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The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, [alpha]-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 [micro]mol x [kg.sup.-1] x [h.sup.-1] for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P< 0.01 ) and norleucine reduced (P< 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (elF) 4E binding protein-1 (4E-BP1) and elF4G, lower (P < 0.04) abundance of the inactive 4E-BP1 x elF4E complex, and greater (P < 0.05) active elF4G x eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P< 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC. doi: 10.3945/jn.110.123042.

Linear growth in children is sensitive to nutritional status. Amino acids, in particular leucine, have been shown to regulate cell growth, proliferation, and differentiation through the mammalian target of rapamycin (mTOR), a nutrient-sensing protein kinase. Having recently demonstrated a role for mTOR in chondrogenesis, we hypothesized that leucine restriction, acting through mTOR, would inhibit growth plate chondrocyte proliferation and differentiation. The effect of leucine restriction was compared with that of the specific mTOR inhibitor, rapamycin. Leucine restriction produced a dose-dependent inhibition of fetal rat metatarsal explant growth. This was accounted by reduced cell proliferation and hypertrophy but not apoptosis, mTOR activity, as reflected by ribosomal protein $6 phosphorylation, was only partially inhibited by leucine restriction, whereas rapamycin abolished S6 phosphorylation. In chondrogenic ATDC5 cells, leucine restriction inhibited cell number, proteoglycan accumulation, and collagen X expression despite minimal inhibition of mTOR. Microarray analysis demonstrated that the effect of leucine restriction on ATDC5 cell gene expression differed from that of rapamycin. Out of 1,571 genes affected by leucine restriction and 535 genes affected by rapamycin, only 176 genes were affected by both. These findings indicate that the decreased chondrocyte growth and differentiation associated with leucine restriction is only partly attributable to inhibition of mTOR signaling. Thus nutrient restriction appears to directly modulate bone growth through unidentified mTOR-independent mechanisms in addition to the well-characterized mTOR nutrient-sensing pathway. mammalian target of rapamycin; rapamycin; gene expression

A study is conducted to show that the Tmt1-dependent methylation of 3-isopropylmalate in Saccharomyces cerevisiae is not directly related to the leucine biosynthetic pathway. Results indicate that 3-isopropylmalate methyl ester signals yeast to switch to invasive growth during amino acid starvation.

We have previously shown that a physiological increase in plasma leucine for 60 and 120 min increases translation initiation factor activation in muscle of neonatal pigs. Although muscle protein synthesis is increased by leucine at 60 min, it is not maintained at 120 min, perhaps because of the decrease in plasma amino acids (AA). In the present study, 7- and 26-day-old pigs were fasted overnight and infused with leucine (0 or 400 [micro]mol x [kg.sup.-1] x [h.sup.-1]) for 120 min to raise leucine within the postprandial range. The leucine was infused in the presence or absence of a replacement AA mixture (without leucine) to maintain baseline plasma AA levels. AA administration prevented the leucine-induced reduction in plasma AA in both age groups. At 7 days, leucine infusion alone increased eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) phosphorylation, decreased inactive 4E-BPI x eIF4E complex abundance, and increased active eIF4G x eIF4E complex formation in skeletal muscle; leucine infusion with replacement AA also stimulated these, as well as 70-kDa ribosomal protein S6 kinase, ribosomal protein S6, and eIF4G phosphorylation. At 26 days, leucine infusion alone increased 4E-BP1 phosphorylation and decreased the inactive 4E-BP1 x eIF4E complex only; leucine with AA also stimulated these, as well as 70-kDa ribosomal protein S6 kinase and ribosomal protein S6 phosphorylation. Muscle protein synthesis was increased in 7-day-old (+60%) and 26-day-old (+40%) pigs infused with leucine and replacement AA but not with leucine alone. Thus the ability of leucine to stimulate eIF4F formation and protein synthesis in skeletal muscle is dependent on AA availability and age. neonate; translation initiation; infusion; parenteral; eukaryotic initiation factor 4G; amino acids

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

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed 'trigger sequence,' that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation. autonomous folding unit | protein folding | trigger sequence | leucine zipper | [alpha]-helix

The transcriptional regulator Cg1486 of Corynebacterium glutamicum ATCC 13032 is a member of the IclR protein family and belongs to the conserved set of regulatory proteins in corynebacteria. A defined deletion in the cg1486 gene, now designated ltbR (leucine and tryptophan biosynthesis regulator), led to the mutant strain C. glutamicum IB1486. According to whole-genome expression analysis by DNA microarray hybridizations, transcription of the leuB and leuCD genes encoding enzymes of the leucine biosynthesis pathway was enhanced in C. glutamicum IB1486 compared with the wild-type strain. Moreover, the genes of the trpEGDCFBA operon involved in tryptophan biosynthesis of C. glutamicum showed an enhanced expression in the cg1486 mutant strain. Bioinformatics pattern searches in the upstream regions of the differentially expressed genes revealed the common 12-bp motif CA(T/C)ATAGTG(A/G)GA that is located downstream of the -10 region of the mapped promoter sequences. DNA band shift assays with a streptavidin-tagged LtbR protein demonstrated the specific binding of the purified protein to 40-mers containing the 12-bp motif localized in front of leuB, leuC, and trpE, thereby confirming the direct regulatory role of LtbR in the expression of the leucine and tryptophan biosynthesis pathway genes of C. glutamicum. Genes homologous with ltbR were detected upstream of the leuCD genes in almost all sequenced genomes of bacteria belonging to the taxonomic class Actinobacteria. The ltbR-like genes of Corynebacterium diphtheriae, Corynebacterium jeikeium, Mycobacterium bovis, and Bifidobacterium longum were cloned and shown to complement the deregulation of leuB, leuCD, and trpE gene expression in C. glutamicum IB1486.

Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis. Leucine treatment upregulates mTOR signaling, which enhances protein synthesis; however, the mechanisms are not well understood. Herein, treatment of C2C12 myoblast cells with leucine enhanced the phosphorylation of mTOR and ribosomal protein S6 kinase. Leucine treatment also decreased the adenosine monophosphate/ATP ratio in myoblasts by 36.4 [+ or -] 9.1% (P < 0.05) and reduced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) a subunit at [Thr.sup.172] (28.6 [+ or -] 4.9% reduction, P < 0.05) and inhibited AMPK activity (43.6 [+or -] 3.5% reduction, P < 0.05). In addition, leucine increased the phosphorylation of mTOR at [Ser.sup.2448] by 63.5 [+ or -] 10.0% (P < 0.05) and protein synthesis by 30.6 [+ or -] 6.1% (P < 0.05). Applying 5-aminoimidazole-4-carboxamide 1-beta-d-ribonucleoside, an activator of AMPK, abolished the stimulation of mTOR signaling by leucine, showing that AMPK negatively controls mTOR signaling. To further show the role of AMPK in mTOR signaling, myoblasts expressing a dominant negative AMPKa subunit were employed. Negative myoblasts had very low AMPK activity. The activation of mTOR induced by leucine in these cells was abated, showing that AMPK contributed to mTOR activation. In conclusion, leucine stimulates mTOR signaling in part through AMPK inhibition. This study implicates AMPK as an important target for nutritional management to enhance mTOR signaling and protein synthesis in muscle cells, thereby increasing muscle growth. Key words: adenosine monophosphate-activated protein kinase, leucine, myoblast cell, mammalian target of rapamycin, protein synthesis

In hippocampal pyramidal cells, dopamine acts at D1 receptors to reduce peak [Na.sup.+] currents by activation of phosphorylation by PKA anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 anchors PKA to neuronal [Na.sup.+] channels is not known. By using a strategy of coimmunoprecipitation from transfected tsA-201 cells, we have found that AKAP15 directly interacts with [Na.sub.v]1.2a channels via the intracellular Loop between domains I and II. This loop contains key functional phosphorylation sites. Mutagenesis indicated that this interaction occurs through a modified leucine zipper motif near the N terminus of the loop. Whole-cell patch clamp recordings of acutely dissociated hippocampal pyramidal cells revealed that the D1 dopamine receptor agonist SKF 81297 reduces peak [Na.sup.+] current amplitude by 20.5%, as reported previously. Disruption of the leucine zipper interaction between [Na.sub.v]1.2a and AKAP15 through the inclusion of a small competing peptide in the patch pipette inhibited the SKF 81297-induced reduction in peak [Na.sup.+] current, whereas a control peptide with mutations in amino acids important for the leucine zipper interaction did not. Our results define the molecular mechanism by which G protein-coupled signaling pathways can rapidly and efficiently modulate neuronal excitability through local protein phosphorylation of [Na.sup.+] channels by specifically anchored PKA. sodium channel

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation [K.sup.+], cytoplasmic [K.sup.+] in bacteria is required for diverse processes. Here, we show that enzyme II[A.sup.Ntr] (EII[A.sup.Ntr]) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli [K.sup.+] transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EII[A.sup.Ntr] was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EII[A.sup.Ntr] for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the [K.sup.+] level in culture media is lowered. We demonstrate that dephosphorylated EII[A.sup.Ntr], but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of [K.sup.+]. High cellular [K.sup.+] levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EII[A.sup.Glc] regulates a variety of transport systems for carbon sources, whereas dephosphorylated EII[A.sup.Ntr] regulates the transport system for [K.sup.+], which has global effects related to nitrogen metabolism. leucine toxicity | nitrogen-metabolic phosphotransferase system (PTS) | potassium transporter TrkA | protein-protein interaction | signal transduction

Nucleotide binding site--leucine-rich repeat (NBS-LRR) proteins mediate pathogen recognition in both mammals and plants. The molecular mechanisms by which pathogen molecules activate NBS-LRR proteins are poorly understood. Here we show that RPS5, a NBS-LRR protein from Arabidopsis, is activated by AvrPphB, a bacterial protease, via an indirect mechanism. When transiently expressed in Nicotiana benthamiana leaves, full-length RPS5 protein triggered programmed cell death, but only when coexpressed with AvrPphB and a second Arabidopsis protein, PBS1, which is a specific substrate of AvrPphB. Using coimmunoprecipitation analysis, we found that PBS1 is in a complex with the N-terminal coiled coil (CC) domain of RPS5 before exposure to AvrPphB. Deletion of the RPS5 LRR domain caused RPS5 to constitutively activate programmed cell death, even in the absence of AvrPphB and PBS1, and this activation depended on both the CC and NBS domains. The LRR and CC domains both coimmunoprecipitate with the NBS domain but not with each other. Thus, the LRR domain appears to function in part to inhibit RPS5 signaling, and cleavage of PBS1 by AvrPphB appears to release RPS5 from this inhibition. An amino acid substitution in the NBS site of RPS5 that is known to inhibit ATP binding in other NBS-LRR proteins blocked activation of RPS5, whereas a substitution thought to inhibit ATP hydrolysis constitutively activated RPS5. Combined, these data suggest that ATP versus ADP binding functions as a molecular switch that is flipped by cleavage of PBS1. AvrPphB | disease resistance | NOD domain | Pseudomonas syringae | RPS5

Various atrophic stimuli increase two muscle-specific E3 ligases, muscle RING finger 1 (MuRF1) and atrogin-1, and knockout mice for these 'atrogenes' display resistance to denervation-induced atrophy. The present study determined whether increased atrogin-1 and MuRF1 mRNA are mediated by overproduction of endogenous glucocorticoids or inflammatory cytokines in adult rats and whether atrogene expression can be downregulated by anabolic agents such as insulin-like growth factor (IGF)-I and the nutrient-signaling amino acid leucine. Both atrogin-1 and MuRF1 mRNA in gastrocnemius was upregulated dose and time dependently by endotoxin. Additionally, peritonitis produced by cecal ligation and puncture increased atrogin-1 and MuRF1 mRNA in gastrocnemius (but not soleus or heart) by 8 h, which was sustained for 72 and 24 h, respectively. Whereas the sepsis-induced increase in atrogin- 1 expression was completely prevented by IGF-I, the increased MuRF1 was not altered. In contrast to the IGF-I effect, the sepsis-induced increased mRNA of both atrogenes was unresponsive to either acute or repetitive administration of leucine. Whereas exogenous infusion of TNF-[alpha] increased atrogin-1 and MuRF1 in gastrocnemius, pretreatment of septic rats with the TNF antagonist TNF-binding protein did not prevent increased expression of either atrogene. Similarly, whereas dexamethasone increased atrogene expression, pretreatment with the glucocorticoid receptor antagonist RU-486 failed to ameliorate the sepsis-induced increase in atrogin-1 and MuRF1. Thus, under in vivo conditions in mature adult rats, the sepsis-induced increase in muscle atrogin-1 and MuRF1 mRNA appears both glucocorticoid and TNF independent and is unresponsive to leucine. muscle RING finger-1; muscle atrophy F-box protein; ubiquitin ligase; muscle wasting; insulin-like growth factor I; leucine

Prostate apoptosis response-4 (Par-4) is a leucine zipper protein linked to apoptotic cell death in prostate cancer and neuronal tissues. The leucine zipper domain of Par-4 (Leu.zip) mediates protein-protein interactions that are essential for sensitization of cells to apoptosis, and overexpression of Leu.zip blocks Par-4 activity in a dominant negative fashion. Ischemiareperfusion-induced renal injury (IRI) is clinically important because it typically damages renal tubular epithelial cells and glomerular cells, and it is the most common cause of acute renal failure (ARF). We now report that Par-4 is expressed in renal tubule cells and that aberrant expression of Par-4 activity plays a crucial role in activating apoptotic pathways in well-characterized models of renal IRI. Increased levels of Par-4 were observed following chemical ischemia-reperfusion in HK-2 cells in vitro and in mouse renal tubular cells following bilateral clamping of renal pedicles in vivo. Inhibition of Par-4 expression by specific par-4 antisense oligonucleotides largely prevented HK-2 cell apoptosis induced by IRI. Overexpression of Par-4 in these cells exacerbated mitochondrial dysfunction and caspase activation and conferred increased sensitivity to IRI-induced apoptosis. Expression of Leu.zip, a dominant negative regulator of Par-4, largely prevented mitochondrial dysfunction and caspase activation and significantly inhibited IRI-induced apoptosis in HK-2 cells. In addition, transfection of Par-4 increased while transfection of Leu.zip decreased necrosis in HK-2 cells following prolonged IRI. These results identify Par-4 as a novel and early mediator of renal tubule cell injury following IRI and provide a potential target for developing new therapeutic strategies for renal IRI and ARF. prostate apoptosis response-4; apoptosis; necrosis; mitochondria; caspase

The present study determined whether thermal injury increases the expression of the ubiquitin (Ub) E3 ligases referred to as muscle ring finger (MuRF)-1 and muscle atrophy F-box (MAFbx; aka atrogin-1), which are muscle specific and responsible for the increased protein breakdown observed in other catabolic conditions. After 48 h of burn injury (40% total body surface area full-thickness scald burn) gastrocnemius weight was reduced, and this change was associated with an increased mRNA abundance for atrogin-1 and MuRF-1 (3.1- to 8-fold, respectively). Similarly, burn increased polyUb mRNA content in the gastrocnemius twofold. In contrast, there was no burn-induced atrophy of the soleus and no significant change in atrogin-l, MuRF-1, or polyUb mRNA. Burns also did not alter E3 ligase expression in heart. Four hours after administration of the anabolic agent insulin-like growth factor (IGF)-I to burned rats, the mRNA content of atrogin-1 and polyUb in gastrocnemius had returned to control values and the elevation in MuRF-1 was reduced 50%. In contrast, leucine did not alter E3 ligase expression. In a separate study, in vivo administration of the proteasome inhibitor Velcade prevented burn-induced loss of muscle mass determined at 48 h. Finally, administration of the glucocorticoid receptor antagonist RU-486 did not prevent burn-induced atrophy of the gastrocnemius or the associated elevation in atrogin-1, MuRF-1, or polyUb. In summary, the acute muscle wasting accompanying thermal injury is associated with a glucocorticoid-independent increase in the expression of several Ub E3 ligases that can be down-regulated by IGF-I. muscle atrophy F-box; muscle wasting; leucine

Familial periodic fever syndromes, otherwise known as hereditary autoinflammatory syndromes, are inherited disorders characterized by recurrent episodes of fever and inflammation. The general hypothesis is that the innate immune response in these patients is wrongly tuned, being either too sensitive to very minor stimuli or turned off too late. The genetic background of the major familial periodic fever syndromes has been unraveled, and through research into the pathophysiology, a clearer picture of the innate immune system is emerging. After an introduction on fever, interleukin-1[beta] and inflammasomes, which are involved in the majority of these diseases, this manuscript offers a detailed review of the pathophysiology of the cryopyfin-associated periodic syndromes, familial Mediterranean fever, the syndrome of pyogenic arthritis, pyoderma gangrenosum and acne, Blau syndrome, TNF-receptor-associated periodic syndrome and hyper-IgD and periodic fever syndrome. Despite recent major advances, there are still many questions to be answered regarding the pathogenesis of these disorders. inflammasome; interleukin-1[beta]; nucleotide-binding oligomerization domain-leucine rich repeat proteins; tumor necrosis factor receptor; isoprenoid pathway

The innate immune system includes microbial pattern recognition receptors that detect bacteria and viral products at the cell surface, in vesicles, and within the cytoplasm. Transmembrane signaling occurs through Toll-like receptors (TLRs). Cytoplasmic receptors are generally members of the nucleotide-binding domain (NOD)-leucine-rich repeat (LRR) family (CATERPILLER family). They influence the effects of other family members and of TLRs. Most NOD-LRR members enhance signal transduction, but Monarch-1 counterbalances TLR activity. NOD-LRR family members also act within the adaptive immune system. The class II transactivator regulates major histocompatibility complex class II expression. In the intestine, it is developmentally regulated, and its expression depends on weaning and, independently, on age. CATERPILLER; nucleotide-binding domain; leucin-rich repeat; Toll-like receptor; class II transactivator; leucin-rich repeat- and pyrin domain-containing protein 3

During nitric oxide signaling, type I[alpha] cGMP-dependent protein kinase (PKGI[alpha]) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGI[alpha] interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the N[H.sub.2]-terminal LZ of PKGI[alpha] (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGI[alpha] interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGI[alpha] is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772-14777, 2000), and there is an RK motif within the aa 888-928 sequence of MYPT1 in LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGI[alpha] interaction, we designed four MYPT1 fragments that contained both the aa 888-928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888-928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888-928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888-928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGI[alpha] in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 ([R.sup.916][K.sup.917]) to AA decreased binding of MYPT1 to PKGI[alpha] in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of [R.sup.916][K.sup.917] to [E.sup.916][E.sup.917] eliminated binding, suggesting that one factor important for the PKGI[alpha]-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGI[alpha]-MYPT1 interaction. myosin light chain phosphatase; nitric oxide; smooth muscle; calcium desensitization; cGMP-dependent protein kinase; cGMP

The plant hormone auxin can be regulated by formation and hydrolysis of amide-linked indole-3-acetic acid (IAA) conjugates. Here, we report the characterization of the dominant Arabidopsis iaa-leucine resistant3 (ilr3-1) mutant, which has reduced sensitivity to IAA-Leu and IAA-Phe, while retaining wild-type responses to free IAA. The gene defective in ilr3-1 encodes a basic helix-loop-helix leucine zipper protein, bHLH105, and the ilr3-1 lesion results in a truncated product. Overexpressing ilr3-1 in wild-type plants recapitulates certain ilr3-1 mutant phenotypes. In contrast, the loss-of-function ilr3-2 allele has increased IAA-Leu sensitivity compared to wild type, indicating that the ilr3-1 allele confers a gain of function. Microarray and quantitative real-time PCR analyses revealed five downregulated genes in ilr3-1, including three encoding putative membrane proteins similar to the yeast iron and manganese transporter Ccc1p. Transcript changes are accompanied by reciprocally misregulated metal accumulation in ilr3-1 and ilr3-2 mutants. Further, ilr3-1 seedlings are less sensitive than wild type to manganese, and auxin conjugate response phenotypes are dependent on exogenous metal concentration in ilr3 mutants. These data suggest a model in which the ILR3/bHLH105 transcription factor regulates expression of metal transporter genes, perhaps indirectly modulating IAA-conjugate hydrolysis by controlling the availability of metals previously shown to influence IAA-amino acid hydrolase protein activity.

We studied the effect of acute hyperinsulinemia on amino acid (AA) utilization and oxidation rates independent of insulin-enhanced glucose metabolism in fetal sheep. Metabolic studies were conducted in each fetus (n = 11) under three experimental periods. After control period (C) study, a fetal hyperin-sulinemic-euglycemic-euaminoacidemic (HI-euG-euAA) clamp was established, followed by a hyperinsulinemic-hypoglycemic-euamino-acidemic (HI-hypoG-euAA) clamp to decrease glucose metabolic rates toward C values. Infusions of [sup.3][H.sub.2]O, L-[[l-.sup.13]C]leucine, and [[sup.14]C(U)]glucose were administered to measure blood flow, leucine oxidation, and fetal glucose uptake, utilization, and oxidation in each period. Fetal glucose utilization rate increased 1.7-fold with hyperinsulinemia (C 5.8 [+ or -] 0.8 mg * [kg.sup.-1] * [min.sup.-1], HI-euG-euAA 10 [+ or -] 1.3 mg * [kg.sup.-1] * [min-1], p < 0.000l), returning to rates not different from C with hypoglycemia (HI-hypoG-euAA 7.1 [+ or -] 0.9 mg * [kg.sup.-1] * [min.sup.-1] vs. C value, P = 0.15). Fetal glucose oxidation rate increased 1.7-fold with hyperinsulinemia (C 3.1 [+ or -] 0.2 mg * [kg.sup.-1] * [min.sup.-1] HI-euG-euAA 5.4 [+ or -] 0.4 mg * [kg.sup.-1] * [min.sup.-1], P < 0.0001) and decreased to near control rates with hypoglycemia (4.0 [+ or -] 0.3 HI-hypoG-euAA vs. C value, P = 0.006). AA utilization rates increased with hyperinsulinemia for all essential and most nonessential AAs (P < 0.001) and did not change when insulin-induced increases in glucose utilization returned to control rates. Leucine oxidation rate increased 1.7-fold with hyperinsulinemia (C 1.0 [+ or -] 0.3 [micro]mol * [min.sup.-1] * [kg.sup.-1], HI-euG-euAA 1.7 [+ or -] 0.3 [micro]mol * [min.sup.-1] * [kg.sup.-1], P < 0.002) and did not change when glucose oxidation rate was decreased with hypoglycemia. These results demonstrate that, in fetal sheep, insulin promotes AA utilization and oxidation independent of its simultaneous effects on glucose metabolism. In acute hyperinsulinemic conditions, AA oxidation does not change when insulin-induced glucose utilization is prevented. insulin; leucine; fetal sheep

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

We measured in vivo and in vitro nutrient-stimulated insulin secretion in late gestation fetal sheep to determine whether an intrinsic islet defect is responsible for decreased glucose-stimulated insulin secretion (GSIS) in response to chronic hypoglycemia. Control fetuses responded to both leucine and lysine infusions with increased arterial plasma insulin concentrations (average increase: 0.13 [+ or -] 0.05 ng/ml leucine; 0.99 [+ or -] 0.26 ng/ml lysine). In vivo lysine-stimulated insulin secretion was decreased by chronic (0.37 [+ or -] 0.18 ng/ml) and acute (0.27 [+ or -] 0.19 ng/ml) hypoglycemia. Leucine did not stimulate insulin secretion following acute hypoglycemia but was preserved with chronic hypoglycemia (0.12 [+ or -] 0.09 ng/ml). Isolated pancreatic islets from chronically hypoglycemic fetuses had normal insulin and DNA content but decreased fractional insulin release when stimulated with glucose, leucine, arginine, or lysine. Isolated islets from control fetuses responded to all nutrients. Therefore, chronic late gestation hypoglycemia causes defective in vitro nutrient-regulated insulin secretion that is at least partly responsible for diminished in vivo GSIS. Chronic hypoglycemia is a feature of human intrauterine growth restriction (IUGR) and might lead to an islet defect that is responsible for the decreased insulin secretion patterns seen in human IUGR fetuses and low-birth-weight human infants. hypoglycemia; glucose doi:10.1152/ajpendo.00643.2005

In 2 experiments, 6 ruminally cannulated Holstein steers (205 [+ or -] 23 and 161 [+ or -] 14 kg initial BW in Exp. 1 and 2, respectively) housed in metabolism crates were used in 6 x 6 Latin squares to study the effects of excess AA supply on Met (Exp. 1) and Leu (Exp. 2) use. All steers received a diet based on soybean hulls (DMI = 2.66 and 2.45 kg/d in Exp. 1 and 2, respectively); ruminal infusions of 200 g of acetate/d, 200 g of propionate/d, and 50 g of butyrate/d, as well as abomasal infusion of 300 g of glucose/d to provide energy without increasing the microbial protein supply; and abomasal infusions of a mixture of all essential AA except Met (Exp. 1) or Leu (Exp. 2). Periods were 6 d, with 2-d adaptations and 4 d to collect N balance data. All treatments were abomasally infused. In Exp. 1, treatments were arranged as a 2 x 3 factorial, with 2 amounts of L-Met (0 or 4 g/d) and 3 AA supplements (no additional AA, control; 100 g/d of nonessential AA + 100 g/d of essential AA, NEAA + EAA; and 200 g/d of essential AA, EAA). Supplemental Met increased (P < 0.01) retained N and decreased (P < 0.01) urinary N and urinary urea N. Retained N increased (P < 0.01) with NEAA + EAA only when 4 g/d of Met was provided, but it increased (P < 0.01) with EAA with or without supplemental Met. Both AA treatments increased (P < 0.01) plasma urea and serum insulin. Plasma glucose decreased (P = 0.03) with supplemental Met. In Exp. 2, treatments were arranged as a 2 x 3 factorial with 2 amounts of L-Leu (0 or 4 g/d) and 3 AA supplements (control, NEAA + EAA, and EAA). Supplemental Leu increased (P < 0.01) retained N and decreased (P < 0.01) urinary N and urinary urea N. Both AA treatments increased (P < 0.01) retained N, and they also increased (P < 0.01) urinary N, urinary urea N, and plasma urea. Serum insulin increased (P = 0.06) with supplemental Leu and tended (P = 0.10) to increase with both AA treatments. Supplementation with excess AA improved Met and Leu use for protein deposition by growing cattle. Key words: amino acid, cattle, growth, leucine, methionine, utilization

Feeding stimulates pancreatic digestive enzyme synthesis at the translational level, and this is thought to be mediated by hormones and neurotransmitters. However, BCAAs, particularly leucine, stimulate protein synthesis in several tissues. We investigated whether BCAA stimulated the translational machinery in murine pancreas and whether their effects were independent of hormones. Rats and mice were administered (i.g. gavage) individual BCAA at 1.35 mg/g (body weight) and rat isolated pancreatic acini were incubated with BCAA under different conditions. Activation of translation initiation factors and total protein synthesis were analyzed. BCAA gavage stimulated the phosphorylation of the initiation factor 4E (elF4E) binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase (S6K), with leucine being the most effective. Leucine also increased the association of the initiation factors elF4E and elF4G, but did not affect the activity of the guanine nucleotide exchange factor elF2B, nor total protein synthesis. BCAA acted independently of insulin signaling on isolated pancreatic acini from diabetic rats. The ability of leucine to promote phosphorylation of 4E-BP1 and S6K as well as enhance the assembly of the elF4F complex was unimpaired in CCK-deficient mice. Finally, rapamycin (0.75 mg/kg) administered to rats 2 h before leucine gavage inhibited the phosphorylation of S6 and 4E-BP1 induced by leucine. We conclude that leucine may participate, as a signal as well as a substrate, in activating the translational machinery in pancreatic acinar cells independently of hormonal effects and that this action is through the mTOR pathway. KEY WORDS: * pancreas * leucine * translation * cholecystokinin * insulin

Leucine modulates protein translation in higher eukaryotes by affecting phosphorylation and the function of proteins that regulate the initiation and/or elongation steps. These include the initiation factor 4E binding protein 1 (4E-BP1), initiation factor 4E (elF4E), initiation factor 2 (elF2[alpha]), ribosomal S6 kinases (S6K1/2), and elongation factor 2 (eEF2). The alteration of protein translation by leucine starvation was studied during myogenic differentiation using the mouse C2C12 cell line as well as the role of rapamycin-sensitive mTOR (mammalian target of rapamycin) in the signaling of leucine in myotubes. A time course study showed that 1 h of leucine starvation decreased protein synthesis and S6K1 phosphorylation in myoblasts, whereas 3-5 h of starvation were necessary to induce such an alteration in myotubes. Although S6K1 phosphorylation was reduced in leucine-deprived myotubes, S6K2 and S6 phosphorylation were not affected. In contrast, rapamycin decreased the phosphorylation of S6K2 and S6 in myotubes. It is therefore likely that under the conditions present, the rapamycin-sensitive mTOR was not affected by leucine starvation. S6K1 dephosphorylation may thus be mTOR independent, and the functional mTOR/ S6K2 pathway may maintain S6 phosphorylation. An increased phosphorylation of eEF2 in myoblasts and myotubes indicated that global protein synthesis was reduced via a decrease in translation elongation. An increased association between 4E-BP1 and elF4E, and increased phosphorylation of elF2[alpha] also contributed to decreasing protein synthesis in leucine-starved myoblasts. In contrast, in leucine-starved myotubes, there were no change in the 4E-BPI-elF4E association or elF2[alpha] phosphorylation, suggesting that these factors were not rate limiting for decreasing protein synthesis in leucine-deprived myotubes. J. Nutr. 136: 1466-1471, 2006. KEY WORDS: * myogenic differentiation * leucine starvation * protein translation * mTOR * S6K1

The purpose of this study was to determine the effects of resistance training in combination with a leucine and whey protein supplement or a carbohydrate placebo on strength and muscle cross-sectional area (CSA). Thirty-three men (mean age [+ or -] SD = 22.4 [+ or -] 2.4 years) were assigned to 1 of 3 groups: (1) supplementation group (SUPP), (2) placebo group (PL), or (3) control group (CON). The SUPP and PL performed unilateral training of the leg extensor muscles with the nondominant limb for 8 weeks. The strength of each limb, muscle CSA of the quadriceps femoris (QF), and body composition were assessed pre-training and posttraining. The results indicated significant increases in strength for both limbs in the SUPP but only the trained limb in the PL. The increase in strength for the trained limb of the SUPP was greater than that for the trained limb of the PL. There was no significant increase in strength for either limb in the CON. There were significant increases in the CSA of all muscles of the QF of the trained limb for the SUPP and PL, and of the vastus lateralis of the untrained limb for the SUPP. The increases in QF CSA did not differ between the SUPP and PL. No significant CSA changes were found for either limb in the CON. There were no significant changes in body composition for the SUPP, PL, or CON. The current findings suggest that leucine and whey protein supplementation may provide an ergogenic effect which enhances the acquisition of strength beyond that achieved with resistance training and a carbohydrate placebo. KEY WORDS. amino acids, muscular strength, quadriceps femoris, strength training, cross-training, cross-education

The present study was carried out to assess the effects of protease inhibitor (PI) therapy on basal whole body protein metabolism and its response to acute amino acid-glucose infusion in 14 human immunodeficiency virus (HIV)-infected patients. Patients treated with PIs (PI+, 7 patients) or without PIs (PI-, 7 patients) were studied after an overnight fast during a 180-min basal period followed by a 140-min period of amino acid-glucose infusion. Protein metabolism was investigated by a primed constant infusion of L-[1-[sup.13]C]leucine. Dual-energy X-ray absorptiometry for determination of fat-free mass (FFM) and body fat mass measured body composition. In the postabsorptive state, whole body leucine balance was 2.5 times (P < 0.05) less negative in the PI+ than in the PI- group. In HIV-infected patients treated with PIs, the oxidative leucine disposal during an acute amino acid-glucose infusion was lower (0.58 [+ or -] 0.09 vs. 0.81 [+ or -] 0.07 [micro]mol x kg FF[M.sup.-1] x [min.sup.-1] using plasma [[sup.13]C]leucine enrichment, P = 0.06; or 0.70 [+ or -] 0.10 vs. 0.99 [+ or -] 0.08 [micro]mol x kg FF[M.sup.-1] x [min.sup.-1] using plasma [[sup.13]C]ketoisocaproic acid enrichment, P = 0.04 in PI+ and PI-groups, respectively) than in patients treated without PIs. Consequently, whole body nonoxidative leucine disposal (an index of protein synthesis) and leucine balance (0.50 [+ or -] 0.10 vs. 0.18 [+ or -] 0.06 [micro]mol x kg FF[M.sup.-1] x [min.sup.-1] in PI+ and PI- groups respectively, P < 0.05) were significantly improved during amino acid-glucose infusion in patients treated with PIs. However, whereas the response of whole body protein anabolism to an amino acid-glucose infusion was increased in HIV-infected patients treated with PIs, any improvement in lean body mass was detected. leucine kinetics; protease inhibitor therapy; human immunodeficiency virus infection; amino acid requirements

Leucine tracer has been widely used for examining whole-body protein turnover in humans, but has not been evaluated as an indicator to be used in the indicator amino acid oxidation (IAAO) method. The goal of this study was to determine whether the L-[1-[sup.13C]leucine isotope is an acceptable indicator by comparing it with an established tracer, L-[1-[sup.13]C]lysine. Healthy men (n = 7; 29.9 [+ or -] 4.8 y old) were fed in random order a diet with 7 graded intakes of phenylalanine without tyrosine. In the first study (n = 5), subjects were administered an excess leucine intake of 65 mg/(kg*d), and in the second study (n = 5), they were given the mean requirement of 45 mg/ (kg*d) to determine whether leucine intake affected the pattern of response. Previous IAAO studies using lysine and phenylalanine demonstrated a clear pattern in [sup.13]C[O.sub.2] production, i.e., increasing test amino acid intake resulted in a linear decrease to plateau, with a readily discernable breakpoint indicating the requirement. This pattern of production of [sup.13]C[O.sub.2], indicates clear partitioning of the indicator amino acid between oxidation and protein synthesis. This was not observed with leucine at an intake of 65 mg/(kg*d). Conversely, at the lower leucine intake of 45 mg/(kg*d), a breakpoint was seen and a total aromatic amino acid requirement estimate that did not differ from that obtain using lysine as the indicator was obtained. In conclusion, leucine may be used as the indicator in the IAAO technique only when the daily intake leucine is given at its mean requirement level and the potential metabolic effects of other variables are taken into consideration. KEYWORDS: * indicator amino acid oxidation * leucine * aromatic amino acids * phenylalanine * tyrosine

Malnutrition is prevalent in children with chronic cholestatic liver disease. Using the noninvasive indicator amino acid oxidation (IAAO) technique, we recently determined that mild-to-moderate chronic cholestatic (MCC) liver disease increases the need for branched-chain amino acids (BCAA) in children. To examine the underlying mechanisms responsible for this increased need for BCAA in liver disease, we measured L-[1-[sup.13]C]-Ieucine oxidation in the postabsorptive and fed states in 10 children with MCC liver disease (8.8 [+ or -] 3.5 y) and in 11 healthy children (9.4 [+ or -] 2.2 y). The oxidation of L-[sup.13]C]-Ieucine to [sup.13]C[O.sub.2] [[F.sup.13]C[O.sub.2] in [micro]mol/(kg*h)] was determined after a primed, continuous oral administration of the tracer. Total BCAA in diet was provided at 300 mg/(kg*d) to ensure that leucine oxidation was measured when leucine intake was in excess of requirements. In the postabsorptive state, the rate of release of [sup.13]C[O.sub.2] from [sup.13]C-leucine oxidation ([F.sup.13]C[O.sub.2)] and whole-body leucine oxidation were significantly higher in children with MCC liver disease (P < 0.05). However, [F.sup.13]C[O.sub.2] and whole-body leucine oxidation did not differ in the fed state. We conclude that the increased need for dietary BCAA in MCC liver disease is mediated in part by increased leucine oxidation in the postabsorptive state. KEY WORDS: * leucine metabolism * children * liver disease

Regions with tandemly arranged leucine-rich repeats (LRRs) have been found in many prokaryotic and eukaryotic proteins, in which they provide a remarkably versatile framework for the formation of ligand-binding sites. Bacterial LRR proteins include the recently described Sir protein of Streptococcus pyogenes, which is related to internalin A of Listeria monocytogenes. Here, we show that strains of the human pathogen Streptococcus agalactiae express a protein, designated Blr, which together with Slr defines a family of internalin A-related streptococcal LRR proteins. Analysis with specific antibodies demonstrated that Blr is largely inaccessible on S. agalactiae grown in vitro, but surface exposure was increased ~100-fold on mutants lacking polysaccharide capsule. In S. pyogenes, surface exposure of Slr was not affected in a mutant lacking hyaluronic acid capsule but was increased >20-fold in mutants lacking M protein or protein F. Thus, both Blr and Slr are efficiently camouflaged by other surface structures on bacteria grown in vitro. When Blr and Slr exposed on the bacterial surface were compared, they exhibited only little immunological cross-reactivity, in spite of extensive residue identity, suggesting that their surface-exposed parts have been under evolutionary pressure to diverge functionally and/or antigenically. These data identify a family of immunologically diverse streptococcal LRR proteins that show unexpected complexity in their interactions with other bacterial surface components.

The three branched-chain amino acids (BCAAs) are the most hydrophobic of the amino acids and play crucial roles in determining the structures of globular proteins as well as the interaction of the transmembrane domains of membranous proteins with phospholipid bilayers. However, the three BCAAs do not behave identically. In terms of protein secondary structure, valine and isoleucine exhibit a definite preference for the [beta]-structure, whereas leucine has a higher preference for the [alpha]-helix. Although mutation of one BCAA to another is commonly regarded as conservative, there are well-documented examples of such substitutions that have a significant effect on protein function. The occurrence of BCAA in nature is, therefore, attributable to their primary role in protein structure, not to their secondary metabolic roles. These functions are important for almost all proteins; therefore, BCAA commonly account for ~20-25% of most dietary proteins. Dietary BCAA largely escape first-pass splanchnic metabolism. The first steps in their catabolism are common to all three, involving the BCAA aminotransferase (BCAT) and branched-chain [alpha]-keto acid dehydrogenase (BCKD). Their further metabolism employs distinct pathways to different end-products (glucose and/or ketone bodies). However, the fact that the flux-generating step for the catabolism of the three BCAAs occurs at one of the common steps indicates that the production of these downstream products are not individually regulated and, hence, may not play important individual roles. The catabolism of the BCAAs is highly regulated by both allosteric and covalent mechanisms. BCKD is inhibited by phosphorylation and activated by dephosphorylation. Allosteric inhibition of the kinase by the branched-chain keto acids (BCKA) (particularly by [alpha]-ketoisocaproate) serves both as a mechanism for promoting the catabolism of excess quantities of these amino acids as well as for conserving low concentrations of these dietary essential amino acids. Cytosolic and mitochondrial isoenzymes of BCAT have been identified. They are thought to play an important role in brain neurotransmitter metabolism. KEY WORDS: * leucine * isoleucine * valine * protein structure * muscle metabolism

Citrobacter rodentium is a murine pathogen that is now widely used as an in vivo model for gastrointestinal infections due to its similarities with human enteropathogens, such as the possession of a locus for enterocyte effacement (the LEE island). We studied the lrp gene of C. rodentium and found that it encodes a product highly similar to members of the Lrp (leucine-responsive regulatory protein) family of transcriptional regulators, able to recognize leucine as an effector and to repress the expression of its own structural gene. In enterobacteria, Lrp is a global regulator of gene expression, as it controls a large variety of genes, including those coding for cell appendages and other potential virulence factors. Based on the well-established role of Lrp on the expression of pilus genes in Escherichia coli, we also studied the role of Lrp in controlling the formation of the type I pilus in C. rodentium. Type I pili, produced by the fim system, are virulence factors of uropathogens, involved in mediating bacterial adhesion to bladder epithelial cells. Yeast agglutination assays showed that Lrp is needed for type I pilus formation and real-time PCR experiments indicated that Lrp has a strong leucine-mediated effect on the expression of the fim AICDFGH operon. Mutant studies indicated that this positive action is exerted mainly through a positive control of Lrp on the phase variation mechanism that regulates fimAICDFGH expression. A quantitative analysis of its expression suggested that this operon may also be negatively regulated at the level of transcription.

Six ruminally cannulated Holstein steers (initial BW = 189 [+ or -] 11 kg) housed in metabolism crates were used in a 6 x 6 Latin square to study effects of ruminal ammonia load on Leu utilization. All steers received a diet based on soybean hulls (2.7 kg of DM/ d), ruminal infusions of 200 g of acetate/d, 200 g of propionate/d, and 50 g of butyrate/d, as well as an abomasal infusion of 300 g of glucose/d to provide energy without increasing microbial protein supply and an abomasal infusion of a mixture (238 g/d) of all essential AA except Leu. Treatments were arranged as a 3 x 2 factorial and included Leu (0, 4, or 8 g/d) infused abomasally and urea (0 or 80 g/d) infused ruminally. Abomasal Leu infusion linearly decreased (P < 0.05) both urinary and fecal N excretions and linearly increased (P < 0.05) retained N, but the decreases in urinary N excretion in response to Leu tended (P = 0.07) to be greater, and the increases in retained N in response to Leu were numerically greater in the presence of the urea infusion. Although urea infusions increased (P < 0.05) plasma urea concentrations, urinary N excretions, and urinary urea excretions, retained N also was increased (P < 0.05). The efficiency of deposition of supplemental Leu ranged from 24 to 43% when steers received 0 or 80 g of urea/d, respectively. Under our experimental conditions, increasing ammonia load improved whole-body protein deposition in growing steers when Leu supply was limiting. Key Words: Amino Acids, Ammonia, Cattle, Growth, Leucine, Utilization

Three weeks of caloric restriction alters protein metabolism in normal-weight young men. Am J Physiol Endocrinol Metab 289: E446-E455, 2005. First published May 3, 2005; doi:10.1152/ajpendo.00001.2005.--The effects of prolonged caloric restriction (CR) on protein kinetics in lean subjects has not been investigated previously. The purpose of this study was to test the hypotheses that 21 days of CR in lean subjects would 1) result in significant losses of lean mass despite a suppression in leucine turnover and oxidation and 2) negatively impact exercise performance. Nine young, normal-weight men [23 [+ or -] 5 y, 78.6 [+ or -] 5.7 kg, peak oxygen consumption (V[O.sub.2] peak) 45.2 [+ or -] 7.3 ml x [kg.sup.-1] x [min.sup.-1], mean [+ or -] SD] were underfed by 40% of the calories required to maintain body weight for 21 days and lost 3.8 [+ or -] 0.3 kg body wt and 2.0 [+ or -] 0.4 kg lean mass. Protein intake was kept at 1.2 g x [kg.sup.-1] x [day.sup.-1]. Leucine kinetics were measured using [alpha]-ketoisocaproic acid reciprocal pool model in the postabsorptive state during rest and 50 min of exercise (EX) at 50% of V[O.sub.2] peak. Body composition, basal metabolic rate (BMR), and exercise performance were measured throughout the intervention. At rest, leucine flux ([approximately equal to] 131 [micro]mol x [kg.sup.-1] x [h.sup.-1]) and oxidation ([R.sub.ox]; [approximately equal to] 19 [micro]mol x [kg.sup.-1] x [h.sup.-1]) did not differ pre- and post-CR. During EX, leucine flux (129 [+ or -] 6 vs. 121 [+ or -] 6) and [R.sub.ox] (54 [+ or -] 6 vs. 46 [+ or -] 8) were lower after CR than they were pre-CR. Nitrogen balance was negative throughout the intervention ([approximately equal to] 3.0g N/day), and BMR declined from 1,898 [+ or -] 262 to 1,670 [+ or -] 203 kcal/day. Aerobic performance (V[O.sub.2] peak, endurance cycling) was not impacted by CR, but arm flexion endurance decreased by 20%. In conclusion, 3 wk of caloric restriction reduced leucine flux and [R.sub.ox] during exercise in normal-weight young men. However, despite negative nitrogen balance and loss of lean mass, whole body exercise performance was well maintained in response to CR. energy intake; energy expenditure; leucine flux; exercise; nitrogen balance; lean mass

Contraction and relaxation of heart muscle cells is regulated by cycling of calcium between cytoplasm and sarcoplasmic reticulum. Human phospholamban (PLN), expressed in the sarcoplasmic reticulum membrane as a 30-kDa homopentamer, controls cellular calcium levels by a mechanism that depends on its phosphorylation. Since PLN was discovered [approximately equal to to]30 years ago, extensive studies have aimed to explain how it influences calcium pumps and to determine whether it acts as an ion channel. We have determined by solution NMR methods the atomic resolution structure of an unphosphorylated PLN pentamer in dodecylphosphocholine micelles. The unusual bellflower-like assembly is held together by leucine/isoleucine zipper motifs along the membrane-spanning helices. The structure reveals a channel-forming architecture that could allow passage of small ions. The central pore gradually widens toward the cytoplasmic end as the transmembrane helices twist around each other and bend outward. The dynamic N-terminal amphipathic helices point away from the membrane, perhaps facilitating recognition and inhibition of the calcium pump. leucine/isoleucine zipper | membrane channel | NMR | dipolar couplings

Studies both in vivo and in vitro have shown that leucine at a very high dose can stimulate muscle protein synthesis, an effect that is enhanced in vivo by insulin secreted in response to the leucine dose. High leucine can also inhibit protein degradation in skeletal muscle, as well as in liver. In contrast, at normal physiological levels, increasing leucine concentration by infusion stimulates muscle protein synthesis by enhancing its sensitivity to insulin. It is concluded that the role of leucine in vivo is to provide a signal that amino acids are available, which in combination with the signal of energy availability from insulin, stimulates muscle protein synthesis. KEY WORDS: * leucine * isoleucine * valine * branched-chain amino acids * protein synthesis * insulin * muscle

Maple syrup urine disease (MSUD) is caused by a deficiency in the branched-chain [alpha]-ketoacid dehydrogenase complex. Accumulations of branched-chain amino acids (BCAAs) and branched-chain [alpha]-ketoacids (BCKAs) in patients with MSUD induce ketoacidosis, neurological disorders, and developmental disturbance. BCAAs and BCKAs influence on the nervous system can be estimated by analyzing these patients. According to clinical investigations on MSUD patients, leucine levels over 400 [micro]mol/L apparently can cause any clinical problem derived from impaired function of the central nervous system. Damage to neuronal cells found in MSUD patients are presumably because of higher concentrations of both blood BCAAs or BCKAs, especially [alpha]-ketoisocapronic acids. These clinical data from MSUD patients provide a valuable basis on understanding leucine toxicity in the normal subject. KEY WORDS: * maple syrup urine disease * leucine * branched-chain amino acid * branched-chain [alpha]-ketoacid * branched-chain [alpha]-ketoacid dehydrogenase

There is no good evidence for establishing branched-chain amino acid (BCAA) tolerance levels for humans. With pigs, chicks, and rats, data are available concerning excessive intake levels of BCAA, but most of the information is for growing animals instead of for adults. Estimates of maintenance requirements for (high-quality) protein and BCAA in pigs weighing between 43 and 140 kg are 350 mg * [kg.sup.-1] * [d.sup.-1] for protein and 28.7 mg * [kg.sup.-1] * [d.sup.-1] for total BCAA. In contrast, human adult maintenance requirement estimates are much higher, i.e., 660 mg * [kg.sup.-1] * [d.sup.-1] for good quality protein and a range of 68 to 144 mg * [kg.sup.-1] * [d.sup.-1] for total BCAA. The human maintenance BCAA requirement estimates range from 10.3 to 22% of the maintenance protein requirement. Whole-body protein of 45-kg pigs contains 14.2 g BCAA/100 g protein, but the maintenance requirement (based on nitrogen balance) for total BCAA is only 8.2% of the total maintenance protein requirement. Conversely, sulfur amino acid (methionine + cysteine), threonine, and tryptophan maintenance requirements of pigs as a percentage of the maintenance protein requirement are much higher than whole-body protein levels of these amino acids. This suggests that the efficiency of using absorbed amino acids of dietary origin or of reusing endogenous amino acids arising from body protein catabolism may vary considerably among the indispensable amino acids. Additionally, work with pigs points to the conclusion that whole-body amino acid concentrations are poor predictors of both maintenance requirements and ideal amino acid profiles. Based on studies with young experimental animals, a rather large dietary excess (above requirement) of an individual BCAA is well tolerated when consumed in diets containing surfeit levels of protein and the other 2 BCAA. KEY WORDS: * leucine * isoleucine * valine * pig * chick * rat * human

Changes were investigated in plasma metabolites and physiological and toxicological variables in rats fed for 2 wk on a basal diet or diets with 1.5, 5, 10, 15, and 30% added leucine. In the same experiment, the changes in gene expression in livers of rats fed the basal diet or diets with 5% and 15% added leucine were investigated using DNA microarrays. Cluster analysis of multivariate correlations of metabolites and physiological and toxicological variables indicated that the variables associated with excess nitrogen clustered together with leucine and [alpha]-ketoisocaproate. The gene expression data, although preliminary, indicated that there was little change in the expression of enzymes of the catabolic pathways for leucine but that there were changes in enzymes associated with nitrogen metabolism and other pathways downstream of leucine catabolism. The data seem consistent with excess leucine exerting its effects through the overloading of nitrogen metabolism and that urea or [alpha]-ketoisocaproate could be an early marker for the upper limit of adequate intake. KEY WORDS: * gas chromatography-mass spectrometry * DNA microarray * dietary reference intake * tolerable upper level * acceptable daily intake

Since the in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. In 1978, Sherwin demonstrated in humans an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the BCAAs have been administered in varying amounts and durations, and the effect upon protein metabolism has been measured. Measurements of changes in muscle amino acid metabolism were made by arteriovenous difference measurements and by biopsies. An anabolic effect of leucine and the branched-chain amino acids (BCAAs) on reduction of muscle protein breakdown was found in these studies, with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole-body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect, produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis, has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition, no untoward effects have been reported in any of these studies from infusion of the BCAAs at upward 3 times basal flux or 6 times normal dietary intake during the fed portion of the day. KEY WORDS: * protein synthesis * protein breakdown * leucine

Glutamic acid is an important excitatory neurotransmitter of the brain. Two key goals of brain amino acid handling are to maintain a very low intrasynaptic concentration of glutamic acid and also to provide the system with precursors from which to synthesize glutamate. The intrasynaptic glutamate level must be kept low to maximize the signal-to-noise ratio upon the release of glutamate from nerve terminals and to minimize the risk of excitotoxicity consequent to excessive glutamatergic stimulation of susceptible neurons. The brain must also provide neurons with a constant supply of glutamate, which both neurons and glia robustly oxidize. The branched-chain amino acids (BCAAs), particularly leucine, play an important role in this regard. Leucine enters the brain from the blood more rapidly than any other amino acid. Astrocytes, which are in close approximation to brain capillaries, probably are the initial site of metabolism of leucine. A mitochondrial branched-chain aminotransferase is very active in these cells. Indeed, from 30 to 50% of all [alpha]-amino groups of brain glutamate and glutamine are derived from leucine alone. Astrocytes release the cognate ketoacid [[alpha]-ketoisocaproate (KIC)] to neurons, which have a cytosolic branched-chain aminotransferase that reaminates the KIC to leucine, in the process consuming glutamate and providing a mechanism for the 'buffering' of glutamate if concentrations become excessive. In maple syrup urine disease, or a congenital deficiency of branched-chain ketoacid dehydrogenase, the brain concentration of KIC and other branched-chain ketoacids can increase 10- to 20-fold. This leads to a depletion of glutamate and a consequent reduction in the concentration of brain glutamine, aspartate, alanine, and other amino acids. The result is a compromise of energy metabolism because of a failure of the malate-aspartate shuttle and a diminished rate of protein synthesis. KEY WORDS: * brain metabolism * leucine * glutamate * maple syrup urine disease * inborn errors of metabolism * amino acids

Naphthalene dioxrygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the a-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotolnene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced (14-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases.

The membrane topology of proprotein convertase (PC3) and of a PC3 construct containing a conventional transmembrane segment of 19 leucines was analyzed. Alkaline extraction was performed to assess membrane integration.

Naturally occurring mutations in insulin associated with diabetes mellitus identify critical determinants of its biological activities. The crystal structure of insulin Wakayama, a clinical variant in which a conserved valine in the A chain is substituted by leucine, is described.

Analysis of transcripts of 75 genes encoding putative basic leucine zipper (bZIP) transcription factors in the Arabidopsis genome identified AtbZIP60, which was induced by tunicamycin. AtbZIP60 encodes a predicted protein of 295 aa with a putative transmembrane domain near its C terminus after a bZIP domain. A truncated form of AtbZIP60 without a transmembrane domain (AtbZIP60[DELTA]C) fused with GFP localized to the nucleus, suggesting translocation of native protein to the nucleus by release from the membrane. AtbZIP60 was also induced by DTT and azetidine-2-carboxylate, which induce the endoplasmic reticulum (ER) stress response (also called the unfolded protein response). Expression of AtbZIP60[DELTA]C clearly activated any of three BiP and two calnexin promoters in a dual luciferase assay using protoplasts of cultured cells. The induction was considered to be through cis-elements plant-specific unfolded protein response element and ER stress-response element. Interestingly, AtbZIP60[DELTA]C also appeared to induce the expression of AtbZIP60 through an ER stress-response element-like sequence in the promoter of AtbZIP60. These characteristics of AtbZlP60 imply a signal transduction pathway of the ER stress response unique to plants. Arabidopsis thaliana | BiP | tunicamycin | unfolded protein response

Oral administration of a single bolus of leucine in an amount equivalent to the daily intake (1.35 g/kg body wt) enhances skeletal muscle protein synthesis in food-deprived rats. To elucidate whether smaller amounts of leucine can also stimulate protein synthesis, rats were administered the amino acid at oncentrations ranging from 0.068 to 1.35 g/kg body wt by oral gavage. Thirty minutes following the administration of doses of leucine as low as 0.135 g/kg body wt, skeletal muscle protein synthesis was significantly greater than control values. The increase in protein synthesis was associated with changes in the regulation of biomarkers of mRNA translation initiation as evidenced by upregulated phosphorylation of the translational repressor, eukaryotic initiation factor (elF)4E-binding protein 1 (4E-BP1), the association of elF4G with the mRNA cap binding protein elF4E, and the phosphorylation of the 70-kDa ribosomal protein S6 kinase. Alterations in the phosphorylation of elF4G, as well as the association of 4E-BP1 with elF4E, were observed following leucine administration; however, these changes appeared to be biphasic with maximal changes occurring when circulating insulin concentrations were elevated. Thus it appears that leucine administration affects mRNA translation and skeletal muscle protein synthesis through modulation of multiple biomarkers of mRNA translation. The ability of small doses of leucine to stimulate skeletal muscle protein synthesis suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans. KEY WORDS: * translation initiation * gastrocnemius muscle * mTOR