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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.sup.+/-] mice was observed compared with WT mice; whereas whole body lean body mass was reduced. Gastrocnemius weight was decreased in [mTOR.sup.+/-] mice, which was attributable in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and [mTOR.sup.+/-] mice to the same extent. Reduced muscle protein synthesis in [mTOR.sup.+/-] 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 ([Ser.sup.307]) to the same extent in WT and [mTOR.sup.+/-] mice. Muscle atrogin-1 and MuRF1 mRNA content was elevated in [mTOR.sup.+/-] 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-I as well as tissue expression of TNF[alpha], IL-6, or NOS2 did not differ between WT and [mTOR.sup.+/-] mice. Finally, whereas LPS impaired the ability of leucine to stimulate muscle protein synthesis and 4E-BP1 phosphorylation in WT mice, this inflammatory state rendered [mTOR.sup.+/-] 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. mammalian target of rapamycin; endotoxin; protein synthesis; eukarotic initiation factor 4E-binding protein-1; ribosomal protein S6 kinase-1; leucine doi: 10.1152/ajpendo.00676.2009.

Introduction The branched-chain amino acids (BCAA), including leucine, isoleucine, and valine, are essential amino acids that participate in the de novo synthesis and structural maintenance of nascent protein and biosynthesis [...], The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-α-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1α subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1α dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain [alpha]-keto acid dehydrogenase (BCKD E1[alpha] subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1[alpha] or BCKD kinase, in liver BCKD E1[alpha] was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1[alpha]. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1[alpha] in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity. obesity; mitochondrial branched chain amino acid transaminase; branched chain keto acid dehydrogenase; branched chain keto acid dehydrogenase kinase; ob/ob mice; Zucker rats; bariatric surgery; humans

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 (roTOR) 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 [gamma]-form. In contrast, the feeding-induced increase in phosphorylation of PKCe 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. translation initiation; eukaryotic initiation factor 4G; protein kinase C[epsilon]; mammalian target of rapamycin; ribosomal protein S6 kinase 1; leucine; hindlimb perfusion doi:10.1152/ajpendo.00037.2007

In vitro, leptin secretion is regulated at the level of mRNA translation by the rapamycin-sensitive mammalian target of rapamycin (mTOR) and its agonist leucine (Leu). Studies were conducted on meal-trained rats to evaluate the potential physiological relevance of these in vitro findings and the role of Leu in affecting rises in plasma leptin observed after a meal. In the first study, we correlated changes in plasma insulin and Leu to mTOR-signaling pathway activation and plasma leptin at different times during meal feeding. Rapid rises in plasma insulin and Leu, along with mTOR signaling (phosphorylation of eIF4G, S6K1, rpS6, and 4E-BP1) in adipose tissue were observed during the 3-h meal and declined thereafter. Plasma leptin rose more slowly, peaking at 3 h, and was inhibited by rapamycin (0.75 mg/kg) pretreatment. In another experiment, oral Leu or norleucine was provided instead of a meal. Leu and norleucine stimulated a rise in plasma leptin; however, the magnitude was less than the response to a complete meal. In a third study, rats were provided a meal that lacked Leu, branched-chain amino acids, or all amino acids. Stimulation of leptin secretion was reduced ~40% in animals provided the Leu-deficient meal. Further reductions were not observed by removing the other amino acids. Thus Leu appears to regulate most of the effects of dietary amino acids on the postprandial rise in plasma leptin but is responsible only for part of the leptin response to meal feeding. protein synthesis; translation initiation; obesity; rats; meal feeding; meal trained doi:10.1152/ajpendo.00462.2005

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 (elF)4G ([Ser.sup.1108]), which returned to basal levels within 3 h of removal of food. Likewise, meal feeding was associated with an increase in phosphorylation of elF4E binding protein-1 (4EBP1) in the [gamma]-form during feeding. Phosphorylation of mammalian target of rapamycin (mTOR) on [Ser.sup.2448] or [Ser.sup.2481] or 70-kDa ribosomal protein S6 kinase (S6K1) on [Thr.sup.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.sup.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 elF4G and 4EBP1 during meal feeding and results in sustained increases in phosphorylation of PKB and PKC-[epsilon].

Feeding promotes protein accretion in skeletal muscle through a stimulation of the mRNA translation initiation phase of protein synthesis either secondarily to nutrient-induced rises in insulin or owing to direct effects of nutrients themselves. The present set of experiments establishes the effects of meal feeding on potential signal transduction pathways that may be important in accelerating mRNA translation initiation. Gastrocnemius muscle from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled before, during, and after the meal. Meal feeding enhanced the assembly of the active eIF4GxeIF4E complex, which returned to basal levels within 3 h of removal of food. The increased assembly of the active eIF4G*eIF4E complex was associated with a marked 10-fold rise in phosphorylation of eIF4G([Ser.sup.1108]) and a decreased assembly of inactive 4E-BPI-eIF4E complex. The reduced assembly of 4E-BPI-eIF4E complex was associated with a 75-fold increase in phosphorylation of 4E-BP1 in the [gamma]-form during feeding. Phosphorylation of S6KI on [Ser.sup.789] was increased by meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of mammalian target of rapamycin (roTOR) on [Ser.sup.2448] or [Ser.sup.2481], an upstream kinase responsible for phosphorylating both S6KI and 4E-BP1, was increased at all times during meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than alter 1 h. Phosphorylation of PKB, an upstream kinase responsible for phosphorylating roTOR, was elevated only after 0.5 h of meal feeding for [Thr.sup.308, whereas phosphorylation [Ser.sup.473] was significantly elevated at only 0.5 and 1 h after initiation of feeding. We conclude from these studies that meal feeding stimulates two signal pathways in skeletal muscle that lead to elevated eIF4G*eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E. amino acids; nutrition; feeding; ribosomal protein S6 kinase; eukaryotic initiation factor-4E; eukaryotic initiation factor-4E-binding protein-1; eukaryotic initiation factor-4G; eukaryotic initiation factor-4F

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[epsilon] and PKC[delta]) in the gastrocnemius of rats by use of modification state-dependent phosphopeptide-specific antibodies. The phosphorylation of PKC[epsilon] on the catalytic domain autophosphorylation site ([Ser.sup.729]) 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 PKC[delta] on either the catalytic domain autophosphorylation site ([Ser.sup.643]) or activation loop site ([Thr.sup.505]) 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[epsilon] in response to meal feeding. Elevating the leucine concentration stimulated the phosphorylation of PKC[epsilon] in gastrocnemius from perfused hindlimb and caused a shift in the distribution of PKC[epsilon] from the membrane fraction to the cytosolic fraction. The results indicate that leucine leads to an activation (autophosphorylation) and subcellular redistribution of PKC[epsilon], but not PKC[delta], 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[epsilon], suggesting that multiple signaling pathways become activated with leucine. amino acids; gastrocnemius; protein kinase C isoforms; ribosomal protein S6 kinase-1; eukaryotic initiation factor 4E-binding protein-1

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 [alpha]-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1[alpha]-subunit at [Ser.sup.293] (S293), which inactivates the complex. Because the [alpha]-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 mTOR 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. mammalian target of rapamycin; ribosomal protein S6 kinase-1; branched-chain [alpha]-keto acid dehydrogenase

Acute administration of leucine and norleucine activates the mammalian target of rapamycin (mTOR) cell-signaling pathway and increases rates of protein synthesis in a number of tissues in fasted rats. Although persistent stimulation of mTOR signaling is thought to increase protein synthetic capacity, little information is available concerning the effects of chronic administration of these agonists on protein synthesis, mTOR signal transduction, or leucine metabolism. Hence, we developed a model of chronic leucine/norleucine supplementation via drinking water and examined the effects of chronic (12 days) supplementation on protein synthesis in adipose tissue, kidney, heart, liver, and skeletal muscle from ad libitum-fed rats. The relative concentration of proteins involved in mTOR signaling and the two initial steps in leucine oxidation were also examined. Leucine or norleucine supplementation was accompanied by increased rates of protein synthesis in adipose tissue, liver, and skeletal muscle, but not in heart or kidney. Supplementation was not associated with increases in the anabolic hormones insulin or insulin-like growth factor I. Chronic supplementation did not cause apparent adaptation in either components of the mTOR cell-signaling pathway that respond to leucine (mTOR, ribosomal protein S6 kinase, and eukaryotic initiation factor 4E-binding protein-1) or the first two steps in leucine metabolism (the mitochondrial isoform of branched-chain amino acid transaminase, branched-chain keto acid dehydrogenase, and branched-chain keto acid dehydrogenase kinase), which may be involved in terminating the signal from leucine. These results suggest that provision of leucine or norleucine supplementation via the drinking water results in stimulation of postprandial protein synthesis in adipose tissue, skeletal muscle, and liver without notable adaptive changes in signaling proteins or metabolic enzymes. mammalian target of rapamycin; adipose tissue; eukaryotic initiation factor 4E-binding protein-1; branched-chain keto acid dehydrogenase kinase

In freshly isolated rat adipocytes, leucine or its analog norleucine activates the mammalian target of rapamycin (mTOR)-signaling pathway. This results in phosphorylation of the ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), two proteins involved in the initiation phase of protein synthesis. The purpose of the studies reported herein was to address the question of whether or not these in vitro effects of leucine and norleucine on adipocytes could be extended to the intact animal and to other tissues. To accomplish this, food-deprived (18 h) male Sprague-Dawley rats were orally administered solutions (2.5 ml/100 g body wt) containing normal saline (0.9% NaCl), a carbohydrate mixture (26.2% D-glucose and 26.2% sucrose), leucine (5.4%), or norleucine (5.4%). The protein synthetic responses of adipose tissue were measured and compared with those of other tissues. In addition, S6K1 and 4E-BP1 phosphorylation was measured, as was the plasma concentration of insulin and tissue ATP concentrations. Leucine administration stimulated protein synthesis in adipose tissue, gastrocnemius, and kidney but not in liver and heart. Norleucine stimulated protein synthesis in all of the tissues tested but, in contrast to leucine, without affecting plasma insulin concentrations. The carbohydrate meal had no effect on protein synthesis in any tissue tested but elicited a robust increase in plasma insulin. These findings provide support for a role of leucine as a direct-acting nutrient signal for stimulation of protein synthesis in adipose tissue as well as other select tissues. In adipose tissue, the effects of the different treatment conditions on the acute regulation of protein synthesis closely correlated with changes in phosphorylation of S6K1 and 4E-BP1; however, this correlation did not exist in all tissues examined. This result implies that leucine or norleucine may acutely stimulate protein synthesis, at least in some tissues, by a mechanism that is independent of both S6K1 and 4E-BP1 phosphorylation.

Research was conducted to examine the physiological role and the mechanism involved in the effects of amino acids on protein metabolism, target of rapamycin (TOR) activation and other events such as the multicellular clustering of adipocytes. The individual amino acids responsible for these effects were identified by focusing on the TOR substrate and translational repressor 4E-BP1. Results indicate that the mechanism used by amino acids to stimulate phosphorylation involved the interaction of leucine with a specific binding site.

Research was conducted to study the phosphorylation of insulin-regulated 70-kDa ribosomal protein S6 kinase (p70(super S6k)) in rat adipocytes. Adipocytes were isolated from Sprague-Dawley rats and were suspended in growth factor-reduced Matrigel for primary culture while the phosphorylation of p70(super S6k) was investigated using a gel shift assay. Results indicated that the cell signaling pathways utilized by insulin receptors stimulated the multicellular clustering of adipocytes.

The role of extracellular matrix metalloproteinase (MMP) in cellular migration, multicellular organization and cluster formation were analyzed by zymography in primary cultures of adipocytes. Analysis of primary adipocyte cultures indicated the presence of MMP-2 which was secreted by basement membrane components. The secretion of MMP-2 by differentiated adipocytes also mediated the formation of several peptides from fibronectin.

Analysis of the influence of diabetes on the rate of protein synthesis in vivo reveals that reduced cellular content of elongation factor 2 (EF-2) results in the inhibition of peptide-chain elongation. In diabetic hearts, decreased rates of protein synthesis are due to the reductions in the number of ribosomes and the restriction of peptide-chain elongation. The EF-2 content of diabetic rates rises to control values by insulin therapy.

Carbonic anhydrase (CA) II and III express themselves in adipocytes 3T3-L1 and 3T3-F442A, as a function of differentiation. The concentration of CA III in 3T3 adipocytes is lowered by 65% to 90%, which is attributed to the adverse influence of insulin content-present in the culture media. Insulin is believed to be responsible for metabolic regulation of adipose CAIII. The pH of the media falls rapidly, causing a decline in the bicarbonate (Co2)-buffered media. The pH value and the bicarbonate (Co2) present in the media play an important role in maintaining CA II and CA III concentrations. The concentration of pyruvate carboxylase and ATP citrate lyase, the two determining factors of adipocyte-differentiation, are unaffected by pH or GibCo2-I media effects.

The role of carbonic anhydrase III (CA III) in obesity was studied in Zucker rats by separating proteins using one-dimensional sodium dodecyl sulfate (SDS) and two-dimensional SDS isoelectric fcousing polyacrylamide gel electrophoresis. Laser densitometry, microsequencing, isoform-specific antisera and enzyme activity measurements revealed an obesity associated decrease in 28-kDa cytosolic adipocyte protein identified as CA III. This suggests that CA III may be associated with hyperinsulinemia and hyper-responsiveness of adipocytes.

Computational models and simulations often involve representations of decision-making processes. Numerous methods exist for representing decision-making at varied resolution levels based on the objectives of the simulation and the desired level of fidelity for validation. Decision making relies on the type of decision and the criteria that is appropriate for making the decision; therefore, decision makers can reach unique decisions that meet their own needs given the same information. Accounting for personalized weighting scales can help to reflect a more realistic state for a modeled system. To this end, this article reviews and summarizes eight multi-criteria decision analysis (MCDA) techniques that serve as options for reaching unique decisions based on personally and individually ranked criteria. These techniques are organized into a taxonomy of ratio assignment and approximate techniques, and the strengths and limitations of each are explored. We compare these techniques potential uses across the Agent-Based Modeling (ABM), System Dynamics (SD), and Discrete Event Simulation (DES) modeling paradigms to inform current researchers, students, and practitioners on the state-of-the-art and to enable new researchers to utilize methods for modeling multi-criteria decisions. [ABSTRACT FROM AUTHOR]