Gino Castriota

Merck, Whitehouse Station, New Jersey, United States

Are you Gino Castriota?

Claim your profile

Publications (9)32.76 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. These results support the proposition that the proteins encoded by the genes identified in our cell-based druggable genome siRNA screen hold the potential to serve as novel pharmacological targets for the treatment of T2D.
    PLoS ONE 01/2012; 7(5):e36384. · 3.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A series of benzimidazolone carboxylic acids and oxazolidinediones were designed and synthesized in search of selective PPARγ modulators (SPPARγMs) as potential therapeutic agents for the treatment of type II diabetes mellitus (T2DM) with improved safety profiles relative to rosiglitazone and pioglitazone, the currently marketed PPARγ full agonist drugs. Structure-activity relationships of these potent and highly selective SPPARγMs were studied with a focus on their unique profiles as partial agonists or modulators. A variety of methods, such as X-ray crystallographic analysis, PPARγ transactivation coactivator profiling, gene expression profiling, and mutagenesis studies, were employed to reveal the differential interactions of these new analogues with PPARγ receptor in comparison to full agonists. In rodent models of T2DM, benzimidazolone analogues such as (5R)-5-(3-{[3-(5-methoxybenzisoxazol-3-yl)benzimidazol-1-yl]methyl}phenyl)-5-methyloxazolidinedione (51) demonstrated efficacy equivalent to that of rosiglitazone. Side effects, such as fluid retention and heart weight gain associated with PPARγ full agonists, were diminished with 51 in comparison to rosiglitazone based on studies in two independent animal models.
    Journal of Medicinal Chemistry 11/2011; 54(24):8541-54. · 5.61 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To investigate the impact of reduced adipocyte fatty acid-binding protein 4 (FABP4) in control of body weight, glucose and lipid homeostasis in diet-induced obese (DIO) mice. We applied RNA interference (RNAi) technology to generate FABP4 germline knockdown mice to investigate their metabolic phenotype. RNAi-mediated knockdown reduced FABP4 mRNA expression and protein levels by almost 90% in adipocytes of standard chow-fed mice. In adipocytes of DIO mice, RNAi reduced FABP4 expression and protein levels by 70 and 80%, respectively. There was no increase in adipocyte FABP5 expression in FABP4 knockdown mice. The knockdown of FABP4 significantly increased body weight and fat mass in DIO mice. However, FABP4 knockdown did not affect plasma glucose and lipid homeostasis in DIO mice; nor did it improve their insulin sensitivity. Our data indicate that robust knockdown of FABP4 increases body weight and fat mass without improving glucose and lipid homeostasis in DIO mice.
    International journal of obesity (2005) 02/2011; 35(2):217-25. · 5.22 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.
    Bioorganic & medicinal chemistry letters 10/2010; 20(20):6088-92. · 2.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: GLUT4 (glucose transporter 4) plays important roles in glucose homoeostasis in vivo. GLUT4 expression and function are diminished in diabetic human and animal subjects. The goal of the present study is to develop a cell-based assay for identifying negative regulators of GLUT4 translocation as potential targets for the treatment of Type 2 diabetes. Traditional GLUT4 translocation assays performed in differentiated myocytes or adipocytes are difficult to perform, particularly in HTS (high-throughput screening) mode. In the present study, we stably co-expressed c-Myc and eGFP [enhanced GFP (green fluorescent protein)] dual-tagged recombinant GLUT4 with recombinant IRS1 (insulin receptor substrate 1) in HEK-293 cells (human embryonic kidney cells) (HEK-293.IRS1.GLUT4 cells). Insulin treatment stimulated both glucose uptake and GLUT4 translocation in these cells. GLUT4 translocation is quantified by a TRF (time-resolved fluorescence) assay in a 96-well HTS format. TRF assays confirmed insulin-stimulated GLUT4 translocation, which can be inhibited by PI3K (phosphoinositide 3-kinase) or Akt [also called PKB (protein kinase B)] inhibitors. Treatment with palmitate increased IRS1 serine phosphorylation and reduced insulin-stimulated Akt phosphorylation and GLUT4 translocation, indicating insulin resistance. Knockdown of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and PTP1B (protein tyrosine phosphatase 1B) gene expression by siRNA (small interfering RNA) treatment significantly increased GLUT4 translocation only in cells treated with palmitate but not in untreated cells. Similar results were obtained on treatment with siRNA of JNK1 (c-Jun N-terminal kinase 1), S6K1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and PKC(theta) (protein kinase C theta). In summary, we have established and validated a novel GLUT4 translocation assay that is optimal for identifying negative regulators of GLUT4 translocation. In combination with more physiologically relevant secondary assays in myotubes and adipocytes, this assay system can be used to identify potential novel therapeutic targets for the treatment of Type 2 diabetes.
    Biochemical Journal 12/2008; 418(2):413-20. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Despite their proven antidiabetic efficacy, widespread use of peroxisome proliferator-activated receptor (PPAR)gamma agonists has been limited by adverse cardiovascular effects. To overcome this shortcoming, selective PPARgamma modulators (SPPARgammaMs) have been identified that have antidiabetic efficacy comparable with full agonists with improved tolerability in preclinical species. The results of structural studies support the proposition that SPPARgammaMs interact with PPARgamma differently from full agonists, thereby providing a physical basis for their novel activities. Herein, we describe a novel PPARgamma ligand, SPPARgammaM2. This compound was a partial agonist in a cell-based transcriptional activity assay, with diminished adipogenic activity and an attenuated gene signature in cultured human adipocytes. X-ray cocrystallography studies demonstrated that, unlike rosiglitazone, SPPARgammaM2 did not interact with the Tyr473 residue located within helix 12 of the ligand binding domain (LBD). Instead, SPPARgammaM2 was found to bind to and activate human PPARgamma in which the Tyr473 residue had been mutated to alanine (hPPARgammaY473A), with potencies similar to those observed with the wild-type receptor (hPPARgammaWT). In additional studies, we found that the intrinsic binding and functional potencies of structurally distinct SPPARgammaMs were not diminished by the Y473A mutation, whereas those of various thiazolidinedione (TZD) and non-TZD PPARgamma full agonists were reduced in a correlative manner. These results directly demonstrate the important role of Tyr473 in mediating the interaction of full agonists but not SPPARgammaMs with the PPARgamma LBD, thereby providing a precise molecular determinant for their differing pharmacologies.
    Molecular pharmacology 02/2008; 73(1):62-74. · 4.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: alpha1-Acid glycoprotein (alpha1-AGP) is an acute phase protein that can potentiate cytokine secretion by mononuclear cells and may induce thrombosis by stabilizing the inhibitory activity of plasminogen activator inhibitor-1. Thus, alpha1-AGP may promote pathobiologies associated with type 2 diabetes mellitus (T2DM) including insulin resistance and cardiovascular disease. Here, we demonstrate that antidiabetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists inhibited expression of 3T3-L1 adipocyte alpha1-AGP in a concentration- and time-dependent manner via an apparent PPARgamma-mediated mechanism. As a result, synthesis and secretion of the glycoprotein was reduced. While PPARgamma agonist regulation of genes with functional peroxisome proliferator response elements in their promoter such as phosphoenolpyruvate carboxykinase were unaffected when cellular protein synthesis was inhibited, downregulation of alpha1-AGP mRNA was ablated thereby supporting the proposition that PPARgamma activation inhibits alpha1-AGP expression indirectly. These results suggest a potential novel adipocytic mechanism by which PPARgamma agonists may ameliorate T2DM-associated insulin resistance and cardiovascular disease.
    Cell Biology International 07/2007; 31(6):586-91. · 1.64 Impact Factor
  • Source
    01/2007;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma modulates the expression of numerous genes involved in glucose and lipid homeostasis and plays a critical role in adipocyte differentiation. Expression of uncoupling protein (UCP)1, which is necessary for thermogenesis, is strongly stimulated by PPARgamma agonists but without an increase in energy expenditure. This study was designed to assess whether PPARgamma-induced UCP1 has any functional impact and, if so, whether it involves sympathetic activity. In a first phase, obese ob/ob C57BL/6J mice and lean controls were treated for 2 wk with the PPARgamma agonist [2-(2-[4-phenoxy-2-propylphenoxy]ethyl)indole-5-acetic acid] (COOH). COOH induced UCP1 expression in brown and white adipose tissues as well as that of other genes associated with substrate oxidation and thermogenesis. However, UCP1 induction did not increase energy expenditure, as assessed by indirect calorimetry and other energy balance measurements. In a second phase, mice received for an additional 2 wk a combination of COOH and the beta(3)-adrenergic receptor (beta(3)-AR) agonist CL-316243 to stimulate the adrenergic signaling pathway and assess whether COOH-induced UCP1 was physiologically functional. The beta(3)-AR agonist stimulated thermogenesis in lean and ob/ob mice, an effect that was much stronger in COOH-pretreated mice, which exhibited lower respiratory quotient, higher oxygen consumption, and marked weight and fat mass loss, compared with mice not pretreated with COOH. These results demonstrate that PPARgamma agonism increases the thermogenic potential of white and brown adipose depots in lean and obese mice. This enhanced capacity leads to increased thermogenesis under beta-adrenergic stimulation, suggesting that the sympathetic drive is blunted by PPARgamma agonism.
    Endocrinology 09/2004; 145(8):3925-34. · 4.72 Impact Factor