RESUMO
Islet transplantation is an effective treatment for type 1 diabetes (T1D). However, a shortage of donors and the need for immunosuppressants are major issues. The ideal solution is to develop a source of insulin-secreting cells and an immunoprotective method. No bioartificial pancreas (BAP) devices currently meet all of the functions of long-term glycemic control, islet survival, immunoprotection, discordant xenotransplantation feasibility, and biocompatibility. We developed a device in which porcine islets were encapsulated in a highly stable and permeable hydrogel and a biocompatible immunoisolation membrane. Discordant xenotransplantation of the device into diabetic mice improved glycemic control for more than 200 days. Glycemic control was also improved in new diabetic mice "relay-transplanted" with the device after its retrieval. The easily retrieved devices exhibited almost no adhesion or fibrosis and showed sustained insulin secretion even after the two xenotransplantations. This device has the potential to be a useful BAP for T1D.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Transplante das Ilhotas Pancreáticas , Animais , Camundongos , Suínos , Diabetes Mellitus Tipo 1/cirurgia , Transplante Heterólogo , Diabetes Mellitus Experimental/cirurgia , PâncreasRESUMO
Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics lankacidin and lankamycin, and their biosynthesis is tightly controlled by butenolide-type signaling molecules SRB1 and SRB2. SRBs are synthesized by SRB synthase SrrX, and induce lankacidin and lankamycin production at 40 nM concentration. We here investigated the role of a P450 monooxygenase gene srrO (orf84), which is located adjacent to srrX (orf85), in SRB biosynthesis. An srrO mutant KA54 accumulated lankacidin and lankamycin at a normal level when compared with the parent strain. To elucidate the chemical structures of the signaling molecules accumulated in KA54 (termed as KA54-SRBs), this mutant was cultured (30 L) and the active components were purified. Two active components (KA54-SRB1 and KA54-SRB2) were detected in ESI-MS and chiral HPLC analysis. The molecular formulae for KA54-SRB1 and KA54-SRB2 are C15H26O4 and C16H28O4, whose values are one oxygen smaller and two hydrogen larger when compared with those for SRB1 and SRB2, respectively. Based on extensive NMR analysis, the signaling molecules in KA54 were determined to be 6'-deoxo-SRB1 and 6'-deoxo-SRB2. Gel shift analysis indicated that a ligand affinity of 6'-deoxo-SRB1 to the specific receptor SrrA was 100-fold less than that of SRB1. We performed bioconversion of the synthetic 6'-deoxo-SRB1 in the Streptomyces lividans recombinant carrying SrrO-expression plasmid. Substrate 6'-deoxo-SRB1 was converted through 6'-deoxo-6'-hydroxy-SRB1 to SRB1 in a time-dependent manner. Thus, these results clearly indicated that SrrO catalyzes the C-6' oxidation at a final step in SRB biosynthesis.
Assuntos
4-Butirolactona/análogos & derivados , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , 4-Butirolactona/biossíntese , 4-Butirolactona/química , Antibacterianos/biossíntese , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/genética , Eritromicina/análogos & derivados , Eritromicina/biossíntese , Eritromicina/química , Genes Bacterianos , Macrolídeos/química , Macrolídeos/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutação , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Streptomyces/genéticaRESUMO
Preclinical Research & Development MR1704 is a selective G protein-coupled receptor 40/free fatty acid receptor 1 agonist, which exhibited favorable pharmacokinetic profiles and glucose-lowering effects in animal models. We studied the effects of MR1704 in a sulfonylurea-desensitized Sprague-Dawley rat model and evaluated the risk of pancreatic ß-cell exhaustion compared to that of glibenclamide in Zucker fatty rats. Rats fed ad libitum a diet containing 0.03% glibenclamide exhibited lower non-fasting blood glucose levels compared to those in rats fed a control diet during the first 6 days. However, the response to glibenclamide disappeared on day 9. In a rat oral glucose tolerance test (OGTT), MR1704 reduced the plasma glucose excursion, whereas glibenclamide did not show this effect. In Zucker fatty rats, oral administration of MR1704 reduced glucose excursion during the OGTT, and the effects of MR1704 were maintained after 2-week treatment. In contrast, the glucose-lowering effects of glibenclamide were diminished, and glucose tolerance was aggravated after 2-week treatment. These results indicated that MR1704 provided more sustainable effects compared to those of the sulfonylurea, glibenclamide suggesting that MR1704 may be an attractive therapeutic option for diabetic patients who are unresponsive to sulfonylurea treatment.
Assuntos
Hipoglicemiantes/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Tiazóis/farmacologia , Animais , Glicemia/efeitos dos fármacos , Resistência a Medicamentos , Teste de Tolerância a Glucose , Glibureto/uso terapêutico , Masculino , Ratos Sprague-Dawley , Ratos ZuckerRESUMO
Activation of G protein-coupled receptor 40/Free fatty acid receptor 1 (GPR40/FFAR1), which is highly expressed in pancreatic ß cells, is considered an important pharmacologic target for the treatment of type 2 diabetes mellitus. The aim of this study was to determine the effect of MR1704, a novel GPR40/FFAR1 agonist, on glucose homeostasis in rats. MR1704 is a highly potent and selective, orally bioavailable agonist with similar in vitro potencies among humans, mice, and rats. Treatment of rat islets with MR1704 increased glucose-dependent insulin secretion. Augmentation of glucose-dependent insulin secretion was abolished by adding a GPR40/FFAR1 antagonist. In mouse, insulinoma MIN6 cells, palmitic acid induced the activity of caspase 3/7 after a 72-h exposure, while pharmacologically active concentrations of MR1704 did not. In an oral glucose tolerance test in normal Sprague-Dawley rats, orally administered MR1704 (1-10 mg·kg-1 ) reduced plasma glucose excursion and enhanced insulin secretion, but MR1704 did not induce hypoglycemia, even at 300 mg·kg-1 , in fasted Sprague-Dawley rats. In addition, orally administered MR1704 reduced plasma glucose excursion and enhanced insulin secretion in diabetic Goto-Kakizaki rats. Oral administration of MR1704 once daily to Goto-Kakizaki rats reduced their blood glucose levels during a 5-week treatment period without reducing pancreatic insulin content; as a result, hemoglobin A1C levels significantly decreased. These results suggest that MR1704 improves glucose homeostasis through glucose-dependent insulin secretion with a low risk of hypoglycemia and pancreatic toxicity. MR1704 shows promise as a new, glucose-lowering drug to treat type 2 diabetes mellitus.
RESUMO
The transcription factor NF-E2-related factor 2 (Nrf2) is a key regulator of cellular defense mechanisms against oxidative stress. Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system, is characterized by progressive demyelination and neurodegeneration induced by inflammation and oxidative stress. The induction of Nrf2 signaling has been shown to inhibit disease development and progression in the experimental autoimmune encephalomyelitis (EAE) model of MS in mice. In the present study, we performed a high-throughput screening assay using a chimeric construct of the N-terminal portion of Nrf2 fused to LacZ. Using this approach, we identified the novel Nrf2 inducer TFM-735. Using human primary cell profiling systems, we found that TFM-735 inhibited T cell proliferation and exerted immuno-modulatory effects by inhibiting the production of IL-6 and IL-17. TFM-735 also inhibited IL-17 secretion from human peripheral blood mononuclear cells stimulated with anti-CD3 and anti-CD28. In EAE mice treated with TFM-735, the expression of the Nrf2 target gene Nqo1 increased in the brain and spleen, disease severity was ameliorated, and plasma IL-17 levels decreased. Furthermore, TFM-735 inhibited luciferase activity in Wim-6 transgenic EAE mice expressing the human interleukin 6-luciferase (hIL6-BAC-Luc) reporter. Therefore, these findings indicate that TFM-735 is a potent Nrf2 inducer that inhibits inflammatory cytokine production and disease progression in mice with EAE and that TFM-735 is a promising therapeutic agent for MS.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Pirazóis/farmacologia , Tiazóis/farmacologia , Animais , Células HEK293 , Humanos , Interleucina-17/metabolismo , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/uso terapêutico , Tiazóis/uso terapêuticoRESUMO
OBJECTIVE: Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final committed step in triglyceride biosynthesis. DGAT1 null mice are known to be resistant to diet-induced obesity, and more insulin sensitive relative to the wild-type; however, the mice exhibit abnormalities in the skin. This work determined whether the intestine-targeted DGAT1 inhibitor could improve obesity and insulin resistance without skin aberrations in mice. DESIGN AND METHODS: We synthesized 2 DGAT1 inhibitors: Compound A, described in the patent application from the Japan Tobacco, and Compound B (A-922500), reported by Abbott Laboratories. Both compounds were evaluated for inhibitory activities against DGAT1 enzymes and effects on the skin in mice in vivo. Compound B was further investigated for effects on obesity and insulin resistance in diet-induced-obese (DIO) mice. RESULTS: The 2 compounds comparably inhibited the DGAT1 enzyme activity and the cellular triglyceride synthesis in vitro, while they showed different distribution patterns in mice in vivo. Compound A, which distributed systemically, caused skin aberrations, while Compound B, which preferentially distributed to the intestine, improved obesity and insulin resistance without skin aberrations in DIO mice. CONCLUSIONS: Our results suggest that the intestine is the key tissue in which DGAT1 plays a role in promoting obesity and insulin resistance.
Assuntos
Compostos de Bifenilo/uso terapêutico , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Resistência à Insulina , Intestinos/efeitos dos fármacos , Obesidade/tratamento farmacológico , Compostos de Fenilureia/uso terapêutico , Animais , Compostos de Bifenilo/efeitos adversos , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacocinética , Diacilglicerol O-Aciltransferase/metabolismo , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Células HT29 , Células Hep G2 , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos de Fenilureia/efeitos adversos , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacocinética , Pele/efeitos dos fármacos , Distribuição TecidualRESUMO
New signaling molecules that induce lankacidin and lankamycin production in Streptomyces rochei were extracted from the culture filtrate and purified by Sephadex LH20 and silica gel chromatography with the help of bioassay. Chiral HPLC and ESI-MS analyses indicated the presence of two active components--SRB1 and SRB2--and their molecular formulas were established to be C15H24O5 and C16H26O5, respectively. By extensive NMR analysis, SRB1 and SRB2 were determined to be 2-(1'-hydroxy-6'-oxo-8'-methylnonyl)-3-methyl-4-hydroxybut-2-en-1,4-olide and 2-(1'-hydroxy-6'-oxo-8'-methyldecyl)-3-methyl-4-hydroxybut-2-en-1,4-olide, respectively. These structures were finally confirmed by chemical synthesis and the absolute configuration at C-1' was determined to be R in each case. The synthetic 1'R isomers induced production of lankacidin and lankamycin at around 40 nM concentrations. SRB1 and SRB2 are therefore distinct from the well-known 2,3-disubstituted γ-butyrolactone molecules such as A-factor, virginia butanolide, and SCB1 and and belong, like avenolide, recently isolated from Streptomyces avermitilis, to the γ-butenolide family.
Assuntos
4-Butirolactona/análogos & derivados , Eritromicina/análogos & derivados , Macrolídeos/metabolismo , Streptomyces/metabolismo , 4-Butirolactona/síntese química , 4-Butirolactona/química , Eritromicina/química , Eritromicina/metabolismo , Isomerismo , Macrolídeos/química , Espectroscopia de Ressonância Magnética , Conformação MolecularRESUMO
OBJECTIVE: We have provided evidence that saturated fatty acids, which are released from adipocytes via macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for the Toll-like receptor (TLR) 4 complex in macrophages, thereby aggravating obesity-induced adipose tissue inflammation. The aim of this study was to identify the molecule(s) activated in adipose tissue macrophages in obesity. RESEARCH DESIGN AND METHODS: We performed a cDNA microarray analysis of coculture of 3T3-L1 adipocytes and RAW264 macrophages. Cultured adipocytes and macrophages and the adipose tissue of obese mice and humans were used to examine mRNA and protein expression. RESULTS: We found that macrophage-inducible C-type lectin (Mincle; also called Clec4e and Clecsf9), a type II transmembrane C-type lectin, is induced selectively in macrophages during the interaction between adipocytes and macrophages. Treatment with palmitate, a major saturated fatty acid released from 3T3-L1 adipocytes, induced Mincle mRNA expression in macrophages at least partly through the TLR4/nuclear factor (NF)-κB pathway. Mincle mRNA expression was increased in parallel with macrophage markers in the adipose tissue of obese mice and humans. The obesity-induced increase in Mincle mRNA expression was markedly attenuated in C3H/HeJ mice with defective TLR4 signaling relative to control C3H/HeN mice. Notably, Mincle mRNA was expressed in bone-marrow cell (BMC)-derived proinflammatory M1 macrophages rather than in BMC-derived anti-inflammatory M2 macrophages in vitro. CONCLUSIONS: Our data suggest that Mincle is induced in adipose tissue macrophages in obesity at least partly through the saturated fatty acid/TLR4/NF-κB pathway, thereby suggesting its pathophysiologic role in obesity-induced adipose tissue inflammation.
Assuntos
Tecido Adiposo/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Obesidade/metabolismo , Células 3T3-L1 , Tecido Adiposo/efeitos dos fármacos , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Humanos , Inflamação/genética , Inflamação/metabolismo , Lectinas Tipo C/genética , Modelos Lineares , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Obesidade/genética , Ácido Palmítico/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
In this study, a number of 2',4'-BNA- and 2',4'-BNA(NC)-modified siRNAs were designed and synthesized. Their thermal stability, nuclease resistance and gene silencing properties against cultured mammalian cells were evaluated and compared with those of natural siRNAs. The 2',4'-BNA- and 2',4'-BNA(NC)-modified siRNAs (named siBNA and siBNA(NC), respectively) showed very high T(m) values, were remarkably stable in serum sample and showed promising RNAi properties equal to those exhibited by natural siRNAs. Thermally stable siBNAs composed of slightly modified sense and antisense strands were capable of suppressing gene expression equal to that of natural siRNA. A number of modifications on the sense strand by 2',4'-BNA or 2',4'-BNA(NC), either consecutively or separated by natural RNA nucleotides, is tolerable in RNAi machinery. Modifications at the Argonauate (Ago2) cleavage site of the sense strand (9-11th positions from the 5'-end of the sense strand) produced variable results depending on siRNA composition. Mostly, modification at the 10th position diminished siRNA activity. In moderately modified siRNAs, modification at the 11th position displayed usual RNAi activity, while modification at the 9th position showed variable results depending on siRNA composition.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Nucleotídeos/química , Interferência de RNA/fisiologia , RNA Interferente Pequeno/química , RNA/química , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Conformação de Ácido NucleicoRESUMO
Antigene strategy is promising technology to regulate gene expression. We have previously reported that 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotides (TFOs) significantly enhanced the binding affinity towards the target dsDNA. In spite of its usefulness, the TFO-binding site may not completely overlay the protein-binding site because of the limitation of TFOs targeting sequences. To overcome this problem, we developed an antigene-based new methodology called "antigene-block" strategy. In this methodology, the TFOs bearing a bulky hairpin tail are used for efficient inhibition of protein-DNA interaction. The antigene-block TFOs having 2',4'-BNA modifications formed stable triplexes with the homopurine-homopyrimidine sequence which partially overlap the transcription factor NF-kappaB binding site. In addition, the antigene-block TFOs significantly reduced the expression level of the target-gene in living cells, while conventional 2',4'-BNA-modified or unmodified TFOs showed no effect on the target-gene expression.