Department of Biochemistry, Medical School, University of Tasmania, Hobart, Tasmania 7001, Australia
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ABSTRACT |
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Triglyceride hydrolysis by the perfused
rat hindlimb is enhanced with serotonin-induced nonnutritive flow (NNF)
and may be due to the presence of nonnutritive route-associated
connective tissue fat cells. Here, we assess whether NNF influences
muscle uptake of 0.55 mM palmitate in the perfused hindlimb.
Comparisons were made with insulin-mediated glucose uptake. NNF induced
during 60 nM insulin infusion inhibited hindlimb oxygen uptake from
22.0 ± 0.5 to 9.7 ± 0.8 µmol · g1 · h
1
(P < 0.001), 1-methylxanthine metabolism (indicator of
nutritive flow) from 5.8 ± 0.4 to 3.8 ± 0.4 nmol · min
1 · g
1
(P = 0.004), glucose uptake from 29.2 ± 1.7 to
23.1 ± 1.8 µmol · g
1 · h
1
(P = 0.005) and muscle 2-deoxyglucose uptake from
82.1 ± 4.6 to 41.6 ± 6.7 µmol · g
1 · h
1
(P < 0.001). Palmitate uptake, unaffected by insulin
alone, was inhibited by NNF in extensor digitorum longus, white
gastrocnemius, and tibialis anterior muscles; average inhibition was
from 13.9 ± 1.2 to 6.9 ± 1.4 µmol · g
1 · h
1
(P = 0.02). Thus NNF impairs both fatty acid and
glucose uptake by muscle by restricting flow to myocytes but, as shown
previously, favors triglyceride hydrolysis and uptake into nearby
connective tissue fat cells. The findings have implications for lipid
partitioning in limb muscles between myocytes and attendant adipocytes.
acid uptake, nutritive flow, oxygen consumption, perfusion pressure
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INTRODUCTION |
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REPORTS BY A NUMBER OF LABORATORIES that vasoconstrictors substantially influence metabolism of the constant-flow perfused rat hindlimb (12, 17, 30, 34) have drawn attention to the possibility that flow redistribution may be responsible for these effects. Consistent with this, a number of our studies (see review in Ref. 6) and those of others (24, 32) have shown that oxygen uptake by the hindlimb is markedly affected by the proportion of nutritive to nonnutritive blood flow. The balance of flow between the two routes, nutritive and nonnutritive, is controlled by vasoconstrictors that act at different sites in the vascular tree to regulate relative flow distribution (6, 7). Vasoconstrictors that mediate such redistribution have been categorized into those that recruit nutritive flow (e.g., low-dose norepinephrine; angiotensins I, II, and III; and vasopressin) and those that recruit nonnutritive flow (e.g., serotonin) in a constant total flow pump-perfused preparation (6).
Physical evidence for the concept of two vascular routes in muscle comes from a number of studies that we have undertaken and includes vascular casting (21), FITC-dextran entrapment as a marker for recruited and derecruited vascular space (21), surface fluorescence measurement of tendon vessels when perfused with a fluorescent vascular marker (22), microdialysis out-to-in ratio of 3H2O and [14C]ethanol (23), laser Doppler flowmetry (5), and microsphere embolism with use of latex microspheres of different sizes (38). However, the precise anatomic nature of the nonnutritive route of muscle is still unresolved, despite knowledge of its presence for 70 years (24). There is evidence that it has access to connective tissue of tendons (1, 15), and a recent laser Doppler flowmetry study that used randomly placed microprobes into various muscles suggests that the nonnutritive route is homogeneously distributed within each muscle (5).
In a previous study (9), we showed that, when flow was predominantly nonnutritive during a state of high vascular resistance induced by serotonin, overall chylomicron triglyceride hydrolysis by the perfused rat hindlimb was increased. This may be due to flow being directed to lipoprotein lipase-rich fat cells located on the connective tissue vessels that constitute some of the nonnutritive route of muscle. This implies that fat cells distributed on the interfibrillar connective tissue of the endomysium, perimysium, and epimysium receive nutrient from the nonnutritive route of skeletal muscle, even though their metabolic activity is small compared with muscle. It follows that, when the proportion of nutritive to nonnutritive flow is altered, fuel and hormones are differently partitioned. Thus, in the present study, we examine whether fatty acid and glucose uptake by the constant-flow perfused rat hindlimb in the presence of insulin is influenced by a decrease in the nutritive-to-nonnutritive ratio.
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MATERIALS AND METHODS |
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Animals. Animals were cared for in accordance with the principles of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990, Australian Government Publishing Service, Canberra). Experimental procedures were approved by the Animal Ethics Committee of the University of Tasmania. Males of a local strain of hooded Wistar rats (140-160 g) were housed at 22°C on a 12:12-h light-dark cycle and allowed free access to water and a commercial rat chow (Gibsons, Hobart, Australia) containing 21.4% protein, 4.6% lipid, 68% carbohydrate, and 6% crude fiber with added vitamins and minerals. Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (5-6 mg/100 g body wt) before all surgical procedures.
Free fatty acid solution.
Palmitic acid (369.2 mg), 1.4 ml of 1 M NaOH, 28 ml of distilled
H2O, and, when required, 80 µCi
[14C]palmitic acid (55 mCi/mmol, Amersham) were heated at
75-90°C for 20 min or until saponified. The solution was then
cooled to 50-60°C before the addition of Krebs-Ringer
bicarbonate buffer (90.6 ml) containing 6% (wt/vol) BSA. Finally, the
mixture was passed through a 1.2-µm filter and stored at 20°C
until used. [3H]mannitol was used as a marker for the
extracellular space for the palmitate uptake perfusions. A stock
solution of 65 mM mannitol and 40 µCi [3H]mannitol
(19.7 Ci/mmol, NEN) was prepared. Analysis of the filtered palmitate
solution indicated it to be ~4.9 mM, with a fatty acid-to-albumin ratio of 7.2.
2-Deoxy-[3H]glucose solution. Although the arteriovenous difference in glucose may be used to calculate hindleg glucose uptake, this measurement does not exclude the amounts taken up by bone, skin, fat, and other tissues present in minimal amounts in the preparation. Similarly, the uptake of 2-deoxyglucose measures glucose uptake into the excised hindlimb muscles, which can include uptake by attendant fat cells, even though this may be relatively trivial because of the mass difference favoring muscle. To measure 2-deoxyglucose uptake, a solution of 90 µCi 2-deoxy-[3H]glucose (2-DG; 43 Ci/mmol, Amersham), 2 mM sucrose, and 50 µCi [14C]sucrose (565 mCi/mmol, Amersham) was prepared in saline. [14C]sucrose was used in these perfusions to correct for volume in the extracellular space.
Hindlimb perfusions. Hindlimb surgery was essentially as described by others (33), with additional details given previously (11). The left hindlimb was perfused in a nonrecirculating mode with 6% (wt/vol) Ficoll (Amersham Pharmacia Biotech) containing Krebs-Ringer bicarbonate buffer and 1.27 mM CaCl2 at 8 ml/min; the total flow was always maintained constant at this value. The buffer was continually gassed via a Silastic tube oxygenator with carbogen (95% O2-5% CO2), and the temperature was maintained at 37°C in a heat-exchanger coil. The rat and apparatus were in a temperature-controlled cabinet at 37°C. Details for continuous monitoring of perfusion pressure and for determining oxygen uptake from continuous recording of venous PO2 are given elsewhere (9).
2-DG uptake.
After completion of the surgical procedure, the rat hindlimb was
perfused for 40 min with 6% Ficoll-Krebs buffer, pH 7.4, containing
8.3 mM glucose as well as other components to be described. Insulin
(Humulin, Aza Research) or vehicle infusions were then commenced and
maintained for the entire experiment (Fig.
1A). The actual insulin
concentration as determined by ELISA (Mercodia, Uppsala, Sweden) was 60 nM and deliberately chosen to achieve maximal response. The infusion of
serotonin (5-HT; 3 µM final concentration) or vehicle was commenced
at 50 min and continued for the entire experiment. At 60 min, the
buffer reservoir was changed and then, while the infusion of insulin
and 5-HT or vehicle was maintained, the rat hindlimb was perfused with
medium containing the following constituents: 26 ml unlabeled palmitic
acid solution, 1.3 ml 2-DG solution, 232 ml gassed 6% Ficoll-Krebs
buffer, and 0.65 ml of 10 mM 1-methylxanthine (1-MX). The final
concentration of palmitic acid was 0.55 ± 0.04 mM, and the fatty
acid-to-albumin ratio was 7.2, although this may be lower as the 6%
Ficoll may also bind some fatty acid. In support of this, there was no
loss of radioactive palmitate after several passages of the perfusion mix through the apparatus in the absence of a hindlimb, despite an
abundance of lipophilic binding surfaces. The hindlimb was perfused
with this mixture for 30 min (from 60 to 90 min, Fig. 1A),
and venous samples of 1.5 ml were taken every 10 min. The total
perfusion time was 90 min. Thus the three types of experiments conducted when measuring 2-DG uptake were control, insulin, and 5-HT + insulin. Because the uptake of 2-DG in the absence of
insulin is minimal, experiments in which only 5-HT was infused were not conducted. It should be noted that the presence of NaOH in the fatty
acid solution did not alter the pH of the perfusion medium.
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Free fatty acid uptake. Perfusion details were essentially as for 2-DG uptake. After completion of the surgical procedure, the rat hindlimb was perfused for 40 min with 6% Ficoll-Krebs buffer. Insulin or vehicle infusions were commenced and maintained for the remainder of the experiment (90 min). 5-HT or vehicle infusion was commenced at 50 min, and at 60 min (while the infusion of insulin and 5-HT or vehicle was maintained), the buffer reservoir was changed to one containing 26 ml [14C]palmitic acid solution (0.55 mM final), 2 ml [3H]mannitol solution, 232 ml gassed 6% Ficoll-Krebs buffer, and 0.65 ml of 10 mM 1-MX. The hindlimb was perfused with this mixture for 30 min (from 60 to 90 min in Fig. 1B), and venous samples of 1.5 ml were taken every 10 min. The total perfusion time was 90 min. Thus the four types of experiments conducted when measuring [14C]palmitic acid uptake were control, insulin, 5-HT, and 5-HT + insulin.
Analytic and radioactivity determinations. The concentration of glucose in each venous perfusate sample (from venous samples indicated in Fig. 1) was determined with a YSI 2300 Stat Plus glucose analyzer.
Perfusate samples containing radioactivity were immediately centrifuged. Samples (200 µl) were then added to 6 ml of Amersham Biodegradable Counting Scintillant and counted using a dual-label counting system (Beckman Coulter LS 6500). After perfusion, the soleus, plantaris, extensor digitorum longus (EDL), gastrocnemius red (G. Red), gastrocnemius white (G. White), and tibialis anterior muscles of the left hindlimb were removed. Excised muscles were freeze-dried overnight to obtain dry weight and later rehydrated with 1 ml of water and digested with 1 ml of Soluene (tissue solubilizer; Packard). When digestion was complete, 100 µl of acetic acid were added, together with 14 ml of Amersham Biodegradable Counting Scintillant. The average uptake of [14C]palmitic acid or 2-DG was calculated from the uptake of radioactivity into the individual muscles and the contributing mass of each muscle to the total mass of those tested, as shown by the equation
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Determination of 1-MX conversion. Perfusate samples of 100 µl were deproteinated with 25 µl of 2 M perchloric acid and centrifuged, and an aliquot of 40 µl was injected onto a reverse-phase HPLC column of Ultrasphere ODS column (25 cm; 5 µm particles, Beckman) under isocratic conditions at 1.0 ml/min, with a buffer of 0.3 M KH2PO4-0.5% methanol-0.5% acetonitrile-0.2% tetrahydrofuran, pH 4.0, and detection at a wavelength of 268 and 282 nm. Values obtained from the HPLC were used to calculate the metabolism of 1-MX.
Statistical analysis.
Statistically significant differences for oxygen
uptake (O2) and perfusion pressure
(PP) between groups over the last 30-min perfusion period were assessed
using two-way, repeated-measures ANOVA. All other tests were conducted
using one-way ANOVA. All comparisons were made using the
Student-Newman-Keuls multiple comparison test. P values
<0.05 were considered to be significant. One, two, or three symbols (*
to show significant difference from control and + to show
significant difference from insulin perfusions) were used to denote
P values of <0.05,
0.01, and
0.001, respectively.
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RESULTS |
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In all experiments, O2, PP, 1-MX
metabolism, and glucose uptake were determined. The inclusion of either
[14C]palmitic acid or 2-DG for uptake measurement (last
30 min) in one or the other one-half of the experiments had no apparent
effect on any of these parameters (not shown), and thus all data were pooled. Values for
O2 and PP are shown
in Fig. 2, A and B,
respectively. Insulin (60 nM) had no effect on either
O2 or PP, with 0.55 mM palmitic acid
present. However, the vasoconstrictor 3 µM serotonin markedly
inhibited
O2 in close association with
pressure rise, but there was no effect of insulin to moderate either
effect of 5-HT. Figure 2 also shows that the maximal effects of 5-HT,
with or without insulin, occurred soon after addition, with
O2 decreasing from 22.0 ± 0.5 to
9.7 ± 0.8 µmol · g
1 · h
1
(P < 0.001) at the maximum. This was accompanied by an
increase in PP from 41.6 ± 1.3 to 167.7 ± 13 mmHg
(P < 0.001) at the same time point (10 min). At
subsequent time points, there was a gradual decline so that, at 90 min,
only approximately one-half of the maximal response remained.
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The metabolism of 1-MX, by the capillary endothelial enzyme xanthine
oxidase, has been used previously by us as an indicator of nutritive
flow (27, 41). Figure 3
shows the effect of insulin, 5-HT, and the combination of insulin + 5-HT on 1-MX metabolism, determined at the 80-min time point, when
steady-state conditions have been attained (27). Whereas
insulin had no effect on 1-MX metabolism, 5-HT with or without insulin
was inhibitory. Thus 1-MX metabolism decreased from 5.8 ± 0.4 to
4.5 ± 0.4 nmol · min1 · g
1
(P = 0.024) with 5-HT alone and to 3.8 ± 0.4 nmol · min
1 · g
1
(P = 0.004) with 5-HT + insulin. The trend for
insulin to further increase the inhibitory effect of 5-HT was not
significant (Fig. 3).
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The effects of insulin, 5-HT, and the combination of insulin + 5-HT on hindlimb glucose uptake were assessed and shown in Fig.
4. Uptake was determined from
arteriovenous differences, total flow rate, and the perfused muscle
mass. Glucose uptake across the total perfused mass was stimulated
approximately threefold (from 12.2 ± 0.7 to 29.2 ± 1.7 µmol · g1 · h
1;
P < 0.001). 5-HT reduced the insulin-mediated glucose
uptake across the entire hindlimb (from 29.2 ± 1.7 to 23.1 ± 1.8 µmol · g
1 · h
1;
P = 0.005, and the insulin-mediated increment from 17.0 to 10.9; i.e., 36%) but was without effect on its own compared with
control. A subset of experiments using the nonmetabolizable glucose
tracer 2-DG was conducted to assess the effect of 5-HT on
insulin-mediated glucose uptake by individual hindlimb muscles. Insulin
significantly increased 2-DG uptake in all hindlimb muscles tested and
was particularly evident in the EDL and tibialis muscles (Fig.
5A). The increase was greater
than sixfold (from 13.6 ± 1.4 to 82.1 ± 4.6 µmol · g
1 · h
1;
P < 0.001) when the uptake into all muscles was
averaged (Fig. 5B). The addition of 5-HT with insulin
decreased 2-DG uptake in all muscles, and for the average this was
reflected by a decrease from 82.1 ± 4.6 to 41.6 ± 6.7 µmol · g
1 · h
1
(P < 0.001; Fig. 5B). Measuring the uptake
of 2-DG into individual muscles is a more accurate indicator of muscle
glucose uptake (although a contribution from attendant fat cells cannot
be ruled out) than arteriovenous hindlimb differences, because
measurements of glucose uptake across the entire hindlimb may also have
contributions from other tissues, including fat, bone, and skin.
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A second subset of experiments was conducted to assess the effects of
insulin and 5-HT on the uptake of [14C]palmitic acid by
the hindlimb muscles. Figure
6A shows that, whereas insulin
tended to increase the uptake of [14C]palmitic acid,
particularly by soleus and G. Red, this was not significant. When all
muscles were averaged, there was also no significant effect of 5-HT.
However, when insulin was coinfused with 5-HT, there was a significant
reduction in fatty acid uptake by the muscles with predominantly white
fibers (EDL, G. White, and tibialis). Thus 5-HT decreased
insulin-mediated [14C]palmitic acid uptake from 19.9 ± 1.3 to 5.8 ± 2.1 µmol · g1 · h
1
in the EDL (P < 0.001), 7.6 ± 1.1 to 3.1 ± 0.9 µmol · g
1 · h
1
in the G. White (P = 0.036), and 17.5 ± 0.3 to
5.3 ± 1.9 µmol · g
1 · h
1
in the tibialis (P < 0.001). When the uptake across
the selected muscles was averaged (Fig. 6B), the combination
of 5-HT and insulin resulted in a significant reduction compared with
insulin alone (i.e., from 13.9 ± 1.2 to 6.9 ± 1.4 µmol · g
1 · h
1;
P = 0.02).
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DISCUSSION |
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The main findings from this study were the reduced muscle fatty acid and insulin-mediated glucose uptake across the hindlimb when nonnutritive flow predominated. This contrasted with our previous study, in which chylomicron triglyceride uptake was enhanced under the same conditions (9). Together, the findings underscore the importance of flow distribution within muscle that controls nutrient and hormone partitioning, with implications for the accumulation of nutrients in both plasma and tissue in the longer term if these conditions prevail.
The reduced capacity to take up both glucose and unesterified fatty acids when flow is predominantly nonnutritive may also contribute in the longer term to hyperglycemia and hyperlipidemia. Elevated free fatty acids are stimulants of both hepatic glucose production and VLDL synthesis (2). Thus a cyclic process is facilitated with hypertriglyceridemia, interfibrillar fat cell enlargement, hyperglycemia, and muscle insulin resistance.
The data for this and the previous study (9) were obtained from individual muscles that were dissected from the hindlimb. It is generally assumed by most researchers that dissected muscle is predominantly pure and free of other contributing tissues. Our previous findings suggested that this may not be valid, as fat cells and associated lipoprotein lipase (LPL) removed with the muscles and situated between the fibers are likely to have contributed to the enhanced uptake of chylomicron triglyceride previously reported (9). Although LPL is known to be distributed in the skeletal muscle myocytes, Camps et al. (4) also found high levels in the muscle connective tissue, and this was the explanation by us (9) for the enhanced uptake of LPL-associated fatty acid when nonnutritive flow was predominant in red muscles. In the present study, we have focused on the uptake of albumin-bound free fatty acid, which is not dependent on LPL activity and is likely to be more dependent on endothelial surface area and fatty acid transporters (35, 36). Therefore, free fatty acid uptake was expected to decrease with reduced nutritive (capillary) perfusion (5-HT infusion) in all muscles. Although this appears to have occurred only in muscles with predominantly white fibers (40), it is likely to have occurred also in the red muscles. The uptake into the myocytes of the red muscle may be masked by free fatty acid extraction into the associated white fat cells in these muscles. There is the additional possibility that the estimations of 2-DG uptake are to some degree distorted by a background of associated fat cells, which are more concentrated in connective tissue of red muscles (40).
Reduced muscle uptake of palmitate during hemodynamic insulin resistance implies that triglyceride deposits within the myocyte will be reduced. Classically, it is thought that elevated plasma free fatty acids during insulin resistance are reassembled into lipoproteins in the liver for metabolism in the periphery, leading to increased skeletal muscle triglyceride. Consistent with this notion, a correlation between insulin resistance and muscle triglyceride levels (14, 20, 25, 26) and increased lipid oxidation (16) has been recorded. Despite this, there are reports showing that fatty acid uptake is decreased with impaired glucose tolerance (37), type 2 diabetes (3), and in women with visceral obesity (10). In addition, reduced lipid oxidation has been recorded in subjects with non-insulin-dependent diabetes mellitus (19) and visceral obesity (10). The findings reported in this study may in part explain those reported by Turpeinen et al. (37), showing that the uptake of fatty acid was reduced in states of insulin resistance. Therefore, intracellular accumulation of triglyceride may be a result of reduced lipid oxidation rather than increased fatty acid uptake.
Impaired insulin-mediated glucose uptake during nonnutritive flow may imply that access to the more metabolically active muscle for both fuel and hormone is reduced. For muscle cells, this translates as a state of acute insulin resistance (albeit "hemodynamic insulin resistance"). However, glucose and insulin access to the less metabolically active adipocytes on the nonnutritive or connective tissue route is enhanced, because flow not entering the nutritive route in a constant-flow system must enter the nonnutritive route. As pointed out previously (9), this will favor fat accretion through enhanced triglyceride delivery to fat cells via LPL and insulin-mediated glycerophosphate production from adipocyte glycolysis. In the long term, this could provide the basis for "marbling" of muscle, visible at the micro or macro level as the interfibrillar accumulation of fat (40).
In the present study, nutritive (capillary) flow was determined by metabolism of infused 1-MX, a substrate for the capillary endothelial enzyme xanthine oxidase. In vivo insulin acts to increase 1-MX metabolism, but this is markedly inhibited when fatty acids are elevated due to coinfusion of Intralipid and heparin (8). Thus failure of insulin to increase 1-MX metabolism and, therefore, nutritive flow in the present experiments involving constant-flow perfusions may be attributable to an inhibitory effect of palmitate. There is also the possibility that the hindlimb under basal conditions, with no vasconstrictor present, is essentially fully dilated (31); thus insulin is unable to vasodilate further and recruit nutritive flow. Within this context, insulin does not dilate the hindlimb vasculature when preconstricted by 5-HT2 agonists either in vivo (28) or in perfusion (29). We have previously shown that 5-HT reduces hindlimb lactate output, possibly due to reduced uptake of glucose, fatty acids, and oxygen, reflecting a state of metabolic hibernation rather than leading to an anaerobic state in which breakdown of glycogen stores is stimulated.
Finally, it is important to note that the vasoconstrictor 5-HT has been
used herein as a model substance to induce an acute state of
predominantly nonnutritive flow. Estimates from the
O2 data of Fig. 2 and 1-MX metabolism of
Fig. 3 suggest that, at the dose of 5-HT used, there was a decrease in
nutritive flow to 57% of basal initially (Fig. 2 at 60 min) declining
to 29% (Fig. 2) or 35% (Fig. 3). We have shown previously that 5-HT
has no direct effects on muscle metabolism or contractility independent of its vascular effects. Thus isolated incubated muscles showed no
effect of 5-HT addition on insulin-mediated glucose uptake (29) or contractility from field stimulation
(13). Although 5-HT-mediated vasoconstriction to induce
nonnutritive flow represents only an acute state of insulin resistance
with decreased free fatty acid uptake, long-term nonnutritive flow may
occur in vivo during extended periods of low physical activity or
during stress when sympathetic outflow is increased. There are data to
show that high frequency sympathetic nervous system activity mediates a
pattern of nonnutritive flow in the constant flow perfused rat hindlimb
(18).
In summary, the predominantly nonnutritive flow pattern induced in these experiments by the vasoconstrictor 5-HT resulted in significant reduction in free fatty acid and insulin-mediated glucose uptake across the hindlimb. Thus reduced access for glucose and free fatty acid to myocytes is offset by enhanced access for triglycerides to attendant connective tissue adipocytes. Such findings illustrate a role of nutritive and nonnutritive blood flow in controlling nutrient partitioning. The findings also have particular implications for glucose uptake in insulin-resistant states, such as Intralipid/heparin infusions (8) and the genetically obese Zucker rat (39). In both examples, insulin-mediated capillary recruitment (nutritive flow) is impaired and may contribute to the decrease in insulin-mediated glucose uptake by muscle in vivo.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the (Australian) National Health and Medical Research Council, Australian Research Council, and the National Heart Foundation of Australia.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. G. Clark, Dept. of Biochemistry, Medical School, Univ. of Tasmania, Hobart, Tasmania 7001, Australia
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 November 26, 2002;10.1152/ajpendo.00153.2002
Received 10 April 2002; accepted in final form 21 November 2002.
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