guanosine-triphosphate and Diabetes-Mellitus--Type-2

Surface levels of membrane proteins are determined by a dynamic balance between exocytosis-mediated surface delivery and endocytosis-dependent retrieval from the cell surface. Imbalances in surface protein levels perturb surface protein homeostasis and cause major forms of human disease such as type 2 diabetes and neurological disorders. Here, we found a Reps1-Ralbp1-RalA module in the exocytic pathway broadly regulating surface protein levels. Reps1 and Ralbp1 form a binary complex that recognizes RalA, a vesicle-bound small guanosine triphosphatases (GTPase) promoting exocytosis through interacting with the exocyst complex. RalA binding results in Reps1 release and formation of a Ralbp1-RalA binary complex. Ralbp1 selectively recognizes GTP-bound RalA but is not a RalA effector. Instead, Ralbp1 binding maintains RalA in an active GTP-bound state. These studies uncovered a segment in the exocytic pathway and, more broadly, revealed a previously unrecognized regulatory mechanism for small GTPases, GTP state stabilization.

Topics: ATP-Binding Cassette Transporters; Calcium-Binding Proteins; Diabetes Mellitus, Type 2; Exocytosis; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Membrane Proteins; ral GTP-Binding Proteins

Elevated γ-glutamyl transferase (γ-GTP) level is associated with metabolic syndrome, impaired glucose tolerance, and insulin resistance, which are risk factors for type 2 diabetes. We aimed to investigate the association of cumulative exposure to high γ-GTP level with risk of diabetes.. Using nationally representative data from the Korean National Health Insurance system, 346,206 people who were free of diabetes and who underwent 5 consecutive health examinations from 2005 to 2009 were followed to the end of 2018. High γ-GTP level was defined as those in the highest quartile, and the number of exposures to high γ-GTP level ranged from 0 to 5. Hazard ratio (HR) and 95% confidence interval (CI) for diabetes were analyzed using the multivariable Cox proportional-hazards model.. The mean follow-up duration was 9.2±1.0 years, during which 15,183 (4.4%) patients developed diabetes. There was a linear increase in the incidence rate and the risk of diabetes with cumulative exposure to high γ-GTP level. After adjusting for possible confounders, the HR of diabetes in subjects with five consecutive high γ-GTP levels were 2.60 (95% CI, 2.47 to 2.73) in men and 3.05 (95% CI, 2.73 to 3.41) in women compared with those who never had a high γ-GTP level. Similar results were observed in various subgroup and sensitivity analyses.. There was a linear relationship between cumulative exposure to high γ-GTP level and risk of diabetes. Monitoring and lowering γ-GTP level should be considered for prevention of diabetes in the general population.

Topics: Diabetes Mellitus, Type 2; Female; Follow-Up Studies; gamma-Glutamyltransferase; Glucose Intolerance; Guanosine Triphosphate; Humans; Male

The epithelial-mesenchymal transition (EMT) plays an important role in diabetic renal fibrosis. The ARAP1 gene is located near risk alleles for Type 2 diabetes, and its function has been linked to cytoskeleton rearrangement, Golgi apparatus remodeling, and endocytic trafficking of membrane receptors. The role of ARAP1 and its antisense RNA, ARAP1-AS2, in the pathogenesis of diabetes is unclear. To clarify the roles of ARAP1 and its antisense RNA in diabetes and related complications, we examined if the expression of these transcripts changed under high glucose (HG) conditions. To do this, we examined transcript levels in HK-2 cells, and explored the roles of ARAP1 and ARAP1-AS2 in the EMT process in HK-2 cells. We found increased expression of ARAP1-AS2 and ARAP1 in HK-2 cells under HG condition, and observed that the overexpression of ARAP1-AS2 significantly increased the EMT process. In addition, HG upregulated Cdc42-GTP levels in HK-2 cells, and increased cytoskeleton rearrangement, cell viability, and migration. After knockdown of ARAP1, the level of Cdc42-GTP was decreased; cytoskeleton reorganization, cell viability, and migration processes were decreased; and EMT and expression of fibrosis marker protein. Overall, our results indicated that ARAP1-AS2/ARAP1 may participate in cytoskeleton rearrangement and EMT processes in HK-2 cells through increased Cdc42-GTP levels.

Topics: Alleles; Carrier Proteins; cdc42 GTP-Binding Protein; Cell Movement; Cytoskeleton; Diabetes Mellitus, Type 2; Epithelial Cells; Epithelial-Mesenchymal Transition; Glucose; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Kidney Tubules; Risk Factors; RNA, Long Noncoding

Transglutaminase 2 (TG2) is an enzyme with diverse biological functions. TG2 catalyzes transamidation reactions, has intrinsic kinase activity, and acts as a G-protein in intracellular signaling. TG2 (Tgm2)-null mice are glucose intolerant and have impaired glucose-stimulated insulin secretion (GSIS). Furthermore, three naturally occurring missense mutations in the human TGM2 gene, corresponding to amino acid substitutions of Met330Arg, Ile331Asn, and Asn333Ser in the TG2 protein, have been reported and found to be associated with early-onset type 2 diabetes. However, their effect on TG2 function is not fully understood. To determine this, we have reproduced naturally occurring mutations in TG2 using site-directed mutagenesis. Overexpression of Myc-TG2 mutants in INS-1E cells resulted in a reduction of GSIS in comparison with cells overexpressing wild-type Myc-TG2 (WT-TG2). The maximum reduction was found in cells overexpressing Ile331Asn-TG2 (32%) followed by Met330Arg-TG2 (20%), and the least in Asn333Ser-TG2 (7%). Enzymatic analysis revealed that TG2 mutants have impaired transamidation and kinase activities in comparison with WT-TG2. GTP-binding assays showed that TG2 mutants also have altered GTP-binding ability, which is found to be modulated in response to glucose stimulation. Collectively, these data suggest that naturally occurring mutations in TG2 affect transamidation, kinase, and GTP-binding functions of TG2. While reduced insulin secretion, as a result of naturally occurring mutations in TG2, is due to the impairment of more than one biological function of TG2, it is the transamidation function that appears to be impaired during the first phase, whereas the GTP-binding function affects the second phase of insulin secretion.

Topics: Age of Onset; Animals; Cell Line; Cell Survival; Diabetes Mellitus, Type 2; Enzyme Activation; Gene Expression; GTP-Binding Proteins; Guanosine Triphosphate; Insulin; Insulin Secretion; Islets of Langerhans; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Protein Transport; Rats; Reactive Oxygen Species; Recombinant Fusion Proteins; Transglutaminases

At present, the data obtained by us and other authors give evidence that disturbances in hormonal signaling systems are the main causes of development of pathological changes and complications under the diabetes. However, the molecular mechanisms of these disturbances remain obscure, especially in the case of insulin-independent type II diabetes. Using neonatal streptozotocin model of 80- and 180-days type II diabetes the changes in functional activity of hormone-regulated adenylyl cyclase (AC) signaling systems components in the myocardium and the brain striatum of diabetic rats in comparison with the control animals were found. The transduction of AC inhibitory hormonal signal meditated through Gi proteins was shown to by disturbed under diabetes. This was manifested in both the decrease of hormone inhibitory effect on AC activity and weakening of hormone stimulation of G-protein GTP-binding activity. In the case of noradrenaline (myocardium) the inhibitory pathway of AC regulation by the hormone was vanished and the stimulation pathway, in contrary, was protected. Prolongation of diabetes from 80 up to 180 days led to some weakening of Gi-protein-mediated hormonal signal transduction. Stimulating effect of biogenic amines and relaxin on the AC activity and GTP-binding in the myocardium and brain of diabetic rats were weakly changed in the case of both 80- and 180-days diabetes. To sum up, the experimental type II diabetes caused disturbances mainly in Gi-coupled signaling cascades participating in hormone inhibition of AC activity.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Animals, Newborn; Biogenic Amines; Corpus Striatum; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Disease Models, Animal; Down-Regulation; Epinephrine; GTP-Binding Proteins; Guanosine Triphosphate; Heart; Hormones; Male; Myocardium; Norepinephrine; Protein Binding; Rats; Rats, Wistar; Relaxin; Serotonin; Signal Transduction; Somatostatin

We recently described novel regulatory roles for protein histidine phosphorylation of key islet proteins (e.g., nucleoside diphosphate kinase and succinyl thiokinase) in insulin secretion from the islet beta-cell (Kowluru A. Diabetologia 44: 89-94, 2001; Kowluru A, Tannous M, and Chen HQ. Arch Biochem Biophys 398: 160-169, 2002). In this context, we also characterized a novel, ATP- and GTP-sensitive protein histidine kinase in isolated beta-cells that catalyzed the histidine phosphorylation of islet (endogenous) proteins as well as exogenously added histone 4, and we implicated this kinase in the activation of islet endogenous G proteins (Kowluru A. Biochem Pharmacol 63: 2091-2100, 2002). In the present study, we describe abnormalities in ATP- or GTP-mediated histidine phosphorylation of nucleoside diphosphate kinase in islets derived from the Goto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes. Furthermore, we provide evidence for a marked reduction in the activities of ATP- or GTP-sensitive histidine kinases in GK rat islets. On the basis of these observations, we propose that alterations in protein histidine phosphorylation could contribute toward insulin-secretory abnormalities demonstrable in the diabetic islet.

Topics: Adenosine Triphosphate; Animals; Diabetes Mellitus, Type 2; Female; Guanosine Triphosphate; Histidine; Islets of Langerhans; Male; Nucleoside-Diphosphate Kinase; Phosphorus Radioisotopes; Phosphorylation; Radionuclide Imaging; Rats; Rats, Mutant Strains; Rats, Wistar

The first non-substrate like inhibitors of human cytosolic phosphoenolpyruvate carboxykinase (PEPCK) competitive with GTP are reported. An effort to discover orally active compounds that improve glucose homeostasis in Type 2 diabetics by reversibly inhibiting PEPCK led to the discovery of 1-allyl-3-butyl-8-methylxanthine (5). We now report modifications at N-1 and C-8 that improved the in vitro activity of the initial xanthine HTS hit by 100-fold and a developing SAR for this class of inhibitor.

Topics: Diabetes Mellitus, Type 2; Enzyme Inhibitors; Glucose; Guanosine Triphosphate; Homeostasis; Humans; Phosphoenolpyruvate Carboxykinase (GTP); Structure-Activity Relationship

In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109-121, 178-195, 254-271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTP gamma S, and GDP beta S but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109-121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109-121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis.

Topics: Actin Cytoskeleton; Amino Acid Sequence; Animals; Arizona; Asian People; Diabetes Mellitus, Type 2; Epitope Mapping; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Indians, North American; Molecular Sequence Data; Molecular Weight; Muscle, Skeletal; Myocardium; ras Proteins; Rats; Rats, Sprague-Dawley; Rats, Zucker; RNA, Messenger; Structure-Activity Relationship; Tissue Distribution