J. Anim. Sci. 2004. 82:2558-2567
© 2004 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Inhibition of endogenous nitric oxide production influences ovine hindlimb metabolism independently of insulin concentrations1
J. J. Cottrell*,
,
R. D. Warner*,,
M. B. McDonagh and
F. R. Dunshea,
,2
* Victoria University, Werribee, Australia;
and
Department of Primary Industries, Werribee, Australia; and
and
Institute of Land and Food Resources, University of Melbourne, Australia
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Abstract
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The hindlimb arteriovenous difference (AVD) model was used to determine whether 30 mg/kg of the nitric oxide synthase (NOS) inhibitor L-NG-nitroarginine methyl ester (hydrochloride; L-NAME) inhibited ovine NO synthesis and influenced muscle metabolism. Eight Border Leicester x Merino cross lambs (50 to 55 kg BW) were infused with saline (control) or saline containing L-NAME via an indwelling jugular vein catheter in a balanced randomized crossover design with 3 d between treatments. The abdominal aorta and deep femoral vein were catheterized for assessment of AVD of hind limb metabolism. Arterial hematocrit and insulin concentration and both arterial and venous concentrations of nitrate/nitrite (NOx), glucose, lactate, NEFA, and urea were determined. Infusion of L-NAME decreased arterial NOx concentrations (P = 0.049), indicating inhibition of systemic NO synthesis. Treatment had no effect on arterial (3.5 vs. 3.6 ± 0.19 mmol/L for control and L-NAME lambs, respectively; P = 0.39) or venous (3.3 vs. 3.4 ± 0.16 mmol/L, P = 0.55) plasma glucose concentrations or on glucose AVD (0.19 vs. 0.27 ± 0.065 mmol/L, P = 0.20). There was an interaction (P = 0.038) between time and treatment, such that L-NAME initially increased the AVD of glucose (up to 180 m) divergent from control lambs. The response was then decreased before a possible inflection beyond 240 min. Infusion of L-NAME increased hindlimb venous NEFA (222 vs. 272 ± 13.2 µmol/L, P = 0.007) and NEFA AVD (79.4 vs. –13.3 ± 31.5 µmol/L, P = 0.018). These metabolic changes were independent of plasma insulin concentrations, which were not affected by L-NAME infusion (25.3 vs. 27.8 ± 3.62 mU/L, P = 0.85). The increase in hindlimb lipolysis after L-NAME infusion does not seem to be due to increased lipolysis of plasma triacylglycerol because circulating arterial (155 vs. 142 ± 20.8 µmol/L, P = 0.58), venous (154 vs. 140 ± 20.5 µmol/L, P = 0.50), and AVD (1.0 vs. 2.9 ± 3.17 µmol/L, P = 0.38) triacylglycerol concentrations were unaffected by L-NAME infusion. In conclusion, these data indicate that infusion of 30 mg of L-NAME/kg inhibits NO synthesis, which in turn influences fat and carbohydrate metabolism in the ovine hindlimb independently of plasma insulin concentrations.
Key Words: Glucose • Insulin • L-NG-Nitroarginine Methyl Ester (Hydrochloride) • Nitric Oxide • Nonesterified Fatty Acids • Ovine
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Introduction
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The influence of muscle metabolism on meat quality is highlighted by dark cutting and dark firm dry meat, which occur due to decreased conversion of glycogen to lactate after slaughter (Lawrie, 1958
; Pethick et al., 1995). Glycogen depletion can occur as a result of endocrine responses to stress including adrenaline release or increased physical activity (Lacourt and Tarrant, 1985). Because NO influences many physiological pathways in skeletal muscle, including contraction and metabolism, it is hypothesized that the response to stress is partly mediated by NO. The involvement of endogenous NO in influencing muscle contraction has been demonstrated by increases in muscle force on inhibition of endogenous NO production (Kobzik et al., 1994; King-Vanvlack et al., 1995). Other experiments have shown increased skeletal muscle NO synthase (NOS) activity and expression after exercise (Roberts et al., 1999; Tatchum-Talom et al., 2000). Due to the close relationship between muscle contraction and NOS activity, it is likely that increases in NOS activity are coupled with contractile and metabolic responses associated with livestock stress.
Nitric oxide has been demonstrated to have multiple regulatory roles in muscle metabolism. This can occur on a cellular level because NO influences muscle metabolism via endogenous synthesis of NOS within the muscle fiber. This has been demonstrated in hepatic and endothelial mitochondria (Giulivi, 1998; Clementi et al., 1999), rat hepatic glycogenolysis (Moy et al., 1991; Borgs et al., 1996), and muscle glycolysis (Young and Leighton, 1998, Cottrell et al., 2002). Nitric oxide also influences muscle metabolism via affecting insulin sensitivity and increased tissue perfusion (Baron et al., 1995; Sadri and Lautt, 1999). The aims of this experiment were to investigate the effect of i.v. infusion of the NOS inhibitor, L-NG-nitroarginine methyl ester (hydrochloride; L-NAME; Cayman Chemical Co., Ann Arbor, MI), on NO synthesis and ovine hindlimb metabolism before investigating the involvement of NO during stress and meat quality.
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Material and Methods
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Lambs and Surgery
All procedures were approved by the Victorian Institute of Animal Science Animal Ethics Committee. Eight Border Leicester x Merino wethers (50 to 55 kg BW) were housed in individual metabolism crates with ambient lighting. Lambs were fed ad libitum on a pelleted feed (Table 1) dispensed at 3-h intervals via an autofeeder, with supplementation of approximately 100 g of lucerne hay. Catheters were placed in a jugular vein, a medial saphenous artery, and a lateral saphenous vein (Oddy et al., 1987; Mc Donagh et al., 1999) 2 to 3 d preinfusion. A 12-gauge "Dwellcath" (catalog No. 351-365, Sutherland Medical, Melbourne, Australia) was inserted into the jugular vein of conscious lambs, while hindlimb catheterization was performed under halothane (Rhone Merieux, Athens, GA) gas anesthesia (approximately 3% halothane, 0.5 L/min in air) after i.v. administration of 10 to 15 mg of thiobarbital/kg (thiopentone sodium; Durax Pty Ltd., Australia). Catheters were introduced to the lateral saphenous vein, and the tips were placed approximately 40 cm (measured from the insertion point to the pin bone) in the deep femoral vein. Arterial blood was collected from the abdominal aorta via the lateral saphenous artery with an insertion distance of approximately 20 cm (Teleni and Annison, 1986). All lambs were given approximately 15 mg/kg of Engemycin (oxytetracycline; Intervet Australia Pty Ltd., Melbourne, Australia) by intramuscular injection after completion of surgery. Catheter material was 1.50 mm o.d. x 1.00 mm i.d. polyethylene tubing (Dural Plastics and Engineering Pty Ltd., Sydney, Australia).
L-NAME Challenge and Blood Sampling
Saline or 30 mg/kg of L-NAME in saline was administered in a 10-mL bolus via the jugular catheter. The concentration of 30 mg/kg was determined from literature values (Griffith and Lilbourne, 1996) and from a preliminary study wherein concentrations of L-NAME between 10 and 30 mg/kg were infused and effects on plasma glucose and lactate concentrations determined (data not presented). Blood samples (approximately 3 mL) were simultaneously removed from the hindlimb arterial and venous catheters at –60, –30, –15, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, and 360 min relative to the infusion. Hematocrit (%) was determined by removal of a small sample of whole arterial blood by capillary action into a 75-µL heparinized hematocrit tube (Hirschman Laborgerate, Eberstadt, Germany) and centrifugation for 5 min at 3,000 x g (Haematokrit, Hettich Zentrifugen, Tuttlingen, Germany). The remaining blood was dispensed into a heparinized blood tube and centrifuged for 10 min at 4,000 x g (JB-40, Beckman, Fullerton, CA). The plasma was then removed, separated into aliquots, and frozen at –20°C until analyses. Each lamb was used for each treatment (control and L-NAME) by bleeding on two separate experimental days separated by a 3-d "washout" period. Patency of indwelling catheters was maintained between experimental days by daily flushing with heparinized saline solution (50,000 U/L; David Bull Laboratories, Warwick, U.K.). Since heparin increases the activity of lipoprotein lipase, it was not used on the day of infusion (Gartner and Vahouny, 1966; Olivecrona and Egelrud, 1974). Instead, the catheters were flushed with 12.5 g of K2EDTA/L of 0.9% (wt/vol) NaCl after each blood sample to prevent clot formation. Infusions were randomized for each animal and day (refer to statistical design section below).
Biochemical Analysis of Plasma
Plasma nitrate/nitrite (NOx), glucose, lactate, and urea were analyzed as per the manufacturers’ instructions using enzymatic kits (catalog No. 7810001, Cayman Chemical Co., catalog No. 510-A, 735-10 and 640-B, Sigma Aldrich, St. Louis, MO, respectively). Before NOx assay, approximately 400 µL of plasma was ultrafiltered using centrifugal filter units with 10,000-molecular-weight cut offs (Ultrafree MC, Millipore, Billerica, MA) to remove plasma proteins that interfere with the assay (Cayman Chemical Co., catalog No. 781001 kit booklet). The centrifugal filter units were centrifuged for approximately 5 h at 2°C and 6,500 x g with a microcentrifuge (EBA 12, Hettich Zentrifugen). Ultrafiltered plasma was frozen and stored at –20°C until analysis. Plasma NEFA were determined using kits (catalog No. 279-75401, Waco, TX), modified to conduct extra assays by a fivefold dilution of all reagents in 0.025 M phosphate buffer (pH 7.8) (Dunshea and King, 1995). Plasma triacylglycerol (TAG) concentrations were determined spectrophotometrically after incubation with triglyceride infinity reagent (catalog No. 343-25P, Sigma Aldrich) and COBAS MIRA S autoanalyzer (Roche, Basel, Switzerland). Arterial plasma from each lamb was pooled within baseline (–60 to 0 min), acute (15 to 120 min), and semi-acute (150 to 360 min) phases, and insulin concentrations were determined using a kit as per manufacturers guidelines (Amersham Pharmacia, Buckinghamshire, U.K.). Recombinant human insulin was used as a standard and bound I125-insulin was determined with a gamma counter (1277 Gamma Master LKB Wallac, Turku, Finland). Glucose, lactate, NOx, and NEFA assays were conducted using microtiter plates and reader (Titretek Multiscan, Laboratory Systems, Joensuu, Finland). Urea assays were measured using a spectrophotometer with sipper attachment (U-2000, Hitachi Ltd., Tokyo, Japan).
Statistical Analyses
Lambs (n = 8) were given a bolus of saline (control) or L-NAME on two separate days in a balanced randomized block design. The pre-infusion samples (baseline) were averaged to obtain the baseline mean between –60 and 0 min relative to infusion. Data from the response period (15 to 360 min after infusion) were analyzed using ANOVA, using the baseline mean as a covariate and blocks on day of infusion and individual lambs. From these analyses, the effects of time, treatment (control vs. L-NAME), and the interaction between time and treatment were obtained. An exception was the arterial insulin data, which were pooled within baseline (–60 to 0 min), acute (15 to 120 min), and semi-acute (150 to 360 min) phases relative to infusion. These data were analyzed as above with the acute and semi-acute phases as the response periods. All errors were calculated as the standard error of the differences of the means and all statistical analyses performed in Genstat 5.41 (VSN Int. Ltd., Herts, U.K.; Payne and Lane, 1993).
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Results
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Arterial NOx concentrations were decreased by L-NAME treatment (19.0 vs. 16.7 ± 0.94 µM for control and L-NAME respectively, P = 0.049; Figure 1A), whereas venous NOx concentrations were unaffected (19.7 vs. 18.1 ± 1.60, P = 0.37; Figure 1B). There was no effect of treatment on hindlimb NOx AVD (0.62 vs. –1.50 ± 3.016, P = 0.19; Figure 1C). No effects of time or interaction between treatment and time were observed on arterial or venous NOx concentrations. There was no main effect of L-NAME treatment on arterial (3.5 vs. 3.6 ± 0.19 mmol/L, P = 0.39) or venous (3.3 vs. 3.4 ± 0.16 mmol/L, P = 0.55) plasma glucose concentrations and glucose AVD (0.19 vs. 0.27 ± 0.065 mmol/L, P = 0.20; Figures 2A, B). The interaction between L-NAME treatment and time was not significant for arterial (P = 0.54) or venous plasma glucose concentrations (P = 0.99). However, an interaction (P = 0.038) between L-NAME treatment and time was observed, such that the glucose AVD was initially increased in the L-NAME-infused lambs (up to 180 min), whereas control concentrations decreased in the corresponding period. The divergence between control and L-NAME responses diminished or even reversed beyond 240 min (Figure 2C). Arterial plasma insulin concentration was unaffected by L-NAME treatment (25.3 vs. 27.8 ± 3.62 mU/L, P = 0.85) and time (P = 0.85), and no interaction between treatment and time was observed (P = 0.37).
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Figure 1. Responses in A) arterial and B) venous plasma nitrate and nitrite (NOx) or C) arteriovenous difference (AVD) after control or 30 mg/kg L-NG-nitroarginine methyl ester (L-NAME) infusions. A) P = 0.20, 0.049, and 0.82 ± 0.256 for time, treatment, and time x treatment, respectively. B) P = 0.54, 0.37, and 0.71. C) P = 0.026, 0.19, and 0.51.
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Figure 2. Responses in A) arterial and B) venous plasma glucose or C) arteriovenous difference (AVD) after control or 30 mg/kg L-NG-nitroarginine methyl ester (L-NAME) infusions. A) P = 0.98, 0.39, and 0.54 for time, treatment, and time x treatment, respectively. B) P = 0.88, 0.55, and 0.99. C) P = 0.90, 0.20, and 0.038.
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Arterial (0.66 vs. 0.66 ± 0.101 mmol/L, P = 0.99) and venous (0.60 vs. 0.62 ± 0.100 mmol/L, P = 0.83) concentrations and the plasma lactate AVD (0.024 vs. 0.088 ± 0.0487 mmol/L, P = 0.24; Figure 3) were unaffected by L-NAME treatment. Although there was no effect of time on either arterial plasma lactate concentrations (P = 0.65) or lactate AVD (P = 0.38), venous plasma lactate tended (P = 0.060) to increase with time. A short-term increase in arterial lactate concentrations was observed with L-NAME treatment approximately 30 min after infusion, followed by a return to control concentrations as indicated by a moderate interaction (P = 0.072) between time and treatment. Venous (P = 0.41) and AVD (P = 0.11) plasma lactate concentrations were unaffected by the acute increase in arterial plasma lactate concentrations.
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Figure 3. Responses in A) arterial and B) venous plasma lactate or C) arteriovenous difference (AVD) after control or 30 mg/kg L-NG-nitroarginine methyl ester (L-NAME) infusions. A) P = 0.65, 0.99 and 0.072 for time, treatment, and time x treatment, respectively. B) P = 0.060, 0.83, and 0.41. C) P = 0.38, 0.24, and 0.11.
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Whereas arterial plasma NEFA concentrations were unaffected by L-NAME treatment (288 vs. 273 ± 19.0 µmol/L, P = 0.42), L-NAME treatment increased venous plasma NEFA concentrations (222 vs. 272 ± 13.2 µmol/L, P = 0.007) and plasma NEFA AVD (79.4 vs. –13.3 ± 31.5 µmol/L, P = 0.018; Figure 4). There were no effects of time on arterial (P = 0.67) and venous (P = 0.86) plasma concentrations or plasma NEFA AVD (P = 0.75).
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Figure 4. Responses in A) arterial and B) venous plasma NEFA or C) arteriovenous difference (AVD) after control or 30 mg/kg L-NG-nitroarginine methyl ester (L-NAME) infusions. A) P = 0.67, 0.42 and 0.73 for time, treatment, and time x treatment, respectively. B) P = 0.86, 0.007, and 0.063. C) P = 0.75, 0.018, and 0.91.
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Venous NEFA concentrations tended to initially increase then plateau following L-NAME treatment, whereas control plasma NEFA concentrations decreased with time, as indicated by a moderate interaction between treatment and time (Figure 4B
). The interaction between L-NAME treatment and time was not significant for arterial (P = 0.73) or NEFA AVD concentrations (P = 0.91).
Overall, arterial (156 vs. 142 ± 20.8 µmol/L, P = 0.58) and venous (154 vs. 140 ± 20.5 µmol/L, P = 0.50) plasma TAG concentrations were unaffected by L-NAME treatment (Figure 5A, 5B). An acute increase in plasma TAG concentration was observed approximately 15 min following L-NAME treatment (P < 0.001), after which concentrations returned to control levels. The acute increase in arterial TAG concentrations with L-NAME treatment was also reflected in venous TAG concentrations (P < 0.001, Figure 5B). No effects of L-NAME treatment (–1.0 vs. 2.9 ± 3.17 µmol/L, P = 0.23) or time (P = 0.38) were observed on plasma TAG AVD concentrations (Figure 5C). The interaction between time and treatment for the AVD of TAG approached significance (P = 0.088), possibly reflecting a slight inversion with time.
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Figure 5. Responses in A) arterial and B) venous plasma triacylglycerol (TAG) or C) arteriovenous difference (AVD) after control or 30 mg/kg L-NG-nitroarginine methyl ester (L-NAME) infusions. A) P < 0.001, 0.58, and 0.001 for time, treatment, and time x treatment, respectively. B) P = 0.003, 0.50, and <0.001. C) P = 0.38, 0.23, and 0.088.
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Arterial plasma urea concentrations were decreased by L-NAME treatment (7.7 vs. 7.3 ± 0.15 mmol/L, P = 0.011; Figure 6A). There was also a weak interaction (P = 0.099) such that arterial urea concentrations tended to decrease with time following L-NAME infusion. Venous urea concentrations were unaffected by L-NAME treatment (7.8 vs. 7.6 ± 0.19, P = 0.35). There were no effects of L-NAME treatment on plasma urea AVD (–0.10 vs. –0.31 ± 0.188 mmol/L, P = 0.28).
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Figure 6. Responses in A) arterial and B) venous plasma urea or C) arteriovenous difference (AVD) after control or 30 mg/kg L-NG-nitroarginine methyl ester (L-NAME) infusions. A) P = 0.097, 0.011 and 0.099 for time, treatment, and time x treatment, respectively. B) P = 0.14, 0.35, and 0.58. C) P = 0.54, 0.28, and 0.85.
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Arterial plasma hematocrit was not affected by L-NAME (27.4 vs. 27.9 ± 0.79, P = 0.55) but tended to increase with time after infusion (0.21%/h, P = 0.10; data not shown).
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Discussion
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Infusion of 30 mg of L-NAME/kg inhibited systemic NO synthesis, as demonstrated by the decrease in arterial NOx concentrations. Although the data do not indicate decreases in hindlimb NO synthesis, metabolic alterations in the hind limb were observed during systemic NO inhibition. The short-term increase in hindlimb glucose AVD is indicative of increased hindlimb glucose uptake. Venous plasma NEFA concentrations were increased by NOS inhibition, suggesting an increase in lipolysis and/or a decrease in hindlimb NEFA utilization. Increases in glucose uptake and lipolysis were not due to increased insulin concentrations, which were unaffected by NOS inhibition. Changes in systemic metabolism also included decreased urea concentrations, most likely due to altered hepatic amino acid catabolism and/or gluconeogenesis. Although the AVD model used in the current study is an indicator of hindlimb metabolism, further research is required to accurately determine the effect of L-NAME on hindlimb net balance. This would include quantification of blood flow and isotopic tracers to account for metabolite uptake and release.
Increased muscle glucose uptake after infusion of arginine analogs as observed in the current study is not unique. For example, Butler et al. (1998) observed increased glucose uptake and leg blood flow with the arginine analog NG-monomethyl-L-arginine (L-NMMA) in human calf muscle. Also, Balon et al. (1999) observed that chronic oral ingestion of L-NAME for 14 d by rats increased muscle glucose uptake after an insulin challenge, but not under basal conditions. In the same animals, a decreased insulin response to oral glucose challenges was observed, indicating that whereas chronic NOS inhibition increased peripheral insulin responsiveness, insulin production was inhibited. Lajoix et al. (2001) found that pancreatic ß-cells have the neuronal NOS isoform (nNOS) localized in insulin secretory granules and insulin release was stimulated by inhibition of NOS with L-NAME (rather than inhibited), whereas inhibition was observed with the NO donor sodium nitroprusside (SNP). Therefore, the apparent increase in hindlimb glucose uptake in the current study may be a function of increased insulin sensitivity, rather than increased insulin concentrations since the latter were unchanged.
The present data contrast with other experiments that found inhibition of NOS with L-NMMA decreased rat muscle glucose uptake (Baron et al., 1995, 2000; Balon and Nadler, 1997). Also, Young et al. (1997) found that NO donors increased insulin- or contraction-mediated glucose uptake. They proposed that muscle glucose uptake was increased due to increased muscle blood flow mediated by insulin or pharmacological vasodilators. According to Baron et al. (1996), as much as 30% of insulin-dependent glucose uptake can be attributed to increased muscle blood flow. However, Bradley et al. (1999) observed that L-NMMA decreased leg glucose AVD in the exercising human independently of blood flow. In a similar study, Higaki et al. (2001) showed that L-NMMA did not change contraction-induced glucose uptake, whereas SNP increased glucose uptake. From this, Higaki et al. (2001) hypothesized that NO increases glucose uptake in skeletal muscle by a mechanism independent of insulin or contraction. Although our data indicates that there was an increase in hindlimb glucose uptake with NOS inhibition (rather than a NO donor), both experiments indicate the involvement of NO in regulating limb glucose uptake is independent of insulin concentrations.
Although some of these findings may seem contradictory, one must be careful when making direct comparisons between different NOS inhibitors. For example, the arginine analog L-NAME is a more specific and longer-acting inhibitor of nNOS than L-NMMA (Klatt et al., 1996). Studies by Baron et al. (1995, 1996) and Bursztyn et al. (1997) investigated hemodynamic effects of insulin on glucose uptake, mediated by endothelial NOS during L-NMMA-induced vasoconstriction. The choice of L-NAME for the present experiment reflects a different aim: to measure metabolic changes in skeletal muscle after perturbation of metabolic homeostasis by inhibition of nNOS, the most prevalent skeletal muscle isoform of NOS (Nakane et al., 1993; Kobzik et al., 1994).
As discussed previously, an acute increase in hindlimb glucose uptake may be due to increased noninsulin-concentration-dependent glucose uptake by skeletal muscle or increased insulin sensitivity rather than to altered hemodynamics. Although, given that blood flow was not measured the current study, the latter cannot be discounted. In the sheep, approximately 80% of whole body glucose utilization is not insulin dependent (Petterson et al., 1993), and even in tissues that are reliant on insulin, such as skeletal muscle, there is a component of glucose uptake that is not insulin dependent. This component of glucose utilization would presumably increase when glycemia is increased during L-NAME treatment (Gottesman et al., 1983). In addition, expression of glucose transporters on the surface membrane of muscle cells is NO sensitive. Etgen et al. (1997) observed increased glucose uptake due to increased surface expression of glucose transporter-4 glucose receptors in isolated rat epitrochlearis muscles preincubated with the NO donor SNP for 90 min. Although this observation runs counter to our data, it demonstrates that regulation of glucose uptake can be influenced at the cellular level by NO and that experiments in live animals can yield different results than those in isolated muscles.
Arterial and venous plasma lactate concentrations and hindlimb lactate AVD were not significantly affected by infusion of 30 mg of L-NAME/kg, indicating that NOS inhibition did not affect the rate of glucose oxidation in resting, lambs fed ad libitum. Likewise, Licker et al. (1998) observed concentrations up to 100 mg of L-NAME/kg had no influence on circulating lactate concentrations in anaesthetized pigs, indicating that NO does not influence glucose oxidation under basal conditions. However, infusion of L-NAME has been observed to increase lactate concentrations in skeletal muscle postmortem (Cottrell et al., 2002) and in plasma from exercising horses (Mills et al., 1999). Therefore, it is likely that NO inhibits lactate production when glucose oxidation is stimulated.
Venous plasma NEFA concentrations were significantly increased by NOS inhibition, particularly 180 min after infusion. Plasma NEFA are derived from lipolysis of triglycerides in adipose tissue or from hydrolysis of circulating triglycerides (Pethick and Dunshea, 1993). Although the surgical hindlimb preparation is used primarily as a model for skeletal muscle metabolism (Dunshea et al., 1995), the hindlimb of sheep also contains (depending on age, breed, and nutrition) between 5 (Oddy et al., 1984) and 30% (Ulyatt and Barton, 1963) fat tissue. The mechanism initiating increases in venous NEFA concentrations is unlikely to involve plasma TAG hydrolysis since plasma TAG concentrations were not decreased by NOS inhibition. Therefore, it is likely that increased venous NEFA concentrations are due to increased adipose tissue or skeletal muscle TAG hydrolysis or decreased NEFA utilization.
Chronic feeding of rats with an arginine analog has been observed to inhibit the activity of the rate-limiting enzyme of fatty acid oxidation, carnitine palmitoyltransferase, whereas fatty acid synthesis was unaffected. This finding indicated that increases in circulating triglyceride concentrations associated with NOS inhibition were due to decreased fatty acid oxidation (Khedara et al., 1999). Conversely, Picard et al. (2001) observed that decreased fatty acid oxidation during endotoxin induced hypertriglyceridemia was due to inhibition of lipoprotein lipase (LPL) activity in rat skeletal muscle by NO overproduction. In a demonstration that lipolysis is redox sensitive, lipolysis in white adipose tissue was increased and decreased by pharmacological donors of NO yielding the nitrosonium cation (NO+) and NO (Gaudiot et al., 1998), respectively. Other experiments conducted in vivo have linked NO to lipolysis. Fruhbeck and Gomez-Ambrosi (2001) observed elevated levels of plasma NOx during leptin-stimulated lipolysis. It is unknown whether NOS inhibition resulted in substantial LPL inhibition in this experiment, or whether LPL inhibition alone could result in the acute increase in arterial TAG observed or the apparent decrease in NEFA utilization. Likewise the involvement of hormone sensitive lipase cannot be excluded, particularly due to the NO-mediated effects on insulin sensitivity and tissue perfusion reported in other studies.
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Implications
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These results indicate that nitric oxide infusion of 30 mg/kg of L-NG-nitroarginine methyl ester (hydrochloride) is sufficient to inhibit endogenous nitric oxide production in lambs. Furthermore, infusion of L-NG-nitroarginine methyl ester (hydrochloride) altered hindlimb metabolism, specifically resulting in an apparent decrease in nonesterified fatty acid utilization and/or an increase in hindlimb lipolysis and possible decreases in glucose uptake. The effects of L-NG-nitroarginine methyl ester (hydrochloride) may be mediated via a variety of mechanisms, including metabolic changes at a cellular level, altered hormonal sensitivity, or by decreasing hindlimb blood flow. Collectively, these results indicate that infusion of 30 mg/kg of L-NG-nitroarginine methyl ester (hydrochloride) is likely to influence meat quality.
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Footnotes
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1 The funding from Meat and Livestock Australia and the technical assistance of H. Oddy, M. Kerr, B. Doughton, D. Kerton, and K. Perkins are gratefully acknowledged. 
2 Correspondence: Victorian Institute of Animal Science, 600 Sneydes Rd., Werribee 3030, Australia (phone: +61 3 9742 0438; fax: +61 3 9742 0400; e-mail: frank.dunshea{at}nre.vic.gov.au).
Received for publication January 26, 2004.
Accepted for publication April 26, 2004.
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Abstract
Introduction
