Atypical antipsychotics rapidly and inappropriately switch peripheral fuel utilization to lipids, impairing metabolic flexibility in rodents.

Patients taking atypical antipsychotics are frequented by serious metabolic (eg, hyperglycemia, obesity, and diabetes) and cardiac effects. Surprisingly, chronic treatment also appears to lower free fatty acids (FFAs). This finding is paradoxical because insulin resistance is typically associated with elevated not lower FFAs. How atypical antipsychotics bring about these converse changes in plasma glucose and FFAs is unknown. Chronic treatment with olanzapine, a prototypical, side effect prone atypical antipsychotic, lowered FFA in Sprague-Dawley rats. Olanzapine also lowered plasma FFA acutely, concomitantly impairing in vivo lipolysis and robustly elevating whole-body lipid oxidation. Increased lipid oxidation was evident from accelerated losses of triglycerides after food deprivation or lipid challenge, elevated FFA uptake into most peripheral tissues (∼2-fold) except heart, rises in long-chain 3-hydroxylated acyl-carnitines observed in diabetes, and rapid suppression of the respiratory exchange ratio (RER) during the dark cycle. Normal rises in RER following refeeding, a sign of metabolic flexibility, were severely blunted by olanzapine. Increased lipid oxidation in muscle could be explained by ∼50% lower concentrations of the negative cytoplasmic regulator of carnitine palmitoyltransferase I, malonyl-CoA. This was associated with loss of anapleurotic metabolites and citric acid cycle precursors of malonyl-CoA synthesis rather than adenosine monophosphate-activated kinase activation or direct ACC1/2 inhibition. The ability of antipsychotics to lower dark cycle RER in mice corresponded to their propensities to cause metabolic side effects. Our studies indicate that lipocentric mechanisms or altered intermediary metabolism could underlie the FFA lowering and hyperglycemia (Randle cycle) as well as some of the other side effects of atypical antipsychotics, thereby suggesting strategies for alleviating them.

Overexpression of ornithine decarboxylase decreases ventricular systolic function during induction of cardiac hypertrophy.

Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to ß-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to ß-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.

Molecular characterization of skeletal muscle atrophy in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD), a neurodegenerative disorder caused by mutant huntingtin, is characterized by a catabolic phenotype. To determine the mechanisms underlying muscle wasting, we examined key signal transduction pathways governing muscle protein metabolism, apoptosis, and autophagy in R6/2 mice, a well-characterized transgenic model of HD. R6/2 mice exhibited increased adiposity, elevated energy expenditure, and decreased body weight and lean mass without altered food intake. Severe skeletal muscle wasting accounted for a majority of the weight loss. Protein synthesis was unexpectedly increased 19% in gastrocnemius muscle, which was associated with overactivation of basal and refeeding-stimulated mammalian target of rapamycin (mTOR) signaling, elevated Akt expression and Ser(473) phosphorylation, and decreased AMPK Thr(172) phosphorylation. Moreover, mRNA abundance of atrogenes muscle ring finger-1 and atrophy F-box, was markedly attenuated during fasting and refeeding, and the urinary excretion of 3-methylhistidine was decreased, arguing against a role for the ubiquitin proteasome-mediated proteolysis in the atrophy. In contrast, mRNA expression of several caspase genes and genes involved in the extrinsic or intrinsic apoptotic pathway, caspase-3/7, -8, and -9 activity, protein abundance of caspase-3 and -9, Fas, and Fadd, and cytochrome c release were elevated. Protein expressions of LC3B-I and -II, beclin-I, and atg5 and -7 in muscle were upregulated. Thus, mutant huntingtin in skeletal muscle results in increased protein synthesis and mTOR signaling, which is countered by activation of the apoptotic and autophagic pathways, contributing to an overall catabolic phenotype and the severe muscle wasting.

Functional proteomic analysis reveals sex-dependent differences in structural and energy-producing myocardial proteins in rat model of alcoholic cardiomyopathy.

Long-term ethanol exposure leads to a sexually dimorphic response in both the susceptibility to cardiac pathology (protective effect of the female heart) and the expression of selected myocardial proteins. The purpose of the present study was to use proteomics to examine the effect of chronic alcohol consumption on a broader array of cardiac proteins and how these were affected between the sexes. Male and female rats were maintained for 18 wk on a 40% ethanol-containing diet in which alcohol was provided in drinking water and agar blocks. Differences in the content of specific cardiac proteins in isopycnic centrifugal fractions were determined using mass spectrometry on iTRAQ-labeled tryptic fragments. A random effects model of meta-analysis was developed to combine the results from multiple iTRAQ experiments. Analysis of a network of proteins involved in cardiovascular system development and function showed that troponins were oppositely regulated by alcohol exposure in females (upregulated) vs. males (downregulated), and this effect was validated by Western blot analysis. Pathway analysis also revealed that alcohol-consuming males showed increased expression of proteins involved in various steps of oxidative phosphorylation including complexes I, III, IV, and V, whereas females showed no change or decreased content. One implication from these findings is that females may be protected from the toxic effects of alcohol due to their ability to maintain contractile function, maintain efficiency of force generation, and minimize oxidative stress. However, the alcohol-induced insult may lead to increased production of reactive oxygen species and structural abnormalities in male myocardium.

Inhibition of glycogen synthase kinase 3[beta] activity with lithium in vitro attenuates sepsis-induced changes in muscle protein turnover.

Loss of lean body mass is a characteristic feature of the septic response, and the mechanisms responsible for this decrease and means of prevention have not been fully elucidated. The present study tested the hypothesis that in vitro treatment of skeletal muscle with lithium chloride (LiCl), a glycogen synthase kinase (GSK) 3 inhibitor, would reverse both the sepsis-induced increase in muscle protein degradation and inhibition of protein synthesis. Sepsis decreased GSK-3[beta] phosphorylation and increased GSK-3[beta] activity, under basal conditions. Sepsis increased muscle protein degradation, with a concomitant increase in atrogin 1 and MuRF1 mRNA and 26S proteosome activity. Incubation of septic muscle with LiCl completely reversed the increased GSK-3[beta] activity and decreased proteolysis to basal nonseptic values, but only partially reduced proteosome activity and did not diminish atrogene expression. Lithium chloride also did not ameliorate the sepsis-induced increase in LC3-II, a marker for activated autophagy. In contrast, LiCl increased protein synthesis only in nonseptic control muscle. The inability of septic muscle to respond to LiCl was independent of its ability to reverse the sepsis-induced increase in eukaryotic initiation factor (eIF) 2B[varepsilon] phosphorylation, decreased eIF2B activity, or the reduced phosphorylation of FOXO3, but instead was more closely associated with the continued suppression of mTOR (mammalian target of rapamycin) kinase activity (e.g., reduced phosphorylation of 4E-BP1 and S6). These data suggest that in vitro lithium treatment, which inhibited GSK-3[beta] activity, (a) effectively reversed the sepsis-induced increase in proteolysis, but only in part by a reduction in the ubiquitin-proteosome pathway and not by a reduction in autophagy; and (b) was ineffective at reversing the sepsis-induced decrease in muscle protein synthesis. This lithium-resistant state seems mediated at the level of mTOR and not eIF2/eIF2B. Hence, use of GSK-3[beta] inhibitors in the treatment of sepsis may not be expected to fully correct the imbalance in muscle protein turnover.

BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival in septic mice.

Endotoxin (LPS) and sepsis decrease mammalian target of rapamycin (mTOR) activity in skeletal muscle, thereby reducing protein synthesis. Our study tests the hypothesis that inhibition of branched-chain amino acid (BCAA) catabolism, which elevates circulating BCAA and stimulates mTOR, will blunt the LPS-induced decrease in muscle protein synthesis. Wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout mice were studied 4 h after Escherichia coli LPS or saline. Basal skeletal muscle protein synthesis was increased in knockout mice compared with WT, and this change was associated with increased eukaryotic initiation factor (eIF)-4E binding protein-1 (4E-BP1) phosphorylation, eIF4E.eIF4G binding, 4E-BP1.raptor binding, and eIF3.raptor binding without a change in the mTOR.raptor complex in muscle. LPS decreased muscle protein synthesis in WT mice, a change associated with decreased 4E-BP1 phosphorylation as well as decreased formation of eIF4E.eIF4G, 4E-BP1.raptor, and eIF3.raptor complexes. In BCATm knockout mice given LPS, muscle protein synthesis only decreased to values found in vehicle-treated WT control mice, and this ameliorated LPS effect was associated with a coordinate increase in 4E-BP1.raptor, eIF3.raptor, and 4E-BP1 phosphorylation. Additionally, the LPS-induced increase in muscle cytokines was blunted in BCATm knockout mice, compared with WT animals. In a separate study, 7-day survival and muscle mass were increased in BCATm knockout vs. WT mice after polymicrobial peritonitis. These data suggest that elevating blood BCAA is sufficient to ameliorate the catabolic effect of LPS on skeletal muscle protein synthesis via alterations in protein-protein interactions within mTOR complex-1, and this may provide a survival advantage in response to bacterial infection.

Ectopic expression of eIF2Bepsilon in rat skeletal muscle rescues the sepsis-induced reduction in guanine nucleotide exchange activity and protein synthesis.

Eukaryotic initiation factor 2B (eIF2B) is a guanine nucleotide exchange factor (GEF) whose activity is both tightly regulated and rate-controlling with regard to global rates of protein synthesis. Skeletal muscle eIF2B activity and expression of its catalytic epsilon-subunit (eIF2Bepsilon) have been implicated as potential contributors to the altered rates of protein synthesis in a number of physiological conditions and experimental models. The objective of this study was to directly examine the effects of exogenously expressed eIF2Bepsilon in vivo on GEF activity and protein synthetic rates in rat skeletal muscle. A plasmid encoding FLAG-eIF2Bepsilon was transfected into the tibialis anterior (TA) of one leg, while the contralateral TA received a control plasmid. Ectopic expression of eIF2Bepsilon resulted in increased GEF activity in TA homogenates of healthy rats, demonstrating that the expressed protein was catalytically active. In an effort to restore a deficit in eIF2B activity, we utilized an established model of chronic sepsis in which skeletal muscle eIF2B activity is known to be impaired. Ectopic expression of eIF2Bepsilon in the TA rescued the sepsis-induced deficit in GEF activity and muscle protein synthesis. The results demonstrate that modulation of eIF2Bepsilon expression may be sufficient to correct deficits in skeletal muscle protein synthesis associated with sepsis and other muscle-wasting conditions.

Impact of chronic alcohol ingestion on cardiac muscle protein expression.

BACKGROUND: Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. METHODS: The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT). Following the reaction with the ICAT reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. RESULTS: Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. CONCLUSIONS: Based on the changes in proteins, we speculate modulation of cardiac muscle protein expression represents a fundamental alteration induced by chronic alcohol consumption, consistent with changes in myocardial wall thickness measured under the same conditions.

Skeletal muscle protein balance in mTOR heterozygous mice in response to inflammation and leucine.

Sepsis and lipopolysaccharide (LPS) may decrease skeletal muscle protein synthesis by impairing mTOR (mammalian target of rapamycin) activity. The role of mTOR in regulating muscle protein synthesis was assessed in wild-type (WT) and mTOR heterozygous (+/-) mice under basal conditions and in response to LPS and/or leucine stimulation. No difference in body weight of mTOR(+/-) mice was observed compared with WT mice; whereas whole body lean body mass was reduced. Gastrocnemius weight was decreased in mTOR(+/-) mice, which was attributable in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and mTOR(+/-) mice to the same extent. Reduced muscle protein synthesis in mTOR(+/-) mice under basal and LPS-stimulated conditions was associated with lower 4E-BP1 and S6K1 phosphorylation. LPS also decreased PRAS40 phosphorylation and increased phosphorylation of raptor and IRS-1 (Ser(307)) to the same extent in WT and mTOR(+/-) mice. Muscle atrogin-1 and MuRF1 mRNA content was elevated in mTOR(+/-) mice under basal conditions, implying increased ubiquitin-proteasome-mediated proteolysis, but the LPS-induced increase in these atrogenes was comparable between groups. Plasma insulin and IGF-I as well as tissue expression of TNFalpha, IL-6, or NOS2 did not differ between WT and mTOR(+/-) mice. Finally, whereas LPS impaired the ability of leucine to stimulate muscle protein synthesis and 4E-BP1 phosphorylation in WT mice, this inflammatory state rendered mTOR(+/-) mice leucine unresponsive. These data support the idea that the LPS-induced reduction in mTOR activity is relatively more important in regulating skeletal muscle mass in response to nutrient stimulation than under basal conditions.

Alcohol-induced IGF-I resistance is ameliorated in mice deficient for mitochondrial branched-chain aminotransferase.

Acute alcohol intoxication decreases skeletal muscle protein synthesis by impairing mammalian target of rapamycin (mTOR). In 2 studies, we determined whether inhibition of branched-chain amino acid (BCAA) catabolism ameliorates the inhibitory effect of alcohol on muscle protein synthesis by raising the plasma BCAA concentrations and/or by improving the anabolic response to insulin-like growth factor (IGF)-I. In the first study, 4 groups of mice were used: wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout (KO) mice orally administered saline or alcohol (5 g/kg, 1 h). Protein synthesis was greater in KO mice compared with WT controls and was associated with greater phosphorylation of eukaryotic initiation factor (eIF)-4E binding protein-1 (4EBP1), eIF4E-eIF4G binding, and 4EBP1-regulatory associated protein of mTOR (raptor) binding, but not mTOR-raptor binding. Alcohol decreased protein synthesis in WT mice, a change associated with less 4EBP1 phosphorylation, eIF4E-eIF4G binding, and raptor-4EBP1 binding, but greater mTOR-raptor complex formation. Comparable alcohol effects on protein synthesis and signal transduction were detected in BCATm KO mice. The second study used the same 4 groups, but all mice were injected with IGF-I (25 microg/mouse, 30 min). Alcohol impaired the ability of IGF-I to increase muscle protein synthesis, 4EBP1 and 70-kilodalton ribosomal protein S6 kinase-1 phosphorylation, eIF4E-eIF4G binding, and 4EBP1-raptor binding in WT mice. However, in alcohol-treated BCATm KO mice, this IGF-I resistance was not manifested. These data suggest that whereas the sustained elevation in plasma BCAA is not sufficient to ameliorate the catabolic effect of acute alcohol intoxication on muscle protein synthesis, it does improve the anabolic effect of IGF-I.

Regulation of TNF-induced oxygen radical production in human neutrophils: role of delta-PKC.

In human neutrophils, TNF-elicited O(2)(-) production requires adherence and integrin activation. How this cooperative signaling between TNFRs and integrins regulates O(2)(-) generation has yet to be fully elucidated. Previously, we identified delta-PKC as a critical early regulator of TNF signaling in adherent neutrophils. In this study, we demonstrate that inhibition of delta-PKC with a dominant-negative delta-PKC TAT peptide resulted in a significant delay in the onset time of TNF-elicited O(2)(-) generation but had no effect on Vmax, indicating an involvement of delta-PKC in the initiation of O(2)(-) production. In contrast, fMLP-elicited O(2)(-) production in adherent and nonadherent neutrophils was delta-PKC-independent, suggesting differential regulation of O(2)(-) production. An important step in activation of the NADPH oxidase is phosphorylation of the cytosolic p47phox component. In adherent neutrophils, TNF triggered a time-dependent association of delta-PKC with p47phox, which was associated with p47phox phosphorylation, indicating a role for delta-PKC in regulating O(2)(-) production at the level of p47phox. Activation of ERK and p38 MAPK is also required for TNF-elicited O(2)(-) generation. TNF-mediated ERK but not p38 MAPK recruitment to p47phox was delta-PKC-dependent. delta-PKC activity is controlled through serine/threonine phosphorylation, and phosphorylation of delta-PKC (Ser643) and delta-PKC (Thr505) was increased significantly by TNF in adherent cells via a PI3K-dependent process. Thus, signaling for TNF-elicited O(2)(-) generation is regulated by delta-PKC. Adherence-dependent cooperative signaling activates PI3K signaling, delta-PKC phosphorylation, and delta-PKC recruitment to p47phox. delta-PKC activates p47phox by serine phosphorylation or indirectly through control of ERK recruitment to p47phox.

Castration differentially alters basal and leucine-stimulated tissue protein synthesis in skeletal muscle and adipose tissue.

Reduced testosterone as a result of catabolic illness or aging is associated with loss of muscle and increased adiposity. We hypothesized that these changes in body composition occur because of altered rates of protein synthesis under basal and nutrient-stimulated conditions that are tissue specific. The present study investigated such mechanisms in castrated male rats (75% reduction in testosterone) with demonstrated glucose intolerance. Over 9 wk, castration impaired body weight gain, which resulted from a reduced lean body mass and preferential sparing of adipose tissue. Castration decreased gastrocnemius weight, but this atrophy was not associated with reduced basal muscle protein synthesis or differences in plasma IGF-I, insulin, or individual amino acids. However, oral leucine failed to normally stimulate muscle protein synthesis in castrated rats. In addition, castration-induced atrophy was associated with increased 3-methylhistidine excretion and in vitro-determined ubiquitin proteasome activity in skeletal muscle, changes that were associated with decreased atrogin-1 or MuRF1 mRNA expression. Castration decreased heart and kidney weight without reducing protein synthesis and did not alter either cardiac output or glomerular filtration. In contradistinction, the weight of the retroperitoneal fat depot was increased in castrated rats. This increase was associated with an elevated rate of basal protein synthesis, which was unresponsive to leucine stimulation. Castration also decreased whole body fat oxidation. Castration increased TNFα, IL-1α, IL-6, and NOS2 mRNA in fat but not muscle. In summary, the castration-induced muscle wasting results from an increased muscle protein breakdown and the inability of leucine to stimulate protein synthesis, whereas the expansion of the retroperitoneal fat depot appears mediated in part by an increased basal rate of protein synthesis-associated increased inflammatory cytokine expression.

Leucine supplementation of drinking water does not alter susceptibility to diet-induced obesity in mice.

Branched-chain amino acids (BCAA), Leu, and the signaling pathways they regulate have been reported to either improve or worsen adiposity and insulin sensitivity. Therefore, it is unclear whether dietary supplementation of Leu would be beneficial. To help address this question, we examined the effect of adding Leu (150 mmol/L; Expt. 1 and Expt. 2) or BCAA (109 mmol/L of each; Expt. 3) to the drinking water on diet-induced obesity (induced with a 60-kJ% fat diet) in singly housed C57BL6/J male mice for at least 14 wk. Liquid and solid food intakes were evaluated weekly along with body weight. During the last few weeks, several blood samples were taken at different times for plasma glucose, total cholesterol, or Leu measurements. Metabolic rate by indirect calorimetry, locomotor activity by light beam breaking, body composition by H1-NMR, and insulin tolerance were also determined. Compared with control, supplementation did not affect body weight, food intake, oxygen consumption, locomotor activity, body composition, insulin tolerance, or total cholesterol. In fed mice, this method of Leu supplementation only increased plasma Leu by 76% when the supplemented group was compared with control. On the other hand, after overnight food deprivation, the plasma Leu did not differ between these 2 groups, even though the mice in the supplemented group had continuous access to Leu-containing water during the solid food deprivation. Taken together, the results do not provide evidence that either Leu or BCAA supplementation of drinking water ameliorates diet-induced obesity in mice, although it may improve glycemia.

Alcohol-induced decrease in muscle protein synthesis associated with increased binding of mTOR and raptor: Comparable effects in young and mature rats.

BACKGROUND: Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation. However, these studies have been performed in relatively young rapidly growing rats in which muscle protein accretion is more sensitive to growth factor and nutrient stimulation. Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner. The hypothesis tested in the present study was that young rats will show a more pronounced decrement in muscle protein synthesis than older mature rats in response to acute EtOH intoxication. METHODS: Male F344 rats were studied at approximately 3 (young) or 12 (mature) months of age. Young rats were injected intraperitoneally with 75 mmol/kg of EtOH, and mature rats injected with either 75 or 90 mmol/kg EtOH. Time-matched saline-injected control rats were included for both age groups. Gastrocnemius protein synthesis and the activity of the mTOR pathway were assessed 2.5 h after EtOH using [³H]-labeled phenylalanine and the phosphorylation of various protein factors known to regulate peptide-chain initiation. RESULTS: Blood alcohol levels (BALs) were lower in mature rats compared to young rats after administration of 75 mmol/kg EtOH (154 ± 23 vs 265 ± 24 mg/dL). However, injection of 90 mmol/kg EtOH in mature rats produced BALs comparable to that of young rats (281 ± 33 mg/dL). EtOH decreased muscle protein synthesis similarly in both young and high-dose EtOH-treated mature rats. The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E.eIF4G and inactive eIF4E.4EBP1 complex. Moreover, EtOH increased the binding of mTOR with raptor in a manner which appeared to be AMPK- and TSC-independent. In contrast, although muscle protein synthesis was unchanged in mature rats given low-dose EtOH, compared to control values, the phosphorylation of rpS6 and eIF4G was decreased. CONCLUSION: These data indicate that muscle protein synthesis is equally sensitive to the inhibitory effects of EtOH in young rapidly growing rats and older mature rats which are growing more slowly, but that mature rats must be given a relatively larger dose of EtOH to achieve the same BAL. Based on the differential response in mature rats to low- and high-dose EtOH, the decreased protein synthesis was associated with a reduction in mTOR activity which was selectively mediated via a reduction in 4E-BP1 phosphorylation and an increase in mTOR.raptor formation.

Activation of AMP-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside prevents leucine-stimulated protein synthesis in rat skeletal muscle.

Several stress conditions are characterized by activation of 5'-AMP-activated protein kinase (AMPK) and the development of leucine resistance in skeletal muscle. In the present study, we determined whether direct activation of the AMPK by 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) prevents the characteristic leucine-induced increase in protein synthesis by altering mammalian target of rapamycin (mTOR) signal transduction. Rats were injected with AICAR or saline (Sal) and 1 h thereafter received an oral gavage of leucine (or Sal). Efficacy of AICAR was verified by increased AMPK phosphorylation. AICAR decreased basal in vivo muscle (gastrocnemius) protein synthesis and completely prevented the leucine-induced increase, independent of a change in muscle adenine nucleotide concentration. AICAR also prevented the hyperphosphorylation of eukaryotic initiation factor (eIF) 4E binding protein (4E-BP1), ribosomal protein S6 kinase (S6K1), S6, and eIF4G in response to leucine, suggesting a decrease in mTOR activity. Moreover, AICAR prevented the leucine-induced redistribution of eIF4E from the inactive eIF4E.4E-BP1 to the active eIF4E.eIF4G complex. This ability of AICAR to produce muscle leucine resistance could not be attributed to a change in phosphorylation of tuberous sclerosis complex (TSC)2, the formation of a TSC1.TSC2 complex, the binding of raptor with mTOR, or the phosphorylation of eukaryotic elongation factor-2. However, the inhibitory actions of AICAR were associated with reduced phosphorylation of proline-rich Akt substrate-40 and increased phosphorylation of raptor, which represent potential mechanisms by which AICAR might be expected to inhibit leucine-induced increases in mTOR activity and protein synthesis under in vivo conditions.

Skeletal and cardiac myopathy in HIV-1 transgenic rats.

The mechanism by which human immunodeficiency virus (HIV)-1 infection in humans leads to the erosion of lean body mass is poorly defined. Therefore, the purpose of the present study was to determine whether transgenic (Tg) rats that constitutively overexpress HIV-1 viral proteins exhibit muscle wasting and to elucidate putative mechanisms. Over 7 mo, Tg rats gained less body weight than pair-fed controls exclusively as a result of a proportional reduction in lean, not fat, mass. Fast- and slow-twitch muscle atrophy in Tg rats did not result from a reduction in the in vivo-determined rate of protein synthesis. In contrast, urinary excretion of 3-methylhistidine, as well as the content of atrogin-1 and the 14-kDa actin fragment, was elevated in gastrocnemius of Tg rats, suggesting increased muscle proteolysis. Similarly, Tg rats had reduced cardiac mass, which was independent of a change in protein synthesis. This decreased cardiac mass was associated with a reduction in stroke volume, but cardiac output was maintained by a compensatory increase in heart rate. The HIV-induced muscle atrophy was associated with increased whole body energy expenditure, which was not due to an elevated body temperature or secondary bacterial infection. Furthermore, the atrophic response could not be attributed to the development of insulin resistance, decreased levels of circulating amino acids, or increased tissue cytokines. However, skeletal muscle and, to a lesser extent, circulating insulin-like growth factor I was reduced in Tg rats. Although hepatic injury was implicated by increased plasma levels of aspartate and alanine aminotransferases, hepatic protein synthesis was not different between control and Tg rats. Hence, HIV-1 Tg rats develop atrophy of cardiac and skeletal muscle, the latter of which results primarily from an increased protein degradation and may be related to the marked reduction in muscle insulin-like growth factor I.

Partial dissociation of TSC2 and mTOR phosphorylation in cardiac and skeletal muscle of rats in vivo.

Insulin promotes protein accretion in cardiac and skeletal muscles through a stimulation of the mRNA translation initiation phase of protein synthesis. The present set of experiments examined the regulatory TSC2 signaling pathway that potentially contributes to the myocardial responsiveness of protein synthesis to insulin in post-absorptive male Sprague-Dawley rats in vivo. Heart and skeletal muscles were sampled from rats up to 1 h following intravenous injection of various doses of insulin. In cardiac muscle, TSC2 phosphorylation was elevated only at the highest plasma insulin concentration (386 ng/ml). In contrast, the extent of mTOR phosphorylation either on Ser((2448)) or Ser((2481)) was raised at 24-fold less concentration of insulin and corresponded with increased phosphorylation of PKB(Thr(308)) or PKB(Ser(473)). In gastrocnemius, TSC2 phosphorylation was elevated at plasma insulin concentrations (16 ng/ml) lower than that observed in cardiac muscle (386 ng insulin/ml). The increased TSC2 phosphorylation corresponded with a marked stimulation of PKB phosphorylation. However, mTOR(Ser(2448)) or mTOR(Ser(2481)) phosphorylation was not elevated until the plasma insulin concentration reached 97 ng/ml. The results indicate there is a dissociation of TSC2 and mTOR phosphorylation in vivo.

Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle.

Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., "atrogenes"). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss of muscle mass/protein in response to chronic alcohol abuse appears to result primarily from a decrement in muscle protein synthesis, not an increase in degradation.

Acute alcohol intoxication increases REDD1 in skeletal muscle.

BACKGROUND: The mechanism by which acute alcohol (EtOH) intoxication decreases basal muscle protein synthesis via inhibition of the Ser/Thr kinase mammalian target of rapamycin (mTOR) is poorly defined. In this regard, mTOR activity is impaired after over expression of the regulatory protein REDD1. Hence, the present study assessed the ability of REDD1 as a potential mediator of the EtOH-induced decrease in muscle protein synthesis. METHODS: The effect of acute EtOH intoxication on REDD1 mRNA and protein was determined in striated muscle of rats and mouse myocytes using an RNase protection assay and Western blotting, respectively. Other components of the mTOR signaling pathway were also assessed by immunoblotting. For comparison, REDD1 mRNA/protein was also determined in the muscle of rats chronically fed an alcohol-containing diet for 14 weeks. RESULTS: Intraperitoneal (IP) injection of EtOH increased gastrocnemius REDD1 mRNA in a dose- and time-dependent manner, and these changes were associated with reciprocal decreases in the phosphorylation of 4E-BP1, which is a surrogate marker for mTOR activity and protein synthesis. No change in REDD1 mRNA was detected in the slow-twitch soleus muscle or heart. Acute EtOH produced comparable increases in muscle REDD1 protein. The EtOH-induced increase in gastrocnemius REDD1 was independent of the route of EtOH administration (oral vs. IP), the nutritional state (fed vs. fasted), gender, and age of the rat. The nonmetabolizable alcohol tert-butanol increased REDD1 and the EtOH-induced increase in REDD1 was not prevented by pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole. In contrast, REDD1 mRNA and protein were not increased in the isolated hindlimb perfused with EtOH or in C2C12 myocytes incubated with EtOH, under conditions previously reported to decrease protein synthesis. Pretreatment with the glucocorticoid receptor antagonist RU486 failed to prevent the EtOH-induced increase in REDD1. Finally, the EtOH-induced increase in REDD1 was not associated with altered formation of the TSC1*TSC2 complex or the phosphorylation of TSC2 which is down stream in the REDD1 stress response pathway. In contradistinction to the changes observed with acute EtOH intoxication, REDD1 mRNA/protein was not changed in gastrocnemius from chronic alcohol-fed rats despite the reduction in 4E-BP1 phosphorylation. CONCLUSIONS: These data indicate that in fast-twitch skeletal muscle (i) REDD1 mRNA/protein is increased in vivo by acute EtOH intoxication but not in response to chronic alcohol feeding, (ii) elevated REDD1 in response to acute EtOH appears due to the production of an unknown secondary mediator which is not corticosterone, and (iii) the EtOH-induced decrease in protein synthesis can be dissociated from a change in REDD1 suggesting that the induction of this protein is not responsible for the rapid decrease in protein synthesis after acute EtOH administration or for the development of alcoholic myopathy in rats fed an alcohol-containing diet.

Acute and chronic ethanol consumption differentially impact pathways limiting hepatic protein synthesis.

This review identifies the various pathways responsible for modulating hepatic protein synthesis following acute and chronic alcohol intoxication and describes the mechanism(s) responsible for these changes. Alcohol intoxication induces a defect in global protein synthetic rates that is localized to impaired translation of mRNA at the level of peptide-chain initiation. Translation initiation is regulated at two steps: formation of the 43S preinitiation complex [controlled by eukaryotic initiation factors 2 (eIF2) and 2B (eIF2B)] and the binding of mRNA to the 40S ribosome (controlled by the eIF4F complex). To date, alcohol-induced alterations in eIF2 and eIF2B content and activity are best investigated. Ethanol decreases eIF2B activity when ingested either acutely or chronically. The reduced eIF2B activity most likely is a consequence of twofold increased phosphorylation of the alpha-subunit of eIF2 on Ser(51) following acute intoxication. The increase in eIF2alpha phosphorylation after chronic alcohol consumption is the same as that induced by acute ethanol intoxication, and protein synthesis is not further reduced by long-term alcohol ingestion despite additional reduced expression of initiation factors and elongation factors. eIF2alpha phosphorylation alone appears sufficient to maximally inhibit hepatic protein synthesis. Indeed, pretreatment with Salubrinal, an inhibitor of eIF2alpha(P) phosphatase, before ethanol treatment does not further inhibit protein synthesis or increase eIF2alpha phosphorylation, suggesting that acute ethanol intoxication causes maximal eIF2alpha phosphorylation elevation and hepatic protein synthesis inhibition. Ethanol-induced inhibition of hepatic protein synthesis is not rapidly reversed by cessation of ethanol consumption. In conclusion, sustained eIF2alpha phosphorylation is a hallmark of excessive alcohol intake leading to inhibition of protein synthesis. Enhanced phosphorylation of eIF2alpha represents a unique response of liver to alcohol intoxication, because the ethanol-induced elevation of eIF2alpha(P) is not observed in skeletal muscle or heart.