1Division of Nutritional Sciences, Departments of 5Food Science and Human Nutrition, 2Animal Sciences, and 4Pathology, University of Illinois Urbana-Champaign, Urbana, Illinois; and 3Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Evansville, Indiana
Submitted 23 June 2004 ; accepted in final form 20 August 2004
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ABSTRACT |
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glucose; insulin receptor substrate-1; initiation factors; phosphatidylinositol 3-kinase
Interaction of Leu and insulin as regulators of the insulin signaling/phosphatidylinositol 3-kinase (PI 3-kinase) phosphorylation cascade suggests a unique link between dietary protein content and peripheral actions of insulin (28). The PI 3-kinase signaling cascade is central to regulation of many metabolic processes, such as glucose uptake and protein synthesis. The signaling pathway is initiated when insulin binds its membrane-bound receptor. The insulin receptor contains an intrinsic tyrosine protein kinase activity that triggers a series of phosphorylations on the insulin receptor substrate-1 (IRS-1). IRS-1 serves as a docking protein to mediate downstream signals through phosphotyrosine-SH-2 domain interactions with PI 3-kinase. PI 3-kinase plays a major role in regulation of the metabolic actions of insulin signaling, including stimulation of glucose transport via phosphorylation of Akt or protein kinase C- and -
, resulting in stimulation of the GLUT4 glucose transporter and stimulation of protein synthesis through activation of the protein kinase mTOR (mammalian target of rapamycin; see Ref. 8 for review). Increased intracellular Leu triggers mTOR-dependent translational stimulation of protein synthesis (3) via increased phosphorylation of p70S6 kinase and the inhibitory protein 4E-BP1, causing it to dissociate from the translation initiation factor eIF4E, resulting in increased translation initiation (17, 26).
Although these findings suggest an anabolic role for Leu, there are other reports that Leu can decrease glucose disposal, induce hyperinsulinemia, and potentially lead to insulin resistance (1, 15). Studies using euglycemic clamps and infusions of Leu in human forearm during fasted conditions resulted in increased plasma glucose concentrations, decreased glucose uptake, and elevated plasma insulin levels in a dose-dependent manner (29). Likewise, Ferrannini et al. (9) found that intravenous administration of a complete mixture of amino acids stimulated insulin secretion and reduced glucose use in normal humans. Boden and Tappy (6) found that infusion of a large dose of amino acids stimulated plasma insulin levels but did not alter insulin-stimulated glucose uptake.
At the molecular level, amino acids appear to influence upstream events in the insulin signaling pathway via mTOR phosphorylation of IRS-1 (24, 32). Addition of amino acids at increased concentrations decreases association of PI 3-kinase with IRS-1 and reduces PI 3-kinase activity (24, 32), suggesting that supplemental amino acids may impair insulin signaling and contribute to insulin resistance. Although high levels of amino acids reduce PI 3-kinase activity, they appear to have no effect on insulin-mediated activation of PI 3-kinase activity but instead accelerate the rate of deactivation (32). L6 muscle cells exposed to elevated concentrations of amino acids had increased Ser/Thr phosphorylation and degradation of IRS-1/PI 3-kinase complex. Similar results exist in 3T3-L1 adipocytes (30).
In total, high levels of amino acids (presumably Leu) stimulate mTOR, resulting in increased downstream signaling pathway activity at eIF4E and p70S6 kinase, whereas mTOR phosphorylation of IRS-1 appears to decrease upstream PI 3-kinase activity, hence, an apparent paradox with amino acids stimulating downstream muscle protein synthesis, while at the same time reducing insulin signaling activity and glucose transport. The report by Tremblay and Marette (32) appears to provide a hypothesis to explain both findings. These investigators suggest that Leu stimulation of mTOR does not prevent the initial activation of PI 3-kinase but instead accelerates the degradation of the active IRS-1/PI 3-kinase complex. Hence, mTOR may be a component of the feedback regulation of the signaling pathway.
As a first step to evaluate this hypothesis, we examined the time course of the Leu and insulin effects on PI 3-kinase activity after a meal and examined physiological outcomes associated with upstream activation (i.e., glucose transport) and downstream activation (i.e., translational initiation). Specifically, to isolate effects of insulin vs. Leu on regulation of the insulin/PI 3-kinase signaling pathway, we designed the following three acute feeding treatments: 1) a high-carbohydrate (CHO) meal designed to produce elevated blood insulin with basal levels of Leu, 2) a high-Leu meal designed to produce high blood Leu with basal levels of insulin, and 3) a combined meal of CHO plus Leu to produce elevated levels of both insulin and Leu.
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MATERIALS AND METHODS |
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Before (12 h) the experiment, food was removed from all cages, and animals were randomly assigned to one of the following three treatments: CHO, Leu, or a combined meal of CHO plus Leu (CHO + Leu). The CHO treatment provided 2.63 g CHO (mixture of 262.5 g glucose/l and 262.5 g sucrose/l in distilled water) accounting for 15% of daily energy intake for this age and strain of rat (14). The Leu treatment provided 0.27 g Leu (54.0 g Leu/l in distilled water) equivalent to the amount of Leu consumed by rats during 24 h of free access to AIN-93 powdered diet. The CHO + Leu treatment was a combination of the CHO and Leu treatments described above. These treatments were designed to represent potential dietary effects of CHO and Leu. Treatment consisted of 5 ml oral gavage of the designated test meal.
The combined treatment of CHO + Leu could be designed as levels of CHO and Leu equal to the individual treatments or as an isocaloric treatment with substitution of Leu for an equal energy level of sucrose from the CHO treatment. A preliminary experiment comparing these approaches showed that the decrease in CHO in the isocaloric treatment resulted in decreases in the plasma concentrations of glucose and insulin and PI 3-kinase activity. To ensure these differences were because of Leu and not the varying levels of CHO, we kept the level of CHO the same in the combined CHO + Leu test meal.
After oral gavage of the test meal, animals were killed at 0, 15, 30, 60, or 90 min after gavage. At the time of the oral gavage, animals killed at 60 min also received an intraperitoneal injection of 20 µCi 2-[14C]deoxyglucose (Amersham Biosciences, Piscataway, NJ) to measure glucose uptake by tissues.
Because of potential differences in the rate of gastric emptying associated with the presence of amino acids (20), we conducted an initial study to determine if oral administration of Leu interfered with the rate of glucose absorption from the gut. Animals were assigned to either CHO or CHO + Leu treatments identical to those described above. In addition, 20 µCi 2-[14C]deoxyglucose were included in the gavage solution. Animals were killed at 15, 30, and 60 min. Plasma specific activity of glucose was determined to compare treatment effects on glucose entry in the bloodstream from the gut.
Sample collection. Animals were killed via decapitation. Trunk blood was collected and centrifuged at 1,800 g for 10 min at 4°C to obtain plasma. Gastrocnemius and plantaris muscles were removed and immediately frozen in liquid nitrogen.
Plasma measurements. Plasma glucose concentrations were determined by the glucose oxidase method (Infinity Glucose Reagent; Sigma, St. Louis, MO). Plasma insulin was measured using a commercial radioimmunoassay kit for rat insulin (Linco Research, St. Charles, MO).
Determination of PI 3-kinase activity.
Gastrocnemius muscles from each animal were homogenized using a Polytron homogenizer in 7 vol buffer containing 50 mM HEPES, 150 mM NaCl, 2 mM sodium vanadate, 10 mM sodium pyrophosphate, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 5 µg/ml leupeptin, 1 µg/ml aprotonin, 1% IGEPAL (Sigma), and 10% glycerol. Immediately after homogenization, samples were centrifuged at 35,000 g for 60 min at 4°C. Supernatant was collected and stored at 80°C until analysis. PI 3-kinase was immunoprecipitated from supernatants with anti-IRS-1 antibodies (Upstate Biotechnology, Charlottesville, VA), and resultant immune complexes were washed extensively. Kinase reactions were performed in a buffer containing 0.33 mg/ml L--phosphatidylinositol, 10 mM MgCl2, 0.4 mM EGTA, 0.4 mM NaHPO4, 7.5 µM [
-32P]ATP (13 µCi/nmol), and 20 mM HEPES, pH 7.1 for 15 min. Phospholipids were extracted with 1:1 chloroform-methanol and resolved on silica gel plates by TLC in chloroform-methanol-ammonium hydroxide (4 M; 75:58:17). Results were analyzed by autoradiography on a Molecular Dynamics Typhoon phosphorimage system (22).
Quantification of IRS-1 and PI 3-kinase. Muscle supernatants, prepared as described above, were immunoprecipitated with an IRS-1-specific antibody and resolved on 7.5% SDS-PAGE to determine total IRS-1 content in each tissue. Total PI 3-kinase content was quantified using a PI 3-kinase antibody (Upstate Biotechnology) and running the samples on a 10% SDS-PAGE.
Glucose uptake.
Animals received an intraperitoneal injection of 2-deoxy-D-[2,6-3H]glucose (Amersham Biosciences) immediately after the oral gavage. For determination of glucose uptake, plantaris muscles (200 mg) were homogenized in 5 ml double-distilled H2O and boiled for 3 min at 100°C. Samples were centrifuged at 12,000 g for 5 min. Sample supernatant (1.5 ml) was applied to an equilibrated anion-exchange resin (Dowex 2 x 8, 400 mesh; Sigma) according to methods described by Ohishima et al. (23).
Quantification of 4E-BP1·eIF4E complex.
Gastrocnemius muscles were homogenized using a Polytron homogenizer in 7 vol of homogenization buffer consisting of 20 mM HEPES, 100 mM potassium chloride, 0.2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 50 mM sodium fluoride, 50 mM -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.5 mM sodium vanadate, and 1 µM Microcystin LR (Sigma). The homogenate was immediately centrifuged at 10,000 g for 10 min at 4°C. The supernatant was used for measurement of 4E-BP1 bound to eIF4E according to methods described by Gautsch et al. (14).
Statistics. Data analyses were performed using SAS version 8.2 (SAS Institute, Cary, NC). Data were analyzed using a one-way ANOVA with treatment group as the independent variable and statistical significance set at P < 0.05.
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RESULTS |
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DISCUSSION |
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To our knowledge, this is the first study to demonstrate the potential of Leu to modulate upstream elements of the insulin signaling pathway under physiological conditions. Studies using in vitro methods report that amino acids inhibit upstream events in the insulin signaling pathway (24, 32). These studies conclude that addition of amino acids at increased concentrations decreases the association of PI 3-kinase to IRS-1, resulting in inhibition of insulin-stimulated PI 3-kinase activity. In the present study, we found that an oral dose of Leu had no effect on PI 3-kinase activity compared with fasted control animals. However, when Leu and CHO were administered together, Leu appears to blunt the increased PI 3-kinase activity seen in animals administered CHO alone.
Although Leu alters PI 3-kinase activity, it did not affect glucose uptake in skeletal muscle under sedentary conditions compared with CHO-treated animals. Blood glucose concentrations (Fig. 1), absorption data (Fig. 3), and rate of glucose uptake in muscle (Fig. 5) are in agreement that Leu had minimal effect on glycemic control. In addition, glucose and insulin levels did not differ between the CHO + Leu and CHO treatments. These data indicate that administration of a physiological dose of Leu by oral gavage has minimal effect on plasma insulin and no effect on the in vivo glycemic response.
We propose that the decreases in PI 3-kinase activity seen in animals receiving CHO + Leu vs. CHO alone could be attributed to the following two possible mechanisms: 1) increased IRS-1 Ser/Thr phosphorylation, inhibiting formation of the IRS-1/PI 3-kinase complex, or 2) increased degradation of the IRS-1/PI 3-kinase complex. Tremblay and Marette (32) proposed that Leu accelerates degradation of the active IRS-1/PI 3-kinase complex. The importance of the distinction between these two potential mechanisms is that, in the former scenario, the signal is blocked and does not appear, whereas in the later scenario, the signal appears but the magnitude or duration of the signal is shortened. For example, if the signal was blocked, loss of PI 3-kinase activity would be expected to reduce insulin-dependent glucose transport. However, if the signal is initiated, the signal cascade could activate physiological outcomes, such as glucose transport or protein synthesis, and the subsequent deactivation may be more important to the duration of the signal or feedback regulations. The present study supports the second scenario, with findings of normal levels of glucose transport and normal activation of the initiation factor eIF4E.
There are numerous reports that amino acids such as Leu and arginine can induce hyperinsulinemia and potentially lead to insulin resistance (15). Studies using euglycemic clamps and infusions of Leu in the human forearm, under fasted conditions, resulted in increased plasma glucose concentrations, decreased glucose uptake, and elevated plasma insulin levels in a dose-dependent manner (29). In addition, intravenous administration of mixed amino acids stimulates insulin secretion and inhibits glucose use in normal humans (9, 25). Additional studies conclude that, after infusion with a large dose of amino acids, plasma insulin levels increase; however, insulin-stimulated total body glucose uptake is unaltered (6). An important consideration with these studies is that they used intraveneous euglycemic clamp methods conducted at supraphysiological concentrations.
In the present study, Leu, when given alone via oral gavage, produced an initial rise in plasma insulin concentration that returned to baseline within 30 min after the gavage. When Leu was given in combination with CHO, the early insulin response was indistinguishable from the response to CHO alone; however, at 60 and 90 min after the gavage, the CHO + Leu group had a somewhat higher level of insulin. Responses of plasma glucose to the Leu treatment were small. In the Leu-only treatment, there was a 15% decrease in the blood glucose concentration at 60 min after the gavage; whereas, in the CHO + Leu group, plasma glucose was 15% lower than the CHO alone treatment at 90 min after the gavage. These decreases in blood glucose appear to follow the Leu-induced peaks in plasma insulin.
These data confirm that Leu is an insulin secretagogue (11, 12, 19); however, using direct comparisons with oral CHO, a large physiological dose of Leu produced minimal insulin response. The impact of Leu on insulin release and glycemic response appears to differ widely based on route of administration and experimental conditions. These findings are consistent with the previous reports by Floyd et al. (11, 12). These investigators evaluated the insulin response to intravenous infusion of amino acids or glucose (12) and also examined the insulin response to oral intake of protein (11). They found that infusion of 30 g amino acids produced a threefold higher insulin response (180 µU/ml) than infusion of 30 g glucose (
50 µU/ml), suggesting a dramatic hyperinsulinemic effect of amino acids. However, these investigators also examined the same measurements after subjects consumed a meal of 500 g beef liver and found that the peak insulin response to the protein meal was only 30 µU/ml. Assuming that Leu is one of the most potent insulin secretagogues, the intravenous infusion provided <5 g Leu, whereas the beef meal provided one >14 g Leu (11). These data suggest that amino acids have substantially less impact on plasma insulin concentrations when entering the body via the gastrointestinal tract.
Similarly, Nuttall and Gannon and colleagues (13, 16, 21) reported minimal and colleagues effect of increased dietary protein on plasma insulin and glucose. Using isoenergetic meals, they demonstrated that substituting dietary protein for CHO reduced the meal responses of both plasma glucose and insulin (21). Likewise, they reported that, with consumption of a test meal containing 50 g protein (consumed as lean beef) vs. 50 g glucose, the protein intake alone had essentially no impact on basal blood glucose concentrations, and the insulin response to the meal was <20% of the response with a comparable energy intake from glucose (13). Although these results are intuitively obvious, they directly contradict the findings from studies using euglycemic clamp methods.
In summary, Leu participates in modulation of the insulin/PI 3-kinase signaling pathway. Leu appears to increase mTOR kinase activity, resulting in stimulation of translation control of protein synthesis (5). In the present study, we demonstrate that Leu also modifies upstream activity by modulating PI 3-kinase activity. Specifically, Leu appears to blunt the duration of the insulin-induced PI 3-kinase activity. Although Leu modifies PI 3-kinase activity in the presence of high blood glucose, there was no effect of Leu on net glucose transport in muscle or on stability of blood glucose concentration. These data suggest that Leu interacts with the insulin/PI 3-kinase signaling pathway at multiple steps but that a high Leu meal has no effects on acute glycemic control.
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GRANTS |
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FOOTNOTES |
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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.
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REFERENCES |
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