Proteins that coat the lipid droplets (also known as PAT proteins or perilipin (PLIN) family proteins) have diverse functions that are not well elucidated in many tissues. In skeletal muscle, there is even less known about the functions or characteristics of these proteins or how they might change in response to perturbations that alter both intramyocellular lipid (IMCL) content and fat utilization and oxidation. Therefore, the purpose of this study was to examine the human muscle content and gene expression of the four skeletal muscle PLIN proteins in both lean and obese men and women and how this was changed following a 12-week endurance training protocol. PLIN2-PLIN5 proteins were all more abundant in women than in men (p = 0.037 and p < 0.0001, respectively), consistent with higher IMCL content observed in female skeletal muscle. PLIN5 (previously known as OXPAT) is of particular interest because it has previously been associated primarily with oxidative tissues that rely heavily on fat oxidation for energy production. Although PLIN5 was not different between lean and obese subjects, it was the only PLIN protein to increase in response to endurance training in both sexes. PLIN5 correlated with IMCL volume (p < 0.0001), but in general, the other PLIN proteins did not correlate well with IMCL volume, suggesting that the relationship between lipid accumulation and PLIN family protein content is not a simple one. Although more work is necessary, it is clear that PLIN5 likely plays an important role in IMCL accumulation and oxidation, both of which increase with endurance training in human skeletal muscle.

MacNeilLG, Baker SK, Stevic I, Tarnopolsky MA. 17␤-estradiol attenuates exercise-induced neutrophil infiltration in men. Am J Physiol Regul Integr Comp Physiol 300: R1443-R1451, 2011. First published March 2, 2011; doi:10.1152/ajpregu.00689.2009 attenuates exercise-induced muscle damage and inflammation in some models. Eighteen men completed 150 eccentric contractions after random assignment to placebo (Control group) or E2 supplementation (Experimental group). Muscle biopsies and blood samples were collected at baseline, following 8-day supplementation and 3 h and 48 h after exercise. Blood samples were analyzed for sex hormone concentration, creatine kinase (CK) activity and total antioxidant capacity. The mRNA content of genes involved in lipid and cholesterol homeostasis [forkhead box O1 (FOXO1), caveolin 1, and sterol regulatory element binding protein-2 (SREBP2)] and antioxidant defense (SOD1 and -2) were measured by RT-PCR. Immunohistochemistry was used to quantify muscle neutrophil (myeloperoxidase) and macrophage (CD68) content. Serum E2 concentration increased 2.5-fold with supplementation (P Ͻ 0.001), attenuating neutrophil infiltration at 3 h (P Ͻ 0.05) and 48 h (P Ͻ 0.001), and the induction of SOD1 at 48 h (P ϭ 0.02). Macrophage density at 48 h (P Ͻ 0.05) and SOD2 mRNA at 3 h (P ϭ 0.01) increased but were not affected by E2. Serum CK activity was higher at 48 h for both groups (P Ͻ 0.05). FOXO1, caveolin 1 and SREBP2 expression were 2.8-fold (P Ͻ 0.05), 1.4-fold (P Ͻ 0.05), and 1.5-fold (P Ͻ 0.001) and higher at 3 h after exercise with no effect of E2. This suggests that E2 attenuates neutrophil infiltration; however, the mechanism does not appear to be lesser oxidative stress or membrane damage and may indicate lesser neutrophil/endothelial interaction. mRNA expression; sex differences; leukocytes; oxidative stress SKELETAL MUSCLE IS A HETEROGENEOUS and adaptable tissue, able to function across a spectrum of physiological and metabolic conditions and remodel in response to changing demands. When subjected to unaccustomed exercise, particularly contractions that forcibly lengthen muscle (also known as eccentric contractions), myofibers are stressed beyond their functional capacity and damage may occur (16,51). This damage can be measured in 48 h following exercise as structural disturbances at cellular and subcellular levels (19, 52), muscle protein appearance in blood (46, 52), tissue inflammatory cell infiltration (31, 52), and increased soreness and impaired muscle function (17, 21). Many genes involved in postexercise membrane homeostasis, stress management, and growth are also affected, changing the myofiber transcriptome (46, 53). Because of the substantial effect on muscle tissue, eccentric contractions are commonly used for exercise-induced muscle damage and/or inflammation research.Most animal studies report differences in measures of exercise-induced muscle damage, inflammation, and repair between males and females (3, 39, 61). Manipulation of the female sex hormone 17␤-estradiol (E2) ind...

Factor-Xa assembly into the prothrombinase complex decreases its availability for inhibition by antithrombin + unfractionated heparin (AT + UFH). We have developed a novel covalent antithrombin-heparin complex (ATH), with enhanced anticoagulant actions compared with AT + UFH. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase. Discontinuous inhibition assays were performed to determine final k(2) values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor-prothrombinase interactions. The k(2) for inhibition of prothrombinase versus free Xa by AT + UFH was lower, whereas for ATH were much higher. Relative to intact prothrombinase, rates for Xa inhibition by AT + UFH in complexes devoid of prothrombin/vesicles/factor-Va were higher. For ATH, exclusion of prothrombin decreased k(2), removal of vesicles increased k(2) and exclusion of factor-Va gave no effect. While UFH may displace Xa from prothrombinase, Xa is detained within prothrombinase during ATH reactions. We confirm prothrombinase hinders inhibitory action of AT + UFH, whereas ATH is less affected with prothrombin being a key component in the complex responsible for the opposing effects. Overall, the results suggest that covalent linkage between AT-heparin assists access and neutralization of complexed Xa, with concomitant inhibition of prothrombinase function compared with conventional non-conjugated heparin.

Heparin is a major prophylactic and treatment agent for thrombosis. Structurally, this anticoagulant is a polydisperse, highly negatively charged polysaccharide mixture that contains a variable density of sulfate group substituents per molecule. Previous study has shown that heparin molecules have a high affinity for a wide range of metal ions with varying oxidation states. However, reports in literature on binding of heparin to metals have investigated only a small sampling of heparin-metal ion interactions. Since interaction of heparin with fluid phase and cell surface macromolecules in vivo is dependent on the heparin structure when bound in a metal ion complex, a survey of the physical parameters for heparin binding to metals is imperative. Atomic absorption and spectrophotometry experiments were performed for metal quantification, and in this study, the relative values for affinity constants and number of binding sites for heparin binding to several alkaline, alkaline earth, main group, and transition metals in their most common oxidation states are reported. We found an overall trend for heparin-metal affinity to be Mn(2+) > Cu(2+) > Ca(2+) > Zn(2+) > Co(2+) > Na(+) > Mg(2+) > Fe(3+) > Ni(2+) > Al(3+)> Sr(2+), with the trend in N (b) being opposite compared with the K (a).