the ectopic accumulation of lipids in tissues such as the skeletal muscle and liver is highly implicated in the pathogenesis of insulin resistance and glucose intolerance . in skeletal muscle , elevated levels of intramyocellular lipids are strongly associated with impaired rates of insulin - stimulated whole - body glucose disposal . in the liver , excessive intracellular lipid levels are associated with impaired insulin - mediated suppression of glucose production . however , there are settings where elevated tissue lipid levels do not confer insulin resistance , most notably the  athlete 's paradox  where highly trained athletes have elevated levels of intramyocellular triglyceride yet are highly insulin sensitive [ 3 , 4 ] . a similar paradox is presented in subjects with loss of function mutations in the adipose triglyceride lipase ( atgl ) gene . atgl is the rate - limiting enzyme for triglyceride catabolism in most cells and tissues and a lack of atgl activity results in a dyslipidemic phenotype with augmented visceral adipose tissue and extensive ectopic triglyceride accumulation in pancreas and skeletal muscle . these subjects , though glucose intolerant on account of a limited insulin secretory response , nevertheless show normal whole - body insulin sensitivity . these mice show augmented adipose tissue mass , as well as extensive ectopic triglyceride accumulation in heart , liver , and skeletal muscle [ 6 , 7 ] . the excessive myocardial triglyceride accumulation is associated with the development of severe cardiac insufficiency at about 12 weeks of life . despite this excessive adiposity , atgl mice are glucose tolerant and insulin sensitive and the basis of this paradox remains poorly understood . in the basal fasting state , glucose homeostasis is dependent on a tight control of endogenous glucose production ( egp ) and efficient peripheral glucose disposal . the curtailment of triglyceride hydrolysis in atgl mice can potentially modify both liver and muscle glucose metabolism , ultimately contributing to improved glucose tolerance . in skeletal muscle , a reduction in free - fatty acid ( ffa ) levels due to attenuation of triglyceride hydrolysis can potentially favor glucose oxidation in accord with the postulations of the randle cycle , thereby promoting peripheral glucose disposal . meanwhile , a restriction in availability of systemic ffa and glycerol results in restricted gluconeogenesis from glycerol as well as pyruvate , the latter being highly dependent on -oxidation for atp and reducing equivalents . it is not known to what extent glucose tolerance in the atgl mouse is explained by improved peripheral glucose oxidation versus restriction of gluconeogenesis . therefore , the aim of this study was to compare egp and peripheral glucose disposal in atgl versus wild - type ( wt ) mice . this was achieved by c - isotopomer analysis of plasma glucose following a primed - infusion of [ u - c6]glucose : a tracer that provides a measurement of egp by isotope dilution , as well as a measure of glucose carbon recycling via the cori cycle , a measure of the sparing of glucose oxidation by peripheral tissues as illustrated by figure 1 . plasma glucose c - isotopomers from blood spots were quantified by a novel and sensitive isotopomer - targeted lc - ms / ms analysis . unlike gc - ms methods that require sample derivatization , this analysis was achieved following a simple and rapid sample extraction procedure . all experimental procedures were approved beforehand by the local animal ethics committee of the radboud university nijmegen medical center ( runmc ) ( nijmegen , netherlands ) . the mice used in this project were a kind gift of professor rudolf zechner from graz university ( graz , austria ) . atgl mice were bred from heterozygous atgl ( atgl ) in a mixed genetic background c57bl/6j and 129ola as described previously . mice were fed ad libitum a standard chow diet and were housed in 12  h/12  h light - dark cycle with controlled temperature ( 2224c ) . six whole - body knockout atgl mice and seven atgl littermates ( referred to hereafter as wild - type ( wt ) mice ) were used for this study . animals were fasted for 6  h and anesthetized with 1.5% isoflurane given by a gas mixture ( 2  :  1 o2/n2o ) through a facial mask . they were administered intravenously a primed - constant infusion of [ u - c6]glucose consisting of a 6.25  mol / kg priming dose followed by 5  mol / kg / min constant infusion for 120  min . blood spots were collected directly onto whatman number 6 filter paper at 105 , 110 , and 115  min . for a subgroup of animals , blood spots were collected before the start of tracer infusion in order to obtain background c levels . six mm blood spot disks were punched ; then 50  l of internal standard ( is ) solution consisting of 30  m [ u - c6,u - h7]glucose in distilled water and 450  l of etoh were added . samples were sonicated in a bath for 45  min at room temperature and then centrifuged for 5  min at 13,900  g . samples were sonicated with a cup - horn at 40% , for 2  min , with pulses of 1  sec and pauses of 1  sec . samples were subsequently purified by solid phase extraction ( spe ) using reversed phase packed zip - tips and acn / h2o as the mobile phase . the lc system used for all analyses was an ultimate 3000 lc system ( thermo scientific , dionex ) . the ms used was a hybrid triple quadrupole / linear ion trap 4000 qtrap lc - ms / ms system equipped with an electrospray ionization ( esi ) turbo v ion spray source ( absciex ) . the software operating the lc system was the chromeleon 6.80 ( thermo scientific , dionex ) and the ms system was controlled by the analyst 1.5.1 ( absciex ) . chromatographic separation was performed on a luna nh2 3  m , 100   , 150  2.00  mm column . the flow rate was 150  l / min and running time for each sample was 15  min . samples were eluted with an acn / h2o gradient starting at 80%/20% , reaching 60%/40% at 5  min and 20%/80% by 15  min . after sample injection , one blank ( h2o ) of 4  min running and afterwards a second blank ( h2o ) of 8  min running were injected . the sample injection was 1  l , while for the blanks 19  l was injected . samples were analyzed at 150  l / min by esi . the ionization source operated in the negative mode at an ion spray voltage of 4500  v , with nebulizer gas 1 pressure set to 35  psi , nebulizer gas 2 set to 30  psi , and a temperature of 450c . the 4  min blank was operated in the positive mode , while the 8  min blank program was operated in the negative mode . glucose enrichments were quantified using the multiple reaction monitoring ( mrm ) triple quadrupole scan mode . the following ms parameters were maintained for all transitions : collision gas 6  psi , curtain gas 30  psi , collision cell exit potential 8  ev , entrance potential 4  ev , dwell time 100  ms , and declustering potential 50  ev . the collision energy was optimized for each transition . mrm transitions for monitoring [ u - c6]glucose were 179/89 , for [ 1,2-c2]glucose 181/89 and 181/91 , for [ 1,2,3-c3]glucose 182/89 and 182/92 , and for [ u - c6]glucose 185/92 . finally , for the [ u - c6 , u - h7]glucose internal standard , the selected transition was 192/94 . excess enrichments of glucose c - isotopomers were quantified from calibration curves prepared with known ratios of unenriched glucose to [ 1,2-c2]glucose , [ 1,2,3-c3]glucose , and [ u - c6]glucose . the amount of glucose c - isotopomers measured from the calibration solutions spanned the range of measured concentrations from the blood samples . analysis of single and combined c - isotopomer standards indicated no significant cross - contamination of m+6 , m+3 , and m+2 c - mass isotopomer between the different c - enriched glucose standards . enrichment of plasma glucose from infused [ u - c6]glucose ( ep ) was determined from the m+6 abundance of plasma glucose . the rate of appearance ( ra ) of plasma glucose and egp were calculated as follows:(1)glucose  ra = eiepi , egp = rai , where i is infusion rate of [ u - c6]glucose in mol / kg / min and ei is the percent enrichment of infusate [ u - c6]glucose . the units of ra and egp were reported as mol glucose / kg body weight / min . the rate of peripheral glucose disposal ( rd ) was assumed to be equal to ra . for quantifying cori cycle activity , the following equations were used:(2)fraction  of  glucose  ra  undergoing  cori  cycling=1.50.5m+2+m+3m+6,absolute  cori  cycling  flux = fractional  cori  cycle  fluxrd , where the fraction of glucose rd undergoing cori cycling is given as a percentage and the absolute cori cycle flux is reported as mol / kg / min . plasma glucose m+6 enrichment was considered as the precursor pool while the sum of plasma glucose m+2 and m+3 isotopomer enrichments was assumed to be the products following passage of the m+6 species through the cori cycle . the m+3 species detected by the 182/92 lc - ms / ms transition was assumed to represent the sum of [ 1,2,3-c3]glucose and [ 4,5,6-c3]glucose isotopomers while the m+2 species detected by the 181/91 transition was assumed to represent the sum of [ 1,2-c2]glucose and [ 5,6-c2]glucose isotopomers ( figure 1 ) . these four species represent ~8590% of glucose isotopomers that are formed during recycling of [ u - c6]glucose [ 10 , 11 ] . gluconeogenesis also results in an approximately ~1.5-fold dilution of c - enrichment via krebs cycle carbon exchange ; hence a correction factor of 1.5 was incorporated into the equation [ 10 , 11 ] . finally , since one m+6 glucose generates two recycled molecules containing one or the other of the m+3 and m+2 daughter isotopomers , for calculating the recycling fluxes , the sum of m+3 and m+2 isotopomer enrichments was divided by 2 to maintain the correct stoichiometry between the plasma glucose precursor and recycled glucose product . both groups of mice were statistically not different in age , but the atgl group tended to be younger . this was due to some spontaneous deaths due to heart failure occurring in this group at about 8 weeks of age in addition to some mortalities during the tracer administration protocol , also likely related to heart failure . after 6  h of fasting , both groups had equivalent fasting plasma glucose and plasma triglyceride levels but atgl mice had significantly lower levels of plasma ffa at 6  h of fasting compared to wt mice . the glucose m+6 , m+3 , and m+2 isotopomer distributions are summarized in figure 2 . for a subset of animals ( 4 atgl and 2 wt ) , a blood sample was collected immediately before [ u - c6]glucose infusion to determine background levels of m+2 , m+3 , and m+6 . based on our detection limit criteria ( signal - to - noise ratio 3 ) , no signals from any of these isotopomers were observed in any of the samples . for blood that was collected after [ u - c6]glucose infusion , most of the variance in c - isotopomer enrichments reported for each group arose from differences between animals , with atgl mice showing somewhat higher variance than wt mice . thus , the variability in glucose mass isotopomer enrichments between the 105- , 110- , and 115-minute blood samples for each animal was low , with the mean coefficient of variation being 7% for the parent m+6 isotopomer and 8% and 11% for the daughter m+2 and m+3 isotopomers , respectively . for both atgl and wt mice , the distribution of the recycled glucose c - isotopomers was heavily skewed toward m+2 over m+3 species ( figure 2 ) . an excess of m+2 over m+3 isotopomer distribution within the triose moiety of glucose was also previously reported in rats infused with [ u - c]lactate . also , an excess of [ 1,2-c2]glucose over [ 1,2,3-c3]glucose isotopomers has been quantified by c nmr in mice administered with [ u - c]propionate [ 13 , 14 ] , an alternative gluconeogenic substrate that , like [ u - c]lactate , is also metabolized to glucose via the hepatic krebs cycle and anaplerosis . no differences in either glucose ra or egp rates were found between wt and atgl mice . our estimated egp values are higher compared to values of ~50  mol / kg / min that were previously reported for fasted wt mice that were chronically catheterized and fasted for 26  h . it is known that egp declines during the progression of fasting ; hence a 6  h fasted mouse would be expected to have higher egp rates compared to a 26  h fasted mouse . wt and atgl mice also had similar rates of fractional and absolute cori cycle fluxes . cori cycling accounted for the majority ( ~80% ) of glucose utilization indicating that peripheral glucose oxidation was highly spared in both groups of mice . there is a strong association between elevated ectopic lipid levels and insulin resistance in both humans and rodents [ 1719 ] . metabolites related to triglyceride - fatty acid interconversion such as fatty - acyl coa , diacylglycerol , and ceramides have been shown to disrupt insulin signaling [ 2023 ] and have therefore been hypothesized as the causative agents of insulin resistance in the setting of elevated ectopic lipid . however , this view is challenged by the atgl mouse since it has extensive levels of ectopic lipids in insulin - sensitive tissues such as skeletal muscle , heart , and liver but is nevertheless glucose tolerant . in contrast , when ectopic lipid depots are induced in wt mice via high - fat and/or high - sugar feeding , they develop insulin resistance and glucose intolerance [ 24 , 25 ] . in the basal fasted state , insulin sensitivity is primarily defined by the matching of hepatic glucose production to peripheral glucose uptake . with diet - induced dyslipidemia and ectopic lipid accumulation , there is a well characterized upregulation of fasting egp [ 26 , 27 ] , primarily due to the impaired control of hepatic gluconeogenesis . this so - called hepatic insulin resistance is a prominent and early feature of diet - induced glucose intolerance and is strongly associated with elevated hepatic lipid levels . meanwhile , the accumulation of lipid in skeletal muscle , albeit slower than the development of hepatic steatosis , is nevertheless accompanied by a reduction in insulin - stimulated glucose disposal . the features of fasting glucose production and peripheral disposal that explain the high glucose tolerance in atgl mouse remain unclear . in the initial study on the effect of atgl knockout on metabolic status and insulin sensitivity by haemmerle et al . , atgl and wt controls had similar levels of fasting plasma glucose and insulin . this suggests that atgl and wt mice had similar insulin sensitivity under fasting conditions , at least as assessed by fasting insulin and glucose levels . however under fed conditions and also following a glucose load , atgl mice revealed higher insulin sensitivity compared to wt mice . since insulin - stimulated disposal of glucose into peripheral tissues assumes a much greater role in glycemic control under fed compared to fasted states , this suggests that the higher insulin sensitivity of atgl mice is more related with peripheral rather than hepatic insulin actions . in support of this , kienesberger et al . reported that in vivo atgl skeletal muscle insulin signaling was improved with increased insulin receptor substrate 1 and akt phosphorylation , pi3k and akt activities , and glut4 protein expression . interestingly , hepatic insulin signaling was unchanged or impaired in atgl mice , suggesting a more prominent role of peripheral over hepatic insulin actions in explaining their high insulin sensitivity . the role of extrahepatic tissues in determining whole - body insulin sensitivity of atgl mice becomes even more prominent under increased energetic demand since they are more dependent on glucose as an oxidative fuel . thus at rest , atgl skeletal muscle atp and other high - energy phosphate levels were comparable to the littermates and even upon high intense electrostimulation , the muscle oxidative capacity was not compromised . however , atgl mice showed significantly lower muscle glycogen levels both at rest and after electrostimulation , further supporting an increased demand for carbohydrate oxidation . similarly , schoiswohl et al . showed that exercised atgl mice presented hepatic glycogen reserves which were severely depleted . this is consistent with an increased mobilization of glucose for skeletal muscle oxidation in response to insufficient fatty acid availability . conversely , the muscle - specific atgl knockout , which recapitulates the high intramyocellular triglyceride level of the global atgl , did not have differences in oxidative substrate selection , glucose homeostasis , or peripheral insulin sensitivity compared to control mice   while resting muscle oxidative phosphorylation and oxidative capacity was not compromised in global atgl mice . nevertheless , when exercised , atgl mice showed limited generation of ffa while at the same time hepatic glycogen reserves were severely depleted . this is consistent with an increased mobilization of glucose for skeletal muscle oxidation in response to insufficient fatty acid availability . furthermore , atgl mice showed significantly lower muscle glycogen levels both at rest and after electrostimulation , further supporting an increased demand on carbohydrate reserves for muscle energy utilization . mice that underwent liver - selective atgl knockdown developed steatosis following both normal and high - fat feeding but were protected against glucose intolerance and hyperinsulinemia during high - fat feeding . while hepatic insulin signaling in response was not modified by hepatic atgl knockdown , expression of gluconeogenic enzymes was decreased in both normal and high - fat feeding settings   suggesting reduced capacity for gluconeogenesis . moreover , hepatic fatty acid oxidation was found to be impaired in atgl mice , which , by restricting the availability of atp and reducing equivalents , would also constrain gluconeogenesis from pyruvate precursors . how do our measurements of fasting glucose kinetics reconcile with these previous studies ? at 6  h of fasting , we found a significantly reduced level of plasma ffa in atgl mice compared to wt , suggesting impairment of fasting whole - body lipolysis . the concentration of plasma ffa has been shown to exert strong and acute control of gluconeogenic flux [ 3436 ] , but the reduced availability of ffa in atgl mice did not appear to compromise egp fluxes , at least at 6 hours of fasting . we found that peripheral glucose metabolism was largely directed towards the cori cycle , with the majority of glucose carbons being recycled . this suggests that skeletal muscle glucose oxidation , at least at rest , was not significantly enhanced in atgl mice but was instead highly spared to the same extent as in wild - types . presumably , the production of ffa by other lipases , for example , spillover , or the catabolism of other substrates as amino acids was sufficient to maintain the energy demands of skeletal muscle , at least in the resting state . a possible confounding factor in our study is the fact that the body weights of the atgl mice were not significantly higher than the wt ( due primarily to increased adiposity ) , as has been consistently reported in previous studies , for example , [ 6 , 7 ] . this may be related to the tendency for the atgl mice to be younger than their wt littermates as previously described . intramyocellular lipid ( imcl ) levels were assayed in vivo using h magnetic resonance spectroscopy in the tibialis anterior muscle of 3 atgl and 4 atgl mice of this study . atgl mice had levels of imcl relative to total creatine of 3.6  0.44 versus 0.71  0.80 in atgl mice . although the number of mice used for this analysis was limited , the data supports the characteristic ectopic lipid accumulation in atgl mice . whether cori cycle and egp fluxes are similarly sustained in atgl mice during longer fasting periods or during high - energy demanding states , for example , exercise , remains to be investigated . presumably , the production of ffa by lipases other than atgl was sufficient to maintain the energy demands of skeletal muscle , at least in the resting state . in addition to providing estimates of egp from isotope dilution of [ u - c6]glucose , lc - ms / ms also provided precise quantification of m+3 and m+2 triose isotopomers associated with cori cycling . their abundances were quantified from specific calibration curves prepared with [ 1,2-c2]glucose and [ 1,2,3-c3]glucose . the fragmentation mechanism that yields the 181/91 and 182/92 ms / ms transitions cleaves the glucose molecule into two fragments of identical mass representing carbons [ 1,2 , 3 ] and [ 4,5 , 6 ] . thus , the abundance of m+2 and m+3 isotopomers from both triose halves of the glucose molecule was reported . for both wt and atgl mice , we observed a significantly higher abundance of the glucose m+2 over the m+3 isotopomer . as illustrated by figure 3 , this can be explained by an excess production of [ 2,3-c2]triose - p over [ 1,2,3-c3]triose - p resulting from pyruvate cycling and is concordant with high activities of pyruvate cycling that have been described in fasted mice [ 13 , 14 ] , rats , and humans [ 3840 ] . c - isotopomer studies of glucose enriched from [ u - c]propionate have shown that the formation of other possible m+2 triose isotopomers ( i.e. , [ 1,2-c2]triose - p and [ 1,3-c2]triose - p ) is low relative to that of [ 2,3-c2]triose - p , [ 13 , 14 ] . therefore , the observed excess of m+2 over m+3 primarily involves the  first pass  isotopomers described in figures 1 and 3 . previous estimates of m+2 and m+3 enrichments from [ u - c6]glucose recycling in rats and humans have revealed variable levels of m+2 and m+3 isotopomer products following [ u - c6]glucose infusion . in 24 h fasted rats infused intraduodenally with substrate levels of [ u - c6]glucose , a ~40% excess of m+2 to m+3 was found for both glucose and glycogen . however , in another study where rats were infused intragastrically with [ u - c6]glucose , a small excess of m+3 over m+2 glucose was reported in both plasma glucose and hepatic glycogen . a study of healthy humans fasted for periods ranging from 12 to 40  h showed an excess of m+3 to m+2 in 12 and 20  h fasted individuals but a 2-fold excess of m+2 over m+3 in 40  h fasted subjects . in a study of three 65  h fasted subjects , there was a modest excess of m+2 over m+3 for two of the subjects , while the third subject had a modest excess of m+3 over m+2 . however , these human measurements were acknowledged to have limited precision and specificity due to low c - enrichment levels   while specific glucose m+2 and m+3 isotopomers were not independently calibrated in any of these aforementioned human or animal studies . as with previous gc - ms and nmr - based analyses of [ u - c]glucose enrichment and cori cycling , our approach is ultimately limited by the precision of the isotopomer quantifications , in particular the recycled m+2 and m+3 species whose levels are typically lower than the parent [ u - c]glucose while at the same time background contributions , in particular to the m+2 species , are higher . also , this approach does not provide information on other gluconeogenic precursors such as fructose and glycerol , which , in addition to glycogenolysis , could influence the fractional contribution of cori cycling to egp . in summary , we quantified glucose appearance and recycling fluxes in 6  h fasted atgl mice . as for wt controls , the majority of glucose that underwent peripheral metabolism was spared from oxidation . this indicates that , under these resting conditions , there was sufficient availability of other substrates , presumably including fatty acids , for muscle energy generation . finally , resolution of recycled glucose isotopomers by ms / ms revealed a distribution consistent with active hepatic pyruvate cycling for both atgl and wt mice .
mice deficient in adipose triglyceride lipase ( atgl/ ) present elevated ectopic lipid levels but are paradoxically glucose - tolerant . 
 measurement of endogenous glucose production ( egp ) and cori cycle activity provide insights into the maintenance of glycemic control in these animals . 
 these parameters were determined in 7 wild - type ( atgl+/ ) and 6 atgl/ mice by a primed - infusion of [ u-13c6]glucose followed by lc - ms / ms targeted mass - isotopomer analysis of blood glucose . 
 egp was quantified by isotope dilution of [ u-13c6]glucose while cori cycling was estimated by analysis of glucose triose 13c - isotopomers . 
 fasting plasma free fatty - acids were significantly lower in atgl/ versus control mice ( 0.43  0.05  mm versus 0.73  0.11  mm , p < 0.05 ) . 
 six - hour fasting egp rates were identical for both atgl/ and control mice ( 79  11 versus 71  7  mol / kg / min , resp . ) . peripheral glucose metabolism was dominated by cori cycling ( 80  2% and 82  7% of glucose disposal for atgl/ and control mice , resp . ) indicating that peripheral glucose oxidation was not significantly upregulated in atgl/ mice under these conditions . 
 the glucose 13c - isotopomer distributions in both atgl/ and control mice were consistent with extensive hepatic pyruvate recycling . 
 this suggests that gluconeogenic outflow from the krebs cycle was also well compensated in atgl/ mice .