Lou Ann Friend

Stimulation of both aerobic glycolysis and Na+-K+-ATPase activity in skeletal muscle by epinephrine or amylin

Epinephrine and amylin stimulate glycogenolysis, glycolysis, and Na(+)-K(+)-ATPase activity in skeletal muscle. However, it is not known whether these hormones stimulate glycolytic ATP production that is specifically coupled to ATP consumption by the Na(+)-K(+) pump. These studies correlated glycolysis with Na(+)-K(+)-ATPase activity in resting rat extensor digitorum longus and soleus muscles incubated at 30 degrees C in well-oxygenated medium. Lactate production rose three- to fourfold, and the intracellular Na(+)-to-K(+) ratio (Na(+)/K(+)) fell with increasing concentrations of epinephrine or amylin. In muscles exposed to epinephrine at high concentrations (5 x 10(-7) and 5 x 10(-6) M), ouabain significantly inhibited glycolysis by approximately 70% in either muscle and inhibited glycogenolysis by approximately 40 and approximately 75% in extensor digitorum longus and soleus, respectively. In the absence of ouabain, but not in its presence, statistically significant inverse correlations were observed between lactate production and intracellular Na(+)/K(+) for each hormone. Epinephrine had no significant effect on oxygen consumption or ATP content in either muscle. These results suggest for the first time that stimulation of glycolysis and glycogenolysis in resting skeletal muscle by epinephrine or amylin is closely linked to stimulation of active Na(+)-K(+) transport.Epinephrine and amylin stimulate glycogenolysis, glycolysis, and Na+-K+-ATPase activity in skeletal muscle. However, it is not known whether these hormones stimulate glycolytic ATP production that is specifically coupled to ATP consumption by the Na+-K+pump. These studies correlated glycolysis with Na+-K+-ATPase activity in resting rat extensor digitorum longus and soleus muscles incubated at 30°C in well-oxygenated medium. Lactate production rose three- to fourfold, and the intracellular Na+-to-K+ratio (Na+/K+) fell with increasing concentrations of epinephrine or amylin. In muscles exposed to epinephrine at high concentrations (5 × 10-7 and 5 × 10-6 M), ouabain significantly inhibited glycolysis by ∼70% in either muscle and inhibited glycogenolysis by ∼40 and ∼75% in extensor digitorum longus and soleus, respectively. In the absence of ouabain, but not in its presence, statistically significant inverse correlations were observed between lactate production and intracellular Na+/K+for each hormone. Epinephrine had no significant effect on oxygen consumption or ATP content in either muscle. These results suggest for the first time that stimulation of glycolysis and glycogenolysis in resting skeletal muscle by epinephrine or amylin is closely linked to stimulation of active Na+-K+transport.

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.

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.

Adrenergic Antagonists Reduce Lactic Acidosis in Response to Hemorrhagic Shock

BACKGROUND Hemorrhagic shock is associated with lactic acidosis and increased plasma catecholamines. Skeletal muscle increases lactate production under aerobic conditions in response to epinephrine, and this effect is blocked by ouabain, a specific inhibitor of the cell membrane Na+/K+ pump. In this study, we tested whether adrenergic antagonists can block lactate production during shock. METHODS Male Sprague-Dawley rats (250-300 g) were pretreated with phenoxybenzamine (2 mg/kg, i.v.) and/or propranolol (0.5 mg/kg, i.p.) before hemorrhaging to a mean arterial pressure of 40 mm Hg for 1 hour. Skeletal muscle perfusion, plasma lactate, and catecholamines were measured at baseline, 55 minutes after shock, and 1 hour after resuscitation. In a separate study, extensor digitorum longus and soleus muscles were incubated in Krebs buffer (95:5, O2:CO2) with 10 mmol/L glucose. One of each muscle pair was incubated in the absence or presence of epinephrine and of one or both adrenergic blockers. Medium lactate concentration was then measured. RESULTS The combination of alpha- and beta-blockers significantly reduced plasma lactate levels during hemorrhage. In contrast, beta-blockade alone was associated with a significant increase in plasma lactate and epinephrine. None of the blockers altered tissue perfusion. Epinephrine stimulation of muscle lactate production in vitro was completely blocked by propranolol. CONCLUSION Epinephrine release in response to hypotension is a primary stimulus for muscle lactate production in this model of hemorrhagic shock. Hypoxia alone does not explain the increased lactate levels because tissue perfusion was not altered by the adrenergic antagonists. These observations challenge the rationale behind lactate clearance as an end point for resuscitation after hemorrhagic shock.

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-3β 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.

Resuscitation with fresh whole blood ameliorates the inflammatory response after hemorrhagic shock.

BACKGROUND Hemorrhagic shock is the leading cause of potentially preventable death after traumatic injury. Hemorrhage and subsequent resuscitation may result in a dysfunctional systemic inflammatory response and multisystem organ failure, leading to delayed mortality. Clinical evidence supports improved survival and reduced morbidity when fresh blood products are used as resuscitation strategies. We hypothesized that the transfusion of fresh whole blood (FWB) attenuates systemic inflammation and reduces organ injury when compared with conventional crystalloid resuscitation after hemorrhagic shock. METHODS Male mice underwent femoral artery cannulation and hemorrhage to a systolic blood pressure of 25 mm Hg +/- 5 mm Hg. After 60 minutes, the mice were resuscitated with either FWB or lactated Ringers solution (LR). Mice were decannulated and killed at intervals for tissue histology, serum cytokine analysis, and vascular permeability studies. Separate groups of mice were followed for survival studies. RESULTS When compared with FWB, mice resuscitated with LR required increased resuscitation fluid volume to reach goal systolic blood pressure. When compared with sham or FWB-resuscitated mice, LR resuscitation resulted in increased serum cytokine levels of macrophage inflammatory protein-1alpha, interleukin-6, interleukin-10, macrophage-derived chemokine, KC, and granulocyte macrophage colony stimulating factor as well as increased lung injury and pulmonary capillary permeability. No survival differences were seen between animals resuscitated with LR or FWB. CONCLUSIONS Resuscitation with LR results in increased systemic inflammation, vascular permeability, and lung injury after hemorrhagic shock. Resuscitation with FWB attenuates the inflammation and lung injury seen with crystalloid resuscitation. These findings suggest that resuscitation strategies using fresh blood products potentially reduce systemic inflammation and organ injury after hemorrhagic shock.

Increased skeletal muscle Na+, K+-ATPase activity as a cause of increased lactate production after hemorrhagic shock.

BACKGROUND Lactate production after hemorrhagic shock may be produced by aerobic glycolysis, which has been linked to activity of the Na+/K+ pump in smooth muscle and other tissues. We tested whether increased muscle Na+/K+ pump activity after shock was linked to increased lactate production. METHODS Male Sprague-Dawley rats were subjected to 1 or 2 hours of hemorrhagic shock and then resuscitated with shed blood and normal saline. After 24 hours, pairs of extensor digitorum longus muscles were preincubated for 30 minutes in Krebs buffer (95:5, O2:CO2) with 10 mmol/L glucose. One muscle served as a control and was incubated in buffer alone; the other was incubated in buffer with 1 mmol/L ouabain, an inhibitor of the Na+, K+-ATPase. Lactate, ADP, ATP, glycogen, and creatinine-phosphate were determined. RESULTS Under these well-oxygenated conditions, muscles from shocked rats produced about twice as much lactate as sham muscles. Inhibition of the Na+/K+ pump by ouabain significantly reduced lactate production. CONCLUSIONS Hypoxia is unlikely to account for increased muscle lactate production after resuscitated hemorrhagic shock, because high lactate production persists under well-oxygenated incubation conditions. Inhibition of shock-induced lactate production by ouabain indicates energetic coupling of glycolysis to the Na+, K+-ATPase.

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.

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.

Hypobaric hypoxia exacerbates the neuroinflammatory response to traumatic brain injury.

OBJECTIVE To determine the inflammatory effects of time-dependent exposure to the hypobaric environment of simulated aeromedical evacuation following traumatic brain injury (TBI). METHODS Mice were subjected to a blunt TBI or sham injury. Righting reflex response (RRR) time was assessed as an indicator of neurologic recovery. Three or 24 h (Early and Delayed groups, respectively) after TBI, mice were exposed to hypobaric flight conditions (Fly) or ground-level control (No Fly) for 5 h. Arterial blood gas samples were obtained from all groups during simulated flight. Serum and cortical brain samples were analyzed for inflammatory cytokines after flight. Neuron specific enolase (NSE) was measured as a serum biomarker of TBI severity. RESULTS TBI resulted in prolonged RRR time compared with sham injury. After TBI alone, serum levels of interleukin-6 (IL-6) and keratinocyte-derived chemokine (KC) were increased by 6 h post-injury. Simulated flight significantly reduced arterial oxygen saturation levels in the Fly group. Post-injury altitude exposure increased cerebral levels of IL-6 and macrophage inflammatory protein-1α (MIP-1α), as well as serum NSE in the Early but not Delayed Flight group compared to ground-level controls. CONCLUSIONS The hypobaric environment of aeromedical evacuation results in significant hypoxia. Early, but not delayed, exposure to a hypobaric environment following TBI increases the neuroinflammatory response to injury and the severity of secondary brain injury. Optimization of the post-injury time to fly using serum cytokine and biomarker levels may reduce the potential secondary cerebral injury induced by aeromedical evacuation.