microRNA-15b-5p shuttled by mesenchymal stem cell-derived extracellular vesicles protects podocytes from diabetic nephropathy via downregulation of VEGF/PDK4 axis.

Diabetic nephropathy (DN) is a severe complication of diabetes lethal for end-stage renal disease, with less treatment methodologies and uncertain pathogenesis. In the current study, we determined the role of mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) containing microRNA (miR)-15b-5p in DN. After extraction and identification of MSC-derived EVs, mouse podocyte line MPC5 was selected to establish an in vitro high-glucose (HG) cell model, where expression of miR-15b-5p, pyruvate dehydrogenase kinase 4 (PDK4) and VEGFA expression in tissues and cells were determined. The loss- and gain- function assays were conducted to determine the roles of miR-15b-5p, PDK4 and VEGFA. MPC5 cells were then co-cultured with MSC-derived EVs and their biological behaviors were detected by Western blot, CCK-8 assay, and flow cytometry. The binding relationship between miR-15b-5p and PDK43 by dual luciferase reporter gene assay. The expression of miR-15b-5p was downregulated in podocytes under HG environment, but highly expressed in mouse MSCs-derived EVs. EVs-derived miR-15b-5p could protect MPC5 cell apoptosis and inflammation. miR-15b-5p inhibited the expression of PDK4 by directly bound to the 3'UTR region of PDK4 gene. miR-15b-5p inhibits VEGF expression by binding to PDK4. Inhibition of PDK4 decreased VEGFA expression and reduced apoptosis and inflammation. Collectively, miR-15b-5p shuttled by MSC-derived EV can play protective roles in HG-induced mouse podocyte injury, possibly by targeting PDK4 and decreasing the VEGFA expression.

Overexpression of E3 ubiquitin ligase Cbl attenuates endothelial dysfunction in diabetes mellitus by inhibiting the JAK2/STAT4 signaling and Runx3-mediated H3K4me3.

BACKGROUND: Diabetes mellitus (DM), a most common chronic disease, is featured with impaired endothelial function and bioavailability of nitric oxide (NO), while E3 ubiquitin ligase appears to alleviate endothelial dysfunction as a promising option for DM treatment. Herein, we aimed to determine whether E3 ubiquitin ligase casitas B-lineage lymphoma (Cbl) alleviates endothelial dysfunction in DM rats by JAK2/STAT4 pathway. METHODS: A rat model of DM was developed through intraperitoneal injection of streptozotocin, followed by collection of aortic tissues to determine the expression of Cbl, JAK2, runt-related transcription factor 3 (Runx3) and STAT4. Human umbilical vein endothelial cells (HUVECs) were cultured in high glucose (HG) condition to induce DM as an in vitro model. With gain- and loss-function method, we assessed the aberrantly expressed Cb1 on endothelial dysfunction, NO production and apoptosis of HUVECs. RESULTS: Cbl was reduced in DM rat tissues and HG-induced HUVECs, where JAK2, Runx3 and STAT4 were elevated. It was found that overexpression of Cbl alleviated endothelial dysfunction by increasing NO production and restoring vasodilation and suppressing apoptosis of HUVECs. Mechanistically, Cb1 enhanced JAK2 ubiquitination and decreased JAK2 and STAT4 expression, where STAT4 improved Runx3 expression by regulating histone H3 lysine 4 trimethylation level. Overexpression of JAK2 and STAT4, or Runx3 increased apoptosis of HUVECs, abrogating the effect of Cb1 on endothelial function. CONCLUSION: In conclusion, Cbl alleviates endothelial dysfunction by inactivation of the JAK2/STAT4 pathway and inhibition of Runx3 expression in DM. These evidence might underlie novel Cbl-based treatment against DM in the future.

PIAS1 impedes vascular endothelial injury and atherosclerotic plaque formation in diabetes by blocking the RUNX3/TSP-1 axis.

The protein PIAS1 functions as a type of ubiquitin-protease, which is known to play an important regulatory role in various diseases, including cardiovascular diseases and cancers. Its mechanism of action primarily revolves around regulating the transcription, translation, and modification of target proteins. This study investigates role and mechanism of PIAS1 in the RUNX3/TSP-1 axis and confirms its therapeutic effects on diabetes-related complications in animal models. A diabetic vascular injury was induced in human umbilical vein endothelial cells (HUVECs) by stimulation with H2O2 and advanced glycation end product (AGE), and a streptozotocin (STZ)-induced mouse model of diabetes was constructed, followed by detection of endogenous PIAS1 expression and SUMOylation level of RUNX3. Effects of PIAS1 concerning RUNX3 and TSP-1 on the HUVEC apoptosis and inflammation were evaluated using the ectopic expression experiments. Down-regulated PIAS1 expression and SUMOylation level of RUNX3 were identified in the H2O2- and AGE-induced HUVEC model of diabetic vascular injury and STZ-induced mouse models of diabetes. PIAS1 promoted the SUMOylation of RUNX3 at the K148 site of RUNX3. PIAS1-mediated SUMOylation of RUNX3 reduced RUNX3 transactivation activity, weakened the binding of RUNX3 to the promoter region of TSP-1, and caused downregulation of TSP-1 expression. PIASI decreased the expression of TSP-1 by inhibiting H2O2- and AGE-induced RUNX3 de-SUMOylation, thereby arresting the inflammatory response and apoptosis of HUVECs. Besides, PIAS1 reduced vascular endothelial injury and atherosclerotic plaque formation in mouse models of diabetes by inhibiting the RUNX3/TSP-1 axis. Our study proved that PIAS1 suppressed vascular endothelial injury and atherosclerotic plaque formation in mouse models of diabetes via the RUNX3/TSP-1 axis.

Adipocytes-Derived Exosomal microRNA-1224 Inhibits M2 Macrophage Polarization in Obesity-Induced Adipose Tissue Inflammation via MSI2-Mediated Wnt/ß-Catenin Axis.

SCOPE: Phenotypic switch of macrophage polarization in adipose tissue has been associated with obesity-induced adipose tissue inflammation (OATI). Therefore, this study aims to explore the possible mechanism of adipocytes-derived exosomes (ADEs) carrying microRNA-1224 (miR-1224) in M2 macrophage polarization of OATI. METHODS AND RESULTS: miR-1224-knockout (miR-1224-KO) mice for this study, and isolated primary adipocytes from high-fat diet (HFD) or normal diet (SD)-fed mice are developed. ADEs are extracted and cocultured with bone marrow-derived macrophages (BMDMs). The macrophagic crown-like structures (CLS) and M1 and M2 phenotype macrophages in epididymal white adipose tissue (epiWAT) are observed by immunohistochemistry and flow cytometry. The obtained data indicate that miR-1224 is highly expressed in adipose tissues and adipocytes of obese mice. miR-1224 knockout decreases CLS number and increases M2 macrophages polarization in epiWAT. In addition, miR-1224 can be transferred to BMDMs via ADEs, which targeted musashi RNA binding protein 2 (MSI2) expression and inactivated Wnt/ß-catenin pathway, inhibiting macrophage M2 polarization and promoting inflammatory factor release. CONCLUSION: Exosomal miR-1224 derived by adipocytes targets MSI2 and blocks the Wnt/ß-catenin pathway, which inhibits macrophage M2 polarization and promotes inflammatory factor release, ultimately promoting OATI.

Mutational Analysis of Mitochondrial tRNA Genes in 200 Patients with Type 2 Diabetes Mellitus.

OBJECTIVE: Previous studies showed that variants in mitochondrial DNA (mtDNA) are associated with type 2 diabetes mellitus (T2DM). However, the relationships between mitochondrial tRNA (mt-tRNA) variants and T2DM remain poorly understood. METHODS: In this study, we performed a mutational screening of 22 mt-tRNA genes in a cohort of 200 Han Chinese subjects with T2DM and 200 control subjects through PCR-Sanger sequencing. The identified mt-tRNA variants were assessed for their pathogenicity via the phylogenetic approach, structural and functional analysis. Furthermore, two Han Chinese pedigrees with maternally inherited diabetes and deafness (MIDD) were reported by clinical and genetic assessments. RESULTS: A total of 49 genetic variants in mt-tRNA genes were identified; among them, 31 variants (17 pathogenic/likely pathogenic) were absent in controls, located at extremely conserved nucleotides, may have potential structural and functional significance, thereby considered to be T2DM-associated variants. In addition, sequence analysis of entire mitochondrial genomes of the matrilineal relatives from two MIDD pedigrees revealed the occurrence of tRNALeu(UUR) A3243G and T3290C mutations, as well as sets of polymorphisms belonging to mitochondrial haplogroups F2 and D4. However, the lack of any functional variants in connexin 26 gene (GJB2) and tRNA 5-methylaminomethyl-2-thiouridylate (TRMU) suggested that nuclear genes may not play active roles in clinical expression of MIDD in these pedigrees. CONCLUSION: Our data indicated that mt-tRNA variants were associated with T2DM, screening for mt-tRNA pathogenic mutations was recommended for early detection and prevention of mitochondrial diabetes.

Effect of a 12-Week Aerobic Exercise Training on Serum Fetuin-A and Adipocytokine Levels in Type 2 Diabetes.

OBJECTIVE: We aimed to evaluate the effect of 12-week aerobic exercise training on fetuin-A levels in type 2 diabetes mellitus and examine the relationships between fetuin-A and adipocytokine levels and cardiovascular risk factors. METHODS: The study included 32 patients with type 2 diabetes mellitus who were assigned to an exercise or a control group. The exercise group underwent 12 weeks of exercise (consisting of a 5-min warm-up, 60-min aerobic bicycle training performed at 70% of the maximal heart rate, a cool-down period, 5 times/week). Adiponectin, resistin, and fetuin-A serum levels were measured using enzyme-linked immunosorbent assay. Leptin serum levels were measured by a radioimmunoassay. RESULTS: Exercise for 12 weeks significantly reduced serum fetuin-A (643.1±109.4 to 448.7±92.5 µg/mL, P<0.05), leptin (11.9±7.2 to 8.6±5.7 ng/dL, P<0.05), and resistin (3.2±1.5 to 2.2±1.4 ng/mL, P<0.05) levels, but increased adiponectin (6.9±1.9 to 8.1±1.7 µg/mL, P<0.05) levels. In the exercise group, Δfetuin-A positively correlated with differences in weight (r=0.654, P=0.046), body mass index (r=0.725, P=0.002), waist circumference (r=0.898, P=0.013), and adiponectin levels (r=0.662, P=0.035). CONCLUSIONS: Aerobic exercise significantly decreased serum fetuin-A levels in type 2 diabetes mellitus, which can be attributed to weight loss and related to increased adiponectin levels.

Association between resistin +299A/A genotype and nonalcoholic fatty liver disease in Chinese patients with type 2 diabetes mellitus.

OBJECTIVE: To investigate the relationship between the resistin intronic +299G/A polymorphism and nonalcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM). METHODS: We selected 738 T2DM patients, including 395 with NAFLD and 343 without fatty liver disease, as well as 279 healthy control individuals, and analyzed their resistin +299G/A polymorphism genotype by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Plasma resistin levels in T2DM patients with NAFLD were at the highest (P<0.05). The frequency of AA genotype at the +299 site of the resistin gene in patients with concurrent T2DM combined with NAFLD was significantly different from that in the control (P<0.05). The AA genotype was found to be associated with a 1.80-fold increased risk for T2DM combined with NAFLD, 2.05-fold increased risk for obesity and 2.37-fold increased risk for obesity of abdominal type compared to the GG (P<0.05, respectively). The multivariate non-conditional logistic regression model analysis further shows that the AA genotype is a risk factor for the development of NAFLD in T2DM patients (OR, 2.32; 95% CI, 1.05-4.68; P<0.05). CONCLUSION: The resistin +299AA genotype may be associated with increases in the risk of the NAFLD development in T2DM patients.