Diabetes mellitus is caused by deficiency of insulin secretion from the pancreatic islet beta cells and/or insulin resistance in liver, muscle and adipocytes, resulting in glucose intolerance and hyperglycemia. Several protein tyrosine phosphatases, such as PTP1B (PTPN1), TCPTP (PTPN2), LYP (PTPN22), PTPIA-2, PTPMEG2 (PTPN9) or OSTPTP are involved in insulin signaling pathway, insulin secretion and autoreactive attack to pancreatic beta cells. Genetic mutation or overexpression of these phosphotases has been found to cause or increase the risk of diabetes mellitus. Some population with high risk for type 2 diabetes has overexpressed PTP1B, a prototypical tyrosine phosphatase which down-regulates insulin and leptin signal transduction. Animal PTP1B knockout model and PTP1B specific inhibitor cellular studies indicate PTP1B may serve as a therapeutic target for type 2 diabetes. TCPTP shares more than 70% sequence identity with PTP1B in their catalytic domain. TCPTP dephosphorylates tyrosine phosphorylated substrates overlapping with PTP1B but also has its own distinct dephosphorylation sites and functions. Recent research indicates TCPTP may have role in type 1 diabetes via dysregultaion of cytokine-mediated immune responses or pancreatic beta cell apoptosis. The tyrosine phosphatase LYP, which down-regulates LCK activity in T cell response, can become mutated as R620W which is highly correlated to type 1 diabetes. LYP R620W may be a gain of function mutation which suppresses TCR signaling. Patients bearing the R620W mutant have impaired T cell responses and increased populations of (CD45RO+CD45RA-) CD4+ T cells. A detailed elucidation of mechanism of R620W in type 1 diabetes and specific LYP inhibitor development will help characterize LYP R620W as a therapeutic target. A receptor tyrosine phosphatase, PTPIA-2/beta is a major autoantigen of type 1 diabetes. A diagnosis kit identifying PTPIA-2/beta autoantibodies is valuable in early detection and prevention of type 1 diabetes. In addition, other phosphatase like OSTPTP and PTPMEG2 are involved in type 2 diabetes via regulation of insulin production, beta cell growth or insulin signaling. Research into understanding the mechanism of these tyrosine phosphatases in diabetes, such as their precise functions in the regulation of insulin secretion, the insulin response and the immune response will strengthen our knowledge of diabetes pathophysiology which may result in new diagnostic and therapeutic strategies for diabetes.
Animals ; Diabetes Mellitus ; enzymology ; Diabetes Mellitus, Type 1 ; enzymology ; Diabetes Mellitus, Type 2 ; enzymology ; Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; genetics ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 2 ; genetics ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 22 ; genetics ; metabolism ; Protein Tyrosine Phosphatases, Non-Receptor ; classification ; genetics ; metabolism

OBJECTIVETo study the protein tyrosine phosphatase-1B (PTP1B) inhibitory activity of natural products from algae aiming at searching for new way for the treatment of type 2 diabetes mellitus (T2DM) and obesity.

METHODBromophenols derivatives from algae were screened against the PTP1B by the colorimetric assay with GST/PTP1B fusion protein. The Me2SO was distributed as the full enzyme activity, and Na3VO4 (IC50 2 micromol L(-1)) was distributed as the positive control. Inhibition rate was assayed and IC50 were calculated by LOGIT method.

RESULTThree bromophenols from Rhodomela confervoides and Leathesia nana, 3, 4-dibromo-5-(methoxymethyl)-1, 2-benzenediol (1), 2-methyl-3-(2, 3-dibromo4, 5-dihydroxy)-propylaldehyde (2) and 3-(2, 3-dibromo-4, 5-dihydroxy-phenyl)-4-bromo-5, 6-dihydroxy-1, 3-dihydroiso-benzofuran (3) showed significant inhibitory activity against PTP1B. IC50 values were 3.4 +/- micromol L(-1), 4.5 micromol L(-1) and 2.8 micromol L(-1), respectively.

CONCLUSIONThe results prove that three bromophenol derivatives from algae with significant inhibitory activity against PTP1B were potential and effective therapeutic agents for treatment of T2DM and obesity.

Diabetes Mellitus, Type 2 ; drug therapy ; metabolism ; Eukaryota ; chemistry ; Phaeophyta ; chemistry ; Phenols ; chemistry ; therapeutic use ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; Rhodophyta ; chemistry

OBJECTIVETo explore if the hypoxia/reoxygenation-induced increased activity and expression of PTP-1B in neonatal rat cardiomyocytes are mediated by nitric oxide (NO).

METHODSNeonatal rat cardiomyocytes were isolated and randomly divided into 4 groups: normal group (N group); hypoxia/reoxygenation group (H/R group); N(omega)-nitro-l-arginine methylester treated group (L-NAME group); hypoxia/reoxygenation plus L-NAME group (L-NA + H/R group). PTP-1B activity in cardiomyocytes was determined spectrophotometrically at 405 nm, PTP-1B expression in cardiomyocytes was detected by Western blot.NO and LDH concentrations in cell medium were also assayed.

RESULTSPTP-1B activity and expression in cardiomyocytes was significantly higher in the H/R group as compared to the N group and this increase could be blocked by cotreatment with L-NAME. As compared to H/R group, nitric oxide and LDH concentrations in cell medium were significantly decreased in the L-NA + H/R group (NO concentration: H/R group, 368% +/- 13% and L-NA + H/R group, 61% +/- 7%, P < 0.005; LDH concentration: H/R group, 41.2 +/- 6.7 and L-NA + H/R group, 23.6 +/- 4.8, P < 0.05).

CONCLUSIONSThis study showed that pretreatment with L-NAME, a non-selective inhibitor of NOS, prevented the hypoxia/reoxygenation-induced increase in PTP-1B activity and expression in cardiomyocytes, suggesting PTP-1B activation during hypoxia/reoxygenation was mediated by nitric oxide.

Animals ; Cell Hypoxia ; Cells, Cultured ; Myocytes, Cardiac ; cytology ; metabolism ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; metabolism ; Rats

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

The amino acid sequence (1-301aa) coding the human PTP1B catalytic domain (PTP1Bc) was obtained from the GenBank. The PTP1Bc gene was constructed by overlapping PCR, then was inserted into vector pET-22b(+) and expressed efficiently in E. coli BL21(DE3) under optimum condition after IPTG induction. The proteins were expressed mainly as inclusion bodies with the yield of more than 30% of total bacterial proteins. The expressed products were purified through Ni(2+)-affinity chromatographic column. After purification, the purity of the proteins was more than 95%. Western blotting analysis suggested that the purified proteins could specially combine with anti-PTP1B antibody. Enzyme activity assay showed that the protein has phosphatase activities.
Catalysis ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Inclusion Bodies ; metabolism ; Polymerase Chain Reaction ; methods ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

To screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors, a high-throughput screening (HTS) model in 384-well microplate with total volume of 50 microL was established. Recombinant PTP1B was cloned and expressed in E. coli. with its specific substrate 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP). The HTS model was based on enzyme reaction rate with enhanced sensitivity and specificity (Z' = 0.78). A total of 24,240 samples were screened, among them 80 samples with inhibition greater than 70% were selected for further rescreening. Finally, six compounds with high inhibitory activity were identified, whose IC50 values were 21.58, 18.39, 15.37, 11.92, 37.27, and 36.61 microg x mL(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.
Drug Evaluation, Preclinical ; methods ; Enzyme Inhibitors ; analysis ; pharmacology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; methods ; Humans ; Inhibitory Concentration 50 ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; metabolism ; Sensitivity and Specificity ; Vanadates ; pharmacology

Protein tyrosine phosphatase (PTP) 1B is a potential target for the treatment of diabetes and obesity. Phosphotyrosine (pTyr) is the substrate for PTP1B dephosphorylation. Malonic acid moiety was used herein as a mimic of the phosphate group in pTyr, and novel malonic acid derivatives 1-7 were designed, synthesized and evaluated as PTP1B inhibitors. Results from enzymatic assays indicated that compounds 3 and 4 exhibited potent inhibition against human recombinant PTP1B with IC50 values of 7.66 and 1.88 micromol x L(-1), respectively.
Drug Design ; Enzyme Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Humans ; Inhibitory Concentration 50 ; Malonates ; chemical synthesis ; chemistry ; pharmacology ; Molecular Structure ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; metabolism ; Structure-Activity Relationship

Animals ; Cell Survival ; radiation effects ; Light ; adverse effects ; Mice ; Mice, Knockout ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; metabolism ; Retinal Rod Photoreceptor Cells ; cytology ; enzymology ; radiation effects

Protein tyrosine phosphatase 1B (PTP1B) has led to an intense interest in developing its inhibitors as anti-diabetes, anti-obesity and anti-cancer agents. The fruits of Rubus chingii (Chinese raspberry) were used as a kind of dietary traditional Chinese medicine. The methanolic extract of R. chingii fruits exhibited significant PTP1B inhibitory activity. Further bioactivity-guided fractionation resulted in the isolation of three PTP1B inhibitory ursane-type triterpenes: ursolic acid (1), 2-oxopomolic acid (2), and 2α, 19α-dihydroxy-3-oxo-urs-12-en-28-oic acid (3). Kinetics analyses revealed that 1 was a non-competitive PTP1B inhibitor, and 2 and 3 were mixed type PTP1B inhibitors. Compounds 1-3 and structurally related triterpenes (4-8) were further analyzed the structure-activity relationship, and were evaluated the inhibitory selectivity against four homologous protein tyrosine phosphatases (TCPTP, VHR, SHP-1 and SHP-2). Molecular docking simulations were also carried out, and the result indicated that 1, 3-acetoxy-urs-12-ene-28-oic acid (5), and pomolic acid-3β-acetate (6) bound at the allosteric site including α3, α6, and α7 helix of PTP1B.
Enzyme Inhibitors ; chemistry ; metabolism ; Fruit ; chemistry ; Humans ; Kinetics ; Methanol ; chemistry ; Molecular Docking Simulation ; Molecular Structure ; Plant Extracts ; chemistry ; Protein Binding ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; metabolism ; Protein Tyrosine Phosphatases ; antagonists & inhibitors ; Rubus ; chemistry ; Structure-Activity Relationship ; Triterpenes ; chemistry ; metabolism

OBJECTIVETo investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials.

METHODSThe wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay.

RESULTSATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively).

CONCLUSIONSBoth SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

Adenosine Triphosphate ; pharmacology ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Dual-Specificity Phosphatases ; Genetic Vectors ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase Phosphatases ; Neoplasm Invasiveness ; Phosphorylation ; Prostatic Neoplasms ; metabolism ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Protein Tyrosine Phosphatases ; genetics ; metabolism ; Receptors, Purinergic P2 ; physiology ; Signal Transduction ; Transfection ; p38 Mitogen-Activated Protein Kinases ; metabolism