Search

Enhanced fibroblast growth factor 21 (FGF21) production and circulation has been linked to the metabolic adaptation to starvation. Here, we demonstrated that hepatic FGF21 expression is induced by dietary protein restriction, but not energy restriction. Circulating FGF21 was increased 10-fold in mice and rats fed a low-protein (LP) diet. In these animals, liver Fgf21 expression was increased within 24 hours of reduced protein intake. In humans, circulating FGF21 levels increased dramatically following 28 days on a LP diet. LP-induced increases in FGF21 were associated with increased phosphorylation of eukaryotic initiation factor 2a (eIF2α) in the liver, and both baseline and LP- induced serum FGF21 levels were reduced in mice lacking the eIF2α kinase general control nonderepressible 2 (GCN2). Finally, while protein restriction altered food intake, energy expenditure, and body weight gain in WT mice, FGF21-deficient animals did not exhibit these changes in response to a LP diet. These and other data demonstrate that reduced protein intake underlies the increase in circulating FGF21 in response to starvation and a ketogenic diet and that FGF21 is required for behavioral and metabolic responses to protein restriction. FGF21 therefore represents an endocrine signal of protein restriction, which acts to coordinate metabolism and growth during periods of reduced protein intake., Introduction Nutrient restriction engages a variety of metabolic and behavioral adaptations, and in the case of energy restriction, these adaptations are well described as including alterations in feeding behavior, energy [...]

Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was [...]

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.

The uptake of branched-chain amino acids (BCAA) from plasma by lactating porcine mammary gland substantially exceeds their output in milk, whereas glutamine output is 125% greater than its uptake from plasma. In this study, we tested the hypothesis that BCAA are catabolized for glutamine synthesis in mammary tissue. Mammary tissue slices from sows on d 28 of lactation were incubated at 37[degrees]C for 1 h in Krebs buffer containing 0.5 or 2 mmol/L L-[1-[.sup.14]C]- or L-[U-[.sup.14]C]-labeled leucine, isoleucine, or valine. Rates of BCAA transport and degradation in mammary tissue were high, with ~60% of transaminated BCAA undergoing oxidative decarboxylation and the remainder being released as branched-chain [alpha]ketoacids (BCKA). Most (~70%) of the decarboxylated BCAA were oxidized to C[O.sub.2]. Rates of net BCAA transamination were similar to rates of glutamate, glutamine, aspartate, asparagine, and alanine synthesis. Consistent with the metabolic data, mammary tissue expressed BCAA aminotransferase (BCAT), BCKA decarboxylase, glutamine synthetase (GS), glutamate-oxaloacetate aminotransferase, glutamate-pyruvate aminotransferase, and asparagine synthetase, but no phosphate-activated glutaminase, activity. Western blot analysis indicated relatively high levels of mitochondrial and cytosolic isoforms of BCAT, as well as BCKA dehydrogenase and GS proteins in mammary tissue. Our results demonstrate that glutamine and aspartate (abundant amino acids in milk protein) were the major nitrogenous products of BCAA catabolism in lactating porcine mammary tissue and provide a biochemical basis to explain an enrichment of glutamine and aspartate in sow milk. J. Nutr. 139: 1502-1509, 2009.

Decline in muscle mass, protein synthesis, and mitochondrial function occurs with age, and amino acids are reported to enhance both muscle protein synthesis and mitochondrial function. It is unclear whether increasing dietary protein intake corrects postabsorptive muscle changes in aging. We determined whether a 10-day diet of high [HP; 3.0 g protein x kg fat-free mass [(FFM).sup.-1] x [day.sup.-1]] vs. usual protein intake (UP; 1.5 g protein x kg [FFM.sup.-1] x [day.sup.-1]) favorably affects mitochondrial function, protein metabolism, and nitrogen balance or adversely affects insulin sensitivity and glomerular filtration rate (GFR) in 10 healthy younger (24 [+ or -] 1 yr) and 9 older (70 [+ or -] 2 yr) participants in a randomized crossover study. Net daily nitrogen balance increased equally in young and older participants, but postabsorptive catabolic state also increased, as indicated by higher whole body protein turnover and leucine oxidation with no change in protein synthesis. Maximal muscle mitochondrial ATP production rate was lower in older people, with no change occurring in diet. GFR was lower in older people, and response to HP was significantly different between the two groups, with a significant increase occurring only in younger people, thus widening the differences in GFR between the young and older participants. In conclusion, a short-term high-protein diet increased net daily nitrogen balance but increased the postabsorptive use of protein as a fuel. HP did not enhance protein synthesis or muscle mitochondrial function in either young or older participants. Additionally, widening differences in GFR between young and older patients is a potential cause of concern in using HP diet in older people. dietary protein; protein metabolism; mitochondrial function; kidney function; aging

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

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

There are several features of the metabolism of the indispensable BCAAs that set them apart from other indispensable amino acids. BCAA catabolism involves 2 initial enzymatic steps that are common to all 3 BCAAs; therefore, the dietary intake of an individual BCAA impacts on the catabolism of all 3. The first step is reversible transamination followed by irreversible oxidative decarboxylation of the branched-chain [alpha]-keto acid transamination products, the branched chain [alpha]-keto acids (BCKAs). The BCAA catabolic enzymes are distributed widely in body tissues and, with the exception of the nervous system, all reactions occur in the mitochondria of the cell. Transamination provides a mechanism for dispersing BCAA nitrogen according to the tissue's requirements for glutamate and other dispensable amino acids. The intracellular compartmentalization of the branched-chain aminotransferase isozymes (mitochondrial branched-chain aminotransferase, cytosolic branched-chain aminotransferase) impacts on intra- and interorgan exchange of BCAA metabolites, nitrogen cycling, and net nitrogen transfer. BCAAs play an important role in brain neurotransmitter synthesis. Moreover, a dysregulation of the BCAA catabolic pathways that leads to excess BCAAs and their derivatives (e.g., BCKAs) results in neural dysfunction. The relatively low activity of catabolic enzymes in primates relative to the rat may make the human more susceptible to excess BCAA intake. It is hypothesized that the symptoms of excess intake would mimic the neurological symptoms of hereditary diseases of BCAA metabolism. KEY WORDS: * branched-chain aminotransferases * nitrogen cycles * glutamate * alanine * mitochondria

A study was done to explore the mechanistic details of the redox-active CXXC motif, by mutating C315, C318, or both to alanine or serine using site-directed mutagenesis. The findings suggest that C315 is the peroxide-reactive thiol(ate) functioning as the redox sensor in the regulation of hBCATm and the thiol group at position C318 functions to prevent overoxidation of C315 beyond the sulfenic acid state by forming a more stable, readily reducible disulfide bond.

We have examined the localization of the first two enzymes in the branched-chain amino acid (BCAA) catabolic pathway: the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) and the branched-chain [alpha]-keto acid dehydrogenase (BCKD) enzyme complex. Antibodies specific for BCATm or BCATc were used to immunolocalize the respective isozymes in cryosections of rat tissues. BCATm was expressed in secretory epithelia throughout the digestive tract, with the most intense expression in the stomach. BCATm was also strongly expressed in secretory cells of the exocrine pancreas, uterus, and testis, as well as in the transporting epithelium of convoluted tubules in kidney. In muscle, BCATm was located in myofibrils. Liver, as predicted, was not immunoreactive for BCATm. Unexpectedly, BCATc was localized in elements of the autonomic innervation of the digestive tract, as well as in axons in the sciatic nerve. The distributions of BCATc and BCATm did not overlap. BCATm-expressing cells also expressed the second enzyme of the BCAA catabolic pathway, BCKD. In selected monkey and human tissues examined by immunoblot and/or immunohistochemistry, BCATm and BCATc were distributed in patterns very similar to those found in the rat. The results show that BCATm is in a position to regulate BCAA availability as protein precursors and anabolic signals in secretory portions of the digestive and other organ systems. The unique expression of BCATc in neurons of the peripheral nervous system, without coexpression of BCKD, raises new questions about the physiological function of this BCAT isozyme. digestive system; human; leucine; monkey; rat

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

Substrate binding of the human mitochondrial branched chain aminotransferase with substrate amino acids, isoleucine ketimine and pyridoxamine phosphate, does not involve domain or loop closure as seen in other proteins. Furthermore, cysteine residues 315 and 318 are important for the enzyme structure and form a thiol-thiolate hydrogen bond.

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

There is evidence that during lactation, uptake of the essential branched-chain amino acids (BCAA) by mammary glands exceeds their output in milk protein. In this study, we have measured the potential of lactating rats to catabolize BCAA. The activity, relative protein and specific mRNA levels of the first two enzymes in the BCAA catabolic pathway, branched-chain aminotransferase (BCAT) and branched-chain [Alpha]-keto acid dehydrogenase (BCKD), were measured in mammary gland, liver and skeletal muscle obtained from rat dams at peak lactation (12 d), from rat dams 24 h after weaning at peak lactation and from age-matched virgin controls. Western analysis showed that the mitochondrial BCATm isoenzyme was found in mammary gland. Comparison of lactating and control rats revealed that tissue BCATm activity, protein and mRNA were at least 10-fold higher in mammary tissue during lactation. Values were 1.3- to 1.9-fold higher after 24 h of weaning. In mammary gland of lactating rats, the BCKD complex was fully active. In virgin controls and weaning dams, only about 20% of the complex was in the active state. Hypertrophy of the liver and mammary gland during lactation resulted in a 73% increase in total oxidative capacity in lactating rats. The results are consistent with increased expression of the BCATm gene in the mammary gland during lactation, whereas oxidation appears to be regulated primarily by changes in activity state (phosphorylation state) of BCKD. KEY WORDS: branched-chain amino acids; aminotransferase; lactation; rats; branched-chain [Alpha]-keto acid dehydrogenase

The mitochondrial inorganic phosphate signal was characterized in isolated rat liver mitochondria respiring on succinate and glutamate. The effects of Ca2+ and Mn2+, external pH, temperature and media composition were investigated to determine factors affecting limited nuclear magnetic resonance visibility in the cell. Moreover, the relationship of pH and phosphate gradients was assessed in addition to the ability of matrix inorganic phosphates to monitor intramitochondrial pH.