the amp - activated protein kinase ( ampk ) is emerging as a chief regulator of whole - body energy balance ( bolster et  al . , 2002 ; holmes et  al .
, 1999 ; kahn et  al . , 2005 ; kimura et  al . , 2003 ;
, 2002 ; winder  et  al . , 1997 , 2000 ) . in skeletal muscle ,
once ampk becomes activated , it exerts control in part by regulating fatty - acid oxidation through the phosphorylation of acetyl - coa carboxylase 2 ( acc2 ) and mitochondrial biogenesis through increasing the expression of proteins vital for proper mitochondrial function such as citrate synthase and succinate dehydrogenase ( bergeron et  al . , 2001 ;
merrill et  al . , 1997 ; winder et  al . , 1997 , 2000 ; zong et  al . ,
reductions in mitochondrial oxidative - phosphorylation activity , which contribute to dysregulated intracellular lipid metabolism , have recently been implicated in the increased incidence of insulin resistance and type 2 diabetes that occurs with aging ( petersen et  al . ,
given the critical role that ampk plays in the regulation of mitochondrial biogenesis and fatty - acid oxidation , we examined the effect of aging on the ampk signaling pathway and mitochondrial biogenesis during acute pharmacological stimulation , exercise , and chronic pharmacological stimulation .
5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ( aicar ) is an acute activator of ampk , which is taken up into cells and converted into 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside monophosphate ( zmp ) , which mimics the effects of amp on ampk ( corton et  al . , 1995 ) .
when bound to ampk , amp or zmp induces a conformational change that makes ampk a better substrate for activation by the upstream kinase lkb1 ( hawley et  al . , 2003 ) .
in vivo aicar infusions in 3-month - old (  young  ) and 28-month - old (  old  ) fisher 344 male rats increased ampk-2 activity by 44% in the extensor digitalis longus ( edl ) muscles of the young aicar - treated rats versus young saline - treated ( control ) rats ( figure  1a ) .
in contrast , aicar infusions did not increase ampk-2 activity in the old rats , demonstrating reduced ampk activation with aging .
furthermore , ampk phosphorylation of one of its key substrates in skeletal muscle , acc2 , at serine 79 ( ser79 ) was increased by 120% in the young aicar - treated rats , whereas there was no difference in the old aicar - treated rats ( figure  1b ) .
ampk activity is regulated by the phosphorylation of threonine 172 ( thr172 ) on the catalytic  subunit of the enzyme ( hawley et  al . , 1996 ) ,
and studies in tissue - specific mouse knockouts suggest that lkb1 is the major upstream kinase in skeletal muscle , at least for 2 ( sakamoto et  al . , 2005 )
. the reduced aicar - stimulated ampk-2 activity could be attributed to reduced phosphorylation since thr172 phosphorylation increased by 162% in the young aicar - treated rats and was unaltered in the old aicar - treated rats ( figure  1c ) .
these aging - associated differences in ampk-2 activity and thr172 phosphorylation between aicar - treated and saline - treated rats were independent of any changes in ampk-1 activity or in the expression of the ampk-1 and 2 subunits ( see figure  s1 in the supplemental data available with this article online ) .
they also were not associated with changes in the expression ( figure  1d ) or activity of lkb1 ( figure  1e ) or in the phosphorylation of ser491 on ampk-2 ( data not shown ) , which has been reported to antagonize phosphorylation at  thr172 ( horman et  al . , 2006 ) .
2003 ) , which have recently been identified as splice variants ( s.a.h . and d.g.h .
although the two forms were expressed in almost equal amounts in rat skeletal muscle by western blotting , their expression was not different in the old rats .
exercise or contraction rapidly depletes atp , which leads to the increase in the amp : atp ratio that is responsible for causing ampk activation ( sakamoto et  al . , 2005 ;
groups of young and old fisher 344 male rats that had been exercise trained for 5 days completed a 5-day treadmill exercise regimen that resulted in a 110% increase in ampk-2 activity in the young rats .
in contrast , the old rats displayed severely blunted ampk-2 activity in response to exercise ( figure  2a ) .
additionally , phosphorylation of ser79 on acc2 was blunted in the old rats despite a 127% increase in the young rats ( figure  2b ) .
lastly , thr172 phosphorylation increased 55% in the young exercising rats and did not change in  the old exercising rats compared to their controls ( figure  2c ) .
these aging - associated differences in ampk-2 activity and thr172 phosphorylation between exercising rats and sedentary rats were independent of any changes in ampk-1 activity or in the expression of the ampk-1 and 2 subunits ( figure  s1 ) .
recent studies have demonstrated that ampk serves an important role in regulating mitochondrial biogenesis during chronic energy deprivation and is associated with increased mrna expression of peroxisome proliferator - activated receptor  coactivator 1 ( pgc-1 ) , the transcriptional coactivator responsible for mitochondrial biogenesis in skeletal muscle . to investigate how aging impacts ampk - stimulated mitochondrial biogenesis , we chronically fed young and old rats a diet with 1% -guanidinopropionic acid ( -gpa ) for 8 weeks .
since -gpa has a lower km for creatine kinase than creatine , its presence results in lower intramyocellular concentrations of atp and phosphocreatine and thus chronically stimulates ampk activity .
accordingly , ampk-2 activity ( figure  3a ) increased 146% in the edl muscles of the young rats fed the -gpa diet .
a similar increase was detected in ampk-2 activity in the soleus tissue , an oxidative fiber , demonstrating the broad effect of -gpa on ampk-2 activity in young rats .
pgc-1 mrna expression ( figure  3b ) also increased , by 86% , in the edl muscles of the young rats fed the -gpa diet .
these increases in the young rats were associated with a 38% increase in mitochondrial density as assessed by electron microscopy ( figure  3c ) , a 289% increase in -ala synthase ( -alas ) mrna expression ( figure 
3d ) , and a 76% increase in cytochrome c protein expression ( figure  3e ) . in contrast , we observed no increases in ampk-2 activity , pgc-1 mrna expression , mitochondrial density , -alas mrna expression , or cytochrome c protein expression in the edl muscles of the old rats .
these results suggest that aging - associated reductions in ampk - stimulated activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging - induced insulin resistance and type 2 diabetes .
we studied fisher 344 male rats aged 3 months ( young ) and 28 months ( old ) ( national institute on aging ) .
the rats were maintained on standard rat chow ( ralston purina ) and housed in an environmentally controlled room with a 12/12 hr light / dark cycle .
rats were chronically catheterized via the right jugular vein and allowed to recover ( 58 days ) until they regained their preoperative weight .
weight - matched young and old animals were infused with isotonic saline ( control ) or the ampk activator 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ( aicar ) ( bolus , 100 mg / kg ; constant , 10 mg / kg / min ; toronto research chemicals inc . ) for 60 min . during the aicar experiments ,
plasma glucose concentrations were maintained constant at basal concentrations ( 100 mg / dl ) using a variable infusion of 20% ( w / v ) dextrose solution in order to prevent hypoglycemia .
blood was sampled at  0 min and every 15 min for glucose measurements . at the end of the infusions ,
rats were anesthetized with intravenous pentobarbital ( 50 mg / kg ) , and the skeletal muscle was rapidly excised and freeze clamped in liquid nitrogen .
we studied fisher 344 male rats aged 3 months ( young ) and 28 months ( old ) .
the rats were maintained on standard rat chow ( ralston purina ) and housed in an environmentally controlled room with a 12/12 hr light / dark cycle .
the rats were introduced to the treadmill ( columbus instruments ) with a 10 min run at 10 m / min once per day for 4 days .
a performance test to determine the maximum running capacity for each rat was performed on the fifth day by running the rats for 10 min at 10 m / min and then increasing the speed 1 m / min every minute until exhaustion .
the young rats ' maximum capacity was determined to be 38  m / min , and the old rats ' maximum capacity was 20 m / min .
the speed at which all of the rats reached exhaustion was noted , and the rats were run at 85% of that speed for 5 more days after a 2 day break for 30 min each day .
thus , the young rats were run at 32 m / min , and the old rats were run at 17 m / min . on the fifth day , immediately after exercise , the rats were anesthetized with isoflurane , and the skeletal muscle was rapidly excised and freeze clamped in liquid nitrogen . throughout this experiment , control rats remained sedentary in their cages .
we studied fisher 344 male rats aged 3 months ( young ) and 28 months ( old ) .
the rats were maintained on standard rat chow ( ralston purina ) with or without 1% -gpa and were housed in an environmentally controlled room with a 12/12 hr light / dark cycle .
body weight and food consumption were measured every 23 days . on the eighth week of feeding , the rats were chronically catheterized via the right jugular vein and allowed to recover ( 58 days ) until they regained their preoperative weight .
the rats were then anesthetized with intravenous pentobarbital ( 50 mg / kg ) , and the skeletal muscle was rapidly excised and freeze clamped in liquid nitrogen for analytical procedures . other skeletal muscle was prepared separately for mitochondrial quantification using electron microscopy .
individual skeletal muscle samples were prepared by immersion in 2.5% glutaraldehyde ( electron microscopy sciences ) in 0.1 m cacodylate buffer ( ph 7.4 ) at 4c overnight .
skeletal muscles were then washed three times in 0.1 m cacodylate buffer ( ph 7.4 ) and postfixed for 1 hr in 1% osmium tetroxide ( embed-812 epoxy resin ) in the same buffer at room temperature .
after three washes in water , the samples were stained for 1 hr at room temperature in 2% uranyl acetate , washed again in water , and dehydrated in a graded series of ethanol dilutions ( 50%100% ) .
ultrathin ( 60 nm ) sections were cut using a reichert ultracut ultramicrotome , collected on formvar- and carbon - coated grids , stained with 2% uranyl acetate and lead citrate , and examined in a philips 410 electron microscope . only cross - sections of skeletal muscle
were examined for quantification of mitochondrial density . for each individual rat and skeletal muscle ,
six random pictures were taken at a magnification of 7,100 and printed at a final magnification of 18,250. the volume density of mitochondria was estimated using the point - counting method .
the average volume density was calculated for each individual rat and skeletal muscle and was used to calculate the average volume density for each treatment . at the end of the experiments , the rats were anesthetized as described above .
the edl muscles from the left and right legs were quickly freeze clamped in situ .
edl was ground with a mortar and pestle and mixed with 1 ml of lysis buffer ( 50 mm tris - hcl buffer [ ph 7.5 at 4c ] , 50 mm naf , 5 mm nappi , 1 mm edta , 1 mm egta , 1 mm dithiothreitol [ dtt ] , 1 mm benzamidine , 1 mm phenylmethanesulfonyl fluoride [ pmsf ] , glycerol [ 10% v / v ] , triton x-100 [ 1% v / v ] ) .
homogenates were spun at 20,800  g for 10 min at 4c , and protein concentrations were determined .
ampk-2 were immunoprecipitated overnight from cell lysates containing 1 mg of protein using 1 l of either ampk-1 antibody or ampk-2 antibody ( santa cruz biotechnology ) .
skeletal muscle ampk-1 and ampk-2 activity were determined by following the incorporation of [ p]atp into a synthetic peptide containing the amara sequence on the following day .
edl muscle samples were frozen in liquid nitrogen and later ground with a mortar and pestle . for each sample ,
homogenates were spun at 12,900 rpm for 10 min at 4c , and protein concentration was estimated .
lkb1 was immunoprecipitated from lysates containing 3  mg of protein using the n - terminal antipeptide antibody ( boudeau et  al . , 2003 ) .
lkb1 assays ( 1 mg of lysate per assay ) were conducted using purified gst - ampk-1 catalytic subunit as substrate in shaking incubators as described previously for immunoprecipitate kinase assays of ampk
total rna was isolated from the edl muscle using the rneasy kit ( qiagen ) .
cdna was prepared from 2 g of rna using the stratascript rt - pcr kit ( strategene ) with random hexamer primers according to the manufacturer 's instructions .
the resulting cdna was diluted , and a 4 ng aliquot was used in a 40 l pcr reaction using sybr green and selected primers .
the primers for cytochrome c were 5-tcttgacttcctgaccttgggctt-3 ( forward ) and 5-actcccgcttcttgtagctttcca-3 ( reverse ) .
pcr reactions were run in duplicate and quantitated with an abi prism 7700 sequence detection system ( applied biosystems ) .
for all western blots except for lkb1 expression , 30 g of cell lysate was diluted in lds laemmli sample buffer before sds - page .
sds gel electrophoresis was performed using precast bis - tris 4%12% gradient polyacrylamide gels in the mops buffer system ( invitrogen ) .
after transfer to nitrocellulose membranes , membranes were incubated in blocking buffer ( 5% milk ) for 1 hr and immunoblotted with anti - p - ampk ( thr172 ) antibody ( cell signaling ) , anti - p - acc ( ser79 ) antibody ( cell signaling ) , anti - ampk-1 antibody ( cell signaling ) , anti - ampk-2 antibody ( cell signaling ) , or cytochrome c antibody ( santa cruz biotechnology ) at 1:1000 dilution in 5% bovine serum albumin .
the membranes were washed three times for 15 min with tbs ( 10 mm tris - hcl [ ph 7.4 ] , 0.5 m nacl ) plus tween 20 ( 0.2% v / v ) ( tbst ) .
the membranes were immersed in blocking buffer and a corresponding igg - conjugated secondary antibody and were shaken for 1  hr .
proteins were then detected with enhanced chemiluminescence , and autoradiographs were quantified using densitometry . for western blotting of lkb1 , 15 g of tissue extract was diluted in lds sample buffer ( invitrogen ) .
sds gel electrophoresis was performed using precast bis - tris 4%12% gradient polyacrylamide gels in the mops buffer system ( invitrogen ) .
proteins were transferred to nitrocellulose membranes ( bio - rad ) using the xcell ii blot module ( invitrogen ) .
membranes were incubated in odyssey blocking buffer ( li - cor ) for 1  hr .
the membrane was subsequently incubated with 1:1000 anti - lkb1 n - terminal antibody ( raised against residues 2436 ) , which had been diluted in odyssey blocking buffer containing 0.2% tween 20 .
the membranes were immersed in blocking buffer containing tween 20 ( 0.2% v / v ) and 1 g / ml anti - sheep igg conjugated to ir dye 680 ( molecular probes ) and shaken for 1 hr , protected from light .
the membranes were then washed six times for 5 min using tbst and once for 5 min in pbs .
the membranes were scanned in the 700 nm channel using the odyssey ir imager , and lkb1 protein levels were quantified using odyssey ir imaging system software .
 summaryrecent studies have demonstrated a strong relationship between aging - associated reductions in mitochondrial function , dysregulated intracellular lipid metabolism , and insulin resistance . 
 given the important role of the amp - activated protein kinase ( ampk ) in the regulation of fat oxidation and mitochondrial biogenesis , we examined ampk activity in young and old  rats and found that acute stimulation of ampk-2 activity by 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ( aicar ) and exercise was blunted in skeletal muscle of old rats . furthermore , mitochondrial biogenesis in response to chronic activation of ampk with -guanidinopropionic acid ( -gpa ) feeding was also diminished in old rats . 
 these results suggest that aging - associated reductions in ampk activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging . 
Introduction
Results and Discussion
Experimental Procedures