Contrasting effects of exercise and NOS inhibition on tissue-specific fatty acid and glucose uptake in mice

Jeffrey N. Rottman1,2,5, Deanna Bracy4,5, Carlo Malabanan4,5, Zou Yue1, Jeff Clanton3, and David H. Wasserman4,5

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

After recovery from surgery, mice received either normal water ad libitum or water containing 1 mg/ml (approx 100-120 mg · kg-1 · day-1) of Nomega -nitro-L-arginine methyl ester (L-NAME; Sigma) for 3 days. This dose and duration were chosen to effect near-maximal but reversible inhibition of NO synthesis (1, 24, 53) but not result in the change in body temperature associated with acute intravenous NO synthase (NOS) inhibition (5). Animal weight, activity, and grooming were monitored, and no difference was observed between control and L-NAME-fed mice. The day before studies, mice were acclimated to treadmill running at the intensity used during experiments with one 10-min bout of exercise.

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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Experimental protocol (top) and relative serum activity (bottom). Tracers 125I-labeled beta -methyl-p-iodophenylpentadecanoic acid ([125I]BMIPP) and deoxy-[2-3H]glucose ([3H]DG) were injected at 5 min and sampled as shown. NEFA, nonesterified fatty acids. Serum tracer values are relative to the maximal measurement per animal. Curves did not differ significantly between protocol conditions and are therefore aggregated. Data shown are means ± SE.

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 gamma -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 beta -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Basal metabolic paramters of mice by L-NAME feeding

[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 protein-1 · 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).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Tissue-specific [125I]BMIPP extraction


                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Calculated tissue-specific fatty acid flux

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).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Tissue-specific [125I]BMIPP uptake. Lines connect similar protocol conditions (rest/exercise), with closed/open symbols representing presence and absence of nitric oxide synthase (NOS) inhibition by nitro-L-arginine methyl ester (L-NAME) feeding, respectively. Ex/L-NAME, exercise + L-NAME; Gastroc, gastrocnemius; SVL, superficial vastus lateralis. Means ± SE are shown. *** P < 0.001, ** P < 0.01, * P < 0.06=5 for exercise effect by ANOVA; ++P < 0.01, +P < 0.05 for L-NAME effect by ANOVA.

To test whether the observed increase in tissue [125I]BMIPP activity could be attributed to an exercise-induced increase in blood volume, tissue hemoglobin content was also measured. Measurement of tissue hemoglobin content in conjunction with measurement of blood tracer concentrations demonstrated that a negligible fraction (<5%) of the changes in flux that were observed could be attributed to a change in tissue blood volume.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Tissue-specific [3H]DGP uptake

In contrast to BMIPP, no significant changes occurred in [2-3H]DG uptake in sedentary mice fed L-NAME. The changes in [2-3H]DG uptake with exercise and L-NAME were similar to those occurring with exercise alone (no significant L-NAME effect and no significant interaction with exercise by ANOVA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Fold stimulation of [125I]BMIPP and [3H]DG extraction. * P < 0.05 vs. sedentary [125I]BMIPP uptake; +P < 0.05 vs. sedentary [3H]DG uptake.

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 beta -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 omega -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arnal, JF, Warin L, and Michel JB. Determinants of aortic cyclic guanosine monophosphate in hypertension induced by chronic inhibition of nitric oxide synthase. J Clin Invest 90: 647-652, 1992[ISI][Medline].

2.   Balon, TW. Integrative biology of nitric oxide and exercise. Exerc Sport Sci Rev 27: 219-253, 1999[Medline].

3.   Balon, TW, and Nadler JL. Evidence that nitric oxide increases glucose transport in skeletal muscle. J Appl Physiol 82: 359-363, 1997[Abstract/Free Full Text].

4.   Barger, PM, and Kelly DP. PPAR signaling in the control of cardiac energy metabolism. Trends Cardiovasc Med 10: 238-245, 2000[ISI][Medline].

5.   Bernstein, RD, Ochoa FY, Xu X, Forfia P, Shen W, Thompson CI, and Hintze TH. Function and production of nitric oxide in the coronary circulation of the conscious dog during exercise. Circ Res 79: 840-848, 1996[Abstract/Free Full Text].

6.   Binas, B, Danneberg H, McWhir J, Mullins L, and Clark AJ. Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization. FASEB J 13: 805-812, 1999[Abstract/Free Full Text].

7.   Coburn, CT, Knapp FF, Jr, Febbraio M, Beets AL, Silverstein RL, and Abumrad NA. Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice. J Biol Chem 275: 32523-32529, 2000[Abstract/Free Full Text].

8.   Colwell, DR, Higgins JA, and Denyer GS. Incorporation of 2-deoxy-D-glucose into glycogen. Implications for measurement of tissue-specific glucose uptake and utilisation. Int J Biochem Cell Biol 28: 115-121, 1996[ISI][Medline].

9.   Corbett, JR. Fatty acids for myocardial imaging. Semin Nucl Med 29: 237-258, 1999[ISI][Medline].

10.   Depre, C, Gaussin V, Ponchaut S, Fischer Y, Vanoverschelde JL, and Hue L. Inhibition of myocardial glucose uptake by cGMP. Am J Physiol Heart Circ Physiol 274: H1443-H1449, 1998[Abstract/Free Full Text].

11.   Drexler, H. Nitric oxide synthases in the failing human heart: a doubled-edged sword? Circulation 99: 2972-2975, 1999[Free Full Text].

12.   Duplain, H, Burcelin R, Sartori C, Cook S, Egli M, Lepori M, Vollenweider P, Pedrazzini T, Nicod P, Thorens B, and Scherrer U. Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase. Circulation 104: 342-345, 2001[Abstract/Free Full Text].

13.   Etgen, GJ, Jr, Fryburg DA, and Gibbs EM. Nitric oxide stimulates skeletal muscle glucose transport through a calcium/contraction- and phosphatidylinositol-3-kinase-independent pathway. Diabetes 46: 1915-1919, 1997[Abstract].

14.   Fernando, P, Bonen A, and Hoffman-Goetz L. Predicting submaximal oxygen consumption during treadmill running in mice. Can J Physiol Pharmacol 71: 854-857, 1993[ISI][Medline].

15.   Furler, SM, Cooney GJ, Hegarty BD, Lim-Fraser MY, Kraegen EW, and Oakes ND. Local factors modulate tissue-specific NEFA utilization: assessment in rats using 3H-(R)-2-bromopalmitate. Diabetes 49: 1427-1433, 2000[Abstract].

16.   Goodyear, LJ, and Kahn BB. Exercise, glucose transport, and insulin sensitivity. Annu Rev Med 49: 235-261, 1998[ISI][Medline].

17.   Hajri, T, Ibrahimi A, Coburn CT, Knapp FF, Jr, Kurtz T, Pravenec M, and Abumrad NA. Defective fatty acid uptake in the spontaneously hypertensive rat is a primary determinant of altered glucose metabolism, hyperinsulinemia, and myocardial hypertrophy. J Biol Chem 276: 23661-23666, 2001[Abstract/Free Full Text].

18.   Halseth, AE, Bracy DP, and Wasserman DH. Overexpression of hexokinase II increases insulin and exercise-stimulated muscle glucose uptake in vivo. Am J Physiol Endocrinol Metab 276: E70-E77, 1999[Abstract/Free Full Text].

19.   Higaki, Y, Hirshman MF, Fujii N, and Goodyear LJ. Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle. Diabetes 50: 241-247, 2001[Abstract/Free Full Text].

20.   Hirai, T, Visneski MD, Kearns KJ, Zelis R, and Musch TI. Effects of NO synthase inhibition on the muscular blood flow response to treadmill exercise in rats. J Appl Physiol 77: 1288-1293, 1994[Abstract/Free Full Text].

21.   Horowitz, JF, and Klein S. Lipid metabolism during endurance exercise. Am J Clin Nutr 72: 558S-563S, 2000[Abstract/Free Full Text].

22.   Huang, PL. Mouse models of nitric oxide synthase deficiency. J Am Soc Nephrol 11, Suppl16: S120-S123, 2000[ISI][Medline].

23.   Jones, NL, and Killian KJ. Exercise limitation in health and disease. N Engl J Med 343: 632-641, 2000[Free Full Text].

24.   Kaikita, K, Fogo AB, Ma L, Schoenhard JA, Brown NJ, and Vaughan DE. Plasminogen activator inhibitor-1 deficiency prevents hypertension and vascular fibrosis in response to long-term nitric oxide synthase inhibition. Circulation 104: 839-844, 2001[Abstract/Free Full Text].

25.   Kantor, PF, Lucien A, Kozak R, and Lopaschuk GD. The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase. Circ Res 86: 580-588, 2000[Abstract/Free Full Text].

26.   Kapur, S, Bedard S, Marcotte B, Cote CH, and Marette A. Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action. Diabetes 46: 1691-1700, 1997[Abstract].

27.   Kemp, BE, Mitchelhill KI, Stapleton D, Michell BJ, Chen ZP, and Witters LA. Dealing with energy demand: the AMP-activated protein kinase. Trends Biochem Sci 24: 22-25, 1999[ISI][Medline].

28.   Khedara, A, Kawai Y, Kayashita J, and Kato N. Feeding rats the nitric oxide synthase inhibitor, L-N(omega)nitroarginine, elevates serum triglyceride and cholesterol and lowers hepatic fatty acid oxidation. J Nutr 126: 2563-2567, 1996[ISI][Medline].

29.   Kingwell, BA. Nitric oxide-mediated metabolic regulation during exercise: effects of training in health and cardiovascular disease. FASEB J 14: 1685-1696, 2000[Abstract/Free Full Text].

30.   Knapp, FF, Jr, and Kropp J. BMIPP-design and development. Int J Card Imaging 15: 1-9, 1999[Medline].

31.   Knapp, FF, Jr, Kropp J, Franken PR, Visser FC, Sloof GW, Eisenhut M, Yamamichi Y, Shirakami Y, Kusuoka H, and Nishimura T. Pharmacokinetics of radioiodinated fatty acid myocardial imaging agents in animal models and human studies. Q J Nucl Med 40: 252-269, 1996[Medline].

32.   Kobzik, L, Reid MB, Bredt DS, and Stamler JS. Nitric oxide in skeletal muscle. Nature 372: 546-548, 1994[ISI][Medline].

33.   Kraegen, EW, Cooney GJ, Ye J, and Thompson AL. Triglycerides, fatty acids and insulin resistance. Exp Clin Endocrinol Diabetes 109: 516-526, 2001.

34.   Kropp, J, Eisenhut M, Ambrose KR, Knapp FF, Jr, and Franke WG. Pharmacokinetics and metabolism of the methyl-branched fatty acid (BMIPP) in animals and humans. J Nucl Med 40: 1484-1491, 1999[Abstract].

35.   Leaf, A. Transepithelial transport and its hormonal control in toad bladder. Ergeb Physiol Biol Chem Exp Pharmakol 56: 216-263, 1965[Medline].

36.   Lewandowski, ED. Metabolic mechanisms associated with antianginal therapy. Circ Res 86: 487-489, 2000[Free Full Text].

37.   Mäki, MT, Haaparanta M, Nuutila P, Oikonen V, Luotolahti M, Eskola O, and Knuuti JM. Free fatty acid uptake in the myocardium and skeletal muscle using fluorine-18-fluoro-6-thia-heptadecanoic acid. J Nucl Med 39: 1320-1327, 1998[Abstract].

38.   Mäki, MT, Haaparanta MT, Luotolahti MS, Nuutila P, Voipio-Pulkki LM, Bergman JR, Solin OH, and Knuuti JM. Fatty acid uptake is preserved in chronically dysfunctional but viable myocardium. Am J Physiol Heart Circ Physiol 273: H2473-H2480, 1997[Abstract/Free Full Text].

39.   Maxwell, AJ, Ho HV, Le CQ, Lin PS, Bernstein D, and Cooke JP. L-Arginine enhances aerobic exercise capacity in association with augmented nitric oxide production. J Appl Physiol 90: 933-938, 2001[Abstract/Free Full Text].

40.   Menahan, LA, and Sobocinski KA. Comparison of carbohydrate and lipid metabolism in mice and rats during fasting. Comp Biochem Physiol B Biochem Mol Biol 74: 859-864, 1983[ISI].

41.   Morgan, CR, and Lazarow AL. Immunoassay of insulin: two antibody system plasma insulin of normal, subdiabetic, and diabetic rats. Am J Med Sci 257: 415-419, 1963.

42.   Mu, J, Brozinick JT, Jr, Valladares O, Bucan M, and Birnbaum MJ. A role for AMP-activated protein kinase in contrac. Mol Cell 7: 1085-1094, 2001[ISI][Medline].

43.   Niswender, KD, Shiota M, Postic C, Cherrington AD, and Magnuson MA. Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism. J Biol Chem 272: 22570-22575, 1997[Abstract/Free Full Text].

44.   Recchia, FA, McConnell PI, Bernstein RD, Vogel TR, Xu X, and Hintze TH. Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog. Circ Res 83: 969-979, 1998[Abstract/Free Full Text].

45.   Roberts, CK, Barnard RJ, Jasman A, and Balon TW. Acute exercise increases nitric oxide synthase activity in skeletal muscle. Am J Physiol Endocrinol Metab 277: E390-E394, 1999[Abstract/Free Full Text].

46.   Shankar, RR, Wu Y, Shen HQ, Zhu JS, and Baron AD. Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance. Diabetes 49: 684-687, 2000[Abstract].

47.   Stadler, J, Billiar TR, Curran RD, Stuehr DJ, Ochoa JB, and Simmons RL. Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am J Physiol Cell Physiol 260: C910-C916, 1991[Abstract/Free Full Text].

48.   Tada, H, Thompson CI, Recchia FA, Loke KE, Ochoa M, Smith CJ, Shesely EG, Kaley G, and Hintze TH. Myocardial glucose uptake is regulated by nitric oxide via endothelial nitric oxide synthase in Langendorff mouse heart. Circ Res 86: 270-274, 2000[Abstract/Free Full Text].

49.   Tikunov, B, Levine S, and Mancini D. Chronic congestive heart failure elicits adaptations of endurance exercise in diaphragmatic muscle. Circulation 95: 910-916, 1997[Abstract/Free Full Text].

50.   Torizuka, K, Yonekura Y, Nishimura T, Tamaki N, Uehara T, Ikekubo K, and Hino M. [A Phase 1 study of beta-methyl-p-(123I)-iodophenyl-pentadecanoic acid (123I-BMIPP)]. Kaku Igaku 28: 681-690, 1991[Medline].

51.   Virkamaki, A, Rissanen E, Hamalainen S, Utriainen T, and Yki-Jarvinen H. Incorporation of [3-3H]glucose and 2-[1-14C]deoxyglucose into glycogen in heart and skeletal muscle in vivo: implications for the quantitation of tissue glucose uptake. Diabetes 46: 1106-1110, 1997[Abstract].

52.   Young, ME, Radda GK, and Leighton B. Nitric oxide stimulates glucose transport and metabolism in rat skeletal muscle in vitro. Biochem J 322: 223-228, 1997[ISI][Medline].

53.   Zatz, R, and Baylis C. Chronic nitric oxide inhibition model six years on. Hypertension 32: 958-964, 1998[Free Full Text].

54.   Zhao, G, Bernstein RD, and Hintze TH. Nitric oxide and oxygen utilization: exercise, heart failure and diabetes. Coron Artery Dis 10: 315-320, 1999[ISI][Medline].


Am J Physiol Endocrinol Metab 283(1):E116-E123
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society