C-reactive protein induces phosphorylation of insulin receptor substrate-1 on Ser307 and Ser 612 in L6 myocytes, thereby impairing the insulin signalling pathway that promotes glucose transport.

AIMS/HYPOTHESIS: C-reactive protein (CRP) is associated with insulin resistance and predicts development of type 2 diabetes. However, it is unknown whether CRP directly affects insulin signalling action. To this aim, we determined the effects of human recombinant CRP (hrCRP) on insulin signalling involved in glucose transport in L6 myotubes. MATERIALS AND METHODS: L6 myotubes were exposed to endotoxin-free hrCRP and insulin-stimulated activation of signal molecules, glucose uptake and glycogen synthesis were assessed. RESULTS: We found that hrCRP stimulates both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2 activity. These effects were paralleled by a concomitant increase in IRS-1 phosphorylation at Ser(307) and Ser(612), respectively. The stimulatory effects of hrCRP on IRS-1 phosphorylation at Ser(307) and Ser(612) were partially reversed by treatment with specific JNK and ERK1/2 inhibitors, respectively. Exposure of L6 myotubes to hrCRP reduced insulin-stimulated phosphorylation of IRS-1 at Tyr(632), a site essential for engaging p85 subunit of phosphatidylinositol-3 kinase (PI-3K), protein kinase B (Akt) activation and glycogen synthase kinase-3 (GSK-3) phosphorylation. These events were accompanied by a decrease in insulin-stimulated glucose transporter (GLUT) 4 translocation to the plasma membrane, glucose uptake and glucose incorporation into glycogen. The inhibitory effects of hrCRP on insulin signalling and insulin-stimulated GLUT4 translocation were reversed by treatment with JNK inhibitor I and the mitogen-activated protein kinase inhibitor, PD98059. CONCLUSIONS/INTERPRETATION: Our data suggest that hrCRP may cause insulin resistance by increasing IRS-1 phosphorylation at Ser(307) and Ser(612) via JNK and ERK1/2, respectively, leading to impaired insulin-stimulated glucose uptake, GLUT4 translocation, and glycogen synthesis mediated by the IRS-1/PI-3K/Akt/GSK-3 pathway.

Control of mouse cumulus cell-oocyte complex integrity before and after ovulation: plasminogen activator synthesis and matrix degradation.

During the preovulatory period, cumulus cells (CCs) form a hyaluronan-protein extracellular matrix (cumulus expansion) that positively influences oocyte fertilization. Degradation of this matrix and CC-oocyte complex (COC) dissociation occurs within a few hours of ovulation and parallels the aging of oocytes. Modulation of CC proteolytic activity by gonadotropins and oocyte soluble factors has been hypothesized to determine such cumulus matrix changes. In the present study, we investigated plasminogen activator (PA) synthesis by COCs during the expansion and disassembly processes. Our results show that the secretion of tissue type PA and urokinase type PA (uPA) by oocytes and CCs, respectively, does not change significantly during expansion but dramatically increases thereafter. Compact COCs were isolated from immature mice, primed 48 h earlier with 5 IU PMSGs, and were induced to expand in vitro with 100 ng/ml FSH in the presence of 1% FCS. Full expansion was achieved at 16 h, when hyaluronan synthesis ceased. Release of hyaluronan and CCs from the COC matrix began between 18 and 20 h of culture, which indicates that matrix degradation started at this time. PA activities in culture media were determined by SDS-PAGE, followed by a zymography at various time intervals between 4 and 32 h of culture. Secreted tissue type PA and uPA activity abruptly increased between 16 and 20 h after FSH stimulation. Slot blot hybridization of CC messenger RNA showed that uPA messenger RNA levels correlated with the increase in uPA activity. Similar temporal patterns of PA synthesis and matrix degradation were found in COCs induced to expand in vivo by injection of 5 IU human CG into PMSG-primed mice. Cultures of CCs, both in the presence and absence of oocytes, revealed that uPA synthesis is repressed in FSH-stimulated CCs by an oocyte-soluble factor for the first 16 h of culture, whereas CC responsiveness to this factor is lost thereafter. In conclusion, the data show that a sophisticated interplay between oocyte and CCs causes the two cell types to simultaneously secrete PA activity after ovulation. The fact that matrix degradation parallels PA production strongly supports the hypothesis that these enzymes may destabilize the expanded COC matrix.

Hyaluronan synthesis by mouse cumulus cells is regulated by interactions between follicle-stimulating hormone (or epidermal growth factor) and a soluble oocyte factor (or transforming growth factor beta1).

Expansion of the cumulus cell-oocyte complex (COC) in the preovulatory mammalian follicle requires a transient induction of hyaluronan (HA) synthesis by the cumulus cells. We studied the interactions of known factors that regulate this process by isolating compact COCs from mice and inducing their expansion in vitro. Maximum HA synthesis requires either follicle-stimulating hormone (FSH) or epidermal growth factor (EGF) in combination with either a soluble factor(s) produced by the oocyte or transforming growth factor beta1. FSH (or EGF) exerts its effects during the first 2 h of incubation, before HA synthesis actually begins. The oocyte factor(s) (or transforming growth factor beta1) exerts its effects from 2 h onwards and must be continuously present throughout the subsequent approximately 10 h to achieve a maximum level of HA synthesis. FSH stimulates intracellular cAMP synthesis, which correlates with net HA production up to approximately 14 fmol/COC at 5 ng/ml FSH; however, higher concentrations of FSH increase cAMP levels approximately 10-fold higher with no additional effect on HA synthesis. EGF at saturating concentrations for HA synthesis does not stimulate cAMP above basal levels. Tyrosine kinase inhibitors genistein and tyrphostin AG18 nearly abolish the HA synthesis response to EGF and inhibit the response to FSH by approximately 60%, suggesting that a tyrosine kinase activity is involved for both factors, whereas FSH also operates partially through another signaling pathway. Actinomycin D abolishes HA synthesis if added at the beginning of culture and reduces HA synthesis by approximately 50% if added between 6-12 h when HA synthesis is normally maximal. The results suggest that regulation of HA synthesis is primarily controlled at the transcriptional level.

Hyaluronic acid and proteoglycan accumulation in the cumulus oophorus matrix.

A factor produced by the oocytes induces in vitro FSH-treated cumulus and mural granulosa cells to synthesize hyaluronic acid. In the present study we examined hyaluronic acid synthesis by the two cell populations in vivo. After injection of hCG into PMSG-primed immature mice, cumulus and mural granulosa cells adjacent to the antrum synthesized a large amount of such glycosaminoglycan, while the outermost layers layers of mural granulosa cells did not. The results suggest that in vivo differences in hyaluronic acid synthesis between follicle cell subpopulations depend on a diffusion gradient of the oocyte factor. We also identified a proteoglycan species synthesized by cumulus cells which may contribute to hyaluronic acid organization in the intercellular spaces.