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AIMS/HYPOTHESIS: Mutations in Isl1, encoding the insulin enhancer-binding protein islet-1 (ISL1), may contribute to attenuated insulin secretion in type 2 diabetes mellitus. We made an Isl1E283D mouse model to investigate the disease-causing mechanism of diabetes mellitus. METHODS: The ISL1E283D mutation (c. 849A>T) was identified by whole exome sequencing on an early-onset type 2 diabetes family and then the Isl1E283D knockin (KI) mouse model was created and an IPGTT and IPITT were conducted. Glucose-stimulated insulin secretion (GSIS), expression of Ins2 and other ISL1 target genes and interacting proteins were evaluated in isolated pancreas islets. Transcriptional activity of Isl1E283D was evaluated by cell-based luciferase reporter assay and electrophoretic mobility shift assay, and the expression levels of Ins2 driven by Isl1 wild-type (Isl1WT) and Isl1E283D mutation in rat INS-1 cells were determined by RT-PCR and western blotting. RESULTS: Impaired GSIS and elevated glucose level were observed in Isl1E283D KI mice while expression of Ins2 and other ISL1 target genes Mafa, Pdx1, Slc2a2 and the interacting protein NeuroD1 were downregulated in isolated islets. Transcriptional activity of the Isl1E283D mutation for Ins2 was reduced by 59.3%, and resulted in a marked downregulation of Ins2 expression when it was overexpressed in INS-1 cells, while overexpression of Isl1WT led to an upregulation of Ins2 expression. CONCLUSIONS/INTERPRETATION: Isl1E283D mutation reduces insulin expression and secretion by regulating insulin and other target genes, as well as its interacting proteins such as NeuroD1, leading to the development of glucose intolerance in the KI mice, which recapitulated the human diabetic phenotype. This study identified and highlighted the Isl1E283D mutation as a novel causative factor for type 2 diabetes, and suggested that targeting transcription factor ISL1 could offer an innovative avenue for the precise treatment of human type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Proteínas con Homeodominio LIM , Mutación Missense , Factores de Transcripción , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Humanos , Masculino , Insulina/metabolismo , Femenino , Ratas , Secreción de Insulina/genética , Islotes Pancreáticos/metabolismoRESUMEN
Precise differentiation of glucokinase (GCK) monogenic diabetes from gestational diabetes mellitus (GDM) is critical for accurate management of the pregnancy outcome. We screened GCK-MODY complicating pregnancies in Chinese GDM patients, explored the pathogenesis of novel GCK mutations, and evaluated the patients' pregnancy outcome and management. The GCK gene from 411 GDM patients was screened with PCR-direct sequencing and multiplex ligation-dependent probe amplification (MLPA) and 15 GCK mutations were identified. We also retrospectively analyzed a total of 65 pregnancies from 21 GCK-MODY families, wherein 41 were from 15 maternal families and 24 were from six paternal families. Bioinformatic analysis and biochemical functional study were conducted to identify novel GCK mutations. In total, we identified 21 GCK mutations: 15 from the 411 GDM patients and six from 24 fathers. Of th Asp78Asn (GAC â AAC), Met87Arg (ATG â AGG), Leu451Val (CTT â GTT), Leu451Pro (CTG â CCG) and 1019 + 20G > A e mutations, five, i.e., were novel and deleterious, with markedly decreased enzyme activity and thermal stability. The unaffected offspring of GCK mutation-affected mothers were heavier than affected offspring (p < 0.001). Of 21 insulin-treated affected mothers, 10 had maternal hypoglycemia (47.6%) and seven had perinatal complications (33.3%), and the affected offspring of the insulin-treated affected mothers had significantly lower birth weights than that of the 20 diet-control affected mothers (p = 0.031). In this study, the prevalence of GCK-MODY complicating pregnancy in Chinese GDM patients was 3.6% (15/411). The defective GCK may contribute to the hyperglycemia in GCK-MODY. Insulin therapy is not beneficial for GCK-MODY complicating pregnancy and therefore should not be recommended.
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Diabetes Mellitus Tipo 2 , Diabetes Gestacional , Embarazo en Diabéticas , China , Diabetes Mellitus Tipo 2/genética , Diabetes Gestacional/genética , Femenino , Glucoquinasa/genética , Humanos , Insulina/genética , Mutación , Embarazo , Resultado del Embarazo , Embarazo en Diabéticas/epidemiología , Embarazo en Diabéticas/genética , Embarazo en Diabéticas/terapia , Estudios RetrospectivosRESUMEN
TSPAN8 mediates signal transduction from extracellular cues and regulates cell development, activation, growth, and motility. However, whether TSPAN8 is involved in the progression of diabetic nephropathy (DN) remains unclear. This study aimed to explore the potential functional roles of TSPAN8 in regulating autophagy and apoptosis of HK-2 cells induced by high glucose (HG). RT-PCR and western blot analysis (WB) were employed to detect TSPAN8 levels in the blood samples of DN patients as well as in HG-induced HK-2 cells. Cell proliferation of HK-2 cells was examined by CCK-8 assay, and apoptosis was analyzed by flow cytometry. The functional role of TSPAN8 was evaluated by the transfection of TSPAN8 expression plasmid. Results showed that TSPAN8 level was significantly reduced in the blood samples of DN patients and HG-induced HK-2 cell lines. TSPAN8 overexpression rescued HG-induced apoptosis in HK-2 cells. TSPAN8 could form a complex with Rictor and mTORC2. TSPAN8 overexpression suppressed HG-induced autophagy in HK-2 cells, which was dependent on mTOR activity. In conclusion, the present study showed that TSPAN8 mitigates HG-induced autophagy and apoptosis in HK-2 cells, which may serve as candidate target for DN treatment.
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Nefropatías Diabéticas , Diana Mecanicista del Complejo 2 de la Rapamicina , MicroARNs , Tetraspaninas , Apoptosis , Autofagia , Nefropatías Diabéticas/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , MicroARNs/metabolismo , Tetraspaninas/metabolismoRESUMEN
BACKGROUND: Diabetic nephropathy (DN) is one of the most common and serious complications of diabetes, which can lead to renal failure and fatality. miRNAs are an important class of endogenous non-coding RNAs implicated in a wide range of biological processes and pathological conditions. This study aims to investigate the potential functional roles of miR-543 in DN and its underlying mechanisms. METHODS: qRT-PCR was performed to detect the expression levels of miR-543 and TSPAN8 in kidney tissues of mice with DN. Western blot (WB) was used to measure the protein levels. CCK8 assay was employed to evaluate the proliferation of HK2 cells. Dual luciferase reporter assay was conducted to verify the functional interaction between miR-543 and TSpan8. RESULTS: The downregulation of miR-543 and upregulation of TSPAN8 were observed in kidney tissues of mice with DN. miR-543 mimic significantly decreased cell proliferation and autophagy in high-glucose (HG)-induced HK2 cells, and promoted cell fibrosis. We further identified a putative binding site between miR-543 and TSPAN8, which was validated by Dual luciferase reporter assay. The treatment of miR-543 mimic and miR-543 inhibitor could reduce or increase TSPAN8 protein level respectively. We further showed that the overexpression of TSPAN8 could attenuate HG-induced cell injury by reducing fibrosis and increase autophagy. The effects of miR-543 mimic in proliferation, fibrosis, and autophagy were rescued by TSPAN8 overexpression. CONCLUSIONS: Our study indicate that miR-543 mediates high-glucose induced DN via targeting TSPAN8. Interfering miR-543/TSPAN8 axis could serve as potential approach to ameliorate DN.
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Diabetes Mellitus , Nefropatías Diabéticas , MicroARNs , Animales , Autofagia/genética , Nefropatías Diabéticas/patología , Femenino , Fibrosis , Glucosa/toxicidad , Humanos , Masculino , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Tetraspaninas/genéticaRESUMEN
BACKGROUND: Diabetes mellitus is often associated with microvascular and macrovascular lesions, and hyperglycemia-induced vascular endothelial cell damage is a key factor. METHODS: We investigated long non-coding RNAs (lncRNAs) and mRNAs that are affected by hyperglycemia-induced damage using human umbilical vein endothelial cells (HUVECs) as a model. HUVECs were cultured under high (25 mmol/L) or normal (5 mmol/L) glucose conditions for 6 d, and then lncRNAs and protein-coding transcripts were profiled by RNA-seq. RESULT: Among 40,379 lncRNAs screened, 214 were upregulated (log2 [fold-change] > 1, FDR < 0.05) and 197 were downregulated (log2 [fold-change] < - 1, FDR < 0.05) in response to high-glucose. Furthermore, among 28,431 protein-coding genes screened, 778 were upregulated and 998 were downregulated. A total of 945 lncRNA/mRNA pairs were identified, including 126 differentially expressed lncRNAs predicted to target 201 mRNAs, among which 26 were cis-regulatory interactions. The corresponding lncRNA-mRNA network was composed of 354 lncRNA nodes, 1167 mRNA nodes and 9735 edges. Dozens of lncRNAs with high degree may play important roles in high-glucose-induced HUVEC damage, including ENST00000600527, NONHSAT037576.2, NONHSAT135706.2, ENST00000602127, NONHSAT200243.1, NONHSAT217282.1, NONHSAT176260.1, NONHSAT199075.1, NONHSAT067063.2, NONHSAT058417.2. CONCLUSION: These observations may provide novel insights into the regulatory molecules and pathways of hyperglycemia-related endothelial dysfunction in diabetes-associated vascular disease.
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Biomarcadores/análisis , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Hiperglucemia/genética , ARN Largo no Codificante/genética , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Hiperglucemia/inducido químicamente , Hiperglucemia/patología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Whether the Arg913Gln variation (rs11643718, G/A) of SLC12A3 contributes to diabetic nephropathy (DN) remains controversial. We undertook a case-control study to evaluate the association of the SLC12A3-Arg913Gln variation with the risk of end-stage renal disease (ESRD) in Chinese type 2 diabetes mellitus (T2DM) patients undergoing hemodialysis, and analyzed the genotype-phenotype interaction. Unrelated Chinese T2DM patients (n = 372) with diabetic retinopathy were classified into the non-DN (control) group (n = 151; duration of T2DM >15 years, no signs of renal involvement) and the DN-ESRD group (n = 221; ESRD due to T2DM, receiving hemodialysis). Polymerase chain reaction-direct sequencing was used to genotype the SLC12A3-Arg913Gln variation for all participants. The frequency of the GA+AA genotype in the DN-ESRD group was significantly higher than that of the non-DN group (23.1 vs. 9.9%; adjusted OR 2.2 (95% CI 1.3-4.5), P = 0.019). In the non-DN group, GA+AA carriers had a significantly higher urinary albumin excretion rate (UAER) and diastolic blood pressure compared with GG carriers (both P < 0.05). The SLC12A3-Arg913Gln variation may be associated with increased blood pressure and UAER and, therefore, could be used to predict the development and progression of DN-ESRD in Chinese T2DM patients undergoing hemodialysis.
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Diabetes Mellitus Tipo 2/genética , Nefropatías Diabéticas/genética , Fallo Renal Crónico/genética , Mutación Missense , Anciano , Sustitución de Aminoácidos , Pueblo Asiatico/genética , China , Diabetes Mellitus Tipo 2/terapia , Nefropatías Diabéticas/terapia , Genotipo , Humanos , Fallo Renal Crónico/terapia , Persona de Mediana Edad , Diálisis Renal , Miembro 3 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
Conflicting associations between define (KCNJ11) variations and susceptibility to late-onset (>40 years old) type 2 diabetes mellitus (T2DM) have been reported in different ethnic groups. We investigated whether the E23K (GâA, rs5219) or A190A (CâT, rs5218) variations in KCNJ11 are associated with early-onset T2DM and blood pressure in the Chinese population. Case-control study of 175 unrelated Chinese patients with early-onset T2DM (age of onset <40 years old) who receive (ins+, n = 57) or do not receive insulin (ins-, n = 118), and 182 non-diabetic control subjects. PCR-direct sequencing was performed to genotype E23K and A190A; the genotypic frequencies and associations with clinical characteristics were analyzed. The genotypic frequencies of E23K-GA+AA were higher and A190A-TT was lower in the early-onset T2DM group, especially the T2D-ins+ group, compared to the non-diabetic control group (p < 0.01 or 0.05, respectively). In non-diabetic subjects, E23K-AA carriers had significantly higher 2 h plasma glucose and lower 2 h insulin than E23K-GG carriers (both p < 0.05). A190A-TT or E23K-GG carriers had higher systolic blood pressure (SBP) than CC or AA carriers in the non-diabetic control and T2DM groups (both p < 0.05). In the T2DM ins+ group, E23K-AA carriers had lower onset age and duration of diabetes and higher BMI than GG carriers, and A190A-TT carriers had higher SBP than CC carriers (all p < 0.05). The E23K-GA or AA genotypes may increase the susceptibility to early-onset T2DM, while A190A-TT may protect against early-onset T2DM. On the other hand the A190A-TT or E23K-GG genotypes may increase the risk of hypertension in the Chinese population.
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Diabetes Mellitus Tipo 2/genética , Estudios de Asociación Genética , Hipertensión/genética , Canales de Potasio de Rectificación Interna/genética , Adulto , Edad de Inicio , Anciano , Presión Sanguínea/genética , China , Diabetes Mellitus Tipo 2/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Hipertensión/patología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
The preproghrelin (GHRL) Leu72Met polymorphism (rs 696217) is associated with obesity, reduced glucose-induced insulin secretion in healthy or diabetic subjects, and reduced serum creatinine (Scr) levels in type 2 diabetes. We evaluated the association of the Leu72Met polymorphism with measures of insulin sensitivity in non-diabetic control individuals and type 2 diabetics, and whether this variation contributes to the development of diabetic nephropathy (DN) in type 2 diabetes. A case-control study was performed of 291 non-diabetic control subjects and 466 patients with type 2 diabetes, of whom 238 had DN with overt albuminuria (DN group; albuminuric excretion rate [AER] ≥ 300 mg/24 h) and 228 did not have DN, but had diabetes for more than 10 years (non-DN group). Genotyping was performed using a TaqMan PCR assay. The Leu/Leu, Leu/Met, and Met/Met genotype frequencies were significantly different between the non-DN and DN groups (p = 0.011). The frequency of the variant genotypes (Leu/Met, Met/Met) was significantly lower in the DN group than the non-DN group (23.5 vs. 36.0 %, p = 0.003). Met/Met non-diabetic control subjects had lower BMI and Scr levels and higher eGFR level than Leu/Leu or Leu/Met individuals (p < 0.05). Leu/Met and Met/Met type 2 diabetics had significantly lower AER and Scr levels and higher eGFR level than Leu/Leu type 2 diabetics (all p < 0.001). The GHRL Leu72Met polymorphism may help to maintain normal renal function and may protect against the development of DN by reducing albuminuria and improving renal function in Chinese patients with type 2 diabetes.
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Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/genética , Ghrelina/genética , Polimorfismo de Nucleótido Simple , Precursores de Proteínas/genética , Anciano , Sustitución de Aminoácidos , Estudios de Casos y Controles , China , Diabetes Mellitus Tipo 2/genética , Resistencia a la Enfermedad/genética , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Pseudohypoparathyroidism (PHP) is a rare genetic disease characterized by hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) in serum. Here, we report a case of a patient with pseudohypoparathyroidism type IB (PHPIB) and subclinical hypothyroidism, analyze the clinical and genetic data of his family members, review the relevant literature, and classify and discuss the pathogenesis and clinical characteristics of each subtype. Finally, we discuss the treatment approach to improve clinicians' understanding of the disease.
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AIMS/HYPOTHESIS: More than 90% of Chinese familial early-onset type 2 diabetes mellitus is genetically unexplained. To investigate the molecular aetiology, we identified and characterised whether mutations in the KCNJ11 gene are responsible for these families. METHODS: KCNJ11 mutations were screened for 96 familial early-onset type 2 diabetic probands and their families. Functional significance of the identified mutations was confirmed by physiological analysis, molecular modelling and population survey. RESULTS: Three novel KCNJ11 mutations, R27H, R192H and S116F117del, were identified in three families with early-onset type 2 diabetes mellitus. Mutated KCNJ11 with R27H or R192H markedly reduced ATP sensitivity (E23K>R27H>C42R>R192H>R201H), but no ATP-sensitive potassium channel currents were detected in the loss-of-function S116F117del channel in vitro. Molecular modelling indicated that R192H had a larger effect on the channel ATP-binding pocket than R27H, which may qualitatively explain why the ATP sensitivity of the R192H mutation is seven times less than R27H. The shape of the S116F117del channel may be compressed, which may explain why the mutated channel had no currents. Discontinuation of insulin and implementation of sulfonylureas for R27H or R192H carriers and continuation/switch to insulin therapy for S116F117del carriers resulted in good glycaemic control. CONCLUSIONS/INTERPRETATION: Our results suggest that genetic diagnosis for the KCNJ11 mutations in familial early-onset type 2 diabetes mellitus may help in understanding the molecular aetiology and in providing more personalised treatment for these specific forms of diabetes in Chinese and other Asian patients.
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Pueblo Asiatico/genética , Diabetes Mellitus Tipo 2/genética , Canales KATP/genética , Mutación Missense , Canales de Potasio de Rectificación Interna/genética , Adolescente , Adulto , Anciano , Sustitución de Aminoácidos , Niño , Análisis Mutacional de ADN , Diabetes Mellitus Tipo 2/sangre , Femenino , Hemoglobina Glucada/genética , Humanos , Canales KATP/sangre , Masculino , Persona de Mediana Edad , LinajeRESUMEN
Diabetic nephropathy (DN) represents a major diabetes-related complication, which could undermine renal function. CircCOL1A2 has been previously reported to show abnormal expression during DN. However, its functional role in the progression of DN, as well as the potential molecular mechanisms, remains unclear. The present work examined the expression of circCOL1A2 in the plasma of DN patients, and employed high glucose (HG)-challenged HK-2 cells as the in vitro cell model of hyperglycemia (HG)-induced DN. CircCOL1A2 was silenced using siRNA in HK-2 cells to clarify the functional engagement of circCOL1A2 in HG-induced DN. We examined the roles of circCOL1A2 in regulating oxidative stress by measuring reactive oxygen species (ROS), lipid peroxidation, and superoxide dismutase (SOD) levels. Besides, the effects of circCOL1A2 silencing on pyroptosis were investigated by RT-qPCR, western blot (WB), and ELISA assays. StarBase (version 2.0) was used to identify the downstream effector of circCOL1A2, and their interactions were further verified through dual-luciferase reporter analysis, RNA pull-down assays, and RNA immunoprecipitation (RIP) assay. CircCOL1A2 was highly expressed in DN patients and HG-induced HK-2 cells. Knocking down circCOL1A2 alleviated oxidative stress and pyroptosis upon HG treatment. In addition, we demonstrated that circCOL1A2 knockdown could promote miR-424-5p expression while inhibiting Serum/Glucocorticoid Regulated Kinase 1 (SGK1) level. Furthermore, miR-424-5p inhibitor or SGK1 overexpression impaired the effects of circCOL1A2 knockdown on HG-induced oxidative stress and pyroptosis. Hence, our results demonstrated that the circCOL1A2 mediates HG-exposed pyroptosis and oxidative stress through modulating miR-424-5p/SGK1 axis in diabetic nephropathy, indicating that silencing circCOL1A2 is a potential intervention strategy for DN management.
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Diabetes Mellitus , Nefropatías Diabéticas , MicroARNs , Humanos , Colágeno Tipo I , Nefropatías Diabéticas/genética , Glucocorticoides , Glucosa/toxicidad , MicroARNs/genética , Estrés Oxidativo , Piroptosis/genética , ARN Circular/genéticaRESUMEN
Diabetic nephropathy (DN) is a common clinical syndrome in diabetic patients. Functional characterization of non-coding (ncRNAs) involved in the progression of DN can provide insights into the diagnosis and therapeutic management of DN. Human kidney proximal tubular epithelial cells (HK-2) were challenged by high glucose (HG, 50 mM) as a cell model of DN. The expression level of long non-coding RNA (lncRNA) ZFAS1 was quantified by qRT-PCR. The proteins and cytokines related to fibrosis and scortosis in DN (NLRP3, GSDMD-N, IL-1ß and Caspase 1, fibronectin, collagen I, collagen III, IL-1ß, and IL-18) were examined by western blot or ELISA. RNA precipitation and luciferase reporter activity experiments were conducted to assess the molecular associations. ZFAS1 and SGK1 were highly induced in HK-2 cells challenged with HG, while miR-525-5p downregulated upon HG treatment. ZFAS1 knockdown attenuated HG-induced fibrosis and scortosis in HK-2 cells by reducing the levels of NLRP3, GSDMD-N, Caspase 1, fibronectin, collagen I/III, IL-1ß, and IL-18. Mechanically, ZFAS1 knockdown protected HK-2 cells from HG-induced injury by upregulating miR-525-5p and repressing SGK1 expression. Overall, our results suggest that knocking down ZFAS1 may be formulated as a protective strategy in ameliorating DN progression through regulating miR-525-5p/SGK1 pathway. Targeting ZFAS1 could be further explored as a potential approach for the management of DN.
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The aims of the present study were to evaluate the expression of prolyl 4-hydroxylase subunit alpha 3 (P4HA3) in adipocytes and adipose tissue and to explore its effect on obesity and type 2 diabetes mellitus (T2DM). We initially demonstrated that P4HA3 was significantly upregulated in the subcutaneous adipose tissue of obesity and T2DM patients, and its functional roles in adipocyte differentiation and insulin resistance were investigated using in vitro and in vivo models. The knockdown of P4HA3 inhibited adipocyte differentiation and improved insulin resistance in 3T3-L1 cells. In C57BL/6J db/db mice fed with a high fat diet (HFD), silencing P4HA3 significantly decreased fasting blood glucose and triglycerides (TG) levels, with concomitant decrease of body weight and adipose tissue weight. Further analysis showed that P4HA3 knockdown was correlated with the augmented IRS-1/PI3K/Akt/FoxO1 signaling pathway in the adipose and hepatic tissues of obese mice, which could improve hepatic glucose homeostasis and steatosis of mice. Together, our study suggested that the dysregulation of P4HA3 may contribute to the development of obesity and T2DM.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Procolágeno-Prolina Dioxigenasa , Animales , Ratones , Dieta Alta en Grasa , Ratones Endogámicos C57BL , Obesidad/metabolismo , Fosfatidilinositol 3-Quinasas , Procolágeno-Prolina Dioxigenasa/metabolismoRESUMEN
Objective: The aim of this study was to analyze the percentages of T helper 17 cells (Th17s) and T regulatory cells (Tregs) in autoimmune Hashimoto's thyroiditis (HT), and the expression of the checkpoint molecules programmed death receptor 1/programmed death ligand 1 (PD-1/PD-L1) on these cells. Methods: This is a case-control study involving 53 initially diagnosed HT patients (HT group) and 21 normal controls (NC group). The peripheral blood mononuclear cells from the individuals of the two groups were isolated and restimulated ex vivo; the percentage of Th17s, Tregs, PD-1+ Th17s, PD-L1+ Th17s, PD-1+ Tregs, and PD-L1+ Tregs was assessed by flow cytometric analysis. Results: (1) The percentage of Th17s in the peripheral blood of the HT group was significantly higher than that of the NC group [(6.38 ± 1.32)% versus (3.12 ± 0.66)%; t = 14.110, P < 0.001], while the percentage of peripheral blood Tregs was significantly lower [(3.82 ± 1.48)% versus (5.61 ± 1.60)%; t = -4.599, P < 0.001]. (2) HT patients' Th17s expressed PD-1 at a significantly lower frequency than their counterparts in the NC [(6.46 ± 2.77)% versus (18.51 ± 3.96)%; t = -14.842, P < 0.001], while no difference was observed for PD-L1 between the two groups. (3) In contrast, both PD-1 and PD-L1 were expressed at significantly higher frequency on HT patients' Tregs than on NC [respectively: (17.01 ± 3.04)% versus (10.23 ± 2.77)%; t = 8.850, P < 0.001 for PD-1; (16.60 ± 9.58)% versus (11.36 ± 10.14)%; t = 2.089, P < 0.005, for PD-L1]. Conclusion: (1) The increased percentage of Th17s and decreased percentage of PD-1+ Th17s in the HT group suggest that a loss of control on Th17 activity through the checkpoint inhibitory axis PD-1/PD-L1 may participate in disease pathogenesis. (2) While the decreased percentage of Tregs in HT patients may explain a lack of regulatory functions able to prevent the autoimmune destruction of the thyroid, the significance of the increased frequency of Tregs expressing PD-1 and PD-L1, previously reported to boost Tregs differentiation, remains to be established. Elucidating this apparent contradiction may reveal important mechanisms underlying HT pathogenesis.
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Enfermedad de Hashimoto , Tiroiditis Autoinmune , Antígeno B7-H1/metabolismo , Estudios de Casos y Controles , Humanos , Leucocitos Mononucleares/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T Reguladores , Células Th17/metabolismoRESUMEN
BACKGROUND: The intervention of circular RNA HIPK3 (circHIPK3) in diabetes has drawn increasing attention in recent years. However, the underlying mechanism of circHIPK3 in diabetic nephropathy (DN) has not been fully elucidated. Thus, the current study aims to investigate the role of circHIPK3 in high glucose (HG)-induced toxicity to human renal tubular epithelial HK-2 cells. METHODS: The expression of circHIPK3 in HK-2 cells induced by HG was determined by qRT-PCR and Western blot. The regulatory effects of circHIPK3 and miR-326/miR-487a-3p on cells proliferative and apoptosis were evaluated by CCK-8 and flow cytometry. Dual-luciferase reporter assay was applied to predict the target genes of miR-326 or miR-487a-3p. RESULTS: Expression level of circHIPK3 in HK-2 cells was remarkably decreased after the treatment of HG. The overexpression of circHIPK3 effectively reversed the HG-induced HK-2 cell proliferation inhibition and apoptosis. Furthermore, SIRT1 was confirmed to be the target gene of miR-326 and miR-487a-3p, which were showed to be the downstream genes of circHIPK3. The silencing of miR-326 or miR-487a-3p was also proved to induce proliferation and reduce apoptosis in HG-induced HK-2 cells. CONCLUSION: Our data suggest that overexpression of circHIPK3 can attenuate the proliferation inhibition of HK-2 induced by HG and inhibit apoptosis through sponging miR-326 or miR-487a-3p to regulate SIRT1.
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Inflammatory response is closely related with the development of many serious health problems worldwide including diabetes mellitus (DM). Ubiquitin-fold modifer 1 (Ufm1) is a newly discovered ubiquitin-like protein, while its function remains poorly investigated, especially in inflammatory response and DM. In the present study, we analyzed the role of Ufm1 on inflammatory response in DM, and found that the proinflammatory cytokine levels (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß) and Ufm1 expression were highly increased both in the peritoneal macrophages of db/db mice and Raw264.7 cells induced by lipopolysaccharide (LPS). Western blot and luciferase reporter assay showed that NF-κB pathway was obviously activated in macrophages and the expression of LZAP, an inhibitor of NF-κB pathway, was down-regulated. With the LZAP knockdown plasmid and activation plasmid, we demonstrated that NF-κB/p65 activation was inhibited by LZAP in macrophages. The interaction of Ufm1 and LZAP was further proved with co-immunoprecipitation assay in HEK293 and Raw264.7 cells. The LZAP expression was also related with the presence of Ufm1 demonstrated by Ufm1 knockdown plasmid and activation plasmid. Besides that, we finally proved that the expression and activation of Ufm1 induced by LPS were regulated by JNK/ATF2 and JNK/c-Jun pathway with the use of SP600125. In conclusion, the present study demonstrated that Ufm 1 could activate NF-κB pathway by down-regulating LZAP in macrophage of diabetes, and its expression and activation were regulated by JNK/ATF2 and c-Jun pathway.
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Proteínas de Ciclo Celular/metabolismo , Diabetes Mellitus/metabolismo , Regulación hacia Abajo/fisiología , Macrófagos Peritoneales/metabolismo , FN-kappa B/metabolismo , Proteínas/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD/metabolismo , Células RAW 264.7 , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinas/metabolismoRESUMEN
Soluble suppression of tumorigenicity 2 (sST2) is a free form of membrane-bound ST2, which is a member of the interleukin-1 receptor family. Previous research has shown that sST2 is associated with diabetes, but cardiovascular risk factors have not been established.To analyze the relationship between sST2 and carotid intima-media thickness (CIMT) in patients with type 2 diabetes mellitus (T2DM).After screening, a total of 118 subjects with T2DM were divided into 2 groups according to the measurement of CIMT (normal CIMT (NCIMT), nâ=â58; abnormal CIMT (ACIMT), nâ=â60), and 60 healthy subjects (normal control (NC), nâ=â60) were recruited in this study. CIMT was measured by a color Doppler ultrasound, and sST2 and other metabolic parameters were measured as well.The median concentration of sST2 was elevated in the ACIMT group (31.30âng/ml) compared with the NCIMT group (28.29âng/ml, Pâ<â.01) and the NC group (20.15âng/ml, Pâ<â.01). After adjustment for age and sex, log sST2 was strongly associated with smoking history (ßâ=â0.197, 95% CI, 0.084-0.311, Pâ<â.01), FPG level (ßâ=â0.302, 95% CI, 0.162-0.442, Pâ<â.01) and HbA1c level (ßâ=â0.296, 95% CI, 0.165-0.426, Pâ<â.01) and negatively correlated with HDL level (ßâ=â-0.153, 95% CI, -0.259 to -0.046, Pâ<â.01). Furthermore, sST2 level was a risk factor for increased CIMT in patients with T2DM.Increased sST2 level not only was associated with indicators of glucose and lipid metabolism but also was a risk factor for increased CIMT in patients with T2DM. Thus, sST2 may be a potential novel marker to assess the progression of diabetic macrovascular complications.
Asunto(s)
Grosor Intima-Media Carotídeo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/diagnóstico por imagen , Proteína 1 Similar al Receptor de Interleucina-1/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
BACKGROUND: Ubiquitin-fold modifier-1 (Ufm1) is a recently identified ubiquitin-like protein. We previously confirmed that Ufm1 expression was increased in diabetic mice. However, its role in the development of diabetes remains undefined. METHODS: Lentivirus-mediated gene knockdown and overexpression techniques were used to observe the effect of Ufm1 on the expression of inflammatory factors, adhesion molecules and chemokines, as well as the transcriptional activity of nuclear factor kappa-B (NF-κB) in macrophages. Western blot and immunofluorescence analyses were used to analyse the mechanism by which Ufm1 affects the transcriptional activity of NF-κB. Finally, the effects of Ufm1 on inflammation and pancreatic, renal and myocardial damage were observed in db/db mice. RESULTS: Knockdown of Ufm1 by lentivirus shRNA targeting Ufm1 (Lv-shUfm1) led to decreased secretion of IL-6, IL-1ß, ICAM-1, VCAM-1, MCP-1 and CXCL2 in RAW264.7 cells that were exposed to LPS and TNF-α, while lentiviral overexpression of Ufm1 (Lv-Ufm1) caused the opposite effect. Interestingly, further investigation indicated that Ufm1 induced NF-κB p65 nuclear translocation in RAW264.7 cells via increasing the ubiquitination and degradation of IκBα. In an in vivo experiment, pretreatment of db/db mice with Lv-shUfm1 reduced the mRNA levels of TNF-α, IL-6, IL-1ß, ICAM-1, VCAM-1, MCP-1 and CXCL2 in resident peritoneal macrophages (RPMs) and decreased the plasma levels of TNF-α, IL-6, IL-1ß, ICAM-1, VCAM-1, MCP-1 and CXCL2. Additionally, in Lv-Ufm1-treated mice, the inverse results were observed. Following treatment with Lv-shUfm1 and Lv-Ufm1, NF-κB p65 nuclear translocation in RPMs was decreased and increased, respectively. Importantly, we observed that Lv-shUfm1 injection led to a decrease in plasma glycaemia, a reduction in urinary albuminuria and cardiomyocyte hypertrophy and an improvement in the histopathological appearance of pancreatic, kidney and myocardial tissue. Pretreatment of the mice with Lv-shUfm1 inhibited macrophage infiltration in the pancreas, kidney and myocardial tissue. CONCLUSION: Our data elucidate a new biological function of Ufm1 that mediates inflammatory responses. Ufm1-mediated p65 nuclear translocation occurs by modulating the ubiquitination and degradation of IκBα. Moreover, downregulating Ufm1 is an effective strategy to prevent the development of type 2 diabetes and its complications.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Inflamación/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Proteínas/metabolismo , Ubiquitinación , Animales , Células Cultivadas , Células HEK293 , Humanos , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Masculino , Ratones , Proteínas/antagonistas & inhibidores , Proteínas/genética , Células RAW 264.7 , ARN Interferente Pequeño/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Diabetic nephropathy (DN) is a common complication of diabetes that is the dominant cause of end-stage renal disease. However, the pathological mechanism of DN is yet to be elucidated. Serum and glucocorticoid induced kinase (SGK) 1, a ubiquitously expressed kinase, was employed in the current study to assess its effect on DN in vivo and in vitro. Male BALB/C mice and a human tubular epithelial cell line (HK-2) were utilized for experimentation. Male BALB/C mice and a human tubular epithelial cell line (HK-2) were utilized for experimentation. Pathological changes were measured via HE and staining and immunohistochemistry was performed to measure the expression of SGK 1. An SGK1 inhibitor, GSK650394, was applied to analyze the role of SGK1 in HK-2 cell epithelial-mesenchymal transition (EMT). Associated protein expressions were assessed via western blotting. In addition, migration was measured using a scratch wound healing assay. 3-methyladenine (3-MA), an autophagy inhibitor, was used to determine the variation of autophagy following SGK1 inhibition. The expression of autophagy proteins were analyzed. Furthermore, the expression of PI3K, AKT, mTOR and their levels of phosphorylation were measured. The results revealed that the ultrastructure of renal tissue suffered damage and that the expression of SGK1 was markedly increased. After SGK1 inhibition, HK-2 cell EMT was suppressed and cell migration was attenuated. Furthermore, the autophagy of HK-2 cells was promoted, an increased expression of Beclin-1 and LC3 II was detected, and a decreased expression of p62 was observed. Additionally, the phosphorylation of PI3K, AKT and mTOR were markedly upregulated. The results indicated that blocking autophagy signaling via 3-MA muted SGK1-protected against HG-evoked cell injury. Our study demonstrated that SGK1 inhibition promoted autophagy and suppressed renal tubular epithelial cell EMT in DN, indicating that SGK1 may serve as a potential therapeutic target of DN.
RESUMEN
The current study was designed to investigate the protective role of geniposide (GE) in liver injury in diabetic C57BL/KsJ-db/db mice and to explore the underlying mechanisms. The oral glucose tolerance test was performed, and the levels of insulin, alanine aminotransferase (ALT), aspartate aminotransaminase (AST), total cholesterol (TC) and triglyceride (TG) were determined. The levels of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and tumour necrosis factor-α were decreased by GE, metformin and fasudil in diabetic db/db mice. Western blotting analysis showed that the expression levels of Rho, ROCK1, ROCK2, p-NF-κBp65 and p-IκBα were significantly reversed by GE treatment. These findings demonstrated that GE exhibits a protective effect on diabetic hepatic inflammation.