Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress.

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNAPro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling. Deletion of GCN2 reduced cell survival to HF whereas pharmacological inhibition of mTORC1 afforded protection. HF treatment of mice synchronously activated the GCN2-mediated ISR and mTORC1 in liver whereas Gcn2-null mice allowed greater mTORC1 activation to HF, resulting in liver steatosis and cell death. We conclude that HF causes an amino acid imbalance that uniquely activates both GCN2 and mTORC1. Loss of GCN2 during HF creates a disconnect between metabolic state and need, triggering proteostasis collapse.

Evaluating Ketosis in Primate Field Studies: Validation of Urine Test Strips in Wild Bornean Orangutans (Pongo pygmaeus wurmbii).

The use of urine test strips (e.g., Roche Chemstrip®) has become the standard for quickly assessing the physiological condition and/or health of wild primates. These strips have been used to detect ketosis as a marker of fat catabolism in several primate taxa in their natural environments in response to changing food availability. However, the use of urine strips to determine ketosis has only been validated in human studies, and thus it remains unclear whether these strips accurately detect and quantify ketone bodies in nonhuman primates. We examined variations in ketone body concentrations in urine samples collected from wild Bornean orangutans at the Tuanan Orangutan Research Station. We assessed the accuracy of qualitative results from Chemstrip test strips in the field (i.e., negative, small, moderate, and large) using an enzyme-linked assay in the laboratory to determine the concentrations of acetoacetate of the same urine samples. Urine samples that tested positive for ketones in the field had significantly higher levels of ketones in the enzymatic assay compared to those that tested negative. There was significant variation in acetoacetate concentrations among the 4 Chemstrip values; however, post hoc tests revealed no significant differences between negative and small samples. We conclude that urinary test strips provide a useful tool for determining ketotic state in wild orangutans, but caution should be taken when interpreting results from samples showing only small levels of ketones on these strips.

The Contribution of Intestinal Gluconeogenesis to Glucose Homeostasis Is Low in 2-Day-Old Pigs.

Background: Active gluconeogenesis is essential to maintain blood glucose concentrations in neonatal piglets because of the high glucose requirements after birth. In several adult mammals, the liver, kidney, and possibly the gut may exhibit gluconeogenesis during fasting and insulinopenic conditions. During the postnatal period, the intestine expresses all of the gluconeogenic enzymes, suggesting the potential for gluconeogenesis. Galactose in milk is a potential gluconeogenic precursor for newborns.Objective: Our aim was to quantify the rate of intestinal glucose production from galactose in piglets compared with the overall rate of glucose production.Methods: A single bolus of [U-14C]-galactose was injected into 2-d-old piglets (females and males; mean ± SEM weight: 1.64 ± 0.07 kg) through a gastric catheter. Galactosemia, glycemia, and glucose turnover rate (assessed by monitoring d-[6-3H]-glucose) were monitored. Intestinal glucose production from [U-14C]-galactose was calculated from [U-14C]-glucose appearance in the blood and isotopic dilution. Galactose metabolism was also investigated in vitro in enterocytes isolated from 2-d-old piglets that were incubated with increasing concentrations of galactose.Results: In piglet enterocytes, galactose metabolism was active (mean ± SEM maximum rate of reaction: 2.26 ± 0.45 nmol · min-1 · 106 cells-1) and predominantly oriented toward lactate and pyruvate production (74.0% ± 14.5%) rather than glucose production (26.0% ± 14.5%). In conscious piglets, gastric galactose administration led to an increase in arterial galactosemia (from 0 to 1.0 ± 0.8 mmol/L) and glycemia (35% ± 12%). The initial increase in arterial glycemia after galactose administration was linked to an increase in glucose production rate (33% ± 15%) rather than to a decrease in glucose utilization rate (3% ± 6%). The contribution of intestinal glucose production from galactose was <10% of total glucose production in 2-d-old piglets.Conclusion: Our results indicate that there is a low contribution to glucose homeostasis from intestinal gluconeogenesis in 2-d-old piglets.

The role of leucine and its metabolites in protein and energy metabolism.

Leucine (Leu) is a nutritionally essential branched-chain amino acid (BCAA) in animal nutrition. It is usually one of the most abundant amino acids in high-quality protein foods. Leu increases protein synthesis through activation of the mammalian target of rapamycin (mTOR) signaling pathway in skeletal muscle, adipose tissue and placental cells. Leu promotes energy metabolism (glucose uptake, mitochondrial biogenesis, and fatty acid oxidation) to provide energy for protein synthesis, while inhibiting protein degradation. Approximately 80 % of Leu is normally used for protein synthesis, while the remainder is converted to α-ketoisocaproate (α-KIC) and ß-hydroxy-ß-methylbutyrate (HMB) in skeletal muscle. Therefore, it has been hypothesized that some of the functions of Leu are modulated by its metabolites. Both α-KIC and HMB have recently received considerable attention as nutritional supplements used to increase protein synthesis, inhibit protein degradation, and regulate energy homeostasis in a variety of in vitro and in vivo models. Leu and its metabolites hold great promise to enhance the growth and health of animals (including humans, birds and fish).

Adipocytes Are the Only Site of Glutamine Synthetase Expression Within the Lactating Mouse Mammary Gland.

Background: Glutamine in milk is believed to play an important role in neonatal intestinal maturation and immune function. For lactating mothers, glutamine utilization is increased to meet the demands of the enlarged intestine and milk production. However, the source of such glutamine during lactation has not been studied. Objectives: We aimed to assess the effects of lactation on the expression of glutamine synthetase (GS) in the mammary gland and other tissues of lactating mice. Methods: Mouse tissues were sampled at 4 time points: 8-wk-old (virgin, control), post-delivery day 5 (PD5, early lactation), PD15 (peak lactation), and involution (4 days after weaning at PD21). We examined the gene expression and protein concentrations of GS and the first 2 enzymes of branched-chain amino acid catabolism: branched-chain aminotransferase 2 (BCAT2) and branched-chain ketoacid dehydrogenase subunit E1α (BCKDHA). Results: The messenger RNA (mRNA) expression and protein concentrations of GS in mammary glands were significantly lower at PD5 and PD15 compared with the control but were restored at involution. Within the mammary gland, GS protein was only detected in adipocytes with no evidence of presence in mammary epithelial cells. Compared with the control, mRNA and protein concentrations of BCAT2 and BCKDHA in mammary glands significantly decreased during lactation and involution. No changes in GS protein concentrations during lactation were found in the liver, skeletal muscle, and lung. In non-mammary adipose tissue, GS protein abundance was higher during lactation compared with the virgin. Conclusions: This work shows that, within the mouse mammary gland, GS is only expressed in adipocytes and that the relative GS abundance in mammary gland sections is lower during lactation. This suggests that mammary adipocytes may be a site of glutamine synthesis in the lactating mouse. Identifying the sources of glutamine production during lactation is important for optimizing milk glutamine concentration to enhance neonatal and maternal health.

Leucing weight with a futile cycle.

The essential amino acid leucine serves as a signal that activates protein synthesis. A new study by She et al. (2007) in this issue of Cell Metabolism shows that raising circulating leucine by blocking leucine breakdown drives a futile cycle of protein synthesis and degradation that contributes to higher-energy expenditure, resistance to dietary obesity, and improved insulin sensitivity.

Characterization of methionine adenosyltransferase 2beta gene expression in skeletal muscle and subcutaneous adipose tissue from obese and lean pigs.

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2beta) encodes for a regulatory subunit (MAT II beta) that regulates the activity of the MAT2A-encoded isoenzyme and intracellular S-adenosylmethionine levels. Our previous work identified MAT2beta as a candidate gene for intramuscular fat (IMF) deposition in porcine skeletal muscle by microarray technology. Here, we cloned porcine MAT2beta cDNA and compared its expression pattern in subcutaneous adipose tissue and skeletal muscle from obese (Rongchang Breed) and lean (Pig Improvement Company, PIC) pigs (n = 6). The porcine MAT2beta cDNA was 1,800 bp long and encodes for 334 amino acids sharing high similarity with other species. MAT2beta is expressed at a higher level in liver and duodenum, followed by the stomach, fat and longissinus dorsi muscle. As expected, both subcutaneous fat content and IMF content were higher in obese than in lean pigs (both P < 0.01). MAT2beta mRNA abundance was lower in both subcutaneous adipose tissue and skeletal muscle in obese pigs compared with lean pigs (both P < 0.01). MAT II beta protein content was lower in skeletal muscle in obese than in lean pigs (P < 0.05), whereas the opposite was observed in subcutaneous adipose tissue (P < 0.01). These data demonstrated an obesity-related expression variation of the MAT II beta subunit in skeletal muscle and adipose tissue in pigs, and suggest a novel role for the MAT2beta gene in regulation of IMF deposition in skeletal muscle.

Equine placenta expresses glutamine synthetase.

In most mammalian species the developing fetus utilizes large amounts of glutamine derived both from the maternal circulation and synthesized de novo in the placenta. The present study was designed to determine the role of the placenta in glutamine synthesis in the horse. The placentae from eight Standardbred mares were sampled immediately after parturition together with additional tissues obtained at necropsy from three Standbred mares during diestrous. Glutamine synthetase protein was detectable in the non-pregnant horn of the placenta in amounts similar to those seen in gluteus muscle, but the amount in the pregnant horn was two times greater than in the non-pregnant horn. Glutamine was the second most abundant amino acid in amniotic fluid at a concentration of 310 +/- 26 micromole/L with that of glycine being 535 +/- 48 micromole/L. The most abundant amino acids in placental tissue were glycine (3,732 +/- 194 micromole/Kg), glutamate (3,500 +/- 343 micromole/Kg) and glutamine (2,836 +/- 208 micromole/Kg). The results illustrate the importance of glutamine to the equine fetus and establish that the placenta, particularly the pregnant horn, has considerable capacity for glutamine synthesis.

Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes.

The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

Glutamine metabolism and function in relation to proline synthesis and the safety of glutamine and proline supplementation.

At normal intakes, dietary glutamine and glutamate are metabolized by the small intestine and essentially all glutamine within the body is synthesized de novo through the action of glutamine synthetase. The major sites of net glutamine synthesis are skeletal muscle, lung, and adipose tissue and, under some conditions, the liver. In addition to the small intestine, where glutamine is the major respiratory fuel, other sites of net glutamine utilization include the cells of the immune system, the kidneys, and the liver. The intestine expresses pyrroline 5-carboxylate (P5C) synthase, which means that proline is an end product of intestinal glutamine catabolism. Proline can also be synthesized from ornithine and the exact contribution of the 2 pathways is not certain. Infusion of proline i.v. to increase circulating concentrations is associated with increased proline oxidation and decreased proline synthesis. In contrast, conditions of proline insufficiency, after feeding low-proline diets or in response to high rates of proline catabolism in burn patients, do not result in increased proline synthesis. Glutamine supplementation is widespread and up to 0.57-0.75 g.kg(-1).d(-1) is well tolerated. Similarly, the only study of proline supplementation, in which patients with gyrate atrophy were given 488 mg.kg(-1).d(-1), reported no deleterious side effects. In the absence of controlled trials, it is currently not possible to estimate a safe upper limit for either of these 2 amino acids.

Distribution of glutamine synthetase and an inverse relationship between glutamine synthetase expression and intramuscular glutamine concentration in the horse.

Glutamine plays important roles in the interorgan transport of nitrogen, carbon and energy but little is known about glutamine metabolism in the horse. In this study we determined the tissue distribution of glutamine synthetase expression in three Standardbred mares. Expression of glutamine synthetase was highest in kidney and mammary gland, and relatively high in liver and adipose tissue. Expression was lower in gluteus muscle, thymus, colon and lung, and much lower in small intestine, pancreas and uterus. The pattern of glutamine synthetase expression in the horse is similar to that of other herbivores and it is likely that skeletal muscle, liver, adipose tissue and lungs are the major sites of net glutamine synthesis in this species. Expression did not differ between adipose tissue depots but did vary between different muscles. Expression was highest in gluteus and semimembranous muscles and much lower in diaphragm and heart muscles. The concentration of intramuscular free glutamine was inversely correlated with expression of glutamine synthetase (r=-0.81, p=0.0017). The concentration of free glutamine was much higher in heart muscle (21.6+/-0.9 micromol/g wet wt) than in gluteus muscle (4.19+0.33 micromol/g wet wt), which may indicate novel functions and/or regulatory mechanisms for glutamine in the equine heart.

Protein.

Proteins are polymers of amino acids linked via α-peptide bonds. They can be represented as primary, secondary, tertiary, and even quaternary structures, but from a nutritional viewpoint only the primary (amino acid) sequence is of interest. Similarly, although there are many compounds in the body that can be chemically defined as amino acids, we are only concerned with the 20 canonical amino acids encoded in DNA, plus 5 others-ornithine, citrulline, γ-aminobutyrate, ß-alanine, and taurine-that play quantitatively important roles in the body. We consume proteins, which are digested in the gastrointestinal tract, absorbed as small peptides (di- and tripeptides) and free amino acids, and then used for the resynthesis of proteins in our cells. Additionally, some amino acids are also used for the synthesis of specific (nonprotein) products, such as nitric oxide, polyamines, creatine, glutathione, nucleotides, glucosamine, hormones, neurotransmitters, and other factors. Again, such functions are not quantitatively important for most amino acids, and the bulk of amino acid metabolism is directly related to protein turnover (synthesis and degradation). For an individual in nitrogen balance, an amount of protein equal to that of the daily protein (nitrogen) intake is degraded each day with the nitrogen being excreted as urea and ammonia (with limited amounts of creatinine and uric acid). The carbon skeletons of the amino acids degraded to urea and ammonia are recovered through gluconeogenesis or ketone synthesis, or oxidized to carbon dioxide. Of the 20 amino acids present in proteins, 9 are considered nutritionally indispensable (essential) in adult humans because the body is not able to synthesize their carbon skeletons. These 9 amino acids are leucine, valine, isoleucine, histidine, lysine, methionine, threonine, tryptophan, and phenylalanine. In addition, 2 others are made from their indispensable precursors: cysteine from methionine, and tyrosine from phenylalanine. Although arginine is needed in neonates, it appears that adults, with the possible exceptions of pregnancy in females and spermatogenesis in males, can synthesize sufficient arginine to maintain a nitrogen balance. The others, glutamate, glutamine, aspartate, asparagine, serine, glycine, proline, and alanine, can all be synthesized from glucose and a suitable nitrogen source. Under some conditions, glutamine, glutamate, glycine, proline, and arginine may be considered as conditionally indispensable, meaning that the body is not capable of synthesizing them in sufficient quantities for a specific physiologic or pathologic condition (1). Thus, any discussion of dietary protein must consider not only quantity but also quality (ratio of indispensable amino acids).

Lowered concentrations of branched-chain amino acids result in impaired growth and neurological problems: insights from a branched-chain alpha-keto acid dehydrogenase complex kinase-deficient mouse model.

Excess circulating levels of branched-chain amino acids (BCAA), as seen in maple syrup urine disease, result in severe neuropathology. A new mouse model, deficient in the kinase that controls BCAA catabolism, shows that very low circulating levels of BCAA are also associated with neuropathology, including the development of epileptic seizures. These mice clearly demonstrate the need to control essential amino acid levels within both upper and lower limits.

Novel findings regarding Glut-4 expression in adipose tissue and muscle in horses--a preliminary report.

One of the hallmarks of insulin resistance is a reduction in glucose transporter-4 (Glut-4) expression in adipose tissue but not in skeletal muscle. However, while Glut-4 has been demonstrated in skeletal and cardiac muscles in horses it has not been demonstrated in adipose tissue. The initial objectives of the present study were: (1) to test the hypothesis that Glut-4 expression would vary between selected key skeletal muscles; (2) to test the hypothesis that it would also vary between representative adipose tissue depots, and (3) to see whether expression would be greater in adipose tissue compared to muscle. Glut-4 expression was determined by Western blot using samples obtained from post mortem biopsies obtained from four muscles (gluteus medius, semitendinosus, heart, and diaphragm), and four adipose tissues (subcutaneous, retroperitoneal, mesenteric, and omental) in three horses. There were no differences (P>0.05) in Glut-4 protein expression between the muscles sampled. Likewise there were no differences (P>0.05) in Glut-4 protein expression between fat depots. There was a significant difference (P=0.03) when pooled means for Glut-4 expression in muscle (58.8+/-2.5 densitometry units) were compared with adipose tissue (115.8+/-15.7). This difference in Glut-4 expression in these two tissues with distinctly different metabolic reasons for taking up glucose may warrant further investigation to see if there are more pronounced differences in Glut-4 expression in muscle and adipose tissue in various populations of horses.

High fat diet enriched with saturated, but not monounsaturated fatty acids adversely affects femur, and both diets increase calcium absorption in older female mice.

Diet induced obesity has been shown to reduce bone mineral density (BMD) and Ca absorption. However, previous experiments have not examined the effect of high fat diet (HFD) in the absence of obesity or addressed the type of dietary fatty acids. The primary objective of this study was to determine the effects of different types of high fat feeding, without obesity, on fractional calcium absorption (FCA) and bone health. It was hypothesized that dietary fat would increase FCA and reduce BMD. Mature 8-month-old female C57BL/6J mice were fed one of three diets: a HFD (45% fat) enriched either with monounsaturated fatty acids (MUFAs) or with saturated fatty acids (SFAs), and a normal fat diet (NFD; 10% fat). Food consumption was controlled to achieve a similar body weight gain in all groups. After 8wk, total body bone mineral content and BMD as well as femur total and cortical volumetric BMD were lower in SFA compared with NFD groups (P<.05). In contrast, femoral trabecular bone was not affected by the SFAs, whereas MUFAs increased trabecular volume fraction and thickness. The rise over time in FCA was greater in mice fed HFD than NFD and final FCA was higher with HFD (P<.05). Intestinal calbindin-D9k gene and hepatic cytochrome P450 2r1 protein levels were higher with the MUFA than the NFD diet (P<.05). In conclusion, HFDs elevated FCA overtime; however, an adverse effect of HFD on bone was only observed in the SFA group, while MUFAs show neutral or beneficial effects.

Is the small intestine a gluconeogenic organ.

Gluconeogenesis is responsible for the maintenance of blood glucose levels as hepatic glycogen stores become depleted. Traditionally, only liver and kidney have been believed to be capable of gluconeogenesis, but a gluconeogenic capacity for the small intestine has recently been proposed. This possibility is supported by the expression of key gluconeogenic enzymes and radiolabeled tracer experiments, but these data are not unequivocal and alternative roles can explain the presence of gluconeogenic enzymes in this organ.

Glutamine metabolism in uricotelic species: variation in skeletal muscle glutamine synthetase, glutaminase, glutamine levels and rates of protein synthesis.

High intracellular glutamine levels have been implicated in promoting net protein synthesis and accretion in mammalian skeletal muscle. Little is known regarding glutamine metabolism in uricotelic species but chicken breast muscle exhibits high rates of protein accretion and would be predicted to maintain high glutamine levels. However, chicken breast muscle expresses high glutaminase activity and here we report that chicken breast muscle also expresses low glutamine synthetase activity (0.07+/-0.01 U/g) when compared to leg muscle (0.50+/-0.04 U/g). Free glutamine levels were 1.38+/-0.09 and 9.69+/-0.12 nmol/mg wet weight in breast and leg muscles of fed chickens, respectively. Glutamine levels were also lower in dove breast muscle (4.82+/-0.35 nmol/mg wet weight) when compared to leg muscle (16.2+/-1.0 nmol/mg wet weight) and much lower (1.80+/-0.46 nmol/mg wet weight) in lizard leg muscle. In fed chickens, rates of fractional protein synthesis were higher in leg than in breast muscle, and starvation (48 h) resulted in a decrease in both glutamine content and rate of protein synthesis in leg muscle. Thus, although tissue-specific glutamine metabolism in uricotelic species differs markedly from that in ureotelic animals, differences in rates of skeletal muscle protein synthesis are associated with corresponding differences in intramuscular glutamine content.

Glutamine and glutamate: Nonessential or essential amino acids?

Glutamine and glutamate are not considered essential amino acids but they play important roles in maintaining growth and health in both neonates and adults. Although glutamine and glutamate are highly abundant in most feedstuffs there is increasing evidence that they may be limiting during pregnancy, lactation and neonatal growth, particularly when relatively low protein diets are fed. Supplementation of diets with glutamine, glutamate or both at 0.5 to 1.0% to both suckling and recently weaned piglets improves intestinal and immune function and results in better growth. In addition such supplementation to the sow prevents some of the loss of lean body mass during lactation, and increases milk glutamine content. However, a number of important questions related to physiological condition, species under study and the form and amount of the supplements need to be addressed before the full benefits of glutamine and glutamate supplementation in domestic animal production can be realized.

Functions of Antimicrobial Peptides in Gut Homeostasis.

Antimicrobial peptides (AMPs), produced by several species including bacteria, insects, amphibians and mammals as well as by chemical synthesis and genetically engineered microorganisms, are of great importance in maintaining normal gut homeostasis. AMPs exhibit a broad spectrum of antimicrobial activity and inhibit microbial cells by interaction with their membranes or by other mechanisms, such as inhibition of cell-wall synthesis or suppression of nucleic acid or protein synthesis. In addition to their direct antimicrobial functions, they have multiple roles in the stabilization of epithelial barrier integrity and function as potent immune regulators. The fate of AMPs in vivo is poorly understood, prompting the need for studying AMPs pharmacokinetics. This review summarizes the current knowledge about the basic biology of AMPs and discusses the features of AMPs in gut homeostasis and their relative mechanisms of action.

Not all injury-induced muscle proteolysis is due to increased activity of the ubiquitin/proteasome system: evidence for up-regulation of macrophage-associated lysosomal proteolysis in a model of local trauma.

A characteristic response to injury is a dramatic loss of skeletal muscle protein owing to increased muscle protein breakdown. Over the past decade, numerous studies have indicated that up-regulation of the ubiquitin-proteasome system is a common mechanism underlying such injury-induced muscle proteolysis. However, a recent study using a single-impact trauma to the gastrocnemius muscle found that, although the rate of muscle proteolysis was dramatically increased, the ubiquitin-proteasome system was not involved. Rather, an increase in lysosomal activity, through infiltration of the damaged tissue by mononuclear macrophages, is responsible for the high rates of protein breakdown.