PTP1B Targeted In Screening Hypoglycemic Drugs

Diabetes mellitus (DM) is a chronic disease accompanied by a series ofmetabolic disorders due to insulin deficiency or impaired insulin action. Theprevalence of DM is increasing globally. According to the latest data from theInternational Diabetes Federation (IDF),382million people are living with diabetesin2013and by2035this figure will rise to592million. Type2diabetes mellitus(T2DM) is the most common form and accounts for around90%of all diabetesworldwide, which is characterized by hyperglycemia, dyslipidaemia and insulinresistance in its metabolic target tissues. T2DM may result in severe complications,including renal failure, blindness, slow wound healings and cardiovascular diseases.Protein tyrosine phosphatases (PTP), the regulators of tyrosinephosphorylation-dependent cellular events, play an important role in numerouscritical physiological processes and metabolism. Malfunction of PTP activity hassignificant implications in many human diseases. PTP1B is the first member of PTPsuperfamily isolated from human placenta and characterized as a~50kDa protein(435amino acids), contains a highly conserved catalytic motif, in which Cys215andArg221are crucial for its phosphatase catalytic activity.PTP1B is a key negative-regulator of insulin signaling pathway, which acts byblocking the insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR)and thereby the insulin receptor substrate-1(IRS-1). Normally, insulin evokes acascade of phosphorylation events, starting with the autophosphorylation of IR onmultiple tyrosyl residues, which enhances IR kinase activity and leads to recruitmentof IRS-1, followed by activation of phosphatidylinositol3-kinase (PI3K), proteinkinase B (PKB; also known as AKT), and finally glucose transporter4(GLUT4) andextracellular signal-regulated kinases1/2(Erk1/2). Activated GLUT4couldtranslocate to membrane and thus affect the glucose uptake. ERK could enter cellnucleus and affect the express and translation of gene. Overexpressions of PTP1B or increased activities of this enzyme were found in insulin-resistant and obese patients.PTP1B-null mice are healthy, display an enhanced sensitivity to insulin, and areresistant to type2diabetes and obesity when fed with a high fat diet. Considerableinterest grew in the potential of PTP1B as a therapeutic target for treating diabetesand obesity.Magnolia bark is a traditional Chinese medicine, known under the name houpu(from Magnolia officinalis), which has been used for years to treat a variety ofdisorders including anxiety, nervous disturbances,"stagnation of qi"(low energy),asthma and digestive problems. However, its anti-diabetic potency and mechanism invivo are still unclear. In our preliminary study, we purified the catalytic domain ofhuman PTP1B from recombinant E. coli cells. We screened the inhibitors of PTP1Bin vitro from diverse traditional Chinese drug. We found that magnolia extract wasan efficient inhibitor of PTP1B with the IC50of55.96μg/ml. Moreover, magnoliaextract displayed good selectivity on PTP1B over other PTPs family members,including SHP1(IC50=501.94μg/ml), SHP2(IC50=521.56μg/ml), TCPTP (IC50=395.59μg/ml) and HePTP (IC50=677.88μg/ml). Kinetic analysis confirmed thatmagnolia extract was a reversible competitive inhibitor of PTP1B and the inhibitorconstant (Ki) was calculated to be0.049mg/ml. Hence, magnolia extract wasselected to further investigate the hypoglycemic potency in the followingexperimental section.In order to confirm that whether magnolia extract takes its inhibitory effect onPTP1B in cell level,3T3-L1adipocytes and C2C12myotubes are stimulated withmagnolia extract at various concentrations. We found that magnolia extract enhancedtyrosine phosphorylation levels of cellular proteins in a dose-dependent manner inboth3T3-L1adipocytes and C2C12myotubes. Furthermore, magnolia extractenhanced the insulin-induced tyrosine phosphorylations of IRβ and ERK1/2in adose-dependent manner, without altering the total protein levels. In addition,magnolia extract enhanced insulin-stimulated GLUT4translocation in aconcentration-dependent manner. These findings revealed that magnolia extract improved insulin-sensitivity by inhibiting PTP1B activity and subsequently activatedthe downstream signaling pathway, for instance ERK1/2phosphorylation andGLUT4translocation.To further investigate the hypoglycemic potency of magnolia extract, db/dbmice suffering insulin resistance and severe hyperglycemia were selected as a modelof T2DM. We found that db/db diabetic mice treated orally by0.5g/kg magnoliaextract for5weeks showed an obvious decrease in fasting plasma glucose levelcompared with the diabetic control mice without drug treatment. To explore thepotential toxicity, we treated Chinese Kunming mice with different dose of magnoliaexact. The acute toxicity of magnolia extract is preliminarily evaluated as medianlethal dose with95%confidence (LD50=2.0±0.2g/kg), which indicate thatmagnolia extract has little toxicity and is well tolerated.We conclude that magnolia exact with its competitive inhibitory activity withgood selectivity on PTP1B enhanced insulin signaling pathway and promotedGLUT4translocation, and consequently lowered the blood glucose levelsdramatically in db/db mice. All our results have thus highlighted the potential ofmagnolia exact as an inhibitory targeting on PTP1B for anti-T2DM drug discovery.