GPR119 activation by DA-1241 alleviates hepatic and systemic inflammation in MASH mice through inhibition of NFκB signaling.

BACKGROUND AND PURPOSE: GPR119 activation has been suggested to improve hyperglycemia, dyslipidemia and hepatic steatosis. But its therapeutic potential for metabolic dysfunction-associated steatohepatitis (MASH) are underexplored. Here, we investigated the effects of DA-1241, a novel GPR119 agonist, on MASH and explored its underlying mechanism of anti-inflammatory effects. EXPERIMENTAL APPROACH: The in vivo anti-MASH effect was assessed by examining the preventive effect in MS-MASH and Ob-MASH mice and the therapeutic effect in MASH with severe hyperglycemia and diet-induced obese (DIO)-MASH mice. Histological and biochemical changes in liver tissue were assessed. Both plasma and hepatic biomarkers related to inflammation and fibrosis were comprehensively analyzed. To understand its mode of action, changes in NFκB signaling were determined in HepG2 and THP-1 cells. KEY RESULTS: DA-1241 attenuated MASH progression and alleviated the MASH phenotypes in MASH mouse models with different etiologies, regardless of glucose-lowering activity. In DIO-MASH mice, DA-1241 significantly reduced biochemical parameters related to steatosis, inflammation and fibrosis in the liver with reduced plasma liver enzymes. When used in combination with a dipeptidyl peptidase 4 (DPP4) inhibitor, DA-1241 further improved the MASH phenotype by increasing endogenous glucagon-like peptide-1 effect. Notably, DA-1241 alone and in combination reduced liver inflammation and restored inflammation-related hepatic gene expression, leading to remission of systemic inflammation as assessed by plasma inflammatory cytokines and chemokines. We demonstrated that DA-1241 reduces macrophage differentiation through downregulation of NFκB signaling by activating GPR119. CONCLUSION: Our data suggest the therapeutic potential of DA-1241, alone and in combination with a DPP4 inhibitor, for MASH.

A novel GPR119 agonist DA-1241 preserves pancreatic function via the suppression of ER stress and increased PDX1 expression.

DA-1241 is a novel small molecule G protein-coupled receptor 119 (GPR119) agonist in early clinical development for type 2 diabetic patients. This study aimed to elucidate the pharmacological characteristics of DA-1241 for its hypoglycemic action. DA-1241 potently and selectively activated GPR119 with enhanced maximum efficacy. DA-1241 increased intracellular cAMP in HIT-T15 insulinoma cells (EC50, 14.7 nM) and increased insulin secretion (EC50, 22.3 nM) in association with enhanced human insulin promoter activity. Accordingly, postprandial plasma insulin levels were increased in mice after single oral administration of DA-1241. Postprandial glucose excursion was significantly reduced by single oral administration of DA-1241 in wild-type mice but not in GPR119 knockout mice. GLP-1 secretion was increased by DA-1241 treatment in mice. Thus, upon combined sitagliptin and DA-1241 treatment in high-fat diet/streptozotocin (HFD/STZ)-induced diabetic mice, plasma active GLP-1 levels were synergistically increased. Accordingly, blood glucose and triglyceride levels were significantly lowered both by DA-1241 and sitagliptin alone and in combination. Immunohistochemical analysis revealed that ß-cell mass with reduced PDX1 levels in the islets from HFD/STZ diabetic mice was significantly preserved by DA-1241, whereas increased glucagon and BiP levels were significantly suppressed. In HIT-T15 insulinoma cells subjected to ER stress, decreased cell viability was significantly rescued by treatment with DA-1241. Additionally, increased apoptosis was largely attenuated by DA-1241 by inhibiting BiP and CHOP expression through suppression of p38 MAPK. In conclusion, these studies provide evidence that DA-1241 can be a promising antidiabetic drug by potentially preserving pancreatic functions through suppressing ER stress and increasing PDX1 expression.

Additive effects of evogliptin in combination with pioglitazone on fasting glucose control through direct and indirect hepatic effects in diabetic mice.

Due to very limited preclinical reports, pharmacodynamic interactions between dipeptidyl peptidase 4 (DPP4) inhibitors and peroxisome proliferator-activated receptor γ (PPARγ) agonists are not conclusive yet. This study aimed to evaluate the pharmacological responses from adding evogliptin, a DPP4 inhibitor, to pioglitazone, a PPARγ agonist, in diabetic db/db mice after a 2-week treatment. This combination led to further decrease in both fasting and fed blood glucose levels compared to evogliptin alone (P < 0.05), but combination effects were more dramatic in fasting glucose levels (P < 0.05 vs. each treatment alone). Of note, plasma glucagon and high-molecular-weight (HMW) form of adiponectin were also further altered by the combination (P < 0.05 vs. each treatment alone). In line with these results, hepatic gluconeogenic gene expression was normalized by this combination. However, although evogliptin or pioglitazone directly suppressed glucose output in HepG2 hepatocytes, their combination did not further reduce hepatic glucose output. By contrast, glucose utilization of HepG2 cells was synergistically enhanced by this combination regardless of insulin presence (P < 0.05 vs. each treatment alone). These results suggest that the combination of evogliptin and pioglitazone is more efficacious in fasting glucose control through systemic alterations such as decreasing glucagon and increasing adiponectin, and through enhancing glucose utilization. To our knowledge, this is the first report regarding the significant combination effects of DPP4 inhibitors plus PPARγ agonists on plasma HMW adiponectin and hepatic glucose utilization. Our findings provide insight that the evogliptin and pioglitazone combination therapy may be more beneficial in type 2 diabetic patients characterized by exaggerated glucagon dysregulation.

Reduced metabolic activity of gut microbiota by antibiotics can potentiate the antithrombotic effect of aspirin.

In this study, we investigated the effects of antibiotics on the pharmacological effects of aspirin. The antithrombotic activity of aspirin was evaluated after antibiotic treatment using tail bleeding assay. The pyrosequencing analysis and selective medium culture assay were performed to investigate the alterations in gut microbiota. In addition, the in vitro metabolism assay with fecal suspension and in vivo pharmacokinetic experiments with antibiotic treatment were conducted. Ampicillin treatment significantly prolonged the bleeding time in aspirin-dosed rats. Oral administration of ampicillin significantly reduced gut microbial aspirin-metabolizing activity by 67.0% in rats. Furthermore, systemic exposure to aspirin and its primary metabolite (M1) was significantly increased in ampicillin-treated rats. The results from the pyrosequencing and selective medium culture with rat fecal samples revealed that ampicillin treatment led to the changes of the amounts and composition profile of gut microbiota. These findings suggest that co-administration of antibiotics can modulate the metabolism and pharmacokinetics of aspirin via suppression of metabolic activity of gut microbiota, which could potentiate the therapeutic potency of aspirin.

Hepatic role in an early glucose-lowering effect by a novel dipeptidyl peptidase 4 inhibitor, evogliptin, in a rodent model of type 2 diabetes.

Although multiple dipeptidyl peptidase 4 (DPP4) inhibitors have shown glucose-lowering effects by preserving pancreatic cells in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, the hepatic role in regulation of glucose homeostasis by DPP4 inhibitors in HFD/STZ mice remains elusive. In herein study, parallel comparison of effects on the liver (expression of gluconeogenic genes and the linked signaling molecules) and pancreas (islet morphology and relative area of alpha or beta cells) in combination with glucose-lowering effects were made at the end of 2- and 10-week of evogliptin treatment in HFD/STZ mice. Significant control of hyperglycemia was observed from the second week and persisted during 10-week treatment of 0.3% evogliptin in HFD/STZ mice. This effect was accompanied by increased level of plasma glucagon-like peptide-1 and preserved pancreas islet structure. Furthermore, the hepatic increases in gluconeogenic gene expression in HFD/STZ mice was significantly reduced by evogliptin treatment, which was accompanied by the suppression of cAMP response element-binding protein (CREB) phosphorylation and expression of transducer of regulated CREB protein 2. This hepatic effect of evogliptin treatment was reproduced in 2-week study, however, pancreatic beta-cell area was not altered yet although the expression of pancreatic and duodenal homeobox protein 1 was increased. We conclude that the suppression of hepatic gluconeogenesis by evogliptin is followed by preservation of pancreatic islet, leading to remarkable and persistent glucose-lowering effect in HFD/STZ mice. Our findings provide further insight for the hepatic role in DPP4 inhibitor-mediated glucose control in diabetes.

Beneficial Effects of Evogliptin, a Novel Dipeptidyl Peptidase 4 Inhibitor, on Adiposity with Increased Ppargc1a in White Adipose Tissue in Obese Mice.

Although dipeptidyl peptidase 4 (DPP4) is an adipokine known to positively correlate with adiposity, the effects of pharmacological DPP4 inhibition on body composition have not been fully understood. This study was aimed to assess the effects of DPP4 inhibitors on adiposity for the first time in the established obese mice model. The weight loss effects of multiple DPP4 inhibitors were compared after a 4 week treatment in diet-induced obese mice. In addition, a 2 week study was performed to explore and compare the acute effects of evogliptin, a novel DPP4 inhibitor, and exenatide, a glucagon-like peptide-1 (GLP-1) analogue, on whole body composition, energy consumption, various plasma adipokines and gene expression in white adipose tissue (WAT). After the 4 week treatment, weight loss and blood glucose reductions were consistently observed with multiple DPP4 inhibitors. Moreover, after 2-week treatment, evogliptin dose-dependently reduced whole body fat mass while increasing the proportion of smaller adipocytes. However, insulin sensitivity or plasma lipid levels were not significantly altered. In addition to increased active GLP-1 levels by plasma DPP4 inhibition, evogliptin also enhanced basal metabolic rate without reduction in caloric intake, in contrast to exenatide; this finding suggested evogliptin's effects may be mediated by pathways other than via GLP-1. Evogliptin treatment also differentially increased Ppargc1a expression, a key metabolic regulator, in WAT, but not in skeletal muscle and brown adipose tissue. The increased expression of the downstream mitochondrial gene, Cox4i1, was also suggestive of the potential metabolic alteration in WAT by DPP4 inhibitors. We are the first to demonstrate that pharmacological DPP4 inhibition by evogliptin directly causes fat loss in established obese mice. In contradistinction to exenatide, the fat-loss effect of DPP4 inhibitor is partly attributed to enhanced energy expenditure along with metabolic changes in WAT. These results provide insight into the regulation of energy storage in WAT caused by DPP4 inhibition.

Gut microbiota-mediated drug interactions between lovastatin and antibiotics.

Orally administered drugs may be metabolized by intestinal microbial enzymes before absorption into the blood. Accordingly, coadministration of drugs affecting the metabolic activities of gut microbes (e.g., antibiotics) may lead to drug-drug interactions (DDI). In this study, gut microbiota-mediated DDI were investigated by studying the pharmacokinetics of lovastatin in antibiotic-treated rats. Incubation of lovastatin with human and rat fecalase preparations produced four metabolites, M1 (demethylbutyryl metabolite), M4 (hydroxylated metabolite), M8 (the active hydroxy acid metabolite), and M9 (hydroxylated M8), indicating involvement of the gut microbiota in lovastatin metabolism. The plasma concentration-time profiles of M8 were compared after oral administration of lovastatin to control rats or those treated with either ampicillin (100 mg/kg) or an antibiotic mixture consisting of cefadroxil (150 mg/kg), oxytetracycline (300 mg/kg), and erythromycin (300 mg/kg). Pharmacokinetic analyses indicated that systemic exposure to M8 was significantly lower in antibiotic-treated rats compared with controls. In addition, fecal M8 formation decreased by 58.3 and 59.9% in the ampicillin- and antibiotic mixture-treated rats, respectively. These results suggested that antibiotic intake may reduce the biotransformation of orally administered drugs by gut microbiota and that the subsequent impact on microbiota metabolism could result in altered systemic concentrations of either the intact drug and/or its metabolite(s).

Comparative analysis of the gut microbiota in people with different levels of ginsenoside Rb1 degradation to compound K.

Panax ginseng (family Araliaceae) which contains ginsenoside Rb1 as a main constituent is traditionally used as a remedy for cancer, inflammation, stress, and ageing. The ginsenoside Rb1 in orally administered ginseng is metabolized to bioactive compounds by gut microbiota before their absorptions to the blood. However, its metabolizing activities in individuals are significantly different as we previously demonstrated. Here, we selected 5 samples with fecal activity potently metabolizing ginsenoside Rb1 to compound K (FPG; metabolic activity, 0.058±0.029 pmol/min/mg) and 5 samples with fecal activity non-metabolizing ginsenoside Rb1 to compound K (FNG) from a pool of 100 subjects investigated in a previous study and analyzed fecal microbiota by 16S rRNA gene pyrosequencing. Taxonomy-based analysis showed that the population levels of Firmicutes and Proteobacteria in FPG were lower than in FNG, but those of Bacteroidetes and Tenericutes in FPG were higher than in FNG. At the genus level, the population levels of Clostridiales_uc_g, Oscillibacter, Ruminococcus, Holdemania, and Sutterella in FPG were significantly higher than in FNG, but that of Leuconostoc in FPG was lower than in FNG. The population levels of Bacteroides and Bifidobacterium, which potently metabolizes ginsenoside Rb1 to compound K were dramatically increased in FPG. The gut microbiota compositions of FPG and FNG were segregated on PCO2 by Principal Coordinate Analysis. Intestinal bacterial metabolism of ginseng, particularly ginsenoside Rb1, may be dependent on the composition of gut microbiota, such as Ruminococcus spp., Bacteroides spp. and Bifidobacterium spp.

Ginsenosides Rg5 and Rh3 protect scopolamine-induced memory deficits in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng (family Araliaceae) is traditionally used as a remedy for cancer, inflammation, stress and aging. AIM OF STUDY: To explore whether ginsenosides Rg5 and Rh3, the main constituents of heat-processed ginseng (the root of Panax ginseng), could protect memory deficit. MATERIALS AND METHODS: We isolated ginsenosides Rh3 and Rg5 from heated-processed ginseng treated with and without human feces, respectively. Then we investigated their protective effects on memory impairment using the passive avoidance, Y-maze and Morris water maze tasks in mice. Memory deficit was induced in mice by the intraperitoneal injection of scopolamine. RESULTS: Ginsenosides Rg5 or Rh3 increased the latency time reduced by scopolamine in passive avoidance test. Treatment with ginsenoside Rg5 or Rh3 significantly reversed the lowered spontaneous alteration induced by scopolamine in Y-maze task. Ginsenoisde Rg5 or Rh3 (10 mg/kg) significantly shortened the escape latencies prolonged by treatment with scopolamine on the last day of training trial sessions in Morris water maze task. Furthermore, ginsenosides Rg5 and Rh3 inhibited acetylcholinesterase activity in a dose-dependent manner, with IC50 values of 18.4 and 10.2 µM, respectively. The inhibitory potency of ginsenoside Rh3 is comparable with that of donepezil (IC50=9.9 µM). These ginsenosides also reversed hippocampal brain-derived neurotrophic factor (BDNF) expression and cAMP response element-binding protein (CREB) phosphorylation reduced by scopolamine. Of them, ginsenoside Rh3 more potently protected memory deficit. CONCLUSIONS: Ginsenoside Rg5 and its metabolite ginsenoside Rh3 may protect memory deficit by inhibiting AChE activity and increasing BDNF expression and CREB activation.

Lancemaside A isolated from Codonopsis lanceolata and its metabolite echinocystic acid ameliorate scopolamine-induced memory and learning deficits in mice.

The rhizome of Codonopsis lanceolata (family Campanulaceae), which contains lancemaside A as a main constituent, has been used as herbal medicine to treat inflammation, insomnia, and hypomnesia. Lancemaside A and echinocystic acid, which is its metabolite by intestinal microflora, potently inhibited acetylcholinesterase activity in a dose-dependent manner, with IC50 value 13.6 µM and 12.2 µM, respectively. Its inhibitory potency is comparable with that of donepezil (IC50=10.9 µM). Lancemaside A and echinocystic acid significantly reversed scopolamine-induced memory and learning deficits on passive avoidance task. Lancemaside A orally administered 5h before treatment with scopolamine reversed scopolamine-induced memory and learning deficits more potently than one orally administered 1h before. Echinocystic acid more potently reversed it than lancemaside A. Lancemaside A and echinocystic acid significantly reversed scopolamine-induced memory and learning deficits on the Y-maze and Morris water maze tasks. Lancemaside A and echinocystic acid also increased the expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding protein (p-CREB). Based on these findings, orally administered lancemaside A may be metabolized to echinocystic acid, which may be absorbed into the blood and ameliorate memory and learning deficits by inhibiting AChE activity and inducing BDNF and p-CREB expressions.

Antibiotics attenuate anti-scratching behavioral effect of ginsenoside Re in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Panax ginseng CA Meyer (ginseng) has been used for diabetes, cancer, stress and allergic diseases in the traditional Chinese medicine. AIM OF THE STUDY: To understand the role of intestinal microflora in the pharmacological effect of ginsenoside Re, which is a main constituent of ginseng, we investigated its anti-scratching behavioral effect in the mice treated with or without antibiotics. MATERIALS AND METHODS: Ginsenoside Re was orally administered to the mice treated with antibiotics (cefadroxil, oxytetracycline and erythromycin mixture (COE), streptomycin or/and tetracycline) and then investigated the relationship between ginsenoside Re-metabolizing ß-glucosidase and α-rhamnosidase activities of intestinal microflora and its antiscratching behavioral effect. The anti-scratching behavioral effects of ginsenosides were investigated in the increments of 1 h and 6 h after their oral administrations. The scratching behavioral frequency was measured for 1 h after treatment with histamine. RESULTS: Ginsenoside Re inhibited histamine-induced scratching behavior in mice. The anti-scratching behavioral effect of ginsenoside Re was more potent 6 h after its oral administration than 1 h after. However, its inhibitory effect was significantly attenuated in mice treated with COE, but it nearly was not affected in mice treated with streptomycin and/or tetracycline. Treatment with COE also significantly lowered fecal ginsenoside Re-metabolizing ß-glucosidase and α-rhamnosidase activities in mice, as well as fecal metabolic activity of ginsenoside Re to ginsenoside Rh1. The anti-scratching behavioral effect of ginsenoside Rh1, a metabolite of ginsenoside Re by intestinal microflora, was superior to that of ginsenoside Re. Ginsenoside Rh1 potently inhibited the expression of IL-4 and TNF-α, as well as the activation of NF-κB and c-jun activation in histamine-stimulated scratching behavioral mice. CONCLUSION: Ginsenoside Re may be metabolized to ginsenoside Rh1 by intestinal microflora, which enhances its anti-scratching behavioral effect by inhibiting NF-κB and c-jun activations.

Expression of heparinase I of Bacteroides stercoris HJ-15 and its degradation tendency toward heparin-like glycosaminoglycans.

Recombinant heparinase I was cloned from Bacteroides stercoris HJ-15 (BSrhepI), overexpressed in Escherichia coli, and intensively characterized. The complete gene of BSrhepI was identified by Southern blotting, and was overexpressed as an inclusion body. The inclusion body was solubilized with 4 M guanidine-HCl, and the denatured BSrhepI was easily purified using Ni(2+)-affinity column chromatography. The purified but denatured enzyme was then successfully refolded by dialysis against 50 mM Tris-HCl (pH 7.0) containing 1mM DTT and CaCl(2). BSrhepI was most active in 50mM Tris-HCl buffer containing 300 mM NaCl, 10 mM CaCl(2), and 1 mM DTT (pH 7.0) at 37°C. This enzyme digested not only heparin, but also heparan sulfate. Through comparative HPLC-analysis of each degraded product of heparin and heparan sulfate by digestion with BSrhepI or flavobacterial heparinase I, we verified that BSrhepI has a broader spectrum of substrate specificities than other reported heparinases.

Metabolism of ginsenoside Rb1 by human intestinal microflora and cloning of its metabolizing ß-D-glucosidase from Bifidobacterium longum H-1.

To understand the role of intestinal microflora in expressing the pharmacological effect of ginsenoside Rb1, the metabolic activity of ginsenoside Rb1 by 148 fecal specimens was measured and its metabolizing ß-glucosidase was cloned. The average activities for p-nitrophenyl-ß-D-glucopyranoside and ginsenoside Rb1 were 0.097±0.059 µmol/min/mg and 0.311±0.118 pmol/min/mg, respectively. These enzyme activities were not different between male and female, or between ages. A gene encoding ß-D-glucosidase (BglX) was cloned from Bifidobacterium longum H-1, which transformed ginsenoside Rb1 to compound K. The probe for cloning was synthesized from the genes encoding a ß-D-glucosidase of previously reported B. longum DJO10A. The sequences of the cloned gene revealed 2364 bp open reading frame (ORF) encoding a protein containing 787 amino acids (molecular weight of 95 kDa). The gene exhibited 99% homology (identities) to that of B. longum. The cloned gene was expressed under T7 promoter of the expression vector, pET-39b(+), in Escherichia coli BL21(DE3), and the expressed enzyme was purified by using HiTrap immobilized metal affinity chromatography (IMAC) HP. The enzyme potently biotransformed ginsenoside Rb1, loganin, arctiin and arbutin to ginsenoside Rd, loganetin, arctigenin and hydroquinone, respectively, but was not active in the case of hesperidin, and kakkalide. This is the first report on cloning and expression of ß-D-glucosidase from B. longum. Based on these findings, ginsenoside Rb1 may be metabolized to bioactive compound(s) by exo-ß-D-glucosidase(s) produced from the intestinal bacteria and its pharmacological effects may be dependent on intestinal bacterial exo-ß-D-glucosidase(s) activity.

Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation--the key step of inflammation.

In the preliminary study, ginsenoside Rb1, a main constituent of the root of Panax ginseng (family Araliaceae), and its metabolite compound K inhibited a key factor of inflammation, nuclear transcription factor κB (NF-κB) activation, in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. When ginsenoside Rb1 or compound K were orally administered to 2,4,6-trinitrobenzene sulfuric acid (TNBS)-induced colitic mice, these agents inhibited colon shortening, macroscopic score, and colonic thickening. Furthermore, treatment with ginsenoside Rb1 or compound K at 20mg/kg inhibited colonic myeloperoxidase activity by 84% and 88%, respectively, as compared with TNBS alone (p<0.05), and also potently inhibited the expression of tumor necrosis factor-α, interleukin (IL)-1ß and IL-6, but increased the expression of IL-10. Both ginsenoside Rb1 and compound K blocked the TNBS-induced expressions of COX-2 and iNOS and the activation of NF-κB in mice. When ginsenoside Rb1 or compound K was treated in LPS-induced murine peritoneal macrophages, these agents potently inhibited the expression of the proinflammatory cytokines. Ginsenoside Rb1 and compound K also significantly inhibited the activation of interleukin-1 receptor-associated kinase-1 (IRAK-1), IKK-ß, NF-κB, and MAP kinases (ERK, JNK, and p-38); however, interaction between LPS and Toll-like receptor-4, IRAK-4 activation and IRAK-2 activation were unaffected. Furthermore, compound K inhibited the production of proinflammatory cytokines more potently than did those of ginsenoside Rb1. On the basis of these findings, ginsenosides, particularly compounds K, could be used to treat inflammatory diseases, such as colitis, by targeting IRAK-1 activation.