Gastric vagal afferent neuropathy following experimental spinal cord injury.

Dramatic impairment of gastrointestinal (GI) function accompanies high-thoracic spinal cord injury (T3-SCI). The vagus nerve contains mechano- and chemosensory fibers as well as the motor fibers necessary for the central nervous system (CNS) control of GI reflexes. Cell bodies for the vagal afferent fibers are located within the nodose gangla (NG) and the majority of vagal afferent axons are unmyelinated C fibers that are sensitive to capsaicin through activation of transient receptor potential vanilloid-1 (TRPV1) channels. Vagal afferent fibers also express receptors for GI hormones, including cholecystokinin (CCK). Previously, T3-SCI provokes a transient GI inflammatory response as well as a reduction of both gastric emptying and centrally-mediated vagal responses to GI peptides, including CCK. TRPV1 channels and CCK-A receptors (CCKar) expressed in vagal afferents are upregulated in models of visceral inflammation. The present study investigated whether T3-SCI attenuates peripheral vagal afferent sensitivity through plasticity of TRPV1 and CCK receptors. Vagal afferent response to graded mechanical stimulation of the stomach was significantly attenuated by T3-SCI at 3-day and 3-week recovery. Immunocytochemical labeling for CCKar and TRPV1 demonstrated expression on dissociated gastric-projecting NG neurons. Quantitative assessment of mRNA expression by qRT-PCR revealed significant elevation of CCKar and TRPV1 in the whole NG following T3-SCI in 3-day recovery, but levels returned to normal after 3-weeks. Three days after injury, systemic administration of CCK-8 s showed a significantly diminished gastric vagal afferent response in T3-SCI rats compared to control rats while systemic capsaicin infusion revealed a significant elevation of vagal response in T3-SCI vs control rats. These findings demonstrate that T3-SCI provokes peripheral remodeling and prolonged alterations in the response of vagal afferent fibers to the physiological signals associated with digestion.

Mesenteric vascular dysregulation and intestinal inflammation accompanies experimental spinal cord injury.

Cervical and high thoracic spinal cord injury (SCI) drastically impairs autonomic nervous system function. Individuals with SCI at thoracic spinal level 5 (T5) or higher often present cardiovascular disorders that include resting systemic arterial hypotension. Gastrointestinal (GI) tissues are critically dependent upon adequate blood flow and even brief periods of visceral hypoxia triggers GI dysmotility. The aim of this study was to test the hypothesis that T3-SCI induces visceral hypoperfusion, diminished postprandial vascular reflexes, and concomitant visceral inflammation. We measured in vivo systemic arterial blood pressure and superior mesenteric artery (SMA) and duodenal blood flow in anesthetized T3-SCI rats at 3 days and 3 wk postinjury either fasted or following enteral feeding of a liquid mixed-nutrient meal (Ensure). In separate cohorts of fasted T3-SCI rats, markers of intestinal inflammation were assayed by qRT-PCR. Our results show that T3-SCI rats displayed significantly reduced SMA blood flow under all experimental conditions (P < 0.05). Specifically, the anticipated elevation of SMA blood flow in response to duodenal nutrient infusion (postprandial hyperemia) was either delayed or absent after T3-SCI. The dysregulated SMA blood flow in acutely injured T3-SCI rats coincides with abnormal intestinal morphology and elevation of inflammatory markers, all of which resolve after 3 wk. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) were acutely elevated following T3-SCI. Our data suggest that arterial hypotension diminishes mesenteric blood flow necessary to meet mucosal demands at rest and during digestion. The resulting GI ischemia and low-grade inflammation may be an underlying pathology leading to GI dysfunction seen following acute T3-SCI.

Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

Chronic α-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy.

Muscle disuse atrophy is observed routinely in patients recovering from traumatic injury and can be either generalized resulting from extended bed rest or localized resulting from single-limb immobilization. The present study addressed the hypothesis that a diet containing 5% α-hydroxyisocaproic acid (α-HICA), a leucine (Leu) metabolite, will slow the loss and/or improve recovery of muscle mass in response to disuse. Adult 14-wk-old male Wistar rats were provided a control diet or an isonitrogenous isocaloric diet containing either 5% α-HICA or Leu. Disuse atrophy was produced by unilateral hindlimb immobilization ("casting") for 7 days and the contralateral muscle used as control. Rats were also casted for 7 days and permitted to recover for 7 or 14 days. Casting decreased gastrocnemius mass, which was associated with both a reduction in protein synthesis and S6K1 phosphorylation as well as enhanced proteasome activity and increased atrogin-1 and MuRF1 mRNA. Although neither α-HICA nor Leu prevented the casting-induced muscle atrophy, the decreased muscle protein synthesis was not observed in α-HICA-treated rats. Neither α-HICA nor Leu altered the increased proteasome activity and atrogene expression observed with immobilization. After 14 days of recovery, muscle mass had returned to control values only in the rats fed α-HICA, and this was associated with a sustained increase in protein synthesis and phosphorylation of S6K1 and 4E-BP1 of previously immobilized muscle. Proteasome activity and atrogene mRNA content were at control levels after 14 days and not affected by either treatment. These data suggest that whereas α-HICA does not slow the loss of muscle produced by disuse, it does speed recovery at least in part by maintaining an increased rate of protein synthesis.

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.

Chronic alcohol feeding impairs mTOR(Ser 2448) phosphorylation in rat hearts.

BACKGROUND: Chronic alcohol administration impairs protein synthesis ultimately causing a loss of proteins in cardiac muscle. Inhibition of protein synthesis resides in the process of mRNA translation. The present set of experiments were designed to examine the potential regulatory effect of chronic alcohol consumption on mammalian target of rapamycin (mTOR), a serine/threonine kinase important in controlling signaling cascades in the mRNA translation initiation pathway in rat hearts. METHODS: Rats were fed a diet containing ethanol for 20 to 26 weeks. Pair-fed rats served as controls. Rates of protein synthesis were measured following intravenous infusion of [(3)H]-L-phenylalanine (150 mM, 30 microCi/ml; 1 ml/100 g body weight). The phosphorylation state of mTOR, eukaryotic initiation factor 4G (eIF4G), protein kinase B (PKB) and S6K1 in heart were measured using immunoblot techniques with phospho-specific antibodies. RESULTS: Protein synthesis was reduced by 35% in animals consuming a diet containing ethanol. The fall in protein synthesis was accompanied by diminished S6K1(Thr(389)) and eIF4G (Ser(1108)) phosphorylation, both downstream effectors of mTOR signaling. These changes in phosphorylation of S6K1 and eIF4G were not associated with differences in the distribution of mTOR between TORC1 and TORC2. Instead, phosphorylation of mTOR on Ser(2448) but not on Ser(2481) was significantly reduced following feeding rats an ethanol containing diet. Decreased phosphorylation of mTOR(Ser(2448)) was not associated with a corresponding lessening of tumor suppressor complex 2 phosphorylation or expression of regulated in development and DNA damage 1, both upstream regulators of mTOR. Likewise, phosphorylation of PKB on either Ser(473) or Thr(308) was unaffected by long-term alcohol consumption. CONCLUSIONS: Chronic ethanol consumption does not alter the distribution of mTOR between TORC1 and TORC2, but instead diminishes mTOR phosphorylation on Ser(2448) independent of changes in tumor suppressor complex 2 and PKB phosphorylation. Furthermore, the data suggest that protein synthesis in rats fed a diet containing ethanol is limited by mTOR-dependent reduction in phosphorylation of S6K1(Thr(389)) and eIF4G(Ser(1108)) secondary to reduced phosphorylation of mTOR(Ser(2448)).

Rapamycin limits formation of active eukaryotic initiation factor 4F complex following meal feeding in rat hearts.

Feeding promotes protein synthesis in cardiac muscle through a stimulation of the messenger RNA translation initiation phase of protein synthesis by enhancing assembly of active eukaryotic initiation factor (eIF)4F complex. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in increasing formation of active eIF4G-eIF4E complex during meal feeding. Hearts from male Sprague-Dawley rats fed a meal consisting of rat nonpurified diet were sampled prior to and 3 h following the meal in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1. Rapamycin prevented the meal feeding-induced stimulation of myocardial protein synthesis. Inhibition of mTOR with rapamycin decreased the association of rapamycin-associated TOR protein with mTOR and prevented the feeding-induced assembly of eIF4G-eIF4E complex. In contrast, the abundance of eIF4E binding protein-1 (4E-BP1)-eIF4E complex was unaffected by either meal feeding or rapamycin. Pretreatment with rapamycin completely prevented the feeding-induced phosphorylation of eIF4G(Ser(1108)), whereas the inhibitor only partially attenuated meal feeding-induced 70-kDa ribosomal protein S6 kinase1(Thr(389)) phosphorylation and extent of 4E-BP1 in the gamma-form. Meal feeding-induced phosphorylation of protein kinase B on either Ser(473) or Thr(308) was unaffected by rapamycin. These findings suggest the extent of phosphorylation of eIF4G following meal feeding occurs by a rapamycin-sensitive mechanism in cardiac muscle. Furthermore, the rapamycin-sensitive reductions in phosphorylation of eIF4G may also lead to decreased formation of active eIF4G-eIF4E complex.

Cytokine-triggered decreases in levels of phosphorylated eukaryotic initiation factor 4G in skeletal muscle during sepsis.

Chronic septic abscess formation causes an inhibition of protein synthesis in gastrocnemius that is not observed in rats with a sterile abscess. The inhibition is associated with an impaired translation initiation. The present study was designed to investigate the effects of sepsis on the level of phosphorylated eukaryotic initiation factor (eIF) 4G in gastrocnemius after induction of a chronic intra-abdominal sterile or septic abscess as a possible mechanism to account for the impairment of translation initiation during sepsis. The extent of phosphorylated eIF4G was reduced by more than 50% (P< 0.05) and 68% (P < 0.01) in gastrocnemius after 3 and 5 days, respectively, and returned to control values after 14 days of abscess formation in septic rats compared with sterile inflammatory animals. To examine the mediators of the septic process contributing to the decreased levels of phosphorylated eIF4G, the cytokine response to sepsis was pharmacologically modulated. First, treatment of septic rats with tumor necrosis factor (TNF) binding protein or interleukin (IL) 1 receptor antagonist increased the level of phosphorylated eIF4G. Second, infusion of TNF-alpha for 24 h in control rats resulted in a 70% decrease in phosphorylated eIF4G. Third, infusion of IL-1ra led to an increase in the level of phosphorylation of eIF4G in rats infused with TNF-alpha. Taken together, the data indicate that a cytokine-dependent decrease in the steady state phosphorylation of eIF4G is a possible mechanism accounting for the inhibition of skeletal muscle protein synthesis during sepsis. Furthermore, the findings support a role of IL-1 as the proinflammatory mediator responsible for the reduced level of phosphorylated eIF4G.

Long-term alcohol administration inhibits synthesis of both myofibrillar and sarcoplasmic proteins in heart.

Alcohol decreases the rate of protein synthesis in cardiac muscle. We investigated the effects of feeding rats a diet containing alcohol for 16 weeks on the myocardial synthesis of myofibrillar and sarcoplasmic (non-myofibrillar) proteins. Alcohol administration decreased the overall rate of protein synthesis in cardiac muscle by 22% compared with controls (P < .05). The rate of synthesis of proteins in the myofibrillar and sarcoplasmic fractions was diminished proportionately after feeding a diet containing alcohol (P < .05). We examined the effects of diminished rates of protein synthesis on the expression of myofibrillar and non-myofibrillar proteins. The cellular content of actin and alpha -myosin heavy chain isoform was significantly reduced and there was an increase in the beta -myosin heavy chain isoform after feeding rats a diet containing alcohol. The reduced expression of myosin heavy chain isoform and actin did not result from a decreased abundance of messenger RNA for either of these proteins. The myocardial content of troponin C and T was unchanged whereas that of troponin I was increased. Ethanol administration reduced the expression of eEF2 and the inducible form of the 70-kDa heat shock protein, whereas the cognate form of the 70-kDa heat shock protein was unaffected in a non-myofibrillar-enriched fraction of cardiac muscle. These results suggest that (1) the reduced protein content observed in the heart after feeding a diet containing alcohol is a consequence of reduced synthesis of both myofibrillar and sarcoplasmic proteins, and (2) the expression of both actin and alpha-myosin heavy chain isoform is affected independently of the messenger RNA content of the proteins. We conclude that translational control mechanisms appear to be important in regulating the expression of myocardial proteins during long-term ethanol intoxication.

Acute alcohol intoxication enhances myocardial eIF4G phosphorylation despite reducing mTOR signaling.

Acute alcohol intoxication impairs myocardial protein synthesis in rats, secondary to a diminished mRNA translational efficiency. Decreased mRNA translational efficiency occurs through altered regulation of peptide chain initiation. The purpose of the present set of experiments was to determine whether acute alcohol intoxication alters the phosphorylation state of eukaryotic initiation factor (eIF) 4G, eIF4G.eIF4E complex formation, and the mammalian target of rapamycin (mTOR) signaling pathway in the heart. Acute alcohol intoxication was induced by injection of alcohol (75 mmol/kg body wt ip). Control animals received an equal volume of saline. Alcohol administration enhanced phosphorylation of eIF4G (Ser(1108)) approximately threefold. Alcohol administration lowered formation of the active eIF4G.eIF4E complex by >90%, whereas it increased the abundance of the inactive 4E-binding protein 1 (4E-BP1).eIF4E complex by approximately 160%. Phosphorylation of mTOR on Ser(2448) and Ser(2481) was decreased by 50%. Reduced mTOR phosphorylation did not result from decreased phosphorylation of PKB. Phosphorylation of 4E-BP1 and S6 kinase 1 (Thr(389)), downstream targets of mTOR, were also reduced after acute alcohol administration. These data suggest that acute alcohol-induced impairments in myocardial mRNA translation initiation result, in part, from marked decreases in eIF4G.eIF4E complex formation, which appear to be independent of changes in phosphorylation of eIF4G but dependent on mTOR.

Diminished ERK 1/2 and p38 MAPK phosphorylation in skeletal muscle during sepsis.

Sepsis induces weight loss and the loss of skeletal muscle proteins, in part through an inhibition of protein synthesis secondary to an inhibition of the key steps controlling mRNA translation in skeletal muscle. We have previously shown that sepsis decreases the phosphorylation of eIF4E. The present study examines the phosphorylation of Erk 1/2 MAPK and p38 MAPK in skeletal muscle of rats with a chronic (5-day) intra-abdominal septic abscess. Mnk1 catalyzes the phosphorylation of eIF4E, and Mnk1 is activated by phosphorylation via Erk1/2 MAPK and p38 MAPK. Sepsis resulted in a significant decrease in the steady-state phosphorylation of Erk 1/2 and p38 MAPKs compared with sterile inflammation. To examine the mediators responsible for decreased phosphorylation of Erk 1/2 and p38 MAPKs, rats were treated with TNF binding protein (TNFbp) or infused for 24 h with TNF. Treatment of septic rats with TNFbp resulted in an increase in the phosphorylation of both Erk 1/2 and p38 MAPKs in skeletal muscle. This was associated with enhanced phosphorylation of eIF4E. In contrast, constant intravenous infusion of TNF-alpha for 24 h resulted in a complete inhibition of p38 MAPK phosphorylation while Erk 1/2 MAPK phosphorylation was increased. The net effect was a modest increase in eIF4E phosphorylation. The results suggest altered regulation of Erk 1/2 and p38 MAPK signal translation pathways by endogenously produced TNF, or some compound dependent on TNF may modulate, in part, the phosphorylation state of eIF4E in skeletal muscle during sepsis.

Differential effects of alcohol consumption on eukaryotic elongation factors in heart, skeletal muscle, and liver.

BACKGROUND: Acute and chronic alcohol administration diminishes rates of protein synthesis in a variety of tissues including skeletal muscle, heart, and liver, through a diminished translational efficiency rather than a reduction in the number of ribosomes. METHODS: The purpose of the present study was to examine the effect of chronic alcohol exposure (8, 12, or 16 weeks) on elongation factors (eEF) as a potential mechanism for controlling mRNA translation in psoas, soleus, heart, and liver. The cellular content of eEF1A and eEF2 and the phosphorylation state of eEF2 in each tissue was measured using immunoblot techniques. RESULTS: The protein content of eEF1A was reduced in psoas, heart, and liver (but not soleus) from rats fed a diet containing alcohol for 16 weeks, but not for 8 or 12 weeks, compared with time-matched pair-fed controls. eEF2 content was only reduced in myocardium after feeding rats an alcohol-containing diet for 16 weeks. In other tissues, no change in eEF2 content was observed. The decreases in eEF protein content were not associated with a concomitant reduction in the mRNA abundance for eEF1A or eEF2. The phosphorylation state of eEF2 was not affected by chronic alcohol consumption in the skeletal muscle or heart. In contrast, the level of eEF2 phosphorylation in the liver was reduced after 8, 12, and 16 weeks of feeding rats an alcohol-containing diet. In contrast, acute alcohol intoxication failed to modulate the content of eEF1A or eEF2 in any of the tissues examined. The phosphorylation state of eEF2 was reduced in psoas following acute alcohol intoxication. CONCLUSIONS: A decreased eEF1A protein content could account, in part, for the inhibition of translational efficiency following chronic (16 weeks) alcohol feeding but not the response to acute alcohol intoxication.

Phosphorylation of eukaryotic initiation factor eIF2Bepsilon in skeletal muscle during sepsis.

We reported that the inhibition of protein synthesis in skeletal muscle during sepsis correlated with reduced eukaryotic initiation factor eIF2B activity. The present studies define changes in eIF2Bepsilon phosphorylation in gastrocnemius of septic animals. eIF2B kinase activity was significantly elevated 175% by sepsis compared with sterile inflammation, whereas eIF2B phosphatase activity was unaffected. Phosphorylation of eIF2Bepsilon-Ser(535) was significantly augmented over 2-fold and 2.5-fold after 3 and 5 days and returned to control values after 10 days of sepsis. Phosphorylation of glycogen synthase kinase-3 (GSK-3), a potential upstream kinase responsible for the elevated phosphorylation of eIF2Bepsilon, was significantly reduced over 36 and 41% after 3 and 5 days and returned to control values after 10 days of sepsis. The phosphorylation of PKB, a kinase thought to directly phosphorylate and inactivate GSK-3, was significantly reduced approximately 50% on day 3, but not on days 5 or 10, postinfection compared with controls. Treatment of septic rats with TNF-binding protein prevented the sepsis-induced changes in eIF2Bepsilon and GSK-3 phosphorylation, implicating TNF in mediating the effects of sepsis. Thus increased phosphorylation of eIF2Bepsilon via activation of GSK-3 is an important mechanism to account for the inhibition of skeletal muscle protein synthesis during sepsis. Furthermore, the study presents the first demonstration of changes in eIF2Bepsilon phosphorylation in vivo.