in humans , skeletal muscle accounts for nearly 40% of the body 's mass and serves as the main tissue involved in glucose uptake during insulin stimulation . several researchers have established that glucose consumption in skeletal muscle decreases with type 2 diabetes mellitus . this reduced glucose consumption is a result of impaired transduction of insulin signals , such as insulin receptor substrate-1 phosphorylation , phosphatidylinositol 3-kinase ( pi3k ) activity , mitogen - activated protein kinase ( mapk ) activity , insulin - responsive glucose transporters , namely , glucose transporter-4 ( glut4 ) , and/or other insulin - independent mechanisms . therefore , a detailed analysis of insulin signaling at the cellular and molecular level is critical to understand the pathogenesis of type 2 diabetes associated with obesity . heterozygous atp10c mice present with the disease states of insulin resistance and obesity , as well as a host of other related disorders , including hyperlipidemia and hyperinsulinemia . previous research using these mice indicates that the atp10c gene appears to be a strong candidate gene for diet - induced obesity and type 2 diabetes mellitus . atp10c maps to the p - locus on mouse chromosome 7 , to a region of a quantitative trait locus associated with body weight , body fat , and diabetic phenotypes . the human ortholog , atp10c , maps to the syntenic region on chromosome 15q12 and is also associated with an elevated body mass index [ 3 , 4 ] . moreover , microarray gene profiling on atp10c heterozygous mice indicated significant changes in the mrna expression of factors involved in insulin - dependent and insulin - independent glucose uptake . although flippases , like atp10c , have been studied for many years , their exact character and function remain unclear . these proteins are believed to maintain the asymmetry of the lipid bilayer by translocating specific phospholipids from one leaflet to the other and vice versa , but they may also participate in the formation of transport vesicles . moreover , deficiencies in these proteins have been shown to cause defects in lipid metabolism and have been implicated in the disease states of obesity , type 2 diabetes , and nonalcoholic fatty liver disease . not much is known about the role of atp10c in regulating insulin resistance in skeletal muscle , if any , and its possible molecular and cellular targets have not been investigated . in view of the above literature , we hypothesized that the type iv p - type atpase , atp10c , has an important role in glucose metabolism . since atp10c is a transmembrane protein it might exert its effect via multiple signaling pathways : ( 1 ) acting solely at the plasma membrane to maintain the nonrandom distribution of phospholipids , thus contributing to a proper membrane environment for normal protein sequestration and function , ( 2 ) acting at the plasma membrane affecting the biogenesis of membrane vesicles important for plasma membrane delivery and/or retrieval of glucose transporter proteins in basal and insulin - stimulated states , and ( 3 ) acting directly on the expression , translocation , and/or function of glucose transporter proteins themselves . since the mapk pathway is known to be a key signaling cascade which mediates glucose clearance / uptake by the skeletal muscle in presence or absence of insulin , we tested whether the mapks , in general , are the targets of atp10c . to prove our hypothesis , specific objectives were to ( a ) establish a tissue culture system of mouse skeletal muscle wherein endogenous expression of atp10c could be monitored , ( b ) alter the endogenous level of atp10c expression by sirna , and ( c ) measure glucose uptake and assess changes in expression of mapks involved in this process . mouse skeletal muscle cell line c2c12 , a commercially available cell line , was kindly provided by dr . seung baek , college of veterinary medicine , the university of tennessee , knoxville , tn , usa . dulbecco 's modified eagle medium ( dmem ) containing 4.5  mg / l glucose and 4.5  mm / l l - glutamine , antibiotics ( 100  iu / ml penicillin and 100  g / ml streptomycin ) , absolute blue sybr green rox quantitative pcr mix , bovine calf serum ( bcs ) , and radioimmunoprecipitation assay ( ripa ) buffer were from thermo fisher scientific ( waltham , ma ) . dmem with 1% antibiotics and 10% bcs is furthermore referred to as the complete growth media . horse serum , 2-deoxy [ h ] glucose ( 2-dog ) , protease inhibitor cocktail in dmso solution , mapk inhibitor sb203580 , and human insulin solution ( 10  mg / ml in hepes , ph 8.2 ) were from sigma aldrich ( st . louis , mo ) . a bicinchoninic acid kit ( bca ) and an enhanced chemiluminescence ( ecl ) western blotting detection kit were purchased from pierce biotech inc . ( rockford , il ) and used in protein experiments . primary antibodies ( p38 , phospho - p38 , jnk , phospho - jnk , p44/42 , and phospho - p44/42 ) as well as the secondary antibody , horseradish peroxidase- ( hrp- ) conjugated anti - rabbit igg , were obtained from cell signaling technology ( danvers , ma ) . caveolin-1 was used as an immunoblot control and was purchased from santa cruz biotechnologies ( santa cruz , ca ) . the secondary antibody , hrp - conjugated anti - goat igg , was also obtained from santa cruz biotechnologies . hiperfect transfection reagent , atp10c - specific sirna constructs , rneasy mini kit , and quantitect primer assays for myod and atp10c were from qiagen ( valencia , ca ) . quantitative pcr primers specific for mouse glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) were designed using the primer 3 program ( http://primer3.sourceforge.net/ ) and were commercially obtained from operon ( huntsville , al ) . the iscript cdna synthesis kit was acquired from bio - rad laboratories ( hercules , ca ) . samuel cushman , national institute of diabetes and digestive and kidney diseases , national institutes of health , bethesda , md . all immunofluorescence materials ( protein blocks [ normal rabbit ] , negative control [ normal rabbit ] , and antibody diluent ) were purchased from biogenex ( san ramon , ca ) . millicell ez slides from millipore ( billerica , ma ) were used for immunofluorescence experiments . secondary antibody specific for immunofluorescence application , alexa fluor 568 donkey anti - rabbit , as well as prolong gold antifade reagent was purchased from invitrogen ( carlsbad , ca ) . roughly 2.0  10 cells were seeded in a 60  mm dish or a single well of a 6-well plate . cells at 70% confluency were differentiated in the presence of 2% horse serum - enriched media for 35 days . completely differentiated myotubes ( days 35 ) were either subjected to various treatments described in the relevant sections or harvested for subsequent experiments . three different sirna oligonucleotides against atp10c were commercially obtained ( qiagen ) ; one was generated from the sequence at the 3 end of the atp10c gene , si00906220 ( sense : r[ccu ggg uau uga aac caa a]dtdt and antisense : r[uuu ggu uuc aau acc cag g]dtdg ) , and the second , si00906213 , and third , si00906206 , were generated from the sequence at the 5 end ( sense : r[cgu cuu ugc ugc aau gaa a]dtdt and antisense : r[uuu cau ugc agc aaa gac g]dgda ) . c2c12 myotubes were transiently transfected with atp10c - specific and scrambled sirna using hiperfect transfection reagent ( qiagen ) according to the manufacturer 's instructions . myotubes transfected with sirna were either harvested or treated with the reagents indicated in the relevant sections and/or used to measure glucose uptake . in the first experiment , the optimum concentration and time of knockdown for each sirna used were determined . briefly , hiperfect transfection regent and sirnas were mixed at various concentrations ( 0 , 50 , 100 , and 200  nm ) to form a complex . the transfection complexes were then applied to designated cells and incubated for 24 , 48 , or 72  h before subsequent analysis . c2c12 myotubes demonstrating efficient atp10c knockdown and , therefore , used in all further experiments were designated as c210c/. mock - transfected ( i.e. , transfected with hiperfect only ) c2c12 myotubes ( c2wt ) were used as corresponding controls . the following procedures were performed as described elsewhere [ 5 , 10 , 11 ] . cells were washed with ice cold 1x pbs , and total rnas were isolated using rneasy mini rna kit ( qiagen ) , according to the manufacturer 's instructions . single - stranded cdna was synthesized using the iscript cdna synthesis kit ( bio - rad ) and amplified using gene - specific primers by quantitative pcr ( qpcr by quantitect primer assays ) , with mouse gapdh as the housekeeping gene . all mrna expressions were achieved by qpcr using absolute sybr green rox quantitative pcr mix on the strategene mx3005p with mxpro analysis software under the following pcr conditions : 1 cycle of 50c for 15  min and 95c for 2  min , followed by 40 cycles of 95c for 25  s , 52c for 25  s , and 72c for 1  min . the relative abundance of target gene expression was calculated using the 2-ct and standard curve method , with ct being the difference between ct of the target gene normalized with respect to the gapdh ct . total cell lysates were isolated using ripa buffer according to standard methods [ 13 , 14 ] . briefly , cells were washed twice with 1x pbs and lysed in ripa buffer containing protease inhibitor cocktails at 4c for 30  min . protein estimation was performed using the bca kit ( pierce biotech ) , according to the manufacturer 's instructions . equal concentrations ( 25100  gs ) of proteins were resolved on 10% sds - page , using 5x laemmli sample buffer containing tris - hcl ( 375  mm , ph 6.8 ) , glycerol ( 48% ) , sds ( 6% ) , beta - mercaptoethanol ( 6% ) , and bromophenol ( 0.03% ) . after electrophoresis , proteins were transferred to nitrocellulose membranes , blocked for 1  h in blocking solution ( 15% bsa in tbst buffer ) , and incubated with specific primary antibodies overnight at 4c . primary antibodies were detected with hrp - conjugated secondary antibodies , and antibody - protein complexes were visualized using ecl ( pierce biotech ) . results are expressed as the ratio of target protein expression to that of an internal loading control , caveolin-1 . for immunofluorescence , 2.0  10 cells were seeded onto 4-well chamber slides ( millipore ) and subjected to the appropriate treatments as described in the relevant sections . immunofluorescence assays were carried out according to standard methods as described by others [ 8 , 15 ] . briefly , cells were fixed with 1% paraformaldehyde in 0.1  m sodium phosphate buffer , ph 7.3 for 10  min at room temperature . cells were washed in 1x pbs , permeabilized by incubating with 0.01% tween-20/pbs for 10  min , and then washed again with 1x pbs . after the last wash , cells were blocked using blocking buffer ( 1% bsa , 2% normal serum , 0.1% tween-20 in pbs ) for 30  min . blocking solution once blocking was complete , the cells were incubated with specific primary antibodies overnight at 4c . bound antibody was visualized under the microscope ( nikon ti - e eclipse , nikon instruments , melville , ny ) by incubating for 1  h with secondary antibody labeled with alexa fluor 568 ( tritc ) . to visualize the nucleus , cells were exposed for 5  min at room temperature to a concentration of 300  nm dapi ( 4 , 6-diamidino-2-phenylindole , dilactate ) in pbs . additionally , both positive and negative controls were prepared and imaged alongside the samples to correct for any background fluorescence and to serve as controls for quantitative analysis . images were captured using an epifluorescence microscope ( nikon instruments ) with a 60x objective lens ( na 1.49 ) and an automated stage . briefly , myoblasts were plated in 6-well cell culture plates at a density of 2  10 cells / well and allowed to differentiate under normal conditions . after differentiation , cells were washed with ix pbs and serum - starved in dmem only for 35  h. cells were stimulated with 100  nm insulin in dmem for 30  min at 37c . each 6-well plate was setup so that wells 1 , 2 , and 3 did not contain insulin and wells 4 , 5 , and 6 did contain insulin . insulin induction was stopped by washing the cells twice with 1  ml krebs - ringer hepes ( krp ) ( 121  mm nacl , 4.9  mm kcl , 1.2  mm mgso4 , 0.33  mm cacl2 , 12  mm hepes ) minus glucose at room temperature . cytochalasin b ( 5  l of 1  mm stock/1  ml cocktail ) was used to normalize for nonspecific glucose uptake . glucose uptake was determined after the addition of  h-2-deoxyglucose ( 1  l of 10  ci / mmol stock/1  ml cocktail ) in krp buffer at 37c for 5  min . incorporation was terminated by washing the cells twice with 1  ml ice cold krp plus 25  mm glucose . cells were lysed on ice for 30  min with ripa buffer . following incubation , 0.5  ml cell lysates were mixed with 10  ml scintillation fluid ( scintiverse ) and subjected to liquid scintillation counting . for protein quantitation ( bca method , pierce biotech ) , 250  l of the lysate was used and processed according to the manufacturer 's instructions . glucose uptake was expressed as disintegrations per minute per microgram of total protein ( dpm/g ) . data is reported as the glucose uptake stimulation expressed as the ratio of dpm/g of total protein in presence of insulin to that in the absence of insulin . relative densitometry analyses of the immunoblots were determined using scion ( http://scion-image.software.informer.com/ ) and image j ( http://rsb.info.nih.gov/ij/index.html ) analysis software . by giving an arbitrary value of 1.0 to the respective control sample ( caveolin-1 ) of each experiment , immunofluorescence images were analyzed using nis - elements software ( ar v. 3.1 ) ( nikon instruments ) . for each sample , 9 dual - channel images were captured and stitched together to form a large image ( 3  3 ) . mean fluorescence intensity per cell was calculated ( mfi per cell = total image fluorescence / cell count ) . cell count was determined using a nuclear stain ( 300  nm dapi in pbs ) . the data are expressed as mean  se . for comparison of two groups , p  values were calculated using the two - tailed paired student t test . in all cases p 0.05 was considered statistically significant , and p  0.10 was indicative of a trend . to alleviate whole - animal complexities , c2c12 , a mouse skeletal muscle cell line , was selected as an in vitro model to identify molecular and cellular targets of atp10c and then assess its biological role , if any , in insulin signaling and glucose metabolism [ 16 , 17 ] . under permissive conditions , differentiation of c2c12 myoblasts to myotubes was confirmed by visual inspection ( see supplemental figure 1 in supplementary material available online at doi.10.1155/2012/152902 ) , as well as by monitoring the expression of skeletal muscle - specific mrnas for myod and myogenin . cells were collected on days 1 , 3 , and 5 during the differentiation process . cells collected at day 1 represent the myoblasts , which are then stimulated to differentiate into myotubes by days 35 . the activation of cell differentiation is characterized by the expression of myogenic regulatory factors including myod and myogenin . this is followed by the expression of additional factors including but not limited to myf-5 and mrf4 and permanent exit from the normal growth and proliferation cycles . as expected , the expression of myogenin increased as myoblasts were stimulated to differentiate ( 30% to 52% ) , and decreased when differentiation was complete ( 20% ) . similarly , qpcr showed that myod expression increased dramatically on day 1 ( 172% ) , then steadily declined on days 3 and 5 ( 64% and 24% , resp . ) . interestingly , atp10c was expressed in both cell types , and its expression increased upon differentiation ( 42% to 64% ) and steadily decreased as myotubes were formed ( 47% on day 5 ) . changes in atp10c expression with differentiation were similar , but , not as striking as that of myogenin and myod ( supplemental figure 2 ) , ramifications of which are clearly beyond the scope of this study . the data thus demonstrated the expression of atp10c mrna in c2c12 cells , and gave us an opportunity to modulate its expression in both myoblasts and myotubes , both of which can have significant consequences in insulin signaling pathways . one of the limitations of global gene - targeting and whole - animal approach is that adaptations over time might occur , possibly producing secondary phenotypes that are not directly linked to the mutation . in this case , the role of atp10c could be shown either by generating knockout mice or using specific inhibitors against atp10c . since no inhibitors are available , and to avoid whole - animal complexities in transgenics , we modulated atp10c expression in vitro in c2c12 cells by transiently transfecting them with three independent and commercially available atp10c - specific sirnas ( qiagen ) . atp10c is a putative transmembrane domain protein , and as such a good antibody against atp10c has yet to be generated , making experiments to study atp10c challenging . therefore , in this analysis , changes in atp10c expression were determined solely at the mrna level by quantitative pcr using quantitect primer assays ( qiagen ) . out of the three sirnas tested , only one , si00906220 , resulted in a significant knockdown of 70% ( data not shown ) . as shown in figure 1 , significant knockdowns ( 70 to 100% ) were observed in all samples with the exception of 200  nm , 72  h. higher concentrations and longer time periods resulted in cell death and poor quality of cells ( as judged visually ) . taking these criteria into consideration as well as the optimization of concentration and time of transfection , 50  nm of sirna ( si00906220 ) and a time point of 24  h were selected for all subsequent experiments . gapdh was used as the housekeeping gene , and no significant mrna changes in its corresponding expression in transfected cells were observed . results thus suggest that changes in atp10c expression after transfection with sirna ( si00906220 ) were not an artifact , and there was no deleterious effect on c2c12 myotubes due to atp10c silencing . all further experiments were carried out under these conditions in wild - type c2c12 ( c2wt ) and atp10c - silenced c2c12 myotubes ( c210c/ ) simultaneously . since these transfections are transient , atp10c mrna knockdown was confirmed in each and every experiment . previous experiments in our laboratory have shown that on a high fat diet , there is a 35% decrease in glucose uptake in soleus muscle in atp10c heterozygotes . based on this finding , we next determined whether downregulation of atp10c expression had a similar effect in vitro . glucose uptake was measured in c2wt and c210c/ myotubes in basal ( without insulin ) and stimulated ( 100  nm insulin , 30  min ) states . fold change representing the glucose uptake stimulation / reduction between the c2wt and c210c/ was compared . as shown in figure 2 , insulin stimulation caused a significant 2.54-fold decrease in 2-dog uptake in c210c/ cells ( p < 0.05 ) . data thus complement the in vivo findings , suggesting that atp10c is necessary for insulin - stimulated glucose uptake in skeletal muscle , and its knockdown renders the myotubes insulin resistant . silencing atp10c rna decreases cellular glucose uptake , which might be of consequence to impaired insulin signaling . insulin - induced glucose uptake into muscle and adipose tissue involves a series of intracellular signaling cascades , culminating in glucose disposal and metabolism [ 1924 ] . the possible mechanisms include insulin - mediated activation of the insulin receptor and/or its downstream molecules , ultimately effecting glut4 expression and translocation . since these processes are a complex interplay of a variety of proteins and because their changes have not been studied in atp10c silencing , we sought to identify changes in the key proteins of one such signaling cascade , the mapk pathway in the absence of any insulin stimulation . specifically , in the present study , the effect of atp10c silencing was considered on three essential mapks : p38 ( mapk14 ) , jnk , and p44/42 , ( erk1/2 ) . total proteins isolated from c2wt and c210c/ myotubes were subjected to a combination of immunoblot and immunofluorescence analysis ( figures 3(a)3(c ) ) . for mapk pathway analysis , results indicated significant upregulation of p38 ( p = 0.02 ) and p44/42 ( p = 0.04 ) and a significant downregulation of jnk ( p = 0.001 ) and phospho - p44/42 ( p = 0.05 ) in c210c/ cells . while not significant , results indicated a trend for an increase in phospho - p38 ( p = 0.1 ) ( figure 3(a ) ) and phospho - jnk ( p = 0.06 ) ( figure 3(c ) ) . beside these signal transduction proteins , there was significant upregulation of myod ( p = 0.01 ) , actin ( p = 0.02 ) ( supplemental figures 3(a ) and 3(b ) ) , and glut1 ( p = 0.03 ) . most importantly , there was no significant change in glut4 expression ( figure 4 ) . this is such a complex process that both in vitro and in vivo data are inconclusive . mapks , specifically , p38 and p44/42 proteins , regulate glucose uptake via insulin - dependent and independent pathways [ 2527 ] . our results suggest that when myotubes become insulin resistant by atp10c silencing , as expected there is a significant increase in the expression of native mapks ; however , they are not activated into their phosphorylated forms at a significant level . data demonstrates that p38 and p44/42 are responsive to changes in atp10c with increased expression , whereas there is no similar effect on jnk , suggesting that it is not a target at least in this pathway . the importance of p38 in this process is further supported by the fact that there are significant changes in glucose transporter proteins as well . glut1 and glut4 have been demonstrated to be the key players of glucose clearance in peripheral tissues . glut1 is responsible for glucose uptake in the basal state whereas glut4 is insulin responsive . defective uptake of glucose mediated by the gluts is a central feature of obesity and type 2 diabetes . interestingly , microarray gene profiling of peripheral tissues obtained from atp10c heterozygotes which were fed a high fat diet for 4 weeks demonstrated upregulation of p38 in soleus muscle . of relevance to our study , mapk protein expression , specifically p38 , has been shown to affect the expression of glut1 and glut4 , which can subsequently affect glucose uptake in peripheral tissues . the mapk protein , p38 , reportedly upregulates the expression of glut1 , thereby altering glucose transport at the basal level , while also involved in an insulin - induced enhancement of intrinsic glut4 activity on the cell surface . since , we do not see any increase in the basal glucose uptake , we strongly feel that the upregulation of glut1 is more of a compensatory mechanism against insulin resistance in c210c/ , since there is no change in glut4 ; the precise underlying mechanism , however , remains to be clarified . this result may prove to be the most crucial , as glut4 is the main player in insulin - stimulated glucose metabolism . reports additionally indicate that the activation of p38 reduces insulin responsiveness , so the exact mechanism by which the mapk pathway is involved in glucose metabolism still remains controversial . up - regulation of myod and actin in transfected cells ( c210c/ ) suggests a regulatory mechanism by which the myotubes are trying to combat the stressful state of insulin resistance . by aiding with glut4-containing vesicle membrane movement and/or fusion , there is considerable evidence that actin is essential for insulin - regulated glucose transport . therefore , these cells may be overexpressing actin acutely in preparation for insulin - stimulated glucose uptake . to confirm the involvement of p38 , the p38-specific inhibitor sb203580 was added to c210c/ cells at a concentration of 10  m for 60  min [ 30 , 31 ] . protein samples were subjected to immunoblot analysis with p38 andphospho - p38 antibodies ( figure 5 ) . our results indicate that c210c/ cells treated with 10  nm sb203580 for 60  min effected the expression of both p38 ( p = 0.1 ) and phospho - p38 ( p = 0.08 ) ( figure 5 ) . while not significant , it appears that the inhibitor was able to partially restore the expression of all the proteins tested suggesting its action on the mapks . this was further confirmed by the glucose uptake assay performed on c210c/ and mapk - inhibited cells . results from the assay showed that glucose uptake remained unchanged , confirming that the inhibitors are acting directly on the mapk proteins and are not affected by changes in atp10c expression ( figure 6 ) . a phenomenon known as phospholipid randomization affects the structure and function of many channels , transporters , and signal transducing proteins and has been implicated in several pathophysiology processes . thus , maintaining the organization and activity of the lipid bilayer is essential for normal cell function . one class of proteins that performs this action is the flippases or type - iv atpases . in yeast studies , these proteins cause the translocation of glycerophospholipids , and this movement is necessary for intracellular membrane and protein trafficking . atp10c is one such phospholipid translocase which encodes for a type iv p - type atpase . our laboratory has demonstrated that atp10c heterozygous mice are insulin resistant and have an altered insulin - stimulated response in peripheral tissues . our obesity mouse model is dietinduced and shows insulin resistance characterized by hyperinsulinemia , hyperglycemia , hyperlipidemia , and obesity in association with glucose intolerance [ 2 , 3 , 11 ] . in fact , a recent publication has sited atp10c as a potential biomarker for obesity and related metabolic disorders . in the present study , we report , for the first time , a direct correlation between atp10c mrna expression levels and glucose metabolism , at least in part via the mapk pathway in c2c12 skeletal muscle cells . our results showed no significant change in the basal glucose uptake suggesting that when atp10c is silenced using atp10c - specific sirna , an acute state of insulin resistance is observed . the mapk protein , specifically p38 , is potentially influencing this system by exerting an effect on the glucose transporter proteins . results from these experiments are interesting and lead to future experiments that will involve other signaling cascades ( pi3k ) , insulin receptors , and substrates as well as other downstream factors , including glut4 translocation .
atp10c is a strong candidate gene for diet - induced obesity and type 2 diabetes . to identify molecular and cellular targets of atp10c 
 , atp10c expression was altered in vitro in c2c12 skeletal muscle myotubes by transient transfection with an atp10c - specific sirna . 
 glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen - activated protein kinases ( mapks ) , p38 , and p44/42 . additionally , glucose transporter-1 ( glut1 ) was significantly upregulated ; no changes in glucose transporter-4 ( glut4 ) expression were observed . 
 the involvement of mapks was confirmed using the specific inhibitor sb203580 , which downregulated the expression of native and phosphorylated mapk proteins in transfected cells without any changes in insulin - stimulated glucose uptake . 
 results indicate that atp10c regulates glucose metabolism , at least in part via the mapk pathway , and , thus , plays a significant role in the development of insulin resistance and type 2 diabetes .