the incidences were getting higher and higher . increased intramyocellular lipid accumulation plays a very important role in obesity and diabetes , as well as their complications [ 2 , 3 ] .
this attracted great interest in the effect of accumulated lipid intermediates on insulin resistance [ 4 , 5 ] .
lipotoxicity was regarded as the link of high levels of lipids with impaired insulin signaling [ 6 , 7 ] .
this sparked the interest in how excessive lipid affects -oxidation and mitochondrial biogenesis , which may have a great impact on glucose utilization and insulin resistance [ 710 ] .
carnitine palmitoyl transferase 1 ( cpt1 ) is an important rate - limiting enzyme of mitochondrial -oxidation , by controlling the mitochondrial uptake of long - chain acyl - coas .
its muscle isoform , cpt1b , is the predominant isoform rich in the heart and skeletal muscles .
it was suggested that inhibiting cpt1 activity by specific cpt1 inhibitors alleviates insulin resistance in diet - induced obese mice .
however , there is no study investigating if specific cpt1b deficiency in skeletal muscles had significant impact on lipids and insulin sensitivity .
therefore , in this study , by establishing a mouse model , we directly explored this issue . with specific knockout of carnitine palmitoyl transferase-1b ( cpt1b ) in skeletal muscles of mice ,
cpt1b m/ mouse model was established as per previously described [ 12 , 16 , 17 ] by using male c57/bl6 mice ( 12 weeks old ; 2025  g ) with cre - lox technology [ 17 , 18 ] .
mice were genotyped by standard pcr of tail dna with pcr master mix ( applied biosystems inc . ) .
twenty mice from each group ( cpt1band cpt1b m/ ) were kept on a 12  h/12  h light / dark cycle , in temperature - steady rooms , and had ad libitum access to water and chow diet .
all experimental procedures were conducted according to the care and use guide of laboratory animals and were approved by the iaec of the huzhou central hospital .
general and metabolic profiles were measured at 12 , 18 , 24 , and 30 weeks of age , including the following : body weight , lipids in serum , food intake , body mass , and fat mass , as per related protocols [ 19 , 20 ] .
skeletal muscle mrna and protein were extracted and purified with standard protocols as per our previous protocols [ 7 , 21 ] .
rt - pcr was performed for mrna quantization per standard protocols with the reagents from abi system inc .
quantitative real - time rt - pcr analyses were carried out by using step one real - time pcr system ( applied biosystems inc . ) .
cycles were 50c for 2  min , a 2  min 95c denaturing step , followed by 50 cycles of 95c denaturation , incubated at 60c for 2  min , and denatured at 95c for 1 sec for the final step .
the sequences of the primers are as follows : for cpt1b : forward : gac cat agaggc act tct cagcat gg , reverse : gcagca gcttca ggg tttgt ; -actin : forward : gtc ctc tcc caa gtc cac ac , reverse : ggg aga cca aaa gcc ttc at ; pakt forward : taa tac gac tca cta tag ggc caa ggagatcatgc , reverse : gatttaggtgacactatagctccaagctatcgtcc ; glut4 forward : accgtggtccttigctgtgtt , reverse : acc cca atg ttg tac cca aac t. as described previously [ 16 , 22 , 23 ] , a modified mitochondrial cpt1 assay was performed to measure cpt1 activity .
briefly , quantified mitochondria ( 100  g protein ) from skeletal muscles were incubated with reaction buffer ( 117 mm tris - hcl , 0.28  mm reduced glutathione , 4.4  mm atp , 4.4  mm mgcl2 , 16.7  mm kcl , 2.2  mm kcn , 40  mg / l rotenone , 0.1% bsa , and 50  mm palmitoylcoa ) at 37c for 5  min . initiate the reaction by adding 2  mm [ c]-carnitine ( 0.1  ci ) and queued it 10  min later with 50  l 1.2  mm ice cold hcl .
[ c]-palmitoyl carnitine was extracted with water saturated butanol and measured via liquid scintillation counting .
as previously described [ 24 , 25 ] , fao was examined with radiolabeled palmitate fao assay .
skeletal muscle tissues were homogenized , and mitochondria were isolated and quantified ( 100  g proteins ) .
palmitate oxidation assay was performed with reaction buffer ( 75.5  mm sucrose , 12.5  mm k2hpo4 , 100  mm kcl , 1.75  mm mgcl2 - 6h2o , 1.75  mm l - carnitine , 0.125  mm l(- ) malic acid , 1.75  mm dtt , 0.07  mm nad+ , 2  mm atp , 10 mm tris - hcl , and 0.07  mm coenzyme a ) .
the reaction started when 200m [ c]-palmitate-15% bsa ( 1  :  6 ) complex ( 0.04  ci / reaction mixture ) was added and stopped by 3.5  m perchloric acid after incubation at 37c for 30  min .
co2-trapping medium ( naoh , 0.1  m ) for c radioactivity was measured by liquid scintillation to calculate palmitate oxidation rate .
after incubation , co2 and c - asps were measured ( ls 6500 ; beckman coulter ) .
calculations were then performed accordingly .   as previously reported [ 2628 ] , tags , dags , and ceramides were tested with kc - esi - ms and gas chromatography ( applied biosystems inc . ) .
after the solution was evaporated dry and reconstituted , the samples were analyzed with mass spectrometry ( ms ) .
the analysis was performed in positive ion mode electrospray ion ( esi - ms ) source and precursor ion scans m / z 264 and 282 ( ceramides ) .
ceramides were quantified by taking the ratios of the integrated intensity for each subspecies to the intensity of c17:0 .   as described [ 2931 ] ,
an intraperitoneal glucose tolerance test ( igtt ) and insulin tolerance test ( itt ) were performed on nonanaesthetized mice after 8 hours ' fasting .
blood glucose was measured with lancet glucometer ( johnson and johnson ) . for igtt , 20% glucose ( 2.0 
blood samples were collected from tail vein at 30 , 60 , 90 , and 120  min for glucose levels . for itt
, glucose blood levels were sampled at 5 , 10 , 15 , 20 , 25 , and 30  min following intraperitoneal injection of human insulin ( 0.75  u 4.5  nmol / kg ; novolin r , novo nordisk ) .   as described previously [ 32 , 33 ]
, skeletal muscles were homogenized in ice - cold buffer ( 250  mm sucrose , 10  mm tris - hcl , 2  mm edta , and 1  mm atp ( ph 7.4 ) ) . for glucose oxidation ,
fresh skeletal muscle tissues were homogenized in ice - cold buffer ( 5  mm kcl , 2  mm tris - hcl , 0.5  mm tris base , 0.25  mm mgcl2 - 6h2o , 0.05  mm edta , and 0.05  mm atp ( ph 7.4 ) ) , 400  l homogenate was used and the reaction started when 200  m [ c]-glucose ( 0.1  ci / reaction mixture ) was added .
co2-trapping medium ( naoh , 0.1  m ) for c radioactivity was measured by liquid scintillation to calculate glucose oxidation and quantified by weight of the skeletal muscle tissue homogenate .
student 's t - test was used to evaluate the statistical significance of differences between knockout and controls .
as shown in figure 1(a ) , the mrna expression of cpt1b decreased specifically in skeletal muscles ( rt - pcr ) but not in the heart muscle in cpt1b m/ mice .
cpt1 activity is barely detectable ( dtnb assay at 412  nm ) in mitochondria ( figure 1(b ) ) for the knockout mice , even up to 10 minutes .
compared to the mice in cpt1b group , those mice in cpt1b m/ group had similar daily food intake . however , as shown in table 1 , their body weights were lower , starting about 12 weeks of age , along with lower fat mass ( p < 0.05 , resp . ) .
their lipids levels were higher ( p < 0.05 ) , but their glucose and insulin levels were similar .
as shown in figure 2(a ) , fao was decreased in isolated mitochondria ( radiolabeled palmitate ) in skeletal muscles of cpt1b m/ mice .
this was accompanied by elevation of ceramides , tags , and dags ( figures 2(b ) and 2(c ) ) .
interestingly , for the ceramides , c16 , c18 , and c18:1 had significant increases for cpt1b m/ , but not for c24 or c24:1 .
both dags and tags are dramatically elevated in cpt1b m/ mice .   as shown in figure 3(a ) , cpt1b m/ mice maintain insulin sensitivity ( insulin tolerance test ) . compared with wild type , glucose tolerance test of cpt1b m/ mice had improved significantly ( as shown in figure 3(b ) ) .
pyruvate oxidation went up ( figure 3(c ) ) and so did insulin - stimulated pakt and glut4 ( figure 4 ) .
the relationship of fatty acid oxidation , lipids accumulation , and insulin sensitivity in skeletal muscles has been quite interesting .
imbalanced fatty acid uptake and fatty acid oxidation ( fao ) have been linked to insulin resistance in muscles , which in turn worsens and complicates obesity and diabetes , as well as their complications [ 7 , 34 , 35 ] .
our study demonstrated that in mice with conditional knockout cpt1b in skeletal muscles , mitochondrial fatty acid oxidation was depressed dramatically .
this was accompanied by increased accumulation of lipids in skeletal muscles , such as dags , tags , and ceramides .
regardless of these changes , insulin tolerance test , glucose tolerance test , and pyruvate oxidation all proved better insulin sensitivity for the knockout mice .
this is partially consistent with the recent results in high - fat diet - induced obese mice , when given cpt1 inhibitor oxfenicine , as far as the major findings are concerned .
another study done with different cpt1 inhibitor , etomoxir , suggested that lipids intermediates increased .
70% of total insulin - induced glucose disposal occurs in skeletal muscles , this may indicate that increased fatty acid intermediates in skeletal muscles could damage insulin sensitivity .
our study showed that regardless of the characteristics of lipids accumulation , the mice are still insulin sensitive .
first , due to the knockout of cpt1b in skeletal muscles , the major fuel sources were in deficit .
this may suggest that signaling pathways related to energy were impaired or injured such as ampk or mtor .
second , abnormal mitochondrial biogenesis and mitochondrial dysfunction may contribute to insulin resistance and diabetes [ 39 , 40 ] .
we explored from these aspects the enzymes involved in mitochondrial activity such as -had and citrate synthase , genes related to transcriptions such as nrf ( nuclear respiratory factor ) , and the major nuclear receptor activator pgc1 .
thirdly , considering the correlation of adaptation of peroxisomal and amino acid to mitochondria , we further explored these changes .
we will report these results in our next paper and further discuss the mechanism involved . in summary ,
it suggested that with specific knockout of cpt1b in skeletal muscles , although there were lipids accumulations in skeletal muscles , the insulin sensitivity still remains .
 
 objective . by specific knockout of carnitine palmitoyl transferase 1b ( cpt1b ) in skeletal muscles 
 , we explored the effect of cpt1b deficiency on lipids and insulin sensitivity . 
 methods . 
 mice with specific knockout of cpt1b in skeletal muscles ( cpt1b m/ ) were used for the experiment group , with littermate c57bl/6 as controls ( cpt1b ) . 
 general and metabolic profiles were measured and compared between groups . 
 mrna expression and cpt1 activity were measured in skeletal muscle tissues and compared between groups . 
 mitochondrial fatty acid oxidation ( fao ) , triglycerides ( tags ) , diglycerides ( dags ) , and ceramides were examined in skeletal muscles in two groups . 
 phosphorylated akt ( pakt ) and glucose transporter 4 ( glut4 ) were determined with real - time polymerase chain reaction ( rt - pcr ) . 
 insulin tolerance test , glucose tolerance test , and pyruvate oxidation were performed in both groups . 
 results . 
 cpt1b m/ model was successfully established , with impaired muscle cpt1 activity . 
 compared with cpt1b mice , cpt1b m/ mice had similar food intake but lower body weight or fat mass and higher lipids but similar glucose or insulin levels . 
 their mitochondrial fao of skeletal muscles was impaired . 
 there were lipids accumulations ( tags , dags , and ceramides ) in skeletal muscle . 
 however , pakt and glut4 , insulin sensitivity , glucose tolerance , and pyruvate oxidation were preserved . 
 conclusion . 
 skeletal muscle - specific cpt1 deficiency elevates lipotoxic intermediates but preserves insulin sensitivity . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion