four - month - old whole - body ampk2 mice ( 29 ) backcrossed to c57bl/6j mice for nine generations , and wild - type littermate controls were fed on either chow , chf , or chf+f diet for nine weeks .
body weight and food consumption were recorded , and edta - plasma and tissues were collected for various analyses as described in the online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1716/dc1 .
male mice were used for all the experiments , except for the measurements of hepatic ampk activity , which were performed on female mice .
the experiments were conducted under the guidelines for the use and care of laboratory animals of the institute of physiology and followed the  principles of laboratory animal care 
( national institutes of health publication no . 85 - 23 , revised 1985 ) .
nonesterified fatty acids ( nefas ) , triglycerides , and total cholesterol were determined in plasma using the following enzymatic photometric tests : nefa - c ( wako chemicals , neuss , germany ) , triacylglycerols liquid , and cholesterol liquid ( pliva - lachema diagnostika , brno , czech republic ) , respectively .
plasma insulin was measured using the sensitive rat insulin ria kit ( linco research , st .
total adiponectin levels and adiponectin multimeric complexes were determined using western blotting ( 31 ) .
the tissue content of triglycerides was estimated in ethanolic koh tissue lysates as described before ( 8) .
the content and fatty acid composition of the phospholipid , diacylglycerol , triglyceride , and ceramide fractions were assessed in tissue lipid extracts ; gene expression was evaluated using real - time rt - pcr ( see online appendix ) .
livers were collected by freeze - clamping , ampk was immunoprecipitated from tissue extracts , and the activity was assayed using a peptide substrate ( 32 ) ; see the online appendix .
five days before the experiment , an indwelling catheter was placed into the left femoral vein under anesthesia ( 33 ) .
mice were allowed to recover for 57 days , followed by a 6-h fast ( 8:00 a.m.2:00 p.m. ) prior to the experiment .
the whole - body glucose turnover was determined under basal ( nonstimulated ) and insulin - stimulated conditions ( hyperinsulinemic - euglycemic clamp ) , using separate groups of mice .
insulin ( actrapid , novo nordisk pharma , denmark ) was infused at a constant rate of 4.8 mu / kgmin for 3 h , while d-[3-h]glucose ( perkin elmer , boston , ma ) was infused at a rate of 15.9 kbq / min . throughout the infusion , glucose concentration and d-[3-h]glucose specific activity ( during the last hour of infusion )
mmol / l ) was maintained by periodically adjusting a variable infusion of 33% glucose ( 33 ) . at the end of a 3-h infusion period ,
mice were first anesthetized by diethylether , exsanguinated through the cervical incision , and then killed by cervical dislocation , and tissues ( liver and quadriceps muscle ) and edta - plasma were collected for biochemical analyses ( see supplementary table 1 and research design and methods of the online appendix for details on basic clamp parameters and methodology ) .
hepatocytes were isolated from livers of fed mice by a modification of the collagenase method ( 34 ) and seeded at a density of 0.5  10 cells per each 35-mm petri dish .
rates of basal and insulin - stimulated de novo lipogenesis and aicar - stimulated fatty acid oxidation were measured using [ 1-c ] acetate and [ 1-c ] palmitate , respectively ( see online appendix ) .
nonesterified fatty acids ( nefas ) , triglycerides , and total cholesterol were determined in plasma using the following enzymatic photometric tests : nefa - c ( wako chemicals , neuss , germany ) , triacylglycerols liquid , and cholesterol liquid ( pliva - lachema diagnostika , brno , czech republic ) , respectively .
plasma insulin was measured using the sensitive rat insulin ria kit ( linco research , st .
total adiponectin levels and adiponectin multimeric complexes were determined using western blotting ( 31 ) .
the tissue content of triglycerides was estimated in ethanolic koh tissue lysates as described before ( 8) . the content and fatty acid composition of the phospholipid , diacylglycerol , triglyceride , and ceramide fractions
were assessed in tissue lipid extracts ; gene expression was evaluated using real - time rt - pcr ( see online appendix ) .
livers were collected by freeze - clamping , ampk was immunoprecipitated from tissue extracts , and the activity was assayed using a peptide substrate ( 32 ) ; see the online appendix .
five days before the experiment , an indwelling catheter was placed into the left femoral vein under anesthesia ( 33 ) .
mice were allowed to recover for 57 days , followed by a 6-h fast ( 8:00 a.m.2:00 p.m. ) prior to the experiment .
the whole - body glucose turnover was determined under basal ( nonstimulated ) and insulin - stimulated conditions ( hyperinsulinemic - euglycemic clamp ) , using separate groups of mice .
insulin ( actrapid , novo nordisk pharma , denmark ) was infused at a constant rate of 4.8 mu / kgmin for 3 h , while d-[3-h]glucose ( perkin elmer , boston , ma ) was infused at a rate of 15.9 kbq / min . throughout the infusion , glucose concentration and d-[3-h]glucose specific activity ( during the last hour of infusion )
mmol / l ) was maintained by periodically adjusting a variable infusion of 33% glucose ( 33 ) . at the end of a 3-h infusion period ,
mice were first anesthetized by diethylether , exsanguinated through the cervical incision , and then killed by cervical dislocation , and tissues ( liver and quadriceps muscle ) and edta - plasma were collected for biochemical analyses ( see supplementary table 1 and research design and methods of the online appendix for details on basic clamp parameters and methodology ) .
hepatocytes were isolated from livers of fed mice by a modification of the collagenase method ( 34 ) and seeded at a density of 0.5  10 cells per each 35-mm petri dish .
rates of basal and insulin - stimulated de novo lipogenesis and aicar - stimulated fatty acid oxidation were measured using [ 1-c ] acetate and [ 1-c ] palmitate , respectively ( see online appendix ) .
specific activities of ampk1 and ampk2 were evaluated in the liver of ad libitum - fed mice after nine weeks of the differential dietary treatment ( fig .
no significant effect of either diet ( chow , chf , and chf+f ) or genotype ( wild - type versus ampk2 ) on ampk1-specific activity was observed , although the ampk1 activity tended to be higher in the ampk2 mice ( fig .
in contrast , ampk2 activity was stimulated by n-3 lc - pufas ( chf+f diet ; fig .
no changes were detected in the activity of ampk1 and ampk2 in the quadriceps muscle in response to n-3 lc - pufas ( not shown ) .
liver ampk1 ( a ) and ampk2 ( b ) activity in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks .
the data are the means  se ( n = 58 ) . in the ampk2 mice ,
* p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf . at four months of age , at the beginning of dietary treatments , wild - type and ampk2 mice fed the chow diet exhibited similar body weights ( table 1 ) . in mice of both genotypes ,
chf - feeding for nine weeks resulted in greater body weight gain compared with the chow - fed mice .
however , this effect was less pronounced in ampk2 mice ( table 1 ) . in both wild - type and ampk2 mice ,
the chf+f diet induced smaller body weight gain than the chf diet ( table 1 and supplementary figure 1 ) .
none of the differences in body weight gain could be explained by caloric intake , which was similar in all experimental groups ( table 1 ) .
the weight of fat depots increased in response to chf feeding , while the chf+f diet partially prevented this increase ( table 1 ) .
triglycerides and nefa levels in plasma of ad libitum - fed mice were similar in the chow- and chf - fed mice , while cholesterol levels were markedly and significantly elevated by the chf diet .
triglycerides and nefa levels were strongly reduced , even below the levels observed in the chow - fed mice ( table 1 ) .
metabolic and plasma parameters in wild - type and ampk2 mice data are means  se of 2730 mice for metabolic parameters and 1315 mice for other measures .
ampk2 and wild - type mice were fed either a chow diet , chf , or chf+f for nine weeks .
body weight gain ( see supplementary fig . 1 ) and plasma parameters were assessed in ad libitum - fed mice after nine weeks .
plasma insulin levels were also assessed in fasted mice after eight weeks . at , adipose tissue ; a.u . , arbitary units ; hmw , total adiponectin , ratio of high molecular weight to total adiponectin ( for levels of all molecular weight forms of adiponectin ; nd , no data ; see supplementary fig .
2 ) ; * p < 0.05 vs. genotype chow ; p < 0.05 vs. genotype chf ; p < 0.05 vs. wild - type on respective diet . after nine weeks of dietary treatment ,
no change was observed in blood glucose , but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes .
however , the increase in plasma insulin levels was less pronounced in ampk2 mice , closely reflecting the genotype - dependent differences in body weight gain ( table 1 ) .
a similar pattern of changes in insulin levels was also observed in fasted mice , in which n-3 lc - pufas significantly reduced insulin levels only in wild - type animals ( table 1 ) . as expected ,
plasma levels of total as well as high molecular weight form of adiponectin , an adipokine associated with increased insulin sensitivity ( 35 ) , were increased 1.4- and 1.2-fold , respectively , in wild - type mice in response to n-3 lc - pufa supplementation ( table 1 and supplementary fig .
2 , available in an online appendix ) ; however , no significant increase of plasma adiponectin by n-3 lc - pufas was observed in ampk2 mice . in further experiments ,
under basal conditions , glucose turnover rate ( gto ; i.e. , glucose uptake in peripheral tissues ) was similar in all groups of mice ( supplementary table 1 ) . under insulin - stimulated conditions ( fig .
f ) , the amount of exogenous glucose required to maintain euglycemia during the clamp , i.e. , the glucose infusion rate ( gir ) , was 1.3-fold lower in ampk2 than in wild - type mice fed the chow diet ( fig .
gir was decreased by the chf diet to a similar level in mice of both genotypes , manifesting diet - induced insulin resistance .
this was attributed to a decreased gto and , in particular , to an impaired suppression of hepatic glucose production ( hgp ) by insulin , with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice ( fig .
2c ) . in wild - type mice , chf+f diet feeding increased gir and gto ( 1.9- and 1.2-fold increase , respectively ) as compared with chf - fed mice , while hgp was lowered to a similar level as in the chow - fed mice .
these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice , namely , at the level of hgp . in contrast
, none of these beneficial effects of n-3 lc - pufas was observed in ampk2 mice , in which neither the gir ( fig .
2b ) differed between the chf+f - fed and the chf - fed mice , whereas the rate of hgp was even higher in the chf+f - fed than in the chf - fed ampk2 mice ( fig . 2c ) .
2d ) , the rate of whole - body glycogen synthesis , which reflects insulin sensitivity of muscle glucose metabolism , was dependent on both diet and genotype ( fig
. 2e ) . in the chow - fed mice , the rate of whole - body glycogen synthesis tended to be higher in wild - type mice than in ampk2 mice . only in the former mice
thus , in wild - type mice , glycogen synthesis was decreased 2.4-fold in response to the chf diet , while n-3 lc - pufas provided a partial protection from this decrease ( fig .
a similar pattern of changes in the glycogen synthesis rate in response to n-3 lc - pufas was observed when measured directly in the skeletal muscle ( fig .
thus , in accordance with the previous study ( 26 ) , the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice
. however , ampk2 mice seemed to be partially protected against chf - induced insulin resistance , while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding .
gir ( a ) , gto ( b ) , hgp ( c ) , whole - body glycolysis ( gl - wb ; d ) , whole - body glycogen synthesis ( gs - wb ; e ) ; and glycogen synthesis in quadriceps muscle ( gs - qm ; f ) were measured in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks .
the data are the means  se ( n = 58 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet .
in addition to the ad libitum - fed mice ( table 1 ) , plasma lipid levels were also measured in fasted mice , as well as in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 2 ) . in contrast to the ad libitum - fed state , chf+f diet did not affect either triglyceride or nefa levels under fasting conditions . under the hyperinsulinemic - euglycemic conditions , both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice ( 1.6-fold and 1.4-fold difference , respectively ) , but no such difference between the diets was observed in ampk2 mice .
cholesterol levels were consistently decreased by n-3 lc - pufas independently of both the metabolic state and genotype ( supplementary table 2 ) . in ad
libitum - fed mice of both genotypes , the hepatic triglyceride content was increased twofold by chf compared with the chow diet , while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes , documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas ( fig .
3a ) . under hyperinsulinemic - euglycemic conditions , n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides . however , this effect was absent in ampk2 mice ( fig .
3b ) . moreover , a strong correlation was found between plasma nefa levels and hepatic triglyceride content assessed under the clamp conditions in the chf+f - fed ampk2 mice ( r = 0.43 , p < 0.05 ) but not in wild - type mice ( r = 0.08 , p = 0.40 ) .
triglyceride concentration in the livers of ad libitum - fed mice ( a ) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp ( b ) .
wild - type and ampk2 mice were fed either a chow diet , chf , or chf+f for 9 weeks .
the data are the means  se ( a , n = 1315 ; b , n = 814 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet .
for the detailed fatty acid composition of triglyceride fractions in the livers of ad libitum - fed mice , see supplementary table 4 .
we sought to determine whether the differential effect of n-3 lc - pufas on accumulation of liver triglycerides in wild - type and ampk2 mice under the clamp conditions could be explained by hepatic lipid metabolism .
in cultured hepatocytes isolated from mice following the different dietary treatments , activities of both fatty acid oxidation and de novo fatty acid synthesis were evaluated .
the stimulatory effects of 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ( aicar ) , an ampk activator , and insulin on fatty acid oxidation and synthesis are shown in fig .
4a and b , respectively ( for corresponding basal metabolic activities , see supplementary table 3 , available in an online appendix ) . in hepatocytes from both chow and chf diet - fed mice ,
the absence of ampk2 was associated with a trend for lower aicar - stimulated fatty acid oxidation . although hepatocytes from chf - fed mice showed reduced stimulatory effect of aicar irrespective of the genotype , chf+f feeding normalized this defect in wild - type but not in ampk2 hepatocytes ( fig .
5a ) , suggesting ampk - dependent induction of capacity for fatty acid oxidation by n-3 lc - pufas in the liver .
the stimulatory effect of insulin on de novo fatty acid synthesis was reduced in hepatocytes from chf - fed wild - type mice , whereas it was retained in the hepatocytes from chf - fed ampk2 mice ( fig .
chf+f feeding tended to restore the stimulatory effect of insulin only in wild - type hepatocytes ( fig .
the effect of differential dietary treatment on the regulation of metabolic fluxes in the liver .
aicar - stimulated fatty acid oxidation ( a ) and insulin - stimulated de novo fatty acid synthesis ( b ) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet , chf , or chf+f . for basal
the expression of scd-1 ( c ) and srebp-1c ( d ) genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp following the differential dietary treatment for 9 weeks .
the data are means  se ( isolated hepatocytes , n = 3 in triplets ; hepatic gene expression , n = 58 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet .
, arbitrary units . the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids ( tfas ; a ) , pufas ( b ) , monounsaturated fatty acids ( mufas ; c ) , and saturated fatty acids ( sfas ; d ) .
animals were fed either a chow diet , chf , or chf+f for 9 weeks .
* p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . for the detailed fatty acid composition of diacylglycerol fractions in the livers of ad libitum fed mice , see supplementary table 4 . to further characterize hepatic effects of differential dietary treatment , the expression of selected genes
was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp ( fig . 4c and d ) .
feeding chf diet suppressed expression of lipogenic genes stearoyl - coa desaturase ( scd-1 ) and srebp-1c in all groups ( except for srebp-1c in ampk2 mice ) .
this suppression was partially counteracted by chf+f diet in wild - type but not ampk2 mice ( fig .
4c and d ) . together with the de novo fatty acid synthesis data , these results further support the ampk2-dependent improvement of liver insulin sensitivity by n-3 lc - pufas . to identify factors predisposing animals to insulin resistance in an ampk2-dependent manner ,
no major genotype - dependent differences in the contents of either ceramides or phospholipids were observed ( supplementary table 4 ) .
in contrast , hepatic content of diacylglycerols was affected in a genotype- and diet - dependent manner ( fig .
wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice , while this effect of the chf+f diet was not observed in ampk2 mice .
moreover , the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids ( fig .
the increase in the pufa content tended to be smaller in the wild - type compared with ampk2 mice ( 1.7-fold and 2.2-fold , respectively ) .
administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice , whereas their level in the ampk2 animals , although decreased , was still significantly higher compared with genotype - matched chow - fed mice ( fig .
-linolenic acid ( 18:3n-3 ) appeared to be by far the most differentially regulated pufa in the diacylglycerol fraction in the two genotypes ( supplementary table 4 ) .
in addition , chf+f diet markedly reduced hepatic content of monounsaturated diacylglycerols in wild - type but not in knockout animals ( fig .
hepatic diacylglycerol levels and their fatty acid composition were also analyzed in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 5 and supplementary fig .
no significant differences among the groups were observed in total diacylglycerols content or in their saturated or monounsaturated fatty acid fractions ( supplementary fig .
specific activities of ampk1 and ampk2 were evaluated in the liver of ad libitum - fed mice after nine weeks of the differential dietary treatment ( fig .
no significant effect of either diet ( chow , chf , and chf+f ) or genotype ( wild - type versus ampk2 ) on ampk1-specific activity was observed , although the ampk1 activity tended to be higher in the ampk2 mice ( fig .
in contrast , ampk2 activity was stimulated by n-3 lc - pufas ( chf+f diet ; fig .
no changes were detected in the activity of ampk1 and ampk2 in the quadriceps muscle in response to n-3 lc - pufas ( not shown ) .
liver ampk1 ( a ) and ampk2 ( b ) activity in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks .
the data are the means  se ( n = 58 ) . in the ampk2 mice ,
at four months of age , at the beginning of dietary treatments , wild - type and ampk2 mice fed the chow diet exhibited similar body weights ( table 1 ) . in mice of both genotypes ,
chf - feeding for nine weeks resulted in greater body weight gain compared with the chow - fed mice .
however , this effect was less pronounced in ampk2 mice ( table 1 ) . in both wild - type and ampk2 mice ,
the chf+f diet induced smaller body weight gain than the chf diet ( table 1 and supplementary figure 1 ) .
none of the differences in body weight gain could be explained by caloric intake , which was similar in all experimental groups ( table 1 ) .
the weight of fat depots increased in response to chf feeding , while the chf+f diet partially prevented this increase ( table 1 ) .
triglycerides and nefa levels in plasma of ad libitum - fed mice were similar in the chow- and chf - fed mice , while cholesterol levels were markedly and significantly elevated by the chf diet .
triglycerides and nefa levels were strongly reduced , even below the levels observed in the chow - fed mice ( table 1 ) .
metabolic and plasma parameters in wild - type and ampk2 mice data are means  se of 2730 mice for metabolic parameters and 1315 mice for other measures .
ampk2 and wild - type mice were fed either a chow diet , chf , or chf+f for nine weeks .
body weight gain ( see supplementary fig . 1 ) and plasma parameters were assessed in ad libitum - fed mice after nine weeks .
plasma insulin levels were also assessed in fasted mice after eight weeks . at , adipose tissue ; a.u .
, arbitary units ; hmw , total adiponectin , ratio of high molecular weight to total adiponectin ( for levels of all molecular weight forms of adiponectin ; nd , no data ; see supplementary fig .
2 ) ; * p < 0.05 vs. genotype chow ; p < 0.05 vs. genotype chf ; p < 0.05 vs. wild - type on respective diet .
after nine weeks of dietary treatment , no change was observed in blood glucose , but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes .
however , the increase in plasma insulin levels was less pronounced in ampk2 mice , closely reflecting the genotype - dependent differences in body weight gain ( table 1 ) .
a similar pattern of changes in insulin levels was also observed in fasted mice , in which n-3 lc - pufas significantly reduced insulin levels only in wild - type animals ( table 1 ) .
as expected , plasma levels of total as well as high molecular weight form of adiponectin , an adipokine associated with increased insulin sensitivity ( 35 ) , were increased 1.4- and 1.2-fold , respectively , in wild - type mice in response to n-3 lc - pufa supplementation ( table 1 and supplementary fig .
2 , available in an online appendix ) ; however , no significant increase of plasma adiponectin by n-3 lc - pufas was observed in ampk2 mice . in further experiments ,
hyperinsulinemic - euglycemic clamps were performed to evaluate whole - body insulin sensitivity . under basal conditions ,
glucose turnover rate ( gto ; i.e. , glucose uptake in peripheral tissues ) was similar in all groups of mice ( supplementary table 1 ) . under insulin - stimulated conditions ( fig .
f ) , the amount of exogenous glucose required to maintain euglycemia during the clamp , i.e. , the glucose infusion rate ( gir ) , was 1.3-fold lower in ampk2 than in wild - type mice fed the chow diet ( fig
gir was decreased by the chf diet to a similar level in mice of both genotypes , manifesting diet - induced insulin resistance .
this was attributed to a decreased gto and , in particular , to an impaired suppression of hepatic glucose production ( hgp ) by insulin , with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice ( fig .
2c ) . in wild - type mice , chf+f diet feeding increased gir and gto ( 1.9- and 1.2-fold increase , respectively ) as compared with chf - fed mice , while hgp was lowered to a similar level as in the chow - fed mice .
these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice , namely , at the level of hgp .
in contrast , none of these beneficial effects of n-3 lc - pufas was observed in ampk2 mice , in which neither the gir ( fig .
2b ) differed between the chf+f - fed and the chf - fed mice , whereas the rate of hgp was even higher in the chf+f - fed than in the chf - fed ampk2 mice ( fig . 2c ) .
2d ) , the rate of whole - body glycogen synthesis , which reflects insulin sensitivity of muscle glucose metabolism , was dependent on both diet and genotype ( fig .
the rate of whole - body glycogen synthesis tended to be higher in wild - type mice than in ampk2 mice . only in the former mice was it significantly affected by dietary treatment .
thus , in wild - type mice , glycogen synthesis was decreased 2.4-fold in response to the chf diet , while n-3 lc - pufas provided a partial protection from this decrease ( fig .
a similar pattern of changes in the glycogen synthesis rate in response to n-3 lc - pufas was observed when measured directly in the skeletal muscle ( fig .
thus , in accordance with the previous study ( 26 ) , the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice
. however , ampk2 mice seemed to be partially protected against chf - induced insulin resistance , while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding .
gir ( a ) , gto ( b ) , hgp ( c ) , whole - body glycolysis ( gl - wb ; d ) , whole - body glycogen synthesis ( gs - wb ; e ) ; and glycogen synthesis in quadriceps muscle ( gs - qm ; f ) were measured in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks .
p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet .
in addition to the ad libitum - fed mice ( table 1 ) , plasma lipid levels were also measured in fasted mice , as well as in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 2 ) .
in contrast to the ad libitum - fed state , chf+f diet did not affect either triglyceride or nefa levels under fasting conditions . under the hyperinsulinemic - euglycemic conditions , both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice ( 1.6-fold and 1.4-fold difference , respectively ) , but
cholesterol levels were consistently decreased by n-3 lc - pufas independently of both the metabolic state and genotype ( supplementary table 2 ) . in ad
libitum - fed mice of both genotypes , the hepatic triglyceride content was increased twofold by chf compared with the chow diet , while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes , documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas ( fig .
3a ) . under hyperinsulinemic - euglycemic conditions , n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides . however
3b ) . moreover , a strong correlation was found between plasma nefa levels and hepatic triglyceride content assessed under the clamp conditions in the chf+f - fed ampk2 mice ( r = 0.43 , p < 0.05 ) but not in wild - type mice ( r = 0.08 , p = 0.40 ) .
triglyceride concentration in the livers of ad libitum - fed mice ( a ) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp ( b ) .
wild - type and ampk2 mice were fed either a chow diet , chf , or chf+f for 9 weeks .
the data are the means  se ( a , n = 1315 ; b , n = 814 ) .
* p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . for the detailed fatty acid composition of triglyceride fractions in the livers of ad libitum - fed mice , see supplementary table 4 .
we sought to determine whether the differential effect of n-3 lc - pufas on accumulation of liver triglycerides in wild - type and ampk2 mice under the clamp conditions could be explained by hepatic lipid metabolism .
in cultured hepatocytes isolated from mice following the different dietary treatments , activities of both fatty acid oxidation and de novo fatty acid synthesis were evaluated .
the stimulatory effects of 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ( aicar ) , an ampk activator , and insulin on fatty acid oxidation and synthesis are shown in fig .
4a and b , respectively ( for corresponding basal metabolic activities , see supplementary table 3 , available in an online appendix ) . in hepatocytes from both chow and chf diet - fed mice ,
the absence of ampk2 was associated with a trend for lower aicar - stimulated fatty acid oxidation . although hepatocytes from chf - fed mice showed reduced stimulatory effect of aicar irrespective of the genotype , chf+f feeding normalized this defect in wild - type but not in ampk2 hepatocytes ( fig .
5a ) , suggesting ampk - dependent induction of capacity for fatty acid oxidation by n-3 lc - pufas in the liver . the stimulatory effect of insulin on de novo fatty acid synthesis was reduced in hepatocytes from chf - fed wild - type mice , whereas it was retained in the hepatocytes from chf - fed ampk2 mice ( fig .
chf+f feeding tended to restore the stimulatory effect of insulin only in wild - type hepatocytes ( fig .
the effect of differential dietary treatment on the regulation of metabolic fluxes in the liver .
aicar - stimulated fatty acid oxidation ( a ) and insulin - stimulated de novo fatty acid synthesis ( b ) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet , chf , or chf+f . for basal
the expression of scd-1 ( c ) and srebp-1c ( d ) genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp following the differential dietary treatment for 9 weeks .
the data are means  se ( isolated hepatocytes , n = 3 in triplets ; hepatic gene expression , n = 58 ) . *
p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . a.u . , arbitrary units .
the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids ( tfas ; a ) , pufas ( b ) , monounsaturated fatty acids ( mufas ; c ) , and saturated fatty acids ( sfas ; d ) .
animals were fed either a chow diet , chf , or chf+f for 9 weeks .
< 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . for the detailed fatty acid composition of diacylglycerol fractions in the livers of ad libitum fed mice , see supplementary table 4 . to further characterize hepatic effects of differential dietary treatment , the expression of selected genes
was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp ( fig .
feeding chf diet suppressed expression of lipogenic genes stearoyl - coa desaturase ( scd-1 ) and srebp-1c in all groups ( except for srebp-1c in ampk2 mice ) .
this suppression was partially counteracted by chf+f diet in wild - type but not ampk2 mice ( fig .
together with the de novo fatty acid synthesis data , these results further support the ampk2-dependent improvement of liver insulin sensitivity by n-3 lc - pufas .
to identify factors predisposing animals to insulin resistance in an ampk2-dependent manner , a detailed analysis of hepatic lipids in ad libitum - fed mice was performed .
no major genotype - dependent differences in the contents of either ceramides or phospholipids were observed ( supplementary table 4 ) .
in contrast , hepatic content of diacylglycerols was affected in a genotype- and diet - dependent manner ( fig .
wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice , while this effect of the chf+f diet was not observed in ampk2 mice .
moreover , the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids ( fig .
the increase in the pufa content tended to be smaller in the wild - type compared with ampk2 mice ( 1.7-fold and 2.2-fold , respectively ) .
administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice , whereas their level in the ampk2 animals , although decreased , was still significantly higher compared with genotype - matched chow - fed mice ( fig .
, -linolenic acid ( 18:3n-3 ) appeared to be by far the most differentially regulated pufa in the diacylglycerol fraction in the two genotypes ( supplementary table 4 ) .
in addition , chf+f diet markedly reduced hepatic content of monounsaturated diacylglycerols in wild - type but not in knockout animals ( fig .
hepatic diacylglycerol levels and their fatty acid composition were also analyzed in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 5 and supplementary fig .
no significant differences among the groups were observed in total diacylglycerols content or in their saturated or monounsaturated fatty acid fractions ( supplementary fig .
previous animal studies demonstrated that n-3 lc - pufas could counteract the development of both hepatic steatosis ( 8,18,36,37 ) and hepatic insulin resistance ( 8,9,16 ) , while suppressing lipogenesis and augmenting lipid catabolism in the liver ( 8,13,19,21 ) . using mice with a whole - body deletion of ampk2 and high - fat feeding , we show for the first time that ampk2
is required for the effect of n-3 lc - pufas to preserve whole - body , muscle , and especially hepatic insulin sensitivity , as well as to suppress hepatic and plasma triglycerides as well as nefa levels under hyperinsulinemic - euglycemic clamp conditions .
in contrast , ampk2 was not required for protection by n-3 lc - pufas from hepatic lipid accumulation and dyslipidemia in ad libitum - fed mice .
in addition to ampk2 , ppar was previously identified as an important determinant of n-3 lc - pufa 's effect on lipid metabolism , especially short - term modulation of hepatic gene expression ( 14 ) and insulin sensitivity ( 16 ) .
thus , the reduction in hepatic triglyceride concentrations by fish oil feeding did not rescue insulin action in ppar-null mice , while hepatic diacylglycerol concentrations were decreased by fish oil in a ppar-dependent manner and were associated with a preserved hepatic insulin sensitivity ( 16 ) .
it is generally accepted that 1 ) diacylglycerols rather than triglycerides or ceramides mediate hepatic insulin resistance in mice fed a high - fat diet ( 19,38,39 ) , 2 ) diacylglycerol - induced insulin resistance depends on activation of protein kinase c , and 3 ) that polyunsaturated diacylglycerols in particular are better protein kinase c activators than saturated diacylglycerol species [ reviewed in refs ( 38,39 ) ] .
also our results showed that chf diet - induced insulin resistance was associated primarily with the accumulation of pufa in hepatic diacylglycerols and that n-3 lc - pufa completely prevented chf diet - induced increase in pufa diacylglycerols in wild - type mice , whereas in ampk2 animals , the content of these lipids was still significantly higher compared with the control .
moreover , it was only in ad libitum - fed mice but not in mice subjected to hyperinsulinemic - euglycemic clamps that the levels of hepatic diacylglycerols and their fatty acid compositions were associated with hepatic insulin sensitivity .
it is possible that under clamp conditions ampk2-dependent effects of n-3 lc - pufas on liver diacylglycerols were masked by metabolic changes occurring during a 3-h infusion of insulin and glucose .
the failure of n-3 lc - pufas to decrease hepatic lipids in ampk2 mice under clamp conditions could be due to primary alterations in metabolic fluxes in the liver , reflecting 1 ) increased de novo fatty acid synthesis , 2 ) decreased secretion of vldl triglycerides , or 3 ) reduced fatty acid oxidation .
de novo fatty acid synthesis was not the responsible factor , because hepatocytes of the n-3 lc - pufa - fed ampk2 mice showed decreased insulin - stimulated de novo fatty acid synthesis and reduced expression of srebp-1c and scd-1 , as compared with hepatocytes isolated from n-3 lc - pufa - fed wild - type mice , reflecting probably low insulin sensitivity of the liver in ampk2 mice .
moreover , aicar - stimulated fatty acid oxidation in hepatocytes from n-3 lc - pufa - fed ampk2 mice was markedly reduced as compared with those from wild - type mice , suggesting decreased hepatic capacity for fatty acid oxidation in the absence of ampk2 , which could contribute to enhanced lipid accumulation .
therefore , these experiments supported a major role of hepatic ampk2 in the regulation of both insulin sensitivity and lipid metabolism by n-3 lc - pufas
. however , the differential modulation of lipid accumulation by n-3 lc - pufas in the livers of wild - type and ampk2 mice under clamp conditions could also be secondary to the ampk2-dependent effects of n-3 lc - pufas in other tissues , resulting in a relatively high hepatic uptake of circulating nefa in ampk2 mice .
this is supported by persistently elevated plasma levels of nefa in ampk2 mice , as well as by a significant correlation between plasma nefa levels and hepatic triglyceride content observed under clamp conditions in ampk2 but not wild - type mice fed n-3 lc - pufa - containing diet .
moreover , it has been shown in humans with nonalcoholic fatty liver disease that most of hepatic triglycerides arise from circulating nefa ( 40 ) . that plasma nefa levels under clamp conditions were reduced only in wild - type but not in ampk2 mice fed n-3 lc - pufas may reflect a role of ampk2 in muscle lipid uptake mediated by lipoprotein lipase ( 41 ) , as well as the antilipolytic effect of ampk in adipose tissue , documented for ampk1 ( 42 ) . in any case , decreased fatty acid oxidation in situ in the liver and , possibly even more importantly
, abundant supply of circulating nefa could be responsible for the lack of the antisteatotic effect of n-3 lc - pufas in ampk2 mice under clamp conditions ( fig .
. putative involvement of ampk2 in antisteatotic action of n-3 lc - pufas in the liver .
dietary intake of n-3 lc - pufas increases the capacity of hepatocytes to oxidize fatty acids in wild - type ( left panels ) but not in ampk2 mice ( right panels ) .
when insulin and glucose levels are high , such as during hyperinsulinemic - euglycemic clamp , wild - type mice fed n-3 lc - pufas exhibit improved hepatic insulin sensitivity and decreased plasma levels of nefas as compared with high - fat diet - fed controls .
this is associated with increased expression of lipogenic genes such as srebp-1c and scd-1 and increased drive for de novo fatty acid synthesis . despite the elevated lipogenic drive under clamp conditions , the livers of wild - type mice fed n-3 lc - pufas show reduced accumulation of triglycerides .
however , in ampk2 mice fed n-3 lc - pufas , hepatic triglyceride content is markedly elevated despite reduced rates of de novo fatty acid synthesis .
this effect could be secondary to persisting elevated nefa levels in circulation and thus better substrate availability in ampk2 mice under clamp conditions .
previous studies reported contradictory results , showing either 1 ) activation of ampk in rat liver ( 22 ) and murine adipose tissue ( 24 ) or 2 ) no changes in ampk activity in the liver , skeletal muscle , and heart of mice ( 43 ) in response to dietary n-3 lc - pufas .
these discrepancies could be related to differences in dietary n-3 lc - pufa intake , nutritional state of animals , and other parameters . in accordance with the involvement of ampk2 in various effects of n-3 lc - pufas ,
our results document activation of ampk2 ( but not ampk1 ) in the liver of mice by long - term n-3 lc - pufa treatment , in the absence of significant changes in either the amp to atp ratio assessed in whole liver extracts [ not shown and ref ( 44 ) ] or the phosphorylation status of lkb1 , an upstream kinase for ampk [ not shown and ref ( 45 ) ] .
in addition , no effect on ampk activity in either cultured hepatocytes ( 21 ) or embryonic kidney cells ( not shown ) of n-3 lc - pufas added to the cell culture medium could be detected .
therefore , the activation of ampk2 by n-3 lc - pufas probably does not depend on a direct interaction between n-3 lc - pufas and ampk . on the other hand , induction of adiponectin by n-3 lc - pufas [ results of this study and refs ( 46,47 ) ]
could be involved , because adiponectin activates ampk in both the liver and skeletal muscle ( 35 ) .
adiponectin is also required for the activation of ampk upon administration of ppar agonists thiazolidinediones , whereas mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to these compounds ( 48 ) .
thus , absence of ampk2 may blunt adiponectin - mediated effects of n-3 lc - pufas . in accordance with the previous study ( 49 )
moreover , the induction of adiponectin by n-3 lc - pufas in ampk2 mice was compromised ( table 1 and supplementary figure 2 ) .
ampk1 and ampk2 contribute equally to total ampk activity in the liver ( 50 ) . in mice with liver - specific ablation of ampk2 ( 27 ) ,
hepatic ampk2 was essential for suppressing hepatic glucose production and maintaining fasting blood glucose levels ; however , the absence of ampk2 did not affect inhibitory action of insulin on hepatic glucose production . in our study ,
although fasting blood glucose levels were unaltered by whole - body ablation of ampk2 , the beneficial effect of dietary n-3 lc - pufas on hepatic insulin sensitivity was clearly ampk2-dependent .
differential regulation of glucose homeostasis in the above transgenic models likely reflects the complexity of whole - body ( 26 ) versus liver - specific ( 27 ) deletion of ampk2 .
in contrast to the previous report , showing induction of adiposity and adipocyte hypertrophy in ampk2 mice fed a lard - based high - fat diet ( 49 ) , our study documented a relatively low weight gain , low adiposity , and smaller fat cells in ampk2 mice fed a corn - oil based high - fat diet .
this discrepancy could be related to the differences in the composition of experimental high - fat diets ; however , our results are consistent with the elevated sympathetic tonus of ampk2 mice ( 26 ) , which may stimulate energy dissipation in these animals . in any case
, lower body weight of chf - fed ampk2 mice as compared with their wild - type counterparts could be related to better insulin sensitivity of the former mice , as suggested by the differences in insulinemia , results of hyperinsulinemic - euglycemic clamp , stimulatory effect of insulin on lipogenesis , and expression of lipogenic genes in the liver . in conclusion ,
the preservation of hepatic insulin sensitivity by n-3 lc - pufas in mice fed a high - fat diet depends on ampk2 .
the accumulation of diacylglycerols , which is regulated in an ampk2-dependent manner , could contribute to the modulation of hepatic insulin sensitivity in response to dietary n-3 lc - pufas .
on the other hand , the ampk2-dependent acute changes in lipid metabolism and hepatic triglyceride accumulation , which are unmasked under insulin - stimulated conditions such as during hyperinsulinemic - euglycemic clamp , largely reflect the extrahepatic action of n-3 lc - pufas .
our results are relevant for the development of novel strategies for prevention and treatment of the metabolic syndrome .
 objectivethe induction of obesity , dyslipidemia , and insulin resistance by high - fat diet in rodents can be prevented by n-3 long - chain polyunsaturated fatty acids ( lc - pufas ) . 
 we tested a hypothesis whether amp - activated protein kinase ( ampk ) has a role in the beneficial effects of n-3 lc-pufas.research design and methodsmice with a whole - body deletion of the 2 catalytic subunit of ampk ( ampk2/ ) and their wild - type littermates were fed on either a low - fat chow , or a corn oil - based high - fat diet ( chf ) , or a chf diet with 15% lipids replaced by n-3 lc - pufa concentrate ( chf+f).resultsfeeding a chf diet induced obesity , dyslipidemia , hepatic steatosis , and whole - body insulin resistance in mice of both genotypes . although chf+f feeding increased hepatic ampk2 activity , the body weight gain , dyslipidemia , and the accumulation of hepatic triglycerides were prevented by the chf+f diet to a similar degree in both ampk2/ and wild - type mice in ad libitum - fed state . 
 however , preservation of hepatic insulin sensitivity by n-3 lc - pufas required functional ampk2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content . under hyperinsulinemic - euglycemic conditions , ampk2 was essential for preserving low levels of both hepatic and plasma triglycerides , as well as plasma free fatty acids , in response to the n-3 lc - pufa treatment.conclusionsour results show that n-3 lc - pufas prevent hepatic insulin resistance in an ampk2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity . 
 ampk2 is also essential for hypolipidemic and antisteatotic effects of n-3 lc - pufa under insulin - stimulated conditions . 
RESEARCH DESIGN AND METHODS
Blood and plasma parameters.
Lipid content and gene expression in the liver.
Activity of 1 and 2 AMPK isoforms.
Hyperinsulinemic-euglycemic clamp.
Primary cultures of hepatocytes.
Statistics.
RESULTS
Enhancement of hepatic AMPK2 activity by n-3 LC-PUFAs.
AMPK2 is not required for antiobesity and hypolipidemic effects of n-3 LC-PUFAs in ad libitum-fed mice.
AMPK2 is essential for the preservation of insulin sensitivity in response to n-3 LC-PUFAs.
Unmasking the role of AMPK2 in the lipid-lowering effect of n-3 LC-PUFAs under hyperinsulinemic-euglycemic conditions.
Dietary n-3 LC-PUFAs increase 5-aminoimidazole-4-carboxamide-1--
Changes in hepatic diacylglycerol levels are associated with insulin-sensitizing effects of n-3 LC-PUFAs.
DISCUSSION
Supplementary Material