AJP - Endo Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 290: E882-E888, 2006. First published December 13, 2005; doi:10.1152/ajpendo.00333.2005
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/E882    most recent
00333.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamaoka, I.
Right arrow Articles by Yoshizawa, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamaoka, I.
Right arrow Articles by Yoshizawa, F.

Intravenous administration of amino acids during anesthesia stimulates muscle protein synthesis and heat accumulation in the body

Ippei Yamaoka,1 Masako Doi,1 Mitsuo Nakayama,1 Akane Ozeki,2 Shinji Mochizuki,2 Kunio Sugahara,2 and Fumiaki Yoshizawa2

1Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Naruto, Tokushima; and 2Department of Animal Science, Utsunomiya University, Utsunomiya, Tochigi, Japan

Submitted 22 July 2005 ; accepted in final form 5 December 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was conducted to determine the contribution of muscle protein synthesis to the prevention of anesthesia-induced hypothermia by intravenous administration of an amino acid (AA) mixture. We examined the changes of intraperitoneal temperature (Tcore) and the rates of protein synthesis (Ks) and the phosphorylation states of translation initiation regulators and their upstream signaling components in skeletal muscle in conscious (Nor) or propofol-anesthetized (Ane) rats after a 3-h intravenous administration of a balanced AA mixture or saline (Sal). Compared with Sal administration, the AA mixture administration markedly attenuated the decrease in Tcore in rats during anesthesia, whereas Tcore in the Nor-AA group became slightly elevated during treatment. Stimulation of muscle protein synthesis resulting from AA administration was observed in each case, although Ks remained lower in the Ane-AA group than in the Nor-Sal group. AA administration during anesthesia significantly increased insulin concentrations to levels ~6-fold greater than in the Nor-AA group and enhanced phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 protein kinase relative to all other groups and treatments. The alterations in the Ane-AA group were accompanied by hyperphosphorylation of protein kinase B and the mammalian target of rapamycin (mTOR). These results suggest that administration of an AA mixture during anesthesia stimulates muscle protein synthesis via insulin-mTOR-dependent activation of translation initiation regulators caused by markedly elevated insulin and, thereby, facilitates thermal accumulation in the body.

thermogenesis; translation initiation; insulin; hypothermia

HYPOTHERMIA CAUSED BY THE ADMINISTRATION of anesthetics occurs commonly during surgery and results from decreased heat production, increased heat loss, and lack of hypothalamic thermoregulation (38). It has been frequently reported that intravenous infusion of an amino acid (AA) mixture increases heat production, which leads to prevention of hypothermia during surgery (26, 35–37). However, the mechanism behind the stimulation of thermogenesis by AA administration during anesthesia remains unknown.

Extra-splanchnic oxygen consumption accounts for ~73 to 75% of the total oxidative metabolism seen in a whole human body given an AA mixture during surgery (37). In alert subjects, intravenous administration of an AA mixture not only increases oxygen uptake but also augments blood flow in extra-splanchnic tissues (7). Although we can infer from these observations that thermogenesis resulting from AA administration occurs mainly in extrasplanchnic organs capable of varying oxygen metabolism, we cannot find reports showing where the rise in energy expenditure occurs and affects heat accumulation in the body.

The AA, as a nutrient, has been shown (14) to be especially effective at increasing energy expenditure in either the degradable pathway (gluconeogenesis and ureagenesis) or the nonoxidative disposal pathway (protein synthesis). In particular, the elevation of protein synthesis may be widely accepted as the most likely explanation for enhanced nutrient-induced thermogenesis (NIT) resulting from AA administration under anesthesia. This explanation is supported by the finding that the AA-induced rise in NIT values is not observed in puromycin (an inhibitor of protein synthesis)-treated rats (43). Also, increases in energy expenditure seen during AA administration are dose dependent and are correlated to AA-induced protein synthesis (21). However, we know of no examination that shows whether AA administration under anesthesia stimulates protein synthesis in some tissues.

The most important stage where nutritional changes play a major role in protein synthesis in skeletal muscle is translation initiation where, as frequently reviewed (25, 27, 29, 39), the translation initiation factors eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 protein kinase (S6K1) play a central role through the mammalian target of rapamycin (mTOR) pathway. Enhancement of these elements of the translation initiation cascade by intravenous or oral AA administration is thought to require both stimulation by AAs and insulin (4, 18, 44, 45, 47).

The present study was conducted to determine the contribution of muscle protein synthesis to the prevention of hypothermia that results from AA administration in rats during anesthesia. First, we examined the rate of protein synthesis (Ks) in skeletal muscle of rats in which a balanced AA mixture was infused intravenously either during propofol anesthesia or in the conscious state. To clarify how the treatment with AA and anesthetics is involved in this process, we measured the phosphorylation states of protein participating in translation initiation control. Furthermore, to elucidate the primary stimuli involved in the stimulation of protein synthesis, we examined the phosphorylation state of protein kinase B (PKB) and mTOR and evaluated plasma insulin and AA concentrations.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Sprague-Dawley rats from Charles River Japan (Yokohama, Japan), weighing 250–310 g, were maintained under conditions of constant humidity and temperature (22 ± 2°C) on a 12:12-h light-dark cycle. They were fed a standard diet and water ad libitum. The following surgical and experimental procedures were approved by the Committee on the Care and Use of Laboratory Animals of Otsuka Pharmaceutical Factory.

Seven days before the experiment, under pentobarbital sodium anesthesia (50 mg/kg), the rats were chronically implanted with electrodes and a transmitter to monitor physiological indexes related to the state of anesthesia. These consisted of an electroencephalogram (EEG) with round-tip miniature screws over the right frontal and left occipital cortices, an electromyogram (EMG) with two stainless-steel wires in the neck muscle, and an intraperitoneal temperature (Tcore) transmitter (TA10TA-F40; Data Science International, St. Paul, MN) in the peritoneal cavity via an abdominal incision. On the day before the experiment, a silicon catheter was inserted into the jugular vein and threaded 2.5 cm proximally from the tip in the rats under diethyl ether anesthesia. This catheter was joined to two plastic tubes via a plastic Y connector, enabling the injection of different solutions from two different pumps. Saline (Sal) was infused continuously at a rate of 1 ml/h per rat via one vinyl tube to prevent blood coagulation; the other tube was filled with Sal and sealed hermetically. Food was withheld from rats for 18 h, but rats were allowed free access to water.

Infusion protocol. An infusion of the test solution (an AA mixture or Sal) and anesthesia were started simultaneously and continued for 180 min (0–180 min). During the treatment, the conscious (Nor) rats in the Nor-Sal group received a fat emulsion preparation (10% soybean oil; Intralipos, Otsuka Pharmaceutical Factory) via one vinyl tube and Sal via the other. The rats in the Nor-AA group received a fat emulsion preparation and a balanced AA mixture (Amiparen, Otsuka Pharmaceutical Factory). The anesthetized (Ane) rats in the Ane-Sal group were anesthetized with a continuous infusion of propofol (1% Diprivan; Zeneca, Milan, Italy) and Sal, and rats in the Ane-AA group received propofol and an AA mixture, all via the same method as the Nor-Sal group. Each test solution was infused at a rate of 14 ml·kg–1·h–1. The fat emulsion preparation and propofol were administered via a bolus injection of >5 s (1.5 ml/kg), and this was followed by a sequential infusion at rates of 4.5 ml·kg–1·h–1 (0–30 min) and 2.25 ml·kg–1·h–1 (30–180 min).

Measurement of intraperitoneal temperature. The rats were connected by a thin cable to an electrical swivel for monitoring EEG and EMG activity, and the rats were placed in the plastic cage that was located on the receiver for temperature transmitter. From 1 h before the start of the test solution infusion to the end of the treatment, the output signal from the transmitter was collected by an antenna in the receiver and then digitized by a Cambridge Electronic Design (CED) 1401 data processor connected to a personal computer (PC-9801NX; NEC, Tokyo, Japan) and stored on a hard disk. Offline data analysis was carried out using a Spike 2 analyzing program (CED). Tcore was continuously sampled every second and was averaged over a 1-min period at 30-min intervals, from 30 min before the start of the test solution infusion to the end of the treatment.

Tissue preparation. At the end of the experiment, pentobarbital sodium (50 mg/kg) was administered via a bolus injection through a catheter, and blood was collected from the abdominal artery and centrifuged at 1,800 g for 20 min at 4°C to obtain the plasma. Skeletal muscle (gastrocnemius muscle) was removed and rinsed in ice-cold saline. Tissues were immediately weighed and homogenized in seven volumes of buffer A [in mM: 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 100 KCl, 0.2 EDTA, 2 ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 dithiothreitol, 50 NaF, 50 beta-glycerophosphate, 0.1 phenylmethylsulfonyl fluoride, 1 benzamidine, and 0.5 sodium vanadate] with a Polytron homogenizer. The homogenates were centrifuged at 10,000 g for 10 min at 4°C. The resulting supernatant was used to examine the phosphorylation of 4E-BP1, S6 protein kinase (S6K1), PKB, and mTOR, as described below.

Measurement of protein synthesis in skeletal muscle. In a separate experiment series, with the animal model treated as described above, the fractional rate of protein synthesis was measured by a flooding-dose method of phenylalanine (19) during the last 10 min of the treatment. The rats were injected with 1 ml/100 g body wt of a solution of L-[2,6-3H]phenylalanine (150 mmol/l, containing 3.70 GBq/l) via the implanted catheter that was used to administer the test solution. After that, the catheter was immediately connected with the plastic Y connector, and the infusion was resumed. Ten minutes after that, the rats were injected with pentobarbital sodium (50 mg/kg) followed immediately by excision of the gastrocnemius muscles, which were quickly frozen in liquid N2 by the clamping method. In all rats the periods between injection of radioisotope and clamping of each tissue were accurately recorded. After fractionation to purified protein and homogenate supernatant, the specific radioactivity of phenylalanine was determined. The fractional rate of protein synthesis (the percentage of protein mass synthesized in 1 day) was calculated as Ks (%) = (Sb/Sa) x 1,440/t x 100, where Sb is the specific radioactivity of the protein-bound phenylalanine, Sa is the specific radioactivity of free tissue phenylalanine, and t is the time of labeling in minutes.

Assessment of phosphorylation state in 4E-BP1. For analysis of the phosphorylation state in 4E-BP1, which is a heat-stable protein, one aliquot of the supernatant was heated for 10 min at 100°C, cooled to room temperature, and centrifuged at 10,000 g for 30 min at 4°C. The supernatant was mixed with an equal volume of 2x SDS sample buffer (2 ml of 0.5 M Tris, pH 6.8, 2 ml of glycerol, 2 ml of 10% SDS, 0.2 ml of beta-melcaptoethanol, 0.4 ml of a 4% solution of bromophenol blue, and 1.4 ml of water to a final volume of 8 ml), reheated for 3 min at 100°C, and subjected to electrophoresis on a 15% polyacrylamide gel. After electrophoresis, the proteins were transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with polyclonal antibodies specific for 4E-BP1 (cat. no. 6936, Santa Cruz Biotechnology). The blots were then developed by use of an enhanced chemiluminescense (ECL) Western blotting kit according to the manufacturer's instructions. The film was scanned (EPSON GT-9500) and analyzed with National Institutes of Health Image 1.61 software. The 4E-BP1 resolves into multiple electrophoretic forms during SDS-PAGE, depending on which and how many sites are phosphorylated, with the more slowly migrating forms representing more highly phosphorylated 4E-BP1.

Assessment of phosphorylation state in S6K1, PKB, and mTOR. The other aliquot of the supernatant was combined with 2x SDS sample buffer in equal proportions, heated for 3 min at 100°C, and then cooled to room temperature. The samples were subjected to electrophoresis on a 7.5% polyacrylamide gel for S6K1, a 12.5% gel for PKB, or a 7.5% gel for mTOR. Proteins were electrophoretically transferred to PVDF membranes. The blots were incubated with primary antibodies to total S6K1 (cat. no. 230, Santa Cruz Biotechnology), phosphorylated (Ser473; cat. no. 9271, Cell Signaling Technology) and total PKB (cat. no. 9272, Cell Signaling Technology), or phosphorylated (Ser2448; cat. no. 2971, Cell Signaling Technology) and total mTOR (cat. no. 2972, Cell Signaling Technology). The blots were then developed by use of an ECL Western blotting kit according to the manufacturer's instructions. Films were scanned and quantitated as described in determination of the phosphorylation state of 4E-BP1. As discussed for 4E-BP1, resolution of S6K1 on SDS polyacrylamide gels results in the separation of the protein into multiple isoelectric forms. The slowest-migrating forms represent hyperphosphorylated forms of the protein, and the fastest migrating forms represent hypo- or dephosphorylated forms of the protein.

Measurement of plasma concentrations of insulin and free AAs. Plasma insulin concentrations were analyzed by the use of a commercial radioimmunoassay kit for rat insulin (DiaSorin, Stillwater, MN). For determination of plasma concentrations of free AAs, plasma was combined with an equal volume of deproteinizing agent (6% sulfosalicylic acid) derivatized with phenylisothiocyanate and followed by high-performance liquid chromatography analysis.

Statistical method. Data for each group are represented as means ± SE. Statistical evaluation of the data was performed by use of a two-way ANOVA followed by the Tukey-Kramer test to determine treatment effect. Differences between the groups were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Changes in Tcore. To examine whether intravenous administration of an AA mixture affects body temperature differently during states of anesthesia (Ane) and consciousness (Nor), we first measured the Tcore of rats during a 3-h infusion of an AA mixture or Sal together with either the anesthetic propofol (consisting of fat emulsion) or a fat emulsion (Fig. 1A). The baseline values of Tcore did not differ among the groups. Tcore in the Nor-AA group increased at 1 h after the onset of infusion, showing the significant difference compared with baseline through the infusion periods (P < 0.01; paired t-test). After a 3-h administration of test solutions, Tcore in the Nor-AA group increased from baseline by 0.45°C. On the other hand, Tcore in the Nor-Sal group did not change during the infusion. Although Tcore in both Ane groups fell dramatically during the infusion, the reduction of Tcore from baseline was 4.6 and 3.3°C in the Sal and AA groups, respectively. At the end of the infusion, Tcore was significantly higher in the Ane-AA group than in the Ane-Sal group.


Figure 1
View larger version (7K):
[in this window]
[in a new window]
  		Fig. 1. Effects of treatment with anesthetic and amino acid (AA) administration on intraperitoneal temperature (Tcore; A), rates of muscle protein synthesis (Ks; B), and plasma insulin concentrations (C). Tcore, Ks, and plasma insulin concentrations were measured in rats infused for 3 h with either a balanced AA mixture (A, {blacktriangleup} or bullet; B and C, filled bars) or saline (Sal) (A, {triangleup} or {circ}; B and C, open bars) either in the conscious (Nor) state (A, {triangleup} and {blacktriangleup}; B and C, left) or under propofol-induced anesthesia (Ane) (A, {circ} and bullet; B and C, right). Values are means ± SE, n = 8 (Tcore and plasma insulin concentrations) and n = 5–6 (Ks). In Tcore: *significant differences (P < 0.05) between the same status group with a different solution by Tukey-Kramer multiple comparisons test. All groups except for the Nor-Sal group represent the significant difference in Tcore compared with baseline through the infusion periods by paired t-test ({dagger}). In Ks and plasma insulin concentrations, means not sharing a superscript are different (P < 0.05).

 
Protein synthesis in skeletal muscle. We next examined the Ks in skeletal muscle of rats infused for 3 h with an AA mixture or Sal during states of anesthesia and consciousness to examine whether AA administration affects muscle protein synthesis differently during the states of anesthesia and consciousness (Fig. 1B). The Ks in skeletal muscle was less in the anesthetized rats than in the normal rats. The Ks was 44% less in the Ane-Sal group than in the Nor-Sal group and 56% less in the Ane-AA group than in the Nor-AA group. The Ks in skeletal muscle was 24% greater in the Nor-AA group (6.8%/day) than in the Nor-Sal group. Under anesthesia, when muscle protein synthesis is markedly repressed, the Ks was 57% greater in the Ane-AA group (3.8%/day) than in the Ane-Sal group. However, the Ks remained lower in the Ane-AA group than in the Nor-Sal group.

Plasma concentrations of insulin and free AAs. We have no information regarding how administration of an AA mixture to rats under anesthesia affects plasma insulin and free AA levels. Therefore, we measured plasma insulin and free AA concentrations in rats infused for 3 h with Sal or AA under the states of anesthesia and consciousness. Plasma insulin levels did not significantly differ between the Nor-Sal group and the Ane-Sal group (Fig. 1C). In contrast, plasma insulin concentrations in the Ane-AA group were highest among the treatment groups, about sixfold greater compared with the Nor-AA group. As shown in Table 1, the treatment with anesthetic did not affect the respective free AA levels in rats given Sal. Total amounts of all AA concentrations in both AA groups were increased; the AA concentrations were greater in the Ane-AA group than in the Nor-AA group. The observations of the individual plasma AA levels in the AA-infused groups showed that plasma concentrations of all AAs excluding tryptophan, asparagine, glutamine, and serine were higher in the Ane-AA group than in the Nor-AA group.


View this table:
[in this window]
[in a new window]
  		Table 1. Plasma AA concentrations

 
Phosphorylation of several components of translation initiation cascade. The mechanism through which AA administration regulates the stimulation of muscle protein synthesis under anesthesia remains to be determined. As shown in Fig. 2A, immunoblots of 4E-BP1 after SDS-PAGE exhibit three electrophoretic forms, termed {alpha}, beta, and {gamma} by the differences in their states of protein phosphorylation. The most highly phosphorylated form, the {gamma}-form, dissociates itself with eIF4E and increases the availability of eIF4E required for the formation of eIF4G·eIF4E complex (41). The proportion of 4E-BP1 in the {gamma}-form did not differ between the Nor-Sal group and the Ane-Sal group. In contrast, greater change in electrophoretic mobility of 4E-BP1 (~6-fold) to the {gamma}-form in the Nor-AA group was shown when compared with the Nor-Sal group. Moreover, we confirmed that the infusion of the AA mixture in rats under anesthesia dramatically accelerated the increase in the amount of 4E-BP1 in the {gamma}-form by 40% compared with those in the conscious state.


Figure 2
View larger version (39K):
[in this window]
[in a new window]
  		Fig. 2. Effects of treatment with anesthetic and AA on phosphorylation states of 4E-binding protein-1 (4E-BP1; A), S6 protein kinase (S6K1; B), PKB (C), and mammalian target of rapamycin (mTOR; D) in skeletal muscle. A–D, top: representative immunoblots; bottom: densitometric analysis of immunoblots as described in METHODS. The proportions of 4E-BP1 in the hyperphosphorylated {gamma}-isoform as a percentage of total 4E-BP1, the proportions of S6K1 in the phosphorylated forms as a percentage of total S6K1 protein, and the amounts of Ser473-phosphorylated PKB and of Ser2448-phosphorylated mTOR were examined in skeletal muscles of rats after 3-h administration with either a balanced AA mixture (filled bars) or Sal (open bars) either in Nor (left) or Ane (right). Values are means ± SE; n = 6. Means not sharing a superscript are different (P < 0.05).

 
We also examined the proportion of the phosphorylated forms of S6K1 in skeletal muscle (Fig. 2B) because the phosphorylation forms of S6K1 are associated with increased activity of the protein for phosphorylating the downstream targets (9). Like 4E-BP1, in the Ane-AA group the proportion of phosphorylated forms of S6K1 was dramatically increased compared with all other groups. Rats in the Nor-AA group also showed significant increases in the phosphorylation forms of S6K1 compared with rats in the Nor-Sal group.

We examined phosphorylation of PKB on Ser473 in skeletal muscle because the phosphorylation of PKB on Ser473 is associated with increased activity of the protein for phosphorylating downstrean targets (2). In the Nor-AA group, the phosphorylation of PKB on Ser473 was markedly enhanced compared with that in both Sal groups (Fig. 2C). The phosphorylation of PKB on Ser473 in the Ane-AA group was further enhanced, and the amounts of the phosphorylated PKB were 53% higher in the Ane-AA group than in the Nor-AA group. However, no significant difference in the phosphorylation of PKB on Ser473 was found between the Nor-Sal group and the Ane-Sal group. Any change in PKB content was not observed under any experimental conditions.

mTOR is activated through the phosphorylation at Ser2448 by PKB in the insulin-signaling pathway (33, 34) and phosphorylates 4E-BP1 and S6K1 (20, 22). Administration of the AA mixture dramatically phosphorylated the residue of mTOR at Ser2448 compared with the two Sal treatment groups (Fig. 2D). The phosphorylation of mTOR was further enhanced by the simultaneous treatment with AA and anesthetic. However, the degree of phosphorylation of mTOR on Ser2448 did not differ between the two Sal groups. Neither the treatment with AA nor the treatment with anesthetic altered mTOR content in the muscle of rats examined.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was conducted to elucidate the contribution of protein synthesis, after intravenous administration of an AA mixture, to the prevention of hypothermia during anesthesia. Using propofol-treated rats, we confirmed that administration of an AA mixture during anesthesia attenuated the marked decrease of Tcore during anesthesia. A significant increase in Tcore was also observed in the Nor-AA group. As speculated in several reports (39, 40), this difference possibly indicates that the feedback regulation in the central nervous system to prevent the temperature of intracranial blood from exceeding a certain value may control the heat production by the AA administration in the conscious state (23) more strictly than in the state under anesthesia (38). According to a recent report (31), even in conscious subjects the AA administration adjusts thermoregulatory defense thresholds upward and increases the resting core temperature.

The present study demonstrated that administration of an AA mixture prominently elevated plasma insulin levels and stimulated protein synthesis in skeletal muscle, even in rats under anesthesia when protein synthesis was markedly depressed. The increase of insulin at supraphysiological blood concentrations or the correction of insulin deficiency, both of which were accomplished by the exogenous addition of insulin, contributed to activate the stimulation of protein synthesis in skeletal muscle (14, 15, 17, 24, 42, 44). These several lines of evidence supported the suggestion that the marked increases in plasma insulin levels seen in the Ane-AA group contributed to the improvement of anabolic response under anesthesia. However, muscle Ks in the Ane-AA group remained lower than in the Nor-Sal group, implying that some systemic alteration during anesthesia limits the effect of insulin on muscle protein synthesis. As a factor, the decline in body temperature itself might play a significant role in lowering the Ks in the Ane-AA group, as is suggested by the finding that a drop of 2°C in body temperature results in an ~20% inhibition of Ks in skeletal muscle and liver of rats (8). The fall in metabolic rate caused by the administration of anesthetics may be also relevant to the negative effect on the protein synthesis due to the fall in metabolic rate, because the treatment with anesthetics depresses metabolic rate 20 to 30% (38) and the amount of oxygen consumption correlates with the Ks (21). Moreover, the decrease in blood flow caused by the hypotension and the diminished heart rate during propofol anesthesia (10) may reduce Ks in the Ane-AA group because muscle protein synthesis is positively correlated with blood flow (5).

The observations shown in the present study suggest that the synergistic increase in insulin levels caused by the administration of an AA mixture and the anesthetic possibly results in the activation of insulin signaling to the translation initiation factors through mTOR in skeletal muscle under conditions of anesthesia and that the phase of translation initiation plays an important role in stimulating muscle protein synthesis by acute nutritional changes during anesthesia as well as in the conscious state (25, 27, 29, 39). However, the phosphorylations of these translation factors are higher in the Ane-AA group than in either of the conscious groups, whereas muscle Ks is less in the Ane-AA group than in either of the conscious groups, implying the existence of other rate-limiting factors in the stimulation of protein synthesis by the treatment with an AA mixture and anesthetic. One potential mechanism is that, unless the delivery of Met-tRNAi to the 40S ribosomal subunit mediated by eIF2 is also appropriately maintained to the delivery of 7-methyl-GTP-capped mRNAs to the 40S ribosomal subunit, the stimulation of muscle protein synthesis by the administration of AA may not be adequately induced during anesthesia. Note that in the brain of the hibernating animal, in which hypothermia is accompanied by decreased protein synthesis, the eIF2 {alpha}-phosphorylated form, which decreases the eIF2B availability, is increased (16). However, an obvious relationship between the stimulation of protein synthesis by acute nutritional change and phosphorylation of eIF2{alpha} or the activity of eIF2B has not been shown in previous in vivo studies (3, 28, 46). Hence, further studies are needed to clarify what the limiting component is in the stimulation of muscle protein synthesis by AA administration during anesthesia.

The finding of synergistic elevation in insulin levels when an AA mixture and anesthetics were coinfused, first demonstrated by the present study, is very intriguing. It is well known that GABAA agonists such as the propofol, used in the present study (13), have reduced the sympathetic tone, decreased the release of norepinephrine (10–12), and, furthermore, inhibited norepinephrine uptake in synaptic transmission at a clinically relevant concentration (40). The reduction in sympathetic tone leads to the exertion of inhibitory effects on insulin release from beta-cells via the {alpha}2-adrenegic pathway (32). In addition, various AAs were highly effective in stimulating insulin secretion (6, 30). Integrating these findings with our present data, we suggest that anesthetics can augment the insulin secretion affected by AA administration in the absence of neuronal-adrenal feedback inhibition. This view is supported by the observation that postprandial plasma insulin levels were increased in quadriplegic patients with complete cervical cord lesions when compared with those in healthy subjects (1).

Note that the observations in this study did not explain completely the mechanisms underlying the prevention of hypothermia by AA administration under anesthesia. Administration of AA under anesthesia contributes not only increased heat production but also a raised threshold for thermoregulatory vasoconstriction (26). How these alterations caused by AA administration are integrated remains unknown, but a role for AA administration in the stimulation of accumulation of heat is likely to gain wide acceptance.

In summary, the present data suggest that the stimulation of protein synthesis in skeletal muscle contributes in some way to the attenuation of anesthesia-induced hypothermia by AA administration. We further demonstrated that administration of both AA and the anesthetic exerts a synergic effect on the phosphorylation of S6K1 and 4E-BP1 via insulin-mTOR signaling, suggesting that an elevation of translation initiation may play a primary role in AA-induced stimulation of protein synthesis under anesthesia. These responses are likely to be triggered by the markedly elevated insulin levels that result from administering AA in rats under anesthesia compared with those in the conscious state.


   ACKNOWLEDGMENTS
 
We thank Yuichi Kawano for assistance in preparing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Ippei Yamaoka, Naruto, Tokushima 772–8601, Japan (e-mail: yamaokih{at}otsukakj.co.jp)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aksnes AK, Brundin T, Hjeltnes N, Maehlum S, and Wahren J. Meal-induced rise in resting energy expenditure in patients with complete cervical spinal cord lesions. Paraplegia 31: 462–472, 1993.[Web of Science][Medline]
  2. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, and Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15: 6541–6551, 1996.[Web of Science][Medline]
  3. Anthony JC, Anthony TG, Kimball SR, Vary TC, and Jefferson LS. Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J Nutr 130: 139–145, 2000.[Abstract/Free Full Text]
  4. Balage M, Sinaud S, Prod'homme M, Dardevet D, Vary TC, Kimball SR, Jefferson LS, and Grizard J. Amino acids and insulin are both required to regulate assembly of the eIF4E·eIF4G complex in rat skeletal muscle. Am J Physiol Endocrinol Metab 281: E565–E574, 2001.[Abstract/Free Full Text]
  5. Biolo G, Tipton KD, Klein S, and Wolfe RR. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. Am J Physiol Endocrinol Metab 273: E122–E129, 1997.[Abstract/Free Full Text]
  6. Blachier F, Mourtada A, Sener A, and Malaisse WJ. Stimulus-secretion coupling of arginine-induced insulin release. Uptake of metabolized and nonmetabolized cationic amino acids by pancreatic islets. Endocrinology 124: 134–141, 1989.[Abstract/Free Full Text]
  7. Brundin T and Wahren J. Effects of i.v. amino acids on human splanchnic and whole body oxygen consumption, blood flow, and blood temperatures. Am J Physiol Endocrinol Metab 266: E396–E402, 1994.[Abstract/Free Full Text]
  8. Caso G, Garlick BA, Casella GA, Sasvary D, and Garlick PJ. Response of protein synthesis to hypercapnia in rats: independent effects of acidosis and hypothermia. Metabolism 54: 841–847, 2005.[CrossRef][Web of Science][Medline]
  9. Dufner A and Thomas G. Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res 253: 100–109, 1999.[CrossRef][Web of Science][Medline]
  10. Ebert TJ. Sympathetic and hemodynamic effects of moderate and deep sedation with propofol in humans. Anesthesiology 103: 20–24, 2005.[CrossRef][Web of Science][Medline]
  11. Elbert TJ and Muzuki M. Propofol and autonomic reflex in humans. Anesth Analg 78: 369–375, 1994.[Web of Science][Medline]
  12. Elbert TJ, Muzuki M, Berens R, Goff D, and Kampine JP. Sympathetic responses to induction of anesthesia in human with propofol or etomidate. Anesthesiology 76: 725–733, 1992.[Web of Science][Medline]
  13. Feng HJ and Macdonald RL. Multiple actions of propofol on alphabetagamma and alphabetadelta GABAA receptors. Mol Pharmacol 66: 1517–1524, 2004.[Abstract/Free Full Text]
  14. Flatt JP. The biochemistry of energy expenditure. In: Recent Advances in Obesity Research, edited by Bray G. London: Newman, 1978, vol. 2, chapt. 22, p. 211–228.
  15. Fluckey JD, Pohnert SC, Boyd SG, Cortright RN, Trappe TA, and Dohm GL. Insulin stimulation of muscle protein synthesis in obese Zucker rats is not via a rapamycin-sensitive pathway. Am J Physiol Endocrinol Metab 279: E182–E187, 2000.[Abstract/Free Full Text]
  16. Frerichs KU, Smith CB, Brenner M, DeGracia DJ, Krause GS, Marrone L, Dever TE, and Hallenbeck JM. Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation. Proc Natl Acad Sci USA 95: 14511–14516, 1998.[Abstract/Free Full Text]
  17. Garlick PJ, Fern M, and Preedy VR. The effect of insulin infusion and food intake on skeletal muscle synthesis in postabsorptive rats. Biochem J 210: 669–676, 1983.[Web of Science][Medline]
  18. Garlick PJ and Grant I. Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Effect of branched-chain amino acids. Biochem J 254: 579–584, 1988.[Web of Science][Medline]
  19. Garlick PJ, McNurlan MA, and Preedy VR. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H] phenylalanine. Biochem J 192: 719–723, 1980.[Web of Science][Medline]
  20. Gingras AC, Kennedy SG, O'Leary MA, Sonenberg N, and Hay N. 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev 12: 502–513, 1998.[Abstract/Free Full Text]
  21. Giordano M and Castellino P. Correlation between amino acid induced changes in energy expenditure and protein metabolism in humans. Nutrition 13: 309–312, 1997.[Web of Science][Medline]
  22. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, and Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 273: 14484–14494, 1998.[Abstract/Free Full Text]
  23. Hayward JN and Baker MA. Role of cerebral arterial blood in the regulation of brain temperature in the monkey. Am J Physiol 215: 389–403, 1968.[Free Full Text]
  24. Jefferson LS. Lilly Lecture 1979: role of insulin in the regulation of protein synthesis. Diabetes 29: 487–496, 1980.[Web of Science][Medline]
  25. Jefferson LS and Kimball SR. Translational control of protein synthesis: implications for understanding changes in skeletal muscle mass. Int J Sport Nutr Exerc Metab 11: S143–S149, 2001.[Web of Science][Medline]
  26. Kasai T, Nakajima Y, Matsukawa T, Ueno H, Sunaguchi M, and Mizobe T. Effect of preoperative amino acid infusion on thermoregulatory response during spinal anaesthesia. Br J Anaesth 90: 58–61, 2003.[Abstract/Free Full Text]
  27. Kimball SR, Farrell PA, and Jefferson LS. Exercise effects on muscle insulin signaling and action. Role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise. J Appl Physiol 93: 1168–1180, 2002.[Abstract/Free Full Text]
  28. Kimball SR and Jefferson LS. Role of amino acids in the translational control of protein synthesis in mammals. Semin Cell Dev Biol 16: 21–27, 2005.[CrossRef][Web of Science][Medline]
  29. Lawrence JC Jr. mTOR-dependent control of skeletal muscle protein synthesis. Int J Sport Nutr Exerc Metab 11: S177–S184, 2001.[Medline]
  30. Malaisse WJ. Branched chain amino and keto acids as regulator of insulin and glucagons release. In: Branched Chain Amino and Keto Acids in Health and Disease, edited by Adibi SA, Fekl W, Langenbeck U, and Schauder P. Basel, Switzerland: Karger, 1984, p. 119–133.
  31. Nakajima Y, Takamata A, Matsukawa T, Sessler DI, Kitamura Y, Ueno H, Tanaka Y, and Mizobe T. Effect of amino acid infusion on central thermoregulatory control in humans. Anesthesiology 100: 634–639, 2004.[CrossRef][Web of Science][Medline]
  32. Nakaki T, Nakadate T, and Kato R. Alpha 2-adrenoceptors modulating insulin release from isolated pancreatic islets. Naunyn Schmiedebergs Arch Pharmacol 313: 151–153, 1980.[CrossRef][Web of Science][Medline]
  33. Nave BT, Ouwens M, Withers DJ, Alessi DR, and Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344: 427–431, 1999.[CrossRef][Web of Science][Medline]
  34. Sekulic A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM, and Abraham RT. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 60: 3504–3513, 2000.[Abstract/Free Full Text]
  35. Sellden E. Nutritional substrate and perioperative hypothermia. Curr Anaesth Crit Care 12: 103–108, 2001.[CrossRef]
  36. Sellden E. Peri-operative amino acid administration and the metabolic response to surgery. Proc Nutr Soc 61: 337–343, 2002.[CrossRef][Web of Science][Medline]
  37. Sellden E, Branstrom R, and Brundin T. Augmented thermic effect of amino acids under general anaesthesia occurs predominantly in extra-splanchnic tissues. Clin Sci (Lond) 91: 431–439, 1996.[Medline]
  38. Sessler DI. Temperature monitoring. In: Anesthesia (4th ed), edited by Miller RD. Philadelphia, PA: Churchill Livingstone, 1994, p. 1363–1382.
  39. Shah OJ, Anthony JC, Kimball SR, and Jefferson LS. 4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle. Am J Physiol Endocrinol Metab 279: E715–E729, 2000.[Abstract/Free Full Text]
  40. Shahani SK, Lingamaneni R, and Hemmings HC Jr. General anesthetic actions on norepinephrine, dopamine, and gamma-aminobutyric acid transporters in stably transfected cells. Anesth Analg 95: 893–899, 2002.[Abstract/Free Full Text]
  41. Sonenberg N. mRNA 5' cap-binding protein eIF4E and control of cell growth. In: Translational Control, edited by Hershey JWB, Mathews MB, and Sonenberg N. Plainview, NY: Cold Spring Harbor Laboratory, 1996, p. 245–269.
  42. Svanberg E, Zachrisson H, Ohlsson C, Iresjo BM, and Lundholm KG. Role of insulin and IGF-I in activation of muscle protein synthesis after oral feeding. Am J Physiol Endocrinol Metab 270: E614–E620, 1996.[Abstract/Free Full Text]
  43. Tsujinaka T, Sakaue M, Iijima S, Ebisui C, Kan K, Kishibuchi M, Morimoto T, and Kido Y. Modulation of thermogenic response to parenteral amino acid infusion in surgical stress. Nutrition 12: 36–39, 1996.[CrossRef][Web of Science][Medline]
  44. Volpi E, Lucidi P, Cruciani G, Monacchia F, Reboldi G, Brunetti P, Bolli GB, and De Feo P. Contribution of amino acids and insulin to protein anabolism during meal absorption. Diabetes 45: 1245–1252, 1996.[Abstract]
  45. Yoshizawa F, Endo M, Ide H, Yagasaki K, and Funabiki R. Translational regulation of protein synthesis in the liver and skeletal muscle of mice in response to refeeding. J Nutr Biochem 6: 130–136, 1995.[CrossRef][Web of Science]
  46. Yoshizawa F, Kimball SR, and Jefferson LS. Modulation of translation initiation in rat skeletal muscle and liver in response to food intake. Biochem Biophys Res Commun 240: 825–831, 1997.[CrossRef][Web of Science][Medline]
  47. Yoshizawa F, Kimball SR, Vary TC, and Jefferson LS. Effect of dietary protein on translation initiation in rat skeletal muscle and liver. Am J Physiol Endocrinol Metab 275: E814–E820, 1998.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/E882    most recent
00333.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamaoka, I.
Right arrow Articles by Yoshizawa, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamaoka, I.
Right arrow Articles by Yoshizawa, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.