While the archetypical role of insulin is that of a metabolic hormone integral for glucose

18 Pages·2007·0.17 MB·English

Diabetes In Press, published online June 11, 2007
Contraction stimulates nitric oxide independent microvascular
recruitment and increases muscle insulin uptake

Received for publication 5 January 2007 and accepted in revised form 1 June 2007.

April C. Inyard, Lucy H. Clerk, Michelle A. Vincent, and Eugene J. Barrett.

Division of Endocrinology and Metabolism, Department of Internal Medicine, University of
Virginia Health System, Charlottesville, VA 22908

Abbreviated title: microvascular recruitment increases muscle insulin

Address correspondence to:
Eugene J Barrett
University of Virginia Health System
P.O. Box 801410
450 Ray C. Hunt Drive
Charlottesville, VA 22908
E-mail: [email protected]

Copyright American Diabetes Association, Inc., 2007
Abstract
Objective: We examined whether contraction-induced muscle microvascular recruitment
would expand the surface area for insulin and nutrient exchange and thereby contribute to
insulin-mediated glucose disposal.
Design/Methods and Results: We measured in vivo rat hindlimb microvascular blood volume
(MBV) using contrast ultrasound and femoral blood flow (FBF) using Doppler ultrasound in
response to a stimulation frequency range. Ten min of 0.1 Hz isometric contraction more than
doubled MBV (P< 0.05; n=6) without affecting FBF (n=7), while frequencies >0.5 Hz increased
both. Specific inhibition of nitric-oxide (NO) synthase with L-NAME (n=5) significantly elevated
mean arterial pressure by ~30 mmHg but had no effect on basal FBF or MBV. We next
examined whether selectively elevating MBV without increasing FBF (0.1 Hz contractions)
increased muscle uptake of albumin-bound Evans Blue dye (EBD). Stimulation at 0.1 Hz (10
min) elicited > 2-fold increases in EBD content (ug EBD/g dry tissue) in stimulated vs
contralateral muscle (n=8; 52.2 ± 3.8 vs 20.0 ± 2.5; P< 0.001). We then measured muscle
uptake of EBD and 125I-insulin (dpm/g dry tissue) with 0.1 Hz stimulation (n=6). Uptake of EBD
(19.1 ± 3.8 vs 9.9 ± 1.0; P< 0.05) and 125I-insulin (5300 ± 800 vs 4244 ± 903; P< 0.05) was
greater in stimulated vs control.
Conclusion: low-frequency contraction increases muscle MBV by a NO-independent pathway
and facilitates muscle uptake of albumin and insulin in the absence of blood flow increases.
This microvascular response may, in part, explain enhanced insulin action in exercising skeletal
muscle.

Keywords: Exercise-muscle contraction, nitric oxide, insulin uptake, capillary recruitment
Exercise reduces the risk of developing skeletal muscle microvasculature by a
insulin resistance and Type 2 diabetes and mechanism similar to insulin is unknown.
improves insulin sensitivity in insulin resistant Dawson et al. (34) examined microvascular
populations (1, 2) through activation of a recruitment in skeletal muscle in response to
number metabolic pathways in skeletal insulin or 2.0 Hz isometric skeletal muscle
muscle. These include AMP-activated kinase contraction and concluded that each can
(AMPK) (3, 4), mitogen-activated protein increase both MBV and total hindlimb femoral
(MAP) kinase (5, 6), Akt (7, 8), and the p70s6k blood flow (FBF). Subsequently, Wheatley et
(9) pathways. These changes potentiate the al. (35) observed that in the obese Zucker rat,
effect of insulin to increase skeletal muscle insulin-mediated recruitment is blunted but
glucose transport (10-13) and uptake (7, 14), 2.0 Hz hindlimb contraction still effectively
increase fatty acid oxidation (15, 16) and recruits microvasculature. This suggests that
increase mitochondrial activity (17, 18). In exercise might recruit microvasculature via a
healthy human volunteers, performing low mechanism that is distinct from that of insulin.
grade exercise during a euglycemic insulin The 2.0 Hz stimulation frequency used in
clamp increases whole body and leg glucose these previous studies (34, 35) increased
uptake to a greater extent than either insulin FBF in addition to recruiting hindlimb
or exercise alone; or even to the sum of their microvasculature. In this study, we
individual effects (19). hypothesized (as had been suggested by
Honig et al. (36) from analysis of tissue
While exercise clearly enhances insulin sections) that low frequency contraction
action on skeletal myocytes, studies suggest effectively increases skeletal muscle MBV
that the rate-limiting step to insulin’s without significantly increasing FBF. This
metabolic actions in skeletal muscle is its would allow us to address the functional
transport from the vasculature to the muscle consequences of microvascular recruitment
interstitium (20, 21). The capillary on the transfer of macromolecules from
endothelium is a physical barrier that plasma into muscle tissue in vivo. We did
macromolecules must cross to access this first using Evans Blue dye (EBD), a
muscle tissue. It is thought insulin is tetrasodium diazo salt (MW 980 Daltons) that
transported in one or more ways including rapidly and irreversibly (37) binds to albumin
passive diffusion (22, 23), vesicular in a 10:1 molar ratio (38) and subsequently
movement (24, 25) and via receptor-mediated by using I-125 labeled insulin as a tracer. In
transcytosis (26). Regardless of the addition, we examined whether either
mechanism of insulin’s transendothelial contraction-induced recruitment, changes in
transport (TET) it remains unknown whether FBF, or muscle EBD uptake were affected by
exercise enhances the uptake of insulin to infusion of the nitric-oxide synthase inhibitor
muscle where it activates multiple metabolic Nω-L-nitro-arginine-methyl ester (L-NAME).
pathways. Research Design and Methods
Animals – Male Sprague-Dawley rats (200–
Insulin increases muscle blood flow and 250 g) were obtained from Charles River
induces microvascular recruitment in both Laboratories (Wilmington, MA), housed at 22
humans (27-29) and animals (30-33). Both ± 2°C with a 12:12 h light:dark cycle and
processes are inhibited by nitric oxide allowed free access to water and standard
synthase (NOS) blockade in rats (33). These chow diet. Rats were fasted overnight prior
insulin-induced increases in microvascular to all experiments. The experimental
blood volume (MBV) expand endothelial protocols followed were approved by the
surface area and may thereby facilitate the Animal Care and Use Committee of the
transfer of nutrients and macromolecules (like University of Virginia.
insulin) from the vasculature to muscle
interstitium. Whether exercise recruits
Surgical Procedures – Animals were hindlimb served as the sham control.
anesthetized with an intraperitoneal (IP) Muscles were stimulated with 2 V square-
injection of sodium pentobarbital (50-55 wave pulse for 0.5 ms resulting in isometric
mg/kg body weight) and placed on a surgical contraction for a very brief duration (Grass
platform. Body temperature was maintained S88 Pulse Generator, Astro-Med, Inc., West
at 38°C with a heating pad. The jugular vein Warwick, RI). The electrical stimulation
and carotid artery were cannulated with frequency was adjusted in a stepwise-manner
polyethylene tubing (PE-50, Intramedic) for every 10 min over a range of 7 frequencies
intravenous infusions, arterial blood sampling (0.0, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 Hz). The
and monitoring of MAP. The animals epigastric vein was cannulated to sample
breathed spontaneously through a blood from the femoral vein of the stimulated
tracheotomy tube for the duration of the leg. Arterial and venous blood samples were
experiment. For experiments where total drawn before and at the end of each bout of
FBF was measured, the femoral vessels in stimulation. Whole blood glucose was
the right hindlimb were exposed as described measured in duplicate using an YSI analyzer
(29, 35) and an ultrasound flow probe (VB (Yellow Springs Instruments, Yellow Springs,
series 0.5 mm; Transonic Systems) was Ohio). Hindlimb glucose uptake was
positioned over the femoral artery. The flow calculated as the arteriovenous (A-V) glucose
probe was interfaced through a flow meter to concentration (mM) difference multiplied by
an IBM-compatible computer. FBF and MAP FBF (mL/min).
were continuously acquired using Windaq
software (Dataq Instruments Inc., Akron OH). Protocol 2: Muscle Evans Blue Dye
Uptake in Response to Low frequency
Protocol 1: Hindlimb Microvascular Contraction – Rats either received a
Recruitment and Glucose Uptake in continuous infusion of saline (n = 8) or L-
Response to Electrical Stimulation – Rats NAME (n = 4) as described for Protocol 1. A
were given either a continuous saline infusion bolus of EBD (25 mg/kg body weight) was
(n = 6) or a continuous infusion of the NOS given intravenously, via jugular vein, and
inhibitor (n = 5), Nω-nitro-L-arginine-methyl allowed to circulate for 5 min before the
ester (L-NAME; 50 ug·min-1·kg-1; Sigma starting low frequency electrical stimulation
Chemicals, St. Louis, MO). We previously (0.1 Hz, 2V, 0.5 ms) of the right hindlimb.
observed that this L-NAME dose raises MAP This frequency was selected because it was
20–30 mmHg above baseline without found in Protocol 1 to increase skeletal
affecting heart rate, and completely inhibits muscle MBV without changing femoral artery
insulin-mediated increases in both FBF and blood flow. After 10 min of low frequency
MBV (33). All animals received a continuous stimulation the animal was sacrificed and
infusion (20 ul/min) of Definity® microbubbles perfused with 120 cc of cold, heparinized
(Bristol-Myers Squibb Medical Imaging Inc., saline retrograde via the carotid artery to
N. Billerica, MA) that began 10 min prior to clear residual intravascular dye. Muscles
and continued throughout the stimulation (gastrocnemius, soleus, gracilis) were
protocol. MBV was measured continuously sampled from both hindlimbs, EBD content
using contrast-enhanced ultrasound (CEU) in measured spectrophotometrically (40) and
response to contraction frequency. In brief, expressed as ug dye per gram of dry tissue.
high-power, pulse-inversion ultrasound (HDI-
5000, Philips Ultrasound) was used to image Protocol 3: Muscle 125I-insulin and Evans
the proximal adductor muscle group Blue Dye Muscle Content in Response to
(adductor magnus and semimembranosus) of Low Frequency Contraction – Rats
the right hindlimb as previously described received a continuous infusion of saline and a
(33, 39). Two metal electrodes were placed bolus of EBD as described in Protocol 2 (n =
in the adductor tendons of the right hindlimb 6). Low-frequency stimulation (0.1 Hz, 2 V,
and the limb was secured in place. The left 0.5 ms) began 5 min after the EBD bolus for
a total duration of 10 min as described for Statistical Analysis – Data are presented as
Protocols 1 and 2. Five min after starting the means ±SEM. Two-way repeated measures
stimulation, rats were given a 1.5 uCi bolus of ANOVA was used to assess the effect of
125I-insulin (Amersham Biosciences, contraction frequency and L-NAME on
Buckinghamshire UK). This mono-iodinated contraction-mediated changes in
species reportedly has insulin receptor microvascular volume. When a significant
binding characteristics similar to native difference of P< 0.05 was found, pair-wise
insulin. This tracer amount of insulin does comparisons were made using Student-
not increase systemic insulin concentrations Newman-Keuls test. Student’s paired t-test
and therefore will not induce muscle was used for statistical comparisons within
microvascular responses. The experiment each treatment group for Protocols 2 and 3.
was terminated as described in Protocol 2 Statistical significance was considered at P<
followed by a systemic ice-cold flush 0.05.
retrograde via the carotid artery clear residual
vascular EBD and 125I-insulin. Results
Gastrocnemius, soleus, and gracilis muscles Protocol 1: Low-frequency electrical
were dissected from the stimulated and the stimulation recruits capillaries by a NO
control contra-lateral hindlimb. Protein bound independent pathway without increasing
125-iodine in blood and muscle samples was femoral blood flow. In response to 10 min of
precipitated using 10% trichloroacetic acid isometric contraction at each of 7 different
and radioactivity measured in a gamma frequencies, we observed a frequency
counter and expressed as dpm per gram of dependent increase in femoral artery blood
dry tissue. EBD muscle content was flow (FBF) in the saline infused animals (n =
measured as described in Protocol 2. 6). FBF was 0.53 ± 0.03 ml/min at baseline
and did not change significantly (0.59 ± 0.05
Immunohistochemistry and Confocal mL/min at 0.05 Hz, 0.64 ± 0.06 mL/min at 0.1
Imaging – Samples of tibialis muscles from Hz) at stimulation frequencies below 0.2 Hz in
protocol 2 animals were snap-frozen in saline infused animals (Fig 1A). However,
isopentane cooled in liquid nitrogen. Frozen FBF increased ~3-fold between 0.2 Hz and
sections (7 µm thickness) were fixed in cold 2.0 Hz (0.77 ± 0.06 mL/min and 2.4 ± 0.26
acetone/methanol and an antibody against mL/min, respectively). There was likewise a
platelet-endothelial adhesion molecule-1 frequency-dependent increase in FBF in the
(PECAM-1; SC-1506 Santa Cruz NOS inhibited group (n = 5) despite the fact
Biosciences, Santa Cruz CA) that L-NAME was infused beginning 30 min
immunocytochemically stained the prior to and continuing throughout the period
vasculature. When exposed to a green laser of electrical stimulation. Mean arterial
(568 nm), EBD fluoresces red. Therefore a pressure (MAP) in the L-NAME group was
green fluorescent secondary antibody ~30 mmHg above (122 vs. 92) the saline
(AlexaFluor 488, Santa Cruz Biosciences, group indicating L-NAME infusion effectively
Santa Cruz CA) was used to visualize inhibited NOS (Fig.1B). With this, there was
PECAM-1 at 488 nm. Immunocytochemical a small (26 beat per min) decline in heart rate
labeling was examined simultaneously using over 45 min which was not statistically
a two-color BX50 WI confocal microscope significant (not shown). In these animals as
(Olympus, Tokyo, Japan) equipped with well, FBF only rose significantly above
krypton and argon laser. The samples were baseline at a frequency of 0.5 Hz (1.95 ± 0.28
scanned at 60x and a series of optical vs. 0.58 ± 0.05 ml/min; P< 0.05, repeated
sections (thickness < 0.1 µm) was acquired measures ANOVA) and increased
from the top to the bottom at a resolution of progressively at higher frequencies. FBF for
1024 x 1024 pixels and stored in 24-bit image the L-NAME group was significantly greater
format. than the saline infused animals at frequencies
of 0.5 Hz and higher resulting in a maximal
6.8-fold as compared to a 3.6-fold increase the frequency dependence for recruitment of
over baseline at 2.0 Hz (Fig.1A). new microvessels is different than that for
Presumably, the higher systemic MAP increases in total femoral flow. This may
contributed to this increase. Baseline indicate that different mechanisms are
hindlimb vascular resistance (mmHg/ml/min) responsible for these two vascular responses.
averaged 171.8 ± 15.4 in the absence of L- Inhibition of NOS with L-NAME was without
NAME and 221.7 ± 29.8 when L-NAME was effect on contraction-induced recruitment.
infused (not shown). In response to electrical Clearly, L-NAME exerted a vascular action
stimulation at 0.1Hz vascular resistance based on the observed increases in MAP
declined in both groups and resistance compared to saline treated animals (122.4 ±
declined further in both groups as stimulation 5.7 vs. 90.0 ± 6.0 mmHg; P< 0.05) and the
frequency increased. higher FBF seen with L-NAME.

The frequency-dependent response for MBV Protocol 2: Low frequency stimulation
(measured by CEU) was markedly different enhances Evans Blue dye uptake by skeletal
from that of FBF (Fig. 2A). There was a muscle. Based on the frequency response
greater than 2-fold increase in MBV in saline observed in Protocol 1, we selected a low
infused animals by 0.1 Hz when compared to stimulation frequency (0.1 Hz) for subsequent
baseline (15.5 ± 4.7 vs. 6.6 ± 1.8 studies of muscle EBD uptake. This low
videointensity units; P< 0.05). MBV only very frequency robustly induced microvascular
modestly increased in response to further recruitment but did not increase FBF
increases in frequency (approximately a 30% compared to baseline (0.59 ± 0.05 vs. 0.53 ±
increase from 0.1 to 2.0 Hz). The frequency- 0.03 ml/min; Fig. 1A). This was separately
dependent response of MBV for the L-NAME confirmed by stimulating the right hindlimb for
group was indistinguishable from that of the 40 min at 0.1 Hz. Again, there was no
saline infused group despite the differences increase in FBF over this longer stimulation
in FBF at contraction frequencies greater duration (0.5 ± 0.1 ml/min at 40 min vs. 0.45
than 0.5 Hz. Microvascular flow velocity ± 0.05 ml/min baseline; data not shown).
(MFV) as estimated from the CEU
replenishment curve indicated that there was In these studies, an intravenous bolus of EBD
no increase at contraction frequencies up to (25 mg/kg) circulated for 5 min before the
0.1 Hz and in the saline infused animals even onset of a 10 min bout of 0.1 Hz stimulation.
a small decline (Fig. 2B). At stimulation Although separate muscle groups were
frequencies > 0.5 Hz, MFV significantly isolated (soleus, tibialis, gracilis) and
increased in both saline and L-NAME infused analyzed, there were no significant
animals compared to baseline (P< 0.05) differences in EBD content noted between
without a significant difference between the fiber types so data for EBD-albumin was
two treatment groups. Inasmuch as MBF is pooled from the 0.1 Hz stimulated samples
approximated by the product of MFV and and compared to contralateral control. As
MBV, these findings are in accord with the shown in Fig. 3 for the saline group (n = 8),
increases in FBF found at higher frequency stimulation at a frequency of 0.1 Hz increased
contractions. Hindlimb glucose uptake did muscle EBD uptake 1.6-fold compared to the
not increase in these studies until higher contralateral control leg (52.2 ± 3.8 vs. 20.0 ±
frequency stimulation and there were no 2.5 ug/g dry tissue; P< 0.001). Electrical
significant differences in glucose uptake stimulation likewise increased EBD content of
between the L-NAME and saline treatment the electrically-stimulated compared to the
groups (2.0 Hz; Fig. 2C). contralateral hindleg in the L-NAME treated
group (n = 4). The muscle EBD content was
These observations suggest that modestly higher with L-NAME infusion in both
microvascular recruitment within skeletal the non-stimulated and stimulated leg when
muscle is stimulated by contraction, and that compared to the saline animals (P = 0.06).
Again, as observed in Protocol 1, the MAP short circulating half-life (<5 min) for intact
was substantially elevated by the L-NAME insulin.
infusion in animals studied under Protocol 2
as compared to saline infused animals (139 ± Discussion
12 vs. 103.± 3 mmHg; P< 0.05), indicating
successful inhibition of NOS by L-NAME. In the present study, we observed that
skeletal muscle contraction increased the
As EBD itself fluoresces red when excited skeletal muscle microvascular volume
with 568 nm frequency laser light, frozen accessible to microbubbles at a lower
sections of skeletal muscle from both 0.1 Hz frequency threshold (0.1 Hz) than that
stimulated and non-stimulated hindlimbs were required to produce sustained increases in
imaged to visualize the location of EBD within total FBF (>0.5 Hz). Our finding that there
the tissue. This was done to ascertain was a 2-3 fold increase in MBV with 10 min of
whether the increased tissue EBD content 0.1 Hz stimulation (Fig. 2B) underscores the
measured in 0.1 Hz stimulated muscle was sensitive nature of microvascular recruitment.
due to increased amounts of EBD remaining Our present data agrees with a previous
in the lumen of recruited microvessels, or report from our laboratory (34) that 2 Hz
represented an increase in EBD taken into stimulation increased MBV ~2-fold in addition
tissue. The vascular endothelium was to increasing FBF. The marked increases in
immunostained with a PECAM-1 antibody. MBV at stimulation frequencies much lower
As shown in Fig. 4, the PECAM-1 antibody than those required to produce any sustained
clearly stains the innermost layer of the increases in total flow suggests that muscle
vessel wall corresponding to the vascular contraction recruits microvessels via a
endothelium. There is little co-localization of mechanism that is distinct from that involving
EBD with PECAM-1. However the red vasodilation of feed arterioles. We previously
fluorescence intensity within muscle fibers (29) reported that incremental increases in
was substantially greater in the stimulated plasma insulin likewise recruits skeletal
versus the non-stimulated skeletal muscle muscle capillaries at insulin concentrations
samples. The dye was primarily lower than that required to increase total
concentrated in the elastin rich perimysium blood flow. However, while insulin-mediated
around the vasculature in non-stimulated increases in MBV are blocked and hindleg
muscle whereas dye reached further into fiber glucose uptake is partially blunted by infusion
bundles in the 0.1 Hz stimulated muscle (data of the nitric oxide inhibitor L-NAME (33), in
not shown). the present study, contraction-mediated
increases are not. While L-NAME at the dose
Protocol 3: low frequency stimulation used inhibits both insulin-induced increases
increases muscle 125I-insulin content. Fig. 5 in MBV and FBF, it had no effect on
compares the skeletal muscle 125I-insulin microvascular recruitment and total flow was
content 5 min after intravenous injection of unchanged or increased in response to low-
1.5 uCi of 125I-insulin in the electrically frequency contraction. We observed a similar
stimulated versus the-lateral leg. Stimulation rise in MAP with L-NAME when given with
at 0.1 Hz significantly increased the tissue systemic insulin (33, 39). Thus, L-NAME
insulin content (5299 ± 800 vs. 4244 ± 903 overcame insulin’s modest dilating action on
dpm/g dry tissue; P< 0.05). In these animals resistance vessels. Compared to insulin,
EBD was also infused beginning 15 min prior exercise is a much more potent stimulus to
to harvesting tissue. We observed a 92% increase flow. It appears the effect of
increase in EBD in the stimulated versus non- exercise to locally vasodilate the exercising
stimulated leg (19.1 ± 3.8 vs. 9.9 ± 1.0 ug/g leg at a time when L-NAME has raised
dry tissue; P< 0.05). The shorter duration of systemic pressure may account for the
insulin infusion was chosen because of the observed flow increase in the stimulated leg.
We cannot exclude other possible systemic
effects (e.g. sympatholysis) of L-NAME which When compared with the non-stimulated
may have contributed to vasodilation in the control leg, it was clear that the 0.1 Hz
stimulated limb. Although differences among stimulated muscle fibers appeared to have a
experimental preparation must be greater amount of EBD within muscle
considered, this agrees with some (41, 42) interstitium. We recognize the empiric nature
but not other (43, 44) previous reports. This of this flushing process. While it is possible
data suggests that low frequency muscle that both the endothelial cell and glycocalyx
contraction acts through a NO-independent of the endothelial lumen could contribute to
mechanism distinct from that induced by some of the dye content measured in muscle
insulin to recruit muscle microvasculature. tissue, some of this may be lost in the
flushing process. However, the confocal
A greater endothelial surface area (i.e. images verified that EBD that entered the
increased MBV) is one of several variables interstitium of skeletal muscle and not dye left
that can promote movement of resident in the expanded microvasculature
macromolecules across the capillary accounted for the increased EBD content.
endothelium. This follows from consideration Our observation that even very low
of Fick’s Law, a simple model for diffusion of frequency, short duration isometric
a solute across a membrane: F=Q(A-I)(1-e- contraction markedly increases microvascular
PS/Q); where F is flux; Q is plasma flow; A and perfusion is consistent with the coupling
I are solute concentrations in the arterial and between motor units and microvascular units
interstitial compartments respectively; P is the proposed by Segal and colleagues (48).
permeability coefficient; and S is the available
surface area (45, 46). From this basic model, Results obtained using 125I-insulin as a tracer
it is apparent that even in the absence of the support the hypothesis that low-frequency
changes of plasma (blood) flow increases in contraction in addition to recruiting capillaries
either surface area or permeability coefficient and promoting albumin movement into
of the endothelial barrier will increase flux. muscle also enhances insulin TET (Fig. 5).
We found that low frequency contraction Recent results from our laboratory indicate
recruitment was accompanied by an that as with albumin, insulin’s movement
approximate 2.5-fold increase in EBD- across the endothelium is a receptor
albumin transfer from the vasculature of the mediated process that may involve caveolae
stimulated compared to the non-stimulated (49). The increase in TET of insulin is less
contralateral hindlimb as measured by dramatic than that for albumin. It is likely that
extravasated EBD content in skeletal muscle this is due at least in part to the shorter time
(Fig. 3). We have no independent measure interval (5 min) selected for the insulin
of the "permeability coefficient" for albumin compared to the 15 min used with EBD. The
movement across the capillary endothelium. 5 min infusion time was selected to minimize
Indeed, the majority of evidence suggests the time available for metabolism of the
that this is a complex process involving a iodinated insulin. We confirmed that at 5 min
receptor-activated, caveolae-mediated post-administration of the 125I-insulin, 90% of
transcytotic process (25, 26, 47) and may not the radioactivity in plasma remained bound to
be well described by the Fick formulation. the insulin (as estimated by TCA
However, the increases we observed in precipitation). This data suggests that low
EBD/albumin transport is consistent with what frequency contraction acutely enhanced the
one would expect based upon the increases transfer of intact iodinated insulin across
in microvascular volume. capillary endothelium as was the case
observed for the EBD-albumin complex.
Immunostaining of frozen skeletal muscle However, our results do not discount the
sections showed that the systemic flush of ice possibility that low frequency contraction may
cold saline was effective in removing EBD also induce changes in the luminal
from the intravascular compartment (Fig. 4). composition of the capillary endothelium (e.g.
altered glycocalyx profile, increased presence contraction might also induce other
of insulin receptors at the membrane, transendothelial transport pathways.
increased numbers of caveolae, etc.), which However, it is clear that low frequency muscle
could also explain the observed increase in contraction can facilitate nutrient and
125I-insulin content in muscle tissue. hormone delivery to skeletal muscle by
providing a greater nutritive flow in vivo. The
In conclusion, our data demonstrates that observation that contraction enhanced insulin
brief (0.5 ms duration) low frequency (0.1 Hz) sensitivity is associated with improved
muscle contraction fully recruits the microcirculation (19) is of potential
microvasculature via a nitric-oxide importance for our understanding of the
independent pathway in the absence of any beneficial effects of acute exercise and this
measurable increase in total blood flow. potential is underscored by recent human
Furthermore, contraction-mediated studies demonstrating dramatic
recruitment promotes the egress of both microvascular recruitment by low-frequency,
albumin and 125I-insulin from the low-intensity exercise (50). These findings
microvasculature to the muscle. L-NAME did may, in part, explain the enhanced insulin-
not inhibit muscle uptake of EBD-bound mediated glucose disposal that accompanies
albumin stimulated by low frequency exercise.
contracted muscle vs. control and even
revealed a modest trend upward. However,
the duration of microvascular recruitment
persistence following acute bouts of exercise Acknowledgements – The University of
or contraction is currently unknown and it Virginia Histology Core assisted in the
remains to be determined whether NOS preparation of frozen sections of muscle
inhibition will have an effect on 125I-insulin tissue. We thank Dr. Brian Duling and Ms.
content in 0.1 Hz stimulated vs. control Katherine Day for help with the confocal
skeletal muscle. While our results suggest imaging. This work was supported by NIH
this enhanced transport of both albumin and grants DK-57878, DK063609 (EJB) and a
125I-insulin is associated with increased pre-doctoral fellowship from the American
capillary surface area (MBV) we cannot Heart Association 0615460U(AI).
exclude the possibility that low frequency
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