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). RESULTS: mRNA expression of several genes involved in glucose and lipid metabolism was higher in skeletal muscle biopsies from female vs. male donors, but unaltered between the sexes in cultured myotubes. Basal and insulin-stimulated glucose uptake, as well as glucose incorporation into glycogen, was similar in myotube cultures derived from male vs. female donors. In males vs. females, insulin increased glucose uptake (1.3 +/- 0.1 vs. 1.5 +/- 0.1-fold respectively) and incorporation into glycogen (2.3 +/- 0.3 vs. 2.0 +/- 0.3-fold respectively) to the same extent. Basal fatty acid oxidation and rate of uptake/accumulation was similar between sexes. In response to the 5'AMP-activated protein kinase activator AICAR, lipid oxidation was increased to the same extent in myotubes established from male vs. female donors (1.6 +/- 0.6 vs. 2.0 +/- 0.3-fold respectively). Moreover, the AICAR-induced rate of uptake/accumulation was similar between sexes. CONCLUSION: Differences in metabolic parameters and gene expression profiles between age-matched men and post-menopausal women noted in vivo are not observed in cultured human skeletal muscle cells. Thus, the sexual dimorphism in glucose and lipid metabolism is likely a consequence of systemic whole body factors, rather than intrinsic differences in the skeletal muscle proper.

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). Leucine (5 mM) reduced glucose uptake and incorporation to glycogen by 13% and 22%, respectively, compared to the scramble siRNA-transfected control at the basal level. Leucine also reduced insulin-stimulated Akt phosphorylation, glucose uptake and glucose incorporation to glycogen (39%, 39% and 37%, respectively), and this reduction was restored after S6K1 silencing. Depletion of S6K1 enhanced basal glucose utilization and protected against the development of impaired insulin action, in response to excessive leucine. In conclusion, S6K1 plays an important role in the regulation of insulin action on glucose metabolism in skeletal muscle.

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. mRNA levels of the glucose transporters GLUT1 and GLUT4, the peroxisomal proliferator activator receptor gamma (PPAR gamma) co-activator 1 (PGC1) and the myocyte-specific enhancer factors (MEF2), MEF2A, MEF2C and MEF2D were determined using real-time PCR analysis after 8 days exposure to the various antidiabetic agents. RESULTS: Insulin-stimulated glycogen synthesis was significantly increased in cultured human myotubes treated with insulin, rosiglitazone or metformin for 8 days, compared with non-treated cells. Furthermore, an 8-day exposure of myotubes to 25 mmol/l glucose impaired insulin-stimulated glycogen synthesis. In contrast, treatment with AICAR was without effect on insulin-mediated glycogen synthesis. Exposure to insulin, rosiglitazone or metformin increased mRNA expression of PGC1 and GLUT4, while AICAR or 25 mmol/l glucose treatment increased GLUT1 mRNA expression. Metformin also increased mRNA expression of the MEF2 isoforms. CONCLUSIONS/INTERPRETATION: Enhanced insulin-stimulated glycogen synthesis in human skeletal muscle cell culture coincides with increased GLUT4 and PGC1 mRNA expression following treatment with various antidiabetic agents. These data show that chronic treatment of human myotubes with insulin, metformin or rosiglitazone has a direct positive effect on insulin action and mRNA expression.

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. No change in expression was observed for big mitogen-activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal-regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin-stimulated PI 3-kinase activity increased with differentiation. Insulin-mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. CONCLUSION: Components of the insulin-signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation.

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. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, cultured myotubes are a useful tool to facilitate biological and molecular validation of novel pharmacological agents aimed to improve glucose metabolism in skeletal muscle.

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<0.05), with further reductions in basal and maximal insulin-stimulated glucose transport after a very low calorie diet (p<0.05). In obese women, IRS-1 associated PI 3-kinase activity was markedly impaired (p<0.05), whereas, IRS-2 associated PI 3-kinase activity was normal. IRS-1 associated PI 3-kinase activity remained blunted after a very low calorie diet, whereas IRS-2 associated PI 3-kinase activity was increased. GLUT4 protein was reduced by 37% in obese versus lean subjects (p<0.05), and decreased further after a very low calorie diet (from 19+/-4 to 14+/-4 arbitrary units; p<0.05). CONCLUSION/INTERPRETATION: IRS-1 signalling to PI 3-kinase is a site of insulin resistance in adipocytes from obese women, whereas insulin action on IRS-2 is normal. Thus, IRS-1 and IRS-2 undergo differential regulation in adipocytes from obese insulin resistant subjects. Finally, a very low calorie diet is associated with a further impairment in glucose transport in adipose tissue. The defect in glucose transport after a very low calorie diet occurs independent of further defects in insulin signalling at the level of the PI 3-kinase.

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. The first assay measures the capacity of antibodies to inhibit the polymerase activity of the RT and the second assay measures the ability of antibodies to capture enzymatically active RT. Twelve clones with the capacity to inhibit at least 50% of the RT activity and 34 clones with high RT-capturing capacity were found. The MAb panel was utilized to evaluate the immunological properties of 18 different RTs representing 9 different HIV1 subtypes. The RT-inhibitory MAbs could be divided into two groups based on their pattern of cross-reactivity toward the different HIV-1 RTs. The degree of diversity recorded among MAbs with RT-capturing capacity was larger. At least seven groups of MAbs with distinct cross-reactivity patterns were identified. Thus, the degree of isoenzyme specificity varied greatly, from MAbs that were quite specific for subtype B RT to one MAb that was able to capture the RTs from all HIV-1 isolates tested except one of the two group O isolates. In conclusion, our study revealed that there exist surprisingly large immunological differences between RTs from different HIV-1 subtypes as well as from the same subtype.

Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera.

IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.

Differences in reverse transcriptase activity versus p24 antigen detection in cell culture, when comparing a homogeneous group of HIV type 1 subtype B viruses with a heterogeneous group of divergent strains.

Failure to detect infection with HIV-1 non-B subtypes in some antibody screening assays has been shown. To date, however, no studies have been published evaluating the capacity of standard tests to quantify replication of divergent HIV-1 in cell culture. Reverse transcriptase (RT) activity and p24 antigen assays are the two methods most commonly used for this purpose. A homogeneous panel of HIV-1 subtype B viruses from northern Italy and a heterogeneous panel of diverse genetic subtypes (A to F and O) from different regions of the world were cultured under identical conditions. A new nonradioactive RT assay was used as a basis for comparison to evaluate the capacity of two p24 assays to quantify viral growth in both panels. Comparison of the p24 amount/RT activity (p24/RT) ratios showed that ratios in the subtype B panel tended to be markedly higher than in the diverse subtype panel. Greatest variation was seen with one of the subtype O isolates, where up to a 400 times lower ratio was obtained compared with the average ratio for the subtype B panel. In addition, one Thai subtype B virus also gave a markedly reduced ratio. Furthermore, comparison between the two p24 assays showed different abilities to detect p24 from different HIV-1 isolates. We discuss limitations for the use of anti-HIV-1 p24 antibodies produced by immunization with subtype B p24 in p24 assays.