CD4+ and CD8+ CD28(null) T Cells Are Cytotoxic to Autologous Muscle Cells in Patients With Polymyositis.

Objective: Inflammatory T cell infiltrates in the skeletal muscle tissue of patients with polymyositis are dominated by CD28-negative effector (CD28(null) ) T cells of both the CD4 and CD8 lineage. These cells are potentially cytotoxic, and the aim of the present study was to develop a fully autologous cell culture system in which to investigate the functional contribution of such CD28(null) T cells to myotoxicity.

Methods: In vitro cocultures of autologous skeletal muscle cells and T cell subsets obtained from 5 polymyositis patients were performed.

Proteasome inhibition in skeletal muscle cells unmasks metabolic derangements in type 2 diabetes.

Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance.

Negative regulation of glucose metabolism in human myotubes by supraphysiological doses of 17β-estradiol or testosterone.

Objective: Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle.

Materials/methods: Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed.

Testosterone or 17{beta}-estradiol exposure reveals sex-specific effects on glucose and lipid metabolism in human myotubes.

Changes in sex hormone levels with aging or illness may lead to metabolic disorders. Moreover, the ratio changes in men versus women may have distinct pathological responses. Since little is known about sex hormone action on muscle metabolism, we examined the role of testosterone or 17β-estradiol (E(2)) in metabolism and investigated whether either hormone may mediate a sex-specific effect.

Regulation of glucose uptake by endothelin-1 in human skeletal muscle in vivo and in vitro.

Context: Expression of the vasoconstrictor and proinflammatory peptide endothelin (ET)-1 is increased in insulin-resistant (IR) subjects.

Objective: The aim of this study was to investigate whether ET-1 regulates skeletal muscle glucose uptake in IR subjects in vivo and in cultured human skeletal muscle cells.

Design And Participants: Eleven subjects participated in three protocols using brachial artery infusion of: A) BQ123 (10 nmol/min) and BQ788 (10 nmol/min) (ET(A) and ET(B) receptor antagonist, respectively), followed by coinfusion with insulin (0.

Evidence against a sexual dimorphism in glucose and fatty acid metabolism in skeletal muscle cultures from age-matched men and post-menopausal women.

Aim: In vivo whole body differences in glucose/lipid metabolism exist between men and women. Thus, we tested the hypothesis that intrinsic sex differences exist in skeletal muscle gene expression and glucose/lipid metabolism using cultured myotubes.

Methods: Myotube cultures were prepared for gene expression and metabolic studies from vastus lateralis skeletal muscle biopsies obtained from age-matched men (n = 11; 59 +/- 2 years) and post-menopausal women (n = 10; 60 +/- 1 years).

Post-transcriptional gene silencing of ribosomal protein S6 kinase 1 restores insulin action in leucine-treated skeletal muscle.

Excessive nutrients, especially amino acids, impair insulin action on glucose metabolism in skeletal muscle. We tested the hypothesis that the branched-chain amino acid leucine reduces acute insulin action in primary myotubes via a negative feedback mechanism involving ribosomal protein S6 kinase 1 (S6K1). The effect of S6K1 on glucose metabolism was determined by applying RNA interference (siRNA).

Glucocorticoid-mediated effects on metabolism are reversed by targeting 11 beta hydroxysteroid dehydrogenase type 1 in human skeletal muscle.

Background: Adipose tissue and liver play important roles in mediating the metabolic actions of glucocorticoids. However, the effects of glucocorticoids on glucose and lipid metabolism in skeletal muscle are not understood completely. Intracellular glucocorticoid action is dependent on 11 beta-hydroxysteroid dehydrogenase 1 (HSD1), an enzyme that converts cortisone to active cortisol.

Effect of serum replacement with plysate on cell growth and metabolismin primary cultures of human skeletal muscle.

Cell- and tissue culture methodology form an important base for biological, biochemical and biomedical research. Most cell culture techniques require the use of animal sera for the successful propagation of cells. However, the varying composition between batches has fuelled the need for alternatives.

Role of AMP kinase and PPARdelta in the regulation of lipid and glucose metabolism in human skeletal muscle.

The peroxisome proliferator-activated receptor (PPAR)delta has been implicated in the regulation of lipid metabolism in skeletal muscle. Furthermore, activation of PPARdelta has been proposed to improve insulin sensitivity and reduce glucose levels in animal models of type 2 diabetes. We recently demonstrated that the PPARdelta agonist GW501516 activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake in skeletal muscle.

Signaling specificity of interleukin-6 action on glucose and lipid metabolism in skeletal muscle.

We identified signaling pathways by which IL-6 regulates skeletal muscle differentiation and metabolism. Primary human skeletal muscle cells were exposed to IL-6 (25 ng/ml either acutely or for several days), and small interfering RNA gene silencing was applied to measure glucose and fat metabolism. Chronic IL-6 exposure increased myotube fusion and formation and the mRNA expression of glucose transporter 4, peroxisome proliferator activated receptor (PPAR)alpha, PPARdelta, PPARgamma, PPARgamma coactivator 1, glycogen synthase, myocyte enhancer factor 2D, uncoupling protein 2, fatty acid transporter 4, and IL-6 (P < 0.

siRNA-based gene silencing reveals specialized roles of IRS-1/Akt2 and IRS-2/Akt1 in glucose and lipid metabolism in human skeletal muscle.

Type 2 diabetes is associated with defects in insulin signaling and the resulting abnormal glucose and lipid metabolism. The complexity of insulin signaling cascades is highlighted by the existence of multiple isoforms of target proteins implicated in metabolic and gene-regulatory events. We utilized siRNA to decipher the specific role of predominant insulin receptor substrates and Akt isoforms expressed in human skeletal muscle.

Cardiotonic steroids stimulate glycogen synthesis in human skeletal muscle cells via a Src- and ERK1/2-dependent mechanism.

The cardiotonic steroid, ouabain, a specific inhibitor of Na(+),K(+)-ATPase, initiates protein-protein interactions that lead to an increase in growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in human skeletal muscle cells (HSMC) and clarified the mechanisms of ouabain signal transduction. In HSMC, ouabain increased glycogen synthesis in a concentration-dependent manner reaching the maximum at 100 nM.

Enhanced insulin-stimulated glycogen synthesis in response to insulin, metformin or rosiglitazone is associated with increased mRNA expression of GLUT4 and peroxisomal proliferator activator receptor gamma co-activator 1.

Aims/hypothesis: The aim of this study was to determine the effect of several antidiabetic agents on insulin-stimulated glycogen synthesis, as well as on mRNA expression.

Methods: Cultured primary human skeletal myotubes obtained from six healthy subjects were treated for 4 or 8 days without or with glucose (25 mmol/l), insulin (400 pmol/l), rosiglitazone (10 micromol/l), metformin (20 micromol/l) or the AMP-activated kinase activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (200 micromol/l). After this, insulin-stimulated glycogen synthesis was determined.

Direct activation of glucose transport in primary human myotubes after activation of peroxisome proliferator-activated receptor delta.

Activators of peroxisome proliferator-activated receptor (PPAR)gamma have been studied intensively for their insulin-sensitizing properties and antidiabetic effects. Recently, a specific PPARdelta activator (GW501516) was reported to attenuate plasma glucose and insulin levels when administered to genetically obese ob/ob mice. This study was performed to determine whether specific activation of PPARdelta has direct effects on insulin action in skeletal muscle.

Prior serum- and AICAR-induced AMPK activation in primary human myocytes does not lead to subsequent increase in insulin-stimulated glucose uptake.

Exposing isolated rat skeletal muscle to 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside [AICAR, a pharmacological activator of AMP-activated protein kinase (AMPK)] plus serum leads to a subsequent increase in insulin-stimulated glucose transport (Fisher JS, Gao J, Han DH, Holloszy JO, and Nolte LA. Am J Physiol Endocrinol Metab 282: E18-E23, 2002). Our goal was to determine whether preincubation of primary human skeletal muscle cells with human serum and AICAR (Serum+AICAR) would also induce a subsequent elevation in insulin-stimulated glucose uptake.

ERK1/2 mediates insulin stimulation of Na(+),K(+)-ATPase by phosphorylation of the alpha-subunit in human skeletal muscle cells.

Insulin stimulates Na(+),K(+)-ATPase activity and induces translocation of Na(+),K(+)-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na(+),K(+)-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na(+),K(+)-ATPase was dependent on ERK1/2 in HSMCs.

Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin-mediated MAPK and PKB phosphorylation.

Aim: We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype.

Methods: We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC).

Results: Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C.

MEF2 activation in differentiated primary human skeletal muscle cultures requires coordinated involvement of parallel pathways.

The myocyte enhancer factor (MEF)2 transcription factor is important for development of differentiated skeletal muscle. We investigated the regulation of MEF2 DNA binding in differentiated primary human skeletal muscle cells and isolated rat skeletal muscle after exposure to various stimuli. MEF2 DNA binding activity in nonstimulated (basal) muscle cultures was almost undetectable.

RNA interference-mediated reduction in GLUT1 inhibits serum-induced glucose transport in primary human skeletal muscle cells.

Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated human skeletal myotube cultures. Serum stimulation of myotubes increases glucose uptake. Using a sensitive photolabeling technique, we demonstrate that this increase in glucose uptake is accompanied by increased cell-surface content of glucose transporter (GLUT) 1.

Insulin action in cultured human skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and GLUT1 content.

In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle.

Phosphorylation of the Na+,K+-ATPase in skeletal muscle: potential mechanism for changes in pump cell-surface abundance and activity.

In skeletal muscle, insulin stimulation leads to phosphorylation of Na(+),K(+)-ATPase alpha-subunits on both serine/threonine and tyrosine residues, translocation of Na(+),K(+)-ATPase molecules to the plasma membrane, and increased Na(+),K(+)-ATPase activity. The molecular nature of the tyrosine kinase that phosphorylates Na(+),K(+)-ATPase is not yet identified. In vitro phosphorylation experiments show that the alpha-subunit of Na(+),K(+)-ATPase from skeletal muscle is a substrate for the tyrosine-specific protein kinase c-src.

Na+,K+-ATPase trafficking in skeletal muscle: insulin stimulates translocation of both alpha 1- and alpha 2-subunit isoforms.

We determined insulin-stimulated Na(+),K(+)-ATPase isoform-specific translocation to the skeletal muscle plasma membrane. When rat muscle plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha(2)- but not alpha(1)-subunits was detected. However, using cell surface biotinylation techniques, an insulin-induced membrane translocation of both alpha(1) and alpha(2)-subunits in rat epitrochlearis muscle and cultured human skeletal muscle cells was noted.

Insulin signal transduction and glucose transport in human adipocytes: effects of obesity and low calorie diet.

Aim/hypothesis: We examined insulin signal transduction at the level of insulin receptor substrates (IRS) 1 and 2, phosphatidylinositol (PI) 3-kinase and glucose transport in isolated subcutaneous adipocytes from obese and lean women.

Methods: Glucose transport and insulin signalling were investigated in isolated adipocytes from six obese women (BMI 36-43 kg/m(2)) (before and after 11 days of very low calorie diet) and from six lean women (BMI 22-26 kg/m(2)).

Results: Insulin sensitivity of glucose transport was reduced in adipocytes from obese women (p<0.

Monoclonal antibodies to native HIV type 1 reverse transcriptase and their interaction with enzymes from different subtypes.

Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT monoclonal antibodies (MAbs). Immunization was done by mixing RT with the ISCOM matrix-forming adjuvant saponin (Quil A). Two different assays, both based on the interaction of native RT and antibodies, were used to monitor the immune response in mice and for screening, selection, and characterization of the MAbs.