Mapping cells and sub-cellular organelles on 2-D gels: ‘new tricks for an old horse’

AbstractNowadays, investigators in all fields are faced with the identification of unknown, up- or down-regulated, modified proteins that they are trying to identify. Two-dimensional (2-D) gel electrophoresis, with its ability to resolve several thousand proteins, is an extremely powerful technique. The current resolution and reproducibility of 2-D gel technology and the establishment of computer assisted 2-D gel protein databases have paved new ways for the identification of proteins

Lifelong physical activity is associated with promoter hypomethylation of genes involved in metabolism, myogenesis, contractile properties and oxidative stress resistance in aged human skeletal muscle

Abstract Lifelong regular physical activity is associated with reduced risk of type 2 diabetes (T2D), maintenance of muscle mass and increased metabolic capacity. However, little is known about epigenetic mechanisms that might contribute to these beneficial effects in aged individuals. We investigated the effect of lifelong physical activity on global DNA methylation patterns in skeletal muscle of healthy aged men, who had either performed regular exercise or remained sedentary their entire lives (average age 62 years). DNA methylation was significantly lower in 714 promoters of the physically active than inactive men while methylation of introns, exons and CpG islands was similar in the two groups. Promoters for genes encoding critical insulin-responsive enzymes in glycogen metabolism, glycolysis and TCA cycle were hypomethylated in active relative to inactive men. Hypomethylation was also found in promoters of myosin light chain, dystrophin, actin polymerization, PAK regulatory genes and oxidative stress response genes. A cluster of genes regulated by GSK3β-TCF7L2 also displayed promoter hypomethylation. Together, our results suggest that lifelong physical activity is associated with DNA methylation patterns that potentially allow for increased insulin sensitivity and a higher expression of genes in energy metabolism, myogenesis, contractile properties and oxidative stress resistance in skeletal muscle of aged individuals

Bimodal Effect on Pancreatic β-Cells of Secretory Products From Normal or Insulin-Resistant Human Skeletal Muscle

OBJECTIVE: Type 2 diabetes is characterized by insulin resistance with a relative deficiency in insulin secretion. This study explored the potential communication between insulin-resistant human skeletal muscle and primary (human and rat) beta-cells. RESEARCH DESIGN AND METHODS: Human skeletal muscle cells were cultured for up to 24 h with tumor necrosis factor (TNF)-alpha to induce insulin resistance, and mRNA expression for cytokines was analyzed and compared with controls (without TNF-alpha). Conditioned media were collected and candidate cytokines were measured by antibody array. Human and rat primary beta-cells were used to explore the impact of exposure to conditioned media for 24 h on apoptosis, proliferation, short-term insulin secretion, and key signaling protein phosphorylation and expression. RESULTS: Human myotubes express and release a different panel of myokines depending on their insulin sensitivity, with each panel exerting differential effects on beta-cells. Conditioned medium from control myotubes increased proliferation and glucose-stimulated insulin secretion (GSIS) from primary beta-cells, whereas conditioned medium from TNF-alpha-treated insulin-resistant myotubes (TMs) exerted detrimental effects that were either independent (increased apoptosis and decreased proliferation) or dependent on the presence of TNF-alpha in TM (blunted GSIS). Knockdown of beta-cell mitogen-activated protein 4 kinase 4 prevented these effects. Glucagon-like peptide 1 protected beta-cells against decreased proliferation and apoptosis evoked by TMs, while interleukin-1 receptor antagonist only prevented the latter. CONCLUSIONS: Taken together, these data suggest a possible new route of communication between skeletal muscle and beta-cells that is modulated by insulin resistance and could contribute to normal beta-cell functional mass in healthy subjects, as well as the decrease seen in type 2 diabetes

Calprotectin — A Novel Marker of Obesity

BACKGROUND: The two inflammatory molecules, S100A8 and S100A9, form a heterodimer, calprotectin. Plasma calprotectin levels are elevated in various inflammatory disorders. We hypothesized that plasma calprotectin levels would be increased in subjects with low-grade systemic inflammation i.e. either obese subjects or subjects with type 2 diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Plasma calprotectin and skeletal muscle S100A8 mRNA levels were measured in a cohort consisting of 199 subjects divided into four groups depending on presence or absence of type 2 diabetes (T2D), and presence or absence of obesity. There was a significant interaction between obesity and T2D (p = 0.012). Plasma calprotectin was increased in obese relative to non-obese controls (p<0.0001), whereas it did not differ between obese and non-obese patients with T2D (p = 0.62). S100A8 mRNA levels in skeletal muscle were not influenced by obesity or T2D. Multivariate regression analysis (adjusting for age, sex, smoking and HOMA2-IR) showed plasma calprotectin to be strongly associated with BMI, even when further adjusted for fitness, CRP, TNF-alpha or neutrophil number. CONCLUSIONS/SIGNIFICANCE: Plasma calprotectin is a marker of obesity in individuals without type 2 diabetes

Plasma and Muscle Myostatin in Relation to Type 2 Diabetes

OBJECTIVE: Myostatin is a secreted growth factor expressed in skeletal muscle tissue, which negatively regulates skeletal muscle mass. Recent animal studies suggest a role for myostatin in insulin resistance. We evaluated the possible metabolic role of myostatin in patients with type 2 diabetes and healthy controls. DESIGN: 76 patients with type 2 diabetes and 92 control subjects were included in the study. They were matched for age, gender and BMI. Plasma samples and biopsies from the vastus lateralis muscle were obtained to assess plasma myostatin and expression of myostatin in skeletal muscle. RESULTS: Patients with type 2 diabetes had higher fasting glucose (8.9 versus 5.1 mmol/L, P<0.001), plasma insulin (68.2 versus 47.2 pmol/L, P<0.002) and HOMA2-IR (1.6 versus 0.9, P<0.0001) when compared to controls. Patients with type 2 diabetes had 1.4 (P<0.01) higher levels of muscle myostatin mRNA content than the control subjects. Plasma myostatin concentrations did not differ between patients with type 2 diabetes and controls. In healthy controls, muscle myostatin mRNA correlated with HOMA2-IR (r = 0.30, P<0.01), plasma IL-6 (r = 0.34, P<0.05) and VO2 max (r = -0.26, P<0.05), however, no correlations were observed in patients with type 2 diabetes. CONCLUSIONS: This study supports the idea that myostatin may have a negative effect on metabolism. However, the metabolic effect of myostatin appears to be overruled by other factors in patients with type 2 diabetes

Plasma YKL-40: A BMI-Independent Marker of Type 2 Diabetes

OBJECTIVE—YKL-40 is produced by macrophages, and plasma YKL-40 is elevated in patients with diseases characterized by inflammation. In the present study, YKL-40 was examined in relation to obesity, inflammation, and type 2 diabetes

GDF15 is an exercise-induced hepatokine regulated by glucagon and insulin in humans

ObjectiveGrowth differentiation factor (GDF)-15 is implicated in regulation of metabolism and circulating GDF15 increases in response to exercise. The source and regulation of the exercise-induced increase in GDF15 is, however not known.MethodPlasma GDF15 was measured by ELISA under the following conditions: 1) Arterial-to-hepatic venous differences sampled before, during, and after exercise in healthy male subjects (n=10); 2) exogenous glucagon infusion compared to saline infusion in resting healthy subjects (n=10); 3) an acute exercise bout with and without a pancreatic clamp (n=6); 4) healthy subjects for 36 hours (n=17), and 5) patients with anorexia nervosa (n=25) were compared to healthy age-matched subjects (n=25). Tissue GDF15 mRNA content was determined in mice in response to exhaustive exercise (n=16).ResultsThe splanchnic bed released GDF15 to the circulation during exercise and increasing the glucagon-to-insulin ratio in resting humans led to a 2.7-fold (P&lt;0.05) increase in circulating GDF15. Conversely, inhibiting the exercise-induced increase in the glucagon-to-insulin ratio blunted the exercise-induced increase in circulating GDF15. Fasting for 36 hours did not affect circulating GDF15, whereas resting patients with anorexia nervosa displayed elevated plasma concentrations (1.4-fold, P&lt;0.05) compared to controls. In mice, exercise increased GDF15 mRNA contents in liver, muscle, and adipose tissue.ConclusionIn humans, GDF15 is a “hepatokine” which increases during exercise and is at least in part regulated by the glucagon-to-insulin ratio. Moreover, chronic energy deprivation is associated with elevated plasma GDF15, which supports that GDF15 is implicated in metabolic signalling in humans