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1.
Obesity (Silver Spring) ; 32(10): 1819-1824, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39238194

RESUMO

OBJECTIVE: The intestinal luminal pH profile varies from stomach to rectum and becomes disrupted in diseases. However, little is known about the pH dependence of incretin hormone secretion, with most in vitro studies having failed to consider this modulatory factor or having used nonphysiological buffer systems. Here, we report the extracellular pH (pHe) dependence of glucagon-like peptide-1 (GLP-1) exocytosis from L cells. METHODS: The pHe dependence of GLP-1 release from GLUTag cells and murine ex vivo primary gut cultures was detected by ELISA. GLP-1 release was measured over a range of pHe under a physiological (CO2/HCO3 -) buffering regime and in its absence (HEPES buffer). The relationship between intracellular pH (pHi) and pHe was mapped given that at least some component of pH sensitivity is likely to be intracellular. RESULTS: GLP-1 secretion from L cells was pHe-dependent and stimulated under alkaline conditions. In the absence of glucose or extracellular calcium, secretion remained at a pHe-insensitive baseline. pHi followed changes in pHe, but the relationship was offset to more alkaline levels in the absence of CO2/HCO3 - buffer and became shallower if [Cl-] changes that normally accompany [HCO3 -] changes were compensated iso-osmotically with gluconate. CONCLUSIONS: GLP-1 secretion is sensitive to pHe and the buffer present. Exploiting this mechanism therapeutically may benefit patients with obesity.


Assuntos
Exocitose , Peptídeo 1 Semelhante ao Glucagon , Concentração de Íons de Hidrogênio , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Animais , Camundongos , Células L , Camundongos Endogâmicos C57BL , Glucose/metabolismo , Cálcio/metabolismo , Bicarbonatos/metabolismo , Mucosa Intestinal/metabolismo , Masculino
2.
World J Microbiol Biotechnol ; 40(7): 199, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38727988

RESUMO

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders.


Assuntos
Akkermansia , Peptídeo 1 Semelhante ao Glucagon , Lactococcus lactis , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Akkermansia/genética , Akkermansia/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Humanos , Células L , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Animais , Camundongos , Linhagem Celular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Viruses ; 16(1)2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38257840

RESUMO

The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK.


Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Fator Regulador 3 de Interferon , Vírus da Hepatite Murina , Tretinoína , Replicação Viral , Animais , Camundongos , Aminoácidos , Antivirais/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Tretinoína/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus da Hepatite Murina/efeitos dos fármacos , Vírus da Hepatite Murina/fisiologia , Células RAW 264.7 , Células L
4.
Int J Mol Sci ; 25(2)2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38256160

RESUMO

Obesity is a risk factor for cardiometabolic diseases. Nutrients stimulate GLP-1 release; however, GLP-1 has a short half-life (<2 min), and only <10-15% reaches the systemic circulation. Human L-cells are localized in the distal ileum and colon, while most nutrients are absorbed in the proximal intestine. We hypothesized that combinations of amino acids and fatty acids potentiate GLP-1 release via different L-cell receptors. GLP-1 secretion was studied in the mouse enteroendocrine STC-1 cells. Cells were pre-incubated with buffer for 1 h and treated with nutrients: alpha-linolenic acid (αLA), phenylalanine (Phe), tryptophan (Trp), and their combinations αLA+Phe and αLA+Trp with dipeptidyl peptidase-4 (DPP4) inhibitor. After 1 h GLP-1 in supernatants was measured and cell lysates taken for qPCR. αLA (12.5 µM) significantly stimulated GLP-1 secretion compared with the control. Phe (6.25-25 mM) and Trp (2.5-10 mM) showed a clear dose response for GLP-1 secretion. The combination of αLA (6.25 µM) and either Phe (12.5 mM) or Trp (5 mM) significantly increased GLP-1 secretion compared with αLA, Phe, or Trp individually. The combination of αLA and Trp upregulated GPR120 expression and potentiated GLP-1 secretion. These nutrient combinations could be used in sustained-delivery formulations to the colon to prolong GLP-1 release for diminishing appetite and preventing obesity.


Assuntos
Aminoácidos , Inibidores da Dipeptidil Peptidase IV , Humanos , Animais , Camundongos , Células L , Triptofano , Antivirais , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Hipoglicemiantes , Nutrientes , Obesidade
5.
Diabetologia ; 67(2): 356-370, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38032369

RESUMO

AIMS/HYPOTHESIS: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo. METHODS: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1-/- mice and immunohistochemistry and gene expression analyses were performed ex vivo. RESULTS: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca2+ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1-/-, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression. CONCLUSIONS/INTERPRETATION: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1.


Assuntos
Diabetes Mellitus Tipo 2 , Derivação Gástrica , Humanos , Animais , Camundongos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Derivação Gástrica/métodos , Células L , Diabetes Mellitus Tipo 2/metabolismo , RNA , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Colesterol , RNA Mensageiro , Glicemia/metabolismo
6.
Diabetes Metab Syndr ; 17(12): 102907, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37980723

RESUMO

AIMS: Glucagon-like peptide 1 (GLP-1) is produced by the L subtype of enteroendocrine cells (EECs). Patients with type 2 diabetes (T2D) exhibit reduced incretin effect, but the pathophysiology and functional change of the L-cells remain unclear. Deciphering the mechanisms of the biological changes in L-cells under T2D conditions may assist in the research of gut-based strategies for T2D therapy. METHODS: We investigated the fasting serum GLP-1 levels and the distribution of colonic L-cells in young and aged participants with and without T2D. Additionally, we established an aged male T2D Wistar rat model subjected to a long-term high-fat and high-fructose (HFHF) diet. Histological investigations and single-cell RNA sequencing (scRNA-seq) analyses were performed to explore the mechanisms underlying functional changes in the colonic EECs. RESULTS: We observed a decline in circulating GLP-1 levels and a reduced number of colonic L-cells in elderly patients with T2D. The mechanisms underlying impaired L-cell formation and disturbed GLP-1 production were revealed using aged T2D rats induced by a long-term HFHF diet. The scRNA-seq results showed that the transcription factors that regulate L-cell commitment, such as Foxa1, were downregulated, and the expression of genes that participate in encoding GLP-1, GLP-1 posttranslational processing, hormone secretion, and nutrient sensing was disturbed. CONCLUSIONS: Taken together, the reduced L-cell lineage commitment and disturbed L-cell functions might be the major cause of the reduced GLP-1 production in aged populations with T2D. Our study provides new insights for identifying novel targets in colonic L-cells for improving endogenous GLP-1 production.


Assuntos
Diabetes Mellitus Tipo 2 , Peptídeo 1 Semelhante ao Glucagon , Humanos , Camundongos , Idoso , Masculino , Ratos , Animais , Células L , Ratos Wistar , Células Enteroendócrinas/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-alfa Nuclear de Hepatócito/farmacologia
7.
Arch Virol ; 168(12): 294, 2023 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-37981594

RESUMO

Congenital tremor (CT) in piglets was first reported in 1922, and although the causative pathogen was unknown for many years, atypical porcine pestivirus (APPV) was recently shown to be the cause. APPV is difficult to isolate, and there have been few reports of APPV isolated from field materials. Here, we successfully isolated infectious particles from a tonsillar emulsion from a CT-affected piglet using the established swine-kidney-derived cell line SK-L. In addition, we produced APPV artificially using these cells. Thus, SK-L cells are useful for both isolation and artificial production of APPV.


Assuntos
Rim , Pestivirus , Animais , Suínos , Camundongos , Células L , Pestivirus/genética , Tonsila Palatina
8.
Arch Razi Inst ; 78(3): 863-871, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-38028862

RESUMO

Infectious bursal disease virus (IBDV) causes a highly contagious disease associated with immunosuppression in young chickens. Production of either egg-based or primary cell-based high-quality vaccines requires time-consuming and costly procedures. To determine a suitable cell line for IBDV replication, L929 cell line was a candidate for the growth kinetics processing of the virus. The L929 cells were proliferated in monolayer, and doubling time was calculated. Replication kinetics an IBDV isolate at the multiplicity of infection 0.1 PFU/cell were determined using virus titration. To adapt IBDV on L929 cells, seven consecutive passages were performed. Virus titer and levels of apoptosis were quantitatively analyzed at each passage. The viral VP2 gene was amplified and sequenced in three passages. An average doubling time of 21 h was estimated for monolayers of L929 cells. Although during early passages, virus growth did not produce a clear cytopathic effect (CPE), an increase in IBDV titers was observed. Serial passages led to the evidence of marked CPEs and an increase in the virus titer in the third passage. During the fourth to seventh passages, consistent CPEs characterized by the formation of granulated and round cells were evident within 24 to 48 hours post-inoculation. The titer of the virus was increased in the third passage onwards to peak in the fourth and constant at 5.9 TCID50 until the end passage. The IBDV replication in connection with DNA fragmentation and FITC, revealed the characteristic picture of apoptosis in a time-dependent manner. We found that the IBDV could easily be adapted to L929 cells, increasing virus yields by about two orders of magnitude. These results indicated that the cell line may be useful in the production of efficient virus particles.


Assuntos
Galinhas , Vírus da Doença Infecciosa da Bursa , Camundongos , Animais , Células L , Linhagem Celular , Sequência de Bases
9.
J Virol ; 97(10): e0082823, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37747236

RESUMO

IMPORTANCE: Reoviruses infect many mammals and are widely studied as a model system for enteric viruses. However, most of our reovirus knowledge comes from laboratory strains maintained on immortalized L929 cells. Herein, we asked whether naturally circulating reoviruses possess the same genetic and phenotypic characteristics as laboratory strains. Naturally circulating reoviruses obtained from sewage were extremely diverse genetically. Moreover, sewage reoviruses exhibited poor fitness on L929 cells and relied heavily on gut proteases for viral uncoating and productive infection compared to laboratory strains. We then examined how naturally circulating reoviruses might adapt to cell culture conditions. Within three passages, virus isolates from the parental sewage population were selected, displaying improved fitness and intracellular uncoating in L929 cells. Remarkably, selected progeny clones were present at 0.01% of the parental population. Altogether, using reovirus as a model, our study demonstrates how the high genetic diversity of naturally circulating viruses results in rapid adaptation to new environments.


Assuntos
Adaptação Fisiológica , Aptidão Genética , Genoma Viral , Interações entre Hospedeiro e Microrganismos , Peptídeo Hidrolases , Reoviridae , Desenvelopamento do Vírus , Animais , Camundongos , Genoma Viral/genética , Genômica , Células L , Peptídeo Hidrolases/metabolismo , Reoviridae/classificação , Reoviridae/genética , Reoviridae/metabolismo , Inoculações Seriadas , Esgotos/virologia
10.
ACS Biomater Sci Eng ; 9(9): 5405-5417, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37638660

RESUMO

Microwave (MW) thermal therapy has been widely used for the treatment of cancer in clinics, but it still shows limited efficacy and a high recurrence rate owing to non-selective heat delivery and thermo-resistance. Regulating glycolysis shows great promise to improve MW thermal therapy since glycolysis plays an important role in thermo-resistance, progression, metabolism, and recurrence. Herein, we developed a delivery nanosystem of shikonin (SK)-loaded and hyaluronic acid (HA)-modified hollow Fe-MOF (HFM), HFM@SK@HA, as an efficient glycolysis-meditated agent to improve the efficacy of MW thermal therapy. The HFM@SK@HA nanosystem shows a high SK loading capacity of 31.7 wt %. The loaded SK can be effectively released from the HFM@SK@HA under the stimulation of an acidic tumor microenvironment and MW irradiation, overcoming the intrinsically low solubility and severe toxicity of SK. We also find that the HFM@SK@HA can not only greatly improve the heating effect of MW in the tumor site but also mediate MW-enhancing dynamic therapy efficiency by catalyzing the endogenous H2O2 to generate reactive oxygen species (ROS). As such, the MW irradiation treatment in the presence of HFM@SK@HA in vitro enables a highly improved anti-tumor efficacy due to the combined effect of released SK and generated ROS on inhibiting glycolysis in cancer cells. Our in vivo experiments show that the tumor inhibition rate is up to 94.75% ± 3.63% with no obvious recurrence during the 2 weeks after treatment. This work provides a new strategy for improving the efficacy of MW thermal therapy.


Assuntos
Ferro , Nanopartículas Metálicas , Estruturas Metalorgânicas , Naftoquinonas , Neoplasias , Estruturas Metalorgânicas/química , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Neoplasias/terapia , Ferro/química , Naftoquinonas/administração & dosagem , Naftoquinonas/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Micro-Ondas/uso terapêutico , Efeito Warburg em Oncologia/efeitos dos fármacos , Células Hep G2 , Linhagem Celular Tumoral , Células L , Feminino , Animais , Camundongos , Humanos
11.
Int J Mol Sci ; 24(10)2023 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-37240166

RESUMO

Thyroid diseases affect a considerable portion of the population, with hypothyroidism being one of the most commonly reported thyroid diseases. Levothyroxine (T4) is clinically used to treat hypothyroidism and suppress thyroid stimulating hormone secretion in other thyroid diseases. In this work, an attempt to improve T4 solubility is made through the synthesis of ionic liquids (ILs) based on this drug. In this context, [Na][T4] was combined with choline [Ch]+ and 1-(2-hydroxyethyl)-3-methylimidazolium [C2OHMiM] + cations in order to prepare the desired T4-ILs. All compounds were characterized by NMR, ATR-FTIR, elemental analysis, and DSC, aiming to check their chemical structure, purities, and thermal properties. The serum, water, and PBS solubilities of the T4-ILs were compared to [Na][T4], as well as the permeability assays. It is important to note an improved adsorption capacity, in which no significant cytotoxicity was observed against L929 cells. [C2OHMiM][T4] seems to be a good alternative to the commercial levothyroxine sodium salt with promising bioavailability.


Assuntos
Líquidos Iônicos , Tiroxina , Tiroxina/síntese química , Tiroxina/farmacocinética , Tiroxina/toxicidade , Disponibilidade Biológica , Solubilidade , Líquidos Iônicos/síntese química , Líquidos Iônicos/farmacocinética , Líquidos Iônicos/toxicidade , Células L , Animais , Camundongos , Permeabilidade
12.
Int J Biol Macromol ; 242(Pt 3): 124885, 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37196725

RESUMO

Although injectable hydrogels with minimally invasive delivery have garnered significant interest, their potential applications have been restricted by a singular property. In this study, a supramolecular hydrogel system with improved adhesion was constructed through host-guest interactions between alginate and polyacrylamide. The maximum tensile adhesion strength between the ß-cyclodextrin and dopamine-grafted alginate/adamantane-grafted polyacrylamide (Alg-ßCD-DA/PAAm-Ad, namely AßCDPA) hydrogels and pigskin reached 19.2 kPa, which was 76 % stronger than the non-catechol-based control hydrogel (ß-cyclodextrin-grafted alginate/adamantane-grafted polyacrylamide, Alg-ßCD/PAAm-Ad). Moreover, the hydrogels demonstrated excellent self-healing, shear-thinning, and injectable properties. The required pressure to extrude the AßCDPA2 hydrogel from a 16G needle at a rate of 2.0 mL/min was 67.4 N. As the polymer concentration and adamantane substitution degree increased, the hydrogels exhibited higher modulus, stronger network structure, and lower swelling ratio and degradation rate. Encapsulating and culturing cells within these hydrogels demonstrated good cytocompatibility. Therefore, this hydrogel can serve as a viscosity extender or bioadhesive, and as a carrier material to deliver encapsulated therapeutic substances into the body through minimally invasive injection methods.


Assuntos
Resinas Acrílicas , Alginatos , Hidrogéis , Adesivos Teciduais , Resistência à Tração , Humanos , Células Endoteliais da Veia Umbilical Humana , Animais , Camundongos , Células L , Linhagem Celular Tumoral
13.
Carbohydr Polym ; 309: 120702, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-36906367

RESUMO

The acidity of high tannic acid (TA) content solution can destroy the structure of protein, such as gelatin (G). This causes a big challenge to introduce abundant TA into the G-based hydrogels. Here, the G-based hydrogel system with abundant TA as hydrogen bonds provider was constructed by a "protective film" strategy. The protective film around the composite hydrogel was first formed by the chelation of sodium alginate (SA) and Ca2+. Subsequently, abundant TA and Ca2+ were successively introduced into the hydrogel system by immersing method. This strategy effectively protected the structure of the designed hydrogel. After treatment with 0.3 w/v TA and 0.06 w/v Ca2+ solutions, the tensile modulus, elongation at break and toughness of G/SA hydrogel increased about 4-, 2-, and 6-fold, respectively. Besides, G/SA-TA/Ca2+ hydrogels exhibited good water retention, anti-freezing, antioxidant, antibacterial properties and low hemolysis ratio. Cell experiments showed that G/SA-TA/Ca2+ hydrogels possessed good biocompatibility and could promote cell migration. Therefore, G/SA-TA/Ca2+ hydrogels are expected to be used in the field of biomedical engineering. The strategy proposed in this work also provides a new idea for improving the properties of other protein-based hydrogels.


Assuntos
Alginatos , Antibacterianos , Antioxidantes , Materiais Biocompatíveis , Gelatina , Hidrogéis , Gelatina/química , Alginatos/química , Hidrogéis/química , Hidrogéis/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Polifenóis , Resistência à Tração , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Movimento Celular/efeitos dos fármacos , Cálcio/química , Cátions Bivalentes/química , Soluções , Staphylococcus aureus/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Animais , Coelhos , Hemólise/efeitos dos fármacos , Células L , Camundongos
14.
Molecules ; 28(3)2023 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-36770747

RESUMO

Theacrine and strictinin of Yunnan Kucha tea prepared from a mutant variety of wild Pu'er tea plants were two major ingredients responsible for the anti-influenza activity. As the COVID-19 outbreak is still lurking, developing safe and cost-effective therapeutics is an urgent need. This study aimed to evaluate the effects of these tea compounds on the infection of mouse hepatitis virus (MHV), a ß-coronavirus serving as a surrogate for SARS-CoV. Treatment with strictinin (100 µM), but not theacrine, completely eliminated MHV infection, as indicated by a pronounced reduction in plaque formation, nucleocapsid protein expression, and progeny production of MHV. Subsequently, a time-of-drug addition protocol, including pre-, co-, or post-treatment, was exploited to further evaluate the possible mechanism of antiviral activity mediated by strictinin, and remdesivir, a potential drug for the treatment of SARS-CoV-2, was used as a positive control against MHV infection. The results showed that all three treatments of remdesivir (20 µM) completely blocked MHV infection. In contrast, no significant effect on MHV infection was observed when cells were pre-treated with strictinin (100 µM) prior to infection, while significant inhibition of MHV infection was observed when strictinin was introduced upon viral adsorption (co-treatment) and after viral entry (post-treatment). Of note, as compared with the co-treatment group, the inhibitory effect of strictinin was more striking in the post-treatment group. These results indicate that strictinin suppresses MHV infection by multiple mechanisms; it possibly interferes with viral entry and also critical step(s) of viral infection. Evidently, strictinin significantly inhibited MHV infection and might be a suitable ingredient for protection against coronavirus infection.


Assuntos
COVID-19 , Vírus da Hepatite Murina , Camundongos , Animais , Vírus da Hepatite Murina/metabolismo , Células L , SARS-CoV-2 , China , Chá/metabolismo
15.
Int Immunopharmacol ; 113(Pt A): 109405, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36461601

RESUMO

BACKGROUND: Incretin impairment refers to L-cell-derived glucagon-like peptide-1 (GLP-1) deficiency, commonly observed in patients with type 2 diabetes mellitus (T2DM). Promoting the enteroendocrine L-cell population to elevate GLP-1 secretory capacity represents a potential therapeutic strategy for T2DM. It has been established that ginsenoside compound K (CK) could stimulate GLP-1 secretion; however, the underlying mechanisms remain elusive. METHODS: CK was intragastrically administered to male db/db mice for 4 weeks that subsequently underwent oral glucose tolerance testing. Serum samples were collected to measure the GLP-1 secretion, insulin level, inflammatory factors, and bile acid (BA) profiles. Ileum epithelial injury was detected by Hematoxylin and Eosin (H&E) and Masson staining. Gene markers associated with L-cell differentiation were evaluated by RT-PCR, and L-cells were labeled by Gcg via immunofluorescence assays. TGR5 and YAP expression was analyzed by immunoblotting and immunofluorescence assays. RESULTS: Compound K attenuated hyperglycemia and inflammation in db/db mice and upregulated TGR5 expression by increasing lithocholic acid (LCA) and deoxycholic acid (DCA) levels in response to ileum epithelium injury. Meanwhile, fibrosis was alleviated, and the crypt architecture was restored, with increased L-cell abundance and serum GLP-1 levels. The upregulation in genes associated with L-cell differentiation promoted transformation into L-cells. Further mechanistic analyses showed that the effects of CK on the L-cell population required YAP activation, which triggered actin cytoskeleton dynamics. CONCLUSIONS: Our results indicate that TGR5 could modulate the abundance of L-cells to enhance GLP-1 release through YAP-driven intestinal regeneration in db/db mice. Accordingly, CK has huge prospects for application to alleviate incretin impairment in T2DM.


Assuntos
Diabetes Mellitus Tipo 2 , Peptídeo 1 Semelhante ao Glucagon , Masculino , Animais , Camundongos , Células L , Incretinas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Camundongos Endogâmicos
16.
Anal Chem ; 94(27): 9648-9655, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35758929

RESUMO

Type 2 diabetes mellitus is a chronic disease associated with obesity and dysregulated human feeding behavior. The hormone glucagon-like peptide 1 (GLP-1), a critical regulator of body weight, food intake, and blood glucose levels, is secreted by enteroendocrine L-cells. The paucity of L-cells in primary intestinal cell cultures including organoids and monolayers has made assays of GLP-1 secretion from primary human cells challenging. In the current paper, an analytical assay pipeline consisting of an optimized human intestinal tissue construct enriched in L-cells paired with standard antibody-based GLP-1 assays was developed to screen compounds for the development of pharmaceuticals to modulate L-cell signaling. The addition of the serotonin receptor agonist Bimu 8, optimization of R-spondin and Noggin concentrations, and utilization of vasoactive intestinal peptide (VIP) increased the density of L-cells in a primary human colonic epithelial monolayer. Additionally, the incorporation of an air-liquid interface culture format increased the L-cell number so that the signal-to-noise ratio of conventional enzyme-linked immunoassays could be used to monitor GLP-1 secretion in compound screens. To demonstrate the utility of the optimized analytical method, 21 types of beverage sweeteners were screened for their ability to stimulate GLP-1 secretion. Stevioside and cyclamate were found to be the most potent inducers of GLP-1 secretion. This platform enables the quantification of GLP-1 secretion from human primary L-cells and will have broad application in understanding L-cell formation and physiology and will improve the identification of modulators of human feeding behavior.


Assuntos
Diabetes Mellitus Tipo 2 , Peptídeo 1 Semelhante ao Glucagon , Animais , Diabetes Mellitus Tipo 2/metabolismo , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Células L , Camundongos
17.
Arq Bras Cir Dig ; 35: e1651, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35730880

RESUMO

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is a disease of global impact that has led to an increase in comorbidities and mortality in several countries. Immunoexpression of the incretin hormones such as glucagon-like peptide-1 (GLP-1) and peptide YY (3-36) (PYY3-36) can be used as a scorer in the gastrointestinal tract to analyze L-cell activity in response to T2DM treatment. This study aimed to investigate the presence, location, and secretion of L cells in the small intestine of patients undergoing the form of bariatric surgery denominated adaptive gastroenteromentectomy with partial bipartition. METHODS: Immunohistochemical assays, quantitative real-time polymerase chain reaction (qPCR), and Western blot analysis were performed on samples of intestinal mucosa from patients with T2DM in both the preoperative and postoperative periods. RESULTS: All results were consistent and indicated basal expression and secretion of GLP-1 and PYY3-36 incretins by L cells. A greater density of cells was demonstrated in the most distal portions of the small intestine. No significant difference was found between GLP-1 and PYY3-36 expression levels in the preoperative and postoperative periods because of prolonged fasting during which the samples were collected. CONCLUSION: The greater number of L cells in activity implies better peptide signaling, response, and functioning of the neuroendocrine system.


Assuntos
Diabetes Mellitus Tipo 2 , Animais , Diabetes Mellitus Tipo 2/cirurgia , Trato Gastrointestinal/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Humanos , Incretinas/metabolismo , Células L , Camundongos , Mucosa/metabolismo
18.
Pharmacol Res ; 180: 106237, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35487405

RESUMO

The significant growth in type 2 diabetes mellitus (T2DM) prevalence strikes a common threat to the healthcare and economic systems globally. Despite the availability of several anti-hyperglycaemic agents in the market, none can offer T2DM remission. These agents include the prominent incretin-based therapy such as glucagon-like peptide-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 inhibitors that are designed primarily to promote GLP-1R activation. Recent interest in various therapeutically useful gastrointestinal hormones in T2DM and obesity has surged with the realisation that enteroendocrine L-cells modulate the different incretins secretion and glucose homeostasis, reflecting the original incretin definition. Targeting L-cells offers promising opportunities to mimic the benefits of bariatric surgery on glucose homeostasis, bodyweight management, and T2DM remission. Revising the fundamental incretin theory is an essential step for therapeutic development in this area. Therefore, the present review explores enteroendocrine L-cell hormone expression, the associated nutrient-sensing mechanisms, and other physiological characteristics. Subsequently, enteroendocrine L-cell line models and the latest L-cell targeted therapies are reviewed critically in this paper. Bariatric surgery, pharmacotherapy and new paradigm of L-cell targeted pharmaceutical formulation are discussed here, offering both clinician and scientist communities a new common interest to push the scientific boundary in T2DM therapy.


Assuntos
Diabetes Mellitus Tipo 2 , Inibidores da Dipeptidil Peptidase IV , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Hipoglicemiantes/uso terapêutico , Incretinas/uso terapêutico , Células L , Camundongos
19.
Endocrinology ; 163(4)2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35266539

RESUMO

Enterotoxigenic Escherichia coli (ETEC)-derived purified heat-stable enterotoxin b (STb) is responsible for secretory diarrhea in livestock and humans. STb disrupts intestinal fluid homeostasis, epithelial barrier function, and promotes cell death. Glucagon-like peptide-2 (GLP-2) is a potent intestinotrophic hormone secreted by enteroendocrine L cells. GLP-2 enhances crypt cell proliferation, epithelial barrier function, and inhibits enterocyte apoptosis. Whether STb can affect GLP-2 producing L cells remains to be elucidated. First, secreted-His-labeled STb from transformed E coli was collected and purified. When incubated with L-cell models (GLUTag, NCI-H716, and secretin tumor cell line [STC-1]), fluorescent immunocytochemistry revealed STb was internalized and was differentially localized in the cytoplasm and nucleus. Cell viability experiments with neutral red and resazurin revealed that STb was toxic in all but the GLUTag cells. STb stimulated 2-hour GLP-2 secretion in all cell models. Interestingly, GLUTag cells produced the highest amount of GLP-2 when treated with STb, demonstrating an inverse relationship in GLP-2 secretion and cell toxicity. To demonstrate a protective role for GLP-2, GLUTag-conditioned media (rich in GLP-2) blocked STb toxicity in STC-1 cells. Confirming a protective role of GLP-2, teduglutide was able to improve cell viability in cells treated with H2O2. In conclusion, STb interacts with the L cell, stimulates secretion, and may induce toxicity if GLP-2 is not produced at high levels. GLP-2 or receptor agonists have the ability to improve cell viability in response to toxins. These results suggest that GLP-2 secretion can play a protective role during STb intoxication. This work supports future investigation into the use of GLP-2 therapies in enterotoxigenic-related diseases.


Assuntos
Enterotoxinas , Peptídeo 2 Semelhante ao Glucagon , Animais , Células Enteroendócrinas/metabolismo , Enterotoxinas/metabolismo , Enterotoxinas/toxicidade , Escherichia coli/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Temperatura Alta , Humanos , Peróxido de Hidrogênio/metabolismo , Células L , Camundongos
20.
Nat Commun ; 13(1): 879, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35169163

RESUMO

Dysregulation of the balance between pro-inflammatory and anti-inflammatory macrophages has a key function in the pathogenesis of Duchenne muscular dystrophy (DMD), a fatal genetic disease. We postulate that an evolutionarily ancient protective mechanism against infection, known as trained immunity, drives pathological inflammation in DMD. Here we show that bone marrow-derived macrophages from a murine model of DMD (mdx) exhibit cardinal features of trained immunity, consisting of transcriptional hyperresponsiveness associated with metabolic and epigenetic remodeling. The hyperresponsive phenotype is transmissible by bone marrow transplantation to previously healthy mice and persists for up to 11 weeks post-transplant. Mechanistically, training is induced by muscle extract in vitro. The functional and epigenetic changes in bone marrow-derived macrophages from dystrophic mice are TLR4-dependent. Adoptive transfer experiments further support the TLR4-dependence of trained macrophages homing to damaged muscles from the bone marrow. Collectively, this suggests that a TLR4-regulated, memory-like capacity of innate immunity induced at the level of the bone marrow promotes dysregulated inflammation in DMD.


Assuntos
Transplante de Medula Óssea , Imunidade Inata/imunologia , Macrófagos/imunologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Receptor 4 Toll-Like/imunologia , Animais , Células da Medula Óssea/imunologia , Linhagem Celular , Modelos Animais de Doenças , Inflamação/imunologia , Células L , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/imunologia , Distrofia Muscular de Duchenne/imunologia , Extratos de Tecidos/farmacologia , Transcrição Gênica/genética
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