Daidzein promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and improves glucose homeostasis in Type 2 diabetic model mice

Daidzein shows estrogenic, antioxidant and antiandrogenic properties as well as cell cycle regulatory activity. However, the antihyperglycemic effect of daidzein remains to be elucidated. In this study, we investigated the in vitro effect of daidzein on glucose uptake, AMPK phosphorylation and GLUT4 translocation on plasma membrane in L6 myotubes and its in vivo antihyperglycmic effect in obese–diabetic model db/db mice. Daidzein was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by daidzein of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a GLUT4 cDNA-coding vector. Daidzein (0.1% in the diet) suppressed the rises in the fasting blood glucose, serum total cholesterol levels and homeostasis model assessment index of db/db mice. In addition, daidzein supplementation markedly improved the AMPK phosphorylation in gastrocnemius muscle of db/db mice. Daidzein also suppressed increases in blood glucose levels and urinary glucose excretion in KK-Ay mice, another Type 2 diabetic animal model. These in vitro and in vivo findings suggest that daidzein is preventive for Type 2 diabetes and an antidiabetic phytochemical.     

D-Pinitol and myo-Inositol Stimulate Translocation of Glucose Transporter 4 in Skeletal Muscle of C57BL/6 Mice

Diabetes mellitus is a complex disease that is characterized by the defection of insulin sensitivity in such peripheral tissues as skeletal muscle, adipose tissue and liver. We have previously demonstrated that certain inositol derivatives stimulated glucose uptake accompanied by the translocation of glucose transporter 4 (GLUT4) to the plasma membrane in L6 myotubes. We investigated in this present study whether an oral intake of D-pinitol (PI) and myo-inositol (MI) would affect GLUT4 translocation in the skeletal muscle of mice. PI or MI at 1 g/kg BW administered orally to mice 30 min before a post-oral injection of glucose at 2 g/kg BW resulted in both PI and MI increasing GLUT4 translocation in the skeletal muscle and lowering the plasma glucose and insulin levels. PI and MI, therefore, have the potential to prevent diabetes mellitus by reducing the postprandial blood glucose level and stimulating GLUT4 translocation in the skeletal muscle.     

The Axin/TNKS complex interacts with KIF3A and is required for insulin-stimulated GLUT4 translocation

Insulin-stimulated glucose uptake by the glucose transporter GLUT4 plays a central role in whole-body glucose homeostasis, dysregulation of which leads to type 2 diabetes. However, the molecular components and mechanisms regulating insulin-stimulated glucose uptake remain largely unclear. Here, we demonstrate that Axin interacts with the ADP-ribosylase tankyrase 2 (TNKS2) and the kinesin motor protein KIF3A, forming a ternary complex crucial for GLUT4 translocation in response to insulin. Specific knockdown of the individual components of the complex attenuated insulin-stimulated GLUT4 translocation to the plasma membrane. Importantly, TNKS2^−/− mice exhibit reduced insulin sensitivity and higher blood glucose levels when re-fed after fasting. Mechanistically, we demonstrate that in the absence of insulin, Axin, TNKS and KIF3A are co-localized with GLUT4 on the trans-Golgi network. Insulin treatment suppresses the ADP-ribosylase activity of TNKS, leading to a reduction in ADP ribosylation and ubiquitination of both Axin and TNKS, and a concurrent stabilization of the complex. Inhibition of Akt, the major effector kinase of insulin signaling, abrogates the insulin-mediated complex stabilization. We have thus elucidated a new protein complex that is directly associated with the motor protein kinesin in insulin-stimulated GLUT4 translocation.     

Design,synthesis and anti-diabetic activity of triazolotriazine derivatives as dipeptidyl peptidase-4 (DPP-4) inhibitors

Type 2 diabetes mellitus (T2DM) is one of the major global metabolic disorders characterized by insulin resistance and chronic hyperglycemia. Inhibition of the enzyme, dipeptidyl peptidase-4 (DPP-4) has been proved as successful and safe therapy for the treatment of T2DM since last decade. In order to design novel DPP-4 inhibitors, various in silico studies such as 3D-QSAR, pharmacophore modeling and virtual screening were performed and on the basis of the combined results of them, total 50 triazolo[5,1-c][1,2,4]triazine derivatives were designed and mapped on the best pharmacophore model. From this, best 25 derivatives were docked onto the active site of DPP-4 enzyme and in silico ADMET properties were also predicted. Finally, top 17 derivatives were synthesized and characterized using FT-IR, Mass, ¹H NMR and ¹³C NMR spectroscopy. Purity of compounds was checked using HPLC. These derivatives were then evaluated for in vitro DPP-4 inhibition. The most promising compound 15q showed 28.05 μM DPP-4 IC₅₀ with 8–10-fold selectivity over DPP-8 and DPP-9 so selected for further in vivo anti-diabetic evaluation. During OGTT in normal C57BL/6J mice, compound 15q reduced blood glucose excursion in a dose-dependent manner. Chronic treatment for 28 days with compound 15q improved the serum glucose levels in type 2 diabetic Sprague Dawley rats wherein diabetes was induced by high fat diet and low dose streptozotocin. This suggested that compound 15q is a moderately potent and selective hit molecule which can be further optimized structurally to increase the efficacy and overall pharmacological profile as DPP-4 inhibitor.     

In vitro anti-hyperglycemic activity of 4-hydroxyisoleucine derivatives

The nonproteinogenic amino acid, 4-hydroxyisoleucine (1) has been isolated in large quantities from the fenugreek (T. foenum-graecum) seeds. Few novel derivatives (3–11 and 13–18) were prepared from the naturally occurring 4-hydroxyisoleucine (1) and screened for their in vitro glucose uptake stimulatory effect in L-6 skeletal muscle cells. The derivatives 6, 7, 8, 10 and 11 exhibited better glucose uptake stimulatory activity than parent compound, 4-hydroxyisoleucine at 5 and 10 µM concentrations and compounds 7 and 11 enhanced translocation of insulin sensitive glucose transporters-4 in skeletal muscle cells.     

Surrogating and redirection of pyrazolo[1,5-a]pyrimidin-7(4H)-one core,a novel class of potent and selective DPP-4 inhibitors

The initial focus on characterizing novel pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as DPP-4 inhibitors, led to a potent and selective inhibitor compound b2. This ligand exhibits potent in vitro DPP-4 inhibitory activity (IC₅₀: 80?nM), while maintaining other key cellular parameters such as high selectivity, low cytotoxicity and good cell viability. Subsequent optimization of b2 based on docking analysis and structure-based drug design knowledge resulted in d1. Compound d1 has nearly 2-fold increase of inhibitory activity (IC₅₀: 49?nM) and over 1000-fold selectivity against DPP-8 and DPP-9. Further in vivo IPGTT assays showed that compound b2 effectively reduce glucose excursion by 34% at the dose of 10?mg/kg in diabetic mice. Herein we report the optimization and design of a potent and highly selective series of pyrazolo[1,5-a]pyrimidin-7(4H)-one DPP-4 inhibitors.     

Isoproterenol inhibits cyclic AMP-mediated but not insulin-mediated translocation of the GLUT4 glucose transporter isoform

Isoproterenol is a beta adrenergic agonist whose effects have been attributed to the generation of cAMP. Previous studies have shown that it inhibits glucose transport in adipocytes without changing the number of insulin-responsive glucose transporters (GLUT4) on the cell surface. However, we have shown previously that cAMP stimulates translocation of GLUT4 to the cell surface in adipocytes (Keladaet al. J Biol Chem 267, 7021–7025, 1992). We therefore further investigated the mechanisms involved in isoproterenol regulation of glucose transport. Consistent with the effects of dibutyryl cAMP, we found that a low concentration of isoproterenol (10 nM) stimulated glucose transport and the translocation of GLUT4 from the low density microsomal fraction to the plasma membrane. By contrast, a higher concentration of isoproterenol (1 M) did not stimulate transport or GLUT4 translocation and furthermore inhibited dibutyryl cAMP-stimulated GLUT4 translocation. This inhibitory effect was specific for cAMP since isoproterenol had no effect on insulin-stimulated GLUT4 translocation. We conclude that isoproterenol has a biphasic effect on glucose transport, mediated by acute translocation of GLUT4 at low concentrations and by inhibition of intrinsic activity at high concentration, both of which may be explained by effects of cAMP. It has a further cAMP-independent effect at high concentration to inhibit cAMP-mediated translocation of GLUT4.This work forms portions of the PhD thesis requirements.     

Epigallocatechin gallate promotes GLUT4 translocation in skeletal muscle

In this study, we investigated whether epigallocatechin gallate (EGCg) affects glucose uptake activity and the translocation of insulin-sensitive glucose transporter (GLUT) 4 in skeletal muscle. A single oral administration of EGCg at 75 mg/kg body weight promoted GLUT4 translocation in skeletal muscle of rats. EGCg significantly increased glucose uptake accompanying GLUT4 translocation in L6 myotubes at 1 nM. The translocation of GLUT4 was also observed both in skeletal muscle of mice and rats ex vivo and in insulin-resistant L6 myotubes. Wortmannin, an inhibitor of phosphatidylinositol 3′-kinase, inhibited both EGCg- and insulin-increased glucose uptakes, while genistein, an inhibitor of tyrosine kinase, failed to inhibit the EGCg-increased uptake. Therefore, EGCg may improve hyperglycemia by promoting GLUT4 translocation in skeletal muscle with partially different mechanism from insulin.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

Chemical constituents from Eucalyptus citriodora Hook leaves and their glucose transporter 4 translocation activities

Bioassay-guided phytochemical investigation of the EtOAc fraction from the leaves of a Chinese medicinal herb, Eucalyptus citriodora Hook, resulted in the isolation of a new compound rhodomyrtosone E (1), along with 12 known compounds (2–13). The structure of the new compound was established by 1D and 2D NMR, MS data and X-ray crystallographic analysis. Betulinic acid (2) and corosolic acid (5) increased glucose transporter 4 (GLUT-4) translocation by 2.38 and 1.78-fold, respectively.     

Dimethyl sulfoxide enhances GLUT4 translocation through a reduction in GLUT4 endocytosis in insulin-stimulated 3T3-L1 adipocytes

Insulin increases muscle and fat cell glucose uptake by inducing the translocation of glucose transporter GLUT4 from intracellular compartments to the plasma membrane. Here, we have demonstrated that in 3T3-L1 adipocytes, DMSO at concentrations higher than 7.5% augmented cell surface GLUT4 levels in the absence and presence of insulin, but that at lower concentrations, DMSO only enhanced GLUT4 levels in insulin-stimulated cells. At a 5% concentration, DMSO also increased cell surface levels of the transferrin receptor and GLUT1. Glucose uptake experiments indicated that while DMSO enhanced cell surface glucose transporter levels, it also inhibited glucose transporter activity. Our studies further demonstrated that DMSO did not sensitize the adipocytes for insulin and that its effect on GLUT4 was readily reversible (t1/2∼12 min) and maintained in insulin-resistant adipocytes. An enhancement of insulin-induced GLUT4 translocation was not observed in 3T3-L1 preadipocytes and L6 myotubes, indicating cell specificity. DMSO did not enhance insulin signaling nor exocytosis of GLUT4 vesicles, but inhibited GLUT4 internalization. While other chemical chaperones (glycerol and 4-phenyl butyric acid) also acutely enhanced insulin-induced GLUT4 translocation, these effects were not mediated via changes in GLUT4 endocytosis. We conclude that DMSO is the first molecule to be described that instantaneously enhances insulin-induced increases in cell surface GLUT4 levels in adipocytes, at least in part through a reduction in GLUT4 endocytosis.     

A high-fat diet catalyzes progression to hyperglycemia in mice with selective impairment of insulin action in Glut4-expressing tissues

Insulin resistance impairs postprandial glucose uptake through glucose transporter type 4 (GLUT4) and is the primary defect preceding type 2 diabetes. We previously generated an insulin-resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in the muscle, adipose, and neuronal subpopulations. However, the rate of diabetes in GIRKO mice remained low prior to 6 months of age on normal chow diet (NCD), suggesting that additional factors/mechanisms are responsible for adverse metabolic effects driving the ultimate progression of overt diabetes. In this study, we characterized the metabolic phenotypes of the adult GIRKO mice acutely switched to high-fat diet (HFD) feeding in order to identify additional metabolic challenges required for disease progression. Distinct from other diet-induced obesity (DIO) and genetic models (e.g., db/db mice), GIRKO mice remained leaner on HFD feeding, but developed other cardinal features of insulin resistance syndrome. GIRKO mice rapidly developed hyperglycemia despite compensatory increases in β-cell mass and hyperinsulinemia. Furthermore, GIRKO mice also had impaired oral glucose tolerance and a limited glucose-lowering benefit from exendin-4, suggesting that the blunted incretin effect contributed to hyperglycemia. Secondly, GIRKO mice manifested severe dyslipidemia while on HFD due to elevated hepatic lipid secretion, serum triglyceride concentration, and lipid droplet accumulation in hepatocytes. Thirdly, GIRKO mice on HFD had increased inflammatory cues in the gut, which were associated with the HFD-induced microbiome alterations and increased serum lipopolysaccharide (LPS). In conclusion, our studies identified important gene/diet interactions contributing to diabetes progression, which might be leveraged to develop more efficacious therapies.     

Synthesis and biological evaluation of pyrrolidine-2-carbonitrile and 4-fluoropyrrolidine-2-carbonitrile derivatives as dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes

A novel series of pyrrolidine-2-carbonitrile and 4-fluoropyrrolidine-2-carbonitrile derivatives was designed, synthesized, and found to act as dipeptidyl peptidase-4 (DPP-4) inhibitors. From this series of compounds, compound 17a was identified as an efficacious, safe, and selective inhibitor of DPP-4. In vivo studies in ICR and KKAy mice showed that administration of this compound resulted in decreased blood glucose in these mice after an oral glucose challenge. Compound 17a showed high DPP-4 inhibitory activity (IC₅₀ = 0.017 μM), moderate selectivity against DPP-4 (selective ratio: DPP-8/DPP-4 = 1324; DPP-9/DPP-4 = 1164), and good efficacy in oral glucose tolerance tests in ICR and KKAy mice. These in vivo anti-diabetic properties and its desirable pharmacokinetic profile in Sprague–Dawley rats demonstrate that compound 17a is a promising candidate for development as an anti-diabetic agent.     

Discovery of triazole-based uracil derivatives bearing amide moieties as novel dipeptidyl peptidase-IV inhibitors

Dipeptidyl peptidase-IV (DPP-4) is a validated target for T2DM treatment. We previously reported a novel series of triazole-based uracil derivatives bearing aliphatic carboxylic acids with potent DPP-4 inhibitory activities in vitro, but these compounds showed poor hypoglycemic effects in vivo. Herein we further optimized the triazole moiety by amidation of the carboxylic acid to improve in vivo activities. Two series of compounds 3a-f and 4a-g were designed and synthesized. By screening in DPP-4, compound 4c was identified as a potent DPP-4 inhibitor with the IC₅₀ value of 28.62 nM. Docking study revealed compound 4c has a favorable binding mode and interpreted the SAR of these analogs. DPP-8 and DPP-9 tests indicated compound 4c had excellent selectivity over DPP-8 and DPP-9. Further in vivo evaluations revealed that compound 4c showed more potent hypoglycemic activity than its corresponding carboxylic acid in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic C57BL/6 mice. The overall results have shown that compound 4c could be a promising lead for further development of novel DPP-4 agents treating T2DM.     

Chemical constituents from Sophora tonkinensis and their glucose transporter 4 translocation activities

Bioassay-guided phytochemical investigation of the EtOAc fraction (ST-EtOAc) from the roots of Sophora tonkinensis resulted in the isolation of a new compound 6aR,11aR-1-hydroxy-4-isoprenyl-maackiain (1), along with 12 known compounds (2–13). The structure of the new compound was established by 1D and 2D NMR, MS data and circular dichroism analysis. Polyprenylated flavonoids 6–9 and 11–13 increased GLUT-4 translocation by the range of 1.35–2.75 folds. Sophoranone (8) exerted the strongest activity with 2.75 folds GLUT-4 translocation enhancement at the concentration of 10 μM. This is the first report of the GLUT-4 translocation activity of the plant Sophora tonkinensis.     

Tetrahydroquinoline and tetrahydroisoquinoline derivatives as potential selective PDE4B inhibitors

Tetrahydroquinoline and tetrahydroisoquinoline derivatives containing 2-phenyl-5-furan moiety were designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. The bioassay results showed that title compounds showed good inhibitory activity against PDE4B and blockade of LPS (lipopolysaccharide) induced TNF-α release, which also exhibited considerable in vivo activity in animal models of asthma/COPD (chronic obstructive pulmonary disease) and sepsis induced by LPS. The bioactivity of compounds containing tetrahydroquinoline (series 4) was higher than that of tetrahydroisoquinoline derivatives (series 3). Compound 4?m with 4-methoxybenzene moiety exhibited the best potential selective activity against PDE4B. The primary structure–activity relationship study and docking results showed that the tetrahydroquinoline moiety of compound 4?m played a key role to form hydrogen bonds and π-π stacking interaction with PDE4B protein while the rest part of the molecule extended into the catalytic domain to block the access of cAMP and formed the foundation for inhibition of PDE4B. Based on LPS induced sepsis model for the measurement of TNF-α inhibition in Swiss Albino mice and neutrophilia inhibition for asthma and COPD in Sprague Dawley rats with the potential molecules, compound 4?m would be great promise as a hit inhibitor in the future study.     

In vivo and in vitro application of black soybean peptides in the amelioration of endoplasmic reticulum stress and improvement of insulin resistance

AimsHepatic endoplasmic reticulum (ER) stress plays a key role in the development of obesity-induced insulin resistance. This study evaluated the effects of peptides from black soybean (BSP) on ER stress and insulin signaling in vitro and in vivo.Main methodsUsing C2C12 myotubes or HepG2 cells, we evaluated the effects of BSP on the expression of proteins involved in insulin signaling and in the ER stress response in insulin-sensitive or insulin-resistant cells. BSP was given orally to db/db mice for 5 weeks to investigate its antidiabetic effects in vivo and the underlying mechanisms.Key findingsBSP increased GLUT4 translocation and glucose transport in myotubes and stimulated Akt-mediated glycogen synthase kinase-3β (GSK-3β) and Foxo1 phosphorylation in HepG2 cells. BSP significantly restored the suppression of insulin-mediated Akt phosphorylation in insulin-resistant cells. BSP significantly inhibited the activation of ER stress-responsive proteins by thapsigargin. BSP also significantly reduced blood glucose and improved glucose tolerance in db/db mice. The serum lipid profile (triglyceride and high-density lipoprotein concentrations) improved concomitantly with the BSP-induced downregulation of hepatic fatty acid synthase expression in db/db mice. Consistent with the results observed in HepG2 cells, BSP downregulated the elevated hepatic ER stress response in diabetic mice concomitantly with an increased expression of phospho-Foxo1.SignificanceA peptide mixture, BSP, showed beneficial effects through multiple mechanisms involving the suppression of hepatic ER stress and restoration of insulin resistance, suggesting that it has potential as an antidiabetic agent.     

Coordinated Regulation of Vasopressin Inactivation and Glucose Uptake by Action of TUG Protein in Muscle

In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake.     

Biomolecular Characterization of Putative Antidiabetic Herbal Extracts

Induction of GLUT4 translocation in the absence of insulin is considered a key concept to decrease elevated blood glucose levels in diabetics. Due to the lack of pharmaceuticals that specifically increase the uptake of glucose from the blood circuit, application of natural compounds might be an alternative strategy. However, the effects and mechanisms of action remain unknown for many of those substances. For this study we investigated extracts prepared from seven different plants, which have been reported to exhibit anti-diabetic effects, for their GLUT4 translocation inducing properties. Quantitation of GLUT4 translocation was determined by total internal reflection fluorescence (TIRF) microscopy in insulin sensitive CHO-K1 cells and adipocytes. Two extracts prepared from purslane (Portulaca oleracea) and tindora (Coccinia grandis) were found to induce GLUT4 translocation, accompanied by an increase of intracellular glucose concentrations. Our results indicate that the PI3K pathway is mainly responsible for the respective translocation process. Atomic force microscopy was used to prove complete plasma membrane insertion. Furthermore, this approach suggested a compound mediated distribution of GLUT4 molecules in the plasma membrane similar to insulin stimulated conditions. Utilizing a fluorescent actin marker, TIRF measurements indicated an impact of purslane and tindora on actin remodeling as observed in insulin treated cells. Finally, in-ovo experiments suggested a significant reduction of blood glucose levels under tindora and purslane treated conditions in a living organism. In conclusion, this study confirms the anti-diabetic properties of tindora and purslane, which stimulate GLUT4 translocation in an insulin-like manner.     

Discovery of 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methylpyridin-2-yl)-benzamide as a novel glucokinase activator (GKA) for the treatment of type 2 diabetes mellitus

Novel heteroaryl-containing benzamide derivatives were synthesized and screened using an in vitro assay measuring increases in glucose uptake and glucokinase activity stimulated by 10 mM glucose in rat hepatocytes. From a library of synthesized compounds, 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methyl pyridin-2-yl)-benzamide (19e) was identified as a potent glucokinase activator with assays demonstrating an EC₅₀ of 315 nM and the induction of a 2.23 fold increase in glucose uptake. Compound 19e exhibited a glucose AUC reduction of 32% (50 mg/kg) in an OGTT study with C57BL/6J mice compared to 28% for metformin (300 mg/kg). Single treatment of the compound in C57BL/J6 and ob/ob mice elicited basal glucose lowering activity, while in a two-week repeated dose study with ob/ob mice, the compound significantly decreased blood glucose levels with no evidence of hypoglycemia risk. In addition, 19e exhibited favorable pharmacokinetic parameters in mice and rats and excellent safety margins in liver and testicular toxicity studies. Compound 19e was therefore selected as a development candidate for the potential treatment of type 2 diabetes.