T-Cell Death Associated Gene 51 Is a Novel Negative Regulator of PPARγ That Inhibits PPARγ-RXRα Heterodimer Formation in Adipogenesis

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.

Encapsulation and Enhanced Delivery of Topoisomerase I Inhibitors in Functionalized Carbon Nanotubes.

The topoisomerase I inhibitors SN-38 and camptothecin (CPT) have shown potent anticancer activity, but water insolubility and metabolic instability limits their clinical application. Utilizing carbon nanotubes as a protective shell for water-insoluble SN-38 and CPT while maintaining compatibility with aqueous media via a carboxylic acid-functionalized surface can thus be a strategy to overcome this limitation. Through hydrophobic-hydrophobic interactions, SN-38 and CPT were successfully encapsulated in carboxylic acid functionalized single-walled carbon nanotubes and dispersed in water. The resulting cell proliferation inhibition and drug distribution profile inside the cells suggest that these drug-encapsulated carbon nanotubes can serve as a promising delivery strategy for water-insoluble anticancer drugs.

Cyp7a1 is continuously increased with disrupted Fxr-mediated feedback inhibition in hypercholesterolemic TALLYHO/Jng mice.

TALLYHO/Jng (TH) mice reveal hypercholesterolemia at an early age before their plasma glucose levels have increased. The increased plasma cholesterol should be related to bile acids (BAs) metabolism, because cholesterol is the precursor of BAs and BAs change cholesterol metabolism in a feedback manner. We analyzed the BAs pool size, BAs composition, and expression levels of several proteins that have key roles in BAs synthesis, excretion, and reabsorption and compared them to those of C57BL/6 (B6) mice to study BAs metabolism in TH mice. TH mice exhibited an increased total BAs pool size, increased BAs content in the cecum feces, and an increased ratio of muricholic acid (MCA)/cholic acid (CA). The mRNA and protein levels of cholesterol 7 alpha-hydroxylase (Cyp7a1) and the ATP-binding cassette sub-family G member 5 (Abcg5) were elevated in the liver but not in the apical sodium bile acid transporter (Asbt) and organic solute transporters (Osts) in the ileum. These results indicate that synthesis and the excretion of BAs from the liver to the gallbladder might be elevated, but the reabsorption rate of BAs in the ileum might be reduced. The declined expression of fibroblast growth factor 15 (Fgf15) and fibroblast growth factor receptor 4 (Fgfr4) was respectively identified in the ileum and the liver, indicating the disrupted feedback inhibition of Cyp7a1. Consequently, hypercholesterolemia in TH mice might increase the BAs amounts via the interrupted Fxr/Fgf15/Fgfr4-mediated feedback regulation of Cyp7a1.

Synthesis and biological evaluation of thiazole derivatives as GPR119 agonists.

A series of 4-(phenoxymethyl)thiazole derivatives was synthesized and evaluated for their GPR119 agonistic effect. Several 4-(phenoxymethyl)thiazoles with pyrrolidine-2,5-dione moieties showed potent GPR119 agonistic activities. Among them, compound 27 and 32d showed good in vitro activity with an EC50 value of 49 nM and 18 nM, respectively with improved human and rat liver microsomal stability compare with MBX-2982. Compound 27 &32d did not exhibit significant CYP inhibition, hERG binding, and cytotoxicity. Moreover, these compounds lowered the glucose excursion in mice in an oral glucose-tolerance test.

Protective effects of carbenoxolone, an 11ß-HSD1 inhibitor, against chemical induced dry eye syndrome.

Dry eye syndrome (DES) is a disorder of the eye due to tear deficiency or excessive evaporation that causes damage to the eye and is associated with discomfort and dryness. 11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) is an enzyme that converts inactive cortisone to active cortisol. Recently, 11ß-HSD1 has been expressed in human and rodent eyes and has been recognized as a target of glaucoma. In this study, the therapeutic effects and underlying mechanisms of topical carbenoxolone, an 11ß-HSD1 inhibitor, were investigated in benzalkonium chloride (BAC)-treated human conjunctival epithelial cells and a rat DES model. In the in vitro study, carbenoxolone dose-dependently inhibited cell death and 11ß-HSD1 activity in BAC-treated human conjunctival epithelial cells. For the in vivo study, carbenoxolone or a solvent was administered to the BAC-induced DES model twice daily. BAC-treated rat eyes showed significant increases in ocular surface damage, a reduction of tears, decrease corneal thickness, corneal basement membrane destruction, apoptosis in the conjunctival epithelium, and expression of pro-inflammatory cytokines (TNF-α and IL-6) and 11ß-HSD1. These effects of BAC were reversed by topical carbenoxolone treatment. These results demonstrate that carbenoxolone can prevent DES by inhibiting pro-inflammatory cytokine expression and cell death of the corneal and conjunctival epithelium via inhibition of both 11ß-HSD1 activity and expression in the eyes of BAC-treated rats. It is suggested that topical 11ß-HSD1 inhibitors may provide a new therapeutic window in the prevention and/or treatment of DES.

Design, synthesis, and biological evaluation of aryl N-methoxyamide derivatives as GPR119 agonists.

A series of N-methoxyamide derivatives was identified and evaluated as GPR119 agonists. Several N-methoxyamides with thienopyrimidine and pyridine scaffolds showed potent GPR119 agonistic activities. Among them, compound 9c displayed good in vitro activity and potency. Moreover, compound 9c lowered glucose excursion in mice in an oral glucose tolerance test and increased GLP-1 secretion in intestinal cells.

Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells.

Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E2. Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways.

Fatty Liver and Insulin Resistance in the Liver-Specific Knockout Mice of Mitogen Inducible Gene-6.

Mitogen inducible gene-6 (Mig-6) is a feedback inhibitor of epidermal growth factor receptor (EGFR) signaling pathway. The liver-specific knockout mice of the Mig-6 gene (Mig-6 d/d ) showed hepatomegaly and increased hypercholesterolemia. In this study, the biomarkers of insulin resistance and the effects of high-fat diets in the wild (Mig-6 f/f ) and Mig-6 d/d mice were analyzed. The fasting plasma concentrations of glucose, triglyceride, cholesterols, free fatty acids, and HOMA-IR were measured and the glucose tolerance and insulin resistance tests were performed in the 25-week-old Mig-6 f/f and the Mig-6 d/d mice. The protein levels of active insulin receptor, glucose 6-phosphatase, and phosphoenolpyruvate carboxykinase were analyzed in the liver and fat. The fasting plasma cholesterol and glucose concentration were higher in the Mig-6 d/d mice than the Mig-6 f/f mice with increased fat deposition in the liver. But the Mig-6 d/d mice had the improved glucose intolerance and insulin resistance without increased amount of phosphoinsulin receptor after insulin infusion in the liver. The hepatic concentration of phosphoenolpyruvate carboxykinase was increased in fasting Mig-6 d/d mice. The feeding of high-fat diet accelerated the plasma lipids profiles and HOMA-IR in the Mig-6 d/d mice but had no differential effects in oral glucose tolerance test and insulin tolerance test in both genotypes. These results suggest that the activated EGFR signaling might increase the fasting plasma glucose concentration through inducing the hepatic steatosis and the improved whole-body insulin resistance in the KO mice be caused by decreased adipogenesis in fat tissues.

Discovery and optimization of adamantane carboxylic acid derivatives as potent diacylglycerol acyltransferase 1 inhibitors for the potential treatment of obesity and diabetes.

We have developed a series of adamantane carboxylic acid derivatives exhibiting potent diacylglycerol acyltransferase 1 (DGAT1) inhibitory activities. Optimization of the series led to the discovery of E-adamantane carboxylic acid compound 43c, which showed excellent in vitro activity with an IC50 value of 5 nM against human and mouse DGAT1, also good druggability as well as microsomal stability and safety profiles such as hERG, CYP and cytotoxicity. Compound 43c significantly reduced plasma triglyceride levels in vivo (in rodents and zebrafish) and also showed bodyweight gain reduction and glucose area under curve (AUC) lowering efficacy in diet-induced obesity (DIO) mice.

Amelioration of hypercholesterolemia by an EGFR tyrosine kinase inhibitor in mice with liver-specific knockout of Mig-6.

Mitogen-inducible gene 6 (Mig-6) is a negative feedback inhibitor of epidermal growth factor receptor (EGFR) signaling. We previously found that Mig-6 plays a critical role in the regulation of cholesterol homeostasis and in bile acid synthesis. In this study, we investigated the effects of EGFR inhibition to identify a potential new treatment target for hypercholesterolemia. We used a mouse model with conditional ablation of the Mig-6 gene in the liver (Albcre/+Mig-6f/f; Mig-6d/d) to effectively investigate the role of Mig-6 in the regulation of liver function. Mig-6d/d mice were treated with either the EGFR inhibitor gefitinib or statin for 6 weeks after administration of a high-fat or standard diet. We then compared lipid profiles and other parameters among each group of mice. After a high-fat diet, Mig-6d/d mice showed elevated serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides and glucose, characteristics resembling hypercholesterolemia in diabetic patients. We observed decreases in serum levels of lipids and glucose in high-fat-diet-fed Mig-6d/d mice after 6 weeks of treatment with gefitinib or statin. Furthermore gefitinib-treated mice showed significantly greater decreases in serum levels of total, HDL and LDL cholesterol compared with statin-treated mice. Taken together, these results suggest that EGFR inhibition is effective for the treatment of hypercholesterolemia in high-fat-diet-fed Mig-6d/d mice, and our findings provide new insights into the development of possible treatment targets for hypercholesterolemia via modulation of EGFR inhibition.

Synthesis and biological evaluation of thienopyrimidine derivatives as GPR119 agonists.

A series of thienopyrimidine derivatives was synthesized and evaluated for their GPR119 agonistic ability. Several thienopyrimidine derivatives containing R(1) and R(2) substituents displayed potent GPR119 agonistic activity. Among them, compound 5d, which is a prototype, showed good in vitro activity with an EC50 value of 3 nM and human and rat liver microsomal stability. Compound 5d exhibited no CYP inhibition and induction, Herg binding, or mutagenic potential. Compound 5d showed increase insulin secretion in beta TC-6 cell and lowered the glucose excursion in mice in an oral glucose-tolerance test.

A novel 11ß-HSD1 inhibitor improves diabesity and osteoblast differentiation.

Selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential as treatment for osteoporosis as well as metabolic syndrome including type 2 diabetes mellitus. Here, we investigated the anti-diabetic, anti-adipogenic, and anti-osteoporotic activity of KR-67500, as a novel selective 11ß-HSD1 inhibitor. Cellular 11ß-HSD1 activity was tested based on a homogeneous time-resolved fluorescence method. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) levels were measured in diet-induced obese (DIO)-C57BL/6 mice administered KR-67500 (50  mg/kg per day, p.o.) for 28 days and, additionally, its anti-diabetic effect was evaluated by OGTT and ITT. The in vitro anti-adipogenic effect of KR-67500 was determined by Oil Red O Staining. The in vitro anti-osteoporotic activity of KR-67500 was evaluated using bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation model systems. KR-67500 improved the in vivo glucose tolerance and insulin sensitivity in DIO-C57BL/6 mice. KR-67500 suppressed cortisone-induced differentiation of 3T3-L1 cells into adipocytes. KR-67500 enhanced BMP2-induced osteoblastogenesis in C2C12 cells and inhibited RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages. KR-67500, a new selective 11ß-HSD1 inhibitor, may provide a new therapeutic window in the prevention and/or treatment of type 2 diabetes, obesity, and/or osteoporosis.

Anti-diabetic and anti-inflammatory effect of a novel selective 11ß-HSD1 inhibitor in the diet-induced obese mice.

It has been reported that the selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential for treating type 2 diabetes mellitus, metabolic syndrome and inflammation. In the present study, we investigated the anti-diabetic and anti-inflammatory effects of N-(5-carbamoyladamantan-2-yl)-3-((2-fluorophenyl) sulfonyl)thiazolidine-2-carboxamide (KR-67105), a novel 11ß-HSD1 inhibitor, in diabetic mice model and preadipocyte model. KR-67105 concentration dependently inhibited 11ß-HSD1 activity in human and mouse 11ß-HSD1 overexpressing cells and mouse 3T3-L1 adipocytes. Furthermore, KR-67105 concentration-dependently inhibited 11ß-HSD1 activity in the ex vivo assay of C57BL/6 mice. In the study with diet-induced obese (DIO) mice, the administration of KR-67105 (100mg/kg/day, orally for 28 days) improved the glucose tolerance and insulin sensitivity as determined by the oral glucose tolerance test and the insulin tolerance test. Anti-diabetic effect by KR-67105 was associated with the suppression of diabetic related genes expression in liver and fat. Furthermore, KR-67105 suppressed 11ß-HSD1 activity in liver and fat of diabetic mice, but showed no effect on adrenal grand weight/body weight ratio and plasma corticosterone concentration in diabetic mice. In 3T3-L1 preadipocytes, cortisone induced the mRNA of inflammatory cytokines and 11ß-HSD1 and reactive oxygen species formation. This effect was abolished by co-incubation with KR-67105 in a concentration-dependent manner. Moreover, KR-67105 attenuated cortisone induced iNOS expression and phosphorylation of NF-κB p65, p38 MAPK, and ERK1/2 in preadipocytes. Taken together, it is concluded that a selective 11ß-HSD1 inhibitor, KR-67105, may provide a new therapeutic window in the prevention and treatment of type 2 diabetes with chronic inflammation without toxicity.

Synthesis and biological evaluation of aminobenzimidazole derivatives with a phenylcyclohexyl acetic acid group as anti-obesity and anti-diabetic agents.

A series of benzimidazole derivatives with a phenylcyclohexyl acetic acid group as DGAT-1 inhibitors was developed. Among the benzimidazole series, compound 5k showed submicromolar in vitro activity toward human and mouse DGAT-1, good selectivity toward DGAT-2, human liver metabolic stability, and pharmacokinetic (PK) and safety profiles such as hERG, CYP and acute toxicity. Additionally, 5k showed good in vivo efficacy in 4weeks study with DIO mouse model.

Anti-diabetic and anti-adipogenic effects of a novel selective 11ß-hydroxysteroid dehydrogenase type 1 inhibitor in the diet-induced obese mice.

Glucocorticoid excess (Cushing's syndrome) causes metabolic syndrome such as visceral obesity, insulin resistance, diabetes mellitus, dyslipidaemia and hypertension. The selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential for treating type 2 diabetes mellitus and metabolic syndrome. In the present study, we investigated the anti-diabetic and anti-adipogenic effects of 4-(2-(1,1-dioxido-6-(2,4,6-trichlorophenyl)-1,2,6-thiadiazinan-2-yl)acetamido)adamantane-1-carboxamide (KR-67183), a novel selective 11ß-HSD1 inhibitor; we also investigated the underlying molecular mechanisms in the cortisone-induced 3T3-L1 adipogenesis model system and diet-induced obese (DIO) mice. KR-67183 concentration-dependently inhibited 11ß-HSD1 activity in human and mouse 11ß-HSD1 over-expressed cells and in the ex vivo assay of C57BL/6 mice. In the study with DIO mice, the administration of KR-67183 (20 and 50mg/kg/day, orally for 28 days) improved the glucose tolerance and insulin sensitivity with suppressed 11ß-HSD1 activity in the liver and fat. However, KR-67183 showed no change in the adrenal gland weight/body weight ratio and plasma corticosterone concentration in DIO mice. Further, KR-67183 suppressed adipocyte differentiation on cortisone-induced adipogenesis in 3T3-L1 cells is associated with the suppression of the cortisone-induced mRNA levels of FABP4, PPARγ2 and GLUT4, and 11ß-HSD1 activity. Taken together, it is suggested that a selective 11ß-HSD1 inhibitor, KR-67183, may provide a new therapeutic window in the prevention and treatment without toxicity in type 2 diabetes with obesity.

Carbenoxolone prevents the development of fatty liver in C57BL/6-Lep ob/ob mice via the inhibition of sterol regulatory element binding protein-1c activity and apoptosis.

Carbenoxolone is the 3-hemisuccinate of glycyrrhetinic acid, the active principal of licorice (Glycyrrhiza glabra). It was reported that carbenoxolone improved glucose tolerance with increased insulin sensitivity in mice with high fat diet-induced obesity. In the present study, we elucidated the protective effect of carbenoxolone in fatty liver animal models of C57BL/6-Lep(ob/ob) mice through inhibition of hepatic lipogenesis and apoptosis. In addition, the potential mechanisms by which carbenoxolone could exert such protection were elucidated. Carbenoxolone was daily administrated by gavage for 28 days in C57BL/6 and C57BL/6-Lep(ob/ob) mice. Carbenoxolone prevented the plasma triglyceride and free fatty acid accumulation associated with the reduction of the expression of sterol regulatory element binding protein-1c, liver X receptor, fatty acid synthase and acethyl-CoA carboxylase in the livers of C57BL/6-Lep(ob/ob) mice. Carbenoxolone also prevented hepatic injury through anti-apoptotic action in the livers of C57BL/6-Lep(ob/ob) mice, accompanied by increased Bcl-2 expression and suppressed Bax and cytochrome c expression. As a mechanism, increased inflammatory cytokine expressions were inhibited by carbenoxolone in the fatty livers of C57BL/6-Lep(ob/ob) mice. Furthermore, carbenoxolone inhibited free fatty acid (oleate/palmitate) induced reactive oxygen species formation and reversed free fatty acid induced mitochondrial membrane depolarization in HepG2 cells. Carbenoxolone prevents the development of fatty liver by inhibiting sterol regulatory element binding protein-1c expression and activity with an anti-apoptotic mechanism via the inhibition of inflammatory cytokine and reactive oxygen species formation in the livers of C57BL/6-Lep(ob/ob) mice. It is suggested that carbenoxolone prevents the development and progression of fatty liver disease in patients with insulin resistance.

Pharmacokinetics and pharmacodynamics of KR-66223, a novel DPP-4 inhibitor.

KR-66223 is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor that is under development for the treatment of type 2 diabetes. We studied the pharmacokinetic and pharmacodynamic characteristics of KR-66223 in rats, monkeys, and dogs to predict PK/PD profiles in humans. KR-66223 exhibited a moderate volume of distribution (0.3-1.8 L/kg), moderate systemic clearance (1-1.76 L/h/kg), long half-life (>3 h), and low oral bioavailability (below 2.5% in all tested species). The EC(50)s for DPP-4 inhibition as calculated by the E(max) model was below 4.25 ng/mL across all species, confirming KR-66223 as a potent DPP-4 inhibitor. In vitro plasma protein binding suggested that it was available (69-89%), correlating with its volume of distribution in animals. Using allometric scaling and the E(max) model, human systemic clearance, volume of the central compartment, volume of the peripheral compartment, and EC50 for DPP-4 inhibition were predicted to be 0.31 L/h/kg, 0.1 L/kg, 2.4 L/kg, and 3 ng/mL, respectively. These results can serve as a valuable foundation for future clinical trials.

Synthesis and biological evaluation of cyclic sulfamide derivatives as 11ß-hydroxysteroid dehydrogenase 1 inhibitors.

A new series of cyclic sulfamide derivatives were synthesized and evaluated for their ability to inhibit 11ß-HSD1. Among this series, 18e showed good in vitro activity toward human 11ß-HSD1, selectivity against 11ß-HSD2, microsomal stability, and pharmacokinetic and safety profiles (hERG, CYP, and acute toxicity). Additionally, 18e exhibited good in vivo efficacy in rat and monkey models.

Inhibitory effect of leptin on rosiglitazone-induced differentiation of primary adipocytes prepared from TallyHO/Jng mice.

The effects of leptin on rosiglitazone-induced adipocyte differentiation were investigated in the primary adipocytes prepared from subcutaneous fat of TallyHO/Jng (TallyHO) mouse, a recently developed model animal for type 2 diabetes mellitus (T2DM). The treatment of leptin inhibited the rosiglitazone-induced adipocyte differentiation with a decreased expression of peroxisome proliferator-activated receptor γ (PPARγ) a key adipogenic transcription factor, both in mRNA and protein levels. Leptin (10 nM) was sufficient to inhibit the adipocyte differentiation, which seemed to come from increased expression of leptin receptor genes in the fat of TallyHO mice. The inhibition of adipogenesis by leptin was restored by the treatment of inhibitors for extracellular-signal-regulated kinase (ERK) (PD98059) and signal transducer and activator of transcription-1 (STAT1) (fludarabine). Furthermore, in vivo intraperitoneal administration of PD98059 and fludarabine increased the PPARγ expression in the subcutaneous fat of TallyHO mice. These data suggest that leptin could inhibit the PPARγ expression and adipocyte differentiation in its physiological concentration in TallyHO mice.

Discovery of ß-aminoacyl containing thiazolidine derivatives as potent and selective dipeptidyl peptidase IV inhibitors.

A series of ß-aminoacyl containing thiazolidine derivatives was synthesized and evaluated for their ability to inhibit DPP-IV. Several thiazolidine derivatives with an acid moiety were found to be potent DPP-IV inhibitors. Among them, compound 2da is the most active in this series with an IC(50) value of 1 nM, and it showed excellent selectivity over DPP-IV related enzymes including DPP-2, DPP-8, and DPP-9. Compound 2da is chemically and metabolically stable, and showed no CYP inhibition, hERG binding or cytotoxicity. Compound 2db, an ester prodrug of 2da, showed good in vivo DPP-IV inhibition after oral administration in rat and dog models.