Insulin-mimetic Compound Research Articles

Identification of Insulin-Mimetic Phytochemicals from Mas Cotek (Ficus deltoidea) for Treatment of Type 2 Diabetes via LC-MS/MS and Molecular Docking Analyses

Type 2 diabetes mellitus, a metabolic syndrome, has become increasingly prevalent in recent years. In the treatment of chronic T2DM, patients are required to take insulin daily, commonly through injections, as the hormone can easily be degraded by the digestive system if taken orally. This can be an uncomfortable experience for the patient. Thus, finding an alternative to insulin, especially from natural compounds, would be beneficial. Ficus deltoidea, which belongs to the Moraceae family, is a medicinal plant known for its anti-diabetic properties. Therefore, this study aimed to identify the phytochemicals from F. deltoidea that mimic insulin by studying their ability to bind to insulin receptors using in silico analysis. A total of 36 phytochemicals were identified in the methanolic extract of F. deltoidea through LC-MS/MS analysis. They were then subjected to molecular docking to determine their binding free energy with the insulin receptor (IR). The results revealed seven phytochemicals with the lowest binding free energy, with 2,3-dihydroxy-N,N'-bis[(E)-1-(4-hydroxy-6-methyl-2-oxochromen-3-yl)ethylideneamino] butanediamide exhibiting the lowest binding free energy at -10.0 kcal/mol. Hence, these phytochemicals demonstrate potential as insulin-mimetic compounds that can be used in the treatment of T2DM.

Cinnamaldehyde supplementation acts as an insulin mimetic compound improving glucose metabolism during adolescence, but not during adulthood, in healthy male rats.

Adolescence is a critical period of increased vulnerability to nutritional modifications, and adolescents may respond differently from adults to dietary intake and nutraceuticals. Cinnamaldehyde, a major bioactive compound of cinnamon, improves energy metabolism, as has been shown in studies conducted primarily in adult animals. We hypothesized that cinnamaldehyde treatment may have a higher impact on the glycemic homeostasis of healthy adolescent rats than on healthy adult rats. Male adolescent (30 days) or adult (90 days) Wistar rats received cinnamaldehyde (40 mg/kg) for 28 days by gavage. The oral glucose tolerance test (OGTT), liver glycogen content, serum insulin concentration, serum lipid profile, and hepatic insulin signaling marker expression were evaluated. Cinnamaldehyde-treated adolescent rats showed less weight gain (P = 0.041), improved OGTT (P = 0.004), increased expression of phosphorylated IRS-1 (P = 0.015), and a trend to increase phosphorylated IRS-1 (P = 0.063) in the liver of adolescent rats in the basal state. None of these parameters was modified after treatment with cinnamaldehyde in the adult group. Cumulative food intake, visceral adiposity, liver weight, serum insulin, serum lipid profile, hepatic glycogen content, and liver protein expression of IRβ, phosphorylated IRβ, AKT, phosphorylated AKT, and PTP-1B in the basal state were similar between both age groups. In a healthy metabolic condition, cinnamaldehyde supplementation affects glycemic metabolism in adolescent rats while promoting no changes in adult rats.

Targeting Breast Cancer and Their Stem Cell Population through AMPK Activation: Novel Insights.

Despite some significant advancements, breast cancer has become the most prevalent cancer in the world. One of the main reasons for failure in treatment and metastasis has been attributed to the presence of cancer initiating cells—cancer stem cells. Consequently, research is now being focussed on targeting cancer cells along with their stem cell population. Non-oncology drugs are gaining increasing attention for their potent anticancer activities. Metformin, a drug commonly used to treat type 2 diabetes, is the best example in this regard. It exerts its therapeutic action by activating 5′ adenosine monophosphate-activated protein kinase (AMPK). Activated AMPK subsequently phosphorylates and targets several cellular pathways involved in cell growth and proliferation and the maintenance of stem-like properties of cancer stem cells. Therefore, AMPK is emerging as a target of choice for developing effective anticancer drugs. Vanadium compounds are well-known PTP inhibitors and AMPK activators. They find extensive applications in treatment of diabetes and obesity via PTP1B inhibition and AMPK-mediated inhibition of adipogenesis. However, their role in targeting cancer stem cells has not been explored yet. This review is an attempt to establish the applications of insulin mimetic vanadium compounds for the treatment of breast cancer by AMPK activation and PTP1B inhibition pathways.

V(III) and V(IV) Schiff base complexes as potential insulin-mimetic compounds – Comparison, characterization and biological activity

The syntheses of five vanadium complexes with ONO donor Schiff bases (L) along with analyses of obtained compounds were described: two [VO(L)(phen)] (phen = 1,10-phenanthroline) with V(IV) and three [V(L)2] with V(III). Ligands were derived from phenylacetic hydrazide and four aldehydes: 3-bromo-5-chloro-salicylaldehyde, 4-(diethylamino)-salicylaldehyde, 3,5-dichloro-salicylaldehyde and 3-metoxy-5-bromo-salicylaldehyde. The single crystal X-ray studies of both V(IV) complexes indicate on octahedral geometry of vanadium. The IR, UV–Vis (reflectance and in solution), cyclic voltammetry and magnetic moment measurements are presented and discussed. The stability of complexes in solutions of different pH was measured. Inhibition of human PTP1B phosphatase assays were performed for all obtained compounds and discussed in terms of their stability.

Local insulin application has a dose-dependent effect on lumbar fusion in a rabbit model.

The purpose of this study was to determine if locally applied insulin has a dose-responsive effect on posterolateral lumbar fusion. Adult male New Zealand White rabbits underwent posterolateral intertransverse spinal fusions (PLFs) at L5-L6 using suboptimal amounts of autograft. Fusion sites were treated with collagen sponge soaked in saline (control, n=11), or with insulin at low (5 or 10 units, n=13), mid (20 units, n=11), and high (40 units, n=11) doses. Rabbits were euthanized at 6weeks. The L5-L6 spine segment underwent manual palpation and radiographic evaluation performed by two fellowship trained spine surgeons blinded to treatment. Differences between groups were evaluated by analysis of variance on ranks followed by post-hoc Dunn's tests. Forty-three rabbits were euthanized at the planned 6weeks endpoint, while three died or were euthanized prior to the endpoint. Radiographic evaluation found bilateral solid fusion in 10%, 31%, 60%, and 60% of the rabbits from the control and low, mid, and high-dose insulin-treated groups, respectively (p<0.05). As per manual palpation, 7 of 10 rabbits in the mid-dose insulin group were fused as compared to 1 of 10 rabbits in the control group (p<0.05). This study demonstrates that insulin enhanced the effectiveness of autograft to increase fusion success in the rabbit PLF model. The study indicates that insulin or insulin-mimetic compounds can be used to promote bone regeneration.

Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores.

Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1–2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment.

Oleanane Triterpenoids from the Leaves of Gymnema inodorum and Their Insulin Mimetic Activities.

During an effort to find insulin mimetic compounds, the leaves of Gymnema inodorum were shown to have a stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation on a 70% ethanol extract of G. inodorum was applied to yield two new (1 and 2) and two known (8 and 9) oleanane triterpenoids with a methyl anthranilate moiety together with five further new oleanane triterpenoids (3-7). The chemical structures of all isolates were determined based on their spectroscopic data, including IR, UV, NMR, and mass spectrometric analysis. The isolated compounds (1-9) were determined for their stimulatory activities on glucose uptake in differentiated 3T3-L1 adipocyte cells using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe. Three compounds (3, 5, and 9) showed stimulatory effects on the uptake of 2-NBDG in 3T3-L1 adipocyte cells. Chemicals with a methyl anthranilate moiety have been considered as crucial contributors of flavor odor in foods, and quantitative analysis showed the content of compound 8 to be 0.90 ± 0.01 mg/g of the total extract. These results suggest that the leaves of G. inodorum have the potential to be used as an antidiabetic functional food or tea.

Molecular Docking Studies and Pharmacophore Modeling of Some Insulin Mimetic Agents from Herbal Sources: A Rational Approach towards Designing of Orally Active Insulin Mimetic Agents

Background:: Many herbal drugs have been found to possess oral insulin mimetic property as evidenced from the literature. Although, to date there is no efficient, synthetic orally active insulin-mimetic drug available clinically. Computer-Aided Drug Design (CADD) may help in the development of such agents through Pharmacophore modeling. Objective:: The present work is aimed at the In-silico designing of Pharmacophore that defines the structural requirements of a molecule to possess oral insulin-mimetic properties. Methods:: A set of 16 orally active insulin-mimetic natural compounds available through literature was used to develop a structure-based pharmacophore in a “three-step filtration process” comprised of Lipinski’s rule of 5, Minimum binding energy with the receptor and Ghose filter to the Lipinski’s rule for oral bioavailability of the drugs. The selected ligands were docked with phosphorylated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog (PDB ID: 1IR3) using Autodock 4.2 and their interaction with the receptor was analyzed followed by the generation of shared and merged feature pharmacophore by Ligandscout 4.2.1. Results:: There are three important structural features that contribute to interaction with the active site of the insulin receptor: these are hydrogen bond donor groups, hydrogen bond acceptor groups and hydrophobic interactions. It is important to note that positive or negative ionizable groups or the presence of aromatic rings are not important for the activity. Conclusion:: Taking a clue from the developed pharmacophore, one may design new lead having necessary groups required for the insulin-mimetic activity that can be elaborated synthetically to get a series of compounds with possible oral insulin-mimetic activity.

Oxovanadium(IV) Coordination Compounds with Kojic Acid Derivatives in Aqueous Solution.

Hydroxypyrone derivatives have a good bioavailability in rats and mice and have been used in drug development. Moreover, they show chelating properties towards vanadyl cation that could be used in insulin-mimetic compound development. In this work, the formation of coordination compounds of oxovanadium(IV) with four kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone) derivatives was studied. The synthetized studied ligands (S2, S3, S4, and SC) have two or three kojic acid units linked through diamines or tris(2-aminoethyl)amine chains, respectively. The chemical systems were studied by potentiometry (25 °C, ionic strength 0.1 mol L−1 with KCl), and UV-visible and EPR spectroscopy. The experimental data were analyzed by a thermodynamic and a chemometric (Multivariate Curve Resolution–Alternating Least Squares) approach. Chemical coordination models were proposed, together with the species formation constants and the pure estimated UV-vis and EPR spectra. In all systems, the coordination of the oxovanadium(IV) starts already under acidic conditions (the cation is totally bound at pH higher than 3–4) and the metal species remain stable even at pH 8. Ligands S3, S4, and SC form three coordination species. Two of them are probably due to the successive insertion of the kojate units in the coordination shell, whereas the third is most likely a hydrolytic species.

Synthesis, structure and properties of V(III,IV and V) complexes with ONO Schiff bases

The synthesis and physicochemical properties of vanadium(III,IV,V) complexes with Schiff base ligands based on 3,5-dibromo-4-methoxy-salicylaldehyde and phenylacetic hydrazide (H2L1), 5-chlorosalicylaldehyde and 4-hydroxybenzhydrazide (H2L2) and 5-chlorosalicylaldehyde and 2-hydroxybenzhydrazide (H2L3) were presented. The formulas of the complexes {[V(L1)(HL1)]·EtOH (1), [VO(L2)(phen)]·2H2O (2) and [VO(L3)(EtO)] (3)} were proposed based on the elemental analysis, IR and UV-Vis spectra. Additionally, the IR and UV-Vis spectra (in solvents as well as in a solid state) have been discussed from the vanadium oxidation state point of view. The single crystal structure of 3 shows triclinic, P-1 space group, structure is stabilized by hydrogen bonds and strong π-π stacking interactions. The oxidation state of the metal centre was also confirmed by the magnetic susceptibility measurements. The stability of the complexes was measured in pH = 7.00 and in pH = 2.00 which allows to evaluate the use of these compounds as insulin mimetic compounds.

Properties, structure and stability of V(IV) hydrazide Schiff base ligand complex

The reaction of vanadyl sulfate, VOSO4·H2O, with Schiff base ligand based on 2-hydroxybenzhydrazide and 5-bromosalicylaldehyde, with addition of 1,10-phenantroline as co-ligand, in ethanolic solution results in isolation of oxovanadium(IV) complex [VO(phen)L]·H2O (1). The complex was characterized by elemental analysis, single crystal X-ray structure measurements, IR, UV–Vis, EPR spectroscopy and cyclic voltammetry. Magnetic susceptibility measurements indicated the +4 vanadium oxidation state. The molecular structure of the complex was confirmed by single-crystal X-ray diffraction, revealing that the central vanadium atom was located in a six-coordinate distorted octahedral geometry. The stability of the complex at native pH and pH = 2.00 was also measured and the results are promising for application the vanadium complex as insulin mimetic compound.

An In Ovo Model for Testing Insulin-mimetic Compounds.

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and β-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An &lt;em&gt;In Ovo&lt;/em&gt; Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and β-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

Evidences for diabetes and insulin mimetic activity of medicinal plants: Present status and future prospects

ABSTRACTDiabetes mellitus (DM) is a considerable systemic metabolic disorder to exhibit various metabolic and cardiovascular disorders, mainly hyperglycemia. The global projected estimate of diabetes in 2030 will be about 439 million adults, out of which 300 million expected are of type-2 diabetes mellitus (T2DM). The present knowledge revealed responsible factors, occurrence and mechanism of these factors involved in the DM diseases. Hence, the aim of this review is to address and summarize the causes, plant resources, importance, present status and future programmes for diabetes control. The present review answers the contemporary present questions raised in the scientific field on DM. Two major problems are explained in detail about the autoimmune attack or dysfunction of β-cell and insulin resistance involved for Type 1 and Type 2 DM, respectively. Though there are various approaches to reduce the ill effects of diabetes and its secondary complications, many preferred herbal formulations due to lesser side effects and low cost. For this reason still it is getting increased attention in searching antidiabetic medicinal plants for hot research and to develop targeted medicine. Recurrence of islet autoimmunity lesson from pancreatic islet cell transplantation to cure T1D was outlined. With these highlights, the review summarizes the current knowledge on diabetes occurrence, factors (environmental and genetics), and types (I, II, gestation, and secondary DM), antidiabetic plants, sources for insulin mimetic plant principle compounds and their target mechanism with current and future trusted research areas for controlling of DM.

A Small Insulinomimetic Molecule Also Improves Insulin Sensitivity in Diabetic Mice.

Dramatic increase of diabetes over the globe is in tandem with the increase in insulin requirement. This is because destruction and dysfunction of pancreatic β-cells are of common occurrence in both Type1 diabetes and Type2 diabetes, and insulin injection becomes a compulsion. Because of several problems associated with insulin injection, orally active insulin mimetic compounds would be ideal substitute. Here we report a small molecule, a peroxyvanadate compound i.e. DmpzH[VO(O2)2(dmpz)], henceforth referred as dmp, which specifically binds to insulin receptor with considerable affinity (KD-1.17μM) thus activating insulin receptor tyrosine kinase and its downstream signaling molecules resulting increased uptake of [14C] 2 Deoxy-glucose. Oral administration of dmp to streptozotocin treated BALB/c mice lowers blood glucose level and markedly stimulates glucose and fatty acid uptake by skeletal muscle and adipose tissue respectively. In db/db mice, it greatly improves insulin sensitivity through excess expression of PPARγ and its target genes i.e. adiponectin, CD36 and aP2. Study on the underlying mechanism demonstrated that excess expression of Wnt3a decreased PPARγ whereas dmp suppression of Wnt3a gene increased PPARγ expression which subsequently augmented adiponectin. Increased production of adiponectin in db/db mice due to dmp effected lowering of circulatory TG and FFA levels, activates AMPK in skeletal muscle and this stimulates mitochondrial biogenesis and bioenergetics. Decrease of lipid load along with increased mitochondrial activity greatly improves energy homeostasis which has been found to be correlated with the increased insulin sensitivity. The results obtained with dmp, therefore, strongly indicate that dmp could be a potential candidate for insulin replacement therapy.

Insulin-mimetic compound hexaquis (benzylammonium) decavanadate is antilipolytic in human fat cells.

AIMTo assess in rodent and human adipocytes the antilipolytic capacity of hexaquis(benzylammonium) decavanadate (B6V10), previously shown to exert antidiabetic effects in rodent models, such as lowering free fatty acids (FFA) and glucose circulating levels.METHODSAdipose tissue (AT) samples were obtained after informed consent from overweight women undergoing plastic surgery. Comparison of the effects of B6V10 and reference antilipolytic agents (insulin, benzylamine, vanadate) on the lipolytic activity was performed on adipocytes freshly isolated from rat, mouse and human AT. Glycerol release was measured using colorimetric assay as an index of lipolytic activity. The influence of B6V10 and reference agents on glucose transport into human fat cells was determined using the radiolabelled 2-deoxyglucose uptake assay.RESULTSIn all the species studied, B6V10 exhibited a dose-dependent inhibition of adipocyte lipolysis when triglyceride breakdown was moderately enhanced by β-adrenergic receptor stimulation. B6V10 exerted on human adipocyte a maximal lipolysis inhibition of glycerol release that was stronger than that elicited by insulin. However, B6V10 did not inhibit basal and maximally stimulated lipolysis. When incubated at dose ≥ 10 μmol/L, B6V10 stimulated by twofold the glucose uptake in human fat cells, but - similarly to benzylamine - without reaching the maximal effect of insulin, while it reproduced one-half of the insulin-stimulation of lipogenesis in mouse fat cells.CONCLUSIONB6V10 exerts insulin-like actions in adipocytes, including lipolysis inhibition and glucose transport activation. B6V10 may be useful in limiting lipotoxicity related to obesity and insulin resistance.

Functional properties of Claramine: A novel PTP1B inhibitor and insulin-mimetic compound

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a “first-in-class” highly selective inhibitor of PTP1B that can cross the blood–brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-β (IRβ), Akt and GSK3β. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.

Local manganese chloride treatment accelerates fracture healing in a rat model.

This study investigated the effects of local delivery of manganese chloride (MnCl2), an insulin-mimetic compound, upon fracture healing using a rat femoral fracture model. Mechanical testing, histomorphometry, and immunohistochemistry were performed to assess early and late parameters of fracture healing. At 4 weeks post-fracture, maximum torque to failure was 70% higher (P<0.05) and maximum torsional rigidity increased 133% (P<0.05) in animals treated with 0.125 mg/kg MnCl2 compared to saline controls. Histological analysis of the fracture callus revealed percent new mineralized tissue was 17% higher (P<0.05) at day 10. Immunohistochemical analysis of the 0.125 mg/kg MnCl2 treated group, compared to saline controls, showed a 379% increase in the density of VEGF-C+ cells. In addition, compared to saline controls, the 0.125 mg/kg MnCl2 treated group showed a 233% and 150% increase in blood vessel density in the subperiosteal region at day 10 post-fracture as assessed by detection of PECAM and smooth muscle α actin, respectively. The results suggest that local MnCl2 treatment accelerates fracture healing by increasing mechanical parameters via a potential mechanism of amplified early angiogenesis leading to increased osteogenesis. Therefore, local administration of MnCl2 is a potential therapeutic adjunct for fracture healing.

Development of a Highly Visual, Simple, and Rapid Test for the Discovery of Novel Insulin Mimetics in Living Vertebrates

Diabetes mellitus is a global epidemic with major impacts on human health and society. Drug discovery for diabetes can be facilitated by the development of a rapid, vertebrate-based screen for identifying new insulin mimetic compounds. Our study describes the first development of a zebrafish-based system based on direct monitoring of glucose flux and validated for identifying novel anti-diabetic drugs. Our system utilizes a fluorescent-tagged glucose probe in an experimentally convenient 96-well plate format. To validate our new system, we identified compounds that can induce glucose uptake via activity-guided fractionation of the inner shell from the Japanese Chestnut (Castanea crenata). The best performing compound, UP3.2, was identified as fraxidin and validated as a novel insulin mimetic using a mammalian adipocyte system. Additional screening using sets of saponin- and triazine-based compounds was undertaken to further validate this assay, which led to the discovery of triazine PP-II-A03 as a novel insulin mimetic. Moreover, we demonstrate that our zebrafish-based system allows concomitant toxicological analysis of anti-diabetic drug candidates. Thus, we have developed a rapid and inexpensive vertebrate model that can enhance diabetes drug discovery by preselecting hits from chemical library screens, before testing in relatively expensive rodent assays.

Pharmacological evaluation of insulin mimetic novel suppressors of PEPCK gene transcription from Paeoniae Rubra Radix

Paeoniae Rubra Radix (root of Paeonia lactiflora) has been frequently employed in Traditional Chinese Medicine (TCM) as and anti-diabetic therapy to enhance blood circulation and dissipate stasis. Previously, we identified a novel hypoglycemic action of a crude extract from Paeoniae Rubra Radix, which also suppressed phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Therefore, the current investigation intended to elucidate potential active bio-constituents of this herb and mechanisms of action. Glucocorticoid receptor (GR) nuclear localization, the PEPCK messenger (m)RNA level, pregnane X receptor (PXR) mRNA expression, cAMP-responsive element-binding protein (CREB) serine phosphorylation and DNA binding were evaluated in dexamethasone (Dex) and 8-bromo-cAMP (CA)-stimulated H4IIE cells, while efficacy of agents was assessed in a stable cell line containing a green fluorescent protein (GFP) reporter driven by the PEPCK promoter. HPLC profiling, colorimetric assays, and NMR analysis were employed for chemical characterization purpose. An extract of Paeoniae Rubra Radix lacking the insulin mimetic compound, 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG), and termed the non-PGG fraction (NPF), consisting of tannin polymers, suppressed PEPCK expression in the presence of an insulin receptor antagonist (HNMPA-AM(3)), suggesting the action of this fraction is independent of the insulin receptor. Furthermore, Dex-stimulated GR nuclear localization and transactivation were prevented by the NPF. Similarly, CA-stimulated CREB serine phosphorylation and DNA binding were also inhibited by the NPF in H4IIE cells. Hence NPF antagonizes both signaling pathways that induce PEPCK gene transcription. In conclusion, the current study proposes that the potent suppressive activity on PEPCK gene transcription observed with Paeoniae Rubra Radix extract, can be attributed to at least two distinct components, namely PGG and NPF.