alzheimer disease ( ad ) is clinically characterized by progressive memory loss and a decline of cognitive functions .
increasing evidence has indicated that global and regional disruptions in brain energy metabolism are critically involved in the pathogenesis of ad , including mitochondrial dysfunctions , decline in glucose uptake , and defects in cholesterol metabolism .
improve global energy metabolism may be effective in preventing cognitive impairment   associated with brain aging and ad .
sirtuin 1 ( sirt1 ) protein is classified as a class iii nad - dependent histone deacetylase , which plays an important role in metabolic function and longevity in mammals .
a sirt1 target is peroxisome proliferator - activated receptor- co - activator-1 ( pgc-1 ) .
pgc-1 has been shown to be a master regulator of mitochondrial biogenesis and energy metabolism [ 15,2022 ] .
a recent study showed that pgc-1 and atp content were decreased in ad hippocampal tissues and appswe m17 cells .
in addition , pgc-1 overexpression could rescue , whereas knockdown of pgc-1 exacerbated , impaired mitochondrial biogenesis and mitochondrial deficits in appswe m17 cells .
the mrna and protein expression levels of sirt1 and pgc-1 in the brains of ad patients were decreased compared with age - matched controls [ 2325 ] .
taken together , increased expression of sirt1 and pgc-1 would exert beneficial effects in ad .
electroacupuncture ( ea ) or acupuncture , a traditional chinese medicine treatment that stimulates specific acupoints , has been shown to improve cognitive deficits in animal models of ad [ 2629 ] .
acupuncture increased blood perfusion and glycol metabolism in ad model rats , which indicated that acupuncture might improve energy metabolism .
our previous study showed that ea increased the activation of amp - activated protein kinase ( ampk ) , a master regulator of cellular energy homeostasis .
activation of sirt1 and pgc-1 improved mitochondrial function . in addition , ea improved insulin sensitivity in obese diabetic mice via activation of sirt1/pgc-1 . however , little is known regarding the effect of ea on the expression sirt1 and pgc-1 in ad . in the present study , we investigated the effect of ea on brain energy metabolism in senescence - accelerated mouse prone 8 ( samp8 ) mice .
furthermore , we examined the effect of ea on sirt1 and pgc-1 expression in samp8 mice .
male samp8 and senescence - accelerated resistant mice 1 ( samr1 ) mice ( 7 months old ) were purchased from the department of laboratory animal science of peking university .
all mice were housed under standard conditions at 222c and a 12 h light / dark cycle with free access to food and water .
all procedures followed the  national institute of health guide for the care and use of laboratory animals  ( nih publications
80 - 23 ) and were approved by the institutional animal care and utilization committee of fujian university of traditional chinese medicine .
male samp8 and male homologous samr1 mice were randomly assigned to the following 3 groups : samr1 normal control group ( rc ) , samp8 control group ( pc ) , and samp8 electroacupuncture group ( pe ) .
briefly , we used nets to fix the mice by an assistant s hands during the entire treatment .
three stainless steel acupuncture needles were inserted at a depth of 5 mm into the  dazhui 
we performed continuous - wave stimulation at a frequency of 2 hz ( intensity 1 ma ) .
an individual ea session was administered daily for 20 min , 8 days , and 2 days of rest , for a period of 30 days .
biochemical and molecular studies were performed in the mouse hippocampus and frontal cortex excised under ether anesthesia after intra - cardiac perfusion with ice - cold normal saline .
we focused on the hippocampus , which is one of the brain areas that is earliest and most severely affected by ad .
atp levels were measured in freshly prepared hippocampal and cortical homogenates samples . due to the high activities of atpase in fresh samples ,
all steps for sample preparation were performed at 4c to avoid the recovery of atpase activity and subsequent degradation of atp in the tissue .
assays were performed using a spectrophotometric plate reader ( paradigm , beckman coulter , usa ) .
tissue atp levels were determined using a luciferin / luciferase atp bioluminescent assay kit ( sigma , st louis , mo , usa ) .
protein samples were isolated from the hippocampus in ice - cold ripa lysis buffer supplemented with protease inhibitors .
after incubation for 40 min at 4c , homogenates were centrifuged at 14 000 rpm for 15 min at 4c and the supernatant were collected and stored in aliquots at 80c until further use .
equivalent amounts ( 30 g ) of each sample , calculated using a bca protein assay kit ( beyotime , haimen , jiangsu , china ) , were separated on 10% sds - polyacrylamide gels .
after electrophoresis , the proteins were transferred onto a nitrocellulose membrane ( millipore ) at 100 v for 60 min on ice .
the membrane was blocked with 5% w / v nonfat dry milk powder in tris - buffered saline with 0.05% tween 20 ( tbs - t ) for 1 h. the membrane was incubated with primary antibody overnight at 4c , followed by secondary antibody for 1 h at room temperature .
the following primary antibodies were used : rabbit anti - sirt1 ( 1:1000 , santa cruz biotechnology , santa cruz , ca , usa ) , rabbit anti- pgc-1 ( 1:1000 , santa cruz biotechnology ) , and anti--actin ( 1:5000 , sigma , st louis , mo , usa ) .
the secondary antibody was hrp - conjugated goat anti - rabbit igg ( 1:1000 , santa cruz biotechnology ) .
the membrane was rinsed with tbs - t and the immunocomplex was visualized using an enhanced chemiluminescence detection kit ( thermo scientific , rockford , il , usa ) .
the signals of the membrane were scanned using the fluorchem scanner and quantified with the nih image j software .
these results were normalized with -actin expression levels and confirmed by triplicate measurements of the same sample .
pgc-1 acetylation was analyzed by immunoprecipitation of pgc-1 followed by western blotting using an acetyl - lysine antibody according to the immunoprecipitation protocol provided by abcam .
the protein samples were pre - cleared with 20 l of protein g ( sigma - aldrich , usa ) for 3 h and then centrifuged to obtain supernatant at 15 000 g for 10 min .
pgc-1 antibody ( 1:1000 , millipore , usa ) and 30 l of protein g were added to the pre - cleared supernatant then incubated for 12 h at 4c .
total rna was extracted from frozen hippocampus using trizol ( invitrogen , ca , usa ) , according to the manufacturer s instructions .
the rna concentrations were determined using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , wilmington , de , usa ) .
next , 0.5 g of rna was processed for cdna synthesis using a high - capacity cdna reverse transcription kit ( invitrogen , ca , usa ) .
the primers used for qrt - pcr were the following : sirt1 forward : 5-tgtgaagttactgcagga gtgtaaa-3 , reverse : 5-gcatagataccgtctcttgatctgaa-3 pgc-1 forward : 5-aagtgtggaactctctggaactg-3 , reverse : 5-gggttatcttgg ttggctttatg-3 ; glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) forward : 5-tggaaagctgtggcgtgat-3 , reverse : 5-tgcttcaccaccttcttgat-3. next , qrt - pcr was performed on a 7300 real - time pcr system ( applied biosystems , ca , usa ) using sybr green pcr master mix ( applied biosystems ) , according to the manufacturer s instructions .
delta ct ( ct ) method and were normalized to gapdh expression in each sample .
the escape latency of mice in the mwm test was analyzed using 2-way analysis of variance ( anova ) for repeated measurement .
tukey s test was further used as a post hoc test to detect between - group differences .
one - way anova was employed to analyze other data obtained in these experiments followed by lsd ( equal variances assumed ) or dunnett s t3 ( equal variances not assumed ) for a post hoc test between groups .
male samp8 and senescence - accelerated resistant mice 1 ( samr1 ) mice ( 7 months old ) were purchased from the department of laboratory animal science of peking university .
all mice were housed under standard conditions at 222c and a 12 h light / dark cycle with free access to food and water .
all procedures followed the  national institute of health guide for the care and use of laboratory animals  ( nih publications
80 - 23 ) and were approved by the institutional animal care and utilization committee of fujian university of traditional chinese medicine .
male samp8 and male homologous samr1 mice were randomly assigned to the following 3 groups : samr1 normal control group ( rc ) , samp8 control group ( pc ) , and samp8 electroacupuncture group ( pe ) .
briefly , we used nets to fix the mice by an assistant s hands during the entire treatment .
three stainless steel acupuncture needles were inserted at a depth of 5 mm into the  dazhui 
we performed continuous - wave stimulation at a frequency of 2 hz ( intensity 1 ma ) .
an individual ea session was administered daily for 20 min , 8 days , and 2 days of rest , for a period of 30 days .
biochemical and molecular studies were performed in the mouse hippocampus and frontal cortex excised under ether anesthesia after intra - cardiac perfusion with ice - cold normal saline .
we focused on the hippocampus , which is one of the brain areas that is earliest and most severely affected by ad .
atp levels were measured in freshly prepared hippocampal and cortical homogenates samples . due to the high activities of atpase in fresh samples ,
were performed at 4c to avoid the recovery of atpase activity and subsequent degradation of atp in the tissue .
assays were performed using a spectrophotometric plate reader ( paradigm , beckman coulter , usa ) .
tissue atp levels were determined using a luciferin / luciferase atp bioluminescent assay kit ( sigma , st louis , mo , usa ) .
protein samples were isolated from the hippocampus in ice - cold ripa lysis buffer supplemented with protease inhibitors .
after incubation for 40 min at 4c , homogenates were centrifuged at 14 000 rpm for 15 min at 4c and the supernatant were collected and stored in aliquots at 80c until further use .
equivalent amounts ( 30 g ) of each sample , calculated using a bca protein assay kit ( beyotime , haimen , jiangsu , china ) , were separated on 10% sds - polyacrylamide gels .
after electrophoresis , the proteins were transferred onto a nitrocellulose membrane ( millipore ) at 100 v for 60 min on ice .
the membrane was blocked with 5% w / v nonfat dry milk powder in tris - buffered saline with 0.05% tween 20 ( tbs - t ) for 1 h. the membrane was incubated with primary antibody overnight at 4c , followed by secondary antibody for 1 h at room temperature .
the following primary antibodies were used : rabbit anti - sirt1 ( 1:1000 , santa cruz biotechnology , santa cruz , ca , usa ) , rabbit anti- pgc-1 ( 1:1000 , santa cruz biotechnology ) , and anti--actin ( 1:5000 , sigma , st louis , mo , usa ) .
the secondary antibody was hrp - conjugated goat anti - rabbit igg ( 1:1000 , santa cruz biotechnology ) .
the membrane was rinsed with tbs - t and the immunocomplex was visualized using an enhanced chemiluminescence detection kit ( thermo scientific , rockford , il , usa ) .
the signals of the membrane were scanned using the fluorchem scanner and quantified with the nih image j software .
these results were normalized with -actin expression levels and confirmed by triplicate measurements of the same sample .
pgc-1 acetylation was analyzed by immunoprecipitation of pgc-1 followed by western blotting using an acetyl - lysine antibody according to the immunoprecipitation protocol provided by abcam .
the protein samples were pre - cleared with 20 l of protein g ( sigma - aldrich , usa ) for 3 h and then centrifuged to obtain supernatant at 15 000 g for 10 min .
pgc-1 antibody ( 1:1000 , millipore , usa ) and 30 l of protein g were added to the pre - cleared supernatant then incubated for 12 h at 4c .
total rna was extracted from frozen hippocampus using trizol ( invitrogen , ca , usa ) , according to the manufacturer s instructions .
the rna concentrations were determined using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , wilmington , de , usa ) .
next , 0.5 g of rna was processed for cdna synthesis using a high - capacity cdna reverse transcription kit ( invitrogen , ca , usa ) .
the primers used for qrt - pcr were the following : sirt1 forward : 5-tgtgaagttactgcagga gtgtaaa-3 , reverse : 5-gcatagataccgtctcttgatctgaa-3 pgc-1 forward : 5-aagtgtggaactctctggaactg-3 , reverse : 5-gggttatcttgg ttggctttatg-3 ; glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) forward : 5-tggaaagctgtggcgtgat-3 , reverse : 5-tgcttcaccaccttcttgat-3. next , qrt - pcr was performed on a 7300 real - time pcr system ( applied biosystems , ca , usa ) using sybr green pcr master mix ( applied biosystems ) , according to the manufacturer s instructions .
delta ct ( ct ) method and were normalized to gapdh expression in each sample .
the escape latency of mice in the mwm test was analyzed using 2-way analysis of variance ( anova ) for repeated measurement .
tukey s test was further used as a post hoc test to detect between - group differences .
one - way anova was employed to analyze other data obtained in these experiments followed by lsd ( equal variances assumed ) or dunnett s t3 ( equal variances not assumed ) for a post hoc test between groups .
the cellular atp levels reflect the state of mitochondria function and brain energy metabolism , thus we determined the atp levels in the hippocampus and frontal cortical tissue to confirm brain energy metabolism .
these results showed that the pc mice showed a significant decrease in atp levels in the hippocampus and frontal cortex compared with the rc mice , indicating impaired brain energy metabolism .
however , ea significantly reversed the aberrant decrease in atp levels in the hippocampus and frontal cortex ( figure 1 ) , indicating an improvement in brain energy metabolism . to further explore the underlying neuroprotective mechanisms of ea treatment on ad , the protein levels of sirt1 and pgc-1 were investigated using western blotting analyses .
we showed that the expression levels of sirt1 and pgc-1 were significantly reduced in the pc group when compared with the rc group ( p<0.05 ; figure 2 ) .
we also found that the expression levels of both sirt1 and pgc-1 were significantly increased in the pe group compared with the pc group ( p<0.05 ; figure 2 ) . in accordance with increased protein levels of sirt1 in the pe group , there was a decrease in the acetylation state of pgc-1 in the pe group ( p<0.05 ; figure 3 ) , suggesting ea increases pgc-1 activity .
we further analyzed the gene expression of both sirt1 and pgc-1 with qrt - pcr .
consistent with the results obtained from western blotting analyses , we also found that the expression of sirt1 and pgc-1 at the mrna level were down - regulated in the pc group compared with the rc group ( p<0.05 ; figure 4 ) .
however , the expression of sirt1 and pgc-1 at mrna level was significantly increased in the pe group compared with the pc group ( p < 0.05 ; figure 4 ) .
the cellular atp levels reflect the state of mitochondria function and brain energy metabolism , thus we determined the atp levels in the hippocampus and frontal cortical tissue to confirm brain energy metabolism .
these results showed that the pc mice showed a significant decrease in atp levels in the hippocampus and frontal cortex compared with the rc mice , indicating impaired brain energy metabolism .
however , ea significantly reversed the aberrant decrease in atp levels in the hippocampus and frontal cortex ( figure 1 ) , indicating an improvement in brain energy metabolism .
to further explore the underlying neuroprotective mechanisms of ea treatment on ad , the protein levels of sirt1 and pgc-1 were investigated using western blotting analyses .
we showed that the expression levels of sirt1 and pgc-1 were significantly reduced in the pc group when compared with the rc group ( p<0.05 ; figure 2 ) .
we also found that the expression levels of both sirt1 and pgc-1 were significantly increased in the pe group compared with the pc group ( p<0.05 ; figure 2 ) . in accordance with increased protein levels of sirt1 in the pe group
, there was a decrease in the acetylation state of pgc-1 in the pe group ( p<0.05 ; figure 3 ) , suggesting ea increases pgc-1 activity .
we further analyzed the gene expression of both sirt1 and pgc-1 with qrt - pcr .
consistent with the results obtained from western blotting analyses , we also found that the expression of sirt1 and pgc-1 at the mrna level were down - regulated in the pc group compared with the rc group ( p<0.05 ; figure 4 ) .
however , the expression of sirt1 and pgc-1 at mrna level was significantly increased in the pe group compared with the pc group ( p < 0.05 ; figure 4 ) .
in this study , we showed that ea treatment improved brain energy metabolism in samp8 mice .
furthermore , ea treatment increased the protein and mrna expression of sirt1 and pgc-1 , and decreased pgc-1 acetylation in samp8 mice .
these data suggest that the effects of ea treatment on brain energy metabolism in ad might be through the sirt1-dependent pgc-1 pathway .
impaired brain metabolism and mitochondrial dysfunction play an important role in the early pathology of ad .
laminin increases the expression of mitochondrial proteins ( cytochrome b and chargerin ii ) during neurite outgrowth .
atp levels in the hippocampus and frontal cortex of samp8 mice were significantly reduced compared with samr1 mice , which was consistent with previous studies .
in addition , our results showed that ea increased atp levels in the hippocampus and frontal cortex , which indicated that ea could improve impaired brain energy metabolism .
our previous study showed that ea improves cognitive deficits in samp8 mice . taken together , ea improves cognitive deficits in ad and may be associated with an improvement in brain energy metabolism , which further support the hypothesis that improvement in brain energy metabolism is effective in preventing the cognitive impairment associated with ad .
therefore , sirt1 acts as a crucial regulator of the network that controls energy homeostasis .
moreover , ad shares characteristics and possible origins with diabetes , and metabolic rundown encountered in type 2 diabetes engenders cerebral vascular insufficiencies that are causally associated with long - term neural degenerative processes in ad .
pgc-1 plays an important role as a master regulator of cellular metabolic homeostasis and has been reported to produce beneficial effects on ad .
pgc-1 has also been suggested to be a potential therapeutic target to increase mitochondrial biogenesis and improve energy metabolism in ad . to elucidate the mechanism for the increase in atp levels following ea treatment , we analyzed the mrna and protein levels of sirt1 and pgc-1 in the hippocampus of samp8 mice .
our results showed that the mrna and protein expression levels of sirt1 and pgc-1 in the hippocampus of samp8 mice were reduced compared with that of samr1 mice .
this finding is consistent with previous studies demonstrating a decrease in the mrna and protein expression levels of sirt1 and pgc-1 in postmortem brain tissue in ad patients [ 2325 ] .
additionally , deacetylation and activation of pgc-1 by enhanced sirt1 activity was involved in neuroprotection in ad .
together , these results suggest that sirt1 and pgc-1 play an important role in ad .
a decrease in the mrna and protein expression levels of sirt1 and pgc-1 may impair brain energy metabolism and may further result in cognitive deficits .
conversely , a recent study reported that pgc-1 overexpression exacerbates a and tau deposition in a transgenic mouse model of ad , which was associated with impairment in proteasome activity .
thus , a more definitive probe of the role of pgc-1 in ad will require further investigation . it has been reported that icariin and alpha - lipoic acid protect against brain injury by enhancing sirt1-dependent pgc-1 expression in ischemic stroke . activating sirt1 and pgc-1
we have previously shown that a2535 suppresses mitochondrial biogenesis and ampk - sirt1-pgc-1 pathway in cultured hippocampal neurons . in the present study
, ea increased the protein and mrna expression of sirt1 and pgc-1. increased pgc-1 expression may regulate energy metabolism by improving atp levels and the production of energy .
together , our data suggest that sirt1 and pgc-1 may be involved in ea treatment for ad .
future studies are planned to determine whether the sirt1-dependent pgc-1 pathway is a molecular target of ea in the treatment of ad .
the sirt1-dependent pgc-1 pathway may be a target for ea treatment or other therapeutic interventions in ad .
 backgroundabnormalities of brain energy metabolism are involved in alzheimer disease ( ad ) . 
 sirtuin 1 ( sirt1 ) is a class iii histone deacetylase and activates peroxisome proliferator - activated receptor- co - activator-1 ( pgc-1 ) , which enhances mitochondrial biogenesis and energy homeostasis . 
 electroacupuncture ( ea ) has been reported to improve brain energy metabolism in ad . 
 however , the effect of ea on sirt1 and pgc-1 in ad remains unclear.material/methodsatp levels were measured using assay kits in the hippocampus and frontal cortex of senescence - accelerated mouse prone 8 ( samp8 ) mice . 
 western blotting analysis and quantitative real - time rt - pcr were performed to measure the expression of sirt1 and pgc-1 in the hippocampus of samp8 mice . 
 pgc-1 acetylation was analyzed using immunoprecipitation.resultscompared with senescence - accelerated resistant mice 1 ( samr1 ) mice , samp8 mice had a decline in atp levels and the expression of sirt1 and pgc-1. ea treatment improved atp levels , upregulated the expression of sirt1 and pgc-1 , and decreased pgc-1 acetylation.conclusionsthese data suggest that ea improved brain energy metabolism , potentially associated with the upregulation of sirt1-dependent pgc-1 expression . 
Background
Material and Methods
Animals
EA treatment
Tissue collection
Assessment of ATP levels
Western blotting analysis
Immunoprecipitation
Quantitative real-time RT-PCR (qRT-PCR)
Statistical analysis
Results
EA increased ATP levels in the hippocampus and frontal cortex
EA upregulated the protein expression of SIRT1 and PGC-1 and decreased PGC-1 acetylation
EA upregulated the gene expression of SIRT1 and PGC-1
Discussion
Conclusions