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Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain -ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1 subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferatoractivated receptor-ɣ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-D-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals. [ABSTRACT FROM AUTHOR]

Sepsis and lipopolysaccharide (LPS) may decrease skeletal muscle protein synthesis by impairing mTOR (mammalian target of rapamycin) activity. The role of mTOR in regulating muscle protein synthesis was assessed in wild-type (WT) and mTOR heterozygous (+/-) mice under basal conditions and in response to LPS and/or leucine stimulation. No difference in body weight of mTOR+/- mice was observed compared with WT mice; whereas whole body lean body mass was reduced. Gastrocnemius weight was decreased in mTOR+/- mice, which was attributable in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and mTOR+/- mice to the same extent. Reduced muscle protein synthesis in mTOR+/- mice under basal and LPS-stimulated conditions was associated with lower 4E-BP1 and S6K1 phosphorylation. LPS also decreased PRAS40 phosphorylation and increased phosphorylation of raptor and IRS-1 (SeFa307) to the same extent in WT and mTOR+/- mice. Muscle atrogin-1 and MuRF1 mRNA content was elevated in mTOR+/- mice under basal conditions, implying increased ubiquitin-proteasome-mediated proteolysis, but the LPS-induced increase in these atrogenes was comparable between groups. Plasma insulin and IGF-1 as well as tissue expression of TNFα, IL-6, or NOS2 did not differ between WT and mTOR+/- mice. Finally, whereas LPS impaired the ability of leucine to stimulate muscle protein synthesis and 4E-BPI phosphorylation in WT mice, this inflammatory state rendered mTOR+/- mice leucine unresponsive. These data support the idea that the LPS-induced reduction in mTOR activity is relatively more important in regulating skeletal muscle mass in response to nutrient stimulation than under basal conditions. [ABSTRACT FROM AUTHOR]

Acute alcohol intoxication decreases skeletal muscle protein synthesis by impairing mammalian target of rapamycin (mTOR). In 2 studies, we determined whether inhibition of branched-chain amino acid (BCAA) catabolism ameliorates the inhibitory effect of alcohol on muscle protein synthesis by raising the plasma BCAA concentrations and/or by improving the anabolic response to insulin-like growth factor (IGF)-l. In the first study, 4 groups of mice were used: wild-type l\NT) and mitochondrial branched-chain aminotransferase (BCATm) knockout (KO) mice orally administered saline or alcohol (5 g/kg, 1 h). Protein synthesis was greater in KO mice compared with \NT controls and was associated with greater phosphorylation of eukaryotic initiation factor (elF)-4E binding protein-i (4EBP1), eIF4E-eIF4G binding, and 4EBP1- regulatory associated protein of mTOR (raptor) binding, but not mTOR-raptor binding. Alcohol decreased protein synthesis in WT mice, a change associated with less 4EBP1 phosphorylation, eIF4E-eIF4G binding, and raptor-4EBP1 binding, but greater mTOR-raptor complex formation. Comparable alcohol effects on protein synthesis and signal transduction were detected in BCATm KO mice. The second study used the same 4 groups, but all mice were injected with IGF-l (25 μg/ mouse, 30 minI. Alcohol impaired the ability of IGF-I to increase muscle protein synthesis, 4EBP1 and 70-kilodalton ribosomal protein S6 kinase-1 phosphory)ation, eIF4E-eIF4G binding, and 4EBP1-raptor binding in WT mice. However, in alcohol-treated BCATm KO mice, this IGF-l resistance was not manifested. These data suggest that whereas the sustained elevation in plasma BCAA is not sufficient to ameliorate the catabolic effect of acute alcohol intoxication on muscle protein synthesis, it does improve the anabolic effect of IGF-l. [ABSTRACT FROM AUTHOR]

Feeding promotes protein synthesis in cardiac muscle through a stimulation of the messenger RNA translation initiation phase of protein synthesis by enhancing assembly of active eukaryotic initiation factor (eIF)4F complex. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in increasing formation of active eIF4G-eIF4E complex during meal feeding. Hearts from male Sprague-Dawley rats fed a meal consisting of rat nonpurified diet were sampled prior to and 3 h following the meal in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1. Rapamycin prevented the meal feeding-induced stimulation of myocardial protein synthesis. Inhibition of mTOR with rapamycin decreased the association of rapamycin-associated TOR protein with mTOR and prevented the feeding-induced assembly of eIF4G-eIF4E complex. In contrast, the abundance of eIF4E binding protein-1 (4E-BP1)-eIF4E complex was unaffected by either meal feeding or rapamycin. Pretreatment with rapamycin completely prevented the feeding-induced phosphorylation of eIF4G(Ser(1108)), whereas the inhibitor only partially attenuated meal feeding-induced 70-kDa ribosomal protein S6 kinase1(Thr(389)) phosphorylation and extent of 4E-BP1 in the gamma-form. Meal feeding-induced phosphorylation of protein kinase B on either Ser(473) or Thr(308) was unaffected by rapamycin. These findings suggest the extent of phosphorylation of eIF4G following meal feeding occurs by a rapamycin-sensitive mechanism in cardiac muscle. Furthermore, the rapamycin-sensitive reductions in phosphorylation of eIF4G may also lead to decreased formation of active eIF4G-eIF4E complex. [ABSTRACT FROM AUTHOR]

Phosphorylation of eukaryotic initiation factor 4G (eIF4G) is hypothesized to be an important contributor to the stimulation of protein synthesis in skeletal muscle following meal feeding. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in mediating the meal feeding-induced elevations in phosphorylation of eIF4G. Gastrocnemius from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled prior to and following 3 h of having the meal provided in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Pretreatment with rapamycin prevented the feeding-induced phosphorylation of mTOR, eIF4G, and S6K1 but only partially attenuated the shift in 4E-BP1 into the γ-form. In contrast, the feeding-induced increase in phosphorylation of PKCε was not reduced by rapamycin. Rapamycin also prevented the augmented association of eIF4G with eIF4E and the decreased association of eIF4E with 4E-BP1. Similar findings were observed in gastrocnemius from animals after oral administration of leucine. Perfusion of gastrocnemius with medium containing rapamycin partially prevented the leucine-induced increase in phosphorylation of eIF4G. Thus, rapamycin attenuated a feeding- or leucine-induced phosphorylation of eIF4G in skeletal muscle both in vivo and in situ. The latter observation implies that the effects observed with rapamycin were the result of modulation of skeletal muscle signaling mechanisms responsible for eIF4G phosphorylation. [ABSTRACT FROM AUTHOR]

Feeding promotes protein synthesis in cardiac muscle through a stimulation of the mRNA translation initiation phase of protein synthesis either secondary to nutrient-induced rises in insulin or because of direct effects of nutrients themselves. The present set of experiments establishes the effects of meal feeding on the potential signal transduction pathways that may be important in accelerating mRNA translation initiation. Hearts were obtained from male Sprague Dawley rats that had been trained to consume a meal consisting of nonpurified diet prior to, during, and following the test meal. Meal feeding raised the extent of phosphorylation of eukaryotic initiation factor (eIF)4G (Ser(1108)), which returned to basal levels within 3 h of removal of food. Likewise, meal feeding was associated with an increase in phosphorylation of eIF4E binding protein-1(4EBP1) in the gamma-form during feeding. Phosphorylation of mammalian target of rapamycin (mTOR) on Ser(2448) or Ser(2481) or 70-kDa ribosomal protein S6 kinase (S6K1) on Thr(389) was not affected by meal feeding or following removal of food. Likewise, the extent of phosphorylation of TSC2, a potential upstream regulator of mTOR, was not significantly altered during meal feeding. Phosphorylation of protein kinase B (PKB) (Thr(308)) was elevated at all time points after initiating meal feeding. Similarly, the phosphorylation of protein kinase C(PKC)-epsilon but not PKC-delta was elevated at all time points after initiating meal feeding. We conclude from these studies that meal feeding stimulates at least 2 signal pathways in cardiac muscle that raises phosphorylation of eIF4G and 4EBP1 during meal feeding and results in sustained increases in phosphorylation of PKB and PKC-epsilon. [ABSTRACT FROM AUTHOR]

Nutrients enhance signaling pathways involved in skeletal muscle growth through an increased rate of protein synthesis. These studies have led to an understanding of the potential role of the mammalian target of rapamycin (mTOR) in this process. However, activation of mTOR cannot account for all the stimulatory effects of nutrients. The purpose of these experiments was to examine the effect of nutrients on the cellular distribution and activation state of novel PKC isoforms (PKC∈ and PKCδ) in the gastrocnemius of rats by use of modification state-dependent phosphopeptide specific antibodies. The phosphorylation of PKC∈ on the catalytic domain autophosphorylation site (Ser729) was elevated during feeding and then returned to basal levels when the feeding period ended. Meal feeding augmented the phosphorylation of the downstream effectors of mTOR, namely S6K1 and 4E-BP1. In contrast, the phosphorylation of PKCδ on either the catalytic domain autophosphorylation site (Ser643) or activation loop site (Thr505) was unaffected. Similar results were obtained when animals were given leucine either acutely via gavage or chronically by dietary supplementations. The effect of leucine was not mimicked by injecting animals with insulin but could be induced by gavage with norleucine, a structural analog of leucine that does not increase plasma insulin concentration. Thus rises in insulin secondary to meal intake or leucine gavage are probably not responsible for increased phosphorylation of PKC∈ in response to meal feeding. Elevating the leucine concentration stimulated the phosphorylation of PKC∈ in gastrocnemius from perfused hindlimb and caused a shift in the distribution of PKC∈ from the membrane fraction to the cytosolic fraction. The results indicate that leucine leads to an activation (autophosphorylation) and subcellular redistribution of PKC∈, but not PKCδ, in gastrocnemius both in vivo and in vitro. Furthermore, activation of the mTOR signaling pathway above basal conditions does not appear to be necessary to induce phosphorylation or translocation of PKC∈, suggesting that multiple signaling pathways become activated with leucine. [ABSTRACT FROM AUTHOR]

Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyperplastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain α-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1α-subunit at Ser[sup 293] (S293), which inactivates the complex. Because the α-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to roTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies, increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr[sup 389], the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR. [ABSTRACT FROM AUTHOR]

Insulin and Leu stimulate a rapamycin sensitive cell-signaling pathway involving TOR complex 1 (TORC1). While the steps leading to insulin activation are known, it is unclear how Leu activates TORC1. It has been proposed that Leu metabolism stimulates mTOR, however this hypothesis has not been rigorously tested except with non-specific inhibitors. To address this question, we generated a line of transgenic mice (BCATm KO) missing the first step in leucine metabolism catalyzed by the mitochondrial branched chain aminotransferase isozyme (BCATm). Muscle and heart TORC1 signaling was activated following insulin injection but not after oral leucine administration. In Langendorf perfused hearts, insulin stimulated S6K1 and 4E-BP1 phosphorylation, whereas leucine was ineffective. To bypass the BCATm block from BCATm, hearts were perfused with the BCATm reaction product, KIC; however it too failed to stimulated S6K phosphorylation. Because BCATm KO animals have elevated plasma BCAAs, TORC1 may be desensitized to Leu; therefore fibroblasts were prepared from newborn mice. Even after five passages in Minimal Essential Media, TORC1 signaling from insulin was observed, however neither Leu nor KIC activated S6K1. BCAAs concentrations were not elevated in conditioned media from KO cells. The results suggest that the BCATm plays a critical role in Leu signaling to TORC1. [ABSTRACT FROM AUTHOR]

Phosphorylation of eukaryotic initiation factor (eIF4G) has been suggested to an important mediator stimulating protein synthesis following meal feeding. We examined the potential role for a rapamycin-sensitive pathway mediating the increased level of eIF4G phosphorylation during meal feeding. Gastrocnemius from male Sprague Dawley rats trained to consume a meal consisting of rat chow, was sampled prior to and following 3h of providing the meal in presence or absence of rapamycin an inhibitor of mammalian Target of Rapamycin (mTOR) complex 1 (TORC 1). Phosphorylation of mTOR was inhibited by rapamycin. Pretreatment with rapamycin prevented the feeding-induced rise in level of phosphorylation of eIF4G and S6K1 but only partially attenuated the shift in 4E-BP1 into the γ-form following meal feeding. Rapamycin also prevented the augmented association of eIF4G with eIF4E, and the decreased the association of eIF4E with 4EBP1. Rapamycin also prevented the leucine-induced rise in level of phosphorylation of eIF4G in perfused hindlimb. In contrast, the feeding-induced increase in phosphorylation of PKC-ε was not reduced by rapamycin following meal feeding. These data imply that the effects we observed with rapamycin are the result of modulation of skeletal muscle cell signaling mechanisms responsible for eIF4G phosphorylation rather than alterations in circulating hormones or metabolites. [ABSTRACT FROM AUTHOR]