1 Diabetes Unit, Section of Endocrinology and Departments of Medicine and Physiology, Boston Medical Center, Boston 02118; and 2 Department of Biochemistry and Molecular Biology, University of Massachusetts, Worcester, Massachusetts 01655
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ABSTRACT |
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Malonyl-CoA acutely regulates fatty acid oxidation in liver in vivo by inhibiting carnitine palmitoyltransferase. Thus rapid increases in the concentration of malonyl-CoA, accompanied by decreases in long-chain fatty acyl carnitine (LCFA-carnitine) and fatty acid oxidation have been observed in liver of fasted-refed rats. It is less clear that it plays a similar role in skeletal muscle. To examine this question, whole body respiratory quotients (RQ) and the concentrations of malonyl-CoA and LCFA-carnitine in muscle were determined in 48-h-starved rats before and at various times after refeeding. RQ values were 0.82 at baseline and increased to 0.93, 1.0, 1.05, and 1.09 after 1, 3, 12, and 18 h of refeeding, respectively, suggesting inhibition of fat oxidation in all tissues. The increases in RQ at each time point correlated closely (r = 0.98) with increases (50-250%) in the concentration of malonyl-CoA in soleus and gastrocnemius muscles and decreases in plasma FFA and muscle LCFA-carnitine levels. Similar changes in malonyl-CoA and LCFA-carnitine were observed in liver. The increases in malonyl-CoA in muscle during refeeding were not associated with increases in the assayable activity of acetyl-CoA carboxylase (ACC) or decreases in the activity of malonyl-CoA decarboxylase (MCD). The results suggest that, during refeeding after a fast, decreases in fatty acid oxidation occur rapidly in muscle and are attributable both to decreases in plasma FFA and increases in the concentration of malonyl-CoA. They also suggest that the increase in malonyl-CoA in this situation is not due to changes in the assayable activity of either ACC or MCD or an increase in the cytosolic concentration of citrate.
acetyl-CoA carboxylase; insulin; glucose; long-chain fatty acyl carnitine; starvation; refeeding
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INTRODUCTION |
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MALONYL-COA
IS AN INHIBITOR of carnitine palmitoyltransferase I (CPT-I), the
enzyme that catalyzes the conversion of long-chain fatty acyl-CoA
(LCFA-CoA) into LCFA-carnitine, in which form it can be transferred
into the mitochondria (20). As shown initially by McGarry
et al. (23), CPT-I catalyzes the rate-limiting step in
hepatic fatty acid oxidation. In liver, insulin and glucose inhibit
fatty acid oxidation, at least in part, by altering malonyl-CoA levels
(37). They appear to act by increasing the activity of the
hepatic isoform of acetyl-CoA carboxylase (ACC), which they do acutely (in minutes to hours) by causing it to be
dephosphorylated (2, 13, 18,
28, 40) and chronically (in hours to days) by
both dephosphorylation and stimulation of its synthesis at the level of
transcription (16, 17). Regulation of hepatic fatty acid oxidation by these mechanisms has been demonstrated in vivo
as early as 1 h after the beginning of carbohydrate refeeding in
starved rats (26, 27, 39,
41).
Whether malonyl-CoA also regulates fatty acid oxidation in skeletal
muscle in response to changes in nutritional state is less clear. In
support of such a notion, malonyl-CoA levels in rat muscle are
decreased after 24-48 h of starvation and are increased after
24 h of refeeding (22, 38). On the other
hand, it is not known whether the increases in malonyl-CoA
concentration during refeeding occur quickly enough to contribute to
the rapid decrease in whole body fat oxidation that occurs after a
carbohydrate meal (9, 10), and if so, how
this increase in malonyl-CoA occurs. To address these questions, the
relationship between malonyl-CoA content and fatty acid oxidation was
examined in starved and starved-refed rats. The present report
describes the changes in respiratory quotient and the concentrations of
malonyl-CoA and LCFA-carnitine in liver and muscle of 48-h-starved rats
and starved rats after various periods of refeeding. In addition, the
activities of the muscle isoform of ACC (ACC) and of
malonyl-CoA decarboxylase in muscle were determined, as were the
concentrations of various allosteric effectors of ACC
. A
preliminary report describing some of this work has appeared
(5).
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MATERIALS AND METHODS |
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Experimental Animals
Male Sprague-Dawley rats weighing ~155-160 g were obtained from Harlan Sprague Dawley (Indianapolis, IN). They were housed in individual cages in a room on a 12:12-h light-dark cycle and fed standard rat chow ad libitum for 6 days. After this, one group of rats was starved for 24 h, after which whole body respiratory quotients (RQs) of the individual rats were measured. Other rats had RQ measurements performed after 48 h of starvation and 1, 3, 12, 18, or 24 h of refeeding. Because all rats did not eat immediately when presented with food, measurements before 1 h of refeeding were not attempted. Food consumption during the refeeding period was determined by subtracting the final weight of the remaining chow bin from that present before feeding.RQ Measurements
RQs were determined using a Perkin-Elmer 1100 Medical Gas Analyzer and a Digital MINC-II computer as described by Flatt (8). Individual rats were placed in a 6-qt aluminum pot covered with a lid. After 1 min to allow for some accumulation of CO2 and consumption of O2, gas samples were taken by introducing a 9-in. metal cannula through a small hole in the lid and connecting it to the gas analyzer's inlet. On each run, gas samples taken over a 5-s intake period were automatically collected by the gas analyzer, and the average RQ value was calculated by the computer. Samples were taken until the average RQ values for three consecutive runs fell within 0.02 units of each other. The listed RQ for each rat was taken as the average of these three values. RQ measurement required ~10 min per rat. For this reason, to keep the time between initiation of feeding and performance of the RQ measurement constant, food was given to individual rats within each group on a 10-min staggered schedule.After RQ determination, rats were injected intraperitoneally with pentobarbital sodium (6 mg/100 g body wt), and samples of blood were taken by orbital sinus puncture. The soleus and gastrocnemius muscles and portions of the liver were then excised and frozen in liquid nitrogen, and the rats were killed by exsanguination.
Assays
Malonyl-CoA in muscle and liver were assayed radioenzymatically in neutralized perchloric acid filtrates by the method of McGarry et al. (23), as described previously (31), and palmitoylcarnitine according to McGarry and Foster (21). Citrate and malate (19) were determined in the same filtrate by standard enzymatic methods. LCFA-carnitine was analyzed by the method of Pace et al. (29). Plasma insulin was determined by radioimmunoassay with a rat insulin standard (Linco, St. Louis, MO) and free fatty acids spectrophotometrically with a commercially available kit (Wako BioProducts, Richmond, VA). Glucose was determined by the hexokinase method (4) and muscle glycogen as described by Passonneau and Lauderdale (30). ACC was determined by 14CO2 fixation after immunoprecipitation of the muscle isoform (37).Malonyl-CoA decarboxylase (MCD) activity in muscle was measured
spectrophotometrically (17) after
(NH4)2SO4 purification with a
Hewlett-Packard (model 8450A) diode array spectrophotometer, set to
measure A335-345 minus A390-400 so as
to subtract out the effect of particulate matter. Partial purification
of MCD was carried out by a modification of the method of Dyck et al.
(6). The (NH4)2SO4
purified fraction (150 µl) from the muscle homogenate was
preincubated in a 700-µl reaction mixture composed of 0.1 M Tris
· HCl, pH 8.0, 0.5 mM dithiothreitol, 10 mM L-malate, 0.5 mM NAD+, and 10 µg malate dehydrogenase (1.0 units) for
10 min at 37°C, in the presence or absence of phosphatase inhibitors
(in mM: 40 -glycerophosphate, 40 NaF, 4 NaPPi, and 1 Na3VO4). Ten micrograms of citrate synthase
(1.7 units) were then added, and the preincubation was continued for 2 min. Malonyl-CoA (0.3 mM) was added to start the MCD reaction, and the
rate of NADH oxidation was remeasured over 7 min. This rate was
corrected for the small rate obtained without added malonyl-CoA.
Protein concentration was determined by the method of Bradford with
bovine serum albumin as the standard (4). Activity is
expressed as nanomoles per minute per milligram of extract protein
subjected to (NH4)2SO4 precipitation.
Statistics
Results are expressed as means ± SE. Statistical differences between multiple groups were determined by analysis of variance followed by the Student-Newman-Keuls multiple comparison test. ![]() |
RESULTS |
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Body Weight, Glucose, and Insulin
Food intake and body weight increased rapidly during refeeding, with substantial increases in both parameters evident as early as 1 h (Table 1). The rats continued to eat and gain weight over the next 2-11 h, after which they ate little and body weight tended to decrease. As expected, plasma insulin and glucose increased during refeeding, with peak values evident at 3 h. Thereafter, insulin levels remained more or less constant, whereas by 18 h the concentration of glucose had begun to decrease.
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RQ
The mean RQ in a 48-h-starved rat at ~5 PM was 0.82 ± 0.004 at 48 h (Fig. 1). After 1 h of refeeding, it increased to 0.93 ± 0.005, by 3 h to 1.00 ± 0.02, and by 12 h to 1.05 ± 0.01. Although the rats consumed little if any chow thereafter (Table 1), at 18 h their mean RQ was 1.09 ± 0.02 and at 24 h, 1.08 ± 0.01, suggesting the occurrence of fatty acid synthesis de novo.
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Plasma Free Fatty Acids
Plasma free fatty acid (FAA) levels were 289 ± 33 µmol/l at 48 h of starvation and decreased during refeeding to 202 ± 32 µmol/l at 1 h and 121 ± 11 µmol/l at 3 h. They remained near the latter value during the remaining 21 h of refeeding (Table 1). The decreases in plasma FFA during refeeding correlated with the increase in RQ, with r = 0.9 (P < 0.05).Malonyl-CoA in Muscle and Liver
After 48 h of starvation, the concentration of malonyl-CoA in the soleus was 0.9 ± 0.1 nmol/g (Fig. 2A) (vs. typical values of 2 nmol/g in a rat fed ad libitum) (33). It increased by ~1 nmol/g after 1 h of refeeding, and more gradually by an equivalent amount over the next 23 h. An identical pattern was observed in the gastrocnemius (Fig. 2B). A plot of the relationship between malonyl-CoA and RQ showed a remarkably tight correlation, with an r of 0.98 (P < 0.01) for the soleus and 0.97 (P < 0.01) for the gastrocnemius (data not shown).
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Malonyl-CoA levels in liver showed a somewhat different pattern of change. At 48 h of starvation, the concentration of malonyl-CoA was 1.9 ± 0.2 nmol/g (Fig. 2C). During refeeding, it increased to 3.1 ± 0.3 nmol/g at 1 h and 5.5 ± 0.3 nmol/g at 3 h. In contrast to muscle, at later times malonyl-CoA levels did not increase further; indeed, they decreased somewhat. After 12, 18, and 24 h of refeeding, the concentrations of malonyl-CoA in the liver were 4.5 ± 0.2, 4.3 ± 0.5, and 4.5 ± 0.3 nmol/g, respectively. Thus the concentration of malonyl-CoA was decreasing in liver at a time when whole body RQ was >1.0 (Fig. 1). Despite this, a strong correlation (r = 0.81, P < 0.05) between hepatic malonyl-CoA and whole body RQ was observed (data not shown).
LCFA-Carnitine
LCFA-carnitine levels were high in both gastrocnemius muscle (Fig. 3A) and liver (Fig. 3B) of the 48-h-starved rat, and they decreased rapidly with refeeding. The decrease in LCFA-carnitine in both tissues was ~50% at 1 h of refeeding, in keeping with the increases in RQ and malonyl-CoA levels and the decrease in plasma FFA at this time. At later times after refeeding, no further decrease in the concentration of LCFA-carnitine was observed; indeed, in muscle after 24 h it had substantially returned to prefeeding values despite the absence of either a decrease in the concentration of malonyl-CoA (Fig. 2) or an increase in plasma FFA (Table 1).
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Possible Bases for the Increase in Malonyl-CoA in Muscle During Refeeding
ACC and MCD activity.
Changes in assayable ACC activity, attributable to covalent
modification or a change in abundance, have not been observed in muscle
as a consequence of starvation or refeeding (38); however,
only a limited number of times after refeeding were examined. Using a
sensitive assay in which interference by other carboxylases is removed
by immunoprecipitating ACC (37), we assayed ACC
activity in immunoprecipitates from gastrocnemius
muscles of 24- and 48-h-starved rats and rats after 1, 3, 12, 18, and 24 h of refeeding. As shown in Table
2, except for a small
increase in activity at the 12-h time point, when the assay was carried out in the presence of 10 mM citrate, no difference in ACC activity was
observed between starved and refed rats.
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Allosteric effectors of ACC.
Cytosolic citrate is both an allosteric activator of ACC
and a precursor of its substrate cytosolic acetyl-CoA, whereas LCFA-CoA
acts as an allosteric inhibitor, which counters the action of citrate
(36). The cytosolic concentration of citrate, as reflected
by the sum of the concentrations of citrate plus malate (34), was not increased in gastrocnemius muscles of
48-h-starved rats after 1, 3, 12, 18, and 24 h of refeeding (Table
2). In general this was due to the fact that the concentration of
malate decreased with refeeding and the concentration of citrate was unchanged. In contrast, as previously reported in these rats
(33), the concentration of LCFA-CoA, an allosteric
inhibitor of ACC, was 7.4 ± 0.6 nmol/g in the 48-h-starved rat
and decreased to 5.8 ± 0.5 and 5.1 ± 0.3 nmol/g after 3 and
24 h of refeeding, respectively (P < 0.01 vs.
value before refeeding).
Glycogen. To determine whether malonyl-CoA levels increase in muscle in parallel with or after repletion of its glycogen stores, the glycogen content of the gastrocnemius muscle was determined at each time point. As shown in Table 2, the concentration of glycogen (as glucose units) was 8.0 ± 0.8 µmol/g (0.13%) in the gastrocnemius of a 48-h-starved rat and increased to 12.4 ± 1.2, 22.3 ± 1.7, and 33.7 ± 3.6 µmol/g after 1, 3, and 12 h, respectively, of refeeding. It then tended to decrease. Thus changes in glycogen levels temporally paralleled those of malonyl-CoA (Fig. 2B) during refeeding.
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DISCUSSION |
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During the transition from the starved to the fed state, increases in plasma insulin and glucose diminish the oxidation of fatty acids by both lowering plasma FFA levels and altering their intracellular metabolism (28, 35, 41). In liver, the intracellular inhibition of fatty acid oxidation is evident within 1 h and appears to be regulated, at least in part, by increases in the concentration of malonyl-CoA, an inhibitor of CPT-I (42). The results presented here show that malonyl-CoA levels are also increased rapidly in muscle during refeeding after a fast. Thus malonyl-CoA levels were increased by 100% in the soleus and by 60% in the gastrocnemius muscle after 1 h of refeeding, and they increased further in both muscles during the succeeding 11 h. That these increases in malonyl-CoA affect fatty acid metabolism is suggested by their close correlation with the increases in whole body RQ. In addition, during the first few hours of refeeding, they correlated with decreases in LCFA-carnitine (Fig. 3), suggesting inhibition of CPT-I.
The mechanism responsible for the increase in malonyl-CoA during refeeding is unclear. In liver, the increase in malonyl-CoA during refeeding has been attributed to increases in the concentrations of insulin and glucose that alter the activities of hepatic kinases and/or phosphatases leading to dephosphorylation and activation of ACC. In contrast, we did not find an increase in assayable ACC activity in muscle during refeeding (Table 2), in agreement with previous reports (38, 39). Likewise, we found no increase in the cytosolic concentration of citrate, an allosteric activator of ACC, which appears to be responsible for the elevated malonyl-CoA levels in muscle produced by sustained increases in insulin and/or glucose in vivo (33) and in vitro (32), nor was MCD activity diminished. The latter is of special interest, because Goodwin and Taegtmeyer (11) recently reported that an increase in MCD activity, rather than a decrease in ACC activity, may account for the decrease in malonyl-CoA in a perfused rat heart preparation when its workload is increased. Furthermore, we have observed that MCD activity is increased in skeletal muscle during contraction (30a).
In the absence of appropriate changes in ACC and MCD activity and
cytosolic citrate, we believe the most likely regulator of malonyl-CoA
during refeeding is the cytosolic concentration of LCFA-CoA, an
allosteric inhibitor of ACC. In support of this contention, decreases
in the whole tissue concentration of LCFA-CoA in muscle have been
observed after both 3 and 24 h of refeeding (see
RESULTS and Ref. 33), events attributable to concurrent decreases in plasma FFA levels (Table 1) and muscle triglyceride hydrolysis (12). Although the results presented here are
consistent with this notion, such a conclusion must be tempered by the
fact that LCFA-CoA is compartmented in cells and its precise
concentration in the cytosol of muscle is not known (7).
If the concentration of LCFA-CoA in the cytosol is decreased as
proposed, however, by virtue of mass action, it would complement the
increase in malonyl-CoA in diminishing CPT-I activity. A hypothetical
schema depicting the interactions between insulin and glucose and
between LCFA-CoA and malonyl-CoA during refeeding is shown in Fig.
4.
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The correlations between changes in RQ and malonyl-CoA and malonyl-CoA and LCFA-carnitine presented here strongly suggest a link between changes in malonyl-CoA concentration in muscle and its rate of fatty acid oxidation. Numerous studies in incubated muscles (1, 24, 34), in which changes in malonyl-CoA concentration and fatty acid oxidation have been closely correlated, also support such a linkage. Despite this, the question of whether malonyl-CoA regulates CPT-I, and secondarily fatty acid oxidation in muscle, is still debated. This is because the CPT-I isoform present in muscle has an inhibitory constant for malonyl-CoA (24-40 nM), ~1/70 of the CPT-I isoform in liver (1,700-2,700 nM) (20, 25). Thus, whereas changes in malonyl-CoA concentration in vivo should modulate CPT-I in liver under physiological conditions, at the concentrations of malonyl-CoA measured in whole muscle (1-4 µM), CPT-I should be inhibited at all times. McGarry and Brown (20) suggested that malonyl-CoA in muscle is either associated with a binding protein that lowers its effective concentration or that a large fraction of it is compartmented in an organelle such as the mitochondria. Preliminary studies (A. Saha, unpublished observations), in which we found that >70% of the malonyl-CoA in a muscle homogenate is present in a 14,000-g fraction that should be enriched in mitochondria, support the latter possibility. Definitive experiments in which the mitochondria are carefully characterized and extraction artifacts ruled out are needed before this can be stated with certainty, however.
The patterns of malonyl-CoA changes in muscle and liver during refeeding were not identical. The increase in malonyl-CoA peaked in the liver at 3 h and then decreased somewhat in keeping with a previous report (27), whereas in both the soleus and gastrocnemius muscles it did not peak until 12 h, and a secondary decrease in malonyl-CoA concentration did not occur. The secondary decrease in malonyl-CoA in liver could be related to the 10-30% increase in liver mass (due to glycogen repletion) that occurs during refeeding or to the fact that the late, but not the early, increase in ACC activity is associated with an increase in fatty acid synthase activity (14). The latter might also account for the finding that whole body RQ increases between 3 and 24 h of refeeding despite a decrease in hepatic malonyl-CoA concentration (Fig. 2).
In conclusion, the results indicate that increases in malonyl-CoA occur very rapidly in muscle during refeeding after a fast and that they are associated with changes in whole body RQ and the concentration of LCFA-carnitine. They also suggest that the increase in malonyl-CoA in muscle in this situation is not due to changes in the assayable activity of either ACC or MCD or an increase in the cytosolic concentration of citrate. The most likely cause is a decrease in the cytosolic concentration of LCFA-CoA, an allosteric inhibitor of ACC.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the expert technical assistance of Ted Kurowski, Virendar Kaushik, and David Tse, and we thank Tomoko Akishino, Jenny Keyerleber, and Christine Waelde for assistance in preparing the manuscript. We also thank Drs. Keith Tornheim and Marc Prentki for their constructive comments.
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FOOTNOTES |
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This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-19514 (N. B. Ruderman and A. K. Saha) and DK-32214 (J. P. Flatt) and a mentor-based grant from the American Diabetes Association (to N. B. Ruderman and D. Dean).
Address for reprint requests and other correspondence: A. K. Saha, Diabetes and Metabolism Unit, Boston Univ. Medical Center, 650 Albany St., EBRC-827, Boston, MA 02118 (E-mail: aksaha{at}bu.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. §1734 solely to indicate this fact.
Received 11 January 2000; accepted in final form 2 March 2000.
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