Fatty acids are an important ligand for peroxisome proliferator-activated receptor (PPAR) activation and transcriptional regulation of metabolic genes. To examine whether reduced plasma free fatty acid (FFA) availability affects the mRNA content of proteins involved in fuel metabolism in vivo, the skeletal muscle mRNA content of various transcription factors, transcriptional coactivators and genes encoding for lipid regulatory proteins were examined before and after 3 h of cycle exercise with (NA) and without (CON) pre-exercise ingestion of nicotinic acid (NA). NA resulted in a marked (3- to 6-fold) increase (P<0.05) in PPARα, PPARδ and PPAR coactivator 1α (PGC1α) mRNA, but was without effect on nuclear respiratory factor-1 and Forkhead transcription factor, fatty acid transcolase/CD36, carnitine palmitoyl transferase 1, hormone sensitive lipase (HSL) and pyruvate dehydrogenase kinase 4. Exercise in CON was associated with increased (P<0.05) PPARα, PPARδ and PGC1α mRNA, which was similar in magnitude to levels observed with NA at rest. Exercise was generally without effect on the mRNA content of lipid regulatory proteins in CON and did not affect the mRNA content of the measured subset of transcription factors, transcriptional co-activators and lipid regulatory proteins during NA. To determine the possible mechanisms by which NA might affect PGC1α expression, we measured p38 MAP kinase (MAPK) and plasma epinephrine. Phosphorylation of p38 MAPK was increased (P<0.05) by NA treatment at rest, and this correlated (r 2=0.84, P<0.01) with increased PGC1α. Despite this close relationship, increasing p38 MAPK in human primary myotubes was without effect on PGC1α mRNA content. Plasma epinephrine was elevated (P<0.05) by NA at rest (CON: 0.27±0.06, NA: 0.72±0.11 nM) and throughout exercise. Incubating human primary myotubes with epinephrine increased PGC1α independently of changes in p38 MAPK phosphorylation. Hence, despite the fact that NA ingestion decreased FFA availability, it promoted the induction of PPARα/δ and PGC1α gene expression to a similar degree as prolonged exercise. We suggest that the increase in PGC1α may be due to the elevated plasma epinephrine levels. Despite these changes in transcription factors/coactivators, the mRNA content of lipid regulatory proteins was generally unaffected by plasma FFA availability.
M J Watt, R J Southgate, A G Holmes, and M A Febbraio
Philippa Davies, Kate Watt, Sharon M Kelly, Caroline Clark, Nicholas C Price, and Iain J McEwan
Poly-amino acid repeats, especially long stretches of glutamine (Q), are common features of transcription factors and cell-signalling proteins and are prone to expansion, resulting in neurodegenerative diseases. The amino-terminal domain of the androgen receptor (AR-NTD) has a poly-Q repeat between 9 and 36 residues, which when it expands above 40 residues results in spinal bulbar muscular atrophy. We have used spectroscopy and biochemical analysis to investigate the structural consequences of an expanded repeat (Q45) or removal of the repeat (ΔQ) on the folding of the AR-NTD. Circular dichroism spectroscopy revealed that in aqueous solution, the AR-NTD has a relatively limited amount of stable secondary structure. Expansion of the poly-Q repeat resulted in a modest increase in α-helix structure, while deletion of the repeat resulted in a small loss of α-helix structure. These effects were more pronounced in the presence of the structure-promoting solvent trifluoroethanol or the natural osmolyte trimethylamine N-oxide. Fluorescence spectroscopy showed that the microenvironments of four tryptophan residues were also altered after the deletion of the Q stretch. Other structural changes were observed for the AR-NTDQ45 polypeptide after limited proteolysis; in addition, this polypeptide not only showed enhanced binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonic acid but was more sensitive to urea-induced unfolding. Taken together, these findings support the view that the presence and length of the poly-Q repeat modulate the folding and structure of the AR-NTD.