1 Department of Physiology and Lipid Research Unit, Laval University Hospital Research Center, Ste-Foy, Québec, Canada
2 Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
3 Department of Surgery, Medical University of Vienna,Vienna, Austria
4 1. Medical Department, Hanusch Hospital, Vienna, Austria
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ABSTRACT |
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Recent data strongly support the notion that nutrient excess plays a predominant role in the development of insulin resistance and manifestation of type 2 diabetes (15). Meat consumption has increased by one third in industrialized countries since the year 1960 (6), resulting in excess nutrient supply of fat and proteins. The hypothesis that increased availability of amino acids might modulate glucose metabolism is supported by recent data showing that a rise in plasma amino acids directly inhibits insulin-stimulated glucose transport/phosphorylation into skeletal muscle, leading to impaired muscle glycogen synthesis and reduced insulin-stimulated whole-body glucose disposal in healthy humans (7). Furthermore, plasma amino acid elevation enhances gluconeogenesis sufficiently to induce hyperglycemia in the presence of impaired insulin secretion (8). Thus, increased availability of amino acids directly modulates hepatic and skeletal muscle glucose metabolism both in the presence of hyperinsulinemia and insulin deficiency. Of note, the effects of short-term amino acid elevation on glucose metabolism in the presence of low peripheral hyperinsulinemia have not yet been evaluated under standardized conditions.
In vitro studies have shown that amino acids generate signals transduced by the serine/threonine kinase mammalian target of rapamycin (mTOR) to regulate protein synthesis and cell proliferation as mediated by S6 kinase 1 (S6K1) and eukaryotic initiation factor 4Ebinding protein 1 (4E-BP1) (9). Thus, mTOR appears to sense the availability of nutrients, most notably amino acids. Interestingly, activation of the mTOR pathway by prolonged insulin stimulation or increased amino acid availability induces insulin resistance in muscle cells and adipocytes in vitro (1012). Activation of mTOR and/or its downstream target, S6K1, leads to serine/threonine phosphorylation and consequently proteosomal degradation of insulin receptor substrate (IRS)-1 (1012). This in turn results in inhibition of phosphoinositide (PI) 3-kinase activity (13,14), an essential step in insulin-mediated glucose metabolism. We further demonstrated that amino acidinduced mTOR/S6K1 activation speeds up insulin-dependent PI 3-kinase deactivation in cultured myocytes even before detectable IRS-1 degradation, suggesting that uncoupling of IRS-1/PI 3-kinase signaling is an early event in the interaction between amino acids and insulin action (13).
This study was therefore designed to determine the effects of short-term elevation of plasma amino acids on whole-body rate of glucose disappearance (Rd) as well as endogenous glucose production (EGP) in the presence of low peripheral hyperinsulinemia as well as during prandial-like peripheral hyperinsulinemia. Furthermore, the insulin-dependent activation of early steps in insulin signaling was simultaneously assessed under these conditions both in biopsies of human skeletal muscle and in cultured L6 skeletal muscle cells.
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RESEARCH DESIGN AND METHODS |
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Seven participants were studied twice during infusion of amino acids and control saline infusion in random order. The two study days were separated by 414 weeks during which time their body weight and lifestyle remained unchanged.
After a 12-h overnight fast, all studies began at 7:30 A.M. (150 min) with the insertion of catheters (Vasofix; Braun, Melsungen, Germany) into one antecubital vein of the left and one of the right arm for blood sampling and infusions, respectively.
Pancreatic clamps were performed as described (7,8,15,16) (Fig. 1). Briefly, somatostatin (UCB Pharma, Vienna, Austria) was infused (5 to 360 min, 0.1 nmol · kg1 · min1) to suppress amino acidinduced secretion of glucoregulatory hormones. From 0 to 180 min, insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) was continuously replaced at a rate of 0.25 mU · kg1 · min1, and from 180 to 360 min, a primed-continuous insulin infusion (1 mU · kg1 · min1) was administered to create standardized conditions of a two-step (peripheral) hyperinsulinemic clamp (0180 min, low peripheral hyperinsulinemia 100 pmol/l; 180360 min, prandial-like peripheral hyperinsulinemia
430 pmol/l). Plasma glucose was maintained at
5.5 mmol/l using a variable D-glucose infusion (20%). Glucose turnover rates were determined using D-[6,6-2H2] glucose (99% enrichment, Cambridge Isotope Laboratories, bolus 16.7 µmol/kg, continuous infusion from 120 min to +360 min, 0.17 µmol · kg1 · min1). To maintain stable enrichments of D-[6,6-2H2]glucose during the clamp tests, the variable glucose infusion was enriched to 2% with D-[6,6-2H2]glucose (7). The enrichment of D-[6,6-2H2]glucose in the variable glucose infusion was adjusted to prevent tracer dilution during the first step (low peripheral hyperinsulinemia) of the clamp, when EGP was expected to rise during amino acid infusion (8). This led to slightly higher enrichment of the variable glucose infusion so that tracer-to-tracee ratios similarly increased within 60 min by
44% (amino acid 42 ± 0.09 vs. control 45 ± 0.04%) and remained stable thereafter during both protocols. On one study day, plasma amino acid concentrations were raised by infusion (0330 min) of a mixture of amino acids (0.18 g · kg1 · h1; Aminoplasmal 10% without electrolytes; Braun, Melsungen, Germany) which is commonly used for parenteral nutrition (7,8). This solution contains the following L-amino acids: isoleucine (5.1 g/l), leucine (8.9 g/l), valine (4.8 g/l), lysine (5.6 g/l), methionine (3.8 g/l), phenylalanine (5.1 g/l), tryptophan (1.8 g/l), arginine (9.2 g/l), histidine (5.2 g/l), N-acetyl-cysteine (0.7 g/l), proline (8.9 g/l), threonine (4.1), glutamate (4.6 g/l), serine (2.4 g/l), glycine (7.9 g/l), alanine (13.7 g/l), asparagine (3.3 g/l), ornithine (3.2 g/l), tyrosine (0.3 g/l), N-acetyl-tyrosine (1.3 g/l), aspartate (1.3 g/l). During control studies, normal saline was infused at identical infusion rates.
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Needle biopsies of the vastus lateralis muscle.
The subjects were supine and resting quietly for at least 60 min, and the right vastus lateralis muscle was prepared sterilely under subcutaneous lidocaine (Xylocain 2%; Astra, Linz, Austria) anesthesia (17). At baseline (120 min), a muscle sample was obtained using a modified Bergström biopsy needle with suction, blotted free of blood, fat, and connective tissue and snap frozen within 30 s in liquid nitrogen. After 30 min of hyperinsulinemia (210 min), a repeat muscle biopsy was taken at a site 4 cm proximal of the first biopsy. All samples were stored in liquid nitrogen until analysis. Muscle biopsies were homogenized with a polytron in six volumes of lysis buffer containing 50 mmol/l HEPES, pH 7.5, 137 mmol/l NaCl, 1 mmol/l CaCl2, 1 mmol/l MgCl2, 10% glycerol, 2 mmol/l EDTA, 10 mmol/l NaF, 2 mmol/l Na3VO4, and protease inhibitor cocktail. Muscle homogenates were solubilized in 1% NP-40 for 1 h at 4°C and centrifuged at 14,000g for 10 min. The supernatant was used for insulin signaling studies as described below.
Cell culture and treatment.
A line of L6 skeletal muscle cells (gift of Dr. Amira Klip, Hospital for Sick Children, Toronto, ON, Canada) clonally selected for high fusion potential was used in the present study. The L6 cell line was derived from neonatal rat thigh skeletal muscle cells and retains many morphological, biochemical, and metabolic characteristics of skeletal muscle. Cells were grown and maintained in monolayer culture in minimum essential medium (MEM) containing 10% (vol/vol) fetal bovine serum and 1% (vol/vol) antibiotic/antimycotic solution (10,000 U/ml penicillin, 10,000 nmol/l streptomycin and 25 nmol/l amphotericin B) in an atmosphere of 5% CO2 at 37°C. L6 myoblasts were allowed to differentiate into myotubes in
-MEM containing 2% (vol/vol) fetal bovine serum and antibiotics for 7 days. Then, serum-deprived (4 h) cells were incubated either in an amino acidfree (Earles balanced salt solution) or amino acidcontaining medium (Earles balanced salt solution + 2x MEM amino acid solution) as previously described (13). Vehicle (0.01% DMSO) or rapamycin (25 nmol/l) was added during the 1-h incubation and stimulated with or without insulin as indicated in the figure legends.
Western blotting.
Muscle homogenates were subjected to SDS-PAGE (18). Antibodies were obtained from Cell Signaling (phospho-IRS-1 [Ser312 and Ser636/639], phospho-GSK-3/ß [Ser21/9] and phospho-S6K1 [Thr421/Ser424]), Santa Cruz (IRS-1 and S6K1), and Upstate Biotechnology (IRS-1 and p85 subunit of PI 3-kinase).
S6K1 activity.
A total of 500 µg muscle homogenates were immunoprecipitated with 2 µg anti-S6K1 coupled to protein A-Sepharose for 2 h at 4°C. Immune complexes were washed three times in 50 mmol/l Tris-acetate, pH 8.0, 5 mmol/l ß-glycerophosphate, 1 mmol/l EDTA, 1 mmol/l EGTA, 0.1% ß-mercaptoethanol, 2 mmol/l Na3VO4, 10 mmol/l NaF, and twice in kinase buffer (50 mmol/l MOPS, pH 7.5, 25 mmol/l ß-glycerophosphate, 5 mmol/l EGTA, 2 mmol/l EDTA, 20 mmol/l MgCl2, 1 mmol/l DTT, 2 mmol/l Na3VO4, and 10 mmol/l NaF). The reaction was started by adding 50 µl kinase buffer (containing 100 µmol/l ATP, 2 µCi [-32P]ATP, and 100 µmol/l S6K1 substrate [Santa Cruz]) for 10 min at 30°C. Reaction product was spotted on p81 filter paper (Whatman) and washed 3 x 15 min with 1% phosphoric acid. Incorporated radioactivity was determined by liquid scintillation counting.
IRS-1associated PI 3-kinase activity.
PI 3-kinase activity was measured in IRS-1 immunoprecipitates based on the technique used for rat muscle (13). In brief, muscle homogenates (1 mg) were incubated with protein A-Sepharose beads (Amersham Bioscience, Uppsala, Sweden) precoupled to IRS-1 antibody (H-165; Santa Cruz Biotechnology, Santa Cruz, CA). Immune complexes were washed, and beads were resuspended in 70 µl kinase buffer (8 mmol/l Tris, pH 7.5, 80 mmol/l NaCl, 0.8 mmol/l EDTA, 15 mmol/l MgCl2, 180 µmol/l ATP, and 5 µCi [-32P]ATP) and 10 µl sonicated PI mixture (20 µg L-
-PI, 10 mmol/l Tris, pH 7.5, and 1 mmol/l EGTA) for 15 min at 30°C. Reaction was stopped by the addition of 20 µl of 8 mol/l HCl, mixed with 160 µl CHCl3:CH3OH (1:1) and centrifuged. Lower organic phases were spotted on oxalate-treated silica gel TLC plate and developed in CHCl3:CH3OH:H2O:NH4OH (60:47:11.6:2). The plate was dried and visualized by autoradiography with intensifying screen at 80°C.
Plasma metabolites and hormones.
Plasma glucose concentrations were measured by the glucose oxidase method (Glucose Analyzer II; Beckman Instruments, Fullerton, CA). Concentrations of individual plasma amino acids were measured by high-performance liquid chromotography (19). Plasma FFA concentrations were determined with a microfluorimetric method (Wako, Richmond, VA). Plasma immunoreactive insulin, C-peptide, glucagon, and growth hormone were measured by commercially available radioimmunoassays (7,8,15,16,20) (insulin, Pharmacia, Uppsala, Sweden; C-peptide, Cis, Gif-Sur-Yvette, France; glucagon, Linco, St. Charles, MO; growth hormone, Sorin Biomedica, Saluggia, Italy). Plasma cortisol was determined following extraction and charcoal-dextran separation by radioimmunoassay (21).
Gas chromatographymass spectrometry for the determination of tracer-to-tracee ratios of 2H in glucose was performed as described (7,8). The glucose-pentaacetate was analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a CP-Sil5 25 m x 0.25 mm x 0.12-µm capillary column (Chrompack, Middelburg, Netherlands) interfaced to a Hewlett-Packard 5971A mass selective detector operating in the electron impact ionization mode. Selective ion monitoring was used to determine tracer enrichments in various molecular mass ion fragments of glucose. 6,6-2H2 (M+2) enrichments in glucose were assessed using fragments of C3-C6 with their masses of 187 and 189, respectively.
Calculations.
Rates of glucose appearance (Ra) and disappearance (Rd) were calculated using Steeles nonsteady-state equations modified for the use of stable isotopes (7,8) with EGP given as the difference between Ra and glucose infusion rates.
Data analysis and statistics.
Autoradiographs were analyzed by laser scanning densitometry using a tabletop Agfa scanner (Arcus II; Agfa-Gavaert, Morstel, Belgium) and quantified with the National Institutes of Health Image program (available from http://rsb.info.nih.gov/nih-image/). Immunoblots and kinase activity determinations were expressed relative to those obtained from a single muscle biopsy to allow comparisons between samples processed during different experiments.
Hormone profiles and glucose turnover data from both protocols (i.e., amino acid and control) were compared by a two-way ANOVA with two within repeated-measures factors for the basal period, the low- and the high-insulin period of the clamp tests. The first within-subject repeated-measures factor with two levels accounted for the paired crossover study design and the second for repetitive measurements during each period of the clamp test. Differences between protocols during each period were assessed from the main effect of the first repeated-measures factor, and differences between amino acid and controls at singular time points were calculated with post hoc Tukey honest significant difference correction for multiple comparisons. Changes of sequential (time-dependent) data within protocols were analyzed by ANOVA for repeated measurements and Dunnetts post hoc testing. Muscle insulin signaling data were analyzed by ANOVA followed by Bonferroni/Dunns post hoc testing. Differences were considered significant at P < 0.05.
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RESULTS |
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Insulin signaling.
To assess the molecular mechanisms underlying amino acidinduced insulin resistance in human skeletal muscle, activation status of mTOR/S6K1 and that of the IRS-1/PI 3-kinase/Akt pathways was measured. Before insulin infusion (basal), S6K1 activity was similar between saline and amino acidinfused subjects (Fig. 4). However, although raising insulinemia to prandial-like peripheral hyperinsulinemia only led to a modest increase in S6K1 activity in saline-infused subjects (1.9-fold above basal), combined hyperinsulinemia and hyperaminoacidemia resulted in a highly significant 3.7-fold increase in the enzymatic activity of S6K1. Given the prominent role of S6K1 in mediating serine phosphorylation of IRS-1 (22), we assessed whether phosphorylation of IRS-1 using phospho-specific antibodies against Ser312 and Ser636/639 (equivalent to Ser307 and Ser632/635 in mice, respectively) was increased in skeletal muscle of amino acidinfused subjects. Whereas immunoreactivity to both antibodies was barely detectable in skeletal muscle isolated from saline-infused subjects, amino acid infusion increased the phosphorylation state of IRS-1 on Ser312 (Fig. 5A) and Ser636/639 (Fig. 5B) in the insulin-stimulated state. Quantification of immunoblots from several muscle biopsies revealed that insulin increased IRS-1 Ser636/639 phosphorylation up to sixfold in amino acidinfused subjects. Furthermore, we found that the heavily serine phosphorylated form of IRS-1 detected in biopsies from amino acidinfused subjects was not prone to a greater rate of degradation because its expression level in muscle homogenates was similar to that of saline-infused humans (Fig. 5C).
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DISCUSSION |
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In the present study, the effects of plasma amino acid elevation on endogenous glucose production and whole-body Rd were evaluated in the presence of low (0180 min) and high (180360 min) peripheral hyperinsulinemia corresponding to fasting portal vein insulin concentrations and prandial peripheral hyperinsulinemia, respectively. This study demonstrates that low peripheral hyperinsulinemia suppressed endogenous glucose production by 70% during control studies. However, this insulin-mediated decline in glucose production was completely blunted during amino acid infusion. This can likely be attributed to direct stimulation of gluconeogenesis by amino acid (8,32) since amino acidinduced endogenous release of glucoregulatory hormones, including glucagon, was inhibited by continuous somatostatin infusion. Impaired postprandial suppression of endogenous glucose production contributes to hyperglycemia in patients with type 2 diabetes (33). Increased amino acid availability could, therefore, play a role in the impairment of insulin-mediated suppression of endogenous glucose production observed in obese patients with type 2 diabetes (34).
In the presence of prandial-like peripheral hyperinsulinemia (180360 min), whole-body Rd was 33% lower during amino acid infusion compared with control studies. Since under such insulin-stimulated conditions the majority of glucose is taken up by skeletal muscle (35), this can be attributed to amino acidinduced skeletal muscle insulin resistance. We have previously shown that amino acids directly inhibit both insulin-stimulated glucose transport/phosphorylation into skeletal muscle and glycogen synthesis reflecting reduced insulin-stimulated whole-body Rd in healthy humans (7). However, the molecular mechanisms involved are still unclear in vivo.
Our first effort to identify the molecular events linked to skeletal muscle insulin resistance under physiological hyperaminoacidemia pointed on the mTOR nutrient-sensing pathway (13). Insulin activation of S6K1, a downstream effector of mTOR, is potentiated by increased amino acid availability, leading to increased inhibitory serine/threonine phosphorylation of IRS-1 and rapid time-dependent deactivation of PI 3-kinase activity (13). In this study, we present evidence that this negative feedback loop is also operative in human skeletal muscle under parenteral nutrient satiation in vivo. Indeed, our data show that amino acid infusion increases activation of S6K1 and phosphorylation of IRS-1 on multiple serine residues, leading to impaired stimulation of PI 3-kinase by insulin in human muscle. Further studies in L6 myocytes confirmed that inhibition of mTOR/S6K1 by rapamycin prevents amino acidinduced hyperphosphorylation of IRS-1 on Ser636/639 and restored insulin activation of PI 3-kinase associated with IRS-1 and glucose transport. In support of these findings is the recent observation that S6K1-deficient mice are protected against diet-induced insulin resistance by a mechanism that involves, at least in part, reduced phosphorylation of IRS-1 on Ser307 and Ser636/639 (22), thereby placing S6K1 as a major player involved in the development of insulin resistance under nutrient abundancy in both animals and humans.
An important aspect of the present study is the observation that amino acidinduced desensitization of insulin action in vivo occurred without any detectable degradation of IRS-1 despite its elevated content in phosphoserine residues. This result was somewhat unexpected because prior in vitro work suggested that ser/thr phosphorylation of IRS-1 is a prerequisite for triggering its degradation (36), notably via the proteasomes (11). Our results suggest therefore that degradation of IRS-1 is not essential for the occurrence of muscle insulin resistance in amino acidinfused human subjects. Moreover, the observation that insulin resistance induced by chronic high-fat feeding (4 months) does not alter IRS-1 protein levels despite its elevated phosphoserine content (22) further suggests that IRS-1 ser/thr phosphorylation, but not its degradation, is the principal mechanism leading to nutrient-induced insulin resistance under both short- and long-term settings in vivo.
Identification of the molecular locus of impaired insulin signaling during the development of insulin resistance is a long-lasting quest. Although it has become obvious that impaired activation of PI 3-kinase is a hallmark of insulin resistance in both animal models and humans, the potential role of Akt in insulin resistance is far from being conclusive. In the present study, we found that Akt was normally activated, despite marked impairment in PI 3-kinase activity. These results are reminiscent of the findings of Kim et al. (37), who found Akt to be normally stimulated by insulin despite reduced PI 3-kinase activity in skeletal muscle of type 2 diabetic subjects. This may be explained by the fact that Akt requires only partial activation of PI 3-kinase to get fully activated. On the other hand, we previously observed that overactivation of the mTOR pathway during prolonged hyperinsulinemia (4 h) reduces Akt phosphorylation concomitantly with degradation of IRS-1 (13). It is therefore possible that a defect in insulin-induced Akt activation occurs only after chronic stimulation of the mTOR/S6K1 pathway by amino acid and marked reduction in IRS-1 content and associated PI 3-kinase activity.
Some limitations of our study must be considered. First, the present study compared hyperaminoacidemic with moderately hypoaminoacidemic conditions. In the present study, total plasma amino acid concentrations observed during amino acid infusion are 40% higher than those seen after ingestion of a large size (50 g) protein meal (38). Furthermore, plasma amino acids were maintained at these elevated concentrations throughout the infusion. The moderate insulin-induced decrease in plasma amino acids during the control study is similar to what has been observed after a carbohydrate-rich meal (39) and to control experiments of other studies (40). Thus, it cannot be ruled out that the differences observed between hyperaminoacidemic and hypoaminoacidemic conditions might be attenuated when fasting plasma amino acid concentrations would be present during the control study. In the present study, an amino acid mixture was infused so that one cannot discriminate between the impact of individual amino acids or certain combinations of amino acids on the obtained results. Furthermore, it cannot be excluded that products of amino acid metabolism contribute to the observed effects. Because it is not feasible to completely control the diet of the participants who are not in-patients for several weeks, differences in dietary habits might have affected the results. We aimed to minimize this possible influence by including volunteers with constant and weight-maintaining dietary habits and by studying them twice during infusion of amino acids and control saline infusion in random order, which allowed intraindividual comparisons.
Second, this study was performed in the absence of endogenous secretion of glucoregulatory hormones. An amino acidinduced rise in plasma glucagon was prevented to exclude its stimulatory effects on EGP, which would likely have induced hyperglycemia in the presence of low peripheral hyperinsulinemia and thereby would have obscured direct amino acid action on hepatic glucose metabolism (8). It is of note that administration of protein or amino acid gives rise to glucagon secretion (8), and hyperglucagonemia is frequently present in type 2 diabetic patients (41), which is different from the hormonal environment created in the present study. Nevertheless, effects of glucagon on skeletal muscle insulin signaling are unlikely because glucagon receptors could not be demonstrated in skeletal muscle (42), and glucagon has no effect on metabolism of forearm tissues in humans (43). However, indirect effects of glucagon on muscle, e.g., by modulation of hepatic glucose metabolism cannot be completely excluded. Of note, the experimental design employed in the present study does not mimic the time course of plasma amino acid and glucagon concentrations in response to ingestion of a protein meal; conclusions on the effects of oral protein intake on skeletal muscle glucose metabolism and insulin signaling have to be drawn with caution.
In summary, we show that increased amino acid availability resulting in overactivation of S6K1 is tightly linked to reduction of insulin-stimulated glucose metabolism in human skeletal muscle. S6K1 seems to operate a feedback loop toward IRS-1 by targeting at least two sets of phosphorylation sites (Ser312 and Ser636/639), causing inhibition of PI 3-kinase and, consequently, muscle glucose uptake. Alternatively, the reported mechanisms could also represent a physiological response to increased amino acid availability. Nevertheless, S6K1 could be an attractive target in the prevention and treatment of nutrient-induced insulin resistance.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the excellent cooperation with G. Pfeiler, F. Garo, A. Hofer, H. Lentner, and the Endocrine Research Laboratory.
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FOOTNOTES |
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F.T. is currently affiliated with the Department of Biochemistry, McGill University, Montréal, Québec, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address correspondencereprint requests to Michael Roden, MD, Department of Internal Medicine III, Währinger Gürtel 1820, A-1090 Vienna, Austria. E-mail: michael.roden{at}meduniwien.ac.at. Or André Marette, PhD, Lipid Research Unit, CHUL Research Center, Ste-Foy, Québec, G1V 4G2, Canada. E-mail: andre.marette{at}crchul.ulaval.ca
Received for publication December 13, 2004 and accepted in revised form May 27, 2005
4E-BP1, eukaryotic initiation factor 4Ebinding protein 1; EGP, endogenous glucose production; FFA, free fatty acid; GSK, glycogen synthase kinase; IRS, insulin receptor substrate; MEM, minimum essential medium; mTOR, mammalian target of rapamycin; PI, phosphoinositide; PKB, protein kinase B; S6K1, S6 kinase 1
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REFERENCES |
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