-amide gradient of 4-20% was used. 3 µl of pooled muscle fiber sample was loaded and compared against a standard curve. Individual gels were compared using the same internal gel standard sample (termed Std. in (A)). The standard curve consisted of data obtained from three different pools of human muscle homogenate samples. Each homogenate sample was loaded in triplicates in the range from 0.25-2 µg of protein. Pooled muscle fiber samples were expected to be and in fact were within this protein range. An example of a stain-free image from one of the standard homogenate samples, loaded in triplicates is shown (A). Each protein lane from the stain-free image was quantified from ~30-250 kDa, due to a higher variation in the bands appearing between type I and II muscle fiber pools outside this range (data not shown). The averaged variation coefficient of each triplicate measure was 2.9% (range: 0.5-5.5%). In (B) the x-axis states the amount of muscle homogenate protein loaded on the gel, while the y-axis states the quantified values of the stain free image in arbitrary units (AU). The value of each muscle homogenate is shown as well as the standard curve from which signal intensity from the muscle fiber pools were compared to. The coefficient of determination of the standard curve was R2=1.00.

SUPPLEMENTARY DATA


Supplementary Figure 2. In order to quantify the glycogen content of individual muscle fiber pools, 4 standard curves (50-400 ng of protein) from 4 different muscle homogenate samples with a known (biochemical determined) glycogen concentration was spotted onto the same PVDF-membrane as the pooled fiber samples. The x-axis states how much glycogen was spotted. The y-axis states the quantified values of the visualized dot-blot in arbitrary units (AU). Each single value of the different homogenate samples is shown as well as the fitted linear curve (R2=0.90). All muscle fiber pools were compared against this standard curve in order to estimate the glycogen content.

SUPPLEMENTARY DATA


Supplementary Figure 3. Actin content in muscle fiber pools from lean, obese and type 2 diabetic subjects. Muscle fiber specific expression of actin was evaluated in the two runs of Western Blotting. No significant differences were observed between muscle fibers, clamp conditions and between groups in western blot #1 (A) and #2 (B). Quantified values are raw data with the basal type I fiber value in the lean group being 100. A representative blot is shown for each protein probed for. White bars represent type I fibers and black bars type II muscle fiber pools. Primary antibody used is stated in supplementary table 1. Data are means±SEM. AU, arbitrary units.

SUPPLEMENTARY DATA


Supplementary Figure 4. Phosphorylation of Akt on sites Thr308 (A) and Ser473 (B) related to the total protein expression of Akt2 as well as phosphorylation of NDRG1 on site Thr346 related to the total content of NDRG1 (C). Phosphorylation of PDH-E1α on site 1 (Ser293) (D) and site 2 (Ser300) (E) was related to PDH-E1α protein expression. Since PDH-E1α expression was determined in a subset of samples the total number of samples is indicated in each bar (D+E). The basal type I fiber value in the lean group is set at 100. White bars represent type I fibers and black bars type II muscle fiber pools. Data are means±SEM. AU, arbitrary units. ***p<0.001 vs basal conditions.

SUPPLEMENTARY DATA


Supplementary Figure 5. Correlation between the MHC 1 content in whole muscle lysate preparation and the glucose disposal rate (A; r=0.53, p=0.002), net glucose oxidation rates (B; r=0.52, p=0.003) and non-oxidative glucose metabolism (C; r=0.44, p=0.01) during insulin stimulated conditions. Circles represent lean controls, squares represent obese controls and triangles represent type 2 diabetic subjects. A Pearson product-moment correlation was performed for all values combined using SigmaPlot (version 12.5, Systat Software, IL).

SUPPLEMENTARY DATA


Supplementary Figure 6. Hexokinase I protein expression was evaluated in a subgroup of type I and II muscle fiber specific pools from lean, obese and type 2 diabetic subjects. The number of samples is indicated in each bar. Quantified values are related to actin with the basal value in the lean group being 100. White bars represent type I fibers and black bars type II muscle fiber pools. Data are means±SEM. AU, arbitrary units.

SUPPLEMENTARY DATA


Supplementary Figure 7. MHC IIx protein expression in type II muscle fiber specific pools from lean, obese and type 2 diabetic subjects. Quantified values are related to actin with the basal value in the lean group being 100. No significant differences in the MHC IIx expression between type II muscle fiber pools were observed. White bars represent basal and black bars insulin-stimulated conditions. Primary antibody used is stated in supplementary table 1. Data are means±SEM. AU, arbitrary units.

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