Regulation of p85alpha phosphatidylinositol-3-kinase expression by peroxisome proliferator-activated receptors (PPARs) in human muscle cells.

Regulation of p85a phosphatidylinositol-3-kinase (p85alphaPI-3K) expression by peroxisome proliferator-activated receptor (PPAR) activators was studied in human skeletal muscle cells. Activation of PPARgamma or PPARbeta did not modify the expression of p85alphaPI-3K. In contrast, activation of PPARalpha increased p85alphaPI-3K mRNA. This effect was potentiated by 9-cis-retinoic acid, an activator of RXR. Up-regulation of p85alphaPI-3K gene expression resulted in a rise in p85alphaPI-3K protein level and in an increase in insulin-induced PI3-kinase activity. According to the role of p85alphaPI-3K in insulin action, these results suggest that drugs with dual action on both PPARgamma and PPARalpha can be of interest for the treatment of insulin resistance.

The regulation of uncoupling protein-2 gene expression by omega-6 polyunsaturated fatty acids in human skeletal muscle cells involves multiple pathways, including the nuclear receptor peroxisome proliferator-activated receptor beta.

Fatty acids have been postulated to regulate uncoupling protein (UCP) gene expression in skeletal muscle in vivo. We have identified, at least in part, the mechanism by which polyunsaturated fatty acids increase UCP-2 expression in primary culture of human muscle cells. omega-6 fatty acids and arachidonic acid induced a 3-fold rise in UCP-2 mRNA levels possibly through transcriptional activation. This effect was prevented by indomethacin and mimicked by prostaglandin (PG) E(2) and carbaprostacyclin PGI(2), consistent with a cyclooxygenase-mediated process. Incubation of myotubes for 6 h with 100 micrometer arachidonic acid resulted in a 150-fold increase in PGE(2) and a 15-fold increase in PGI(2) in the culture medium. Consistent with a role of cAMP and protein kinase A, both prostaglandins induced a marked accumulation of cAMP in human myotubes, and forskolin reproduced the effect of arachidonic acid on UCP-2 mRNA expression. Inhibition of protein kinase A with H-89 suppressed the effect of PGE(2), whereas cPGI(2) and arachidonic acid were still able to increase ucp-2 gene expression, suggesting additional mechanisms. We found, however, that the MAP kinase pathway was not involved. Prostaglandins, particularly PGI(2), are potent activators of the peroxisome proliferator-activated receptors. A specific agonist of peroxisome proliferator-activated receptor (PPAR) beta (L165041) increased UCP-2 mRNA levels in myotubes, whereas activation of PPARalpha or PPARgamma was ineffective. These results suggest thus that ucp-2 gene expression is regulated by omega-6 fatty acids in human muscle cells through mechanisms involving at least protein kinase A and the nuclear receptor PPARbeta.

Triiodothyronine-mediated up-regulation of UCP2 and UCP3 mRNA expression in human skeletal muscle without coordinated induction of mitochondrial respiratory chain genes.

Triiodothyronine (T3) increases mitochondrial respiration and promotes the uncoupling between oxygen consumption and ATP synthesis. T3 effect is mediated partly through transcriptional control of genes encoding mitochondrial proteins. We determined the effect of T3 on mRNA levels of uncoupling proteins (UCP) and proteins involved in the biogenesis of the respiratory chain in human skeletal muscle and on UCP2 mRNA expression in adipose tissue. Ten young, healthy males received 75 to 100 5g of T3 per day for 14 days. The increase in plasma-free T3 levels was associated with an increase of resting metabolic rate and a decrease of respiratory quotient. In skeletal muscle, treatment with T3 induced a twofold increase of both UCP2 and UCP3 mRNA levels (p c oxidase subunits 2 and 4, nuclear respiratory factor 1, mitochondrial transcription factor A, and the co-activator PGC1 did not change during the treatment. In adipose tissue, UCP2 mRNA levels increased threefold. The direct effect of T3 on skeletal muscle an d adipose tissue UCP2 and UCP3 mRNA expression was demonstrated in vitro in human primary cultures. Our data show that T3 induces UCP2 and UCP3 mRNA expression in humans. In skeletal muscle, UCP regulation by T3 is not associated with the transcriptional regulation of respiratory chain proteins.

Variations in the number of uterine angiotensin receptors following changes in plasma angiotensin levels.

3H-labelled angiotensin II binding to receptor sites was studied in plasma membranes isolated from myometrial homogenates of uterine horns. Removal of the kidneys, which results in the disappearance of plasma angiotensin II, was followed 19 h after nephrectomy by an increase in the number of uterine receptor sites without significant variation in the apparent dissociation constant. Acute pressor i.v. injection of angiotensin II into nephrectomized rats immediately before removing uteri, did not affect the number of uterine angiotensin receptors, whereas long-lasting angiotensin infusion did reduce the number of receptors. These changes cannot be accounted for by variations in the occupancy of receptor sites. These results demonstrate that the number of angiotensin receptors, at least in uterine contractile cells, is affected by chronic variations of endogenous angiotensin levels. The relation between the specific supersensitivity to angiotensin II observed in uteri from nephrectomized rats and the variations at the receptor level is discussed.

Variations in the number of uterine angiotensin receptors following changes in plasma angiotensin levels.

3H-labelled angiotensin II binding to receptor sites was studied in plasma membranes isolated from myometrial homogenates of uterine horns. Removal of the kidneys, which results in the disappearance of plasma angiotensin II, was followed by an increase in the number of uterine receptor sites without significant variation in the apparent dissociation constant, which became significant 15 h after nephrectomy. Acute pressor intravenous injection of angiotensin II into nephrectomized rats immediately before removing uteri, did not affect the number of uterine angiotensin receptors, whereas long-lasting angiotensin infusion did reduce the number of receptors. These results provided an explanation for the specific supersensitivity to angiotensin II, observed in uteri excised from nephrectomized rats, which cannot be accounted for by variations in the occupancy of receptor sites. These results also demonstrate that the number of angiotensin receptors, at least in uterine contractile cells, is affected by chronic variations of endogenous angiotensin levels.

Angiotensin-induced variations of receptors in rat uterine membranes.

1. 3H-labelled angiotensin II specfically binds to plasma membranes of rat uterine smooth muscle cells. Two classes of binding sites differing in their affinity for the hormone were demonstrated. The high-affinity binding sites (KD 29 degrees C approximately 2.0 X 10(-8) mol/l) probably correspond to the receptors involved in the biological response. 2. Bilateral nephrectomy significantly increases the concentration of 3H-labelled angiotensin-binding sites, a phenomenon which seems unrelated to the freeing of receptor sites secondary to the suppression of plasma angiotensin. This phenomenon may be responsible for the specific hypersensitivity in vitro to angiotensin of uteri excised in anephric rats as compared with normal rats. 3. Angiotensin II infusion in nephrectomized rats reduced the concentration of 3H-labelled angiotensin-binding sites. 4. It is suggested that the angiotensin receptor concentration is regulated by the concentration of circulating angiotensin.