young , healthy subjects with no history of obesity , hypertension , diabetes , or hyperlipidemia were screened in the general clinical research center ( gcrc ) at the university of virginia . prothrombin time , partial prothrombin time , complete blood counts , lipid profiles , insulin , and glucose were measured . exclusion criteria included pregnancy , smoking , anemia , bleeding tendency , taking medications or supplements that are known to affect endothelial function and/or glucose metabolism , and family history of diabetes in a first - degree relative . a total of 11 ( 8 men , 3 women ) subjects ( age 21.7  0.4 years , bmi 23.2  0.7 kg / m ) were recruited and studied three times in the gcrc . all subjects provided written informed consent at the screening visit , and the study protocol was approved by the university of virginia institutional review board and the gcrc advisory committee . at each study , subjects were admitted the evening before and fasted overnight . on the morning of the study , one intravenous catheter was placed in the right antecubital vein for the infusion of normal saline , intralipid with heparin , microbubbles , insulin , and glucose . a second intravenous catheter was placed in the right hand for blood sampling of glucose , ffas , and insulin . subjects were studied under the following three protocols in random order , with at least 2 weeks between admissions ( fig . study protocol ( a ) and girs during euglycemic - hyperinsulinemic clamps ( b ) . each subject was studied thrice randomly and ceu / mce performed before and after 2-h euglycemic insulin clamp . p < 0.007 vs. control . after obtaining baseline blood samples , baseline skeletal and cardiac muscle microvascular parameter measurements  including microvascular blood volume ( mbv ) and microvascular flow velocity ( mfv)were determined using contrast - enhanced ultrasound ( ceu ) and myocardial contrast echocardiography ( mce ) , and microvascular blood flow ( mbf ) was calculated ( see below ) . each subject then received a 2-h intravenous infusion of regular insulin ( 2 mu / min / kg  10 min and 1 mu / min / kg  110 min ) . plasma glucose was measured every 5 min throughout the insulin infusion , and 20% dextrose was infused at variable rates to maintain euglycemia . skeletal and cardiac muscle mbv and mfv were again measured , and mbf was calculated at the end of the 120 min insulin clamp . plasma insulin and ffas were measured at 0 , 30 , 60 , 90 , and 120 min . the lipid study was carried out in the exact same fashion as the saline control protocol except that all subjects received intravenous infusion of intralipid ( 20% , 45 ml / h ) and heparin ( 0.2 units / kg / min ) for a total of 5 h. heparin was given to activate endothelial lipoprotein lipase to facilitate the conversion of circulating lipids to ffas . the insulin clamp was started 3 h after the initiation of the intralipid / heparin infusion and the ceu / mce measurements were done immediately before and at 120 min of insulin infusion . the salsalate plus lipid study was identical to the lipid protocol except that all subjects received salsalate 25 mg / kg twice a day by mouth for 4 days prior to study . the last salsalate dose was given at time 180 min on the day of study ( fig . plasma salicylate concentrations were 22  1 mg / dl at time 180 min ( prior to the last dose ) and 25  2 mg / dl at time 0 min ( 3 h after the last dose ) , suggesting that the plasma salsalate concentrations had reached a relative steady state . ceu and mce were performed using a sonos 7500 ultrasound system and an s3 phased array transducer ( philips medical systems , andover , ma ) while the subject was in the left decubitus position , as previously described ( 5,7 ) . in brief , the contrast agent ( definity , lantheus medical imaging , north billerica , ma ) was infused intravenously , and after the systemic microbubble concentrations reached steady state ( 3 min ) , intermittent imaging of the forearm was performed in a transaxial plane 5 cm distal to the antecubital fossa using ultraharmonic imaging with pulsing intervals ( pis ) ranging from 1 to 20 s. immediately after the forearm muscle ceu imaging , myocardial microvascular imaging was done using intermittent ultraharmonic imaging from the apical 4- , 2- , and 3-chamber windows , with ultrasound transmitted at 1.3 mhz and received at 3.6 mhz and pis of 1 , 2 , 3 , 4 , 5 , and 8 cardiac cycles . all muscle and cardiac images were blindly analyzed using qlab software ( philips medical systems ) by w.c . and j.l . mbf is derived from the product of mbv and mfv ( i.e. , mbf = mbv  mfv ) . plasma cholesterol , hdl cholesterol , and triglycerides were assayed by the university of virginia clinical chemistry laboratories . plasma glucose was measured using a ysi glucose analyzer ( yellow springs instruments , yellow springs , oh ) . plasma insulin was determined using the immulite 2000 automated immunoassay analyzer ( siemens healthcare diagnostics inc . , deerfield , il ) , and plasma ffas were measured using an in vitro enzymatic colorimetric assay with a wako hr series nefa - hr kit ( wako diagnostics , richmond , va ) . comparisons between measurements made before and after the insulin clamp within each study protocol and across all three studies were done using either paired student t test , one - way anova , or two - way repeated - measures anova where appropriate . young , healthy subjects with no history of obesity , hypertension , diabetes , or hyperlipidemia were screened in the general clinical research center ( gcrc ) at the university of virginia . prothrombin time , partial prothrombin time , complete blood counts , lipid profiles , insulin , and glucose were measured . exclusion criteria included pregnancy , smoking , anemia , bleeding tendency , taking medications or supplements that are known to affect endothelial function and/or glucose metabolism , and family history of diabetes in a first - degree relative . a total of 11 ( 8 men , 3 women ) subjects ( age 21.7  0.4 years , bmi 23.2  0.7 kg / m ) were recruited and studied three times in the gcrc . all subjects provided written informed consent at the screening visit , and the study protocol was approved by the university of virginia institutional review board and the gcrc advisory committee . at each study , subjects were admitted the evening before and fasted overnight . on the morning of the study , one intravenous catheter was placed in the right antecubital vein for the infusion of normal saline , intralipid with heparin , microbubbles , insulin , and glucose . a second intravenous catheter was placed in the right hand for blood sampling of glucose , ffas , and insulin . subjects were studied under the following three protocols in random order , with at least 2 weeks between admissions ( fig . study protocol ( a ) and girs during euglycemic - hyperinsulinemic clamps ( b ) . each subject was studied thrice randomly and ceu / mce performed before and after 2-h euglycemic insulin clamp . p < 0.007 vs. control . after obtaining baseline blood samples , baseline skeletal and cardiac muscle microvascular parameter measurements  including microvascular blood volume ( mbv ) and microvascular flow velocity ( mfv)were determined using contrast - enhanced ultrasound ( ceu ) and myocardial contrast echocardiography ( mce ) , and microvascular blood flow ( mbf ) was calculated ( see below ) . each subject then received a 2-h intravenous infusion of regular insulin ( 2 mu / min / kg  10 min and 1 mu / min / kg  110 min ) . plasma glucose was measured every 5 min throughout the insulin infusion , and 20% dextrose was infused at variable rates to maintain euglycemia . skeletal and cardiac muscle mbv and mfv were again measured , and mbf was calculated at the end of the 120 min insulin clamp . plasma insulin and ffas were measured at 0 , 30 , 60 , 90 , and 120 min . the lipid study was carried out in the exact same fashion as the saline control protocol except that all subjects received intravenous infusion of intralipid ( 20% , 45 ml / h ) and heparin ( 0.2 units / kg / min ) for a total of 5 h. heparin was given to activate endothelial lipoprotein lipase to facilitate the conversion of circulating lipids to ffas . the insulin clamp was started 3 h after the initiation of the intralipid / heparin infusion and the ceu / mce measurements were done immediately before and at 120 min of insulin infusion . the salsalate plus lipid study was identical to the lipid protocol except that all subjects received salsalate 25 mg / kg twice a day by mouth for 4 days prior to study . the last salsalate dose was given at time 180 min on the day of study ( fig . plasma salicylate concentrations were 22  1 mg / dl at time 180 min ( prior to the last dose ) and 25  2 mg / dl at time 0 min ( 3 h after the last dose ) , suggesting that the plasma salsalate concentrations had reached a relative steady state . after obtaining baseline blood samples , baseline skeletal and cardiac muscle microvascular parameter measurements  including microvascular blood volume ( mbv ) and microvascular flow velocity ( mfv)were determined using contrast - enhanced ultrasound ( ceu ) and myocardial contrast echocardiography ( mce ) , and microvascular blood flow ( mbf ) was calculated ( see below ) . each subject then received a 2-h intravenous infusion of regular insulin ( 2 mu / min / kg  10 min and 1 mu / min / kg  110 min ) . plasma glucose was measured every 5 min throughout the insulin infusion , and 20% dextrose was infused at variable rates to maintain euglycemia . skeletal and cardiac muscle mbv and mfv were again measured , and mbf was calculated at the end of the 120 min insulin clamp . plasma insulin and ffas were measured at 0 , 30 , 60 , 90 , and 120 min . the lipid study was carried out in the exact same fashion as the saline control protocol except that all subjects received intravenous infusion of intralipid ( 20% , 45 ml / h ) and heparin ( 0.2 units / kg / min ) for a total of 5 h. heparin was given to activate endothelial lipoprotein lipase to facilitate the conversion of circulating lipids to ffas . the insulin clamp was started 3 h after the initiation of the intralipid / heparin infusion and the ceu / mce measurements were done immediately before and at 120 min of insulin infusion . the salsalate plus lipid study was identical to the lipid protocol except that all subjects received salsalate 25 mg / kg twice a day by mouth for 4 days prior to study . the last salsalate dose was given at time 180 min on the day of study ( fig . plasma salicylate concentrations were 22  1 mg / dl at time 180 min ( prior to the last dose ) and 25  2 mg / dl at time 0 min ( 3 h after the last dose ) , suggesting that the plasma salsalate concentrations had reached a relative steady state . ceu and mce were performed using a sonos 7500 ultrasound system and an s3 phased array transducer ( philips medical systems , andover , ma ) while the subject was in the left decubitus position , as previously described ( 5,7 ) . in brief , the contrast agent ( definity , lantheus medical imaging , north billerica , ma ) was infused intravenously , and after the systemic microbubble concentrations reached steady state ( 3 min ) , intermittent imaging of the forearm was performed in a transaxial plane 5 cm distal to the antecubital fossa using ultraharmonic imaging with pulsing intervals ( pis ) ranging from 1 to 20 s. immediately after the forearm muscle ceu imaging , myocardial microvascular imaging was done using intermittent ultraharmonic imaging from the apical 4- , 2- , and 3-chamber windows , with ultrasound transmitted at 1.3 mhz and received at 3.6 mhz and pis of 1 , 2 , 3 , 4 , 5 , and 8 cardiac cycles . mbf is derived from the product of mbv and mfv ( i.e. , mbf = mbv  mfv ) . plasma cholesterol , hdl cholesterol , and triglycerides were assayed by the university of virginia clinical chemistry laboratories . plasma glucose was measured using a ysi glucose analyzer ( yellow springs instruments , yellow springs , oh ) . plasma insulin was determined using the immulite 2000 automated immunoassay analyzer ( siemens healthcare diagnostics inc . , deerfield , il ) , and plasma ffas were measured using an in vitro enzymatic colorimetric assay with a wako hr series nefa - hr kit ( wako diagnostics , richmond , va ) . statistical analyses were performed using sigmastat 3.1 software ( systat software , inc . , chicago , il ) . comparisons between measurements made before and after the insulin clamp within each study protocol and across all three studies were done using either paired student t test , one - way anova , or two - way repeated - measures anova where appropriate . all subjects were normotensive ( systolic 128  2 mmhg and diastolic 73  2 mmhg ) and had normal total cholesterol ( 172  12 mg / dl ) , triglyceride ( 70  9 mg / dl ) , ldl cholesterol ( 101  8 mg / dl ) , and hdl cholesterol ( 59  5 mg / dl ) concentrations . table 1 shows the plasma glucose , insulin , and ffa concentrations at baseline and after 2-h of insulin infusion . this was associated with a significant decrease in plasma ffa concentrations . in the lipid protocol , although it did not alter basal plasma glucose concentrations , it did increase basal plasma insulin levels by > 60% ( p < 0.001 ) . insulin infusion decreased plasma ffa concentrations by 1.3 meq / l ( 31% , p < 0.001 ) during the lipid study . salsalate pretreatment plus lipid infusion similarly did not alter plasma glucose concentrations . plasma insulin concentrations trended up further ( p = 0.08 ) , but plasma ffa concentrations were significantly lower ( p < 0.04 ) compared with the values obtained in the lipid alone study . plasma ffa concentrations were similarly decreased after insulin infusion ( 24% , p = 0.001 ) . plasma substrate concentrations during insulin clamp * p < 0.0001 , * * p < 0.001 , * * * p = 0.001 vs. respective basal ; # p < 0.001 , # # p < 0.005 vs. control basal ; & p < 0.04 vs. lipid control . during the saline control study , the steady - state glucose infusion rates ( girs ) varied from 2.4 to 8.6 ( average 5.6  0.7 ) mg / kg / min across individuals . figure 1b shows the girs during the entire 120 min of insulin infusion for all three protocols . the steady - state gir decreased from 5.6  0.7 to 4.5  0.5 mg / kg / min ( p < 0.001 ) , and the overall gir area under the curve declined by 20% ( p salsalate treatment did not significantly alter the steady - state gir ( 4.8  0.6 mg / kg / min , p < 0.04 vs. control ) , but it did significantly improve the gir area under the curve ( back to the levels that were not statistically significantly different from the control study [ p = 0.2 ] ) . this improvement in gir was most evident during the first 6090 min when the gir initially tracked that of the control study . however , it then diverged and approached that observed in the lipid study by 120 min . figure 2a  c shows the changes of skeletal muscle microvascular parameters in all three studies . similar to our previous reports ( 6,7 ) , insulin potently increased muscle mbv and mbf without affecting mfv . salsalate pretreatment for 4 days prior to lipid infusion restored insulin - mediated increases near completely in mbv ( p = 0.06 ) and completely in mbf . skeletal muscle mbv ( a ) , mfv ( b ) , and mbf ( c ) at baseline ( open bar ) and at end of 120-min insulin infusion ( black bar ) . compared with respective baseline , * p = 0.01 , * * p = 0.02 . considering the cardiac microcirculation , fig . c shows insulin infusion significantly increased myocardial mbv and mbf without changing mfv . as in skeletal muscle both mbv and mbf values post  insulin infusion were similar to those values seen in the control study . however , in this study there was an elevation of mbv ( p = 0.1 ) and mbf ( p < 0.03 ) seen prior to giving insulin , and there was no statistical significance between the pre- and postinsulin values . myocardial mbv ( a ) , mfv ( b ) , and mbf ( c ) at baseline ( open bar ) and at end of 120-min insulin infusion ( black bar ) . compared with baseline , * p = 0.01 , * * p = 0.001 . compared with control baseline , # the current study examined the effects of salsalate on ffa - induced microvascular and metabolic insulin resistance in humans , using a combination of contrast ultrasonography and hyperinsulinemic - euglycemic clamp methods . our results indicate that high plasma concentrations of ffas induce both microvascular and metabolic insulin resistance and that pretreatment with salsalate prevents ffa - induced muscle microvascular insulin resistance and attenuates whole body metabolic insulin resistance in a group of healthy young adults . ffas cause endothelial insulin resistance via activation of ikk ( 1 ) . in cultured ecs , ffas increase ikk activity and inhibit insulin - mediated enos phosphorylation and no production ( 1 ) . salsalate is a potent anti - inflammatory drug ( 10 ) , and our observation that salsalate prevents ffa - induced microvascular insulin resistance and attenuates whole body metabolic insulin resistance strongly suggests that in humans , ffas cause microvascular insulin resistance via an inflammation - mediated pathway , which contributes to the metabolic insulin resistance . this is clinically significant because treatment with either aspirin or salsalate improves insulin resistance and glycemic control in animals ( 4,11,12 ) , and clinical trials also have shown promising results . indeed , studies in obese humans have shown that salsalate treatment significantly improves glycemia , decreases circulating c - reactive protein , and increases insulin - mediated glucose disposal ( 13,14 ) . salsalate treatment of patients with type 2 diabetes for 14 weeks significantly decreases glycated hemoglobin levels and improves circulating triglyceride and adiponectin concentrations ( 15 ) . in the current study , acute elevation of plasma ffa concentrations abolished insulin - mediated increases in skeletal and cardiac muscle microvascular recruitment and decreased insulin - stimulated whole body glucose disposal this is in line with our previous reports in humans ( 6,7 ) and a previous report in rats ( 9 ) . strong evidence has confirmed that ffas inhibit insulin signaling through the pi3-kinase pathway in both vasculature and muscle . in humans , high plasma concentrations of ffas induce oxidative stress , impair endothelium - dependent vasodilation , blunt insulin - mediated vasodilation ( 1618 ) , and decrease muscle insulin receptor substrate-1-associated pi3-kinase activity and glucose transport ( 19 ) . increased ffa concentrations induce reactive oxygen species generation , which may reduce the bioavailability of no and impair vascular reactivity . in addition , insulin has been shown to induce enos ( 20 ) , and an insulin - resistant state may impair vasodilation by blocking insulin - mediated induction of enos . it is interesting that salsalate treatment prevented the lipid - induced decrease in whole body glucose disposal during the first 6090 min only . toward the last 30 min of clamp , insulin - stimulated glucose disposal rates were not different from the lipid group . that pretreatment with salsalate restored insulin response in muscle mbf suggests salsalate treatment completely prevented lipid - induced microvascular insulin resistance in muscle . thus , the resolution of microvascular insulin resistance may have contributed to the early phase improvement of metabolic insulin sensitivity , whereas later muscle insulin resistance ( without the vascular component ) might have prevailed . indeed , previous studies have demonstrated that insulin - mediated capillary recruitment contributes to 40% of insulin - mediated whole body glucose disposal and that vascular insulin resistance might be more important as a determinant of the rate of onset of insulin action ( 8,21 ) . although salsalate treatment completely restored insulin responsiveness in skeletal muscle microcirculation , in the heart , there was no difference in either mbv or mbf before and after insulin infusion during the intralipid plus salsalate admission . however , the absolute post  insulin infusion values were similar to those seen in the insulin control study . this was most likely secondary to the baseline increases in both mbv ( p = 0.06 ) and mbf ( p = 0.03 ) induced by lipid infusion in the setting of salsalate treatment . in the salsalate plus lipid protocol , basal mbv was 21  9% higher than in the saline control subjects and only 4% less than the insulin - treated control subjects in the saline control protocol . thus , the increase in mbv induced by lipid infusion after salsalate treatment may have preempted any insulin effect . we and others have previously reported that lipid infusion increases basal cardiac ( 6 ) and total leg blood flow ( 18 ) . it is interesting that the addition of salsalate to lipid infusion further enhanced basal cardiac mbf . given that plasma insulin concentrations increased significantly in the basal state , it is likely that both insulin and ffas may have contributed to this increase in basal flow . inasmuch as salsalate is capable of increasing insulin secretion ( 14,22 ) and the plasma insulin trended higher during the salsalate plus lipid study than the lipid alone study , the improved microvascular and metabolic responses could be secondary to increased circulating insulin concentrations . indeed , the significantly lower preclamp ffa concentrations are also suggestive of possible insulin effect . however , this probability is rather low because both the preclamp ( p = 0.08 ) and postclamp ( p = 0.51 ) insulin concentrations were not significantly higher during the salsalate plus lipid admission than the lipid alone admission . in addition , insulin increased muscle mbv only by 25% when its concentrations were raised by 10-fold in the current study . the concentrations of ffas used in the current study are higher than those observed in insulin - resistant states . we opted to use high concentrations of ffas because in the past , we have demonstrated that ffas at these concentrations blocked insulin- and mixed meal  induced muscle microvascular recruitment in healthy humans ( 6,7 ) . thus , it is very much likely that salsalate treatment could be more effective in the setting of low plasma concentrations of ffas . in conclusion , high plasma concentrations of ffas cause muscle microvascular and whole body metabolic insulin resistance in healthy human adults . salsalate treatment blocked ffa - induced microvascular insulin resistance , which was associated with a significant improvement in whole body insulin sensitivity . thus , our data suggest that ffas cause microvascular insulin resistance and dysfunction likely via an inflammation - mediated mechanism and that this contributes to metabolic insulin resistance in humans . treatments aimed at inflammation inhibition may afford an opportunity to improve diabetes control and prevent cardiovascular complications in patients with type 2 diabetes .
objectiveinsulin recruits muscle microvasculature , thereby increasing endothelial exchange surface area . 
 free fatty acids ( ffas ) cause insulin resistance by activating inhibitor of b kinase . elevating plasma ffas impairs insulin s microvascular and metabolic actions in vivo . 
 whether salsalate , an anti - inflammatory agent , prevents ffa - induced microvascular and/or metabolic insulin resistance in humans is unknown.research design and methodseleven healthy , young adults were studied three times in random order . 
 after an overnight fast , on two occasions each subject received a 5-h systemic infusion of intralipid  salsalate pretreatment ( 50 mg / kg / day for 4 days ) . on the third occasion , saline replaced intralipid . 
 a 1 mu / kg / min euglycemic insulin clamp was superimposed over the last 2-h of each study . 
 skeletal and cardiac muscle microvascular blood volume ( mbv ) , microvascular flow velocity ( mfv ) , and microvascular blood flow ( mbf ) were determined before and after insulin infusion . 
 whole body glucose disposal rates were calculated from glucose infusion rates.resultsinsulin significantly increased skeletal and cardiac muscle mbv and mbf without affecting mfv . lipid infusion abolished insulin - mediated microvascular recruitment in both skeletal and cardiac muscle and lowered insulin - stimulated whole body glucose disposal ( p < 0.001 ) . 
 salsalate treatment rescued insulin s actions to recruit muscle microvasculature and improved insulin - stimulated whole body glucose disposal in the presence of high plasma ffas.conclusionshigh plasma concentrations of ffas cause both microvascular and metabolic insulin resistance , which can be prevented or attenuated by salsalate treatment . 
 our data suggest that treatments aimed at inhibition of inflammatory response might help alleviate vascular insulin resistance and improve metabolic control in patients with diabetes .