Amino Acid Deficiency Research Articles

Phosphorylation of eIF2α Is Involved in the Signaling of Indispensable Amino Acid Deficiency in the Anterior Piriform Cortex of the Brain in Rats

Sensing of indispensable amino acid (IAA) deficiency, an acute challenge to protein homeostasis, is demonstrated by rats as rejection of IAA-deficient diets within 20 min. The anterior piriform cortex (APC) of the brain in rats and birds is essential for this nutrient sensing, and is activated by IAA deficiency. Yet the mechanisms that sense and transduce IAA reduction to signaling in the APC, or indeed in any animal cells, are unknown. Because rejection of a deficient diet within 20 min is too rapid to be explained by transcription-derived signals, brain tissue was taken from rats after 20 min access to either a threonine-basal, -devoid, or -corrected diet and examined for proteins associated with early signaling of IAA deficiency in the yeast model. Western blots and immunohistochemistry showed that the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2alpha[Ser51]) and translation of its downstream product, c-Jun, were increased (47%, P < 0.005, and 55%, P < 0.025, respectively) in APC from rats offered devoid, but not corrected diets, compared with those offered basal diets. This was not seen in other brain areas. In cells intensely labeled for cytoplasmic p-eIF2alpha, there was intense fluorescence for c-Jun in the nucleus. Thus, p-eIF2alpha, which is pivotal in the initiation of global protein translation, and its downstream product, the leucine zipper protein, c-Jun, are increased in the mammalian APC within the time frame necessary for the behavioral response. We suggest that p-eIF2alpha and c-Jun participate in signaling nutrient deficiency in the IAA-sensitive neurons of the APC.

Site and extent of digestion and amino acid flow to the small intestine in beef cattle consuming limited amounts of forage.

Eight Angus x Gelbvieh heifers (445 +/- 74.5 kg) fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square double double-crossover designed experiment to assess the effect of restricted forage intake on site and extent of digestion and flow of essential AA amino acids to the small intestine. Heifers were fed chopped (2.54 cm) bromegrass hay (9.2% CP, 64% NDF on an OM basis) at one of four percentages of maintenance (30, 60, 90, and 120%). Experimental periods were 21 d in length, with 17 d of adaptation followed by 4 d of intensive sample collection, after which maintenance requirements and subsequent level of intake were adjusted for BW change. True ruminal OM, NDF, and N digestion (g/d) decreased linearly (P < 0.001) with decreasing forage intake. When expressed as a percentage of OM intake, true ruminal OM and N digestibility were not affected (P = 0.23 to 0.87), whereas ruminal NDF digestibility tended to increase (P = 0.09) as forage intake decreased. Total and microbial essential amino acid flow to the duodenum decreased linearly (P = 0.001) from 496.1 to 132.1 g/d and 329.1 to 96.0 g/d, as intake decreased from 120 to 30% of maintenance intake, respectively. Although the profile of individual essential amino acids in duodenal digesta (P = 0.001 to 0.07) and isolated ruminal microbes differed (P = 0.001 to 0.09) across treatment, the greatest difference noted for total and microbial essential amino acid profile was only 0.3 percentage units. Because total and microbial flow of essential amino acids to the small intestine decreased as OM intake decreased, but true ruminal degradability of individual essential amino acids (P = 0.17 to 0.99) and digesta essential amino acid profile were comparable across treatments, total essential amino acid supply to the small intestine was predicted using OM intake as the independent variable. The resulting simple linear regression equation was: total essential amino acid flow = (0.055 x OM intake) + 1.546 (r2 = 0.91). The model developed in this experiment accounted for more of the variation in the data set than the current beef cattle NRC model, which under-predicted total flow of essential amino acids to the duodenum. The prediction equation developed herein can be used to estimate the supply of essential amino acids reaching the small intestine when formulating supplements to compensate for potential amino acid deficiencies resulting from restricted forage intake.

Site and extent of digestion and amino acid flow to the small intestine in beef cattle consuming limited amounts of forage1

Eight Angus × Gelbvieh heifers (445 ± 74.5 kg) fitted with ruminal and duodenal cannulas were used in a 4 × 4 Latin square double double-cross-over designed experiment to assess the effect of restricted forage intake on site and extent of digestion and flow of essential AA amino acids to the small intestine. Heifers were fed chopped (2.54 cm) bromegrass hay (9.2% CP, 64% NDF on an OM basis) at one of four percentages of maintenance (30, 60, 90, and 120%). Experimental periods were 21 d in length, with 17 d of adaptation followed by 4 d of intensive sample collection, after which maintenance requirements and subsequent level of intake were adjusted for BW change. True ruminal OM, NDF, and N digestion (g/d) decreased linearly (P < 0.001) with decreasing forage intake. When expressed as a percentage of OM intake, true ruminal OM and N digestibility were not affected (P = 0.23 to 0.87), whereas ruminal NDF digestibility tended to increase (P = 0.09) as forage intake decreased. Total and microbial essential amino acid flow to the duodenum decreased linearly (P = 0.001) from 496.1 to 132.1 g/d and 329.1 to 96.0 g/d, as intake decreased from 120 to 30% of maintenance intake, respectively. Although the profile of individual essential amino acids in duodenal digesta (P = 0.001 to 0.07) and isolated ruminal microbes differed (P = 0.001 to 0.09) across treatment, the greatest difference noted for total and microbial essential amino acid profile was only 0.3 percentage units. Because total and microbial flow of essential amino acids to the small intestine decreased as OM intake decreased, but true ruminal degradability of individual essential amino acids (P = 0.17 to 0.99) and digesta essential amino acid profile were comparable across treatments, total essential amino acid supply to the small intestine was predicted using OM intake as the independent variable. The resulting simple linear regression equation was: total essential amino acid flow = (0.055 × OM intake) + 1.546 (r2 = 0.91). The model developed in this experiment accounted for more of the variation in the data set than the current beef cattle NRC. model, which under-predicted total flow of essential amino acids to the duodenum. The prediction equation developed herein can be used to estimate the supply of essential amino acids reaching the small intestine when formulating supplements to compensate for potential amino acid deficiencies resulting from restricted forage intake.

Ammonia toxicity to the brain and creatine

Symptoms of hyperammonemia are age-dependent and some are reversible. Multiple mechanisms are involved. Hyperammonemia increases the uptake of tryptophan into the brain by activation of the L-system carrier while brain glutamine plays a still undefined role. The uptake of tryptophan by the brain is enhanced when the plasma levels of branched-chain amino acids competing with the other large neutral amino acids are low. Hyperammonemia increases the utilization of branched-chain amino acids in muscle when ketoglutarate is low, and this is further enhanced by glutamine depletion (as a result of therapy with ammonia scavengers like phenylbutyrate). Anorexia, most likely a serotoninergic symptom, might further aggravate the deficiency of indispensable amino acids (e.g., branched-chain and arginine). The role of increased glutamine production in astrocytes and the excitotoxic and metabotropic effects of increased extracellular glutamate have been extensively investigated and found to differ between models of acute and chronic hyperammonemia. Using an in vitro model of cultured embryonic rat brain cell aggregates, we studied the role of creatine in ammonia toxicity. Cultures exposed to ammonia before maturation showed impaired cholinergic axonal growth accompanied by a decrease of creatine and phosphocreatine, a finding not observed in mature cultures. By using different antibodies, we have shown that the phosphorylated form of the intermediate neurofilament protein is affected. Adding creatine to the culture medium partially prevents impairment of axonal growth and the presence of glia in the culture is a precondition for this protective effect. Adequate arginine substitution is essential in the treatment of urea cycle defects as creatine is inefficiently transported into the brain.

Effect of alternative pathway therapy on branched chain amino acid metabolism in urea cycle disorder patients

Urea cycle disorders (UCDs) are a group of inborn errors of hepatic metabolism caused by the loss of enzymatic activities that mediate the transfer of nitrogen from ammonia to urea. These disorders often result in life-threatening hyperammonemia and hyperglutaminemia. A combination of sodium phenylbutyrate and sodium phenylacetate/benzoate is used in the clinical management of children with urea cycle defects as a glutamine trap, diverting nitrogen from urea synthesis to alternatives routes of excretion. We have observed that patients treated with these compounds have selective branched chain amino acid (BCAA) deficiency despite adequate dietary protein intake. However, the direct effect of alternative therapy on the steady state levels of plasma branched chain amino acids has not been well characterized. We have measured steady state plasma branched chain and other essential non-branched chain amino acids in control subjects, untreated ornithine transcarbamylase deficiency females and treated null activity urea cycle disorder patients in the fed steady state during the course of stable isotope studies. Steady-state leucine levels were noted to be significantly lower in treated urea cycle disorder patients when compared to either untreated ornithine transcarbamylase deficiency females or control subjects ( P<0.0001). This effect was reproduced in control subjects who had depressed leucine levels when treated with sodium phenylacetate/benzoate ( P<0.0001). Our studies suggest that this therapeutic modality has a substantial impact on the metabolism of branched chain amino acids in urea cycle disorder patients. These findings suggest that better titration of protein restriction could be achieved with branched chain amino acid supplementation in patients with UCDs who are on alternative route therapy.

Phosphorylation of Ca 2+/calmodulin-dependent protein kinase type ii and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet

The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and responds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c- fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related increase in neuronal excitability, but previous studies have shown effects on intracellular Ca 2+ in deficient APC slices in vitro. In the present study we hypothesized that indispensable amino acid deficiency increases intraneuronal Ca 2+, resulting in autophosphorylation of calcium/calmodulin-dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine-devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit returned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex. These findings support the notion that calcium and glutamate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII.

Protected canola meal increases milk protein concentration in dairy cows fed a grass silage-based diet

Low concentrations of protein in milk occur during the summer–autumn in south-west Australia. This is the period, on dryland farms, when the diet of lactating cows typically consists of grass silage and a mixture of crushed lupins and cereal grain. This experiment was conducted to test the hypothesis that supplying protected canola meal would increase the protein concentration of milk and, possibly, milk yield in cows fed grass silage and a lupin–cereal concentrate. Sixty Holstein cows in mid lactation were allocated to 2 equal-sized dietary treatment groups: control (lupin) or protected canola meal. The control diet consisted of 14.5 kg DM grass silage (annual ryegrasses–subterranean clover) and 5.4 kg DM of crushed lupins and barley (4:1) per head per day. For the protected canola meal diet, 2.15 kg DM protected canola meal replaced 2.15 kg lupins. The protected canola meal was produced by treating solvent-extracted canola meal with formaldehyde, to produce a product with an in sacco fractional degradability of 0.29 at a rumen fractional outflow rate of 0.08/h. The equivalent degradability of untreated canola meal was 0.80 and of lupin was 0.83. Cows were individually fed the concentrate ration twice daily, after each milking, then were managed as a single herd in dry lots and fed grass silage. By the end of 8 weeks, cows fed the protected canola meal diet had higher milk protein concentrations (30.7 v. 29.2 g/L; P&lt;0.05) and higher liveweights (604 v. 593 kg; P&lt;0.05). Milk yield (L/day) was increased by 1 L/day, but this effect was not significant (P&gt;0.10). Fat concentration was unaffected by diet (P&gt;0.05). Since the only difference in treatment was the replacement of a portion of lupins with protected canola meal, the results indicate that a deficiency of metabolisable amino acids contributes to the low milk protein concentrations recorded during summer–autumn in south-west Australia. Whether this was acting primarily through a stimulus of appetite, or directly on milk components, could not be determined because silage intakes were not recorded.

Effect of dietary protein on lean body wasting in dogs: correlation between loss of lean mass and markers of proteasome-dependent proteolysis.

To determine the effect of dietary protein intake on lean body wasting in adult canines a study was undertaken to investigate the Ubiquitin Proteasome (UP) pathway and concurrent changes in lean and fat body mass of canines fed variable sources and concentrations of dietary protein. Purpose-bred, intact female canines (56) between the ages of 2 and 3 years were fed either 12 or 28% protein diet for 10 weeks. Each diet contained variable amounts of corn gluten meal and chicken protein sources in ratios of 100 : 0, 67 : 33, 33 : 67 and 0 : 100 per cent (w/w), respectively. All diets were isocaloric with calories coming from protein : fat : carbohydrate at the respective ratios of 12 : 40 : 48% for the 12% diets, and 28 : 40 : 32% for the 28% diets. Standard dual energy X-ray absorptiometry was performed to assess total body lean and fat mass at weeks 0 and 10 of the dietary trial. Muscle biopsies were also taken and processed for protein determination and standard gel electrophoresis with subsequent Western blotting for 20S proteasome and PA700 regulatory cap subunit p31. Statistical analysis revealed a moderate degree of correlation between increasing quantities of corn gluten, which is low in essential amino acids (i.e. lysine, tryptophan), and increasing loss of lean body mass over the 10-week study (R = 0.56). Furthermore, a moderate degree of correlation was observed between increasing concentrations of corn gluten protein and decreased expression of the p31 subunit of the 26S proteasome (R = 0.49). Additionally, the dogs consuming the 12% protein diets had a significant increase in fat mass regardless of the protein source. These findings suggest that lean body wasting in adult canines can be associated with the consumption of low protein diets consisting of predominantly corn gluten, which is likely due to imbalances or subclinical deficiencies of specific essential amino acids, and that low protein diets may augment accumulation of adipose tissue. Although the mechanisms remain unclear, alteration of molecular targets of skeletal muscle proteolysis, specifically involving the UP pathway occur.

The Effect of Glycine Supplementation on Performance of Broilers Fed Sub-marginal Protein with Adequate Synthetic Methionine and Lysine

A study was conducted to determine if synthetic glycine could replace dietary protein and improve performance of commercial broilers. Nine diets were formulated per feed regimen (starter, finisher, and withdrawal). A commercial diet was used as control (Diet A). Amino acids were reduced by 15% from Diet A to obtain Diet B. Synthetic methionine and lysine were added to Diet B to bring their level equal to Diet A, and create Diet C. Three levels of synthetic glycine (0, 0.05 and 0.1%) were added to Diets A, B, and C to obtain the nine dietary treatments. Male chicks (n = 3,150) originating from Ross male x Hubbard female breeder flocks were randomly placed in 63 floor pens in an open-sided house with thermostatically controlled curtains. Floor pens were divided into 7 blocks, each block containing 9 pens. The nine dietary treatments in each feed regimen were randomly assigned to pens in each of the seven blocks on d 1 (starter), 22 (finisher), and 43 (withdrawal) of age. Mortality, feed consumption (FC), body weight (BW) and feed to gain ratio (FG) were determined. Supplementing glycine, or methionine and lysine had no effect on FC. Glycine had no influence on the BW of chicks. The BW decreased with the reduction in dietary amino acids, and increased with an increase in dietary methionine and lysine fed the amino acid deficient diet. Glycine had no beneficial effect on FG, and 0.05% glycine increased (P < 0.05) FG up to 3 weeks. Addition of synthetic glycine (0.05 to 0.1%) to the 15% amino acid deficient diet could not make up for the deficiency of dietary amino acids in broiler diets.

Evaluation of corn gluten meal as a protein source in diets for gilthead sea bream (Sparus aurata L.) juveniles

A 12-week growth trial was conducted to evaluate corn gluten meal as an alternative protein source to fish meal in diets for gilthead sea bream juveniles. The experimental diets were formulated to be isonitrogenous and isoenergetic and to have 20%, 40%, 60% and 80% of fish meal protein, the only protein source in the control diet, replaced by corn gluten meal. At the end of the growth trial only the group fed the diet with 80% corn gluten protein exhibited significantly reduced growth and feed efficiency compared with the fish meal-based diet. This was most likely due to a dietary amino acid deficiency in that diet. A trend was noticed for feed efficiency to improve with the replacement of fish meal protein in the diets up to 60%. There were no significant differences among groups in protein and energy retention (as percentage of intake). At the end of the trial whole body water content of the experimental groups was significantly lower and the lipid content of groups including 60% and 80% corn gluten protein was significantly higher than that in the control. No other differences were observed in whole body composition among groups. Apparent digestibility coefficients (ADC) of the diets were evaluated in a separate trial. The ADC of dry matter of the experimental diets was significantly higher than in the control diet; there were no significant differences among diets in the ADC of energy and protein, except for the ADC of protein of diet with 80% corn gluten protein, which was significantly lower than the control. The results of this study indicate that corn gluten meal can replace up to 60% fish meal protein in diets for gilthead sea bream juveniles with no negative effects on fish performance.

Effects of Amino Acid Deficiency on Monoamines in the Lateral Hypothalamus (LH) in Rats

Animals decrease intake of an indispensable amino acid deficient diet, due in part to decreased dietary limiting amino acid concentrations within the anterior piriform cortex (APC). In addition to studies supporting a primary role for the APC in this phenomenon, recent studies have shown that the lateral hypothalamus (LH), which receives projections from the APC, also mediates the anorectic response to amino acid deficiency. The neurochemical changes within the LH that accompany the anorexia to amino acid deficiency are unclear. We hypothesized that norepinephrine (NE), dopamine (DA) and serotonin, whose levels are altered in response to amino acid deficiency within the APC, also act within the LH to mediate amino acid deficiency-induced anorexia. We determined that ingestion of an amino acid devoid diet increased concentrations of NE and the serotonin metabolite, 5-hydroxyindoleacetic acid in the LH. The 5-hydroxytryptamine metabolite was increased overall, according to analysis by area under the curve. Individual points reached significance at 130 min; NE was elevated at 170 min. These results suggest that the sustained anorectic response following ingestion of an amino acid devoid diet may be associated with increased activity of the NE and 5-hydroxytryptamine systems in the LH.

Effects of Dietary Amino Acid Balance on the Response of Dairy Cows to an Increase of Milking Frequency from Twice to Three Times Daily

An experiment was conducted to examine how the response of dairy cows to a change from twice to three times-daily milking is affected by deficiencies in the dietary supplies of three amino acids, His, Met, and Lys. Six cows were used in a 6×6 Latin square with 14-d periods. The three dietary treatments were: grass silage and a cereal-based supplement containing feather meal as the sole protein supplement; the same silage-cereal diet supplying similar amounts of metabolizable and rumen-undegradable protein but with additional amounts of His, Met, and Lys in the form of fish meal; and the fish meal diet with additional metabolizable energy in the form of an additional 2kg/d of sugar beet pulp. Within each of these dietary treatments, the cows were milked twice and three times daily, making a total of six treatments. When cows were given the feather meal diet, even though dietary metabolizable energy was in considerable excess, a deficiency of specific amino acids prevented any increase in milk yield in response to increasing the frequency of milking from twice to three times daily. In contrast, when cows consumed a similar level of excess metabolizable energy and a similar level of rumen-undegradable protein for which the protein was of better amino acid balance (fish meal), the increased frequency of milking led to increased yield of milk and milk protein.

Feeding ecology and nutrition of Australian lorikeets

Nutritional adequacy is imperative to maintain health and optimize reproductive output of lorikeets. Although lorikeets feed primarily on nectar and pollen, substitution with simple sugars and a high protein diet is inadequate. Although nectar consists primarily of sucrose, the highly indigestible raffinose sugars contained in some plant and insect exudates may promote colonization of beneficial bacteria such as the bifidobacteria, decreasing clostridial populations and possibly influencing infections of eggs with Salmonella enteritidis. Protein content of wild fruits is marginally higher when compared with commercially available fruits commonly fed to birds but fat content of wild fruits can be as high as 29%. Wild figs also have higher fat and calcium content than their domesticated counterparts. While lorikeets can survive on only 2.9% of a high-quality, highly digestible protein source, they may optimize their protein intake by selecting native species with significantly higher protein contents (>24%) and sufficient levels of essential amino acids. Substituting these pollens with inferior sources may result in essential amino acid deficiencies. There are a variety of commercial nectar substitutes on the market but the high vitamin A content of many of these products is excessively high (up to 28,000 IU kg−1), possibly contributing to a number of reproductive failures and poor health in captive lorikeets, including iron storage disease.

Impact of increasing parenteral protein loads on amino acid levels and balance in critically ill anuric patients on continuous renal replacement therapy

Objectives We wanted to establish optimum protein and glucose intakes during total parenteral nutrition by using a constant caloric but changing protein intake in critically ill, ventilated, anuric patients on continuous renal replacement therapy and measuring amino acid and glucose losses across the hemofilter. Methods Eleven consecutive, critically ill patients (eight male, age, 43.5 ± 21.8 y; Acute Physiology and Chronic Health Evaluation II score, 20.5 ± 7.0; Acute Physiology and Chronic Health Evaluation risk of death: 36.5% ± 23.0 and 6 ± 1 impaired organ systems) entered this study. Patients were fed by continuous infusion of a total parenteral mixture consisting of Synthamin (a mixture of essential and non-essential amino acids), 50% dextrose, and intralipid (long-chain triglycerides) to meet caloric requirements as predicted by Schofield's equation corrected by stress factors. The amount of protein infused was varied (1 to 2.5 g · kg −1 · d −1) by increments of 0.25 g · kg −1 · d −1. Patients were stabilized on each feeding regimen for at least 24 h before paired samples of blood and dialysate were taken for amino acid and glucose measurements. Continuous renal replacement therapy was performed by using a blood pump with a blood flow of 100 to 175 mL/min. Dialysate was pumped in and out counter-currently to the blood flow at 2 L/h. A biocompatible polyacrylonitrile hemofilter was used in all cases. Results With protein intakes below 2.5 g · kg −1 · d −1, blood levels of 14% to 57% of the measured amino acids were below the lower limits of the normal range. At 2.5 g · kg −1 · d −1, all measured amino acids were within the normal range. Amino acid balance became more positive as protein input increased ( P = 0.0001). Glucose and amino acid losses were dependent on blood concentration. Overall, 17% (range, 13% to 24%) of infused amino acids and 4% of infused glucose were lost in the dialysate. Conclusions This study of critically ill, ventilated, anuric patients on continuous renal replacement therapy suggested that increases in protein and glucose are required to account for the increased losses across the hemofilter. A protein intake of 2.5 g · kg −1 · d −1 appeared to optimize nitrogen balance and correct amino acid deficiencies.

Tuberous Sclerosis Complex Gene Products, Tuberin and Hamartin, Control mTOR Signaling by Acting as a GTPase-Activating Protein Complex toward Rheb

Background: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear.Results: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently.Conclusions: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.

Rats Rapidly Reject Diets Deficient in Essential Amino Acids

Omnivores must obtain diets balanced with respect to amino acids to support growth and protein synthesis. The standard paradigm used to study behavioral responses to amino acid deficiency combines deficient diets with dietary novelty. The objective of this study was to examine the effects of amino acid deficiency on the first meal of rats without the confounding effects of novelty. We report on a series of five studies of feeding behavior in rats. Rats were fed low protein diets for 5-7 d and then exposed to diets with and without essential amino acids. Rats consistently demonstrated recognition of essential amino acid deficiency within the first meal by a significant reduction in first meal duration, rejecting the deficient diets after just 12-16 min exposure. This is the first report of a rapid effect of amino acid-deficient diets without the confounding effects of dietary novelty.

Threonine Deprivation Rapidly Activates the System A Amino Acid Transporter in Primary Cultures of Rat Neurons from the Essential Amino Acid Sensor in the Anterior Piriform Cortex

Omnivores show recognition of essential (indispensable) amino acid deficiency by changing their feeding behavior within 20 min, yet the cellular mechanisms of amino acid sensation in eukaryotes are poorly understood. The anterior piriform cortex (APC) of the brain in rats or its analog in birds likely houses the in vivo amino acid chemosensor. Because amino acid transporters adapt rapidly to essential amino acid deficiency in several cell models, we hypothesized that activation of electrogenic amino acid transport in APC neurons might contribute to the function of the amino acid sensor. We evaluated transport systems in primary cultures of neurons from the APC, hippocampus and cerebellum, or glia, incubated in complete or threonine-devoid (deficient) medium. After 10 min in deficient medium, uptake of threonine or a system A-selective substrate, methyl amino-isobutyric acid, was increased 60% in APC neurons only (P < 0.05). These results demonstrated upregulation of system A, an electrogenic amino acid-sodium symporter. This depletion-induced activation required sodium, intact intracellular trafficking, and phosphorylation of signal transduction-related kinases. Efflux studies showed that other transporter types were functional in the APC; they appeared to be altered dynamically in threonine-deficient cells in response to rapid increases in system A activity. The present data provided support for the chemical sensitivity of the APC and its role as the brain area housing the indispensable amino acid chemosensor. They also showed a region-specific, phosphorylation-dependent activation of the system A transporter in the brain in response to threonine deficiency.

Branched-chain Ketoacyl Dehydrogenase Deficiency: Maple Syrup Disease.

Classic maple syrup disease can be managed to allow a benign neonatal course, normal growth, and low hospitalization rates. The majority of affected infants that are prospectively managed have good neurodevelopmental outcome; however, acute metabolic intoxication and neurologic deterioration can develop rapidly at any age. Each episode is associated with a risk for cerebral edema, cerebrovascular compromise, and brain herniation. High plasma leucine and, possibly, alpha-ketoisocaproate are the principal neurotoxins in maple syrup disease. Plasma levels rise rapidly in association with net protein catabolism provoked by common infections and injuries. Transient periods of maple syrup disease encephalopathy appear fully reversible, leaving no clinically detectable neurologic sequelae. In contrast, prolonged amino acid imbalance, particularly if occurring during the critical period of brain development, leads to neuronal hypoplasia, a paucity of synapses, and undermyelination. Stagnated maturation and inadequate nutritional maintenance of brain structure have lifelong neurologic and behavioral consequences. Core elements of effective long-term therapy include screening and identification of asymptomatic newborns, frequent plasma amino acid monitoring, careful attention to branched-chain amino acid nurtriture, prevention of cerebral essential amino acid deficiencies, adequate provision of essential omega-3 class fatty acids and micronutrients deficient in commercial formulas, methods for home monitoring of metabolic control, and a commitment to lifelong therapy. Recognizing the risk for acute leucine intoxication depends on anticipating effects of common childhood infection and physiologic stresses on whole body protein turnover. Successful management of metabolic decompensation is based on the use of home sick-day regimens, rapid availability of branched-chain amino acid-free hyperalimentation solutions for hospitalized children, prevention of hyponatremia in patients with leucinosis, and frequent adjustments of intravenous therapies guided by plasma amino acid levels and indices of metabolic and clinical response.

Cadmium-induced non-apoptotic cell death mediated by oxidative stress under the condition of sulfhydryl deficiency

Cadmium (Cd), which accumulates primarily in the liver and the kidney, induces apoptosis and also causes necrotic cell death in certain pathophysiologic situations. Previously, we have shown that Cd activated mitogen-activated protein kinases and that sulfur amino acid deficiency potentiated Cd-induced cytotoxicity via activation of mitogen-activated protein kinases. In the present study, we established the mechanistic basis of apoptotic and non-apoptotic cell death induced by Cd in H4IIE cells a rat-derived hepatocyte cell line. Cd at 0.3–10 μM decreased viability of cells in a concentration-dependent manner. Cd-induced cytotoxicity was enhanced by pretreatment with buthionine sulfoximine (BSO). Cd at 0.3 μM induced translocation of Bad to mitochondria, decreased the level of mitochondrial BcL XL with the release of cytochrome c, and induced procaspase-9 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Sulfhydryl deficiency by BSO, however, blocked PARP cleavage in spite of the decrease in procaspase-9. Cytochrome c release, procaspase-9 activation and PARP cleavage were all increased by 1 μM Cd irrespective of BSO pretreatment. We also used H 2O 2 (10–100 μM) as a source of oxidative stress. Cd (0.3–1 μM) + H 2O 2 (70 μM) resulted in greater extents of cytochrome c release, procaspase-9 activation and PARP cleavage in H4IIE cells than Cd alone. Flow cytometric analysis confirmed apoptotic and non-apoptotic cell death by Cd depending on cellular glutathione (GSH) content. These results provide evidence that Cd at the physiologically obtainable concentration causes non-apoptotic cell death under the condition of sufhydryl deficiency, whereas Cd at the micromolar level induces apoptosis. The cell death mechanism involves cytochrome c release from mitochondria and decrease in the level of procaspase-9, but not PARP cleavage, implying that alterations in cellular sulfhydryls may be the major determining factor for the path of cell death in response to low level of Cd.

White muscle free amino acid concentrations following feeding a maize gluten dietary protein in Atlantic salmon ( Salmo salar L.)

This study aimed to investigate the effect of high maize gluten protein diets on the white muscle free amino acid (FAA) concentrations of Atlantic salmon following feeding and to examine whether these tissue free amino acids profiles highlighted any dietary deficiencies in essential amino acids (EAA). Two isocaloric and isonitrogenous diets (52% protein, 20% oil) were formulated and mixed proportionately to give a total of five feeds in which maize gluten provided 0%, 16%, 32%, 48% and 64% of the total dietary protein (diets 0MG, 16MG, 32MG, 48MG, 64MG, respectively). Triplicate groups of 19 g Atlantic salmon were fed each of the experimental diets for a period of 40 days. At the end of the growth experiment, groups of four salmon from each diet were taken at 4, 8 and 12 h after feeding for the white muscle amino acids analysis. Although fish doubled their weights during the growth period, no significant differences were observed in specific growth rates, feed conversion ratios, protein digestibility and protein growth rates among fish fed the test diets. Total free amino acid (FAA) concentrations did not change at various times after a meal and this was also true for the majority of individual FAAs (except asparagine, which increased significantly 12 h after feeding). The most notable diet-induced changes in the white muscle were an overall reduction in essential FAA concentrations (threonine and lysine) as dietary maize gluten content increased and an increase in histidine and leucine. Correlations between dietary essential amino acids patterns and white muscle protein-bound amino acids were examined. The possibility of partial replacement of fish meal maize gluten up to 50% was demonstrated.