the study protocol was approved by the university of virginia human investigation committee . a total of eight lean ( three men and five women ; bmi < 25 kg / m ) and eight obese ( one man and seven women ; bmi > 30 kg / m ) volunteers with no history of hypertension , diabetes , or hyperlipidemia were recruited and fasted overnight .
subjects were excluded from the study if they had a family history of a first - degree relative with diagnosed diabetes , had a history of smoking , or were taking any medication known to affect either endothelial function or glucose metabolism .
subjects were admitted to the general clinical research center on two separate occasions . on a screening visit serum clinical chemistry values
were measured by the university of virginia clinical chemistry laboratories , and body composition was measured using an air displacement chamber ( bod pod ) ; height , weight , and hip and waist circumferences were also measured . on the day of the study visit
, overnight - fasted subjects were admitted to the general clinical research center on the morning of the study .
subjects remained in bed 1 h before and for the duration of the study and were instructed to refrain from using their arms to minimize effects of movement ( or exercise ) on hemodynamic measurements .
the catheter in the nondominant arm was used for blood sampling of glucose and insulin , and the contralateral arm was used for the infusion of microbubbles .
baseline measurements of total forearm blood flow ( assessed by doppler ultrasound ) and muscle microvascular blood volume ( mbv ) and microvascular flow velocity ( mfv ) ( assessed by ceu ) were performed as described previously ( 4,19 ) .
subjects were then given a liquid meal ( boost ; 480 kcal : 72 from fat , 328 from carbohydrate , and 80 from protein ) .
a mixed meal was chosen to assess the physiological changes that occur in response to a typical meal .
a liquid meal was administered to shorten the amount of time needed for digestion and absorption .
plasma glucose and insulin were measured at 0 , 30 , 60 , 90 , and 120 min .
plasma glucose was measured using a ysi analyzer ( ysi , yellow springs , oh ) .
plasma insulin was measured using an enzyme - linked immunosorbent assay with an interassay coefficient of variation of < 4% . at 60 and
again at 120 min after the mixed meal , brachial artery flow ( doppler ultrasound ) and mbv and mfv ( ceu ) were measured .
for the two - dimensional and doppler ultrasound measurements , an ultrasound system ( hdi 5000 ; philips medical systems , bothell , wa ) with a linear - array transducer was used with a transmit frequency of 12 mhz .
two - dimensional imaging of the brachial artery was performed in the long axis 5 cm proximal to the antecubital fold .
images were triggered to the r wave of the cardiac cycle , and the brachial artery diameter was measured using on - line video calipers .
a pulsed - wave doppler sample blood volume was placed at the same location in the center of the artery and the mean blood velocity was measured using online angle correction and analysis software .
brachial artery mean blood flow was calculated from two - dimensional and doppler ultrasound data using the equation : q = v  ( d/2 ) , where q is brachial blood flow , v is mean brachial artery blood flow velocity , and d is brachial artery diameter .
imaging of the forearm was performed in a transaxial plane 5 cm distal to be antecubital fossa , using power doppler imaging .
a suspension of microbubbles ( definity ; bristol - myers squibb ) was infused intravenously at a rate of 3.0 ml / min for 10 min . once the systemic microbubble concentration reached steady state ( > 2 min ) , intermittent imaging was then performed at pulsing intervals ranging from 0.5 to 25 s , thus allowing progressively greater replenishment of microbubbles in the ultrasound beam elevation .
several background frames at 0.5 s were averaged and digitally subtracted from similarly averaged frames at each pulsing interval .
background - subtracted video intensity at each pulsing interval was measured from a region of interest placed around the deep forearm flexor muscles .
pulsing interval versus video intensity data were fitted to the function y = a ( 1  e ) , where y is video intensity at pulsing interval t , a is plateau video intensity ( mbv , an index of microvascular recruitment ) , and  is the rate constant that provides a measure of mfv ( the rate of microvascular filling ) ( 20 ) .
data are presented as the means  sem , and statistical analysis was performed using sigmastat ( systat software , 2004 ) .
time - series measurements in each group were performed by two - way repeated - measures anova . when data were not normally distributed , repeated - measures anova on ranks was performed .
when a significant difference of p < 0.05 was found , pairwise comparisons by a student - newman - keuls test was used to assess treatment differences .
subjects remained in bed 1 h before and for the duration of the study and were instructed to refrain from using their arms to minimize effects of movement ( or exercise ) on hemodynamic measurements .
the catheter in the nondominant arm was used for blood sampling of glucose and insulin , and the contralateral arm was used for the infusion of microbubbles .
baseline measurements of total forearm blood flow ( assessed by doppler ultrasound ) and muscle microvascular blood volume ( mbv ) and microvascular flow velocity ( mfv ) ( assessed by ceu ) were performed as described previously ( 4,19 ) .
subjects were then given a liquid meal ( boost ; 480 kcal : 72 from fat , 328 from carbohydrate , and 80 from protein ) .
a mixed meal was chosen to assess the physiological changes that occur in response to a typical meal .
a liquid meal was administered to shorten the amount of time needed for digestion and absorption .
plasma glucose and insulin were measured at 0 , 30 , 60 , 90 , and 120 min .
plasma glucose was measured using a ysi analyzer ( ysi , yellow springs , oh ) .
plasma insulin was measured using an enzyme - linked immunosorbent assay with an interassay coefficient of variation of < 4% . at 60 and again at 120 min after the mixed meal , brachial artery flow ( doppler ultrasound ) and mbv and mfv ( ceu ) were measured .
for the two - dimensional and doppler ultrasound measurements , an ultrasound system ( hdi 5000 ; philips medical systems , bothell , wa ) with a linear - array transducer was used with a transmit frequency of 12 mhz .
two - dimensional imaging of the brachial artery was performed in the long axis 5 cm proximal to the antecubital fold .
images were triggered to the r wave of the cardiac cycle , and the brachial artery diameter was measured using on - line video calipers .
a pulsed - wave doppler sample blood volume was placed at the same location in the center of the artery and the mean blood velocity was measured using online angle correction and analysis software .
brachial artery mean blood flow was calculated from two - dimensional and doppler ultrasound data using the equation : q = v  ( d/2 ) , where q is brachial blood flow , v is mean brachial artery blood flow velocity , and d is brachial artery diameter .
imaging of the forearm was performed in a transaxial plane 5 cm distal to be antecubital fossa , using power doppler imaging .
a suspension of microbubbles ( definity ; bristol - myers squibb ) was infused intravenously at a rate of 3.0 ml / min for 10 min . once the systemic microbubble concentration reached steady state ( > 2 min ) , intermittent imaging was then performed at pulsing intervals ranging from 0.5 to 25 s , thus allowing progressively greater replenishment of microbubbles in the ultrasound beam elevation .
several background frames at 0.5 s were averaged and digitally subtracted from similarly averaged frames at each pulsing interval .
background - subtracted video intensity at each pulsing interval was measured from a region of interest placed around the deep forearm flexor muscles .
pulsing interval versus video intensity data were fitted to the function y = a ( 1  e ) , where y is video intensity at pulsing interval t , a is plateau video intensity ( mbv , an index of microvascular recruitment ) , and  is the rate constant that provides a measure of mfv ( the rate of microvascular filling ) ( 20 ) .
data are presented as the means  sem , and statistical analysis was performed using sigmastat ( systat software , 2004 ) .
time - series measurements in each group were performed by two - way repeated - measures anova .
when data were not normally distributed , repeated - measures anova on ranks was performed .
when a significant difference of p < 0.05 was found , pairwise comparisons by a student - newman - keuls test was used to assess treatment differences .
the lean subjects had significantly lower body weight , fat weight , bmi , percent body fat , waist and hip circumferences , and fasting triglycerides compared with the obese subjects .
plasma glucose , total cholesterol , and ldl cholesterol did not differ between the two groups .
the obese group had higher fasting plasma insulin levels ( p < 0.001 ) and lower insulin sensitivity in the fasting state ( estimated using the quantitative insulin sensitivity check index , a surrogate index of insulin sensitivity ) . although the lean group tended to be more active , there were no significant changes in estimated energy expenditure per day or in total physical activity hours per week ( table 1 ) .
figure 1 shows the time course of plasma glucose and insulin levels after the ingestion of the mixed meal . by 30 min after the meal , plasma glucose levels were elevated above baseline for both lean and obese groups ( fig .
1a ) . over the course of the next 90 min , plasma glucose levels declined toward baseline .
there were no significant differences in the time course of changes in plasma glucose between the two groups .
plasma insulin levels rose significantly by 30 min in the lean group and remained elevated for the additional 90 min of the study ( fig .
the obese group had a significantly higher baseline ( fasting ) plasma insulin level ( p < 0.001 ) and a greater postmeal area under the insulin - time curve than the lean group ( p < 0.05 , data not shown ) .
time courses of plasma glucose and insulin concentrations after the ingestion of a mixed meal in lean (  , n = 8) and obese (  , n = 8) volunteers .
measurements were made every 30 min for 2 h. data are means  sem . *
p < 0.05 vs. 0 min ; p < 0.05 vs. lean ; two - way repeated - measures anova and post hoc student - newman - keuls test .
2 . baseline brachial artery diameters in the lean and obese groups were not significantly different from each other ( fig .
after the meal , the brachial artery in the lean group dilated significantly ( p < 0.05 ) by 60 min and remained dilated at 120 min . in the obese group ,
the brachial artery diameter did not change from baseline and was significantly smaller than that for the lean group at 60 min ( p < 0.05 ) and was almost significant at 120 min ( p = 0.055 ) after the meal ( fig .
brachial artery flow velocity was markedly elevated above baseline ( p < 0.05 ) in the lean group by 120 min after the meal ( fig .
brachial artery blood flow increased by 120 min after the meal in the lean group ( p < 0.05 ) but not in the obese group ( ns ) ( fig .
time courses of brachial artery diameter , brachial artery flow velocity , and total brachial artery blood flow measured by doppler ultrasound after the ingestion of a mixed meal in lean (  , n = 8) and obese (  , n = 8) volunteers .
data are means  sem . * p < 0.05 vs. 0 min ; p < 0.05 vs. lean ; # p = 0.055 vs. lean ; two - way repeated - measures anova and post hoc student - newman - keuls test .
figure 3 shows the effect of the mixed meal on forearm microvascular responses . despite hyperglycemia
, muscle mbv rose significantly by 120 min in the lean group indicating muscle microvascular recruitment ( fig .
in contrast , mbv was unchanged in the obese group ( ns ) ( fig .
when the data from the female subjects were compared , we found that the lean group still showed a significant ( p = 0.02 ) increase in mbv , which was absent in the obese group ( p = 0.45 ) .
this finding suggests that it was obesity and not sex that was the distinguishing feature .
the mbv increase in the lean group was accompanied by a trend for mfv to decline by 120 min ( p = 0.065 ) when compared with baseline ( fig .
mfv did not change in the obese group in response to the meal challenge ( fig .
the product of mbv and mfv ( muscle microvascular perfusion ) did not significantly change in response to the mixed meal in either group ( fig .
time course of forearm mbv , mfv , and mbv  mfv measured by contrast - enhanced ultrasound after the ingestion of a mixed meal in lean (  , n = 8) and obese (  , n = 8) volunteers .
* p < 0.05 vs. 0 min ; # p = 0.065 vs. 0 min ; repeated - measures anova on ranks and post hoc student - newman - keuls test .
the present study demonstrated that the ingestion of a mixed meal significantly increased total muscle blood flow and mbv of lean , middle - aged volunteers and that both of these vascular responses are blunted by obesity .
the meal - induced increase in mbv in the lean volunteers reported here is similar to that reported previously in young , lean adults ( 19 ) . in that study ,
mbv increased even earlier ( 60 min ) than reported in the current study ( 120 min ) .
the somewhat slower response noted here may represent an age - related phenomenon ; however , this has not been tested directly .
the most striking finding of the current study is the absence of any increase in mbv in the obese individuals .
we have noted previously that obesity appears to prevent microvascular recruitment in response to euglycemic hyperinsulinemia ( 4 ) . in that study
, plasma insulin levels were increased to high physiological levels , and total forearm blood flow did not change in either lean or obese subjects ( 4 ) . in the current study ,
the ingestion of a mixed meal increased total limb blood flow in lean but not in obese subjects .
the increase in blood flow noted in the lean subjects serves to emphasize the difference in stimulus of a mixed meal relative to infusion of insulin alone under physiological euglycemic conditions .
scognamiglio et al . ( 22 ) demonstrated that the ingestion of a mixed meal increased myocardial microvascular perfusion in healthy control subjects .
however , myocardial microvascular perfusion actually diminished in the patients with type 2 diabetes . in the current study , we also found an increase in microvascular perfusion in skeletal muscle in response to a mixed meal in lean subjects , and this effect was blunted in the obese subjects .
we did not observe a net decline in microvascular perfusion as reported by scognamiglio et al .
the lack of microvascular response in the obese subjects in response to the meal may simply reflect resistance to the vascular action of insulin as was indicated by the insulin clamp studies in obese subjects ( 4 ) .
however , we can not discount the possibility that obesity altered microvascular responses to other hormonal , nutritional , cardiac , and neural factors that change in the postprandial state .
the lack of a significant change in mbv in the obese group is unlikely because of increased muscle adiposity quenching the microbubble signal .
richards , and s. rattigan , unpublished observations ) that high fat  fed , insulin - resistant rats with increased tissue adiposity have a microbubble dose - response curve similar to that in healthy lean rats .
thus , the effects seen herein are due to changes in microvascular perfusion and not to changes in signal characteristics of the microbubble in vivo .
expanding mbv enhances the surface area of the vascular endothelium , which affords nutrients and hormones , such as insulin , increased access for exchange with the muscle interstitium . for insulin , this entry into the muscle interstitium appears to be rate limiting for overall insulin action within skeletal muscle ( 23,24 ) . in this manner insulin , which signals to the body that feeding has occurred ,
may exert a feed - forward signal to enhance muscle blood flow and thereby increase both its own access to the myocyte as well as that of other nutrients such as glucose and amino acids within the meal . in the current study it remains possible that the failure of insulin to exert a vascular action within the skeletal muscle of obese subjects contributed to their
if these increases in insulin and glucose levels occur chronically , they have the potential to increase hepatic vldl production and raise plasma triglyceride concentrations ( 25 ) , as occurs in insulin - resistant obese subjects ( table 1 ) .
a curious aspect of the current study was the observed increase in forearm blood flow in the lean subjects when muscle microvascular blood flow ( mbv  mfv ) was not altered .
first , total forearm blood flow is measured 510 min before the measurement of microvascular flow ( these measurements can not be done simultaneously because of interference of microbubbles with doppler recording ) .
second , the microvascular blood flow measurements are an integrated value of flow occurring over 5 min of data acquisition , whereas doppler flow measurements are made more acutely over the course of 1030 heartbeats .
third , forearm blood flow includes flow to subcutaneous adipose tissue , muscle , bone , and the hand , whereas the microvascular flow assessment is restricted to blood flow within muscle .
a fourth possibility is that microvascular blood flow within muscle is distributed between nutritive and non - nutritive flow routes , with insulin increasing nutritive flow ( 3 ) . in summary ,
the findings reported here suggest a physiological role for microvascular recruitment provoked by meal ingestion in the process of delivery of nutrients and hormones to skeletal muscle .
failure of this microvascular recruitment , particularly in obese individuals , may be one factor that contributes to postprandial hyperinsulinemia associated with insulin resistance / metabolic syndrome .
 objectiveingestion of a mixed meal recruits flow to muscle capillaries and increases total forearm blood flow in healthy young lean people . 
 we examined whether these vascular responses are blunted by obesity.research design and methodswe fed eight middle - aged lean and eight obese overnight - fasted volunteers a liquid mixed meal ( 480 kcal ) . 
 plasma glucose and insulin were measured every 30 min , and brachial artery flow and muscle microvascular recruitment ( contrast ultrasound ) were assessed every 60 min over 2 h after the meal.resultsby 30 min , plasma glucose rose in both the lean ( 5.1  0.1 vs. 6.7  0.4 mmol / l , p < 0.05 ) and the obese groups ( 5.4  0.2 vs. 6.7  0.4 mmol / l , p < 0.05 ) . 
 plasma insulin rose ( 28  4 vs. 241  30 pmol / l , p < 0.05 ) by 30 min in the lean group and remained elevated for 2 h. the obese group had higher fasting plasma insulin levels ( 65  8 pmol / l , p < 0.001 ) and a greater postmeal area under the insulin - time curve ( p < 0.05 ) . 
 brachial artery flow was increased at 120 min after the meal in the lean group ( 38  6 vs. 83  16 ml / min , p < 0.05 ) but not in the obese group . 
 muscle microvascular blood volume rose by 120 min in the lean group ( 14.4  2.2 vs. 24.4  4.2 units , p < 0.05 ) but not in the obese group.conclusionsa mixed meal recruits muscle microvasculature in lean subjects , and this effect is blunted by obesity . 
 this impaired vascular recruitment lessens the endothelial surface available and may thereby impair postprandial glucose disposal . 
RESEARCH DESIGN AND METHODS
Study visit
Doppler ultrasound
CEU
Statistics
RESULTS
CONCLUSIONS