Gene expression profiling in skeletal muscle of Zucker diabetic fatty rats: implications for a role of stearoyl-CoA desaturase 1 in insulin resistance.

AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes. Therefore, we sought to identify and validate genes involved in the development of insulin resistance in skeletal muscle. MATERIALS: Differentially regulated genes in skeletal muscle of male obese insulin-resistant, and lean insulin-sensitive Zucker diabetic fatty (ZDF) rats were determined using Affymetrix microarrays. Based on these data, various aspects of glucose disposal, insulin signalling and fatty acid composition were analysed in a muscle cell line overexpressing stearoyl-CoA desaturase 1 (SCD1). RESULTS: Gene expression profiling in insulin-resistant skeletal muscle revealed the most pronounced changes in gene expression for genes involved in lipid metabolism. Among these, Scd1 showed increased expression in insulin-resistant animals, correlating with increased amounts of palmitoleoyl-CoA. This was further investigated in a muscle cell line that overexpressed SCD1 and accumulated lipids, revealing impairments of glucose uptake and of different steps of the insulin signalling cascade. We also observed differential effects of high-glucose and fatty acid treatment on glucose uptake and long-chain fatty acyl-CoA profiles, and in particular an accumulation of palmitoleoyl-CoA in cells overexpressing SCD1. CONCLUSIONS/INTERPRETATION: Insulin-resistant skeletal muscle of ZDF rats is characterised by a specific gene expression profile with increased levels of Scd1. An insulin-resistant phenotype similar to that obtained by treatment with palmitate and high glucose can be induced in vitro by overexpression of SCD1 in muscle cells. This supports the hypothesis that elevated SCD1 expression is a possible cause of insulin resistance and type 2 diabetes.

The cell surface receptor is a major determinant restricting the host range of the B-lymphotropic papovavirus.

The B-lymphotropic papovavirus (LPV) productively infects only a subset of human B-lymphoma-derived cell lines while transfection of the viral genome yields infectious viral particles in a much wider variety of human hematopoietic cell lines. We have analyzed the contribution of a putative LPV receptor on the cell surface of B-cell lines in restricting the virus host range. In order to establish a quantitative virus binding assay for LPV, infectious virus particles were highly purified by metrizamide equilibrium density centrifugation and used as immunogens to raise seven mouse monoclonal antibodies specific for LPV VP1. Virus particle binding was quantitated in an indirect, nonradioactive assay with an LPV VP1-specific enzyme-linked immunosorbent assay. Binding of LPV particles to permissive human B-lymphoma cell line BJA-B occurred within minutes. Kinetics and capacity of binding were similar at 4 and 37 degrees C. A BJA-B cell was estimated to bind approximately 600 virus particles at conditions under which 50% of the administered virus was bound. The sialidase and trypsin sensitivities of the cellular virus binding moiety show that sialylated and proteinaceous components are necessary components of the LPV receptor on BJA-B cells. Despite a high binding capacity of BJA-B cells for simian virus 40, LPV binding was not significantly affected by a 20-fold excess of simian virus 40 particles, indicating that these related polyomaviruses do not bind to the same receptor on BJA-B cells. Reduction of LPV binding to sialidase-pretreated BJA-B cells was accompanied by a similar reduction of infection, indicating that virus binding may be a limiting factor in the LPV replicative cycle. The two highly LPV-permissive human B-lymphoma cell lines BJA-B and Namalwa displayed high virus binding whereas low and nonpermissive hematopoietic cell lines showed reduced or undetectable virus binding. We conclude that the inability of LPV particles to productively infect the nonpermissive human hematopoietic cell lines analyzed is probably due to the absence or insufficient expression of a functional cell surface receptor.

Synthetic oligonucleotide probes complementary to rRNA for group- and species-specific detection of mycoplasmas.

A variety of genetic probes has been used for the detection of pathogenic bacteria. Here we present a straightforward approach for the development of group- and species-specific oligonucleotide probes complementary to mycoplasmal rRNA. These probes are useful not only for the identification of mycoplasmas in clinical specimens, but also for the detection of Mollicutes in contaminated cell cultures. Radiolabeled rDNA probes detected less than 1 x 10(3) organisms in an RNA-DNA hybridization procedure.