Opposite effects of two PKA inhibitors on cAMP inhibition of IGF-I-induced oligodendrocyte development: a problem of unspecificity?

The stimulatory effect of insulin-like growth factor I (IGF-I) on myelin basic protein (MBP) expression, a parameter for oligodendrocyte development, is mediated by the MAPK and PI3K signaling pathways. We have previously shown that the second messenger cAMP inhibits IGF-I-induced MAPK activation as well as MBP expression. We also showed that the PKA inhibitor Rp-cAMPS reverted the cAMP effect on IGF-I-induced MBP without affecting the cAMP effect on IGF-I-induced MAPK activation. Here we report that, in contrast to Rp-cAMPS, H89 (a PKA inhibitor structurally non-related to Rp-cAMPS) enhances both the inhibitory effect of cAMP on IGF-I-induced MBP expression and the inhibitory effect of cAMP on IGF-I-induced MAPK activation. Likewise, H89 is capable of inhibiting the IGF-I-induced MAPK activation in the absence of PKA stimulation. Thus, we hypothesize that an unspecific action of H89 on a target located upstream MAPK could account for the discrepancies between the effects elicited by Rp-cAMPS and H89.

Differential gene expression profile in omental adipose tissue in women with polycystic ovary syndrome.

CONTEXT: The polycystic ovary syndrome (PCOS) is frequently associated with visceral obesity, suggesting that omental adipose tissue might play an important role in the pathogenesis of the syndrome. OBJECTIVE: The objective was to study the expression profiles of omental fat biopsy samples obtained from morbidly obese women with or without PCOS at the time of bariatric surgery. DESIGN: This was a case-control study. SETTINGS: We conducted the study in an academic hospital. PATIENTS: Eight PCOS patients and seven nonhyperandrogenic women submitted to bariatric surgery because of morbid obesity. INTERVENTIONS: Biopsy samples of omental fat were obtained during bariatric surgery. MAIN OUTCOME MEASURE: The main outcome measure was high-density oligonucleotide arrays. RESULTS: After statistical analysis, we identified changes in the expression patterns of 63 genes between PCOS and control samples. Gene classification was assessed through data mining of Gene Ontology annotations and cluster analysis of dysregulated genes between both groups. These methods highlighted abnormal expression of genes encoding certain components of several biological pathways related to insulin signaling and Wnt signaling, oxidative stress, inflammation, immune function, and lipid metabolism, as well as other genes previously related to PCOS or to the metabolic syndrome. CONCLUSION: The differences in the gene expression profiles in visceral adipose tissue of PCOS patients compared with nonhyperandrogenic women involve multiple genes related to several biological pathways, suggesting that the involvement of abdominal obesity in the pathogenesis of PCOS is more ample than previously thought and is not restricted to the induction of insulin resistance.

The ACAA-insertion/deletion polymorphism at the 3' UTR of the IGF-II receptor gene is associated with type 2 diabetes and surrogate markers of insulin resistance.

OBJECTIVE: The IGF-II receptor gene (IGFIIR) is located at chromosome 6q26, a region that harbors a genetic marker linked to insulin-resistant traits in Mexican-Americans. In the present study conducted in Spaniards, we tested a common polymorphism in IGFIIR for association with type 2 diabetes and insulin-resistant traits. DESIGN: Case-control association study. METHODS: One hundred and forty-five type 2 diabetic patients and 217 non-diabetic controls were genotyped for the ACAA-insertion/deletion polymorphism at the 3' UTR of IGFIIR. Phenotyping included anthropometrics and a metabolic profile, including serum lipid levels and surrogate indexes of insulin resistance whenever possible. RESULTS: Diabetic patients were more frequently homozygous for the wild type 144 bp allele of IGFIIR compared with controls (diabetic patients 77.2%, controls 51.6%, P<0.001) suggesting a potential protective role against type 2 diabetes for the IGFIIR 140 bp variant. Carrying 140 bp alleles was associated with an odds ratio of having diabetes of 0.290 (95% confidence interval 0.109-0.770), and controls homozygous for the wild type 144 bp allele presented with lower insulin and triglyceride levels, which are proxies for insulin resistance. CONCLUSIONS: The ACAA-insertion/deletion polymorphism at the 3' UTR of IGFIIR is associated with type 2 diabetes and influences surrogate markers of insulin resistance in non-diabetic subjects.

Polymorphisms in the interleukin-6 receptor gene are associated with body mass index and with characteristics of the metabolic syndrome.

OBJECTIVE: Low-grade inflammation has been related to obesity, insulin resistance and the metabolic syndrome. The Asp358Ala variant and the CA-repeat polymorphism in the interleukin-6 receptor (IL-6R) gene have been reported to be associated with obesity in Pima Indians and Spanish women, respectively. The aim of this study was to investigate the association between these polymorphisms and obesity in a Mediterranean-Caucasian population, and to determine whether this polymorphism was related to the metabolic syndrome as defined by the National Cholesterol Education Program - Adult Treatment Panel III (NCEP/ATP-III) criteria. DESIGN: Cross-sectional. PATIENTS: Three hundred and ninety subjects from the general population. METHODS: The Asp358Ala and CA-repeat polymorphisms were analysed by polymerase chain reaction (PCR) amplification, followed by restriction fragment length polymorphism or capillary electrophoresis, respectively. RESULTS: Both polymorphisms were in strong linkage disequilibrium, Asp358 alleles being associated with 149 CA-repeat alleles (chi2 = 76.275, P < 0.0001). Therefore, only the association of the Asp358Ala variant with obesity and the metabolic syndrome was assessed in the whole series of subjects. Subjects homozygous for Asp358 alleles had statistically higher body mass index (BMI) compared with Ala358 carriers (27.7 +/- 5.41 vs. 26.6 +/- 4.96 kg/m2; P < 0.05). Moreover, the prevalence of the metabolic syndrome was significantly higher in carriers of the Asp358 allele compared with Ala358 homozygotes (12.7%vs. 0.0%; P = 0.01). This relationship remained significant after adjusting for age, insulin resistance, sex and BMI. CONCLUSIONS: The Asp358Ala and CA-repeat polymorphisms in the IL-6R gene are associated with obesity and characteristics of the metabolic syndrome in our population of Mediterranean subjects.

Polymorphisms in the insulin receptor substrate-1 (IRS-1) gene and the insulin receptor substrate-2 (IRS-2) gene influence glucose homeostasis and body mass index in women with polycystic ovary syndrome and non-hyperandrogenic controls.

BACKGROUND: We aimed to evaluate the influence of the Gly972Arg variant of the insulin receptor substrate-1 gene (IRS-1) and the Gly1057Asp variant in IRS-2 on insulin resistance and glucose tolerance in women with polycystic ovary syndrome (PCOS) and healthy controls. METHODS: Genotypes, allelic frequencies, indexes of insulin resistance, glucose tolerance and hormone profiles were studied in a large sample of Spanish PCOS (n = 103) women compared with a control group (n = 48) of healthy women matched for body mass index. RESULTS: No differences in genotype or allelic frequencies were found between PCOS patients and healthy controls. When considering control subjects and PCOS patients as a whole, IRS-1 Arg972 carriers also presented with increased fasting insulin (133 +/- 60 versus 95 +/- 67 pmol/l, P = 0.008) and insulin resistance measured by homeostasis model assessment (4.3 +/- 2.1 versus 3.1 +/- 2.4, P = 0.009) compared with subjects homozygous for Gly972 alleles. These differences were even higher when restricting the analysis to PCOS patients. Subjects homozygous for the Gly1057 allele of IRS-2 presented with increased 60 and 90 min oral glucose tolerance test (OGTT) glucose levels compared with carriers of one or two Asp1057 alleles (7.9 +/- 2.1 versus 7.1 +/- 2.1 mmol/l, P = 0.042 and 7.0 +/- 2.1 versus 6.0 +/- 1.8 mmol/l, P = 0.014), and a similar tendency was observed for 120 min OGTT glucose levels. CONCLUSIONS: The Gly972Arg in IRS-1 and Gly1057Asp in IRS-2 polymorphisms influence glucose homeostasis in premenopausal women, but are not associated with PCOS.

Association of the polycystic ovary syndrome with genomic variants related to insulin resistance, type 2 diabetes mellitus, and obesity.

We have evaluated the possible association of polycystic ovary syndrome (PCOS) with 15 genomic variants previously described to influence insulin resistance, obesity, and/or type 2 diabetes mellitus. Seventy-two PCOS patients and 42 healthy controls were genotyped for 15 variants in the genes encoding for paraoxonase (three variants), plasma cell differentiation antigen glycoprotein, human sorbin and SH3 domain containing 1, plasminogen activator inhibitor-1, peroxisome proliferator-activated receptor-gamma2, protein tyrosine phosphatase 1B (two variants), adiponectin (two variants), IGF1, IGF2, IGF1 receptor, and IGF2 receptor. Compared with controls, PCOS patients were more frequently homozygous for the -108T variant in paraoxonase (36.6% vs. 9.5%; P = 0.002) and homozygous for G alleles of the ApaI variant in IGF2 (62.9% vs. 38.1%; P = 0.018). Paraoxonase is a serum antioxidant enzyme and, because -108T alleles result in decreased paraoxonase expression, this increase in oxidative stress might result in insulin resistance. G alleles of the ApaI variant in IGF2 may increase IGF2 expression, and IGF2 stimulates adrenal and ovarian androgen secretion. In conclusion, the paraoxonase -108 C-->T variant and the ApaI polymorphism in the IGF2 gene are associated with PCOS and might contribute to increased oxidative stress, insulin resistance, and hyperandrogenism in this prevalent disorder.

Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide.

Polycystic ovary syndrome is a very common disease affecting 6.5% of women. It is frequently associated with obesity with the link between the two disorders being insulin resistance. From morbidly obese women submitted to surgical treatment of obesity, we obtain intra-abdominal adipose tissue samples in order to compare protein patterns between women with and without polycystic ovary syndrome by two-dimensional gel electrophoresis (2-DE) analysis. Adipose tissue is a complex material extremely rich in lipids. To improve protein solubilization a lysis buffer containing 8.4 M urea, 2.4 M thiourea, 5% 3-((3-cholamidoproyl)dimethyl-amino)-1-propanesulfonate and 50 mM dithiothreitol was used and samples were centrifuged to remove fat. Hydroxyethyl disulfide was added to increase resolution in the alkaline region (Olsson, I., Larsson, K., Palmgren, R., Bjellqvist, B., Proteomics 2002, 2, 1630-1632) and it also improved resolution in a wide pH range (3-10). Our work shows for the first time 2-DE maps of human adipose tissue and identifies some of the proteins by mass spectrometry. This information will aid studies on metabolic diseases such as polycystic ovary syndrome, obesity, hypertension and type 2 diabetes.

Serum interleukin-18 concentrations are increased in the polycystic ovary syndrome: relationship to insulin resistance and to obesity.

Low-grade chronic inflammation is involved in the pathogenesis of the metabolic syndrome and atherosclerosis, and serum levels of inflammatory cytokines are useful cardiovascular risk markers. We have studied serum IL-18 concentrations in women with polycystic ovary syndrome (PCOS), focusing on its relationship with obesity and indexes of insulin resistance. Sixty consecutive women with PCOS and 34 healthy women were recruited. Serum levels of IL-18 and lipid and hormone profiles were measured. The insulin sensitivity index was calculated from glucose and insulin concentrations during an oral glucose tolerance test. Data were submitted to a multivariate general linear model introducing age as a covariate. Serum IL-18 levels were increased in PCOS patients compared with controls (P = 0.031) and in obese women compared with lean women (P = 0.018). No interaction between PCOS and obesity was found, suggesting that the influence of PCOS on serum IL-18 concentrations studied here was not different in lean women compared with obese women and that the influence of obesity on serum IL-18 concentrations was the same in the PCOS and control groups. Serum IL-18 levels correlated, after logarithmic transformation, with body mass index (r = 0.38; P < 0.0002), waist-to-hip ratio (r = 0.33; P < 0.001), and total testosterone levels (r = 0.24; P < 0.02), and inversely with the insulin sensitivity index (r = -0.23; P < 0.03). In conclusion, PCOS and obesity induce an increase in serum IL-18 levels, which are also associated with several indexes of global and visceral adiposity and with insulin resistance.

Association of polymorphisms in the interleukin 6 receptor complex with obesity and hyperandrogenism.

OBJECTIVE: Interleukin-6 (IL-6), is an inflammatory cytokine that may influence the pathogenesis of obesity and hyperandrogenism. IL-6 exerts its actions through a heterodimeric receptor consisting of two membrane-bound glycoproteins: an 80-kDa IL-6 binding unit (IL6R-alpha) and a 130-kDa IL-6 signal transducer (gp130). Genetic variability at these loci might contribute to explain the development of obesity and hyperandrogenism. RESEARCH METHODS AND PROCEDURES: We have evaluated the possible association of several polymorphisms in the IL6R-alpha and gp130 genes with obesity and/or hyperandrogenism in a case-control study involving 143 hyperandrogenic patients and 45 healthy women from Spain. RESULTS: A microsatellite CA-repeat polymorphism in the IL6R-alpha locus was associated with obesity. The frequency of the common 149-bp allele was markedly increased in obese women compared with controls when considering patients and controls as a whole (0.41 vs. 0.29, chi(2) = 17.085, p < 0.050). On the other hand, the uncommon Arg148 allele of the Gly148Arg polymorphism in the gp130 gene was more frequent in controls compared with hyperandrogenic patients (0.17 vs. 0.08, chi(2) = 5.605, p = 0.026). Controls carrying Arg148 alleles had lower 11-deoxycortisol and 17-hydroxyprogesterone concentrations, a lower response of androstenedione to 1-24 adrenocorticotropin, and an almost significant decrease in free testosterone levels, suggesting that Arg148 alleles in the gp130 gene have a protective effect against androgen excess and adrenal hyperactivity. DISCUSSION: Polymorphisms in the gp130 and IL6R-alpha loci influence hyperandrogenism and obesity, respectively. Our present results further suggest that proinflammatory genotypes are involved in the pathogenesis of these common metabolic disorders.

Involvement of vasoactive intestinal peptide on insulin-like growth factor I-induced proliferation of rat pituitary lactotropes in primary culture: evidence for an autocrine and/or paracrine regulatory system.

In previous studies we demonstrated that insulin-like growth factor I (IGF-I) induces pituitary vasoactive intestinal peptide (VIP) gene expression and secretion, and that IGF-I-induced prolactin (PRL) release is mediated by VIP. In this study, we investigate the mitotropic action of IGF-I and VIP on pituitary lactotropes, and their possible interplay in this effect. Cultured male rat pituitary cells were treated with rhIGF-I (10(-7)M) and/or VIP (10(-7)M) for 48 h. 5-Bromo-2'-deoxyuridine (BrdU) (10 microM) was added for labeling proliferation of pituitary cells. BrdU-labeling indices indicative of the proliferation rate of lactotropes were determined by double-labeling immunofluorescence staining for PRL and BrdU. Treatment with either IGF-I or VIP increased BrdU-labeling indices of lactotropes, but there was no further increase upon combined incubation with both factors, suggesting an interaction between the signal transduction pathways of IGF-I and VIP. VIP antiserum partially suppressed IGF-I-induced BrdU-labeling indices of lactotropes. We also investigated the intracellular signal transduction pathways in the action of IGF-I and VIP on the proliferation of lactotropes. Treatment of pituitary cells with an inhibitor of the mitogen-activated protein kinase (MAPK) pathway completely abolished IGF-I-induced lactotrope proliferation, whereas it partially suppressed VIP-induced BrdU-labeling indices. The protein kinase A (PKA) inhibitor, which abolished the mitogenic action of VIP, markedly suppressed IGF-I-induced lactotrope proliferation. These results indicate that both IGF-I and VIP stimulate lactotrope proliferation, and that IGF-I-induced lactotrope proliferation is partially mediated by VIP produced locally. Also, this study suggests that interactions between MAPK and cyclic adenosine 3',5'-monophosphate-PKA signaling pathways are implicated in the lactotrope proliferation induced by IGF-I and VIP.

Common single nucleotide polymorphisms in intron 3 of the calpain-10 gene influence hirsutism.

OBJECTIVE: To study three common polymorphisms in intron 3 of the calpain-10 gene (CAPN10) in hyperandrogenic patients. DESIGN: Case-control study. SETTING: Academic hospital. PATIENT(S): Ninety-seven hyperandrogenic patients and 37 healthy controls. INTERVENTION(S): Basal and adrenocorticotropin-stimulated serum samples and genomic DNA samples were obtained during the follicular phase of the menstrual cycle. MAIN OUTCOME MEASURE(S): Genotyping of the UCSNP43, UCSNP44, and UCSNP45 polymorphisms in CAPN10 and serum androgen levels. RESULT(S): Sixteen patients had idiopathic hirsutism, defined as normal serum androgen levels and regular menstrual cycles. Eighty-one hyperandrogenic patients (those presenting with hyperandrogenemic hirsutism or the polycystic ovary syndrome) were analyzed further. UCSNP45 alleles were distributed differently among the study groups. Heterozygosity for the uncommon C allele was increased in patients with idiopathic hirsutism (31.3%) and reduced in hyperandrogenic patients (7.4%) compared with controls (16.2%). The UCSNP44 and UCSNP43 alleles were in linkage disequilibrium, and were distributed equally among patients with idiopathic hirsutism, hyperandrogenism, and controls. However, the uncommon A allele at UCSNP43 was associated with higher hirsutism score (mean [+/- SD], 9.9 +/- 6.8, 12.7 +/- 7.7, and 14.6 +/- 8.2 in GG, GA, and AA participants, respectively). No other differences were observed in clinical and biochemical characteristics, including insulin sensitivity, by CAPN10 variant. CONCLUSION(S): The C allele at the UCSNP45 locus in CAPN10 is associated with idiopathic hirsutism, and UCSNP43 influences the hirsutism score.

The -597 G-->A and -174 G-->C polymorphisms in the promoter of the IL-6 gene are associated with hyperandrogenism.

To evaluate whether genetic variability at the IL-6 gene (IL-6) is associated with hyperandrogenism, we studied four common polymorphisms in the IL-6 promoter (-597G-->A, -572G--> C, -373A(n)T(n), -174G-->C) in 85 hyperandrogenic patients and 25 healthy women. We found 5 different haplotypes when considering the 3 biallelic polymorphisms at positions -597, -572, and -174 of IL-6 (relative frequencies in parentheses): GGG (0.505), AGC (0.377), GGC (0.059), GCG (0.055), and GCC (0.005). The frequencies of the GGG haplotype were 0.559 in patients and 0.320 in controls, whereas those of the AGC haplotype were 0.318 in patients and 0.580 in controls (chi(2) = 12.145; P < 0.02). The -597G-->A and -174G-->C polymorphisms were in linkage disequilibrium (chi(2) = 152.220; P < 0.00001), and were associated with patient or control status. -597G and -174G alleles were more frequent in patients in homozygosity or considering subjects homozygous and heterozygous for G alleles as a whole (P < 0.05 for all analyses). In healthy women G alleles at -597 and -174 were associated with statistically significant higher circulating levels of IL-6 and basal cortisol, 11-deoxycortisol, and 17-hydroxyprogesterone and a tendency (P < 0.10) for higher total T concentrations compared with -597A and -174C alleles. On the contrary, neither the -572G-->C nor the -373A(n)T(n) polymorphism was related to hyperandrogenism or influenced any clinical or biochemical variable. In conclusion, our present results suggest that the -597G-->A and -174G-->C polymorphisms in IL-6 are involved in the pathogenesis of hyperandrogenic disorders.