Targeting peroxisomal fatty acid oxidation improves hepatic steatosis and insulin resistance in obese mice.

Obesity and diabetes normally cause mitochondrial dysfunction and hepatic lipid accumulation, while fatty acid synthesis is suppressed and malonyl-CoA is depleted in the liver of severe obese or diabetic animals. Therefore, a negative regulatory mechanism might work for the control of mitochondrial fatty acid metabolism that is independent of malonyl-CoA in the diabetic animals. As mitochondrial ß-oxidation is controlled by the acetyl-CoA/CoA ratio, and the acetyl-CoA generated in peroxisomal ß-oxidation could be transported into mitochondria via carnitine shuttles, we hypothesize that peroxisomal ß-oxidation might play a role in regulating mitochondrial fatty acid oxidation and inducing hepatic steatosis under the condition of obesity or diabetes. This study reveals a novel mechanism by which peroxisomal ß-oxidation controls mitochondrial fatty acid oxidation in diabetic animals. We determined that excessive oxidation of fatty acids by peroxisomes generates considerable acetyl-carnitine in the liver of diabetic mice, which significantly elevates the mitochondrial acetyl-CoA/CoA ratio and causes feedback suppression of mitochondrial ß-oxidation. Additionally, we found that specific suppression of peroxisomal ß-oxidation enhances mitochondrial fatty acid oxidation by reducing acetyl-carnitine formation in the liver of obese mice. In conclusion, we suggest that induction of peroxisomal fatty acid oxidation serves as a mechanism for diabetes-induced hepatic lipid accumulation. Targeting peroxisomal ß-oxidation might be a promising pathway in improving hepatic steatosis and insulin resistance as induced by obesity or diabetes.

Long-chain dicarboxylic acids play a critical role in inducing peroxisomal ß-oxidation and hepatic triacylglycerol accumulation.

Recent studies provide evidence that peroxisomal ß-oxidation negatively regulates mitochondrial fatty acid oxidation, and induction of peroxisomal ß-oxidation causes hepatic lipid accumulation. However, whether there exists a triggering mechanism inducing peroxisomal ß-oxidation is not clear. Long-chain dicarboxylic acids (LCDAs) are the product of mono fatty acids subjected to ω-oxidation, and both fatty acid ω-oxidation and peroxisomal ß-oxidation are induced under ketogenic conditions, indicating there might be a crosstalk between. Here, we revealed that administration of LCDAs strongly induces peroxisomal fatty acid ß-oxidation and causes hepatic steatosis in mice through the metabolites acetyl-CoA and hydrogen peroxide. Under ketogenic conditions, upregulation of fatty acid ω-oxidation resulted in increased generation of LCDAs and induction of peroxisomal ß-oxidation, which causes hepatic accumulation of lipid droplets in animals. Inhibition of fatty acid ω-oxidation reduced LCDA formation and significantly lowered peroxisomal ß-oxidation and improved hepatic steatosis. Our results suggest that endogenous LCDAs act as triggering molecules inducing peroxisomal ß-oxidation and hepatic triacylglycerol deposition. Targeting fatty acid ω-oxidation might be an effective pathway in treating fatty liver and related metabolic diseases through regulating peroxisomal ß-oxidation.

Thiolutin, a selective NLRP3 inflammasome inhibitor, attenuates cyclophosphamide-induced impairment of sperm and fertility in mice.

OBJECTIVE: The activation of the NLRP3 inflammasome has been implicated in male infertility. Our study aimed to investigate the therapeutic role of Thiolutin (THL), an inhibitor of the NLRP3 inflammasome, on oligoasthenospermia (OA) and to elucidate its mechanisms. MATERIALS AND METHODS: Semen from 50 OA and 20 healthy males were analyzed to assess the sperm quality and levels of inflammatory markers. Their correlation was determined using Pearson's correlation coefficient. The BALB/c mice were intraperitoneal injected by cyclophosphamide at 60 mg/kg/day for five days to induce OA, followed by a two-week treatment with THL or L-carnitine. Reproductive organ size and H&E staining were determined to observe the organ and seminiferous tubule morphology. ELISA and western blotting were utilized to measure sex hormone levels, inflammatory markers, and NLRP3 inflammasome levels. Furthermore, male and female mice were co-housed to observe pregnancy success rates. RESULTS: OA patients exhibited a decrease in sperm density and motility compared to healthy individuals, along with elevated levels of IL-1ß, IL-18 and NLRP3 inflammasome. In vivo, THL ameliorated OA-induced atrophy of reproductive organs, hormonal imbalance, and improved sperm density, motility, spermatogenesis and pregnancy success rates with negligible adverse effects on weight or liver-kidney function. THL also demonstrated to be able to inhibit the activation of NLRP3 inflammasome and associated proteins in OA mice. DISCUSSION: THL can improve sperm quality and hormonal balance in OA mice through the inhibition of NLRP3 inflammasome activation. Thus, THL holds promising potential as a therapeutic agent for OA.

Peroxisome-generated succinate induces lipid accumulation and oxidative stress in the kidneys of diabetic mice.

Diabetes normally causes lipid accumulation and oxidative stress in the kidneys, which plays a critical role in the onset of diabetic nephropathy; however, the mechanism by which dysregulated fatty acid metabolism increases lipid and reactive oxygen species (ROS) formation in the diabetic kidney is not clear. As succinate is remarkably increased in the diabetic kidney, and accumulation of succinate suppresses mitochondrial fatty acid oxidation and increases ROS formation, we hypothesized that succinate might play a role in inducing lipid and ROS accumulation in the diabetic kidney. Here we demonstrate a novel mechanism by which diabetes induces lipid and ROS accumulation in the kidney of diabetic animals. We show that enhanced oxidation of dicarboxylic acids by peroxisomes leads to lipid and ROS accumulation in the kidney of diabetic mice via the metabolite succinate. Furthermore, specific suppression of peroxisomal ß-oxidation improved diabetes-induced nephropathy by reducing succinate generation and attenuating lipid and ROS accumulation in the kidneys of the diabetic mice. We suggest that peroxisome-generated succinate acts as a pathological molecule inducing lipid and ROS accumulation in kidney, and that specifically targeting peroxisomal ß-oxidation might be an effective strategy in treating diabetic nephropathy and related metabolic disorders.

Peroxisomal ß-oxidation stimulates cholesterol biosynthesis in the liver in diabetic mice.

Although diabetes normally causes an elevation of cholesterol biosynthesis and induces hypercholesterolemia in animals and human, the mechanism linking diabetes to the dysregulation of cholesterol biosynthesis in the liver is not fully understood. As liver peroxisomal ß-oxidation is induced in the diabetic state and peroxisomal oxidation of fatty acids generates free acetate, we hypothesized that peroxisomal ß-oxidation might play a role in liver cholesterol biosynthesis in diabetes. Here, we used erucic acid, a specific substrate for peroxisomal ß-oxidation, and 10,12-tricosadiynoic acid, a specific inhibitor for peroxisomal ß-oxidation, to specifically induce and suppress peroxisomal ß-oxidation. Our results suggested that induction of peroxisomal ß-oxidation increased liver cholesterol biosynthesis in streptozotocin-induced diabetic mice. We found that excessive oxidation of fatty acids by peroxisomes generated considerable free acetate in the liver, which was used as a precursor for cholesterol biosynthesis. In addition, we show that specific inhibition of peroxisomal ß-oxidation decreased cholesterol biosynthesis by reducing acetate formation in the liver in diabetic mice, demonstrating a crosstalk between peroxisomal ß-oxidation and cholesterol biosynthesis. Based on these results, we propose that induction of peroxisomal ß-oxidation serves as a mechanism for a fatty acid-induced upregulation in cholesterol biosynthesis and also plays a role in diabetes-induced hypercholesterolemia.

miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

BACKGROUND: Our previous two-dimensional electrophoresis experiment showed that the expression of LASP1 in patients with endometriosis was significantly higher than that of control endometrium. However, the molecular mechanism by which LASP1 is regulated in endometriosis/adenomyosis is unknown. METHODS: Herein, qPCR was performed to analyze the expression levels of LASP1 and miR-218-5p between endometriosis (Ems) cells and control cells. Fluorescence in situ hybridization was carried out to measure the expression level of miR-218-5p in ectopic endometrium versus normal endometrium. After miR-218-5p mimic or inhibitor were transfected, the transwell experiment was carried out to see the effect of miR-218-5p on the migration of endometrial stromal cells (ESCs). EdU was used to measure cell proliferation rate. Dual-luciferase reporter assay was used to verify the binding of hsa-miR-218-5p to the 3'UTR of LASP1. Western blot and immunofluorescence analysis were carried out to identify the protein expression pattern of LASP1 and EMT markers in endometrial tissue. RESULTS: The miR-218-5p is mainly secreted from blood vessels and expressed in the muscle layer around the endometrium, which inhibits the expression level of LASP1 by binding the 3'UTR region of LASP1 in normal ESCs. Overexpression of miR-218-5p impedes the epithelial-to-mesenchymal transition (EMT) and prevents the migration of ESCs and the expression of Vimentin in Ems. CONCLUSIONS: Our findings revealed that miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

11ß-hydroxysteroid dehydrogenases-2 decreases the apoptosis of MC3T3/MLO-Y4 cells induced by glucocorticoids.

The aim of the present study was to confirm the role of 11ß-hydroxysteroid dehydrogenases type 2(11ß-HSD-2) in steroid induced osteonecrosis of the femoral head(SANFH). We cultured mouse bone-like cells (MLO-Y4) and mouse osteoblast-like cells (MC3T3-E1). After overexpressed 11ß-HSD-2 successfully, we induced cell apoptosis by dexamethasone (DXM). The level of cell apoptosis, the expression of Bcl-2 in MLO-Y4 cells and the expression of Fas and caspase8 in MC3T3-E1 cells were detected. Then, we constructed 11ß-HSD-2 siRNA plasmid and represented it on MLO-Y4/MC3T3-E1 Cells, to down-regulate the 11ß-HSD-2 expression. After that, we used dexamethasone to induce cell apoptosis. The level of cell apoptosis, the expression of Bcl-2 in MLO-Y4 cells and the expression of Fas and caspase8 in MC3T3-E1 cells were detected again. In the overexpression model of cells, we found that the amount of cell apoptosis, the expression of Fas and caspase8 in MC3T3-E1 cells are lower than that of control groups. The amount of cell apoptosis, the expression of Fas and caspase8 in MC3T3-E1 cells were more than before when we reduced the expression of 11ß-HSD-2. In our study, we concluded that 11ß-HSD-2 plays an important role in the development of bone or osteoblast cell apoptosis, and the decreased expression of 11ß-HSD-2 may aggravate steroid induced bone/osteoblast cell apoptosis.

Androgen receptor gene mutations in 258 Han Chinese patients with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) affects 8-13% of reproductive-age females worldwide and mutations or aberrant expression of androgen receptor (AR) may cause the onset of this disease. In the present study, 258 samples from Han Chinese patients with PCOS were analyzed for the presence of AR mutations via sequencing of all coding exons of the AR gene. A total of five heterozygous missense mutations, namely p.V3M, p.Q72R, p.S158L, p.S176R and p.G396R, were identified in five of the patients. Among these, p.S158L was a novel mutation that, to the best of our knowledge, has not been reported previously. Although the remaining four mutations have been reported previously, they existed at low frequencies or were absent in the control subjects and in the Exome Aggregation Consortium database. The results of evolutionary conservation and in silico analysis revealed that the p.V3M, p.S158L and p.S176R mutations were pathogenic, whereas the p.Q72R and p.G396R mutations were benign. Compared with the patients with PCOS without AR mutations or with benign AR mutations, markedly lower estrogen levels on the day of human chorionic gonadotropin injection were observed in the three patients with PCOS with potentially pathogenic mutations. In addition, patients with PCOS with pathogenic mutations had lower numbers of oocytes; however, the difference was not statistically significant. Of note, these observations should be interpreted with caution due to the relatively small sample size in the present study. Therefore, a larger number of samples should be collected to validate the results of the present study in future studies. In summary, the present study identified three potential pathogenic mutations in 258 Han Chinese patients with PCOS and these mutations may have an implication in the pathogenesis of PCOS.

cAMP regulates 11ß-hydroxysteroid dehydrogenase-2 and Sp1 expression in MLO-Y4/MC3T3-E1 cells.

11ß-hydroxysteroid dehydrogenase-2 (11ß-HSD2) is one of the key enzymes in glucocorticoid metabolism, which can inactivate local corticosterone and regulate the level of active glucocorticoid in tissues. The expression of 11ß-HSD2 and its regulatory pathway serve an important role in the apoptosis of steroid induced osteonecrosis of the femoral head (SANFH). The present study aimed to identify the regulatory effects of cAMP on the expression of Sp1 transcription factor (Sp1) and 11ß-HSD2 in osteocytes at the cellular level. Murine long bone osteocyte Y4 (MLO-Y4) clone cells and mouse embryo osteoblast-like (MC3T3-E1) cells were cultured in vitro with adenylate cyclase activator or inhibitor (forskolin and SQ22536, respectively) to investigate the effects of alterations to intracellular cAMP levels. mRNA and protein expression levels of Sp1 and 11ß-HSD2 were detected by reverse transcription-quantitative PCR and western blotting, respectively. Compared with the negative control group, the mRNA and protein expression levels of Sp1 were significantly increased in the activation group, whereas Sp1 expression levels were significantly decreased in the inhibition group. Similarly, compared with the negative control group, the mRNA and protein expression levels of 11ß-HSD2 were significantly increased in the activator group, but significantly decreased in the inhibitor group. The aforementioned results indicated that intracellular cAMP levels significantly regulated the expression of Sp1 and 11ß-HSD2 in mouse osteocytes and osteoblasts. Therefore, the present study suggested a potential therapeutic strategy for the prevention of osteonecrosis of the femoral head.

Metabolomics study of serum and urine samples reveals metabolic pathways and biomarkers associated with pelvic organ prolapse.

Pelvic organ prolapse (POP) is a common medical condition among women and involves complicated diagnostics and controversial surgical management. The exact molecular mechanism underlying POP is poorly understood, especially at the metabolism level. To explore the metabolic mechanism underlying POP and discover potential biomarkers for POP diagnosis, we applied a non-targeted metabolomics approach using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Metabolomics study of serum samples from patients with POP (n = 24) and controls (n = 22) revealed a total of 59 metabolites that are significantly different (VIP ≥ 1 and p ≤ 0.05) between the two groups. Between urine samples from POP patients (n = 45) and controls (n = 59), 33 metabolites differed significantly (VIP ≥ 1 and p ≤ 0.05). Metabolic pathways affected by these differentially expressed metabolites were analyzed. In both serum and urine samples, three pathways including arginine biosynthesis and purine metabolism were found to be significantly related to POP. Six metabolites including GPC, 1-methyladenosine, maleic acid, L-pyroglutamic acid, inosine, and citrate are significantly changed (VIP ≥ 1 and p ≤ 0.05) in both serum and urine samples from patients with POP. Receiver operating characteristics (ROC) curve analysis showed that using these six metabolites as a biomarker could distinguish patients with POP from controls with good accuracy in both serum (AUC = 1) and urine samples (AUC = 0.854). Collectively, these results further extended our understanding of key regulatory metabolic pathways involved in the pathophysiology of POP, as well as provided some promising biomarkers for effective POP diagnosis.

FoxM1 promotes the migration of ovarian cancer cell through KRT5 and KRT7.

Forkhead box M1(FoxM1) played an important role in the pathogenesis of ovarian cancer, but its downstream molecular network is mysterious. Here, we combined ChIP-seq with RNA-seq analysis and identified 687 FoxM1-binding regions and 182 genes regulated by FoxM1. The above data pointed out that KRT5 and KRT7 were downstream target genes of FoxM1. Next, we used qPCR and Western blot to verify that FoxM1 knockdown inhibited the expression levels of KRT5 and KRT7. We also demonstrated that FoxM1 regulated KRT5 and KRT7 genes expression through binding a consensus AP-2 cis element, and showed that KRT5 and KRT7 deficiency could prevent the migration but not proliferation of SK-OV-3 cells. Finally, tissue microarray results indicated that KRT5 and KRT7 were highly expressed in ovarian cancer and positively correlated with FoxM1 expression. TCGA database showed that high expression of KRT5 and KRT7 could significantly reduce the survival rate of patients with ovarian cancer. The above results clarify the specific downstream molecular network of FoxM1 to promote the pathogenesis of ovarian cancer, and provide a basis experiment for the judgment of ovarian cancer prognosis and the design of drug targets.

Hardware removal or preservation? Decision making based on a newly developed rating scale.

Hardware exposure following open reduction and internal fixation (ORIF) surgery is a tricky problem. It is always hard for surgeons to decide whether to keep or remove the hardware. In this study, a rating scale and corresponding clinical path is developed based on former published paper as well as our own experience. New admitted patients are first evaluated and scored once they enter the department. Based on the score they get, patients are assigned to different therapeutic schedule, i.e. (1) hardware preservation with pedicel flap transplantation, (2) debridement for further reevaluation and (3) hardware removal with external fixation. Satisfying clinical outcome is achieved that is characterized with high osseous consolidation rate and low complication rate. The result showed that this newly developed rating scale and the related therapeutic schedule could be an available tool to help surgeons to make decisions in the treatment of hardware exposure.