RESUMO
Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 108 s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 108, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Ilhotas Pancreáticas , Humanos , Docetaxel , Diferenciação Celular , Implantação do EmbriãoRESUMO
Neuropeptide Y2 receptor (Y2R) agonism is an important anorectic signal and a target of antiobesity drug discovery. Recently, we synthesized a short-length Y2R agonist, PYY-1119 (4-imidazolecarbonyl-[d-Hyp24,Iva25,Pya(4)26,Cha27,36,γMeLeu28,Lys30,Aib31]PYY(23-36), 1) as an antiobesity drug candidate. Compound 1 induced marked body weight loss in diet-induced obese (DIO) mice; however, 1 also induced severe vomiting in dogs at a lower dose than the minimum effective dose administered to DIO mice. The rapid absorption of 1 after subcutaneous administration caused the severe vomiting. Polyethylene glycol (PEG)- and alkyl-modified derivatives of 1 were synthesized to develop Y2R agonists with improved pharmacokinetic profiles, i.e., lower maximum plasma concentration (Cmax) and longer time at maximum concentration (Tmax). Compounds 5 and 10, modified with 20â¯kDa PEG at the N-terminus and eicosanedioic acid at the Lys30 side chain of 1, respectively, showed high Y2R binding affinity and induced significant body weight reduction upon once-daily administration to DIO mice. Compounds 5 and 10, with their relatively low Cmax and long Tmax, partially attenuated emesis in dogs compared with 1. These results indicate that optimization of pharmacokinetic properties of Y2R agonists is an effective strategy to alleviate emesis induced by Y2R agonism.
Assuntos
Fármacos Antiobesidade/química , Obesidade/tratamento farmacológico , Peptídeo YY/química , Polietilenoglicóis/química , Alquilação , Sequência de Aminoácidos , Animais , Fármacos Antiobesidade/farmacocinética , Fármacos Antiobesidade/uso terapêutico , Cães , Eméticos/química , Eméticos/uso terapêutico , Eméticos/toxicidade , Meia-Vida , Infusões Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/patologia , Peptídeo YY/farmacocinética , Peptídeo YY/uso terapêutico , Receptores de Neuropeptídeo Y/agonistas , Receptores de Neuropeptídeo Y/metabolismo , Vômito/etiologiaRESUMO
The differentiation of pancreatic endocrine cells from human pluripotent stem cells has been thoroughly investigated for their application in cell therapy against diabetes. Although non-endocrine cells are inevitable contaminating by-products of the differentiation process, a comprehensive profile of such cells is lacking. Therefore, we characterized non-endocrine cells in iPSC-derived pancreatic islet cells (iPIC) using single-cell transcriptomic analysis. We found that non-endocrine cells consist of (1) heterogeneous proliferating cells, and (2) cells with not only pancreatic traits but also liver or intestinal traits marked by FGB or AGR2. Non-endocrine cells specifically expressed FGFR2, PLK1, and LDHB. We demonstrated that inhibition of pathways involving these genes selectively reduced the number of non-endocrine cells in the differentiation process. These findings provide useful insights into cell purification approaches and contribute to the improvement of the mass production of endocrine cells for stem cell-derived cell therapy for diabetes.
Assuntos
Células Endócrinas , Células-Tronco Pluripotentes Induzidas , Ilhotas Pancreáticas , Células-Tronco Pluripotentes , Diferenciação Celular , Humanos , Ilhotas Pancreáticas/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismoRESUMO
Monoacylglycerol O-acyltransferase 2 (MGAT2) is one of the key enzymes responsible for triglyceride (TG) re-synthesis in the small intestine. We have previously demonstrated that pharmacological inhibition of MGAT2 has beneficial effects on obesity and metabolic disorders in mice. Here, we further investigate the effects of MGAT2 inhibition on (a) fat-induced gut peptide release and fat intake in normal mice and (b) metabolic disorders in high-fat diet (HFD)-fed ob/ob mice, a model of severe obesity and type 2 diabetes mellitus, using an orally bioavailable MGAT2 inhibitor Compound B (CpdB). CpdB inhibited elevation of plasma TG in mice challenged with an oil-supplemented liquid meal. Oil challenge stimulated the secretion of two gut anorectic hormones (peptide tyrosine-tyrosine and glucagon-like peptide-1) into the bloodstream, and these responses were augmented in mice pretreated with CpdB. In a two-choice test using an HFD and a low-fat diet, CpdB selectively inhibited intake of the HFD in normal mice. Administration of CpdB to HFD-fed ob/ob mice for 5 weeks suppressed food intake and body weight gain and inhibited elevation of glycated hemoglobin. These results indicate that pharmacological MGAT2 inhibition modulates fat-induced gut peptide release and fat intake in normal mice and improves obesity and diabetes in HFD-fed ob/ob mice and thus may have potential for development into a treatment of obesity and its related metabolic diseases.
Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Obesidade/metabolismo , Aciltransferases/antagonistas & inibidores , Animais , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/metabolismo , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Metabolismo Energético , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/genética , Obesidade/fisiopatologia , Triglicerídeos/metabolismo , Aumento de PesoRESUMO
The host environment is a crucial factor for considering the transplant of stem cell-derived immature pancreatic cells in patients with type 1 diabetes. Here, we investigated the effect of insulin (INS)-deficient diabetes on the fate of immature pancreatic endocrine cell grafts and the underlying mechanisms. Human induced pluripotent stem cell-derived pancreatic endocrine progenitor cells (EPCs), which contained a high proportion of chromogranin A+ NK6 homeobox 1+ cells and very few INS+ cells, were used. When the EPCs were implanted under the kidney capsule in immunodeficient mice, INS-deficient diabetes accelerated increase in plasma human C-peptide, a marker of graft-derived INS secretion. The acceleration was suppressed by INS infusion but not affected by partial attenuation of hyperglycemia by dapagliflozin, an INS-independent glucose-lowering agent. Immunohistochemical analyses indicated that the grafts from diabetic mice contained more endocrine cells including proliferative INS-producing cells compared with that from nondiabetic mice, despite no difference in whole graft mass between the two groups. These data suggest that INS-deficient diabetes upregulates the INS-secreting capacity of EPC grafts by increasing the number of endocrine cells including INS-producing cells without changing the graft mass. These findings provide useful insights into postoperative diabetic care for cell therapy using stem cell-derived pancreatic cells.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Animais , Imuno-Histoquímica , CamundongosRESUMO
Glucagon-like peptide-1 (GLP-1) is released from enteroendocrine cells (L cells) in response to food ingestion. The mechanism by which dietary peptides stimulate GLP-1 secretion in the gut is unknown. In the present study, we found that a hydrolysate prepared from zein, a major corn protein [zein hydrolysate (ZeinH)], strongly stimulates GLP-1 secretion in enteroendocrine GLUTag cells. Stimulatory mechanisms of GLP-1 secretion induced by ZeinH were investigated in the rat small intestine under anesthesia. Blood was collected through a portal catheter before and after ZeinH administration into different sites of the small intestine. The duodenal, jejunal, and ileal administration of ZeinH induced dose-dependent increases in portal GLP-1 concentration. GLP-1 secretion in response to the ileal administration of ZeinH was higher than that in the duodenal or jejunal administration. Capsaicin treatment on esophageal vagal trunks abolished the GLP-1 secretion induced by duodenal ZeinH but did not affect the secretion induced by jejunal or ileal ZeinH. These results suggest that ZeinH in the jejunum or ileum directly stimulates GLP-1 secretion but duodenal ZeinH indirectly stimulates GLP-1 secretion via the vagal afferent nerve. A direct blood sampling method from the duodenal vein and ileal mesenteric vein revealed that ZeinH administered into the ligated duodenal loop enhanced GLP-1 concentration in the ileal mesenteric vein but not in the duodenal vein. This confirmed that ZeinH in the duodenum induces GLP-1 secretion from L cells located in the ileum by an indirect mechanism. These results indicate that a potent GLP-1-releasing peptide, ZeinH, induces GLP-1 secretion by direct and indirect mechanisms in the rat intestine.
Assuntos
Duodeno/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Íleo/efeitos dos fármacos , Zeína/farmacologia , Animais , Capsaicina/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Duodeno/inervação , Duodeno/metabolismo , Células Enteroendócrinas/efeitos dos fármacos , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/sangue , Íleo/inervação , Íleo/metabolismo , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Hidrolisados de Proteína , Ratos , Ratos Sprague-Dawley , Fármacos do Sistema Sensorial/farmacologia , Fatores de Tempo , Vagotomia , Nervo Vago/fisiologiaRESUMO
Neuromedin U (NMU) is a neuropeptide that mediates a variety of physiological functions via its receptors, NMUR1 and NMUR2. Recently, there has been an increased focus on NMU as a promising treatment option for diabetes and obesity. A short form of NMU (NMU-8) has potent agonist activity for both receptors but is metabolically unstable. Therefore, we designed and synthesized NMU-8 analogues modified by polyethylene glycol (PEG; molecular weight, 20 kDa; PEG20k) via a linker. 3-(2-Naphthyl)alanine substitution at position 19 increased NMUR2 selectivity of NMU-8 analogues with retention of high agonist activity. Compound 37, an NMUR2-selective PEG20k analogue containing piperazin-1-ylacetyl linker, exhibited a potent body weight-lowering effect with concomitant inhibition of food intake in a dose-dependent manner (body weight loss of 12.4% at 30 nmol/kg) by once-daily repeated dosing for 2 weeks in mice with diet-induced obesity.
Assuntos
Fármacos Antiobesidade/síntese química , Neuropeptídeos/química , Obesidade/tratamento farmacológico , Fragmentos de Peptídeos/síntese química , Polietilenoglicóis/química , Receptores de Neurotransmissores/agonistas , Animais , Fármacos Antiobesidade/farmacocinética , Fármacos Antiobesidade/farmacologia , Peso Corporal/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Naftalenos/síntese química , Naftalenos/farmacocinética , Naftalenos/farmacologia , Obesidade/fisiopatologia , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/farmacologia , Piperazinas/síntese química , Piperazinas/farmacocinética , Piperazinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.
Assuntos
Fármacos Antiobesidade/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Hiperlipidemias/tratamento farmacológico , Hipoglicemiantes/farmacologia , Indóis/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Obesidade/tratamento farmacológico , Sulfonamidas/farmacologia , Animais , Fármacos Antiobesidade/síntese química , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Gorduras na Dieta/metabolismo , Diglicerídeos/antagonistas & inibidores , Diglicerídeos/biossíntese , Inibidores Enzimáticos/síntese química , Jejum , Expressão Gênica , Ensaios de Triagem em Larga Escala , Hiperlipidemias/enzimologia , Hiperlipidemias/patologia , Hipoglicemiantes/síntese química , Indóis/síntese química , Resistência à Insulina , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Obesidade/enzimologia , Obesidade/patologia , Estreptozocina , Sulfonamidas/síntese química , Triglicerídeos/antagonistas & inibidores , Triglicerídeos/biossíntese , Aumento de Peso/efeitos dos fármacosRESUMO
Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has attracted interest as a novel target for the treatment of obesity and metabolic diseases. Starting from N-phenylbenzenesulfonamide derivative 1 with moderate potency for MGAT2 inhibition, we explored an effective location of the hydrophobic group at the 1-position to enhance MGAT2 inhibitory activity. Shifting the hydrophobic group to the adjacent position followed by introduction of a bicyclic central core to restrict the substituent orientation produced N-phenylindoline-5-sulfonamide derivative 10b, which displayed much improved potency, with an IC50 value of 1.0 nM. This compound also exhibited excellent selectivity (greater than 30,000-fold) against related acyltransferases (MGAT3, DGAT1, DGAT2, and ACAT1). Subsequent optimization efforts were directed toward improving pharmacokinetic profiles, which resulted in the identification of 5-[(2,4-difluorophenyl)sulfamoyl]-7-(2-oxopyrrolidin-1-yl)-N-[4-(trifluoromethyl)phenyl]-2,3-dihydro-1H-indole-1-carboxamide (24d) endowed with potent MGAT2 inhibitory activity (IC50 = 3.4 nM) and high oral bioavailability (F = 52%, mouse). In a mouse oral fat tolerance test, oral administration of this compound effectively suppressed the elevation of plasma triacylglycerol levels.
Assuntos
Aciltransferases/antagonistas & inibidores , Indóis/química , Sulfonamidas/química , Administração Oral , Animais , Benzimidazóis/química , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Benzoxazóis/química , Benzoxazóis/farmacocinética , Benzoxazóis/farmacologia , Disponibilidade Biológica , Linhagem Celular , Humanos , Indazóis/química , Indazóis/farmacocinética , Indazóis/farmacologia , Indóis/farmacocinética , Indóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Triglicerídeos/sangueRESUMO
We previously showed that a hydrolysate prepared from corn zein [zein hydrolysate (ZeinH)] strongly stimulates glucagons-like peptide-1 (GLP-1) secretion from the murine GLP-1-producing enteroendocrine cell line and in the rat small intestine, especially in the ileum. Here, we investigated whether ZeinH administered into the ileum affects glucose tolerance via stimulating GLP-1 secretion. To observe the effect of luminal ZeinH itself on GLP-1 secretion and glycemia, ip glucose tolerance tests were performed in conscious rats with ileal and jugular catheters, and plasma glucose, insulin, and GLP-1 (total and active) were measured. In addition, plasma dipeptidyl peptidase-IV activities in the ileal vein were measured after the administration of ZeinH into the ileal-ligated loop in anesthetized rats. The ileal administration of ZeinH attenuated the glucose-induced hyperglycemia accompanied by the enhancement of insulin secretion, whereas meat hydrolysate (MHY) neither induced insulin secretion nor attenuated hyperglycemia. The antihyperglycemic effect was also demonstrated by the oral administration of ZeinH. From these results, it was predicted that the GLP-1-releasing potency of ZeinH was higher than that of MHY. However, both peptides induced a similar increase in total GLP-1 concentration after the ileal administration. In contrast, active GLP-1 concentration was increased only in ZeinH-treated rats. In anesthetized rats, ileal administration of ZeinH, but not MHY, decreased plasma dipeptidyl peptidase-IV activity in the ileal vein. These results indicate that the ileal administration of a dietary peptide, ZeinH, has the dual functions of inducing GLP-1 secretion and inhibiting GLP-1 degradation, resulting in the enhancement of insulin secretion and the prevention of hyperglycemia in rats.