The effect of peripheral neuropathy on lower limb muscle strength in diabetic individuals

Accepted Manuscript The effect of peripheral neuropathy on lower limb muscle strength in diabetic individuals

Jean P. Ferreira, Cristina D. Sartor, Ângela M.O. Leal, Isabel C.N. Sacco, Tatiana O. Sato, Ivana L. Ribeiro, Alice S. Soares, Jonathan E. Cunha, Tania F. Salvini PII: DOI: Reference:

S0268-0033(17)30048-7 doi: 10.1016/j.clinbiomech.2017.02.003 JCLB 4286

To appear in:

Clinical Biomechanics

Received date: Accepted date:

26 August 2016 7 February 2017

Please cite this article as: Jean P. Ferreira, Cristina D. Sartor, Ângela M.O. Leal, Isabel C.N. Sacco, Tatiana O. Sato, Ivana L. Ribeiro, Alice S. Soares, Jonathan E. Cunha, Tania F. Salvini , The effect of peripheral neuropathy on lower limb muscle strength in diabetic individuals. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jclb(2017), doi: 10.1016/j.clinbiomech.2017.02.003

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ACCEPTED MANUSCRIPT The effect of peripheral neuropathy on lower limb muscle strength in diabetic individuals

Jean P. Ferreira1, Cristina D. Sartor2, Ângela M. O. Leal3, Isabel C. N. Sacco2, Tatiana O.

Laboratory of Skeletal Muscle Plasticity, Department of Physical Therapy, Federal

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1

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Sato1, Ivana L. Ribeiro1, Alice S. Soares1, Jonathan E. Cunha1, Tania F. Salvini1

2

Physical Therapy, Speech and Occupational Therapy Department, School of Medicine,

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University of São Paulo, São Paulo, SP, Brazil;

Department of Medicine, Federal University of São Carlos, São Carlos, SP, Brazil.

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3

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University of São Carlos, SP, Brazil;

Address for correspondence:

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Tania F. Salvini, PT, MS, PhD

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Laboratory of Skeletal Muscle Plasticity, Department of Physical Therapy, Universidade Federal de São Carlos, São Carlos, SP, Brazil

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Zip code: 13565-905; Phone: +55 16 33518345

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E-mail: [email protected]

Word Count: 4.145 Figure count: 1 Table count: 4

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ACCEPTED MANUSCRIPT Abstract Background: Skeletal muscle strength is poorly described and understood in diabetic participants with diabetic peripheral neuropathy. This study aimed to investigate the extensor and flexor torque of the knee and ankle during concentric, eccentric, and isometric contractions in men with diabetes mellitus type 2 with and without diabetic

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peripheral neuropathy.

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Methods: Three groups of adult men (n= 92), similar in age, body mass index, and

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testosterone levels, were analyzed: 33 non-diabetic controls, 31 with type 2 diabetes mellitus, and 28 with diabetic peripheral neuropathy. The peak torques in the

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concentric, eccentric, and isometric contractions were evaluated using an isokinetic dynamometer during knee and ankle flexion and extension.

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Findings: Individuals with diabetes and diabetic peripheral neuropathy presented similar low concentric and isometric knee and ankle torques that were also lower than

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contractions, and the joints.

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the controls. However, the eccentric torque was similar among the groups, the

Interpretation: Regardless of the presence of peripheral neuropathy, differences in

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skeletal muscle function were found. The muscle involvement does not follow the same pattern of sensorial losses, since there are no distal-to-proximal impairments. Both knee

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and ankle were affected, but the effect sizes of the concentric and isometric torques were found to be greater in the participants’ knees than in their ankles. The eccentric function did not reveal differences between the healthy control group and the two diabetic groups, raising questions about the involvement of the passive muscle components. Keywords: Muscle strength; polyneuropathy; diabetes; muscle function; low limbs; motor function. 2

ACCEPTED MANUSCRIPT INTRODUCTION Diabetes mellitus type 2 (DM2) and diabetic peripheral neuropathy (DPN) have been correlated to metabolic and inflammatory changes that lead to a reduction in muscle mass and strength (Bouchard and Janssen, 2010; Zamboni et al., 2008). DPN has been associated with the loss of tactile, thermal, and vibratory sensitivities (Boulton

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et al., 2005) in addition to a reduction in motor nerve conduction velocity in latter stages

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leading to changes in the movement of the lower limbs and the dynamic stability during

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locomotion (Fernando et al., 2013). Even when DM2 is not associated with DPN, it is known that it can cause loss of mass and muscle strength (Andreassen et al., 2014;

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Hilton et al., 2008), which may be related to subclinical inflammation (Bouchard and Janssen, 2010; Stenholm et al., 2008; Zamboni et al., 2008) as well as degeneration of

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the sarcoplasmic proteins associated with insulin resistance (Cruz-Jentoft et al., 2010). A few studies (Li et al., 2016; Lee et al., 2013; Peterson et al., 2016) that followed

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patients with DM2 and DPN reported muscle mass losses. Some reported functional

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changes in elderly women with DM2 over a six-year course of the disease (Lee et al., 2013), such as reduced gait velocity, decreased grip strength (Fernando et al., 2013;

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Joule J. et al., 2016), and a 50% reduction in concentric knee extensor torque in men with DM2 over a three-year period (Park et al., 2007). However, the presence of DPN

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was unconfirmed in those studies; the observed functional deficits could also be associated with both DPN and DM2. Individuals that had DM2 for more than 10 years showed less muscle strength, regardless of the presence of DPN (Hatef et al., 2014). Andreassen et al. (2014) found a lower concentric torque of the plantar flexors in individuals with DM2 compared to individuals with diabetes mellitus type 1 (DM1) and non-diabetics, but they reported no difference between diabetics with and without DPN. It has been reported that only 3

ACCEPTED MANUSCRIPT individuals with DM2 had a reduction in the diameter of type I and type II muscle fibers of the gastrocnemius, indicating that torque reduction is associated with atrophy (Andreassen et al., 2014). In a correlational study, Andersen et al. (2004) found that, as DPN became more severe, the flexor and extensor concentric torque at the knee and ankle joints decreased in individuals with DM2. However, in that study, no difference

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was found in knee extension torque between the diabetic individuals with and without

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DPN.

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One of the first studies that evaluated the torque of diabetic individuals that had the disease for more than 20 years noted that the reduction of concentric torque of the

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knee flexors and extensors, at 90°/s, and of the ankle dorsiflexors and plantar flexors at 60º/s, was inversely correlated to DPN (Andersen, 1996). However, individuals of both

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sexes, with DM1 and DM2, were included in the same group (Andersen, 1996). Other results indicated that DM1 and DM2 could affect the skeletal muscle properties via

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different mechanisms (Bouchard and Janssen, 2010; D’Souza et al., 2013). DM1 has

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been associated with hyperglycemia and protein alterations, and DM2 has been associated with inflammation that causes atrophy (D’Souza et al., 2013).

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Gender is another important factor that should be considered in the studies, since there are differences in muscle strength between males and females (Cruz-Jentoft et al.,

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2010). Lowe et al. (2011) reported that estrogen benefits muscle strength, and that estrogen receptors are involved in the underlying mechanism to improve muscle quality. Thus, the influence of gender on muscle performance is highly dependent on the types and levels of hormones. Other studies have suggested that other female hormones could also influence muscle strength, but more research is needed on that topic (Tiidus, 2011). Although some studies have reported torque reduction and functional losses in the lower limbs of diabetic individuals with and without DPN (Andersen et al., 2004; 4

ACCEPTED MANUSCRIPT Andreassen et al., 2014; Bouchard and Janssen, 2010; Fernando et al., 2013; Hatef et al., 2014; Hilton et al., 2008; Park et al., 2007; Petrofsky et al., 2005), so far none of the studies that investigated knee and ankle torques considered all the anthropometric (sex, mass, height, and body mass index [BMI]) and clinical characteristics (type of DM and testosterone and glycated hemoglobin levels) previously discussed. Considering that all

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types of muscle contractions are important for gait and the activities of daily living, our

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aim was to investigate the knee flexor and extensor torques and the ankle dorsiflexion

male subjects with DM2, with and without DPN.

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and plantar flexion torques during concentric, eccentric, and isometric contractions in

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Previous studies (Joule J. et al., 2016; Lee et al., 2013; Peterson et al., 2016, Fernando et al., 2013, Park et al., 2007; Hatef et al., 2014) have found a reduction of

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muscle strength in diabetic individuals, even when DPN was not present, but in several uncontrolled conditions, and this reductions were attributed to be caused by different

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mechanisms. After the onset of DPN, the subsequent nerve damage contributes to an

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even greater decrease in muscle function. Thus, by controlling for sex, age, testosterone and glycated hemoglobin levels, and the clinical characterization of DPN, we

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hypothesized that individuals with DM2 would have lower peak torque than individuals without DM2 and individuals that also had DPN would have even lower torque than

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subjects with DM2 while performing three types of muscle contraction. Moreover we hypothesized that a decrease in the peak torques would be similar during all three different muscle contractions, since they depend on muscle tropism and muscle activation mechanisms.

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ACCEPTED MANUSCRIPT METHODS

Participants Males between the ages of 18 and 65 were included in this study. Participants were recruited at the Endocrinology Clinic at the Health School Unit of the Federal

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University of São Carlos and at the local Medical Specialties Center. Exclusion criteria

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included the presence of cardiovascular diseases with systemic repercussions (unstable

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angina, uncontrolled hypertension); pre-diabetes; renal insufficiency; skeletal muscle diseases (rheumatoid arthritis, osteoarthritis, joint endoprosthesis, tendinitis, bursitis,

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disk herniation, gout, anterior cruciate ligament rupture or cruciate reconstruction, malalignment of the lower limbs); liver disease; hypogonadism (total testosterone level

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of <200 ng / dL); individuals who used anabolic steroids or hormone replacement; BMI <20 kg/m² or >40 kg/m². A total of 287 volunteers were interviewed and invited to

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participate in the assessment of eligibility, and 101 individuals met the inclusion criteria

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and were included in the study. The selected study participants were distributed into three groups: control group, DM2 individuals without DPN group (DPN), and DM2

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individuals with DPN group (DM2) (Figure 1). Figure 1

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The diagnosis of DM2 or the absence of diabetes in the participants was confirmed by an endocrinologist using American Diabetes Association (2014) criteria. The sample size calculation resulted in 99 individuals, (33 per group) to achieve a power of 0.80, with an α of 0.05, within a F-test design, using the mean and standard deviation of the plantar flexors peak torque obtained in a pilot study (Faul et al., 2009). Since the data acquisition protocol did not change after the pilot study, we included all 99 individuals in the data analysis. 6

ACCEPTED MANUSCRIPT Before the data acquisition, we checked for similarities and homogeneous distribution of age, mass, height, BMI, and testosterone levels among groups. The ANOVA and subsequent Tukey post hoc test show only the expected differences relative to some variables. The glycated hemoglobin (HbA1C) levels were higher in the DM2 and DPN groups in comparison to the control group (P <0.01), and no difference

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in these levels was observed between the DM2 and DPN groups (P = 0.75). The degree

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of DPN severity in the DPN group was higher than it was in the DM2 (P <0.01) and the

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control (P <0.01) groups. The mean fuzzy classification was considered moderate in the DPN group, 5.02 (2.7), and absent in the DM2 group, 0.73 (0.2), and the control group,

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0.67 (0.0). Table 1

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This study was approved by the Research Ethics Committee of the Federal

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University of Sao Carlos (protocol number: 797125).

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Clinical assessments

The study participants underwent a clinical assessment, performed by a trained

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physical therapist. The following clinical parameters were evaluated: (i) tactile sensitivity using a 10 g Semmes-Weinstein monofilament; (ii) vibratory perception

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using a 128 Hz tuning fork, and (iii) typical neuropathy symptoms, which were assessed via a questionnaire based on the Michigan Neuropathy Screening Instrument (Bakker and Apelqvist, 2012). To evaluate the presence of DPN, these three groups of variables were used as linguistic inputs in a fuzzy model, as described by Watari et al. (2014). The fuzzy system software combines each fuzzy set of input variables and assigns a score corresponding to the degree of DPN severity. The fuzzy model presented a very strong correlation with the expert’s opinion (Pearson’s coefficient r= 0.943) and a high 7

ACCEPTED MANUSCRIPT accuracy level when classifying real patients that underwent the model’s analysis (Receiver Operating Characteristic (ROC) curve area = 0.91) (Watari et al., 2014). The HbA1C test was used to determine the glycemic control (American Diabetes Association,

2014) , (

using

high-performance

liquid

chromatography

(HPLC).

Chemiluminescence was used to measure the level of total testosterone in the blood

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serum (Furuyama, 1970).

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Strength assessment of the lower limb

Flexion and extension peak torques of the knee and ankle were measured using

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an isokinetic dynamometer (Biodex Medical System 3 Pro, Shirley, NY, USA). The dynamometer calibration was checked before every evaluation session. Concentric and

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eccentric torques at 60º/s, and isometric torques were assessed during knee flexion and extension and ankle dorsiflexion and plantar flexion.

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The participants were positioned according to the manufacturer's specification

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for both joints (Davies, 1992; Eng et al., 2002). For the knee, the participants were seated with the hip flexed at 85°, the axis of dynamometer was aligned with the lateral

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epicondyle, and the dynamometer arm was fixed at the distal third of the leg. For the ankle, the participants were seated with the hip flexed at 70° and with the knee

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maintained at 30° of flexion (Eng et al., 2002). For both joint analyses, the participants were stabilized with a seat belt fastened around the hips, two shoulder straps that crossed the chest, and a strap across the thigh of the tested leg. For the ankle assessment, the foot was fixed with straps across the top of the forefoot and midfoot. The foot was positioned in a bracket such that the axis of rotation of the ankle was aligned with the axis of the lever arm (Nielsen et al., 2014). The ankle range of motion was determined individually to reach a maximum of 10° of dorsiflexion and 35º 8

ACCEPTED MANUSCRIPT of plantar flexion. The total range of motion was settled at 70° for knee flexion and extension, starting from the zero position or 90° of knee flexion. Gravity correction was conducted using Biodex software after weighing each participant’s limb at 30° of knee extension, starting at the zero position and at the neutral ankle position (90º) (Nielsen et al., 2014).

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The torques were assessed in the dominant limb. The dominant limb was

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determined by asking the participants which limb (left or right) they used to kick a ball.

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Five trials of maximal voluntary isokinetic contractions (concentric and eccentric) were conducted for both joints (knee and ankle). Two maximal voluntary contractions were

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obtained for the isometric trials with the knee positioned at 30° of extension and the ankle in the neutral position. Before the maximal protocol, the participants were

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submitted to a familiarization session that included three submaximal voluntary contractions for each of the isokinetic contractions, and one repetition for the isometric

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contractions. A rest period of 1.5 minutes was given between each type of contraction,

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and a rest period of three minutes was given between the familiarization session and the maximal trials (Eng et al., 2002). The orders of the tests (type of joint and type of

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contraction) were randomized using a web randomization site (Dallal, 2008). Only one evaluator conducted all the procedures, and standard encouragement was given to all

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participants (Davies, 1992). The dynamometry data were processed in MATLAB (R2008a version). The peak torques were identified for all types of contractions and normalized by the body weight (N·m/kg x 100) of the participants.

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ACCEPTED MANUSCRIPT Protocol reliability The reliability of the DPN assessment in the fuzzy software was performed on eight participants prior to the data acquisition. The Kappa coefficient for the score was obtained using fuzzy software. The reliability was considered excellent for the categorization of DPN using fuzzy software (inter- and intra-rater: Kappa = 1.00, 100%;

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P <0.01).

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The intraclass correlation coefficient (ICC1, 2) and standard error of measurement

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(SEM) were also tested for the isokinetic dynamometer in five individuals, using the peak torque of the knee and ankle in the concentric, eccentric, and isometric

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contractions. The participants were re-evaluated after a minimal period of 72 hours, following the first assessment. The intra-rater reliability for peak of torque in all

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movements for the concentric, eccentric and isometric contractions for both joints was considered excellent (Table 2).

Statistical Analysis

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Table 2

the

Levene

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The homogeneity of variance and the normality distribution were checked using and

the

Kolmogorov-Smirnov

tests,

respectively.

Demographic

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characteristics and peak knee torque in all three types of contractions met the assumptions for the parametric tests; therefore, a one-way ANOVA test with a Tukey’s post-hoc test for comparisons between the groups were used (alpha=5%). Peak ankle torque data did not present a normal distribution, and they were analyzed using nonparametric tests. Kruskal-Wallis test was used to assess the peak torques between groups and the Mann-Whitney test was used to identify the differences among groups if Kruskal-Wallis results indicated significant differences. For all nonparametric 10

ACCEPTED MANUSCRIPT comparisons among groups using Mann-Whitney test, the alpha level was adjusted according to the number of comparisons (control vs. DM2; control vs. DPN, DPN vs. DM2) or α=0.05/3=0.016: P<0.016. The effect sizes (Hedges'g) of the peak torques among groups were also calculated to demonstrate the size of the difference. Data were analyzed using Statistical Package for the Social Sciences (SPSS) software (version

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17.0).

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RESULTS

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The concentric torques of knee flexion and extension were similar between the

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diabetic groups (flexion: P=0.72; extension: P=0.90), but they were significantly lower in comparison to the controls (control vs. DM2: flexion: P<0.01, extension: P<0.01;

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control vs. DPN: flexion: P<0.01, extension: P<0.01; Table 3). There were no differences in the knee flexion and extension eccentric torques among groups (Table 3).

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For the isometric torque, the DM2 group was found to have lower extension knee torque

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(P=0.01), and there was no difference in the flexion torque (P=0.15) for this group in comparison to the controls. The DPN group was found to have lower isometric knee

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torque in comparison to the controls (flexion: P<0.01, extension: P<0.01). There was no difference in the knee isometric torque between the DM2 and DPN groups (flexion:

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P=0.67; extension: P=0.67; Table 3). The DPN group had lower ankle concentric torque in comparison to the controls (dorsiflexion: P<0.01; plantar flexion: P<0.01), and concentric torque was lower in this group in comparison to the DM2 only for dorsiflexion (P<0.01) and plantar flexion: P=0.07). The DM2 group had a lower concentric ankle torque in comparison to the controls (dorsiflexion: P<0.01; plantar flexion: P<0.01; Table 3). With respect to eccentric torque, no difference in ankle dorsiflexion and plantar flexion was observed 11

ACCEPTED MANUSCRIPT among groups (Table 3). The DPN group was found to have lower isometric ankle torque in comparison to the controls (dorsiflexion: P=0.01; plantar flexion: P<0.01). The DM2 group also had lower plantar flexion torque in comparison to the controls (P <0.01), but no difference was observed in the ankle dorsiflexion between these two groups (P = 0.96). No difference in the isometric ankle torque (dorsiflexion: P = 0.020;

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plantar flexion: P = 0.05; Table 3) was found between DM2 and DPN groups. Despite

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these results, a medium effect size was found between DM2 and DPN groups for

Table 3

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Table 4

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isometric dorsiflexion (Table 4).

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DISCUSSION

This study has two main interesting findings. First, the decrease in concentric

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and isometric peak torques in DM2 participants occurred even before the onset of DPN

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for all of the knee motions and for almost all of ankle motions. Second, the eccentric torque was preserved in all of the joint movements in both DM2 group and DPN group.

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The statistical differences observed in the isometric and concentric peak torques among groups demonstrated that, for almost all the investigated variables, healthy

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individuals are quite different from individuals with DM2, regardless of the presence of advanced DPN. This finding was unexpected, since our assumption was that the peak torques would be lower for participants with more severe clinical signs. Therefore, lower concentric and isometric peak torques are not associated with DPN progression, as believed, at least based on the criteria we applied using decision support software. Our results showed that, before the onset of DPN, it is possible to observe significant losses in muscle function in individuals with DM2, even without the presence of 12

ACCEPTED MANUSCRIPT sensory complications, since the signs and symptoms classified by the fuzzy system were similar between DM2 and the control groups. It is important to highlight that the motor losses did not develop concurrently with the sensory losses; therefore, motor losses

must

be

evaluated

and

considered

independently.

The

Guidelines

recommendation of the International working group on Diabetic Foot (Bakker et al.,

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2016) do not include a specific screening or assessment procedure targeting prevention

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or the treatment of skeletal muscle alteration for diabetic participants, even with the

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growing evidence for muscle impairment reported in the literature in these patients (Andersen et al., 2004; Andreassen et al., 2014; Bouchard and Janssen, 2010; Fernando

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et al., 2013; Hatef et al., 2014; Hilton et al., 2008).

It is important to note that the decreased peak torques for the DM2 group in

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comparison to the controls implies a reduced capability of the muscles to produce isometric and concentric force, which are essential for the stabilization and acceleration

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of movements (Fernando et al., 2013). The only exception was for the concentric torque

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during ankle dorsiflexion, which is mainly held by the tibialis anterior muscle; this was found to be higher for the DM2 group and lower for the DPN group, and both results

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had a great effect size. The reduced function of the anterior tibialis muscle, and its relation to plantar ulceration onset, has been investigated in several studies that have

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reported on gait (Fernando et al., 2013; Sacco and Sartor, 2016; Van Schie et al., 2004), indicating that this muscle dysfunction is associated with plantar ulcers and foot deformities in DPN participants (Van Schie et al., 2004). Our results indicate that the eccentric function was not different among three groups. The fact that eccentric peak torque in all the assessed movements was preserved in individuals with DM2 and DPN was unexpected; this finding suggests that the structures responsible for the eccentric torque might be preserved. It is known that eccentric torque also involves the stiffness 13

ACCEPTED MANUSCRIPT of the structures of the skeletal muscle, ligaments, and tendons (Gajdosik, 2001). Furthermore, regardless of the type of contraction, the myotendinous collagen matrix unit is fundamental for the transmission of strength and muscle function (Gajdosik, 2001). One hypothesis to explain the preserved eccentric torque in diabetics could be that the non-contractile tissues related to passive stress, which also participate in the

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eccentric torque production (Rosenbloom, 2013), would be altered. However, only one

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study that evaluated the passive stiffness of plantar flexors at 60°/s found that diabetic

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individuals with and without DPN had a lower passive stiffness than an age- and anthropometry-matched control group (Salsich et al., 2000).

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Another hypothesis to explain the preservation of eccentric torque in diabetics could be an alteration in the contractile tension of the sarcomere. In the elongated

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position, the sarcomere produces a residual force that contributes to a significant increase in force of contractile tension (Herzog et al., 2015), and, due the calcium

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presence on sarcolemma, the titin is stimulated thereby increasing the total force of the

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muscle fiber (Rassier et al., 2015; Rassier et al., 2003). A recent systematic review identified that calcium accumulation in the heart muscle sarcolemma of rats induced

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diabetic cardiomyopathy and was associated with an increase in heart muscle stiffness (Herzog et al., 2015). However, there is still no evidence for the same alteration in the

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skeletal muscle of diabetic individuals; if there was, we might have another explanation for the findings observed in the present study. In healthy participants, concentric plantar flexion torque at 60º/s is approximately 70% greater than concentric dorsiflexion (Dvir, 2004; Fugl-Meyer et al., 1985). In healthy young adults, the same result was found for eccentric contraction, where dorsiflexion torque was also lower than plantar flexion torque at 90°/s [1.29 (0.57) vs 1.96 (0.66)] (Fox et al., 2008). However, our results did not show the same 14

ACCEPTED MANUSCRIPT trend between ankle dorsiflexion and plantar flexion for eccentric peak torques in all the analyzed groups. Three factors could explain the differences between the findings reported in previous studies (Fox et al., 2008; Fugl-Meyer et al., 1985) and our results: differences in the ages of the participants in the study groups, differences in the dynamometer model that was used to evaluate ankle torque with the knee flexed at 30º,

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and differences in the motion speeds. The lack of standard values for the DM2

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population and for eccentric ankle torque also suggests a need for future investigations.

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We could not identify a distal-to-proximal pattern of skeletal muscle impairments, as commonly described in patients with DPN (Boulton et al., 2005). Our

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results showed that DM2, with and without DPN, presented a similar pattern of torque production in the ankle, but large effect sizes were observed for the differences in the

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knee torque. Therefore, it is necessary to consider the possibility of muscle involvement for the entire lower limb, and not restrict it to distal regions; this suggests that muscle

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impairment is associated with other factors that are independent of the motor nerve

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involvement that leads to loss of muscle strength. Corroborating with this suggestion, some previous studies had observed increased inflammatory factors in individuals with

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DM2 due to metabolic syndrome (Salsich et al., 2000, D’Souza et al., 2013, Zamboni et al., 2008). These factors may also activate the apoptosis pathways of muscles, reducing

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muscle mass and strength, independent of the presence of DPN or the anatomical region (Bouchard and Janssen, 2010). Andreassen et al. (2009) have reported that the gene expression of the neurotrophic factors of the deltoid and gastrocnemius muscles were similar in diabetics (with and without DPN) and non-diabetics, indicating no signs of denervation of both the distal and proximal muscles, which would be identified by the increase in gene expression of these factors. These studies helped us interpret our results since the loss of muscle strength observed in the participants with DM2 was not 15

ACCEPTED MANUSCRIPT associated with the onset of DPN, and it could possibly be associated with other factors that are independent of the motor nerve involvement that leads to loss of muscle strength. Further research on the conditions and mechanisms that might affect the production of muscle strength in diabetic individuals is required. Differences in muscle

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performance could be detected in other conditions, such as during different velocities of

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ankle and knee flexion and extension. Other disease effects could appear during

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submaximal muscle contractions, since they are more closely related to functional activities than maximal muscle contractions. Moreover, the early identification of loss

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of muscle function during clinical evaluation of individuals with DM2 is still a challenge. Further studies are needed to improve the evaluation and contribute to the

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development of more appropriate and reliable instruments. It would also be important to investigate the functional implications associated with the decrease in concentric and

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isometric torques, as well as the preservation of eccentric torque, and the impact on the

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mobility and stability of individuals with DM2. All this knowledge would support prevention and health promotion strategies for the diabetic population.

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CONCLUSIONS

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Regardless of the presence of DPN, we found differences in muscle function, even in the early stages of DM2. This muscle impairment does not follow the same pattern of sensorial losses, since there are no distal-to-proximal impairments. Both the knee and the ankle were affected, but the effect sizes of the concentric and isometric torques were larger for the knee than for the ankle. No difference was observed in the eccentric function between the healthy controls and the two diabetic groups, and this raises questions about the involvement of the passive muscle components. 16

ACCEPTED MANUSCRIPT Acknowledgements The study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Process 2011/22122-5) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). T. F. Salvini, I.C.N. Sacco and C.D. Sartor are funded by the CNPq (Process: 3013442013-2; 305606/2014-0 and 151531/2013-7,

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respectively). J. P. Ferreira has a Master’s scholarship from the Coordenação de

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Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Process: 1406247).

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Andreassen, C.S., Jakobsen, J., Flyvbjerg, A., Andersen, H., 2009. Expression of

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Figure 1 – Flowchart of the study design Control = control group; DM2= diabetic type 2 group; DPN= diabetic type 2 with peripheral neuropathy group.

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ACCEPTED MANUSCRIPT Table l. Clinical and anthropometrical characteristics of the participants.

Height (m)

DM2 (N=31 )

48.00 (31 65) 1.71 (0.07)

53.00 (32 65) 1.72 (0.05)

(N=28 )

56.00 (36 63) 1.72 (0.09) 84.12 85.03 (12.46 (11.71) ) 28.25 28.19 (3.20) (4.01) 27/4 26/3 120 72 (1 (12 360)* 252)* 8.92 8.53 (2.54) (2.56)* * 209.3 198.35 2 (73.58) (72.99 ) 328.5 336.42 0 (111.1 (87.28 3) ) 6.0 1.7 (2.3)* (1.6)* † 5.02 0.73 (2.7)* (0.2) †

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79.97 (12.54)

DPN

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Age (years)

Contr ol (N=33 )

Body Mass Index (kg/m²)

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Time of DM2 onset (months)

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Dominance of the lower limb (Right / Left)

HbA1c (%)

27.21 (3.87) 28/6

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Mass (kg)

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Average Blood Glucose (mg / dL)

5.35 (0.33)

107.94 (9.10) 395.42 (247.8 5)

Symptoms of periferal neuropathy (Questionnaires MNSI)

0.39 (0.4)

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Total testosterone level (ng / dL)

00 (00 - 00)

Degree of periferal neuropathy (Fuzzy score)

0.67 (0.0)

Oral antidiabetic / insulin / oral antidiabetic + insulin (number of patients)

-

24/0/4

15/3/1 0

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F=2.65; P=0.76 F=0.65; P=0.52 F=1.62; P=0.20 F=265; P=0.44 F=40.4 4; P<0.01 F=28.5 8; P<0.01 F=28.5 8; P<0.01 F=1.47; P=0.23 F=93.1 8; P<0.01 F=90.4 7; P<0.01 -

Age and time since diagnosis expressed as median (minimum - maximum), while other data were expressed as mean (standard deviation). ANOVA one way for comparing the variables, with Tukey post hoc test when comparing the groups, * = P <0.05 compared with the Control, † = P <0.05 compared with DM2; m = meters, kg = kilogram, mg = milligrams, dl = deciliter, ng = nanograms. Control = control group; DM2= diabetic type 2 group; DPN= diabetic type 2 with peripheral neuropathy group. 24

ACCEPTED MANUSCRIPT Table 2. Intraclass correlation coefficient (ICC1, 2) and standard error of measurement (SEM) for the knee and ankle peak torques in concentric, eccentric and isometric contractions. Reliability of the peak torque of knee joint Concentric extension

Eccentric flexion

Eccentric extension

Isometric flexion

Isometric extension

ICC= 0,99; SEM= 2.07

ICC= 0,99; SEM= 2.31

ICC= 0,99; SEM= 2.87

ICC= 0,99; SEM= 4.21

ICC= 0,98; SEM= 2.45

ICC= 0,96; SEM= 1.72

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Concentric flexion

ICC = 0.98

ICC = 0.96; SEM = 1.29

SEM = 1.89

Eccentric Eccentric plantar flexion dorsiflexion

Isometric plantar flexion

Isometric dorsiflexion

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Concentric dorsiflexion

ICC = 0.99; SEM = 16.68

ICC = 0.99; SEM = 12.87

ICC = 0.87; SEM = 1.42

ICC = 0.82; SEM = 1.32

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Concentric plantar flexion

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Reliability of peak torque of ankle joint

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ICC value: poor (<0,4); moderate (0,4-0,75); excellent (>0.75). Confidence interval adopted was 95%.

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Table 3. Concentric, eccentric and isometric peak torques of the knee and ankle joints (N-m/ kg x 100; 60º/s).

Type of Contraction

Control (N=33)

Joint Motion

DM2 (N=31)

DPN (N=28)

T P

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Knee Peak Torque

C S U

Flexion Extension

81.46 (31.81) 155.30 (51.13)

60.39 (26.93)* 115.20 (47.91)*

Eccentric

Flexion Extension

167.67 (33.07) 263.45 (74.02)

165.83 (37.84) 230.61 (69.66)

Isometric

Flexion Extension

101.97 (32.43) 219.70 (55.87)

Concentric

54.92 (21.55)* 110.04 (36.55)*

F=8.17; P<0.01 F=9.07; P<0.01

157.98 (34.83) 229.88 (68.82)

F=0.62; P=0.53 F=2.29; P=0.10

71.40 (31.78)* 167.47 (57.48)*

F=6.96; P<0.01 F=7.14; P<0.01

Ankle Peak Torque 24.29 [10.05-42.12] 25.14 [12.99-49.57]* 45.05 [9.09-104.25] 30.06[10.49-73.18]*

17.45 [3.95-29.82]*† 21.37 [10.29-82.76]*

Kruskal-Wallis Test H=13.65; P<0.01 H=15.92; P<0.01

52.27 [31.49-75.19] 45.61 [12.82-93.08]

53.05 [25.11-147.33] 56.28 [21.67-193.32]

H=0.89; P=0.63 H=5.16; P=0.07

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Concentric

Dorsiflexion Plantar Flexion

Eccentric

Dorsiflexion Plantar Flexion

N A

M

86.91 (31.42) 180.05 (57.50)*

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ANOVA

54.06 [35.48-180.34] 56.35 [25.83-191.86]

Dorsiflexion 38.87 [18.09-64.55] 40.00 [14.72-65.84] 34.18 [1.01-57.45]* H=7.63; P=0.02 Plantar Flexion 121.6 [44.64-176.9] 95.23 [11.28-167.86]* 71.87 [38.55-141.43]* H=18.11; P<0.01 Data of knee peak torque are expressed as mean (standard deviation) and data of ankle peak torque are expressed as median [minimum-maximum]. 26 Anova one-way comparing the knee peak torque variables with Tukey post hoc test when comparing the groups. * = P <0.05 compared with the Control, † = P <0.05 compared with DM2. N-m = Newton meters, kg = kilograms. Kruskal Wallis comparing the peak torque variables of the ankle and Mann Whitney test for comparison of peak torque between groups * = P <0.016 compared to Control, † = P <0.016 compared to DM2. Control = control group; DM2= diabetic type 2 group; DPN= diabetic type 2 with peripheral neuropathy group Isometric

ACCEPTED MANUSCRIPT Table 4. Peak torque effect size of the different types of contractions. Type of Contraction

Joint Motion

Control vs DM2

Control vs DPN

DM2 vs DPN

Effect size of knee peak torque Flexion Extension

-0.71 -0.80

-0.96 -1.00

Eccentric

Flexion Extension

-0.05 -0.45

-0.28 -0.46

Isometric

Flexion Extension

-0.47 -0.70

-0.22 -0.12

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Concentric

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-0.95 0.92

-0.21 -0.01 -0.49 -0.21

Dorsiflexion Plantar Flexion

Eccentric

Dorsiflexion Plantar Flexion

0.30 -0.81

-0.78 -1.04

-1.06 -0.36

0.38 0.59

0.20 0.42

-0.18 -0.13

0.05 -0.74

-0.70 -1.21

-0.72 -0.43

Dorsiflexion Plantar Flexion

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Isometric

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Concentric

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Effect size of ankle peak torque

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Negative values mean effect size in relation to the Control, or the DPN compared to DM2. Positive values indicate an inverse difference between the groups. Effect size insignificant (0.00 - 0.19); small 0.20 to 0.39), medium effect size (0.40- 0.79), large effect size (> 0.80). Control = control group; DM2= diabetic type 2 group; DPN= diabetic type 2 with peripheral neuropathy group.

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ACCEPTED MANUSCRIPT Highlights 

This is the first study analyzing knee and ankle torques at different contractions in type 2 diabetes with and without peripheral neuropathy.



Regardless of the presence of peripheral neuropathy, there are differences in



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skeletal muscle strength in the type 2 diabetes. Knee and ankle torques were affected in diabetic subjects, but the knee

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concentric and isometric torques presented even larger effect sizes than the

There were not differences at eccentric torque between both control and the two

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diabetic groups.

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ankle.

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ACCEPTED MANUSCRIPT Conflict of interest Statement

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The authors declare no conflict of interests.

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