Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.

Des-acyl-ghrelin (DAG) normalizes hyperlactacidemia and improves survival in a lethal rat model of burn trauma.

Critical illness, including burn injury, results in elevated plasma lactate levels. Dysregulation of PI3K/Akt signaling has been shown to play a predominant role in the inactivation of skeletal muscle PDC and, hence, in hyperlactacidemia in rat models of sepsis and endotoxemia. This observation, and our previous finding that DAG can reverse burn-induced skeletal muscle proteolysis through the activation of PI3K/Akt pathway, led us to hypothesize that DAG may also attenuate hyperlactacidemia in burn injury. Our investigations revealed that burn injury significantly elevated both skeletal muscle lactate production and plasma lactate levels. Moreover, this was accompanied in skeletal muscle by a 5-7 fold increase in mRNA expression of pyruvate dehydrogenase kinases (PDK) 2 and 4, and a ∼30% reduction in PDC activity. DAG treatment of burn rats completely normalized not only the mRNA expression of the PDKs and PDC activity, but also hyperlactacidemia within 24h of burn injury. DAG also normalized epinephrine-induced lactate production by isolated skeletal muscles from normal rats. Moreover, DAG also improved survival in a lethal rat model of burn trauma. These findings with DAG may have clinical implications because chances of survival for critically ill patients are greatly improved if plasma lactate levels are normalized within 24h of injury.

Phosphodiesterase (PDE) inhibitor torbafylline (HWA 448) attenuates burn-induced rat skeletal muscle proteolysis through the PDE4/cAMP/EPAC/PI3K/Akt pathway.

Treatment of rats after burn-injury with the cyclic AMP phosphodiesterase (PDE) inhibitor, torbafylline (also known as HWA 448) significantly reversed changes in rat skeletal muscle proteolysis, PDE4 activity, cAMP concentrations and mRNA expression of TNFα, IL-6, ubiquitin and E3 ligases. Torbafylline also attenuated muscle proteolysis during in vitro incubation, and this effect was blocked by the inhibitor Rp-cAMPS. Moreover, torbafylline significantly increased phospho-Akt levels, and normalized downregulated phospho-FOXO1 and phospho-4E-BP1 in muscle of burn rats. Similarly, torbafylline also normalized phosphorylation levels of Akt and its downstream elements in TNFα+IFNγ treated C2C12 myotubes. Torbafylline enhanced protein levels of exchange protein directly activated by cAMP (Epac) both in skeletal muscle of burn rats and in TNFα+IFNγ treated C2C12 myotubes. Pretreatment with a specific antagonist of PI3K or Epac significantly reversed the inhibitory effects of torbafylline on TNFα+IFNγ-induced MAFbx mRNA expression and protein breakdown in C2C12 myotubes. Torbafylline inhibits burn-induced muscle proteolysis by activating multiple pathways through PDE4/cAMP/Epac/PI3K/Akt.

Des-acyl ghrelin exhibits pro-anabolic and anti-catabolic effects on C2C12 myotubes exposed to cytokines and reduces burn-induced muscle proteolysis in rats.

Although ghrelin and GHRP-2 have been shown to inhibit skeletal muscle proteolysis in rats with burn injury, the effects of des-acyl ghrelin (DAG) have not been reported. In this paper, we demonstrate that continuous 24h administration of DAG attenuated burn-induced EDL muscle proteolysis, and normalized elevated TNFα mRNA. Combined treatment of cultured C2C12 myotubes with TNFα and IFN-γ (TNF+IFN) inhibited protein synthesis and increased protein breakdown; DAG abolished both effects. PI3 kinase inhibition by LY294002 and mTOR inhibition by rapamycin blocked the reversal of the anti-anabolic effects of TNF+IFN-treated myotubes by DAG. DAG also reversed or attenuated the TNF+IFN-induced reduction in phosphorylation of Akt, FOXO1, 4E-BP-1, and GSK-3ß in myotubes. Furthermore, DAG attenuated the atrophy signal, phospho-NF-κB, and the mRNA expression of MAFbx and MuRF1, upregulated by TNF+IFN in C2C12 myotubes. We conclude that DAG reduces muscle cachexia produced by injury and proinflammatory cytokines, and that DAG or DAG-based compounds may be useful in treating wasting disorders.

Ghrelin receptor agonist, GHRP-2, attenuates burn injury-induced MuRF-1 and MAFbx expression and muscle proteolysis in rats.

Thermal injury results in hypermetabolism, loss of body weight, and skeletal muscle wasting in mice and rats. Our earlier studies have demonstrated that ghrelin injection stimulates food intake and growth hormone release and inhibits skeletal muscle proteolysis in rats with thermal injury. We sought to develop a lower molecular weight, stable and longer acting peptide to combat the catabolic responses caused by thermal injury. Towards this goal, we examined the role of the hexapeptide mimetic of ghrelin, growth hormone-releasing peptide-2 (GHRP-2), on expression of E3 ubiquitin ligases and breakdown of muscle protein in rats with thermal injury. Slow in vivo release of GHRP-2 through minipump for 24h attenuated the thermal injury-induced increase in mRNA expression of IL-6 and of the E3 ubiquitin ligases, MuRF-1 and MAFbx, in rat skeletal muscle. Furthermore, burn-induced increases in total and myofibrillar protein breakdown from rat EDL muscle were attenuated by GHRP-2. These findings suggest that catabolic responses resulting from thermal injury can be attenuated by GHRP-2.

Ghrelin inhibits skeletal muscle protein breakdown in rats with thermal injury through normalizing elevated expression of E3 ubiquitin ligases MuRF1 and MAFbx.

We previously determined that ghrelin synthesis was downregulated after burn injury and that exogenous ghrelin retained its ability both to stimulate food intake and to restore plasma growth hormone levels in burned rats. These observations and the finding that anabolic hormones can attenuate skeletal muscle catabolism led us to investigate whether ghrelin could attenuate burn-induced skeletal muscle protein breakdown in rats. These studies were performed in young rats (50-60 g) 24 h after approximately 30% total body surface area burn injury. Burn injury increased total and myofibrillar protein breakdown in extensor digitorum longus (EDL) muscles assessed by in vitro tyrosine and 3-methyl-histidine release, respectively. Continuous 24-h administration of ghrelin (0.2 mg.kg(-1).h(-1)) significantly inhibited both total and myofibrillar protein breakdown in burned rats. Ghrelin significantly attenuated burn-induced changes in mRNA expression of IGFBP-1 and IGFBP-3 in liver. In EDL, ghrelin attenuated the increases in mRNA expression of the binding proteins, but had no significant effect on reduced expression of IGF-I. Ghrelin markedly reduced the elevated mRNA expression of TNF-alpha and IL-6 in EDL muscle that occurred after burn. Moreover, ghrelin normalized plasma glucocorticoid levels, which were elevated after burn. Expression of the muscle-specific ubiquitin-ligating enzyme (E3) ubiquitin ligases MuRF1 and MAFbx were markedly elevated in both EDL and gastrocnemius and were normalized by ghrelin. These results suggest that ghrelin is a powerful anticatabolic compound that reduces skeletal muscle protein breakdown through attenuating multiple burn-induced abnormalities.

Influence of long chain polyunsaturated fatty acids and ornithine concentrations on complications after renal transplant.

OBJECTIVES: The present study, registered at clinicaltrials.gov with the unique registration number NCT00560014, sought to evaluate the relations between fatty acid concentrations in red blood cells or plasma and amino acid concentrations in plasma on rejection, calcineurin inhibitor toxicity, and new-onset diabetes mellitus. MATERIALS AND METHODS: Lipid profiles on plasma or red blood cell samples were performed preoperatively and postoperatively in 54 patients. Plasma amino acid profiles were obtained in 49 of these patients. RESULTS: High concentrations of total omega-3 fatty acids, eicosapentaenoic and docosahexaenoic acids in red blood cells, and ornithine in plasma, all were associated with a significantly lower incidence of rejection, whereas high total omega-6 fatty acids were associated with a high rejection rate. Calcineurin inhibitor toxicity was associated with low levels of docosahexaenoic acid, ornithine, and the omega-3 index, and high total omega-6 and omega-3/omega-6 ratios. Inhibition of new-onset diabetes mellitus was seen only with high levels of ornithine. Peak concentrations of fatty acids in red blood cells were not obtained until after 30 days. High levels of arginine were not associated with reduced complications. CONCLUSIONS: The levels of selected nutrients in plasma and red blood cell membranes appear to have a profound effect on complications after renal transplant. These preliminary results need confirmation in prospective randomized clinical trials.

GSK-3beta activity is increased in skeletal muscle after burn injury in rats.

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.

Neuropeptide Y (NPY) Y2 receptor-selective agonist inhibits food intake and promotes fat metabolism in mice: combined anorectic effects of Y2 and Y4 receptor-selective agonists.

Peripheral administration of the endogenous Y(2) and Y(4) receptor selective agonists, PYY(3-36) and PP, have been shown to inhibit food intake and body weight gain in rodents, and to reduce appetite and caloric intake in humans. We have previously developed a long-acting, potent and highly selective Y(2) receptor selective agonist, N-alpha-Ac-[Nle(24,28), Trp(30), Nva(31), Psi(35-36)]PYY(22-36)-NH(2) (BT-48). BT-48 (ip) dose-dependently inhibited ad lib food intake and also decreased the respiratory quotient in mice during both the light and dark periods. The latter observation is indicative of enhanced fat metabolism. Moreover, BT-48 also inhibited food intake in fasted mice. Combined ip administration of BT-48 (50nmol/mouse) with a highly potent and selective Y(4) anorectic peptide, BVD-74D (50nmol/mouse), resulted in a powerful and long lasting inhibitory effect on food intake. As expected, this inhibitory effect on food intake was nearly double that exhibited by either peptide (50nmol/mouse) alone. In summary, BT-48, unlike PYY(3-36), exhibits little or no affinity to other "Y" receptors, and may therefore have a better clinical potential than PYY(3-36) for control of food intake. Moreover, it appears that treatment with a combination of Y(2) and Y(4) receptor selective agonists may constitute a more powerful approach to control food intake than treatment with either of these agonists alone.

Ghrelin stimulates food intake and growth hormone release in rats with thermal injury: synthesis of ghrelin.

Ghrelin, a 28-residue octanoylated peptide recently isolated from the stomach, exhibits anti-cachectic properties through regulating food intake, energy expenditure, adiposity, growth hormone secretion and immune response. Burn injury induces persistent hypermetabolism and muscle wasting. We therefore hypothesized that ghrelin may also play a role in the pathophysiology of burn-induced cachexia. Overall ghrelin expression in the stomach over 10 days after burn was significantly decreased (p = 0.0003). Total plasma ghrelin was reduced 1 day after burn. Thus, changes in ghrelin synthesis and release may contribute to burn-induced dysfunctions. Ghrelin (30 nmol/rat, i.p.) greatly stimulated 2 h food intake in rats on five separate days after burn and in control rats. On post-burn day 15, plasma growth hormone levels were significantly lower than in controls, and this was restored to normal levels by ghrelin (10 nmol/rat, i.p.). These observations suggest that ghrelin retains its ability to favorably modulate both the peripheral anabolic and the central orexigenic signals, even after thermal injury despite ongoing changes due to prolonged and profound hypermetabolism, suggesting that long-term treatment with ghrelin may attenuate burn-induced dysfunctions.

Protein breakdown in muscle from burned rats is blocked by insulin-like growth factor i and glycogen synthase kinase-3beta inhibitors.

We reported previously that IGF-I inhibits burn-induced muscle proteolysis. Recent studies suggest that activation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway with downstream phosphorylation of Forkhead box O transcription factors is an important mechanism of IGF-I-induced anabolic effects in skeletal muscle. The potential roles of other mechanisms in the anabolic effects of IGF-I are less well understood. In this study we tested the roles of mammalian target of rapamycin and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation as well as MAPK- and calcineurin-dependent signaling pathways in the anticatabolic effects of IGF-I by incubating extensor digitorum longus muscles from burned rats in the presence of IGF-I and specific signaling pathway inhibitors. Surprisingly, the PI3K inhibitors LY294002 and wortmannin reduced basal protein breakdown. No additional inhibition by IGF-I was noticed in the presence of LY294002 or wortmannin. Inhibition of proteolysis by IGF-I was associated with phosphorylation (inactivation) of GSK-3beta. In addition, the GSK-3beta inhibitors, lithium chloride and thiadiazolidinone-8, reduced protein breakdown in a similar fashion as IGF-I. Lithium chloride, but not thiadiazolidinone-8, increased the levels of phosphorylated Foxo 1 in incubated muscles from burned rats. Inhibitors of mammalian target of rapamycin, MAPK, and calcineurin did not prevent the IGF-I-induced inhibition of muscle proteolysis. Our results suggest that IGF-I inhibits protein breakdown at least in part through a PI3K/Akt/GSK3beta-dependent mechanism. Additional experiments showed that similar mechanisms were responsible for the effect of IGF-I in muscle from nonburned rats. Taken together with recent reports in the literature, the present results suggest that IGF-I inhibits protein breakdown in skeletal muscle by multiple mechanisms, including PI3K/Akt-mediated inactivation of GSK-3beta and Foxo transcription factors.

Elevated blood lactate is not a primary cause of anorexia in tumor-bearing rats.

Tumor-bearing (TB) rats exhibit elevated concentrations of lactate in blood contiguous with the development of anorexia. Continuous intravenous infusion of lactate into non-TB rats reduced food intake at plasma concentrations lower than those observed in anorectic TB rats. Levels of neuropeptide Y (NPY) were elevated in the ventromedial (VMH) and dorsomedial hypothalamic regions of lactate-infused rats. The addition of the enhancer of pyruvate dehydrogenase activity, dichloroacetate (DCA), to the drinking water of TB rats (0.1-0.4%) normalized blood lactate concentration but had no significant effect on anorexia. However, the elevated concentration of NPY in the VMH of anorectic TB rats was also normalized by the DCA treatment. No alterations in regional hypothalamic levels of corticotropin-releasing factor were observed within any treatment conditions. These results suggest that, although hyperlactatemia may be involved in maintaining elevated NPY concentrations in anorectic TB rats, it does not appear to be a significant factor in the etiology of experimental cancer anorexia.

Role of skeletal muscle Na+-K+ ATPase activity in increased lactate production in sub-acute sepsis.

Bacterial sepsis is frequently accompanied by increased blood concentration of lactic acid, which traditionally is attributed to poor tissue perfusion, hypoxia and anaerobic glycolysis. Therapy aimed at improving oxygen delivery to tissues often does not correct the hyperlactatemia, suggesting that high blood lactate in sepsis is not due to hypoxia. Various tissues, including skeletal muscle, demonstrate increased lactate production under well-oxygenated conditions when the activity of the Na+-K+ ATPase is stimulated. Although both muscle Na+-K+ ATPase activity and muscle plasma membrane content of Na+, K+-ATPase subunits are increased in sepsis, no studies in vivo have demonstrated correlation between lactate production and changes in intracellular Na+ and K+ resulting from increased Na+-K+ pump activity in sepsis. Plasma concentrations of lactate and epinephrine, a known stimulator of the Na+-K+ pump, were increased in rats made septic by E. coli injection. Muscle lactate content was significantly increased in septic rats, although muscle ATP and phosphocreatine remained normal, suggesting oxygen delivery remained adequate for mitochondrial energy metabolism. In septic rats, muscle intracellular ratio of Na+:K+ was significantly reduced, indicating increased Na+-K+ pump activity. These data thus demonstrate that increased muscle lactate during sepsis correlates with evidence of elevated muscle Na+-K+ ATPase activity, but not with evidence of impaired oxidative metabolism. This study also further supports a role for epinephrine in this process.

Branched chain amino acids in heptatic encephalopathy.

BACKGROUND: Early theories or hepatic encephalopathy focused on ammonia-driven disruption of the Krebs cycle and cellular energy production. The "false-neurotransmitter" theory directed attention toward the interactions of amino acids, metabolism, the blood-brain barrier and neurotransmission. As they evolved, these studies revealed surprising and subtle effects of ammonia on brain amino acid uptake. DATA SOURCES: Research over a 15-year period in Josef E. Fischer's laboratory explored many aspects of these interactions. Subsequent studies by others have confirmed and extended them into other areas. Insights from this work continue to stimulate attempts to confirm or disprove the clinical utility of branched chain amino acids. CONCLUSIONS: Increased understanding of the factors affecting ammonia, amino acid and neurotransmitter disturbances in chronic liver failure have made a significant and ongoing contribution to the study of metabolism in health and disease.

Hypoxia is not the sole cause of lactate production during shock.

BACKGROUND: Traditionally, elevated blood lactate after hemorrhage is interpreted as tissue hypoperfusion, hypoxia, and anaerobic glycolysis. The severity and duration of the increase in blood lactate correlate with death. Recent in vitro studies indicate that epinephrine stimulates lactate production in well-oxygenated skeletal muscle by increasing activity of the Na+-K+-adenosine triphosphatase (ATPase), which derives a significant amount of adenosine triphosphate from glycolysis. Using in vivo microdialysis, we tested whether inhibiting the Na+-K+ pump with ouabain could reduce muscle lactate production during local exposure, via the microdialysis probe, to epinephrine or during hemorrhage in rats. METHODS: Microdialysis catheters were placed in the muscle of both thighs of pentobarbital-anesthetized male Sprague-Dawley rats (275-350 g) and perfused (1 microL/min) with Krebs-phosphate buffer (pH 7.4) containing ethanol (5 mmol/L) to permit assessment of changes in local blood flow. To inhibit the Na+-K+-ATPase, ouabain (2-3 mmol/L) was added to the perfusate of one leg. In one series of studies, epinephrine was added to the perfusate. In another series, rats were hemorrhaged to a mean arterial pressure of 45 mm Hg for 30 minutes, followed by resuscitation with shed blood and 0.9% sodium chloride. Dialysate fractions were analyzed for lactate and ethanol fluorometrically. RESULTS: Lactate rose during epinephrine exposure or during hemorrhage and resuscitation. Treatment with ouabain reduced dialysate lactate concentration significantly in both series of studies. Local blood flow was reduced by either epinephrine or hemorrhage, but returned toward baseline afterward. Ouabain had no apparent effect on local blood flow. CONCLUSION: Increased Na+-K+ATPase activity during epinephrine treatment or hemorrhage contributes to muscle lactate production. Hypoxia is not necessarily the sole cause of hyperlactatemia during and after hemorrhagic shock.