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1.
Sci Rep ; 12(1): 1429, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-35082341

RESUMO

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.


Assuntos
Descoberta de Drogas , Transportador de Glucose Tipo 3/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/antagonistas & inibidores , Transportador de Glucose Tipo 2/química , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Transportador de Glucose Tipo 5/antagonistas & inibidores , Transportador de Glucose Tipo 5/química , Transportador de Glucose Tipo 5/genética , Transportador de Glucose Tipo 5/metabolismo , Compostos Heterocíclicos com 3 Anéis/química , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/química
2.
J Neurochem ; 160(2): 283-296, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34726780

RESUMO

The retina has the highest relative energy consumption of any tissue, depending on a steady supply of glucose from the bloodstream. Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases, including diabetes and diabetic retinopathy. Here, we used immunofluorescence to show that glucose transporter-2 (GLUT2) is expressed in horizontal cells of the mouse neuroretina in proximity to inner retinal capillaries. To study the function of GLUT2 in the murine retina, we used organotypic retinal explants, cultivated under entirely controlled, serum-free conditions and exposed them to streptozotocin, a cytotoxic drug transported exclusively by GLUT2. Contrary to our expectations, streptozotocin did not measurably affect horizontal cell viability, while it ablated rod and cone photoreceptors in a concentration-dependent manner. Staining for poly-ADP-ribose (PAR) indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA damage. The negative effect of streptozotocin on the viability of rod photoreceptors was counteracted by co-administration of either the inhibitor of connexin-formed hemi-channels meclofenamic acid or the blocker of clathrin-mediated endocytosis dynasore. Remarkably, cone photoreceptors were not protected from streptozotocin-induced degeneration by neither of the two drugs. Overall, these data suggest the existence of a GLUT2-dependent glucose transport shuttle, from horizontal cells into photoreceptor synapses. Moreover, our study points at different glucose uptake mechanisms in rod and cone photoreceptors.


Assuntos
Transportador de Glucose Tipo 2/metabolismo , Glucose/metabolismo , Células Fotorreceptoras/metabolismo , Células Horizontais da Retina/metabolismo , Sinapses/metabolismo , Animais , Transporte Biológico , Camundongos , Retina/metabolismo
3.
Life Sci ; 290: 120261, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34968468

RESUMO

AIMS: We previously showed that hindlimb ischemia-reperfusion (IR) enhanced glucose uptake in the liver through the activation of the parasympathetic nervous system. Although we suggested that the key glucose transporter (GLUT) in this hepatic glucose uptake was GLUT4 by western blotting, the molecular weight of GLUT4 was nearly the same as that of GLUT2, which is predominantly expressed in the liver. We primarily conducted a histological evaluation to determine whether IR specifically accelerates the overexpression of GLUT4, rather than GLUT2, in the hepatocytes in vitro and in vivo. MAIN METHODS: A total of 54 male C57BL/6J mice were used and subjected to 3 min hindlimb ischemia repeated three times with 3 min interval. Focusing on the area connecting portal and central veins, the GLUT4 and GLUT2 expression in the hepatocytes were examined by real-time PCR and immunohistochemically. Moreover, the alteration of GLUT4 and GLUT2 expression by acetylcholine in the primary hepatocytes were examined by immunofluorescence. KEY FINDINGS: IR significantly upregulated the GLUT4, rather than GLUT2, expression in both mRNA and protein in the liver. Histological examination revealed marked glycogen storage in zone1, the periportal area, coincident with the enhanced GLUT4 immunoreactivity, in the IR-treated liver. Incubation of primary hepatocytes with acetylcholine induced the appearance of GLUT4 on the membrane peripheries. SIGNIFICANCE: The overexpression of GLUT4 on the membrane peripheries contributed to increasing glucose uptake found in IR-treated livers. This acceleration of glucose uptake via GLUT4 may induce marked glycogen storage in zone1 through energy production linked with increased glucose preference.


Assuntos
Transportador de Glucose Tipo 4/metabolismo , Glicogênio/metabolismo , Precondicionamento Isquêmico/métodos , Animais , Membrana Celular/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Glucose/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 4/genética , Hepatócitos/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/metabolismo
4.
Dis Markers ; 2021: 2821673, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745386

RESUMO

To elucidate the mechanism of how the liver participates in PM2.5-caused insulin resistance. A novel Wistar rat model was developed in this study by instilling a suspension of lyophilized PM2.5 sample (2.5 mg/kg, 5 mg/kg, or 10 mg/kg) collected from the atmosphere. Systemic insulin resistance indicators, including serum fasting blood glucose (FBG), fasting insulin (FINS), Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), and hemoglobin A1 (HbA1), were upregulated by the PM2.5 instillation. The area under the curve (AUCglu) calculated by intraperitoneal glucose tolerance testing (IPGTT) was also significantly greater in the PM2.5 instillation groups. Additionally, PM2.5 instillation was found to cause liver damage and inflammation. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were significantly elevated by PM2.5 instillation. PM2.5 also triggered IL-6 and TNF-α transcription but inhibited mRNA synthesis and suppressed signaling activation of the insulin-phosphoinositide 3-kinase- (PI3K-) Akt-glucose transporter 2 (GLUT2) pathway in the rat liver by reducing the ratio of phosphorylated Akt to phosphorylated insulin receptor substrate 1 (IRS-1). Thus, PM2.5-induced inflammation activation and insulin signaling inhibition in the rat liver contribute to the development of systemic insulin resistance.


Assuntos
Resistência à Insulina , Fígado/metabolismo , Material Particulado/toxicidade , Transdução de Sinais , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Glicemia/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Hemoglobinas/metabolismo , Insulina/sangue , Interleucina-6/sangue , Fígado/efeitos dos fármacos , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/sangue
5.
Genes (Basel) ; 12(11)2021 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-34828390

RESUMO

Fanconi-Bickel syndrome (FBS) is a very rare but distinct clinical entity with the combined features of hepatic glycogen storage disease, generalized proximal renal tubular dysfunction with disproportionately severe glucosuria, and impaired galactose tolerance. Here, we report five cases (out of 93 diagnosed in our lab) with pathogenic variants on both GLUT2 (SLC2A2) alleles. They come from 3 families and presented with an exceptionally mild clinical course. This course was correlated to data from old and most recent expression and transport studies in Xenopus oocytes. GLUT2 genotype in patients 1 and 2 was p.[153_4delLI];[P417R] with the first variant exhibiting normal membrane expression and partially retained transport activity (5.8%) for 2-deoxyglucose. In patient 3, the very first GLUT2 variant ever detected (p.V197I) was found, but for the first time it was present in a patient in the homozygous state. This variant had also shown unaffected membrane expression and remarkable residual activity (8%). The genotype in patient 4, p.[153_4delLI];[(E440A)], again included the 2-amino-acid deletion with residual transporter function, and patient 5 is the first found to be homozygous for this variant. Our results provide further evidence for a genotype-phenotype correlation in patients with GLUT2 variants; non-functional variants result in the full picture of FBS while dysfunctional variants may result in milder presentations, even glucosuria only, without other typical signs of FBS.


Assuntos
Síndrome de Fanconi/genética , Transportador de Glucose Tipo 2/genética , Mutação , Fenótipo , Adolescente , Adulto , Animais , Síndrome de Fanconi/patologia , Feminino , Genótipo , Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Homozigoto , Humanos , Lactente , Masculino , Linhagem , Xenopus
6.
BMC Cancer ; 21(1): 1026, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34525987

RESUMO

BACKGROUND: Current treatment methods for patients with triple-negative breast cancer (TNBC) are very limited, and the prognosis of TNBC is relatively poor. It has been reported that glucose transporter 1 (GLUT1) is overexpressed in breast cancer cells; however, its association with the prognosis is mostly unclear. Moreover, retinoblastoma gene 1 (RB1) might be used as a biomarker for the sensitivity of breast cancer cells to GLUT1 inhibitors, which brought us to the hypothesis that there might be a close correlation between the expression of GLUT1-4 and the expression of RB1. METHODS: In this study, we systematically analyzed the co-expression of GLUT1-4 and the influence of GLUT1-4 gene expression on the prognosis of breast cancer using data mining methods. We also explored possible relationships between GLUT1-4 and RB1 expression in breast cancer tissues. We used public databases such as ONCOMINE, GEPIA, LinkedOmics, and COEXPEDIA. RESULTS: According to the results, the mRNA expression of SLC2A1 was significantly higher in breast cancer, while the expression levels of SLC2A2-4 were downregulated. The results also indicate that GLUT1 expression does not have significant influence on the overall survival of patients with breast cancer. The mRNA expression of SLC2A1 and RB1 is significantly correlated, which means that tissues with high RB1 mRNA expression might have relatively higher mRNA expression of SLC2A1; however, further study analyzing their roles in the expression regulation pathways with human samples is needed to verify the hypothesis. CONCLUSIONS: The mRNA expression of SLC2A1 was significantly higher in breast cancer. The overall survival of breast cancer patients wasn't significantly correlated with GLUT1-4 expression. The mRNA expression of SLC2A1 and RB1 is significantly correlated according to the analysis conducted in LinkedOmics. It provides reference for future possible individualized treatment of TNBC using GLUT1 inhibitors, especially in patients with higher mRNA expression of RB1. Further study analyzing the roles of these two genes in the regulation pathways is needed.


Assuntos
Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Ubiquitina-Proteína Ligases/metabolismo , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Regulação para Baixo , Feminino , Genes do Retinoblastoma , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 4/genética , Humanos , Prognóstico , RNA Mensageiro/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Ubiquitina-Proteína Ligases/genética , Regulação para Cima
7.
Nutrients ; 13(8)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34444712

RESUMO

Glucagon-like peptide 1 (GLP-1) and PAS kinase (PASK) control glucose and energy homeostasis according to nutritional status. Thus, both glucose availability and GLP-1 lead to hepatic glycogen synthesis or degradation. We used a murine model to discover whether PASK mediates the effect of exendin-4 (GLP-1 analogue) in the adaptation of hepatic glycogen metabolism to nutritional status. The results indicate that both exendin-4 and fasting block the Pask expression, and PASK deficiency disrupts the physiological levels of blood GLP1 and the expression of hepatic GLP1 receptors after fasting. Under a non-fasted state, exendin-4 treatment blocks AKT activation, whereby Glucokinase and Sterol Regulatory Element-Binding Protein-1c (Srebp1c) expressions were inhibited. Furthermore, the expression of certain lipogenic genes was impaired, while increasing Glucose Transporter 2 (GLUT2) and Glycogen Synthase (GYS). Moreover, exendin-4 treatment under fasted conditions avoided Glucose 6-Phosphatase (G6pase) expression, while maintaining high GYS and its activation state. These results lead to an abnormal glycogen accumulation in the liver under fasting, both in PASK-deficient mice and in exendin-4 treated wild-type mice. In short, exendin-4 and PASK both regulate glucose transport and glycogen storage, and some of the exendin-4 effects could therefore be due to the blocking of the Pask expression.


Assuntos
Adaptação Fisiológica , Jejum , Glicogênio Hepático/metabolismo , Fígado/metabolismo , Estado Nutricional , /metabolismo , Animais , Exenatida/metabolismo , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/sangue , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucoquinase/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , /genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Regulação para Cima , Perda de Peso
8.
Cells ; 10(8)2021 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-34440786

RESUMO

The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Soluções para Diálise/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Diálise Renal , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Agregação Celular/efeitos dos fármacos , Agregação Celular/genética , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Linhagem Celular , Meios de Cultura/química , Soluções para Diálise/química , Sistema Endócrino/citologia , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/metabolismo , Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo
9.
J Mol Endocrinol ; 67(3): 71-82, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34223824

RESUMO

Glucose transporter type 2 (GLUT2), encoded by the SLC2A2 gene, is an essential component of glucose-stimulated insulin secretion in pancreatic islet ß-cells. Like that of the gene encoding insulin, expression of the SLC2A2 gene expression is closely linked to ß-cell functionality in rodents, but the mechanism by which ß-cell-specific expression of SLC2A2 is controlled remains unclear. In this report, to identify putative enhancer elements of the mouse Slc2a2 gene, we examined evolutional conservation of the nucleotide sequence of its genomic locus, together with ChIP-seq data of histone modifications and various transcription factors published in previous studies. Using luciferase reporter assays, we found that an evolutionarily conserved region (ECR) located approximately 40 kbp downstream of the transcription start site of Slc2a2 functions as an active enhancer in the MIN6 ß-cell line. We also found that three ß-cell-enriched transcription factors, MafA, NeuroD1, and HNF1ß, synergistically activate transcription through this 3' downstream distal enhancer (ECR3') and the proximal promoter region of the gene. Our data also indicate that the simultaneous binding of HNF1ß to its target sites within the promoter and ECR3' of Slc2a2 is indispensable for transcriptional activation, and that binding of MafA and NeuroD1 to their respective target sites within the ECR3' enhances transcription. Co-immunoprecipitation experiments suggested that MafA, NeuroD1, and HNF1ß interact with each other. Overall, these results suggest that promoter-enhancer communication through MafA, NeuroD1, and HNF1ß is critical for Slc2a2 gene expression. These findings provide clues to help elucidate the mechanism of regulation of Slc2a2 gene expression in ß-cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica , Transportador de Glucose Tipo 2/genética , Fator 1-beta Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Animais , Sequência Conservada , Elementos Facilitadores Genéticos , Transportador de Glucose Tipo 2/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta , Ativação Transcricional
10.
Mar Drugs ; 19(5)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069768

RESUMO

The Red Sea is one of the most biodiverse aquatic ecosystems. Notably, seagrasses possess a crucial ecological significance. Among them are the two taxa Halophila stipulacea (Forsk.) Aschers., and Thalassia hemprichii (Ehrenb. ex Solms) Asch., which were formally ranked together with the genus Enhalus in three separate families. Nevertheless, they have been recently classified as three subfamilies within Hydrocharitaceae. The interest of this study is to explore their metabolic profiles through ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS/MS) analysis in synergism with molecular networking and to assess their chemosystematics relationship. A total of 144 metabolites were annotated, encompassing phenolic acids, flavonoids, terpenoids, and lipids. Furthermore, three new phenolic acids; methoxy benzoic acid-O-sulphate (16), O-caffeoyl-O-hydroxyl dimethoxy benzoyl tartaric acid (26), dimethoxy benzoic acid-O-sulphate (30), a new flavanone glycoside; hexahydroxy-monomethoxy flavanone-O-glucoside (28), and a new steviol glycoside; rebaudioside-O-acetate (96) were tentatively described. Additionally, the evaluation of the antidiabetic potential of both taxa displayed an inherited higher activity of H. stipulaceae in alleviating the oxidative stress and dyslipidemia associated with diabetes. Hence, the current research significantly suggested Halophila, Thalassia, and Enhalus categorization in three different taxonomic ranks based on their intergeneric and interspecific relationship among them and supported the consideration of seagrasses in natural antidiabetic studies.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hydrocharitaceae/química , Hipoglicemiantes/farmacologia , Metaboloma , Animais , Glicemia/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ensaios Enzimáticos , Transportador de Glucose Tipo 2/metabolismo , Hydrocharitaceae/genética , Hidrólise , Hipoglicemiantes/uso terapêutico , Oceano Índico , Insulina/sangue , Masculino , Malondialdeído/metabolismo , Espectrometria de Massas , Óxido Nítrico/sangue , Filogenia , Compostos Fitoquímicos/análise , Ratos Wistar
11.
Nat Commun ; 12(1): 3133, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035238

RESUMO

Heterozygous HNF1A gene mutations can cause maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. However, specific mechanisms of MODY3 in humans remain unclear due to lack of access to diseased human pancreatic cells. Here, we utilize MODY3 patient-derived human induced pluripotent stem cells (hiPSCs) to study the effect(s) of a causal HNF1A+/H126D mutation on pancreatic function. Molecular dynamics simulations predict that the H126D mutation could compromise DNA binding and gene target transcription. Genome-wide RNA-Seq and ChIP-Seq analyses on MODY3 hiPSC-derived endocrine progenitors reveal numerous HNF1A gene targets affected by the mutation. We find decreased glucose transporter GLUT2 expression, which is associated with reduced glucose uptake and ATP production in the MODY3 hiPSC-derived ß-like cells. Overall, our findings reveal the importance of HNF1A in regulating GLUT2 and several genes involved in insulin secretion that can account for the insulin secretory defect clinically observed in MODY3 patients.


Assuntos
Diabetes Mellitus Tipo 2/genética , Transportador de Glucose Tipo 2/genética , Glucose/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Mutação , Células Cultivadas , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Transportador de Glucose Tipo 2/metabolismo , Fator 1-alfa Nuclear de Hepatócito/química , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/citologia , Masculino , Simulação de Dinâmica Molecular , Linhagem , Domínios Proteicos
12.
Mol Ther ; 29(9): 2854-2872, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33895325

RESUMO

The processing, maturation, and secretion of insulin are under precise regulation, and dysregulation causes profound defects in glucose handling, leading to diabetes. Tmem30a is the ß subunit of the phosphatidylserine (PS) flippase, which maintains the membrane asymmetric distribution of PS. Tmem30a regulates cell survival and the localization of subcellular structures and is thus critical to the normal function of multiple physiological systems. Here, we show that conditional knockout of Tmem30a specifically in pancreatic islet ß cells leads to obesity, hyperglycemia, glucose intolerance, hyperinsulinemia, and insulin resistance in mice, due to insufficient insulin release. Moreover, we reveal that Tmem30a plays an essential role in clathrin-mediated vesicle transport between the trans Golgi network (TGN) and the plasma membrane (PM), which comprises immature secretory granule (ISG) budding at the TGN. We also find that Tmem30a deficiency impairs clathrin-mediated vesicle budding and thus blocks both insulin maturation in ISGs and the transport of glucose-sensing Glut2 to the PM. Collectively, these disruptions compromise both insulin secretion and glucose sensitivity, thus contributing to impairments in glucose-stimulated insulin secretion. Taken together, our data demonstrate an important role of Tmem30a in insulin maturation and glucose metabolic homeostasis and suggest the importance of membrane phospholipid distribution in metabolic disorders.


Assuntos
Intolerância à Glucose/genética , Transportador de Glucose Tipo 2/metabolismo , Hiperglicemia/genética , Hiperinsulinismo/genética , Resistência à Insulina/genética , Insulina/metabolismo , Proteínas de Membrana/genética , Obesidade/genética , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Glucose/efeitos adversos , Intolerância à Glucose/metabolismo , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Obesidade/metabolismo , Fosfatidilserinas/metabolismo , Rede trans-Golgi/metabolismo
13.
J Sci Food Agric ; 101(14): 5984-5991, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33856052

RESUMO

BACKGROUND: Nutritional modulations may be considered a strategy to protect mental health. Neuronal homeostasis is highly dependent on the availability of glucose, which represents the primary energy source for the brain. In this study, we evaluated the effects of walnut intake and fructose-rich diet on the expression of glucose transporters (GLUTs) in two rat brain regions: hypothalamus and hippocampus. RESULTS: Our results show that walnut supplementation of fructose-fed animals restored the hypothalamic content of GLUT1 and GLUT3 protein. Furthermore, walnut intake did not affect increased hypothalamic GLUT2 content upon fructose consumption. These effects were accompanied by distinctive alterations of hippocampal GLUTs levels. Specifically, walnut intake increased GLUT1 content, whereas GLUT2 protein was decreased within the rat hippocampus after both individual and combined treatments. CONCLUSION: Overall, our study suggests that walnut supplementation exerted modulatory effects on the glucose transporters within specific brain regions in the presence of developed metabolic disorder. © 2021 Society of Chemical Industry.


Assuntos
Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Juglans/metabolismo , Animais , Frutose/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 3/genética , Masculino , Nozes/metabolismo , Ratos , Ratos Wistar
14.
J Biochem Mol Toxicol ; 35(6): 1-12, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33724628

RESUMO

The current study was done to measure the role of piperlongumine (PL) on hyperglycemia interrelated oxidative stress-mediated inflammation and apoptosis, inflammatory stress, and the diabetic insulin receptor substrate 2 (IRS2), protein kinase B (AKT), and glucose transporter 2 (GLUT-2)/4 signaling pathway in streptozotocin (STZ)-persuaded diabetic animals. Diabetes was initiated in experimental animals via a single dose intraperitoneal inoculation of STZ. Diabetic rats revealed an augmented blood-glucose level with drastically diminished plasma-insulin status. The functions of antioxidants were diminished with enhanced lipid peroxidation, conjugated dienes, and protein carbonyls noticed in diabetic rats'  plasma and pancreatic tissues. An elevation of nuclear factor-κB (NF-κB), tumor necrosis factor-α, and interleukin-6 proteins was noticed in pancreatic tissues as well as IRS2, AKT, GLUT-2, and GLUT-4 marker expressions were quantified in the hepatic tissue of control and diabetic rats. Oral administration of PL for 30 days drastically lowered glucose and higher insulin status in STZ-induced diabetic rats. Impressively, PL oral supplementation considerably restored the antioxidant levels and reduced inflammation and diabetic marker expressions in STZ-diabetic rats. These results were supported through a histological study. Moreover, PL also augmented the level of B-cell lymphoma 2 and diminished the level of Bcl-2-associated X protein in STZ-treated rat's hepatic tissues. Thus, we concluded that PL excellently rescued pancreatic ß cells through mitigating hyperglycemia via dynamic insulin secretion, activating antioxidants, and inhibiting inflammation and apoptosis in the pancreatic and hepatic tissue of diabetic rats.


Assuntos
Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental , Dioxolanos/farmacologia , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Masculino , Ratos , Ratos Wistar
15.
Nutr Res ; 86: 10-22, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33450655

RESUMO

Although human clinical studies have suggested probiotic effects on blood glucose levels, knowledge about molecular mechanisms is still scarce. To test the hypothesis that selected Lactobacillus probiotic bacteria could regulate the activity of enterocyte glucose transporters, we aimed to measure in vitro effects of selected Lactobacillus probiotic bacteria on transcription and translation of intestinal glucose transporters sodium-dependent glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2) as well as transepithelial glucose transport. Lactobacillus plantarum strains (PCS20 and PCS26), Lactobacillus rhamnosus GG (LGG) (ATCC 53103) and Lactobacillus acidophilus (L acidophilus) (ATCC 4356) were co-cultivated with noncarcinogenic porcine enterocytes (CLAB) and human epithelial colorectal adenocarcinoma cells (Caco-2) (ATCC HTB-37). Changes in transcription and expression of SGLT1 and GLUT2 were strain and cell line-specific. In CLAB, LGG was the most potent SGLT1 up-regulator, and PCS26 the most potent down-regulator of GLUT2 transcription, which was also reflected on the protein level. In Caco-2, all tested strains tended to downregulate GLUT2 gene expression, while L acidophilus most effectively reduced GLUT2 protein levels. Statistically significant effect of PCS26 and L acidophilus on GLUT2 molecular and protein levels in CLAB and Caco-2 cell lines, respectively, was also followed by a decreased rate of transepithelial glucose transport. Careful selection of specific Lactobacillus probiotic strains could be used to downregulate glucose absorption in intestinal epithelial cells and thereby could be beneficial as a support treatment of pathologies related to glucose homeostasis.


Assuntos
Enterócitos/metabolismo , Transportador de Glucose Tipo 2/genética , Glucose/metabolismo , Mucosa Intestinal/metabolismo , Lactobacillus , Probióticos , Animais , Transporte Biológico , Células CACO-2 , Linhagem Celular , Regulação para Baixo , Células Epiteliais/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Humanos , Mucosa Intestinal/citologia , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Suínos , Transcrição Genética
16.
Molecules ; 26(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440878

RESUMO

Intestinal transepithelial transport of glucose is mediated by glucose transporters, and affects postprandial blood-glucose levels. This study investigates the effect of wood extracts rich in hydrolyzable tannins (HTs) that originated from sweet chestnut (Castanea sativa Mill.) and oak (Quercus petraea) on the expression of glucose transporter genes and the uptake of glucose and HT constituents in a 3D porcine-small-intestine epithelial-cell model. The viability of epithelial cells CLAB and PSI exposed to different HTs was determined using alamarBlue®. qPCR was used to analyze the gene expression of SGLT1, GLUT2, GLUT4, and POLR2A. Glucose uptake was confirmed by assay, and LC-MS/ MS was used for the analysis of HT bioavailability. HTs at 37 µg/mL were found to adversely affect cell viability and downregulate POLR2A expression. HT from wood extract Tanex at concentrations of 4 µg/mL upregulated the expression of GLUT2, as well as glucose uptake at 1 µg/mL. The time-dependent passage of gallic acid through enterocytes was influenced by all wood extracts compared to gallic acid itself as a control. These results suggest that HTs could modulate glucose uptake and gallic acid passage in the 3D cell model.


Assuntos
Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 4/genética , Taninos Hidrolisáveis/farmacologia , Transportador 1 de Glucose-Sódio/genética , Animais , Linhagem Celular , Fagaceae/química , Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/farmacocinética , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacocinética , Extratos Vegetais/farmacologia , Transportador 1 de Glucose-Sódio/metabolismo , Suínos , Regulação para Cima/efeitos dos fármacos
17.
Food Chem Toxicol ; 149: 112010, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33493636

RESUMO

In this paper, the Caco-2 cell was used to study the glucose absorption regulation and mechanism of kaempferol, caffeic acid and quercetin-3-O-ß-D-galactoside in Lilium lancifolium Thunb in vitro. Glucose oxidase-peroxidase (GOD-POD) method was used to measure glucose consumption in supernatant. The fluorescent D-glucose analog (2-NBDG) was used as a tracer probe to study the changes in the fluorescence intensity of 2-NBDG uptake by Caco-2 cells with an inverted fluorescence microscope. Western blotting and quantitative real-time PCR were used to detect the protein expression and mRNA transcription of SGLT1 and GLUT2. The results showed that caffeic acid and quercetin-3-O-ß-D-galactoside could significantly promote the absorption of glucose by normal Caco-2 cells compared with the control group (P < 0.001). Both caffeic acid and quercetin-3-O-ß-D-galactoside could significantly promote the uptake of glucose tracer 2-NBDG on Caco-2 cells. Caffeic acid and quercetin-3-O-ß-D-galactoside could significantly promote SGLT1 and GLUT2 protein expression levels and mRNA transcription (P < 0.001, P < 0.01, P < 0.05). The mechanism might be related to the promotion of SGLT1 and GLUT2 protein expression levels and mRNA transcription.


Assuntos
Ácidos Cafeicos/farmacologia , Glucose/metabolismo , Quempferóis/farmacologia , Lilium/química , Quercetina/análogos & derivados , Células CACO-2 , Ácidos Cafeicos/química , Sobrevivência Celular , Flores/química , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Humanos , Quempferóis/química , Estrutura Molecular , Quercetina/química , Quercetina/farmacologia , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-32679026

RESUMO

OBJECTIVE: To observe the effects of angiotensin-(1-7) (Ang-(1-7)) on glucose metabolism, islet function and insulin resistance in a rat model of streptozotocin-induced diabetes mellitus (DM) and investigate its mechanism. METHODS: Thirty-four male Wistar rats were randomly divided into 3 groups: control group, which was fed a standard diet, DM group, high-fat diet and injected with streptozotocin, and Ang-(1-7) group receiving an injection of streptozotocin followed by Ang-(1-7) treatment. Blood glucose level, fasting serum Ang II and insulin levels, and homeostasis model assessment of insulin resistance (HOMA-IR) were measured. The pancreases were collected for histological examination, protein and gene expression analysis. RESULTS: Compared with the control group, fasting blood glucose, serum angiotensin II level, and HOMA-IR value increased, while serum insulin level decreased in the DM group. Moreover, islet structure was damaged, ß cells were irregularly arranged, the cytoplasm was loose in the DM group. Expressions of Pancreatic duodenal homeobox-1 (Pdx1), glucose transporter-2 (Glut2) and glucokinase (Gk) were significantly decreased in the DM group compared with the control group. However, the DM-associated changes were dramatically reversed following Ang-(1-7) treatment. CONCLUSION: Ang-(1-7) protects against streptozotocin-induced DM through the improvement of insulin secretion, insulin resistance and islet fibrosis, which is associated with the upregulation of Pdx1, Glut2 and Gk expressions.


Assuntos
Angiotensina I/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Ilhotas Pancreáticas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Angiotensina I/uso terapêutico , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Masculino , Fragmentos de Peptídeos/uso terapêutico , Ratos , Ratos Wistar , Estreptozocina , Transativadores/genética , Transativadores/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
19.
Am J Physiol Gastrointest Liver Physiol ; 320(2): G136-G152, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33237796

RESUMO

Bowel resection accelerates enterocyte proliferation in the remaining gut with suboptimal absorptive and digestive capacity because of a proliferation-associated decrease in functional differentiation markers. We hypothesized that although schlafen 3 (Slfn3) is an important regulator of enterocytic differentiation, Slfn3 would have less impact on bowel resection adaptation, where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers from resected and postoperative bowel of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice. Villus length and crypt depth were increased in WT mice and were even longer in Slfn3KO mice. Mitotic marker, Phh3+, and the proliferation markers Lgr5, FoxL1, and platelet-derived growth factor-α (PDGFRα) were increased after resection in male WT, but this was blunted in male Slfn3KO mice. Cell-shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, whereas Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice, whereas other Slfn family members 1, 2, 5, 8, and 9 had varied expressions that were affected also by sex difference and loss of Slfn3. Differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased, suggesting that enterocytic differentiation effort is incompatible with rapid proliferation shift in intestinal adaptation. Slfn3 absence potentiates villus length and crypt depth, suggesting that the differentiating stimulus of Slfn3 signaling may restrain mucosal mass increase through regulating Villin1, Gelsolin, EGFR, TNFα, and proliferation markers. Therefore, Slfn3 may be an important regulator not only of "normal" enterocytic differentiation but also in response to bowel resection.NEW & NOTEWORTHY The differentiating stimulus of Slfn3 signaling restrains an increase in mucosal mass after bowel resection, and there is a Slfn3-sex interaction regulating differentiation gene expression and intestinal adaptation. This current study highlights the combinatory effects of gender and Slfn3 genotype on the gene expression changes that contribute to the adaptation in intestinal cellular milleu (i.e. villus and crypt structure) which are utilized to compensate for the stress-healing response that the animals display in intestinal adaptation.


Assuntos
Anastomose em-Y de Roux , Proteínas de Ciclo Celular/metabolismo , Animais , Biomarcadores , Proteínas de Ciclo Celular/genética , Proliferação de Células , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Masculino , Camundongos Knockout , RNA/genética , RNA/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores Sexuais , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo
20.
Mol Metab ; 44: 101129, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33246140

RESUMO

OBJECTIVE: The mechanisms behind the efficacy of bariatric surgery (BS) for treating obesity and type 2 diabetes, particularly with respect to the influence of the small bowel, remain poorly understood. In vitro and animal models are suboptimal with respect to their ability to replicate the human intestinal epithelium under conditions induced by obesity. Human enteroids have the potential to accelerate the development of less invasive anti-obesity therapeutics if they can recapitulate the pathophysiology of obesity. Our aim was to determine whether adult stem cell-derived enteroids preserve obesity-characteristic patient-specific abnormalities in carbohydrate absorption and metabolism. METHODS: We established 24 enteroid lines representing 19 lean, overweight, or morbidly obese patients, including post-BS cases. Dietary glucose absorption and gluconeogenesis in enteroids were measured. The expression of carbohydrate transporters and gluconeogenic enzymes was assessed and a pharmacological approach was used to dissect the specific contribution of each transporter or enzyme to carbohydrate absorption and metabolism, respectively. RESULTS: Four phenotypes representing the relationship between patients' BMI and intestinal dietary sugar absorption were found, suggesting that human enteroids retain obese patient phenotype heterogeneity. Intestinal glucose absorption and gluconeogenesis were significantly elevated in enteroids from a cohort of obese patients. Elevated glucose absorption was associated with increased expression of SGLT1 and GLUT2, whereas elevated gluconeogenesis was related to increased expression of GLUT5, PEPCK1, and G6Pase. CONCLUSIONS: Obesity phenotypes preserved in human enteroids provide a mechanistic link to aberrant dietary carbohydrate absorption and metabolism. Enteroids can be used as a preclinical platform to understand the pathophysiology of obesity, study the heterogeneity of obesity mechanisms, and identify novel therapeutics.


Assuntos
Gluconeogênese/fisiologia , Glucose/metabolismo , Intestino Delgado/metabolismo , Obesidade Mórbida/metabolismo , Fenótipo , Células-Tronco/metabolismo , Animais , Cirurgia Bariátrica , Diabetes Mellitus Tipo 2/metabolismo , Carboidratos da Dieta/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 5/metabolismo , Humanos , Absorção Intestinal , Mucosa Intestinal/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo
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