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Abstract: Introduction: Measurement of intracellular triglyceride content is traditionally performed using Oil Red O stain and isopropanol extraction. However, the assay is semi-quantitative and its simplicity needs improving. Therefore, a novel assay is described to address these problems. Methods: Intracellular triglycerides (TG) were extracted directly by organic solvent Dimethyl Sulfoxide (DMSO) without stain. TG concentration dissolved in DMSO was subsequently measured with a commercial assay kit. Results: A series of experiments were conducted to verify the validity of the new protocol such as the extraction efficiency of DMSO, linear range of the kit and sensitivity of the assay. Our results suggest that this novel assay may be applied in the measurement of intracellular triglyceride accumulation induced by pre-adipocyte differentiation or compounds such as peroxisome proliferator-activated receptor γ (PPARγ) agonists. Conclusion: The simplicity and efficiency of the method were validated in this study. Our findings show that this novel assay has potential application in the discovery of anti-diabetic compounds or in the research of cell TG metabolism. [Copyright &y& Elsevier]

Abstract: Introduction: Screening compounds that stimulate liver cell glucose utilization with low cytotoxicity plays an important role in anti-diabetic drug discovery. Existing in vitro assay for the quantitative measurement of cell glucose utilization rate and cytotoxicity of compounds needs to be modified in order to improve the simplicity and reproducibility. An optimized assay was described to addresses these problems. Methods: Compounds were directly mixed with cell suspension. After 20 h incubation, glucose decrement detection in culture medium was performed, followed by the assessment of cell viability using Cell Counting Kit-8 (CCK-8). Results: A series of experiments were conducted to define optimal conditions such as cell inoculation density, incubation time and CCK-8 concentration in cell culture medium. Glucose utilization unit (GUU) was defined for the evaluation of both efficacy and cytotoxicity of a compound. It was found that Rosiglitazone significantly stimulated cell glucose utilization with negligible cytotoxicity at the concentration of 10−5 M. Conclusion: The simplicity and efficiency of the optimized method has been proven in this study. Our findings show that this assay has potential application in anti-diabetic drug discovery such as new peroxisome proliferator-activated receptor γ (PPARγ) agonists. [Copyright &y& Elsevier]

Aim: Non‐alcoholic fatty liver is the most common cause of chronic liver disease. GPR40 is a potential therapeutic target for energy metabolic disorders. GPR40 is a potential therapeutic target for energy metabolic disorders. SZZ15‐11 is a newly synthesized GPR40 agonist. In this study, we estimate the potency of SZZ15‐11 in fatty liver treatment. Methods: In vivo, diet‐induced obese (DIO) mice received SZZ15‐11 (50 mg/kg) and TAK875 (50 mg/kg) for 6 weeks. Blood glucose and lipid, hepatocyte lipid and liver morphology were analysed. In vitro, HepG2 cells and GPR40‐knockdown HepG2 cells induced with 0.3 mM oleic acid were treated with SZZ15‐11. Triglyceride and total cholesterol of cells were measured. At the same time, the AMPK pathway regulating triglycerides and cholesterol esters synthesis was investigated via western blot and quantitative polymerase chain reaction in both liver tissue and HepG2 cells. Results: SZZ15‐11 was found to not only attenuate hyperglycaemia and hyperlipidaemia but also ameliorate fatty liver disease in DIO mice. At the same time, SZZ15‐11 decreased triglyceride and total cholesterol content in HepG2 cells. Whether examined in the liver of DIO mice or in HepG2 cells, SZZ15‐11 upregulated AMPKα phosphorylation and then downregulated the expression of the cholesterogenic key enzyme 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and inhibited acetyl‐CoA carboxylase activity. Furthermore, SZZ15‐11 promotes AMPK activity via [cAMP]i accumulation. Conclusion: This study confirmed that SZZ15‐11, a novel GPR40 agonist, improves hyperlipidaemia and fatty liver, partially via Gs signalling and the AMPK pathway in hepatocytes. [ABSTRACT FROM AUTHOR]

Voglibose is an α-glycosidase inhibitor that improves postprandial hyperglycemia and increases glucagon-like peptide-1 (GLP-1) secretion in patients with type 2 diabetes. Recently, there has been increasing interest in the anti-inflammatory effects of voglibose on the intestine, but the underlying mechanism is not clear. This study evaluated the effects and mechanisms of voglibose on glycemic control and intestinal inflammation. Type 2 diabetic KKAy mice were treated with voglibose (1 mg/kg) by oral gavage once daily. After 8 weeks, glucose metabolism, levels of short-chain fatty acids (SCFAs), systematic inflammatory factors, intestinal integrity and inflammation were evaluated using hematoxylin and eosin staining, immunohistochemistry, immunofluorescence and Western blot analysis. Voglibose ameliorated glucose metabolism by enhancing basal- and glucose-dependent GLP-1 secretion. Several beneficial SCFAs, such as acetic acid and propionic acid, were increased by voglibose in the fecal sample. Additionally, voglibose notably decreased the proportion of pro-inflammatory macrophages and the expression of nuclear factor kappa B but increased the expression of tight junction proteins in the ileum, thus markedly improving intestinal inflammatory damage and reducing the systematic inflammatory factors. Ileal genomics and protein validation suggested that voglibose attenuated inositol-requiring protein 1α-X-box binding protein 1-mediated endoplasmic reticulum stress (ERS). Together, these results showed that voglibose enhanced the secretion of GLP-1, which contributed to the glycemic control in KKAy mice at least in part by regulating intestinal inflammation and the expression of ERS factors. [ABSTRACT FROM AUTHOR]

Hyperglycemia, oxidative stress, and inflammation play key roles in the onset and development of diabetic complications such as diabetic nephropathy (DN). Diphenyl diselenide (DPDS) is a stable and simple organic selenium compound with anti-hyperglycemic, anti-inflammatory, and anti-oxidative activities. Nevertheless, in vitro, the role and molecular mechanism of DPDS on DN remains unknown. Therefore, we investigated the effects of DPDS on tert-butyl hydrogen peroxide (t-BHP)-induced oxidative stress and lipopolysaccharide (LPS)-induced inflammation in rat glomerular mesangial (HBZY-1) cells and explored the underlying mechanisms. DPDS attenuated t-BHP-induced cytotoxicity, concurrent with decreased intracellular ROS and MDA contents and increased SOD activity and GSH content. Moreover, DPDS augmented the protein and mRNA expression of Nrf2, HO-1, NQO1, and GCLC in t-BHP-stimulated HBZY-1 cells. In addition, DPDS suppressed LPS-induced elevations of intracellular content and mRNA expression of interleukin (IL)-6, IL-1β and TNF-α. Furthermore, LPS-induced NFκB activation and high phosphorylation of JNK and ERK1/2 were markedly suppressed by DPDS in HBZY-1 cells. In summary, these data demonstrated that DPDS improves t-BHP-induced oxidative stress by activating the Nrf2/Keap1 pathway, and also improves LPS-induced inflammation via inhibition of the NFκB/MAPK pathways in HBZY-1 cells, suggesting that DPDS has the potential to be developed as a candidate for the prevention and treatment of DN. [ABSTRACT FROM AUTHOR]

A series of novel tetrahydroisoquinoline- C -aryl glucosides has been synthesized and evaluated for the inhibition of human SGLT2. Compared with dapagliflozin, compound 13h exhibited equivalent in vitro inhibitory activity against SGLT2, which might become a promising candidate for the treatment of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single, living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research. [Copyright &y& Elsevier]

Nephropathy is characterized by urinary micro albumin. Rats are usually used in experimental studies. But, there was no specific and simple method for detecting rat urinary albumin. A specific, easily performed, and sensitive method for quantitatively determining rat urinary albumin is needed. Using rabbit anti-rat albumin polyclonal antibody, biotinylated goat anti-rabbit IgG, avidin conjugated peroxidase, and TMB (3,3′,5,5′-tetramethylbenzidine) as substrate, a competitive ELISA for rat albumin in urine was developed. With this method, the urinary albumin in diabetic and normal rats was detected. This method was sensitive to 30 ng/mL and reproducibly quantifies rat urinary albumin levels in the range of 0.05–5 µg/mL. The rabbit anti-rat albumin polyclonal antibody showed no cross reaction with bovine and human serum albumins and rat IgG, and showed little cross-reaction with mouse albumin. The intra-assay CV was less than 10%. The urinary albumin markedly increased in diabetic rats. Since quantifying urinary albumin was very important in the experimental study of diabetic nephropathy, the results from our experiments indicated that this competitive ELISA could be used for quantification of rat urinary albumin. [ABSTRACT FROM AUTHOR]

The effects of the combination of bis (α‐furancarboxylato) oxovanadium (IV) (BFOV) and metformin (Met) on hepatic steatosis were investigated in high‐fat diet‐induced obese C57BL/6J mice (HFC57 mice) for 6 weeks. Oral glucose tolerance test was performed to evaluate glucose metabolism. Moreover, blood and hepatic biochemical and histological indices were detected. Besides, Affymetrix‐GeneChip analysis and Western blot of the liver were performed. Comparing to the monotherapy group, BFOV + Met showed more effective improvement in glucose metabolism, which decreased the fasting blood glucose, insulin levels and improved insulin sensitivity in HFC57 mice. BFOV + Met significantly decreased serum ALT and AST activities and reduced hepatic triglyceride content and iNOS activities, accompanied by ameliorating intrahepatic fat accumulation and hepatocellular vacuolation. Enhanced hepatic insulin signalling transduction and attenuated inflammation pathway were identified as the major pathways in the BFOV + Met group. BFOV + Met significantly down‐regulated the protein expression levels of MMPs, NF‐κB, iNOS and up‐regulated phosphorylation of AKT and AMPK levels. We concluded that a combination of BFOV and metformin ameliorates hepatic steatosis in HFC57 mice via alleviating hepatic inflammation and enhancing insulin signalling pathway, suggesting that the combination of BFOV and metformin is a potential treatment for hepatic steatosis. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to evaluate the efficacy and safety of mulberry twig alkaloids (Sangzhi alkaloids [SZ-A]) in the treatment of type 2 diabetes (T2D).Research Design and Methods: This was a multicenter, randomized, double-blind, double-dummy, and parallel controlled noninferiority clinical trial that was conducted for 24 weeks. A total of 600 patients were randomly allocated to the SZ-A group (n= 360) or acarbose group (n= 240). The primary efficacy end point was the change of glycosylated hemoglobin (HbA1c) compared with baseline. In addition, adverse events (AEs), severe AEs (SAEs), treatment-related AEs (TAEs), and gastrointestinal disorders (GDs) were monitored.Results: After treatment for 24 weeks, the change in HbA1c was -0.93% (95% CI -1.03 to -0.83) (-10.2 mmol/mol [-11.3 to -9.1]) and -0.87% (-0.99 to -0.76) (-9.5 mmol/mol [-10.8 to -8.3]) in the SZ-A and acarbose groups, respectively, and the least squares mean difference was -0.05% (95% CI -0.18 to 0.07) (-0.5 mmol/mol [-2.0 to 0.8]) between the two groups, with no significant difference on the basis of covariance analysis (P > 0.05). The incidence of TAEs and GDs was significantly lower in the SZ-A group than the acarbose group (P < 0.01), but no differences for AEs or SAEs between the two groups were observed (P > 0.05).Conclusions: SZ-A exhibited equivalent hypoglycemic effects to acarbose in patients with T2D. Nevertheless, the incidence of TAEs and GDs was lower following SZ-A treatment than acarbose treatment, suggesting good safety. [ABSTRACT FROM AUTHOR]

The cereal formula powder, Zhengda Jingshan (ZDJS), comprises dietary fiber, multivitamins, fine protein, and various cereal ingredients. The present study evaluated the effects of ZDJS on glucose metabolism and explored the corresponding mechanisms in terms of modulating gut microbiota and the fecal metabolome. Type 2 diabetic db/db mice were given ZDJS (1 g/kg) orally twice daily for 55 days, after which glucose metabolism, inflammation, gut microbiota, and fecal metabolomics were assayed. Repeated administration of ZDJS was associated with a trend toward decreasing fasting blood glucose and a 0.12% decrease in hemoglobin A1c (HbA1c), as well as statistically significant increases in the insulin sensitivity index and decreases in serum levels of tumor necrosis factor (TNF‐α) and ileum expression of mucin‐2. ZDJS also ameliorated the compensatory enlargement of islets and decreased the ratio of the α‐cell area to total islet area; however, this amelioration of impaired oral glucose tolerance became less pronounced as treatment continued. In addition, ZDJS remarkably decreased the abundance of phylum Proteobacteria and the phylum ratio of Firmicutes to Bacteroidetes, as well as altered the fecal metabolic profile. Taken together, our findings demonstrate that ZDJS improved glucose metabolism and reduced inflammation in type 2 diabetic db/db mice, which may be associated with a reshaping of the gut microbiome and fecal metabolome in db/db mice. Thus, our study suggests that ZDJS may represent a complementary therapy for patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Aims: To evaluate a novel tetrahydroisoquinoline derivative YR4‐42 as a selective peroxisome proliferator‐activated receptor γ (PPARγ) modulator (SPPARM) and explore its anti‐diabetic effects in vitro and in vivo. Materials and methods: Using two standard full PPARγ agonists rosiglitazone and pioglitazone as controls, the PPARγ binding affinity and transactivation action of YR4‐42 were evaluated using biochemical and cell‐based reporter gene assays. The capacity of YR4‐42 to recruit coactivators of PPARγ was also assessed. The effects of YR4‐42 on adipogenesis and glucose consumption and PPARγ Ser273 phosphorylation were investigated in 3T3‐L1 adipocytes. The effects of YR4‐42 and pioglitazone, serving as positive control, on glucose and lipids metabolism were investigated in high‐fat diet‐induced obese (DIO) C57BL/6J mice. The expression of PPARγ target genes involved in glucose and lipid metabolism was also assessed in vitro and in vivo. Results: In vitro biochemical and cell‐based functional assays showed that YR4‐42 has much weaker binding affinity, transactivation, and recruitment to PPARγ of the coactivators thyroid hormone receptor‐associated protein complex 220 kDa component (TRAP220) and PPARγ coactivator 1‐α (PGC1α) compared to full agonists. In 3 T3‐L1 adipocytes, YR4‐42 significantly improved glucose consumption without a lipogenesis effect, while blocking tumour necrosis factor α‐mediated phosphorylation of PPARγ at Ser273, thereby upregulating the expression of the PPARγ Ser273 phosphorylation‐dependent genes. Furthermore, in DIO mice, oral administration of YR4‐42 ameliorated the hyperglycaemia, with a similar insulin sensitization effect to that of pioglitazone. Importantly, YR4‐42 also improved hyperlipidaemia‐associated hepatic steatosis without weight gain, which avoids a major side effect of pioglitazone. Thus, YR4‐42 appeared to selectively modulate PPARγ responses. This finding was supported by the gene expression analysis, which showed that YR4‐42 selectively targets PPARγ‐regulated genes mapped to glucose and lipid metabolism in DIO mice. Conclusions: We conclude that YR4‐42 is a novel anti‐diabetic drug candidate with significant advantages compared to standard PPARγ agonists. YR4‐42 should be further investigated in preclinical and clinical studies. [ABSTRACT FROM AUTHOR]

A novel series of GPR40 agonists is designed by introducing nitrogen-containing heterocyclic ring at the terminal phenyl ring of TAK-875 with the aim of decreasing its lipophilicity. Three different β-substituted phenylpropionic acids were investigated as the acidic components. A total of 34 compounds have been synthesized, among which, compound 30 exhibited comparable GPR40 agonistic activity in vitro with TAK-875 and relatively lower lipophilicity through calculation ( 30 , EC 50  = 1.2 μM, cLogP = 1.3; TAK-875: EC 50  = 5.1 μM, cLogP = 3.4). Moreover, compound 30 was able to enhance the insulin secretion of primary islets isolated from normal ICR mice and showed no obvious inhibition against cytochromes P450 in vitro . In vivo , compound 30 exhibited efficacy in oral glucose tolerance test (oGTT) in normal ICR mice. [ABSTRACT FROM AUTHOR]

Abstract Recently, SIRT5 was reported to be a predominant desuccinylase and demalonylase in mitochondria. Ablation of SIRT5 enhances the systemic succinylation and malonylation of mitochondrial proteins, including various metabolic enzymes; however, its function in pancreatic β cells has not yet been clarified. In this study, we evaluated the effects of SIRT5 overexpression on glucolipotoxicity-induced apoptosis in β cell lines. Full-length SIRT5, which preferentially targeted to mitochondria and partially to the nucleus and cytoplasm, was overexpressed in NIT-1 cells. Chronic exposure to excess palmitate and glucose (High-PA-G) induced apoptosis and suppressed glucose-stimulated insulin secretion in β cells. SIRT5 overexpression significantly alleviated apoptosis under the High-PA-G condition, accompanied by suppressed Caspase 3 activity and reduced malondialdehyde levels. SIRT5 overexpression also improved β cell secretory capacity in response to glucose under the High-PA-G condition, suggesting its protective role in β cell function. Furthermore, SIRT5 overexpression reversed the decreasing trend of anti-apoptotic factors BCL-2 and BCL-XL expression under High-PA-G condition. Further regulation mechanisms between SIRT5 and these anti-apoptotic factors remains to be explored in future studies. Our data reveal that SIRT5 is a potentially protective factor for pancreatic β cells against glucolipotoxicity-induced apoptosis and cell dysfunction. Highlights • SIRT5 expression significantly diminished in the pancreas of T2DM animals. • SIRT5 reduced apoptotic cell mass and retained glucose-stimulated insulin secretion in β cell lines upon glucolipotoxicity. • SIRT5 plays an essential role in maintaining the β cell mass and function under glucolipotoxicity condition. [ABSTRACT FROM AUTHOR]

Farnesoid X receptor (FXR) is a promising target for drug discovery against nonalcoholic fatty liver disease (NAFLD). However, no FXR agonist has been approved for NAFLD so far. The R & D of FXR agonists are somewhat hindered by the lack of effective and safe chemotypes. To this end, we developed a multi-stage computational workflow to screen the Specs and ChemDiv chemical library for FXR agonists, which consisted of machine learning (ML)-based classifiers, shape-based and electrostatic-based models, a FRED-based molecular docking protocol, an ADMET prediction protocol and substructure search. As a result, we identified a novel chemotype that has never been reported before, with compound XJ02862 (ChemDiv ID: Y020-6413) as the representative. By designing an asymmetric synthesis strategy, we were able to prepare four isomers of compound XJ02862. Interestingly, one of the isomers, 2-((S)-1-((2S,4R)-2-methyl-4-(phenylamino)-3,4-dihydroquinolin-1(2H)-yl)-1-oxopropan-2-yl)hexahydro-1H-isoindole-1,3(2H)-dione (XJ02862-S2), showed potent FXR agonistic activity in HEK293T cells. The molecular docking, molecular dynamics simulations and site-directed mutagenesis suggested the hydrogen bond between compound XJ02862-S2 and HIS294 of FXR is essential for ligand binding. We further demonstrated that compound XJ02862-S2 had no agonistic effect on TGR5. Further biological experiments have shown that compound XJ02862-S2 could ameliorate hypercholesterolemia, hepatic steatosis, hyperglycemia, insulin resistance (IR) in high-fat-diet induced obese (DIO) mice. In term of molecular mechanism, compound XJ02862-S2 regulates the expression of FXR downstream genes involved in lipogenesis, cholesterol transport and bile acid biosynthesis and transport. Taken together, we have discovered a novel chemotype as potent FXR agonists for NAFLD by computational modeling, chemical synthesis and biological evaluation. [Display omitted] • Compound XJ02862 (ChemDiv ID: Y020-6413) was identified as a novel chemotype of FXR agonists by in silico screening. • The isomer XJ02862-S2 showed potent FXR agonistic activity in HEK293T cells. • Compound XJ02862-S2 had no agonistic effect on TGR5. • Compound XJ02862-S2 ameliorated hypercholesterolemia, hepatic steatosis, hyperglycemia, and insulin resistance in DIO mice. [ABSTRACT FROM AUTHOR]

Glucokinase was glucose sensor in hepatocytes and islet beta cells. It not only promoted glucose metabolism, but also advanced glucose-stimulated insulin secretion. Glucokinase activators had been reported as one kind of new potential drugs for treatment of type 2 diabetes. Compound 6-(5-(3-fluorobenzyloxy)−2-ethoxybenzamido) pyridine-3-carboxylic acid (SHP289-04) was found to increase glucokinase activity effectively by our lab. In order to demonstrate its effect of anti-diabetes, in vitro, human liver carcinoma cell line HepG2 was used to glucose consumption test, and pancreatic cell line NIT-1 was used to assess insulin secretion in response to different concentration of glucose (5 mmol/l and 20 mmol/l). Type 2 diabetes model KKA y mice were chose to evaluate the pharmacodynamics of SHP289-04 in vivo. In hepatocytes, SHP289-04 could accelerate glucose consuming. In NIT cell line, it promoted glucose-stimulated insulin secretion at the 20 mmol/l of glucose. Moreover, it normalized oral glucose tolerance test and down-regulated blood lipid level in KKA y mice. At the same time, it ameliorated function of islets and accelerated the ratio of beta cell/alpha cell mass, and also alleviated the fatty liver of KKA y mice. Therefore, SHP289-04 as a glucokinase activator had the potential effect of diabetes treatment. [ABSTRACT FROM AUTHOR]

Aims Glucagon like-peptide-1 (GLP-1)-based drugs have been proposed as mono- or combined therapy for type 2 diabetes mellitus. Thus we characterized a novel antibody fusion protein engineered by linking the human GLP-1 derivative to a humanized GLP-1 receptor (GLP-1R) antibody via a peptide linker. Materials and methods Glutazumab was characterized by receptor binding and reporter activation assays, and its specificity was investigated with the aid of the cognate receptor antagonist exendin (9-39) and antibody Ab1. Pharmacokinetics was evaluated in Sprague-Dawley (SD) rats and cynomolgus monkeys, and pharmacodynamics was assessed in normal ICR and spontaneous type 2 diabetic KKAy mice. Hypoglycemic effects were evaluated after acute administration and glucose metabolism and β-cell function were assessed with repeated administrations. Dulaglutide was a positive control in all experiments. Results Glutazumab significantly bound and activated GLP-1R, but the receptor antagonist exendin (9-39) did not inhibit the activation except when combined with Ab1. Single injection of glutazumab reduced the blood glucose in ICR mice and KKAy mice, and the half-lives in SD rats and cynomolgus monkeys were 18 h and 33.6 h. Repeated injections of glutazumab controlled glycemic fluctuations and improved β-cell function in KKAy mice. Conclusions As a novel GLP-1R agonist, glutazumab may be a potential treatment for T2DM. [ABSTRACT FROM AUTHOR]

Farnesoid X receptor (FXR), a nuclear receptor mainly distributed in liver and intestine, has been regarded as a potential target for the treatment of various metabolic diseases, cancer and infectious diseases related to liver. Starting from two previously identified chalcone-based FXR antagonists, we tried to increase the activity through the design and synthesis of a library containing chalcones, flavones and chromenes, based on substitution manipulation and conformation (ring closure) restriction strategy. Many chalcones and four chromenes were identified as microM potent FXR antagonists, among which chromene 11c significantly decreased the plasma and hepatic triglyceride level in KKay mice. [ABSTRACT FROM AUTHOR]

Farnesoid X receptor (FXR) is an attractive target for drug discovery against non-alcoholic fatty liver disease (NAFLD). We previously reported an orally active, new-chemotype FXR agonist XJ034 by ensemble learning-driven drug discovery. However, its FXR agonistic activity and the efficacy in vivo remain to be improved. In this study, we designed and synthesized 52 derivatives, and preliminarily evaluated their FXR transactivation activity in HEK293T cells at the concentration of 10 μM. 12 FXR agonists were superior or comparable to compound XJ034 , two of which showed over 9-fold activity of compound XJ034, and were as potent as OCA. The molecular docking and molecular dynamics simulations implied an additional hydrogen bond with TYR383 is involved in FXR transactivation for both compounds. According to EC 50 determined by the confirmatory transactivation assay, we selected adamantan-1-yl(4-(2-amino-5-chlorophenyl)piperazin-1-yl)methanone (10a, EC 50 : 1.05 μM) as our lead compound. Interestingly, compound 10a had no agonistic effect on TGR5 or PPAR, and no cytotoxicity to HepG2 cells. In vivo bioassays with high-fat-diet induced C57BL/6J obese (DIO) mice have shown that compound 10a (100 mg/kg) is more effective than compound XJ034 (200 mg/kg) in improving hyperlipidemia, hepatic steatosis and insulin resistance. We also observed that compound 10a down-regulated the expression of genes involved in liver inflammation in vivo , implying its potential to treat hepatic inflammation. In summary, the present data have proved that our strategy for structural optimization is effective, and compound 10a is a promising lead compound with improved efficacy for NAFLD. [Display omitted] • Compound 10a showed significantly improved activity for FXR, compared with our previously identified hit compound XJ034. • Compound 10a showed high selectivity to FXR and had no cytotoxicity to HepG2 cells. • Compound 10a (100 mg/kg) improved hyperlipidemia, hepatic steatosis and insulin resistance in DIO mice. • Compound 10a (100 mg/kg) down-regulated the expression of genes involved in liver inflammation in vivo. [ABSTRACT FROM AUTHOR]

A series of xanthine derivatives as potent dual ligands targeting DPP-IV and GPR119 was discovered through an approach of the merged pharmacophores of GPR119 agonists and DPP-IV inhibitor linagliptin. Systematic optimization of general structure 5 led to the identification of compound 20i with selective DPP-IV inhibition, good GPR119 agonism activity and favorable metabolic stability. Docking study was performed to elucidate the potent DPP-IV inhibition of 20i . Compound 20i may serve as a tool compound for further design of anti-diabetic drugs targeting both DPP-IV and GPR119. [ABSTRACT FROM AUTHOR]

[Display omitted] • Improving FXR modulating activity by rationally designed modifications on C12 position of oleanolic acid (OA). • Modulating some key FXR downstream genes with respect to glucose and lipid metabolism by these FXR-selective ligands. • Significantly improving hyperglycemia through inhibition of glucogenesis genes PEPCK and G6P in KKay mice fed with high fat diet by 12β-OA compound 7b. Farnesoid X receptor (FXR) ligands have been actively pursued to treat metabolic disorders, liver and bile diseases, among others. Starting from a widely occurring natural product, oleanolic acid (OA), we discovered potent and selective FXR modulator from the 12β-oxygenated OA alkyl esters, with the assistance of molecular modeling. The representative compound 7b modulated some FXR downstream genes involved in glucose and lipid metabolism in cells, and significantly improved hyperglycemia in KKay fat mice fed with high fat diet, through the reduction of mRNA expression of gluconeogenesis genes PEPCK and G6Pase. This study provides a new series of selective FXR modulator, as well as the in vitro and in vivo evidence for their potential to improve hyperglycemia in diabetic mice through FXR antagonism. [ABSTRACT FROM AUTHOR]

To find aldose reductase inhibitors, two previously unreported compounds, grandifolias H and I, and five known compounds, including rosmarinic acid and rosmarinic acid derivatives, were isolated from the roots of Salvia grandifolia. A series of rosmarinic acid derivatives was obtained from rosmarinic acid using simple synthetic methods. The aldose reductase inhibitory activity of the isolated and synthesized compounds was assessed. Seven of the tested compounds showed moderate aldose reductase inhibition (IC 50 = 0.06–0.30 μM). The structure-activity relationship of aldose reductase inhibitory activity of rosmarinic acid derivatives was discussed for the first time. This study provided useful information that will facilitate the development of aldose reductase inhibitors. [ABSTRACT FROM AUTHOR]

A series of novel indole derivatives was designed and synthesized as inhibitors of fructose-1,6-bisphosphatase (FBPase). The most potent compound 14c was identified with an IC 50 value of 0.10 μM by testing the inhibitory activity against recombinant human FBPase. The structure-activity relationships were investigated on the substitution at 4- and 5-position of the indole scaffold. The binding interactions of the title compounds at AMP binding site of FBPase were predicted using CDOCKER algorithm. [ABSTRACT FROM AUTHOR]

Abstract: Motivated by the discovery of a potential ligand that activates both glucokinase (GK) and perioxisome proliferator-activated receptor-γ (PPARγ), this work presents the rational design and synthesis of a series of novel urea derivatives as potent dual-target ligands of GK and PPARγ. The derivatives obtained, particularly compounds 14j, 14m, 15g, 15j, and 15s, showed relatively high enzyme activity and moderate blood glucose-lowering efficacy in normal ICR mice (GK activation fold >1.7, PPARγ activation percentage >38.8%, relative to rosiglitazone). The discovery of a dual-acting agent may provide an effective approach for treating type 2 diabetes mellitus. [Copyright &y& Elsevier]

Abstract: A series of novel indole derivatives was synthesized as inhibitors of fructose-1,6-bisphosphatase (FBPase). Extensive structure–activity relationships were conducted and led to a potent FBPase inhibitor 3.9 with an IC50 of 0.99μM. The binding mode of this series of indoles was predicted using CDOCKER algorithm. The results of this research will shed light on the further design and optimization of novel small molecules as FBPase inhibitors. [Copyright &y& Elsevier]

Morus alba L. (Sangzhi) alkaloids (SZ-A) tablets have been approved by the China National Medical Products Administration for T2DM treatment. Our previous study (Liu et al., 2021) revealed that SZ-A protected against diabetes and inflammation in KKAy mice. However, the mechanism and components in SZ-A exerting anti-inflammatory effects are unclear. Investigate the effects and molecular mechanisms of SZ-A on inflammation, and identify anti-inflammatory active components in SZ-A. The major ingredients in SZ-A were analyzed by HPLC and sulfuric acid - anthrone spectrophotometry. The inhibitory activities of SZ-A on lipopolysaccharide (LPS)-stimulated inflammation were determined in bone marrow-derived macrophage (BMDM) and RAW264.7 cells. The cytokine levels of IL-6 and TNF-α in cell culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression levels of IL-6 and TNF-α were detected by qRT-PCR. The levels of protein phosphorylation of p38 MAPK, ERK, and JNK were analyzed by Western blot. The main components in SZ-A were found to be 1-deoxynojirimycin (DNJ), 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), fagomine (FAG), polysaccharide (APS), and arginine (ARG). SZ-A reduced the levels of IL-6 and TNF-α secreted by LPS-induced RAW264.7 and BMDM cells. Simultaneously, the mRNA expression levels of IL-6 and TNF-α were all significantly suppressed by SZ-A in a concentration-dependent manner. Furthermore, SZ-A inhibited the phosphorylation of p38 MAPK, ERK, and JNK in BMDM and the activation of ERK and JNK signaling in RAW264.7 cells. We also observed that DNJ, DAB, FAG, and ARG markedly downregulated IL-6 and TNF-α cytokine levels, while APS did not have an obvious effect. SZ-A attenuates inflammation at least partly by blocking the activation of p38 MAPK, ERK, and JNK signaling pathways. DNJ, FAG, DAB, and ARG are the main constituents in SZ-A that exert anti-inflammatory effects. [Display omitted] [ABSTRACT FROM AUTHOR]

Berberine is effective for type 2 diabetes mellitus (T2DM), but has limited use in clinic. This study aims to evaluate the effect of berberine combined with stachyose on glycolipid metabolism and gut microbiota and to explore the underlying mechanisms in diabetic rats. Zucker diabetic fatty (ZDF) rats were orally administered berberine, stachyose and berberine combined with stachyose once daily for 69 days. The oral glucose tolerance and levels of blood glucose, insulin, triglyceride and total cholesterol were determined. The gut microbial profile, colonic miRNA and gene expression were assayed using Illumina sequencing. The quantitative polymerase chain reaction was used to verify the expression of differentially expressed miRNAs and genes. Repeated treatments with berberine alone and combined with stachyose significantly reduced the blood glucose, improved the impaired glucose tolerance, and increased the abundance of beneficial Akkermansiaceae , decreased that of pathogenic Enterobacteriaceae in ZDF rats. Furthermore, combined treatment remarkably decreased the abundances of Desulfovibrionaceae and Proteobacteria in comparison to berberine. Combined treatment evidently decreased the expression of intestinal early growth response protein 1 (Egr1) and heparin-binding EGF-like growth factor (Hbegf), and significantly increased the expression of miR-10a-5p, but berberine alone not. Berberine combined with stachyose significantly improved glucose metabolism and reshaped gut microbiota in ZDF rats, especially decreased the abundance of pathogenic Desulfovibrionaceae and Proteobacteria compared to berberine alone, providing a novel strategy for treating T2DM. The underlying mechanisms may be associated with regulating the expression of intestinal Egr1 , Hbegf and miR-10a-5p, but remains further elucidation. Repeated treatments with berberine in combination with stachyose significantly improves glycometabolism and reshaped gut microbiota profile in diabetic ZDF rats, and this is partly mediated by regulating colonic miR-10a-5p, Erg1 and Hbegf expression. [Display omitted] • Berberine plus stachyose improves glycometabolism, shapes microbiota in diabetic rat. • Berberine plus stachyose increases colonic miR-10a-5p expression in diabetic rat. • Berberine plus stachyose decreases colonic Egr1 and Hbegf expression in diabetic rat. • Changes of miR-10a-5p, Egr1 and Hbegf explain effects of berberine plus stachyose. [ABSTRACT FROM AUTHOR]

Abstract: Glucagon-like peptide-1 (GLP-1) is an incretin hormone that decreases postprandial glycemic excursions by enhancing insulin secretion but with short half-life due to rapid inactivation by enzymatic N-terminal truncation. Therefore, efforts are being made to improve the stability of GLP-1 via modifying its structure or inhibiting dipeptidyl-peptidase IV (DPP IV), which is responsible for its degradation. Here we report a novel GLP-1 analog BPI3006 with –NHCO– of Ala8 replaced by –CH(CF3)NH– and features of its metabolic stability, GLP-1 receptor trans-activation and in vivo biological activity. BPI3006 is highly resistant to DPP IV-mediated degradation with 91.1% of parental peptide left after 24h exposure to the enzyme. BPI3006 also effectively activates its target gene promoter through GLP-1 receptor activation by measuring the transiently transfected reporter gene green fluorescence protein (GFP) expression in NIT-1 cells. Furthermore, BPI3006 could well restrain the glycemia variation in fasted normal ICR mice after a single administration followed by an oral glucose loading. In spontaneous type 2 diabetic KKAy mice, BPI3006 injected twice daily could significantly improve the oral glucose tolerance and hyperinsulinemia, as well as ameliorate the food and water consumption. In conclusion, BPI3006 has enhanced resistance to DPP IV leading to improved stability, and shows excellent in vivo biological activity. Thus it may be a new candidate for T2DM treatment and its novel modification may provide valuable guidance for the future development of long-acting GLP-1 analogs. [ABSTRACT FROM AUTHOR]

Abstract: A series of azaindole-α-alkyloxyphenylpropionic acid analogues was synthesized and evaluated for PPAR agonist activities. Structure–activity relationship was developed for PPARα/γ dual agonism. One of the synthesized compound 7a was identified as a potent, selective PPARα/γ dual agonist. [Copyright &y& Elsevier]

For measuring glutamine:fructose-6-phosphate amidotransferase (GFAT) activity in cultured cells, an enzyme method—GDH method—was set up with high-efficiency, high-sensitivity and simple operation by determining the formed glutamate. During the process of making samples, reduced glutathione (GSH, 5 mM) and glucose-6-phophate Na2 (5 mM) were added to the buffer for scraping the cells. The range of protein content in the samples was 80–150 μg. In the GFAT activity assay, the end product reduced acetylpyridine adenine dinucleotide (APADH) was determined at 370 nm directly. The suitable concentrations of the reactants fructose-6-phosphate (F-6-P), glutamine, acetylpyridine adenine dinucleotide (APAD) and glutamate dehydrogenase (GDH) were 0.8, 6 and 0.3 mM and 6 U, respectively. However, the excess of APAD may interfere with the APADH measurement. The reaction time course was 90 min. The GFAT activity in 3T3-L1, L6, HepG2 and HIRc cells were 1.84–8.51 nmol glutamate/mg protein·min. [Copyright &y& Elsevier]

Oxidative stress and inflammation are implicated in the occurrence and progression of diabetic nephropathy (DN). Diphenyl diselenide (DPDS) is a stable and simple diaryl diselenide with anti-hyperglycemic, anti-inflammatory, and antioxidant activities. However, the effects of DPDS on DN are still unclear to date. Herein, we aimed to explore whether DPDS could improve renal dysfunction in streptozotocin (STZ)-induced diabetic rats and its underlying mechanisms. STZ-induced DN rats were administered with DPDS (5 or 15 mg/kg) or metformin (200 mg/kg) once daily by intragastric gavage for 12 weeks. DPDS supplementation significantly improved hyperglycemia, glucose intolerance, dyslipidemia, and the renal pathological abnormalities, concurrent with significantly reduced serum levels of creatinine, urea nitrogen, urine volume, and urinary levels of micro-albumin, β2-microglobulin and N-acetyl-glucosaminidase activities. Moreover, DPDS effectively promoted the activities of antioxidant enzymes, and reduced the levels of MDA and pro-inflammatory factors in serum and the kidney. Furthermore, DPDS supplementation activated the renal Nrf2/Keap1 signaling pathway, but attenuated the high phosphorylation levels of NFκB, JNK, p38 and ERK1/2. Altogether, the current study indicated for the first time that DPDS ameliorated STZ-induced renal dysfunction in rats, and its mechanism of action may be attributable to suppressing oxidative stress via activating the renal Nrf2/Keap1 signaling pathway and mitigating inflammation by suppressing the renal NFκB/MAPK signaling pathways, suggesting a potential therapeutic approach for DN. [Display omitted] • Diphenyl diselenide (DPDS) ameliorated STZ-induced diabetic nephropathy in rats. • DPDS ameliorated systemic and renal oxidative stress in diabetic rats. • DPDS ameliorated systemic and renal inflammation in diabetic rats. • DPDS activated the renal Nrf2/Keap1 signaling pathway in diabetic rats. • DPDS suppressed the renal NFκB and MAPK signaling pathways in diabetic rats. [ABSTRACT FROM AUTHOR]

Diabetic peripheral neuropathy (DPN) is one of the most common microvascular complications occurring in both type 1 and type 2 diabetes mellitus patients. Oxidative stress (OS) plays a key role in the pathogenesis of DPN; thus, antioxidant therapy is considered a promising strategy for treating DPN. Diphenyl diselenide (DPDs) is an organic selenium compound with antioxidant pharmacological activities. This study aimed to evaluate its preventive and therapeutic effects on DPN in rats with streptozotocin (STZ)-induced diabetes and explore the underlying mechanisms. In vitro, RSC96 cells were exposed to high glucose (100 mM) and then treated with different concentrations of DPDs (1, 10, 25 and 50 μM). Notably, DPDs markedly suppressed high glucose-induced cytotoxicity and oxidative stress in Schwann cells by decreasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Furthermore, the DPDs treatment effectively activated Nrf2 signaling and inhibited Keap1 expression. An in vivo DPN model was established in Sprague-Dawley (SD) rats injected with STZ (60 mg·kg−1, ip) and orally administered either different doses of DPDs (5 and 15 mg· kg−1· d−1) for 12 weeks or alpha lipoic acid (ALA, 100 mg kg−1·d−1) as a positive control. The administration of DPDs significantly increased the motor nerve conduction velocity (MNCV), improved thermal and mechanical hyperalgesia and the sciatic nerve morphology, and ameliorated oxidative stress in the serum and the sciatic nerve of rats with DPN. Mechanistically, DPDs reduced the level of Keap1 and stimulated Nrf2 signaling in the sciatic nerve. Taken together, the results of this study indicate that DPDs ameliorates experimental DPN as an antioxidant by activating the Nrf2/Keap1 signaling pathway. DPDs may represent a new alternative treatment for DPN. [ABSTRACT FROM AUTHOR]

The potential therapeutic effect of histone deacetylase 3 (HDAC3) pharmacologic inhibition on diabetes has been focused recently. RGFP966, as a highly-selective HDAC3 inhibitor, its possible roles and underlying mechanism in the treatment of diabetes needs to be clarified. In this study, low-dose streptozotocin (STZ)-induced pre-diabetic mice were used to test the regulatory ability of RGFP966 in blood glucose and insulin. We isolated the islets both from normal C57BL/6 J mice and KKAy mice with spontaneous type 2 diabetes to determine the potency of RGFP966 on glucose-stimulated insulin secretion. NIT-1 pancreatic β-cells induced by sodium palmitate (PA) were applied to identify the protective effects of RGFP966 against β-cell apoptosis. The results showed that administration of RGFP966 in the pre-diabetic mice not only significantly reduced hyperglycemia, promoted phase I insulin secretion, improved morphology of islets, but also increased glucose infusion rate (GIR) during hyperglycemic clamp test. When treated in vitro , RGFP966 enhanced insulin secretion and synthesis in islets of normal C57BL/6J mice and diabetic KKAy mice. In addition, it partially attenuated PA-induced apoptosis in NIT-1 cells. Therefore, our research suggests that RGFP966, probably through selective inhibition of HDAC3, might serve as a novel potential preventive and therapeutic candidate for diabetes. • RGFP966 ameliorated blood glucose level in low-dose of STZ-induced pre-diabetic mice. • RGFP966 improved β cell function in low-dose of STZ-induced pre-diabetic mice. • RGFP966 promoted insulin secretion in vivo and in vitro. • RGFP966 partially attenuated apoptosis induced by PA via HDAC3/Erk1/2 /caspase 3 in vitro. [ABSTRACT FROM AUTHOR]

A series of xanthine compounds derived from the previous hit 20i with modification on the terminal side chain was discovered through ring formation strategy. Systematic optimization of the compounds with rigid heterocycles in the hydrophobic side chain led to the new lead compound HBK001 (21h) with the improved DPP-IV inhibition and moderate GPR119 agonism activity in vitro. As a continuing work to further study the PK and PD profiles, 21h and its hydrochloride (22) were synthesized on grams scale and evaluated on the ADME/T and oral glucose tolerance test (OGTT) in ICR mice. Compound 22 showed the improved bioavailability and blood glucose-lowering effect in vivo compared to its free base 21h probably attributed to its improved solubility and permeability. The preliminary toxicity studies on compound 22 exhibited that the result of mini-Ames was negative and the preliminary acute toxicity LD 50 in mice was above 1.5 g/kg, while it showed moderate inhibition on hERG channel with IC 50 4.9 μM maybe due to its high lipophilicity. These findings will be useful for the future drug design for more potent and safer dual ligand targeting DPP-IV and GPR119 for the treatment of diabetes. Image 1 • Novel xanthine derivatives were designed through ring formation strategy. • Systematic optimization from previous hit 20i led to the new lead compound HBK001. • HBK001 had potent DPP-IV inhibition and moderate GPR119 agonism activity. • HBK001 hydrochloride with superior PK showed potent in vivo glucose-lowering effect. [ABSTRACT FROM AUTHOR]

α-glucosidase inhibitors (AGIs) have been an important category of oral antidiabetic drugs being widely exploited for the effective management of type 2 diabetes mellitus. However, the marketed AGIs not only inhibited the disaccharidases, but also exhibited an excessive inhibitory effect on α-amylase, resulting in undesirable gastrointestinal side effects. Compared to these agents, Ramulus Mori alkaloids (SZ-A), was a group of effective alkaloids from natural Morus alba L., and showed excellent hypoglycemic effect and fewer side effects in the Phase II/III clinical trials. Thus, this paper aims to investigate the selective inhibitory effect and mechanism of SZ-A and its major active ingredients (1-DNJ, FA and DAB) on different α-glucosidases (α-amylase and disaccharidases) by using a combination of kinetic analysis and molecular docking approaches. From the results, SZ-A displayed a strong inhibitory effect on maltase and sucrase with an IC50 of 0.06 μg/mL and 0.03 μg/mL, respectively, which was similar to the positive control of acarbose with an IC50 of 0.07 μg/mL and 0.68 μg/mL. With regard to α-amylase, SZ-A exhibited no inhibitory activity at 100 μg/mL, while acarbose showed an obvious inhibitory effect with an IC50 of 1.74 μg/mL. The above analysis demonstrated that SZ-A could selectively inhibit disaccharidase to reduce hyperglycemia with a reversible competitive inhibition, which was primarily attributed to the three major active ingredients of SZ-A, especially 1-DNJ molecule. In the light of these findings, molecular docking study was utilized to analyze their inhibition mechanisms at molecular level. It pointed out that acarbose with a four-ring structure could perform desirable interactions with various α-glucosidases, while the three active ingredients of SZ-A, belonging to monocyclic compounds, had a high affinity to the active site of disaccharidases through forming a wide range of hydrogen bonds, whose affinity and consensus score with α-amylase was significantly lower than that of acarbose. Our study illustrates the selective inhibition mechanism of SZ-A on α-glucosidase for the first time, which is of great importance for the treatment of type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Summary In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. In vitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin-stimulated glucose uptake in myocytes and 3T3-L1 adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocyte, adipocyte, and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach to treat insulin resistance. [ABSTRACT FROM AUTHOR]