PGC-1 alpha and PGC-1 beta increase protein synthesis via ERR alpha in C2C12 myotubes

The transcriptional coactivators peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and PGC-1β are positive regulators of skeletal muscle mass and energy metabolism; however, whether they influence muscle growth and metabolic adaptations via increased protein synthesis is not clear. This study revealed PGC-1α or PGC-1β overexpression in C2C12 myotubes increased protein synthesis and myotube diameter under basal conditions and attenuated the loss in protein synthesis following the treatment with the catabolic agent, dexamethasone. To investigate whether PGC-1α or PGC-1β signal through the Akt/mTOR pathway to increase protein synthesis, treatment with the PI3K and mTOR inhibitors, LY294002 and rapamycin, respectively, was undertaken but found unable to block PGC-1α or PGC-1β’s promotion of protein synthesis. Furthermore, PGC-1α and PGC-1β decreased phosphorylation of Akt and the Akt/mTOR substrate, p70S6K. In contrast to Akt/mTOR inhibition, the suppression of ERRα, a major effector of PGC-1α and PGC-1β activity, attenuated the increase in protein synthesis and myotube diameter in the presence of PGC-1α or PGC-1β overexpression. To characterize further the biological processes occurring, gene set enrichment analysis of genes commonly regulated by both PGC-1α and PGC-1β was performed following a microarray screen. Genes were found enriched in metabolic and mitochondrial oxidative processes, in addition to protein translation and muscle development categories. This suggests concurrent responses involving both increased metabolism and myotube protein synthesis. Finally, based on their known function or unbiased identification through statistical selection, two sets of genes were investigated in a human exercise model of stimulated protein synthesis to characterize further the genes influenced by PGC-1α and PGC-1β during physiological adaptive changes in skeletal muscle

The measurement of GLUT4 translocation in 3T3-L1 adipocytes

Type 2 diabetes (T2D) is one of the fastest growing threats to human health in westernised and developing countries and is associated with central obesity, atherosclerosis, dyslipidaemia, hyperinsulinaemia and&nbsp; hypertension. Insulin resistance, defined as a diminished response to ordinary levels of circulating insulin in one or more peripheral tissues, is an integral feature of T2D pathophysiology. This includes an impairment of insulin to inhibit hepatic glucose output and to stimulate glucose disposal into muscle and fat. While insulin is responsible for a number of specific biological responses, stimulation of glucose transport is critical for the maintenance of glucose homeostasis. The primary mechanism for insulin stimulation of glucose uptake into muscle and fat is the translocation of glucose transporter 4 (GLUT4) to the cell surface from intracellular storage vesicles within the cell. A major advantage in focussing on insulin regulation of glucose transport is that this represents the endpoint of multiple upstream signalling pathways. This chapter describes the measurement of GLUT4 translocation in cultured cells and its potential application for both&nbsp; mechanistic and therapeutic studies.<br /

3T3-L1 Preadipocytes Exhibit Heightened Monocyte-Chemoattractant Protein-1 Response to Acute Fatty Acid Exposure

<div><p>Preadipocytes contribute to the inflammatory responses within adipose tissue. Whilst fatty acids are known to elicit an inflammatory response within adipose tissue, the relative contribution of preadipocytes and mature adipocytes to this is yet to be determined. We aimed to examine the actions of common dietary fatty acids on the acute inflammatory and adipokine response in 3T3-L1 preadipocytes and differentiated mature adipocytes. Gene expression levels of key adipokines in 3T3-L1 preadipocytes and adipocytes were determined following incubation with palmitic acid, myristic acid or oleic acid and positive inflammatory control, lipopolysaccharide for 2 and 4 h. Inflammatory kinase signalling was assessed by analysis of nuclear factor-κB, p38-mitogen-activated protein kinase and c-jun amino-terminal kinase phosphorylation. Under basal conditions, intracellular monocyte chemoattractant protein-1 and interleukin-6 gene expression levels were increased in preadipocytes, whereas mature adipocytes expressed increased gene expression levels of leptin and adiponectin. Fatty acid exposure at 2 and 4 h increased both monocyte chemoattractant protein-1 and interleukin-6 gene expression levels in preadipocytes to greater levels than in mature adipocytes. There was an accompanying increase of inhibitor of κB-α degradation and nuclear factor-κB (p65) (Ser536) phosphorylation with fatty acid exposure in the preadipocytes only. The current study points to preadipocytes rather than the adipocytes as the contributors to both immune cell recruitment and inflammatory adipokine secretion with acute increases in fatty acids.</p></div

TNFα mRNA levels in 3T3-L1 preadipocytes and adipocytes.

<p>TNFα mRNA expression of 3T3-L1 preadipocytes (hatched bars) and adipocytes (open bars) treated with (A) LPS (10 ng/ml); (B) Palmitic acid (0.5 mM); (C) Myristic acid (0.5 mM); and (D) Oleic acid (0.5 mM) at 0, 2 and 4 h. Data are presented as mean ±SEM (<i>n</i> = 5) normalised to 36B4. *** p&lt;0.001 versus preadipocytes, ^## p&lt;0.01 versus 0 h. Main cell type effect C p&lt;0.05, CC p&lt;0.01.</p

MCP-1 mRNA levels in 3T3-L1 preadipocytes and adipocytes.

<p>MCP-1 gene expression levels of 3T3-L1 preadipocytes (hatched bars) and adipocytes (open bars) treated with (A) LPS (10 ng/ml); (B) Palmitic acid (0.51 mM); (C) Myristic acid (0.5 mM); and (D) Oleic acid (0.5 mM) at 0, 2 and 4 h. Data are presented as mean ±SEM (<i>n</i> = 5) normalised to 36B4. ** p&lt;0.01, *** p&lt;0.001 versus preadipocytes, ^## p&lt;0.01, ^### p&lt;0.001 versus 0 h. Main time effect T p&lt;0.05, main cell type effect CC p&lt;0.01.</p

IL-6 mRNA levels in 3T3-L1 preadipocytes and adipocytes.

<p>IL-6 gene expression levels of 3T3-L1 preadipocytes (hatched bars) and adipocytes (open bars) treated with (A) LPS (10 ng/ml); (B) Palmitic acid (0.5 mM); (C) Myristic acid (0.5 mM); and (D) Oleic acid (0.5 mM) at 0, 2 and 4 h. Data are presented as mean ±SEM (<i>n</i> = 5) normalised to 36B4. *** p&lt;0.001 versus preadipocytes, ^# p&lt;0.05, ^## p&lt;0.01, ^### p&lt;0.001 versus 0 h. Main time effect T p&lt;0.05.</p