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1.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299151

RESUMO

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Túbulos Renais/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais/efeitos dos fármacos , Receptor PAR-2/genética , Edulcorantes/farmacologia
2.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209449

RESUMO

Interactions between endocrine α and ß cells are critical to their secretory function in vivo. The interactions are highly regulated, although yet to be fully understood. In this study, we aim to assess the impact of α and ß cell co-culture on hormone secretion. Mouse clonal cell lines α-TC6-1 (α cell line) and MIN-6 (ß cell line) were cultured independently or in combination in a medium containing 5.5, 11.1, or 25 mM glucose, respectively. After 72 h, hormone release was measured using insulin and glucagon secretion assays, the cell distribution was visualized by inverted microscopy and an immunocytochemistry assay, and changes in gene expressions were assessed using the RT-PCR technique. The co-culture of the two cell lines caused a decrease in glucagon secretion from α-TC1-6 cells, while no effect on insulin secretion from MIN-6 cells was revealed. Both types of cells were randomly scattered throughout the culture flask, unlike in mice islets in vivo where ß cells cluster in the core and α cells are localized at the periphery. During the α-ß cell co-culture, the gene expression of glucagon (Gcg) decreased significantly. We conclude that islet ß cells suppress glucagon secretion from α cells, apparently via direct cell-to-cell contact, of which the molecular mechanism needs further verification.


Assuntos
Comunicação Celular , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Glucose/farmacologia , Secreção de Insulina , Camundongos
3.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209455

RESUMO

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.


Assuntos
Soluções para Diálise/farmacologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Glucose/farmacologia , Inflamação/patologia , Mesoderma/metabolismo , Neovascularização Fisiológica , Diálise Peritoneal , Biomarcadores/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mesoderma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Molecules ; 26(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208534

RESUMO

Endothelial cell dysfunction is considered to be one of the major causes of vascular complications in diabetes. Polyphenols are known as potent antioxidants that can contribute to the prevention of diabetes. Corn silk has been reported to contain polyphenols and has been used in folk medicine in China for the treatment of diabetes. The present study aims to investigate the potential protective role of the phenolic-rich fraction of corn silk (PRF) against injuries to vascular endothelial cells under high glucose conditions in vitro and in vivo. The protective effect of PRF from high glucose toxicity was investigated using human umbilical vein endothelial cells (HUVECs). The protective effect of PRF was subsequently evaluated by using in vivo methods in streptozotocin (STZ)-induced diabetic rats. Results showed that the PRF significantly reduced the cytotoxicity of glucose by restoring cell viability in a dose-dependent manner. PRF was also able to prevent the histological changes in the aorta of STZ-induced diabetic rats. Results suggested that PRF might have a beneficial effect on diabetic patients and may help to prevent the development and progression of diabetic complications such as diabetic nephropathy and atherosclerosis.


Assuntos
Células Endoteliais/efeitos dos fármacos , Polifenóis/farmacologia , Zea mays/metabolismo , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , China , Citoproteção/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Células Endoteliais/metabolismo , Glucose/efeitos adversos , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Masculino , Extratos Vegetais/farmacologia , Polifenóis/química , Ratos , Ratos Sprague-Dawley , Estreptozocina/farmacologia
5.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199046

RESUMO

Hyperglycemia may contribute to the progression of carcinomas by triggering epithelial-to-mesenchymal transition (EMT). Some proteostasis systems are involved in metastasis; in this paper, we sought to explore the mechanism of Hsp70 chaperone in EMT. We showed that knockdown of Hsp70 reduced cell migration capacity concomitantly with levels of mRNA of the Slug, Snail, and Twist markers of EMT, in colon cancer cells incubated in high glucose medium. Conversely, treatment of cells with Hsp70 inducer U-133 were found to elevate cell motility, along with the other EMT markers. To prove that inhibiting Hsp70 may reduce EMT efficiency, we treated cells with a CL-43 inhibitor of the HSF1 transcription factor, which lowered Hsp70 and HSF1 content in the control and induced EMT in carcinoma cells. Importantly, CL-43 reduced migration capacity, EMT-linked transcription factors, and increased content of epithelial marker E-cadherin in colon cancer cells of three lines, including one derived from a clinical sample. To prove that Hsp70 chaperone should be targeted when inhibiting the EMT pathway, we treated cancer cells with 2-phenylethynesulfonamide (PES) and demonstrated that the compound inhibited substrate-binding capacity of Hsp70. Furthermore, PES suppressed EMT features, cell motility, and expression of specific transcription factors. In conclusion, the Hsp70 chaperone machine efficiently protects mechanisms of the EMT, and the safe inhibitors of the chaperone are needed to hamper metastasis at its initial stage.


Assuntos
Glicemia , Transição Epitelial-Mesenquimal , Glucose/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Biomarcadores , Caderinas/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucose/farmacologia , Humanos , Hiperglicemia/etiologia , Hiperglicemia/metabolismo , Ligação Proteica , Fatores de Transcrição da Família Snail/metabolismo
6.
Biochemistry (Mosc) ; 86(7): 878-886, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34284711

RESUMO

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.


Assuntos
Ditiocarb/farmacologia , Glucose/metabolismo , Ervilhas/efeitos dos fármacos , Epiderme Vegetal/efeitos dos fármacos , Espécies Reativas de Oxigênio , Superóxido Dismutase/antagonistas & inibidores , Trientina/farmacologia , Morte Celular , Quelantes/farmacologia , Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Ervilhas/enzimologia , Ervilhas/metabolismo , Ervilhas/fisiologia , Epiderme Vegetal/enzimologia , Epiderme Vegetal/metabolismo , Epiderme Vegetal/fisiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia
7.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205369

RESUMO

The benefits of natural honeybee products (e.g., honey, royal jelly, beeswax, propolis, beevenom and pollen) to the immune system are remarkable, and many of them are involved in the induction of antibody production, maturation of immune cells and stimulation of the immune system. The type of plants in the geographical area, climatic conditions and production method have a significantly influence on the nutritional quality of honey. However, this variability can influence consumer liking by the sensory attributes of the product. The aim of this work was to compare the most popular honeys from Poland in terms of nutritional value, organoleptic properties and antioxidant activity. In the study, five varieties of honey (honeydew, forest, buckwheat, linden and dandelion) from conventional and organic production methods were tested. The nutritional characteristics of honey samples included acidity, content of water, sugars, vitamin C, HMF and phenolics (total and flavonoids), while honey color, taste, aroma and consistency were investigated in the organoleptic characteristics. The antioxidant activity was determined in water- and ethanol-soluble honey extracts using DPPH and ORAC tests. The results showed that organoleptic and nutritional characteristics of popular Polish honeys differ significantly in relation to plant source and production method. The significant effect of honey variety on the content of HMF, saccharose and phenolics, as well as acidity and antioxidant capacity were noted. The impact of variety and variety × production method interaction was significant in the case of the content of vitamin C, glucose and fructose. A visible difference of buckwheat and forest honeys from other samples was observed. The highest content of total phenolics with antioxidant activity based on the SET mechanism was found in buckwheat honeys, while forest honeys were richer in flavonoids.


Assuntos
Antioxidantes/farmacologia , Abelhas/metabolismo , Mel/análise , Nutrientes/farmacologia , Animais , Ácido Ascórbico/farmacologia , Etanol/química , Fagopyrum , Feminino , Flavonoides/farmacologia , Frutose/farmacologia , Glucose/farmacologia , Sistema Imunitário/efeitos dos fármacos , Masculino , Fenóis/farmacologia , Polônia , Água/química
8.
J Biochem Mol Toxicol ; 35(8): e22826, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34060177

RESUMO

Hyperglycemia is considered a risk factor for the enhancement of local anesthetic-induced neurotoxicity. Transient receptor potential melastatin 7 (TRPM7), a kinase-coupled cation channel, has been implicated in a variety of neuropathological processes, including intracellular calcium disturbance and high glucose-induced neuropathy. In this study, we investigated whether TRPM7-related pathophysiology is involved in bupivacaine-induced neurotoxicity in SH-SY5Y cells and how hyperglycemia acts as a risk factor. For initial neurotoxicity evaluation, it was confirmed that cell damage and apoptosis induced by acute exposure to bupivacaine were dependent on its concentration and glucose preconditioning. High glucose preconditioning facilitated the bupivacaine-induced fast and temporary rise in intracellular free calcium concentration ([Ca2+ ]i ), which was attributed to both calcium influx through TRPM7 and calcium store release. Additionally, bupivacaine was shown to increase TRPM7-like currents, particularly in cells preconditioned with high glucose. Bupivacaine-induced neurotoxicity in hyperglycemia was correlated with extracellular signal-regulated kinase (ERK), but not protein kinase B (AKT) activation. Inhibition of TRPM7 and ERK activity alleviates bupivacaine neurotoxicity. These results suggest that therapeutically targeting TRPM7-related pathophysiological changes could be a potential strategy for treating local anesthetic-induced neurotoxicity exacerbated by hyperglycemia.


Assuntos
Bupivacaína/efeitos adversos , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Glucose/farmacologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Bupivacaína/farmacologia , Linhagem Celular Tumoral , Humanos
9.
Am J Physiol Endocrinol Metab ; 321(1): E176-E189, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34121447

RESUMO

Almost 40% of adults worldwide are classified as overweight or obese. Exercise is a beneficial intervention in obesity, partly due to increases in mitochondrial activity and subsequent increases in nicotinamide adenine dinucleotide (NAD+), an important metabolic cofactor. Recent studies have shown that increasing NAD+ levels through pharmacological supplementation with precursors such as nicotinamide mononucleotide (NMN) improved metabolic health in high-fat-diet (HFD)-fed mice. However, the effects of combined exercise and NMN supplementation are unknown. Thus, here we examined the combined effects of NMN and treadmill exercise in female mice with established obesity after 10 wk of diet. Five-week-old female C57BL/6J mice were exposed to a control diet (n = 16) or HFD. Mice fed a HFD were either untreated (HFD; n = 16), received NMN in drinking water (400 mg/kg; HNMN; n = 16), were exposed to treadmill exercise 6 days/wk (HEx; n = 16), or were exposed to exercise combined with NMN (HNEx; n = 16). Although some metabolic benefits of NMN have been described, at this dose, NMN administration impaired several aspects of exercise-induced benefits in obese mice, including glucose tolerance, glucose-stimulated insulin secretion from islets, and hepatic triglyceride accumulation. HNEx mice also exhibited increased antioxidant and reduced prooxidant gene expression in both islets and muscle, suggesting that altered redox status is associated with the loss of exercise-induced health benefits with NMN cotreatment. Our data show that NMN treatment impedes the beneficial metabolic effects of exercise in a mouse model of diet-induced obesity in association with disturbances in redox metabolism.NEW & NOTEWORTHY NMN dampened exercise-induced benefits on glucose handling in diet-induced obesity. NMN administration alongside treadmill exercise enhanced the ratio of antioxidants to prooxidants. We suggest that NMN administration may not be beneficial when NAD+ levels are replete.


Assuntos
Glucose/metabolismo , Mononucleotídeo de Nicotinamida/administração & dosagem , Obesidade/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Dieta Hiperlipídica , Suplementos Nutricionais , Feminino , Glucose/farmacologia , Intolerância à Glucose/terapia , Secreção de Insulina/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NAD/metabolismo , Mononucleotídeo de Nicotinamida/efeitos adversos , Obesidade/etiologia , Obesidade/terapia , Triglicerídeos/metabolismo
10.
Stem Cells Dev ; 30(15): 758-772, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074129

RESUMO

Cytokine storm is recognized as one of the factors contributing to organ failures and mortality in patients with COVID-19. Due to chronic inflammation, COVID-19 patients with diabetes mellitus (DM) or renal disease (RD) have more severe symptoms and higher mortality. However, the factors that contribute to severe outcomes of COVID-19 patients with DM and RD have received little attention. In an effort to investigate potential treatments for COVID-19, recent research has focused on the immunomodulation functions of mesenchymal stem cells (MSCs). In this study, the correlation between DM and RD and the severity of COVID-19 was examined by a combined approach with a meta-analysis and experimental research. The results of a systematic review and meta-analysis suggested that the odd of mortality in patients with both DM and RD was increased in comparison to those with a single comorbidity. In addition, in the experimental research, the data showed that high glucose and uremic toxins contributed to the induction of cytokine storm in human lung adenocarcinoma epithelial cells (Calu-3 cells) in response to SARS-CoV Peptide Pools. Of note, the incorporation of Wharton's jelly MSC-derived extracellular vesicles (WJ-EVs) into SARS-CoV peptide-induced Calu-3 resulted in a significant decrease in nuclear NF-κB p65 and the downregulation of the cytokine storm under high concentrations of glucose and uremic toxins. This clearly suggests the potential for WJ-EVs to reduce cytokine storm reactions in patients with both chronic inflammation diseases and viral infection.


Assuntos
Síndrome da Liberação de Citocina/prevenção & controle , Vesículas Extracelulares/fisiologia , Células-Tronco Mesenquimais/citologia , SARS-CoV-2/fisiologia , Geleia de Wharton/citologia , Adulto , Idoso , COVID-19/sangue , COVID-19/complicações , COVID-19/metabolismo , COVID-19/terapia , Células Cultivadas , Técnicas de Cocultura , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/metabolismo , Síndrome da Liberação de Citocina/virologia , Citocinas/genética , Citocinas/metabolismo , Complicações do Diabetes/sangue , Complicações do Diabetes/metabolismo , Complicações do Diabetes/terapia , Complicações do Diabetes/virologia , Diabetes Mellitus/sangue , Diabetes Mellitus/metabolismo , Diabetes Mellitus/terapia , Diabetes Mellitus/virologia , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Gravidez , Toxinas Biológicas/metabolismo , Toxinas Biológicas/farmacologia , Cordão Umbilical/citologia , Uremia/sangue , Uremia/complicações , Uremia/metabolismo , Uremia/terapia
11.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069257

RESUMO

Candida albicans is an opportunistic pathogen that induces vulvovaginal candidiasis (VVC), among other diseases. In the vaginal environment, the source of carbon for C. albicans can be either lactic acid or its dissociated form, lactate. It has been shown that lactate, similar to the popular antifungal drug fluconazole (FLC), reduces the expression of the ERG11 gene and hence the amount of ergosterol in the plasma membrane. The Cdr1 transporter that effluxes xenobiotics from C. albicans cells, including FLC, is delocalized from the plasma membrane to a vacuole under the influence of lactate. Despite the overexpression of the CDR1 gene and the increased activity of Cdr1p, C. albicans is fourfold more sensitive to FLC in the presence of lactate than when glucose is the source of carbon. We propose synergistic effects of lactate and FLC in that they block Cdr1 activity by delocalization due to changes in the ergosterol content of the plasma membrane.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Fluconazol/farmacologia , Ácido Láctico/farmacologia , Candida albicans/genética , Candida albicans/metabolismo , Membrana Celular/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Ácido Láctico/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Transporte Proteico/efeitos dos fármacos
12.
J Biochem Mol Toxicol ; 35(7): e22797, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33957017

RESUMO

Diabetic nephropathy (DN) is becoming a research hotspot in recent years because the prevalence is high and the prognosis is poor. Lipid accumulation in podocytes induced by hyperglycemia has been shown to be a driving mechanism underlying the development of DN. However, the mechanism of lipotoxicity remains unclear. Increasing evidence shows that acetyl-CoA carboxylase 2 (ACC2) plays a crucial role in the metabolism of fatty acid, but its effect in podocyte injury of DN is still unclear. In this study, we investigated whether ACC2 could be a therapeutic target of lipid deposition induced by hyperglycemia in the human podocytes. Our results showed that high glucose (HG) triggered significant lipid deposition with a reduced ß-oxidation rate. It also contributed to the downregulation of phosphorylated ACC2 (p-ACC2), which is an inactive form of ACC2. Knockdown of ACC2 by sh-RNA reduced lipid deposition induced by HG. Additionally, ACC2-shRNA restored the expression of glucose transporter 4 (GLUT4) on the cell surface, which was downregulated in HG and normalized in the insulin signaling pathway. We verified that ACC2-shRNA alleviated cell injury, apoptosis, and restored the cytoskeleton disturbed by HG. Mechanistically, SIRT1/PGC-1α is close related to the insulin metabolism pathway. ACC2-shRNA could restore the expression of SIRT1/PGC-1α, which was downregulated in HG. Rescue experiment revealed that inhibition of SIRT1 by EX-527 counteracted the effect of ACC2-shRNA. Taken together, our data suggest that podocyte injury mediated by HG-induced insulin resistance and lipotoxicity could be alleviated by ACC2 inhibition via SIRT1/PGC-1α.


Assuntos
Acetil-CoA Carboxilase/metabolismo , Glucose/farmacologia , Resistência à Insulina , Metabolismo dos Lipídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Podócitos/metabolismo , Sirtuína 1/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/genética , Humanos , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Sirtuína 1/genética
13.
Biochem Biophys Res Commun ; 562: 1-8, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34030039

RESUMO

Protein lysine propionylation (Kpr) modification is a novel post-translational modification (PTM) of prokaryotic cells that was recently discovered; however, it is not clear how this modification regulates bacterial life. In this study, the protein Kpr modification profile in Aeromonas hydrophila was identified by high specificity antibody-based affinity enrichment combined with high resolution LC MS/MS. A total of 98 lysine-propionylated peptides with 59 Kpr proteins were identified, most of which were associated with energy metabolism, transcription and translation processes. To further understand the role of Kpr modified proteins, the K168 site on malate dehydrogenase (MDH) and K608 site on acetyl-coenzyme A synthetase (AcsA) were subjected to site-directed mutation to arginine (R) and glutamine (Q) to simulate deacylation and propionylation, respectively. Subsequent measurement of the enzymatic activity showed that the K168 site of Kpr modification on MDH may negatively regulate the MDH enzymatic activity while also affecting the survival of mdh derivatives when using glucose as the carbon source, whereas Kpr modification of K608 of AcsA does not. Overall, the results of this study indicate that protein Kpr modification plays an important role in bacterial biological functions, especially those involved in the activity of metabolic enzymes.


Assuntos
Aeromonas hydrophila/enzimologia , Regulação Enzimológica da Expressão Gênica , Lisina/metabolismo , Propionatos/metabolismo , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/farmacologia , Glucose/farmacologia , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo
14.
J Biol Chem ; 297(1): 100839, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051232

RESUMO

Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.


Assuntos
Metabolismo Energético/genética , Glucose/farmacologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , Linhagem Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pró-Proteína Convertase 1/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
15.
Am J Physiol Endocrinol Metab ; 320(6): E1138-E1147, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33938236

RESUMO

Insulin and insulin-like growth factor-1 (IGF-1) are fetal hormones critical to establishing normal fetal growth. Experimentally elevated IGF-1 concentrations during late gestation increase fetal weight but lower fetal plasma insulin concentrations. We therefore hypothesized that infusion of an IGF-1 analog for 1 wk into late gestation fetal sheep would attenuate fetal glucose-stimulated insulin secretion (GSIS) and insulin secretion in islets isolated from these fetuses. Late gestation fetal sheep received infusions with IGF-1 LR3 (IGF-1, n = 8), an analog of IGF-1 with low affinity for the IGF binding proteins and high affinity for the IGF-1 receptor, or vehicle control (CON, n = 9). Fetal GSIS was measured with a hyperglycemic clamp (IGF-1, n = 8; CON, n = 7). Fetal islets were isolated, and insulin secretion was assayed in static incubations (IGF-1, n = 8; CON, n = 7). Plasma insulin and glucose concentrations in IGF-1 fetuses were lower compared with CON (P = 0.0135 and P = 0.0012, respectively). During the GSIS study, IGF-1 fetuses had lower insulin secretion compared with CON (P = 0.0453). In vitro, glucose-stimulated insulin secretion remained lower in islets isolated from IGF-1 fetuses (P = 0.0447). In summary, IGF-1 LR3 infusion for 1 wk into fetal sheep lowers insulin concentrations and reduces fetal GSIS. Impaired insulin secretion persists in isolated fetal islets indicating an intrinsic islet defect in insulin release when exposed to IGF-1 LR3 infusion for 1 wk. We speculate this alteration in the insulin/IGF-1 axis contributes to the long-term reduction in ß-cell function in neonates born with elevated IGF-1 concentrations following pregnancies complicated by diabetes or other conditions associated with fetal overgrowth.NEW & NOTEWORTHY After a 1-wk infusion of IGF-1 LR3, late gestation fetal sheep had lower plasma insulin and glucose concentrations, reduced fetal glucose-stimulated insulin secretion, and decreased fractional insulin secretion from isolated fetal islets without differences in pancreatic insulin content.


Assuntos
Feto/efeitos dos fármacos , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Animais , Diabetes Gestacional/metabolismo , Esquema de Medicação , Feminino , Doenças Fetais/metabolismo , Macrossomia Fetal/metabolismo , Macrossomia Fetal/patologia , Feto/metabolismo , Idade Gestacional , Bombas de Infusão , Fator de Crescimento Insulin-Like I/administração & dosagem , Ilhotas Pancreáticas/metabolismo , Pancreatopatias/metabolismo , Gravidez , Ovinos
16.
Commun Biol ; 4(1): 594, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34012065

RESUMO

Type 2 diabetes is characterized by ß and α cell dysfunction. We used phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) to monitor oxidative phosphorylation and glycolysis in living islet cells before and after glucose stimulation. In healthy cells, glucose enhanced oxidative phosphorylation in ß cells and suppressed oxidative phosphorylation in α cells. In Type 2 diabetes, glucose increased glycolysis in ß cells, and only partially suppressed oxidative phosphorylation in α cells. FLIM uncovers key perturbations in glucose induced metabolism in living islet cells and provides a sensitive tool for drug discovery in diabetes.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Imagem Molecular/métodos , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Células Secretoras de Glucagon/efeitos dos fármacos , Glicólise , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Microscopia de Fluorescência , Fosforilação Oxidativa , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos
17.
Transplant Proc ; 53(5): 1736-1743, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33934912

RESUMO

BACKGROUND: Ischemia-driven islet isolation procedure is one of the limiting causes of pancreatic islet transplantation. Ischemia-reperfusion process is associated with endothelium dysfunction and the release of pro-senescent microvesicles. We investigated whether pro-senescent endothelial microvesicles prompt islet senescence and dysfunction in vitro. MATERIAL AND METHODS: Pancreatic islets were isolated from male young rats. Replicative endothelial senescence was induced by serial passaging of primary porcine coronary artery endothelial cells, and microvesicles were isolated either from young passage 1 (P1) or senescent passage 3 (P3) endothelial cells. Islet viability was assessed by fluorescence microscopy, apoptosis by flow cytometry, and Western blot. Function was assessed by insulin secretion and islet senescence markers p53, p21, and p16 by Western blot. Microvesicles were stained by the PKH26 lipid fluorescent probe and their islet integration assessed by microscopy and flow cytometry. RESULTS: Regardless of the passage, half microvesicles were integrated in target islets after 24 hours incubation. Insulin secretion significantly decreased after treatment by senescent microvesicles (P3: 1.7 ± 0.2 vs untreated islet: 2.7 ± 0.2, P < .05) without altering the islet viability (89.47% ± 1.69 vs 93.15% ± 0.97) and with no significant apoptosis. Senescent microvesicles significantly doubled the expression of p53, p21, and p16 (P < .05), whereas young microvesicles had no significant effect. CONCLUSION: Pro-senescent endothelial microvesicles specifically accelerate the senescence of islets and alter their function. These data suggest that islet isolation contributes to endothelial driven islet senescence.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Senescência Celular , Ilhotas Pancreáticas/metabolismo , Animais , Apoptose/genética , Sobrevivência Celular , Micropartículas Derivadas de Células/fisiologia , Células Cultivadas , Senescência Celular/genética , Vasos Coronários/citologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Suínos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
18.
Biochimie ; 187: 67-74, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34022290

RESUMO

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.


Assuntos
Escherichia coli/metabolismo , Glucose/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Estresse Fisiológico/genética
19.
Int J Nanomedicine ; 16: 3021-3040, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33935497

RESUMO

Purpose: Silver nanoparticles (AgNPs) are one of the most commonly investigated nanomaterials, especially due to their biomedical applications. However, their excellent cytotoxic and antimicrobial activity is often compromised in biological media due to nanoparticle aggregation. In this work, the aggregation behavior and the related biological activity of three different samples of citrate capped silver nanoparticles, with mean diameters of 10, 20, and 50 nm, respectively, were examined. Methods: Following nanoparticle synthesis and characterization with transmission electron microscopy, their aggregation behavior under various pH values, NaCl, glucose, and glutamine concentrations, furthermore in cell culture medium components such as Dulbecco's Modified Eagle's Medium and fetal bovine serum, was assessed through dynamic light scattering and ultraviolet-visible spectroscopy. Results: The results indicated that acidic pH and physiological electrolyte content universally induce micron-scale aggregation, which can be mediated by biomolecular corona formation. Remarkably, larger particles demonstrated higher resistance against external influences than smaller counterparts. In vitro cytotoxicity and antimicrobial assays were performed by treating cells with nanoparticulate aggregates in differing stages of aggregation. Conclusion: Our results revealed a profound association between colloidal stability and toxicity of AgNPs, as extreme aggregation led to the complete loss of biological activity. The higher degree of aggregation resistance observed for larger particles had a significant impact on the in vitro toxicity, since such samples retained more of their activity against microbes and mammalian cells. These findings lead to the conclusion that aiming for the smallest possible nanoparticles might not be the best course of action, despite the general standpoint of the relevant literature.


Assuntos
Nanopartículas Metálicas/química , Tamanho da Partícula , Prata/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ácido Cítrico/química , Meios de Cultura/química , Difusão Dinâmica da Luz , Fungos/efeitos dos fármacos , Glucose/farmacologia , Glutamina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Cloreto de Sódio/química
20.
Am J Gastroenterol ; 116(4): 780-787, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33982948

RESUMO

INTRODUCTION: The North American Consensus guidelines for glucose breath testing (GBT) for small intestinal bacterial overgrowth (SIBO) incorporated changes in glucose dosing and diagnostic cutoffs. We compared GBT positivity based on hydrogen and methane excretion and quantified symptoms during performance of the North American vs older modified Rome Consensus protocols. METHODS: GBT was performed using the North American protocol (75 g glucose, cutoffs >20 parts per million [ppm] hydrogen increase after glucose and >10 ppm methane anytime) in 3,102 patients vs modified Rome protocol (50 g glucose, >12 ppm hydrogen and methane increases after glucose) in 3,193 patients with suspected SIBO. RESULTS: Positive GBT were more common with the North American vs modified Rome protocol (39.5% vs 29.7%, P < 0.001). Overall percentages with GBT positivity using methane criteria were greater and hydrogen criteria lower with the North American protocol (P < 0.001). Peak methane levels were higher for the North American protocol (P < 0.001). Times to peak hydrogen and methane production were not different between protocols. With the North American protocol, gastrointestinal and extraintestinal symptoms were more prevalent after glucose with both positive and negative GBT (P < 0.04) and greater numbers of symptoms (P < 0.001) were reported. DISCUSSION: GBT performed using the North American Consensus protocol was more often positive for SIBO vs the modified Rome protocol because of more prevalent positive methane excretion. Symptoms during testing were greater with the North American protocol. Implications of these observations on determining breath test positivity and antibiotic decisions for SIBO await future prospective testing.


Assuntos
Infecções Bacterianas/diagnóstico , Testes Respiratórios/métodos , Consenso , Glucose/farmacologia , Enteropatias/diagnóstico , Intestino Delgado/microbiologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Feminino , Humanos , Hidrogênio/análise , Enteropatias/metabolismo , Enteropatias/microbiologia , Masculino , Metano/análise , Pessoa de Meia-Idade , Estudos Retrospectivos
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