Departments of 1 Medicine, 2 Pharmacology, 3 Radiology, 4 Molecular Physiology and Biophysics, and 5 Mouse Metabolic Physiology Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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Isotopic techniques were
used to test the hypothesis that exercise and nitric oxide synthase
(NOS) inhibition have distinct effects on tissue-specific fatty acid
and glucose uptakes in a conscious, chronically catheterized mouse
model. Uptakes were measured using the radioactive tracers
125I-labeled
-methyl-p-iodophenylpentadecanoic acid (BMIPP) and deoxy-[2-3H]glucose (DG) during treadmill exercise with
and without inhibition of NOS. [125I]BMIPP uptake at rest
differed substantially among tissues with the highest levels in heart.
With exercise, [125I]BMIPP uptake increased in both heart
and skeletal muscles. In sedentary mice, NOS inhibition induced by
nitro-L-arginine methyl ester (L-NAME) feeding
increased heart and soleus [125I]BMIPP uptake. In
contrast, exercise, but not L-NAME feeding, resulted in
increased heart and skeletal muscle [2-3H]DG uptake.
Significant interactions were not observed in the effects of combined
exercise and L-NAME feeding on [125I]BMIPP
and [2-3H]DG uptakes. In the conscious mouse, exercise
and NOS inhibition produce distinct patterns of tissue-specific fatty
acid and glucose uptake; NOS is not required for important components
of exercise-associated metabolic signaling, or other mechanisms
compensate for the absence of this regulatory mechanism.
skeletal muscle; fuels; metabolism; lipid; carbohydrate; nitric oxide synthase
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INTRODUCTION |
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EXERCISE IS A MAJOR DETERMINANT of metabolic substrate utilization (23). Nitric oxide (NO) signaling alters metabolism directly and tissue specifically, as well as altering hemodynamic regulation. NO pathways mediate some, but clearly not all, of the tissue-specific metabolic changes occurring with exercise (29). The independent and combined effects of exercise and NO on tissue-specific fatty acid uptake are not well understood.
In this report, we describe concurrent measurement of tissue-specific
fatty acid and glucose uptake in the conscious mouse undergoing rest or
exercise with and without short-term inhibition of NO synthesis. The
interaction of exercise and NO was examined using
125I-labeled
-methyl-p-iodophenylpentadecanoic acid
{[125I]BMIPP, also
[125I]15-(p-iodophenyl)-3(R,S)-methylpentadecanoic
acid}, an agent also used for in vivo studies of fatty acid
uptake in humans, together with deoxy-[2-3H]glucose
([2-3H]DG). The fatty acid analog
[125I]BMIPP is accessible to the cytoplasm,
minimally oxidized in the mitochondria, and not exported from the cell
at an appreciable rate (30, 34). The methodology for
measurement of glucose and fatty acid flux described herein has broad
application to the extensive and continuously expanding set of
genetically altered mouse models.
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METHODS |
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Tracer preparation. BMIPP was a kind gift from Nihon Medi-Physics (Tokyo, Japan). Radioiodination was performed according to the manufacturer's suggested protocol. Briefly, BMIPP was heated in the presence of Na125I solution (740 MBq/200 µl), propionic acid, and copper (II) sulfate. Na2S2O3 was then added and the organic phase ether extracted and sequentially back extracted with saturated NaHCO3 and water. After evaporation, the [125I]BMIPP was solubilized using sonication into ursodeoxycholic acid with a final activity of 775 µCi/ml. An injectate volume of 26 µl (~20 µCi) per mouse was used in these experiments. [2-3H]DG was purchased from New England Nuclear (Boston, MA). Approximately 12 µCi were evaporated and then resuspended in the BMIPP-ursodeoxycholic acid solution immediately before injection to minimize injectate volume.
Mouse maintenance and surgical procedures. All procedures were preapproved by the Vanderbilt University Animal Care and Use Subcommittee. C57BL/6 mice were maintained in microisolater cages with same-gender littermates and fed standard rodent chow ad libitum.
Surgical procedures.
The chronic catheterization procedures are those developed by Niswender
et al. (43) and previously reported from this laboratory (18). Mice were anesthetized with rompun-ketamine (~2
and 2.5 mg/mouse, respectively). The left common carotid artery was
catheterized with a two-part catheter consisting of PE-10 for the
intra-arterial portion and Silastic (0.025 in. OD) for the
extra-arterial portion. The right jugular vein was catheterized with a
one-part Silastic catheter (0.025 in. OD). The free ends of the
catheters were tunneled under the skin to the back of the neck, where
they were attached via stainless steel connectors to lines made of
microrenathane (0.033 in. OD). These lines were exteriorized and sealed
with stainless steel plugs. Lines were cleared daily with saline
containing 200 U/ml heparin and 5 mg/ml ampicillin, meticulously
avoiding air embolus. Animal weight was monitored daily, and animals
were used for experiments once they were within 10% of presurgery
weight and had resumed normal levels of spontaneous activity (3
days). Animals were housed individually after surgery.
Experimental procedures.
Exercise and parallel sedentary studies were performed on 5-h-fasted
mice. Arterial sampling was performed at 0, 10, 15, 20, and 30 min, and
venous injection of the tracers was performed at 5 min (Fig.
1). Mice that underwent exercise did so
on a treadmill at a work rate of 0.6 mph and 0% grade [~80% of
maximal O2 uptake in the mouse(14)] beginning
after the 0-min sample. Immediately after exercise (or at the
corresponding time in the sedentary studies), animals were killed by
injection of phenobarbital. They were then terminally bled by cardiac
puncture, and the intravascular compartment was flushed with a
trans-left ventricular injection of phosphate-buffered saline with a
right atrial exit. Soleus, gastrocneumius, and superficial vastus
lateralis (SVL) muscles and ventricle (heart) and brain were excised
and rapidly freeze-clamped in liquid nitrogen.
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Processing of plasma and muscle samples.
Glucose measurements were made on ~5-µl blood samples (HemoCue,
Mission Viejo, CA). Blood for other measurements was immediately centrifuged and the separated plasma stored at 80°C until analyzed. Serum immunoreactive insulin was measured using a double-antibody method (41). Tissue samples were homogenized into a buffer
containing (in mM) 15 Tris, pH 8, 60 KCl, 15 NaCl, 2 EDTA, 0.15 spermine, 1 dithiothreitol, and 0.4 phenylmethylsulfonyl fluoride.
[125I]BMIPP activity was determined by measuring
-emissions on one aliquot without further treatment, and the
supernatant was recounted after insoluble material was pelleted at
17,000 g for 10 min. Activity was normalized to soluble
protein concentration, determined by a bicinchoninic acid assay (Micro
BCA, Pierce). Hemoglobin concentration was assayed using Drabkin's
Reagent (Sigma Diagnostics, St. Louis, MO). 3H activity was
determined by liquid scintillation counting (Beckman LS 500TD, Beckman
Instruments) with Ecolite+ as scintillant and a dual-channel correction
for
-emissions originating from 125I. One aliquot of
homogenate was counted directly to yield total muscle counts
{[2-3H]DG and 2-deoxy-D-[2-3H]glucose
6-phosphate ([2-3H]DGP)}. A second aliquot was
treated with Ba(OH)2 and ZnSO4 to remove
[2-3H]DGP and was then counted to yield
[2-3H]DG radioactivity. Although most
[2-3H]DG transported into the cell remains in the cell as
[2-3H]DGP, [2-3H]DGP can also serve as a
substrate for glycogen synthesis in skeletal muscle (8,
51). With this particular analytical approach,
[2-3H]DGP incorporated into glycogen is measured in the
same fraction as free [2-3H]DGP, avoiding the
underestimation of [2-3H]DGP that occurs with ion
exchange methods. Nonesterified fatty acid (NEFA) concentrations were
assayed spectrophotometrically (NEFA C Kit, Wako Pure Chemical
Industries, Osaka, Japan).
Calculations. In all experiments, [2-3H]DGP accumulation in tissue was calculated as in Halseth et al. (18). Previous studies have shown that [2-3H]DGP brain levels are not affected by the exercise protocol (18), and we also noted no change in brain [2-3H]DGP with or without L-NAME feeding. Quantitative and qualitative agreement was observed with glucose uptake computed relative to integrated serum concentration and with respect to brain levels (18). Data are presented here normalized to brain for expository simplicity and to avoid the presentation of redundant data. [125I]BMIPP accumulation in tissues was calculated in three ways: 1) disintegrations per minute per microgram of soluble protein, 2) disintegrations per minute per microgram of soluble protein normalized to numerically integrated [125I]BMIPP activity in plasma, and 3) disintegrations per minute per microgram of soluble protein normalized to [125I]BMIPP activity in a fixed volume of infusate. No constant uptake tissue reservoir comparable with brain was available for fatty acid uptake, but the ability to obtain repeated arterial samples enabled accurate quantification of [125I]BMIPP uptake over time. Agreement was observed among these [125I]BMIPP uptake measurements; data are presented normalized to plasma activity integrated over the duration of the experiment, as this should represent the most physiologically meaningful measure. Total tissue NEFA and glucose utilization was calculated by applying a fractional measure of tracer uptake ([125I]BMIPP or [2-3H]DGP) to the integrated serum level of the related substrate (NEFA or glucose, respectively).
Statistical analysis. Differences between groups were determined with Student's t-test or ANOVA (Minitab release 13.20). Unless otherwise noted, data are presented as means ± SE. The significance level was set at P < 0.05.
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RESULTS |
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Characterization of the chronic catheterized mouse model.
Mice ranged from 22 to 32 g. There was no excess mortality or
morbidity associated with the postprocedural administration of
L-NAME. Sampling demonstrated that both metabolic tracers
[2-3H]DG and [125I]BMIPP persisted
throughout the 25-min period of rest or exercise after administration
of the tracers and before the mice were killed (Fig. 1B).
The four groups (control, exercise, L-NAME, exercise + L-NAME), separately or aggregated by L-NAME
treatment, did not differ significantly in weight or gender
distribution. Data showing that L-NAME feeding did not
affect basal glucose, NEFA, or insulin levels are presented in Table
1. NEFA levels at the final measurement were higher in exercising than in sedentary mice (1.06 ± 0.10 vs.
0.77 ± 0.05 mmol/l, P < 0.05). There was a trend
toward higher final insulin levels in exercising mice (0.95 ± 0.22 vs. 0.49 ± 0.12 ng/ml), but differences were not
significant. All animals in the exercise group without
L-NAME completed the full 30-min protocol. However, four of
seven animals in the exercise + L-NAME group
demonstrated exhaustion before completion of the full 30-min treadmill
protocol. The average time to exhaustion in the exercise + L-NAME mice was 26.3 ± 1.5 min, which was
significantly less than the 30-min exercise duration without
L-NAME (P < 0.001).
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[125I]BMIPP uptake and fatty acid flux differed
dramatically among tissues.
[125I]BMIPP uptake at rest, assessed by tissue-specific
activity normalized to soluble protein concentration and corrected for integrated serum activity, differed significantly and substantially among tissues, with a distribution consistent with expected patterns of
fatty acid uptake (P < 0.001 by ANOVA, Table
2). Tissue-specific uptake spanned a
60-fold range. The rates of uptake in brain, which is known to have
minimal fatty acid oxidation, did not differ significantly from
background. The highest rate constants for uptake under sedentary
conditions were observed in heart (77 ± 6 × 10-5 µl serum · µg soluble
protein1 · min
1), a tissue highly
dependent on fatty acid metabolism for energy generation. Levels in
liver and skeletal muscle were intermediate: uptake in soleus, a
predominantly slow muscle, exceeded that in the predominantly
fast-twitch gastrocnemius and SVL muscles. The same pattern was
observed when tissue-specific fatty acid flux was calculated as the
product of fractional tissue-specific BMIPP uptake rate and serum NEFA
levels (Table 3). The intertissue differences remained highly significant (P < 0.001 by
ANOVA).
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Exercise increased tissue [125I]BMIPP uptake and
fatty acid flux.
With exercise, [125I]BMIPP uptake increased more than
twofold in both heart and soleus (Table 2, P < 0.001 and P < 0.002, respectively, and Fig.
2). [125I]BMIPP uptake
increased to a lesser but still statistically significant extent in
gastrocnemius, SVL, and liver (Table 2). The trend toward increased
uptake in liver did not achieve statistical significance. Brain uptake
at rest and with exercise remained negligible. Calculated fatty acid
flux showed a similar magnitude and pattern of effect (Tables 2 and 3).
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NOS inhibition increased [125I]BMIPP uptake and fatty acid flux in muscle. NO signaling has been implicated in tissue-specific direct alterations in metabolic substrate uptake as well as modulation of tissue blood flow (29). Sedentary mice with NOS inhibition induced by L-NAME feeding showed a slightly less than twofold increase in heart and soleus [125I]BMIPP uptake (P < 0.01 and P < 0.05, respectively) compared with sedentary controls (Table 2). In contrast, no statistically significant increase in [125I]BMIPP uptake occurred in liver, and little change occurred in the predominantly fast-twitch gastrocnemius and SVL muscles. The same pattern and relative magnitudes were observed when tissue fatty acid fluxes were calculated, but the higher variance of this derived measurement diminished the significance of the tissue-specific effects (Table 3).
Lack of interaction between L-NAME feeding and exercise. Exercise and L-NAME feeding together produced a greater than threefold increase in heart and soleus [125I]BMIPP uptake compared with sedentary controls not receiving L-NAME (Table 2). Serum NEFA increased modestly under these conditions, consequently increasing calculated fatty acid flux (Table 3). The effects in the gastrocnemius and SVL and liver were less pronounced. ANOVA demonstrated no significant interaction term between L-NAME feeding and exercise in any tissue, consistent with independence of exercise and L-NAME effects.
Exercise, but not L-NAME feeding, resulted in increased
[2-3H]DG uptake.
[2-3H]DG uptake was measured concurrently with tissue
[125I]BMIPP uptake. With exercise, there was an
approximately fourfold increase in [2-3H]DG uptake in
heart and an approximately threefold increase in gastrocnemius
(P < 0.05, Table 4). A
modest increase in [2-3H]DG uptake not reaching
significance was observed in soleus (68%, P = 0.1)
with almost no change in SVL.
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DISCUSSION |
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The metabolic response to exercise in muscle is complex and imperfectly understood (16). One highly conserved component throughout evolution is the accelerated use of alternative energy sources including fatty acids (27). Fatty acids represent a particularly important source of fuel for muscular contraction in heart and skeletal muscle (4, 21). This has therapuetic implications, since substrate preference changes in diabetes and other disease states and there is a higher oxygen cost for generation of energy from fatty acids compared with glucose (25, 36). Ex vivo studies can provide detailed quantitative and mechanistic data regarding metabolic substrate uptake but cannot reproduce the complex interactions underlying the response in vivo.
These data demonstrate a dramatic increase in fatty acid and glucose
tracer uptake in muscle, particularly myocardial and slow-twitch
skeletal muscle, with exercise. The relative magnitude of these changes
may be appreciated in a "polar" presentation (Fig.
3). In this presentation, resting fatty
acid and glucose uptakes, estimated by tissue [125I]BMIPP
and [2-3H]DG, respectively, appear at unity on the
x- and y-axes. The radial spokes then illustrate
fold stimulation in these two variables with exercise,
L-NAME feeding, and exercise with L-NAME
feeding. The experiments in the report did not measure, and this
particular presentation does not reflect, the quantitative importance
of the different substrates in supplying the metabolic energy in each
tissue. However, the polar plot facilitates easy comparison of the
differences in magnitude of stimulation between tissues; lines off the
diagonal represent substrate-specific differences. For example, SVL has
a low capacity for fatty acid use, but exercise increased the amount of
fatty acid uptake to a degree similar to that in soleus. Heart showed a
particularly striking ability to increase both glucose and fatty acid
uptake. The pattern of fatty acid uptake among tissues is very
similar to that reported by Furler et al. (15) using an
alternative tracer agent. The exercise-associated changes in fatty acid
uptake in the mouse are reasonable given corresponding data in humans
(21).
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It is noteworthy that the decreased exercise capacity we observed with L-NAME is consistent with recently reported information on the effects of germline deletion of endothelial-derived NOS, and pharmacological inhibition and augmentation of NO synthesis, on aerobic capacity (39). NO undoubtedly performs several roles at the interface of exercise physiology and metabolism (2, 29, 54). In exercising dogs, Bernstein et al. (5) showed significant elevation of NO production in the coronary circulation, and exercise can increase NO in skeletal muscle (32, 45). Studies in a variety of isolated tissues have consistently demonstrated that increased NO levels can effect a reduction in tissue oxygen consumption, attributed in part to the binding of NO to the heme moiety of cytochrome enzymes (47). Metabolic effects extend beyond this single mechanism. However, the full importance of NO signaling in the overall regulation of exercise metabolic physiology and specific effects on substrate utilization are not well understood. Some studies suggest a surprising divergence of effects in heart and skeletal muscle, whereby NO decreases glucose uptake in heart (5, 10, 44, 48) but increases it in skeletal muscle (3, 13, 52). However, substantial variations in transcardiac glucose measurements have been observed during exercise (5, 44), and in Langendorff mouse hearts little myocardial glucose uptake was noted (48). NO may stimulate glucose uptake through a mechanism that is distinct from both the insulin and contraction pathways (19). Homozygous disruption of the endothelial NOS and neuronal NOS genes in conscious mice resulted in relative insulin resistance, although these studies did not address specific cardiac effects (12, 46).
Our findings obtained in skeletal muscle show that administration of L-NAME did not increase, or even decreased, basal glucose uptake in gastrocnemius and SVL and minimally altered the increase observed with exercise. The lack of change in basal glucose uptake with L-NAME is consistent with earlier ex vivo studies (26). The results with exercise quite possibly reflect a summation of counterbalancing effects, including, for example, the effect of L-NAME on exercise-induced blood flow changes (20). The subacute development of NO inhibition may have also allowed other mechanisms to compensate for the absence of an NO-related regulation. It is unlikely that the difference in exercise duration played a major role, since little difference was observed among those mice that did and did not complete the full 30-min exercise protocol, tissues were obtained immediately after exercise cessation, and the measures of uptake reflect exposure time (18). The changes in fatty acid utilization are a novel and important finding and may play a causal role in the regulation of glucose uptake (or vice versa; reviewed in Ref. 33). The differences among different skeletal muscles could reflect reported differences among different types of muscle fiber types (49). In our study, intermediate-term (72 h) NOS inhibition increased resting cardiac [125I]BMIPP extraction but did not change resting cardiac [2-3H]DG extraction. With addition of exercise, cardiac [125I]BMIPP uptake increased further, whereas the exercise-associated fold increase in [2-3H]DG uptake in heart was somewhat blunted with NOS inhibition. This is similar to the pattern that we observed with skeletal muscle but contrasts with some of the studies cited above (5, 44, 48). Clear differences among these studies include the experimental species, time course of NO inhibition, and, importantly, the model: ex vivo or in vivo and anesthetized or conscious. Although longer-term inhibition of NOS has been associated with alterations in circulating lipids (28), no significant change in basal NEFA was observed in this study. The modest hypertension observed with NO inhibition with the use of the same oral L-NAME dose regimen used this study (24) would be expected to increase cardiac afterload and thus work, affecting metabolic need; its effects on skeletal muscle are less predicable. It is likely that the effects of NOS on muscle metabolism, like those impinging on other aspects of biology, are complex and integrative, arguing strongly for investigating in vivo physiological models (11, 42).
An important consideration in this study is the choice of fatty acid
tracer agent, since "fatty acids" represent a class of compounds.
Cellular utilization and targeting of these specific compounds differ
(35). Long-chain fatty acids are the most important lipid
compounds for energy metabolism under usual dietary conditions; therefore, most tracers represent this class. Even so, commonly used
tracer agents potentially provide readouts of different components of
fatty acid uptake and metabolism. It is notable that, despite significant differences in their tissue retention and -oxidative fates, close agreement in patterns of uptake of various lipid tracers
has been noted, suggesting tight coupling along these lipid metabolic
pathways (7). [125I]BMIPP uptake serves in
this study as the proxy for the physiological variable of fatty acid
uptake, and extensive studies in humans and animals have established
the utility of [125I]BMIPP as a metabolic tracer for
fatty acid utilization (30) (31).
Iodoalkyl-substituted straight-chain fatty acids, such as
17-iodoheptadecanoic acid, are rapidly metabolized in myocardium with
the release of free iodide (30). The radiolabeling of the phenyl group in the
-terminal position opposite the carboxyl prevents nonspecific deiodination, but straight-chain analogs such as
p-iodophenylpentadecanoic acid remain rapidly oxidized and
cleared (9). The inclusion of the methyl group in
addition to iodination of the phenyl group, as in BMIPP, results in
superior tissue retention (30, 34, 50). This has important
practical benefits both for imaging, the most common use of this agent, and for tissue-specific uptake studies such as this report.
[125I]BMIPP may be a particularly advantageous tracer in
the mouse compared with the rat: triglyceride levels are several times
higher in mouse compared with rat heart and have more active turnover (17, 40). This difference means that cellular
esterification of incoming fatty acids in the rat, the cellular process
assessed with BMIPP, is a more sensitive reflection of short-term
changes in fatty acid uptake. It is important that
[125I]BMIPP activity in serum remained readily detectable
throughout the duration of these experiments. The small increase in
serum activity minutes after the bolus injection may reflect some
element of acute redistribution on a finer time scale than typically measured.
Furler et al. (15) employed [3H](R)-2-bromopalmitate ([3H]R-BrP), an alternative labeled fatty acid tracer, in a recent study of tissue-specific NEFA utilization in conscious rats. Like [125I]BMIPP, [3H]R-BrP reflects long-chain fatty acid uptake, and its metabolic products are not rapidly exported (15). These basal patterns of fatty acid flux measured in this study in conscious mice and in Furler et al. in conscious rats show striking similarities (15). Studies that used [125I]BMIPP previously yielded similar organ distribution patterns (6, 7). The methodology used in this study extends these earlier studies in that the chronic catheterization procedures allow for isotope injection and serial [125I]BMIPP measurement without the animal being handled, a potentially major source of stress. This is important because blood [125I]BMIPP is the immediate precursor for the [125I]BMIPP that accumulates in the tissue. Meaningful comparison of tissue [125I]BMIPP uptakes between mice requires that these variables be normalized for circulating radioactivity of this fatty acid analog.
Although there is reason to believe that rough agreement exists among different fatty acid tracers, including [125I]BMIPP (7), direct corroboration would be useful. Such studies could include another class of fatty acid tracer agents, such as fluoro-6-thia-heptadecanoic acid, that primarily measure oxidation (37, 38), although it might be difficult to implement the required positron emission tomographic studies in the mouse. Extrapolation from uptake measurements to absolute measures of tissue energy supply, and comparison among disparate metabolic fuel sources, requires evaluation of the lumped constants for each specific tracer. The limited range of lumped constants for the related tracer [3H]R-BrP in the rat (15) suggests that the rank ordering among the tissues reported here would be maintained after correction, as would the important changes within tissues noted among different conditions. Finally, inhibition of NO synthesis was evaluated at a single time point in this study. Considerable plasticity and homeostatic capacity exists in the NO system (22). To fully understand the interactions of NO signaling and exercise on fatty acid uptake, shorter and longer periods of inhibition will need to be studied, controlling for hemodynamic changes and measuring related variables such as flow.
To conclude, tracer and chronic catheterization approaches allow the measurement of tissue-specific fatty acid and glucose uptake in conscious mice. The methodology measures the expected heart and skeletal muscle increases in fatty acid and glucose uptake with exercise. In contrast, subacute NOS inhibition produced by L-NAME feeding results in increased basal fatty acid uptake in both heart and oxidative skeletal muscle without a substantial increase in glucose uptake. The effects of exercise and NOS inhibition were additive and statistically independent. The approach described here can be applied to the large number of mouse models that are available and can provide further tools for elucidating the regulatory mechanisms governing metabolism during rest and exercise. This, in turn, will provide insights into the pathophysiology of common human disease processes such as insulin resistance and congestive heart failure.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants U24-DK-59637 (Mouse Metabolic Physiology Center) to D. H. Wasserman and J. N. Rottman and DK-54903 to D. H. Wasserman and American Heart Association Grant 9950184N to J. N. Rottman.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. N. Rottman, Div. of Cardiovascular Medicine, Rm. 360 Preston Research Bldg., Vanderbilt Univ. School of Medicine, 23rd Ave. South at Pierce Ave., Nashville, TN 37232-6602 (E-mail: jeff.rottman{at}mcmail.vanderbilt.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 5, 2002;10.1152/ajpendo.00545.2001
Received 7 December 2001; accepted in final form 21 February 2002.
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