Muscle and liver glycogen,protein, and triglyceride in the rat

Summary We have previously found that during exercise net muscle glycogen breakdown is impaired in adrenodemedullated rats, as compared with controls. The present study was carried out to elucidate whether, in rats with deficiencies of the sympatho-adrenal system, diminished exercise-induced glycogenolysis in skeletal muscle was accompanied by increased breakdown of triglyceride and/or protein. Thus, the effect of exhausting swimming and of running on concentrations of glycogen, protein, and triglyceride in skeletal muscle and liver were studied in rats with and without deficiencies of the sympatho-adrenal system. In control rats, both swimming and running decreased the concentration of glycogen in fast-twitch red and slow-twitch red muscle whereas concentrations of protein and triglyceride did not decrease. In the liver, swimming depleted glycogen stores but protein and triglyceride concentrations did not decrease. In exercising rats, muscle glycogen breakdown was impaired by adrenodemedullation and restored by infusion of epinephrine. However, impaired glycogen breakdown during exercise was not accompanied by a significant net breakdown of protein or triglyceride. Surgical sympathectomy of the muscles did not influence muscle substrate concentrations. The results indicate that when glycogenolysis in exercising muscle is impeded by adrenodemedullation no compensatory increase in breakdown of triglyceride and protein in muscle or liver takes place. Thus, indirect evidence suggests that, in exercising adrenodemedullated rats, fatty acids from adipose tissue were burnt instead of muscle glycogen.     

Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo

Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation. After stimulation, the hindlegs were fixed by perfusion. GLUT4-EGFP-positive FDB fibres were isolated and analysed by confocal microscopy. The intracellular distribution of GLUT4-EGFP under basal conditions as well as after translocation to the plasma membrane in response to insulin, contractions, or both, was in accordance with previous studies of endogenous GLUT4. Finally, GLUT4-EGFP trafficking in quadriceps muscle in vivo was studied using time-lapse microscopy analysis in anaesthetised mice and the first detailed time-lapse recordings of GLUT4-EGFP translocation in fully differentiated skeletal muscle in vivo were obtained.     

Hormone-sensitive lipase in skeletal muscle: regulatory mechanisms

AIM: The enzymatic regulation of intramuscular triacylglycerol (TG) breakdown has until recently not been well understood. Our aim was to elucidate the role of hormone-sensitive lipase (HSL), which controls TG breakdown in adipose tissue. METHODS: Isolated rat muscle as well as exercising humans were studied. RESULTS: The presence of HSL was demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying. The content of HSL varies between fibre types, being higher in oxidative than in glycolytic fibres. Analysed under conditions optimal for HSL, neutral lipase activity in muscle can be stimulated by adrenaline as well as by contractions. These increases are abolished by presence of anti-HSL antibody during analysis. Moreover, immunoprecipitation with affinity-purified anti-HSL antibody causes similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions. The immunoreactive HSL in muscle is stimulated by adrenaline via beta-adrenergic activation of protein kinase A (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser563, Ser659 and Ser660. Contraction probably also enhances muscle-HSL activity by phosphorylation, because the contraction-induced increase in HSL activity is increased by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase. A novel signalling pathway in muscle by which HSL activity may be stimulated by protein kinase C (PKC) via extracellular signal regulated kinase (ERK) has been demonstrated. In contrast to previous findings in adipocytes, in muscle activation of ERK is not necessary for stimulation of HSL by adrenaline. However, contraction-induced HSL activation is mediated by PKC, at least partly via the ERK pathway. In fat cells ERK is known to phosphorylate HSL at Ser600. So, phosphorylation of different sites may explain that in muscle the effects of contractions and adrenaline on HSL activity are partially additive. In line with the view that the two stimuli act by different mechanisms, training increases the contraction-mediated, but diminishes the adrenaline mediated HSL activation in muscle. CONCLUSION: The existence and regulation of HSL in skeletal muscle indicate a role of HSL in muscle TG metabolism.     

Additivity of adrenaline and contractions on hormone‐sensitive lipase,but not on glycogen phosphorylase,in rat muscle

Aim: Hormone‐sensitive lipase (HSL) has been proposed to regulate triacylglycerol (TG) breakdown in skeletal muscle. In muscles with different fibre type compositions the influence on HSL of two major stimuli causing TG mobilization was studied. Methods: Incubated soleus and extensor digitorum longus (EDL) muscles from 70 g rats were stimulated by adrenaline (5.5 μm , 6 min) or contractions (200 ms tetani, 1 Hz, 1 min) in maximally effective doses or by both adrenaline and contractions. Results: Hormone‐sensitive lipase activity was increased significantly by adrenaline as well as contractions, and the highest activity (P < 0.05) was seen with combined stimulation [Soleus: 0.40 ± 0.03 (SE) m‐unit mg protein^?1 (basal), 0.65 ± 0.02 (adrenaline), 0.65 ± 0.03 (contractions), 0.78 ± 0.03 (adrenaline and contractions); EDL: 0.18 ± 0.01, 0.30 ± 0.02, 0.26 ± 0.02, 0.32 ± 0.01]. Glycogen phosphorylase activity was always increased more by adrenaline compared with contractions [Soleus: 60 ± 4 (a/a + b)% vs. 46 ± 3 (P < 0.05); EDL: 60 ± 5 vs. 39 ± 6 (P < 0.05)]. After combined stimulation glycogen phosphorylase activity in soleus [59 ± 3 (a/a + b)%] was identical to and in EDL [45 ± 4 (a/a + b)%] smaller (P < 0.05) than the activity after adrenaline only. Conclusions: In slow‐twitch oxidative as well as in fast‐twitch glycolytic muscle HSL is activated by both adrenaline and contractions. These stimuli are partially additive indicating at least partly different mechanisms of action. Contractions may impair the enhancing effect of adrenaline on glycogen phosphorylase activity in muscle.     

Letter to the editor

Medicine, Health Care and Philosophy -     

The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils

The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface- bound plasminogen to plasmin, a process of presumed importance for a number of biologic processes including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor- alpha results in a rapid increase in the expression of uPAR. This process is accompanied by an increased cell-associated plasminogen activation after preincubation of neutrophils with pro-uPA in vitro. By subcellular fractionation of unstimulated neutrophils, 50% of uPAR is recovered in fractions containing latent alkaline phosphatase, corresponding to an intracellular compartment of easily mobilizable secretory vesicles distinct from both primary and specific granules, whereas the remaining 50% of uPAR is associated with a compartment eluting close to the specific granules. In contrast, the ligand pro-uPA is primarily (approximately 80%) found in the specific granules, but small amounts of pro-uPA/uPA (approximately 20%) coelute with latent alkaline phosphatase. Stimulation of neutrophils with FMLP results in translocation of uPAR as well as of pro-uPA from the secretory vesicles, whereas stimulation with PMA is required to translocate material from specific granules. Flow cytometry of neutrophils saturated with exogenous diisopropyl fluorophosphate-uPA shows a large excess (approximately 90%) of unoccupied uPAR on resting as well as FMLP- and PMA-stimulated neutrophils, suggesting a possible role for exogenous pro-uPA in providing neutrophils with a potential for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi.     

The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.     

Evaluation of myocardial iron by magnetic resonance imaging during iron chelation therapy with deferrioxamine: indication of close relation between myocardial iron content and chelatable iron pool

Evaluation of myocardial iron during iron chelation therapy is not feasible by repeated endomyocardial biopsies owing to the heterogeneity of iron distribution and the risk of complications. Recently, we described a noninvasive method based on magnetic resonance imaging. Here, the method was used for repeated estimation of the myocardial iron content during iron chelation with deferrioxamine in 14 adult nonthalassemic patients with transfusional iron overload. We investigated the repeatability of the method and the relationship between the myocardial iron estimates and iron status. The repeatability coefficient (2sD) was 2.8 micromol/g in the controls (day-to-day) and 4.0 micromol/g in the patients (within-day). Myocardial iron estimates were elevated in 10 of all 14 patients at first examination, but normalized in 6 patients after 6 to 18 months of treatment. If liver iron declined below 350 micromol/g all but one of the myocardial iron estimates were normal or nearly normal. At start (R2 = 0.69, P =.0014) and still after 6 months of iron chelation (R2 = 0.76, P =.001), the estimates were significantly and more closely related to the urinary iron excretion than to liver iron or serum ferritin levels. In conclusion, our preliminary data, which may only pertain to patients with acquired anemias, suggest the existence of a critical liver iron concentration, above which elevated myocardial iron is present, but its extent seems related to the size of the chelatable iron pool, as reflected by the urinary iron excretion. This further supports the concept of the labile iron pool as the compartment directly involved in transfusional iron toxicity.