RESUMO
We report the first and enantioselective total syntheses of (+)-1-deacetylcaesalmin C, (+)-δ-caesalpin, (+)-norcaesalpinin MC, and (+)-norcaesalpinin P. Salient features of the synthetic strategy are an exo-selective intramolecular Diels-Alder reaction of a furanoquinone monoketal and subsequent chemoselective reduction of the resulting pentacyclic furfuryl ketal, furnishing a keystone intermediate. The latter enables access to the collection of natural products through implementation of stereoselective oxidations. Having accessed the cassane furanoditerpenoids, we unveil previously unknown bioactivity: (+)-1-deacetylcaesalmin C stimulates respiration in brown adipocytes, which has been suggested to play a central role in treatment of obesity.
Assuntos
Adipócitos Marrons , Produtos Biológicos , Estereoisomerismo , Oxirredução , Respiração CelularRESUMO
We report the first total syntheses of tricyclic mutanobactinsâ A and B, lipopeptides incorporating a thiazepanone, isolated from Streptococcus mutans, a member of the human oral microbiome. A rapid, solid-phase peptide synthesis (SPPS) based route delivers these natural products from a cascade of cyclization reactions. This versatile process was also employed in a streamlined synthesis of mutanobactinâ D. Additionally, we provide an independent synthesis of a truncated mutanobactinâ A analog, utilizing a novel thiazepanone amino acid building block.
Assuntos
Microbiota , Peptídeos Cíclicos , Ciclização , Humanos , Lipopeptídeos/química , Peptídeos Cíclicos/química , Técnicas de Síntese em Fase SólidaRESUMO
Mutanobactin D is a non-ribosomal, cyclic peptide isolated from Streptococcus mutans and shows activity reducing yeast-to-hyphae transition as well as biofilm formation of the pathogenic yeast Candida albicans. We report the first total synthesis of this natural product, which relies on enantioselective, zinc-mediated 1,3-dipolar cycloaddition and a sequence of cascading reactions, providing the key lipidated γ-amino acid found in mutanobactin D. The synthesis enables configurational assignment, determination of the dominant solution-state structure, and studies to assess the stability of the lipopeptide substructure found in the natural product. The information stored in the fingerprint region of the IR spectra in combination with quantum chemical calculations proved key to distinguishing between epimers of the α-substituted ß-keto amide. Synthetic mutanobactin D drives discovery and analysis of its effect on growth of other members of the human oral consortium. Our results showcase how total synthesis is central for elucidating the complex network of interspecies communications of human colonizers.
Assuntos
Antifúngicos/farmacologia , Peptídeos Cíclicos , Antifúngicos/química , Candida albicans/efeitos dos fármacos , Hifas/efeitos dos fármacos , Modelos Moleculares , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologiaRESUMO
We disclose the syntheses of (+)-bromodanicalipinâ A as well as (±)-fluorodanicalipinâ A. The relative configuration and ground-state conformation in solution of both molecules was secured by J-based configuration analysis which revealed that these are identical to natural danicalipinâ A. Furthermore, preliminary toxicological investigations suggest that the adverse effect of danicalipinâ A may be due to the lipophilicity of the halogens.
Assuntos
Bromo/química , Flúor/química , Lipídeos/síntese química , Lipídeos/farmacologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13RESUMO
A total synthesis of the chlorosulfolipid (+)-danicalipin A has been accomplished in 12 steps and 4.4% overall yield. The efficient and scalable synthesis enabled in-depth investigations of the lipid's biological properties, in particular cytotoxicity towards various mammalian cell lines. Furthermore, the ability of (+)-danicalipinâ A to increase the uptake of fluorophores into bacteria and mammalian cells was demonstrated, indicating it may enhance membrane permeability. By comparing (+)-danicalipinâ A with racemic 1,14-docosane disulfate, and the diol precursor of (+)-danicalipinâ A, we have shown that both chlorine and sulfate functionalities are necessary for biological activity.
Assuntos
Lipídeos/farmacologia , Animais , Linhagem Celular , Corantes Fluorescentes/química , Células HT29 , Humanos , Lipídeos/síntese química , CamundongosRESUMO
RATIONALE: Quantitative trait locus mapping of an intercross between C57.Apoeâ»/â» and FVB.Apoeâ»/â» mice revealed an atherosclerosis locus controlling aortic root lesion area on proximal chromosome 10, Ath11. In a previous work, subcongenic analysis showed Ath11 to be complex with proximal (10a) and distal (10b) regions. OBJECTIVE: To identify the causative genetic variation underlying the atherosclerosis modifier locus Ath11 10b. METHODS AND RESULTS: We now report subcongenic J, which narrows the 10b region to 5 genes, Myb, Hbs1L, Aldh8a1, Sgk1, and Raet1e. Sequence analysis of these genes revealed no amino acid coding differences between the parental strains. However, comparing aortic expression of these genes between F1.Apoeâ»/â» Chr10SubJ((B/F)) and F1.Apoeâ»/â» Chr10SubJ((F/F)) uncovered a consistent difference only for Raet1e, with decreased, virtually background, expression associated with increased atherosclerosis in the latter. The key role of Raet1e was confirmed by showing that transgene-induced aortic overexpression of Raet1e in F1.Apoeâ»/â» Chr10SubJ((F/F)) mice decreased atherosclerosis. Promoter reporter constructs comparing C57 and FVB sequences identified an FVB mutation in the core of the major aortic transcription start site abrogating activity. CONCLUSIONS: This nonbiased approach has revealed Raet1e, a major histocompatibility complex class 1-like molecule expressed in lesional aortic endothelial cells and macrophage-rich regions, as a novel atherosclerosis gene and represents one of the few successes of the quantitative trait locus strategy in complex diseases.
Assuntos
Doenças da Aorta/genética , Aterosclerose/genética , Cromossomos de Mamíferos , Proteínas de Membrana/genética , Locos de Características Quantitativas , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Cruzamentos Genéticos , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Fenótipo , Regiões Promotoras Genéticas , Receptores de LDL/genética , Receptores de LDL/metabolismo , Especificidade da EspécieRESUMO
The number of diabetic patients is rising globally and concomitantly so do the diabetes associated complications. The gut secretes a variety of proteins to control blood glucose levels and/or food intake. As the drug class of GLP-1 agonists is based on a gut secreted peptide and the positive metabolic effects of bariatric surgery are at least partially mediated by gut peptides, we were interested in other gut secreted proteins which have yet to be explored. In this respect we identified the gut secreted protein FAM3D by analyzing sequencing data from L- and epithelial cells of VSG and sham operated as well as chow and HFD fed mice. FAM3D was overexpressed in diet induced obese mice via an adeno-associated virus (AAV), which resulted in a significant improvement of fasting blood glucose levels, glucose tolerance and insulin sensitivity. The liver lipid deposition was reduced, and the steatosis morphology was improved. Hyperinsulinemic clamps indicated that FAM3D is a global insulin sensitizer and increases glucose uptake into various tissues. In conclusion, the current study demonstrated that FAM3D controls blood glucose levels by acting as an insulin sensitizing protein and improves hepatic lipid deposition.
Assuntos
Fígado Gorduroso , Resistência à Insulina , Camundongos , Animais , Glicemia/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Fígado Gorduroso/metabolismo , Peptídeos/farmacologia , Lipídeos , Camundongos Endogâmicos C57BL , Glucose/metabolismo , Dieta Hiperlipídica , Citocinas/metabolismoRESUMO
OBJECTIVE: Ath11, an atherosclerosis susceptibility locus on proximal chromosome 10 (0 to 21 cM) revealed in a cross between apolipoprotein E deficient C57BL/6 (B6) and FVB mice, was recently confirmed in congenic mice. The objectives of this study were to assess how Ath11 affects lesion development and morphology, to determine aortic gene expression in congenics, and to narrow the congenic interval. METHODS AND RESULTS: Assessing lesion area over time in congenic mice showed that homozygosity for the FVB allele increased lesion area at 6 weeks persisting through to 24 weeks of age. Staining of aortic root sections at 16 weeks did not reveal obvious differences between congenics. Aortic expression-array analysis at 6 weeks revealed 97 genes that were >2-fold regulated, including 1 gene in the quantitative trait locus interval, Aldh8a1, and 2 gene clusters regulated by Hnf4alpha and Esr1. Analysis of lesion area in 11 subcongenic strains revealed 2 narrowed regions, 10a (21 genes), acting in females, and 10b (7 genes), acting in both genders. CONCLUSIONS: Ath11 appears to act early in lesion formation, with significant effects on aortic gene expression. This quantitative trait locus is genetically complex, containing a female-specific region 10a from 0 to 7.3 megabases (21 genes) and a gender-independent region 10b from 20.1 to 21.9 megabases (7 genes).
Assuntos
Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Cromossomos de Mamíferos , Locos de Características Quantitativas , Fatores Etários , Envelhecimento , Aldeído Desidrogenase/genética , Animais , Aorta/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Cruzamentos Genéticos , Modelos Animais de Doenças , Progressão da Doença , Receptor alfa de Estrogênio/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Predisposição Genética para Doença , Fator 4 Nuclear de Hepatócito/genética , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Fatores SexuaisRESUMO
OBJECTIVE: We have previously identified a quantitative trait locus (QTL) for atherosclerosis susceptibility on proximal chromosome 10 (Chr10) (Ath11) in independent crosses of FVB and C57BL/6 (B6) mice on the apolipoprotein E (ApoE-/-) and LDL receptor (LDLR-/-) deficient backgrounds. The aims of the current study were to (1) test a novel strategy for validating QTLs using interval-specific congenic strains that were heterozygous (F1) across the genome, (2) validate the Chr10 QTL, and (3) to assess whether the phenotype is transferable by bone marrow transplantation. METHODS AND RESULTS: We generated Chr10 (0 to 21 cM) interval-specific mice on the F1.ApoE-/- background by crossing congenic FVB.ApoE-/-Chr10(B6/FVB) with B6.ApoE-/-, and B6.ApoE-/-Chr10(B6/FVB) with FVB.ApoE-/- mice. Lesion size was significantly larger in the resultant F1.ApoE-/-Chr10(FVB/FVB) mice compared to F1.ApoE-/-Chr10(B6/FVB) and F1.ApoE-/-Chr10(B6/B6) mice, validating the Chr10 QTL. The effect of the congenic interval was more robust on the F1.ApoE-/- than on the FVB.ApoE-/- and B6.ApoE-/- backgrounds. Bone marrow transplantation in congenic mice showed that the effect of the proximal Chr10 interval was not transferable by bone marrow-derived cells. CONCLUSIONS: A novel strategy of congenic strains on an F1 background proved useful to validate an atherosclerosis susceptibility QTL on mouse proximal Chr10.
Assuntos
Aterosclerose/genética , Predisposição Genética para Doença/genética , Locos de Características Quantitativas/genética , Animais , Cromossomos de Mamíferos/genética , Cruzamentos Genéticos , Camundongos , Camundongos CongênicosRESUMO
Up-regulation of inflammatory responses is considered a driving force of atherosclerotic lesion development. One key regulator of inflammation is the A20 (also called TNF-alpha-induced protein 3 or Tnfaip3) gene, which is responsible for NF-kappaB termination and maps to an atherosclerosis susceptibility locus revealed by quantitative trait locus-mapping studies at mouse proximal chromosome 10. In the current study, we examined the role of A20 in atherosclerotic lesion development. At the aortic root lesion size was found to be increased in C57BL/6 (BG) apolipoprotein E-deficient (ApoE(-/-)) mice haploinsufficient for A20, compared with B6 ApoE(-/-) controls that expressed A20 normally (60% in males and 23% in females; P < 0.001 and P < 0.05, respectively). In contrast, lesion size was found to be decreased in F(1) (B6 x FVB/N) mice overexpressing A20 by virtue of containing an A20 BAC transgene compared with nontransgenic controls (30% in males, P < 0.001, and 17% in females, P = 0.02). The increase in lesions in the A20 haploinsufficient mice correlated with increased expression of proatherosclerotic NF-kappaB target genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and macrophage-colony-stimulating factor, and elevated plasma levels of NF-kappaB-driven cytokines. These findings suggest that A20 diminishes atherosclerosis by decreasing NF-kappaB activity, thereby modulating the proinflammatory state associated with lesion development.
Assuntos
Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Cisteína Endopeptidases , Citocinas/sangue , Haploidia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Altered GLP-1 secretion from L cells has been implicated in the development of type 2 diabetes mellitus and its resolution following bariatric surgery. However, changes in L cell gene expression, which may form the basis for altered functionality after high fat diet (HFD) or bariatric surgery, have either not been investigated or have given conflicting results. We developed a gcg-DTR-eGFP reporter mouse to isolate ileal and colonic L cells from HFD fed insulin resistant mice and mice showing improved glucose tolerance following vertical sleeve gastrectomy (VSG). Transcriptomic sequencing and identification of genes differentially expressed in response to HFD or VSG revealed small changes with HFD, primarily in immune related genes, but no regulation following VSG. In contrast, large differences were observed between ileal and colonic L cells due to the differential expression of genes involved in nutrient transport and metabolism, reflecting to some extent the differences in the surrounding epithelium. We showed that, in line with the gene expression data, colonic and ileal L cells exhibit differing GLP-1 responses to nutrients (glucose and the gly-sar dipeptide) and hormones (vasopressin). Thus, we hypothesise that colonic and ileal L cells have different physiological roles, with ileal L cells contributing more to postprandial glucose homeostasis by responding to dietary nutrients and colonic cells responding more to non-dietary stimulants.
Assuntos
Glicemia/metabolismo , Colo/metabolismo , Gastrectomia/efeitos adversos , Regulação da Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Íleo/metabolismo , Animais , Cirurgia Bariátrica , Dieta Hiperlipídica , Homeostase , Masculino , Camundongos , Período Pós-Prandial , Análise de Sequência de RNA , TranscriptomaRESUMO
OBJECTIVE: Roux-en-Y gastric bypass (RYGB) surgery produces rapid and persistent reductions in plasma triglyceride (TG) levels associated with fewer cardiovascular events. The mechanisms of the reduction in systemic TG levels remain unclear. We hypothesized that RYGB reduces intestinal TG secretion via altered enterocyte lipid handling. METHODS: RYGB or Sham surgery was performed in diet-induced obese, insulin-resistant male Sprague-Dawley rats. First, we tested whether RYGB reduced test meal-induced TG levels in the intestinal lymph, a direct readout of enterocyte lipid secretion. Second, we examined whether RYGB modified TG enterocyte secretion at the single lipid level and in comparison to other lipid subclasses, applying mass spectrometry lipidomics to the intestinal lymph of RYGB and Sham rats (0-21 days after surgery). Third, we explored whether RYGB modulated the metabolic characteristics of primary enterocytes using transcriptional and functional assays relevant to TG absorption, reesterification, storage in lipid droplets, and oxidation. RESULTS: RYGB reduced overall postprandial TG concentrations compared to Sham surgery in plasma and intestinal lymph similarly. RYGB reduced lymphatic TG concentrations more than other lipid subclasses, and shifted the remaining TG pool towards long-chain, unsaturated species. In enterocytes of fasted RYGB rats, lipid uptake was transcriptionally (Fatp4, Fabp2, Cd36) and functionally reduced compared to Sham, whereas TG reesterification genes were upregulated. CONCLUSION: Our results show that RYGB substantially reduces intestinal TG secretion and modifies enterocyte lipid absorption and handling in rats. These changes likely contribute to the improvements in the plasma TG profile observed after RYGB in humans.
Assuntos
Enterócitos/metabolismo , Derivação Gástrica/métodos , Período Pós-Prandial/fisiologia , Triglicerídeos/sangue , Animais , Glicemia , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Teste de Tolerância a Glucose , Resistência à Insulina , Mucosa Intestinal/metabolismo , Linfa/metabolismo , Masculino , Obesidade/cirurgia , Ratos , Ratos Sprague-DawleyRESUMO
Peptides are important in the drug discovery process. In analogy to nonpeptidic small-molecule counterparts, they can sometimes suffer from disadvantages such as their low bioavailability and poor metabolic stability. Herein, we report the synthesis of new oxetanyl dipeptides and their incorporation into Leu-enkephalin analogues as proof-of-principle studies. The modular approach that is described enables the incorporation of a variety of oxetanyl amino acids into potential peptide therapeutics.
Assuntos
Aminoácidos/química , Dipeptídeos , Encefalina Leucina , Estrutura Molecular , PeptidomiméticosRESUMO
This study documents that chlorinated analogs of leukotoxin diol 1, in which the vic-diol has been replaced with vic-chlorides (2), induce caspase 3 activity and apoptosis on HepG2 cells in a dose-dependent manner in analogy to the parent diol. This suggests that chlorides may substitute for hydroxyls in certain lipids as bioisosteres in defined biological settings.