Browsing by Author "Pardo, Fabian"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Gestational Diabetes Reduces Adenosine Transport in Human Placental Microvascular Endothelium, an Effect Reversed by Insulin
    (PUBLIC LIBRARY SCIENCE, 2012) Salomon, Carlos; Westermeier, Francisco; Puebla, Carlos; Arroyo, Pablo; Guzman Gutierrez, Enrique; Pardo, Fabian; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis
    Gestational diabetes mellitus (GDM) courses with increased fetal plasma adenosine concentration and reduced adenosine transport in placental macrovascular endothelium. Since insulin modulates human equilibrative nucleoside transporters (hENTs) expression/activity, we hypothesize that GDM will alter hENT2-mediated transport in human placental microvascular endothelium (hPMEC), and that insulin will restore GDM to a normal phenotype involving insulin receptors A (IR-A) and B (IR-B). GDM effect on hENTs expression and transport activity, and IR-A/IR-B expression and associated cell signalling cascades (p42/44 mitogen-activated protein kinases (p42/44(mapk)) and Akt) role in hPMEC primary cultures was assayed. GDM associates with elevated umbilical whole and vein, but not arteries blood adenosine, and reduced hENTs adenosine transport and expression. IR-A/IR-B mRNA expression and p42/44(mapk)/Akt ratios ('metabolic phenotype') were lower in GDM. Insulin reversed GDM-reduced hENT2 expression/activity, IR-A/IR-B mRNA expression and p42/44(mapk)/Akt ratios to normal pregnancies ('mitogenic phenotype'). It is suggested that insulin effects required IR-A and IR-B expression leading to differential modulation of signalling pathways restoring GDM-metabolic to a normal-mitogenic like phenotype. Insulin could be acting as protecting factor for placental microvascular endothelial dysfunction in GDM.
  • No Thumbnail Available
    Item
    Glycaemia dynamics in gestational diabetes mellitus
    (2022) Valero, Paola; Salas, Rodrigo; Pardo, Fabian; Cornejo, Marcelo; Fuentes, Gonzalo; Vega, Sofia; Grismaldo, Adriana; Hillebrands, Jan-Luuk; van der Beek, Eline M.; van Goor, Harry; Sobrevia, Luis
    Pregnant women may develop gestational diabetes mellitus (GDM), a disease of pregnancy characterised by maternal and fetal hyperglycaemia with hazardous consequences to the mother, the fetus, and the newborn. Maternal hyperglycaemia in GDM results in fetoplacental endothelial dysfunction. GDM-harmful effects result from chronic and short periods of hyperglycaemia. Thus, it is determinant to keep glycaemia within physiological ranges avoiding short but repetitive periods of hyper or hypoglycaemia. The variation of glycaemia over time is defined as 'glycaemia dynamics'. The latter concept regards with a variety of mechanisms and environmental conditions leading to blood glucose handling. In this review we summarized the different metrics for glycaemia dynamics derived from quantitative, plane distribution, amplitude, score values, variability estimation, and time series analysis. The potential application of the derived metrics from self-monitoring of blood glucose (SMBG) and continuous glucose monitoring (CGM) in the potential alterations of pregnancy outcome in GDM are discussed.
  • Thumbnail Image
    Item
    Insulin-Increased L-Arginine Transport Requires A(2A) Adenosine Receptors Activation in Human Umbilical Vein Endothelium
    (PUBLIC LIBRARY SCIENCE, 2012) Guzman Gutierrez, Enrique; Westermeier, Francisco; Salomon, Carlos; Gonzalez, Marcelo; Pardo, Fabian; Leiva, Andrea; Sobrevia, Luis
    Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1). This process involves the activation of A(2A) adenosine receptors (A(2A)AR) in human umbilical vein endothelial cells (HUVECs). Insulin increases hCAT-1 activity and expression in HUVECs, and A(2A)AR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A(2A)AR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37 degrees C) in the absence or presence of nitrobenzylthioinosine (NBTI, 10 mu mol/L, adenosine transport inhibitor) and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR), and SLC7A1 (for hCAT-1) reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K-m for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1(-1606) or pGL3-hCAT-1(-650) constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1(-1606), and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A(2A)AR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes.
  • No Thumbnail Available
    Item
    Maternal insulin therapy does not restore foetoplacental endothelial dysfunction in gestational diabetes mellitus
    (2017) Subiabre, Mario; Silva, Luis; Villalobos-Labra, Roberto; Toledo, Fernando; Paublo, Mario; Lopez, Marcia A.; Salsoso, Rocio; Pardo, Fabian; Leiva, Andrea; Sobrevia, Luis
    Pregnant women diagnosed with gestational diabetes mellitus subjected to diet (GDMd) that do not reach normal glycaemia are passed to insulin therapy (GDMi). GDMd associates with increased human cationic amino acid transporter 1 (hCAT-1)-mediated transport of L-arginine and nitric oxide synthase (NOS) activity in foetoplacental vasculature, a phenomenon reversed by exogenous insulin. Whether insulin therapy results in reversal of the GDMd effect on the foetoplacental vasculature is unknown. We assayed whether insulin therapy normalizes GDMd-associated foetoplacental endothelial dysfunction. Primary cultures of human umbilical vein endothelial cells (HUVECs) from GDMi pregnancies were used to assay L-arginine transport kinetics, NOS activity, p44/42(mapk) and protein kinase B/Akt activation, and umbilical vein rings reactivity. HUVECs from GDMi or GDMd show increased hCAT-1 expression and maximal transport capacity, NOS activity, and eNOS, and p44/42(mapk), but not Akt activator phosphorylation. Dilation in response to insulin or calcitonin-gene related peptide was impaired in umbilical vein rings from GDMi and GDMd pregnancies. Incubation of HUVECs in vitro with insulin (1 nmol/L) restored hCAT-1 and eNOS expression and activity, and eNOS and p44/42(mapk) activator phosphorylation. Thus, maternal insulin therapy does not seem to reverse GDMd-associated alterations in human foetoplacental vasculature.
  • No Thumbnail Available
    Item
    Over-expression of muscle glycogen synthase in human diabetic nephropathy
    (2015) Gatica, Rodrigo; Bertinat, Romina; Silva, Pamela; Kairath, Pamela; Slebe, Felipe; Pardo, Fabian; Ramirez, Maria J.; Slebe, Juan C.; Campistol, Jose M.; Nualart, Francisco; Caelles, Carme; Yanez, Alejandro J.
    Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes.
  • Thumbnail Image
    Item
    Potential Role of Sodium-Proton Exchangers in the Low Concentration Arsenic Trioxide-Increased Intracellular pH and Cell Proliferation
    (PUBLIC LIBRARY SCIENCE, 2012) Aravena, Carmen; Beltran, Ana R.; Cornejo, Marcelo; Torres, Viviana; Diaz, Emilce S.; Guzman Gutierrez, Enrique; Pardo, Fabian; Leiva, Andrea; Sobrevia, Luis; Ramirez, Marco A.
    Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 mu mol/L, 0-48 hours) in the absence or presence of 5-N, N-hexamethylene amiloride (HMA, 5-100 mu mol/L, NHEs inhibitor), PD-98059 (30 mu mol/L, MAPK1/2 inhibitor), Go6976 (10 mu mol/L, PKC alpha, beta I and mu inhibitor), or Schering 28080 (10 mu mol/L, H+/K(+)ATPase inhibitor) plus concanamycin (0.1 mu mol/L, V type ATPases inhibitor). Incorporation of [H-3]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na+-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk)) were also determined. Lowest ATO (0.05 mu mol/L, similar to 0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Go6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44(mapk) and PKC alpha, beta I and/or mu activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels. Citation: Aravena C, Beltran AR, Cornejo M, Torres V, Diaz ES, et al. (2012) Potential Role of Sodium-Proton Exchangers in the Low Concentration Arsenic Trioxide-Increased Intracellular pH and Cell Proliferation. PLoS ONE 7(12): e51451. doi:10.1371/journal.pone.0051451