duchenne muscular dystrophy ( dmd ) is a recessive x - chromosome - linked disease that affects ~1/36006000 liveborn males . the muscular weakness is progressive , causing loss of ambulation by early adolescence ( between 9 and 12 years of age ) . dmd is caused by mutations in the dmd gene that code for dystrophin , a sarcolemmal cytoskeletal protein [ 24 ] . dmd gene mutations that result in complete loss of dystrophin interrupt their translation , giving rise to dmd . mutations that conserve the open reading frame produce reduced quantities of dystrophin or a dysfunctional or truncated form of dystrophy , resulting in becker muscular dystrophy ( bmd ) , a less severe phenotype . dystrophin is an element of the dystrophin - glycoprotein complex ( dgc ) that provides a mechanical link between the extracellular matrix and cytoskeleton of muscle cells , allowing the plasma membrane of the muscle fiber to resist the mechanical process of the muscle during muscle contraction [ 6 , 7 ] . it has been suggested that the dgc also participates in important cell - signalling processes , functioning as a binding platform for certain ligands   such as neuronal nitric acid synthase , which stimulates glucose transport . skeletal muscle is responsible for > 80% of insulin - stimulated glucose uptake in the body . therefore , destabilization in dgc or their components may generate abnormal signalling of insulin in muscle fibers and lead to alterations in functionality . in fact , it was reported that the disturbance within the surface dgc may contribute to insulin resistance ( ir ) and abnormalities characteristic of the skeletal muscle of diabetic goto - kakizaki rats , because an abnormal subcellular location of glucose transporter 4 ( glut4 ) vesicles in muscle fiber was observed in these rats . however , information on this regard from human studies is lacking . this information led us to propose that dgc alterations may generate changes in glucose metabolism such as ir in skeletal muscle of patients with dmd or bmd . dgc modifications provoke an imbalance in plasma membranes permeability , inducing myofibers through deterioration - regeneration cycles until exhausting their repair capacity . this poses muscle fibers susceptible to necrotic development and to be replaced by both fibrous connective tissue and adipose tissue [ 1 , 13 ] , increasing adiposity . previous body composition studies have showed that dmd patients present greater body fat mass than similarly aged healthy subjects [ 13 , 14 ] . in addition , other investigators have observed that patients with dmd develop overweight or obesity from the age of 7 , reaching a frequency of > 50% at 13 years of age . in addition to the alteration of dgc , overweight and obesity are also risk for ir , which increases the risk for other severe morbidities such as cardiovascular disease and type 2 diabetes in dmd patients . however , the concomitant effect of alterations in dgc and obesity on the risk for insulin resistance has not been properly addressed . thus , the purposes of this investigation were to determine the frequency of ir in patients with dmd / bmd and to evaluate the association of deletions in specific regions of the dmd gene with obesity and ir , as well as exploring molecular mechanisms likely leading to ir , by evaluating molecules involved in glucose metabolism such as insulin receptor , insulin receptor substrate , and glut4 localization in muscle biopsies of dmd / bmd patients . the institutional ( instituto mexicano del seguro social ) ethics committee approved the study prior to the start of patient recruitment . all dmd / bmd patients seen at the outpatient electrodiagnostic muscular dystrophy service at the national institute of rehabilitation were recruited for the cross - sectional study between january 2011 and december 2013 ; 117 patients ( aged 4 years to < 18 years ) were included . subjects with a clinical diagnosis of dmd / bmd were candidates to participate in the study , and confirmatory molecular diagnosis of dystrophy was carried out . patients were eligible for inclusion in this study if they had a deletion in the dmd gene analyzed by multiplex polymerase chain reaction ( mpcr ) . parents and patients received an explanation of study fundamentals , procedures , benefits , right to confidentiality , and the right to withdraw from the study if they wished . all parents provided written informed consent in adherence with the human subjects ' guidelines of the institutional ethics committee . on the day of the study , a peripheral blood sample was collected in vacutainer with and without anticoagulant in a fasting state for genomic dna extraction from leukocytes to determine glucose and insulin , respectively . body composition to measure adiposity was evaluated by dual - energy x - ray absorptiometry ( dexa ) . trained personnel carried out measurements of body weight ( kg ) and height ( mts ) . for subjects who were able to stand erect , height was measured with a wall - mounted stadiometer ( model 208 , seca ) . for subjects unable to stand subjects ' weight was measured ( model bwb-700 , tanita , for ambulatory patients and model 954 seca for wheelchair - bound patients ) wearing light clothing and without shoes . diagnosis of overweight and obesity was obtained using body mass index ( bmi ) , expressed as percentiles . children with bmi  5th percentile were classified as underweight , those with bmi > 5th but < 85th percentile as normal weight , those with bmi  85th but < 95th percentile as overweight , and those with bmi  95th percentile as obese , in accordance with criteria established by the centers for disease control and prevention ( cdc ) in 2009 about bmi for children and teens ( http://www.cdc.gov/healthyweight/assessing/bmi/childrens_bmi/about_childrens_bmi.html ) . body composition measurements were carried out by dexa ( lunar prodigy , ge medical systems , madison , wi ) and encore software , v. 2004 ( lunar corporation ) , was used to analyze whole - body dexa scans . body fat mass was considered high according to the classification as described previously   that used dexa to predict % body fat mass corresponding to the bmi cutoff in male children and adolescents ( 318 years ) as overweight ( range : 18%23% ) and obesity ( range : 2436% ) . one hundred seventeen patients were screened for deletions and duplications by mpcr with the primer sets of chamberlain et al .   and beggs et al .   using the mpcr kit for human dmd / bmd set i + ii ( maxim biotech , san francisco , ca ) according to the protocol recommended by the manufacturer . serum insulin ( u / ml ) was quantified utilizing a commercial kit ( linco research , st . louis , mo ) based on radioimmunoanalysis . a value > 12  u / ml was considered as hyperinsulinemia as was previously reported . serum glucose ( mg / dl ) concentration was measured by the glucose - oxidase method ( glucose - lq , spinreact , s.a . , ir was calculated from insulin and glucose data using the homeostasis model assessment - insulin resistance ( homa - ir ) method ( homa - ir = [ fasting insulin , u / ml ]  [ fasting glucose , mmol / l])/22.5 . a portion was used for immunofluorescence analysis . in this study , we had availability for tissues from only five patients with dmd / bmd of the entire population studied . we also used five biopsies of gastrocnemius muscle from five healthy individuals as controls ( age 4455 years ) to compare cellular localization of dystrophin , insulin receptor , insulin receptor substrate , and glut4 . we analyzed skeletal muscle biopsy from older controls because we have not access to biopsies of muscle from healthy subjects of the same age . nevertheless , older controls allowed the main purpose to evaluate the cellular localization of these molecules in human healthy muscle . specific mouse monoclonal primary antibodies used were glut4 , insulin receptor ( ire ) ( santa cruz biotechnology , santa cruz , ca ) , and dystrophin n - terminus ( dys - n ) and c - terminus ( dys - c ) ( vector laboratories , burlingame , ca ) . polyclonal antibody against insulin receptor substrate ( irs ) ( santa cruz biotechnology ) was also used . afterwards , 7  m cryosections were prepared and added to the coverslip covered with poly - l - lysine . next , nonspecific binding was blocked by incubations of the cryosections with 5% bovine serum albumin in phosphate - buffered solution ( pbs ) for 60  min at 25c . samples were incubated with cy3-secondary antibody ( goat anti - mouse , jackson immunoresearch laboratories , west grove , pa ) for 60  min at 25c in the dark . subsequently , samples were washed with pbs and mounted with dapi ( labeling of nuclei ) and vectashield ( vector laboratories ) . tissues were observed under an olympus bx60 fluorescence microscope ( olympus , tokyo , japan ) . a subjective value was assigned ( normal + + + , decreased + +  , considerably decreased +  , and absent    ) to describe staining , indicating the presence of dystrophin . statistical analysis was performed using the minitab statistical software ( minitab 14 , state college , pa ) . results are presented as median ( minimum , maximum ) ; p value 0.05 was considered significant . pearson correlation analyses were used to evaluate associations between the most frequently deleted exons and fasting insulin , homa - ir , bmi , and body composition . comparisons among nutritional status groups were conducted with one - way anova and dunnett 's method as post hoc test , considering the normal nutritional status group as control . logistic regression models were carried out to identify risk factors for ir , introducing each exon deletion as a predictor and taking into account adiposity . we are reporting the results of 66 subjects identified as carriers in at least one exon of the two deletion - prone regions in the dmd gene . most alterations ( 73.7% ) were clustered in exons 4360 in the major hot spot . deletions of exons 19 , 45 , 47 , 48 , 50 , and 51 were more frequently detected . patient ages were 8.96 ( 4.61 , 17.75 ) years , median ( minimum , maximum ) , and 53% of the population were between 7 and 10 years of age . a low percentage of patients ( 9.1% ) were between 15 and 18 years of age . fasting insulin concentration ranged from 5.2 to 59.9  u / ml , but ~50% present hyperinsulinemia . homa - ir values range from 1.04 to 12.9 , and 36% of the patients had ir ( table 1 ) . most boys presented normal nutritional status ( 59% ) , but 22.7% were overweight / obese and 18.2% were underweight . according to the classification of taylor et al . ( see subjects and methods ) , a higher ( 68% ) prevalence of overweight / obesity was observed in contrast to using bmi ( 22.7% ) . thus , we show group data according to nutritional status from dmd / bmd patients as underweight , normal , and overweight / obese ( table 2 ) . we observed significant differences in insulin concentrations and homa - ir values among nutritional status groups , but no differences in age were detected . overweight / obese boys presented higher glucose , insulin , and homa - ir values as compared to normal bmi group . that group exhibited also a higher proportion ( 80% ) of subjects with ir determined by homa - ir > 3.16 (  : 11.48 , p = 0.004 ) compared to normal nutritional status . interestingly , an important percentage of underweight patients presented hyperinsulinemia and ir ( 27.3% ) . body fat mass was higher in overweight / obese and lower in underweight than in patients with normal nutritional status ( table 2 ) . both fasting insulin and homa - ir were positively correlated with all anthropometric and body composition parameters . however , the highest correlation was found between homa - ir and body fat mass ( figure 2 ) . carriers of deletions in exon 45 ( or = 9.32 ; 95% ci 1.1674.69 ; p = 0.036 ) or exon 50 ( or = 8.73 ; ci95 = 1.1765.10 ; p = 0.035 ) were associated with the risk for ir , even adjusting for adiposity ( figure 3 ) . staining of glut4 vesicles was observed as cytoplasmic aggregates in biopsies from patients 3 , 5 , and 7 ( figure 4 ) , although in patients 3 and 7 there are a smaller number of these aggregates ; the three patients presented hyperinsulinemia and ir ( table 3 ) and patient 5 showed hyperglycemia . staining for ire and irs was similar to control tissue muscle in myofiber sections from five patients . dystrophin staining intensities in myofibers from five patients ( figure 3 ) were lower than those observed in normal biopsy specimens . hyperinsulinemia and ir observed in our patients are important risk factors for developing pathologies such as cardiovascular disease or type 2 diabetes . the frequency of these metabolic alterations increases ( 80% ) importantly when dmd / bmd patients are overweight / obese , with a rate higher than in nondystrophic obese boys reported in our country ( ~50% in children between 3 and 18 years ) . the increased intramuscular body fat mass deposition   in dmd patients and hyperinsulinemia and ir are strong indicators of muscle metabolic defects ; this deposition is present in dgc - related muscular dystrophy . but it is important to consider that almost a third ( 27.3% ) of underweight patients presented hyperinsulinemia and ir which indicates that there is not an intramuscular body fat mass deposition in those patients . therefore , ir in dmd / bmd may be an independent result of fat mass content where alterations of components of dgc such as dystrophin may be involved . to our knowledge , there are no studies that clearly demonstrate whether patients with dmd or bmd present ir or alterations in glucose metabolism . freidenberg and olefsky reported that serum glucose and insulin concentrations after oral glucose administration showed an abnormal increase in the area under their respective curves , suggesting alterations in glucose metabolism from dmd patients . abnormal subcellular accumulation of glut4 vesicles was observed in nonobese , type 2 diabetic skeletal muscle fibers of rats . accordingly , we also observed abnormal cytoplasmic aggregates of glut4 in myofibers from dmd / bmd patients , suggesting a possible alteration of glucose incorporation into the muscle , leading to hyperglycemia . furthermore , we observed that deletions of exons 45 or 50 increase the risk ( ~9.0 times ) for developing ir . in this sense , these exons encode an acting - binding domain and maybe the alteration between f - actin and dystrophin link may disturb some cell - signalling process as binding platform ligands arising in metabolic alteration . anyway , our data suggest that the result of the mutation in the dmd gene may possibly be associated with a metabolic alteration in dmd / bmd patients . the presence of these metabolic alterations in underweight subjects suggests a possible relationship with dmd / bmd . because we included a low number of muscle biopsies from dmd / bmd patients with different grades of damage in the muscle fiber , we detected abnormal cytoplasmic aggregates of glut4 in myofibers only in three patients . at any rate , this information opens an interesting field of study to explore , in muscle biopsies from dmd / bmd patients , cellular signalling pathways involved in glucose metabolism and possibly related to muscle morphology . another limitation of this study is the lack of distinction between dmd and bmd and the lack of appropriate control subjects of the same age . anyway , the main strength of this study is that we demonstrated that those patients develop metabolic alterations , which is essential information for caregivers and physicians in order to prevent other pathologies . we identified differences in nutritional status of dmd / bmd patients according to bmi ( percentiles ) . prevalence of overweight or obesity in these patients was lower ( 22.7% ) than that reported by other authors where ~50% of boys with dmd / bmd were obese by 13 years of age according to bmi [ 13 , 15 ] . for the first time , we present information about the prevalence of obesity in dmd without effect of steroid treatment . the higher total body fat mass observed in boys with dmd is mostly due to increased intramuscular body fat mass deposition in both the central and peripheral regions . in agreement with the classification of taylor et al . , 2002 , body fat mass was higher ( ~68% ) than in healthy boys and in boys with dmd [ 1315 , 25 ] . in conclusion , to our knowledge , these novel results present evidence regarding the presence of metabolic alterations in dmd / bmd patients such as obesity , hyperinsulinemia , and ir , without the effects of steroid treatment . ir had a high frequency and increased significantly when dmd / bmd patients are overweight / obese . deletion of exons 45 or 50 increases the risk ( ~9 times ) for developing ir . abnormal cytoplasmic aggregates of glut4 in myofibers from dmd / bmd patients suggest a possible alteration of glucose incorporation into the muscle .