Primary Cultures Of Human Muscle Research Articles

Glucocorticoid Modulation of Insulin Signaling in Human Subcutaneous Adipose Tissue

Glucocorticoid (GC) excess is characterized by central obesity, insulin resistance, and in some cases, type 2 diabetes. However, the impact of GC upon insulin signaling in human adipose tissue has not been fully explored. We have examined the effect of GC upon insulin signaling in both human sc primary preadipocyte cultures and a novel human immortalized sc adipocyte cell line (Chub-S7) and contrasted this with observations in primary cultures of human skeletal muscle. This is an in vitro study characterizing the impact of GC upon insulin signaling in human tissues. Biopsy specimens were from healthy volunteers who gave their full and informed written consent. Combinations of treatments, including GC, RU38486, and wortmannin, were used. Insulin signaling cascade gene and protein expression and insulin-stimulated glucose uptake were determined. In human adipocytes, pretreatment with GC induced a dose-dependent [1.0 (control); 1.2 +/- 0.1 (50 nm); 2.2 +/- 0.2 (250 nm), P < 0.01 vs. control; 3.4 +/- 0.2 (1000 nm), P < 0.001 vs. control] and time-dependent [1.0 (1 h); 3.2 +/- 2.0 (6 h); 9.1 +/- 5.9 (24 h), P < 0.05 vs. 1 h; 4.5 +/- 2.2 (48 h)] increase in insulin-stimulated protein kinase B/akt phosphorylation. In addition, whereas insulin receptor substrate (IRS)-1 protein expression did not change, IRS-1 tyrosine phosphorylation increased. Furthermore, GC induced IRS-2 mRNA expression (2.8-fold; P < 0.05) and increased insulin-stimulated glucose uptake [1.0 (control) 1.8 +/- 0.1 (insulin) vs. 2.8 +/- 0.2 (insulin + GC); P < 0.05]. In contrast, in primary cultures of human muscle, GC decreased insulin-stimulated glucose uptake [1.0 (control) 1.9 +/- 0.2 (insulin) vs. GC 1.3 +/- 0.1 (insulin + GC); P < 0.05]. We have demonstrated tissue-specific regulation of insulin signaling by GC. Within sc adipose tissue, GCs augment insulin signaling, yet in muscle GCs cause insulin resistance. We propose that enhanced insulin action in adipose tissue increases adipocyte differentiation, thereby contributing to GC-induced obesity.

Mitochondrial Signals for the Nucleus

What's a transcription factor of the basic helix-loop-helix leucine zipper family doing in the cytoplasm? Sans et al. explored why nearly all of the transcription factor MondoA and its binding partner protein called Mlx is found in the cytoplasm but the protein appears to function as a transcription factor in the nucleus. The authors found that in primary cultures of human muscle or erythroblast cells, MondoA and Mlx were associated with the mitochondria. Inhibition of nuclear shuttling of the proteins with an inhibitor of the CRM1 nuclear export factor caused MondoA (detected by confocal immunofluorescence microscopy) to accumulate in the nucleus. Thus, the authors suspected that MondoA could function to carry signals from the mitochondria to the nucleus. They obtained further circumstantial evidence for such a role from microarray experiments designed to detect genes transcribed in response to MondoA and Mlx. MondoA-Mlx regulated a broad range of targets, which included many genes associated with carbohydrate metabolism. Chromatin immunoprecipitation experiments showed that hexokinase II, 6-phospofructo-2-kinase/fructose-2,6-bisphosphatase 3, and lactate dehydrogenase A--three enzymes important for glycolysis--are direct targets for MondoA-Mlx. Indeed, depletion of MondoA by RNA interference caused a decrease in the rate of glycolysis (measured as the rate of conversion of 5-[ 3 H]glucose to 3 H 2 O). Thus, although other functions are not excluded, the authors propose that MondoA may function as an intracellular metabolic sensor. C. L. Sans, D. J. Satterwhite, C. A. Stoltzman, K. T. Breen, D. E. Ayer, MondoA-Mlx heterodimers are candidate sensors of cellular energy status: Mitochondrial localization and direct regulation of glycolysis. Mol. Cell. Biol. 26 , 4863-4871 (2006). [Abstract] [Full Text]

Expression of the intermediate filament‐associated protein related to β‐amyloid precursor protein is developmentally regulated in cultured cells

It was previously reported that a monoclonal antibody to beta-amyloid precursor protein (mab22C11; Boehringer Mannheim, Indianapolis, IN) labels an intermediate filament-associated protein (beta APP-IFAP) in cultured human skin fibroblasts (Dooley et al.: J Neurosci Res 33:60-67, 1992). The time course of its expression and association with different classes of intermediate filaments has been assessed in neurons, Schwann cells, and astrocytes in dissociated cultures of murine brain and spinal cord-dorsal root ganglia; in primary cultures of human muscle; and in the epithelial cell line PtK1. beta APP-IFAP was expressed in all non-neuronal cell types examined. Mab22C11 immunoreactivity was minimal or absent following dissociation or subculture, but gradually increased with time. In fibroblasts, myoblasts, and epithelial cells, the distribution eventually resembled that of vimentin. With the exception of glial fibrillary acidic protein (GFAP), beta APP-IFAP was not associated with the intermediate filament proteins characteristically found in differentiated cells, i.e., desmin, the cytokeratins, and neurofilament proteins. No labeling of neurons by mab22C11 was observed at any stage of in vitro maturation. In sections of Alzheimer's brain, the antibody labeled a subpopulation of reactive astrocytes. It is suggested that beta APP-IFAP may be the product of a member of the beta APP multigene family expressed developmentally in non-neuronal cells.

Interleukin-2 inhibits sodium currents in human muscle cells.

The effect of the T cell growth factor interleukin-2 (IL-2) on muscular Na+ channels was studied in myoballs produced from primary human muscle cultures. The transient Na+ inward currents of the myoballs, elicited by repetitive stimulation at 1 Hz and recorded in the whole-cell mode, were inhibited by IL-2 applied to the external solution, the half maximum effect occurring at 300 U/ml. The effect was complete within 5 s and was totally reversible, the on and off effects having identical time courses. The h infinity curve was shifted in negative direction indicating that the mechanism of IL-2 action is a conversion of the Na+ channels into a state of inactivation. The reaction of the IL-2 solution with an anti IL-2 antibody neutralized the inhibitory effect on the Na+ currents, indicating a specific effect of the peptide growth factor interleukin-2 on muscular Na+ channels. The connection of IL-2 and Na+ channels may be important in inflammatory processes of muscle and nerve.

Vital-freezing of human muscle cultures for storage and reculture.

The vital-freezing of human muscle cultures enabling long-term storage in liquid nitrogen and future reculturing is described. Although human muscle is more fastidious in tissue culture than most cells of other origin, it was successfully frozen and re-established in culture. Cultures re-established after storage in liquid nitrogen differentiated and matured in the same way as primary human muscle cultures, and had the same histochemical and electron microscopic characteristics.