Effects of oxytocin,lipopolysaccharide (LPS), and polyunsaturated fatty acids on prostaglandin secretion and gene expression in equine endometrial explant cultures

Increased secretion of prostaglandin F₂α (PGF₂α) within the uterus because of uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence PG production in many species, although the effects on the mare remain unknown. The present study aimed to determine fatty acid uptake in equine endometrial explants and evaluate their influence on PG secretion and expression of enzymes involved in PG synthesis in vitro. Equine endometrial explants were treated with 100 μM arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid and then challenged with oxytocin (250 nM) or lipopolysaccharide (LPS; 1 μg/mL). Production of PGF₂α and PG E₂ (PGE₂) was measured, and mRNA expression of enzymes involved in PG synthesis was determined with quantitative real-time PCR. Media concentrations of PGF₂α and PGE₂ were higher (P < 0.0001) from endometrial explants challenged with oxytocin or LPS compared with controls despite which fatty acid was added. Only DHA lowered (P < 0.0001) media concentrations of PGF₂α and PGE₂ from explants. Endometrial explants stimulated with oxytocin had increased expression of PG-endoperoxide synthase 1 (PTGS1; P < 0.02), PG-endoperoxide synthase 2 (PTGS2; P < 0.001), PG F₂α synthase (PGFS; P < 0.01), PG E₂ synthase (PGES; P < 0.01), and phospholipase A₂ (PLA₂; P < 0.005) compared with controls and regardless of fatty acid treatment; whereas stimulation with LPS increased expression of PTGS2 (P < 0.004), PGFS (P < 0.03), PGES (P < 0.01), and PLA₂ (P < 0.01) compared with controls and regardless of fatty acid treatment. Treatment with PUFAs, specifically DHA, can influence PG secretion in vitro through mechanisms other than enzyme expression.     

The impairment of IGF-I-stimulated protein synthesis and activation of protein kinase B, p70(S6k), MAP kinase, and p90(rsk) in mouse C2C12 myogenic cells exposed to high glucose and high insulin

The aim of the present study was to examine the effect of high glucose alone and in combination with high insulin on IGF-I-stimulated protein synthesis and the activation of IGF-I signaling pathways in mouse C2C12 myogenic cells. Experiments were performed on mouse C2C12 myoblasts subjected to differentiation under normal glucose (5 mmol/I), high glucose alone (15 mmol/l), or in combination with high insulin (50 nmol/l). Six-day differentiation under high glucose alone or in combination with high insulin resulted in IGF-I resistance, which was manifested by the abolition of the stimulatory effect on protein synthesis. IGF-I caused the activation of protein kinase B (PKB) in control C2C12 myogenic cells. Pretreatment with high glucose did not affect PKB phosphorylation whereas in cells differentiated under high glucose and high insulin PKB activation by IGF-I was markedly decreased as compared with control (differentiation under normal glucose). Neither the p70(S6k) protein content nor the pattern of IGF-I-mediated kinase activation was affected by pretreatment with high glucose, however high glucose and high insulin in combination caused an impairment of the p70(S6k) phosphorylation, in relation to the control. An increase in p42(MAPK) phosphorylation occurred under normal glucose conditions after the stimulation with IGF-I. The MAP kinase was not phosphorylated in response to IGF-I in cells preincubated with high glucose alone or in combination with high insulin. The pattern of p90(rsk) activation by IGF-I was not modified by pretreatment with high glucose, however no activation of p90(rsk) was found in cells pretreated with high glucose and high insulin in combination. In conclusions: 1) high glucose abolishes the stimulatory action of IGF-I on protein synthesis and it does not affect the activation of PKB, p70(S6k), and p90(rsk) in mouse C2C12 myogenic cells, 2) high glucose with high insulin in combination also abolish the stimulatory effect of IGF-I, but this phenomenon is accompanied by attenuated PKB and p70(S6k) activation and the lack of activation of p90(rsk), 3) apart from PKB, p70(S6k) and p90(rsk), other kinases are probably involved in the regulation of IGF-I-mediated protein synthesis in myogenic cells.