Urolithin A exerts anti-tumor effects on gastric cancer via activating autophagy-Hippo axis and modulating the gut microbiota.

Gastric cancer (GC) treatment regimens are still unsatisfactory. Recently, Urolithin A (UroA) has gained tremendous momentum due to its anti-tumor properties. However, the therapeutic effect and underlying mechanisms of UroA in GC are unclear. We explored the effects and related mechanisms of UroA on GC both in vivo and in vitro. A Cell Counting Kit-8 was used to determine the influence of UroA on the proliferation of GC cell lines. The Autophagy inhibitor 3-methyladenine (3MA) was employed to clarify the role of autophagy in the anti-tumor effect of UroA. Simultaneously, we detected the core-component proteins involved in autophagy and its downstream pathways. Subsequently, the in vivo anti-tumor effect of UroA was determined using a xenograft mouse model. Western blotting was used to detect the core protein components of the anti-tumor pathways, and 16S rDNA sequencing was used to detect the effect of UroA on the gut microbiota. We found that UroA suppressed tumor progression. The use of 3MA undermined the majority of the inhibitory effect of UroA on tumor cell proliferation, further confirming the importance of autophagy in the anti-tumor effect of UroA. Invigorating of autophagy activated the downstream Hippo pathway, thereby inhibiting the Warburg effect and promoting cell apoptosis. In addition, UroA modulated the composition of the gut microbiota, as indicated by the increase of probiotics and the decrease of pathogenic bacteria. Our research revealed new anti-tumor mechanisms of UroA, which may be a promising candidate for GC treatment.

Bariatric surgery induces pancreatic cell transdifferentiation as indicated by single-cell transcriptomics in Zucker diabetic rats.

AIMS: Bariatric surgery results in rapid recovery of glucose control in subjects with type 2 diabetes mellitus. However, the underlying mechanisms are still largely unknown. The present study aims to clarify how bariatric surgery modifies pancreatic cell subgroup differentiation and transformation in the single-cell RNA level. METHODS: Male, 8-week-old Zucker diabetic fatty (ZDF) rats with obesity and diabetes phenotypes were randomized into sleeve gastrectomy (Sleeve, n = 9), Roux-en-Y gastric bypass (RYGB, n = 9), and Sham (n = 7) groups. Two weeks after surgery, the pancreas specimen was further analyzed using single-cell RNA-sequencing technique. RESULTS: Two weeks after surgery, compared to the Sham group, the metabolic parameters including fasting plasma glucose, plasma insulin, and oral glucose tolerance test values were dramatically improved after RYGB and Sleeve procedures (p < .05) as predicted. In addition, RYGB and Sleeve groups increased the proportion of pancreatic ß cells and reduced the ratio of α cells. Two multiple hormone-expressing cells were identified, the Gcg+/Ppy + and Ins+/Gcg+/Ppy + cells. The pancreatic Ins+/Gcg+/Ppy + cells were defined for the first time, and further investigation indicates similarities with α and ß cells, with unique gene expression patterns, which implies that pancreatic cell transdifferentiation occurs following bariatric surgery. CONCLUSIONS: For the first time, using the single-cell transcriptome map of ZDF rats, we reported a comprehensive characterization of the heterogeneity and differentiation of pancreatic endocrinal cells after bariatric surgery, which may contribute to the underlying mechanisms. Further studies will be needed to elucidate these results.

Alteration of Ileal lncRNAs After Duodenal-Jejunal Bypass Is Associated With Regulation of Lipid and Amino Acid Metabolism.

Metabolic and bariatric surgery (MBS) can generate a drastic shift of coding and noncoding RNA expression patterns in the gastrointestinal system, which triggers organ function remodeling and may induce type 2 diabetes (T2D) remission. Our previous studies have demonstrated that the altered expression profiles of duodenal and jejunal long noncoding RNAs (lncRNAs) after the duodenal-jejunal bypass (DJB), an investigational procedure and research tool of MBS, can improve glycemic control by modulating the entero-pancreatic axis and gut-brain axis, respectively. As an indiscerptible part of the intestine, the ileal lncRNA expression signatures after DJB and the critical pathways associated with postoperative correction of the impaired metabolism need to be investigated too. High-fat diet-induced diabetic mice were randomly assigned into two groups receiving either DJB or sham surgery. Compared to the sham group, 1,425 dysregulated ileal lncRNAs and 552 co-expressed mRNAs were identified in the DJB group. Bioinformatics analysis of the differently expressed mRNAs and predicted target genes or transcriptional factors indicated that the dysregulated ileal lncRNAs were associated with lipid and amino acid metabolism-related pathways. Moreover, a series of lncRNAs and their potential target mRNAs, especially NONMMUT040618, Pxmp4, Pnpla3, and Car5a, were identified on the pathway. In conclusion, DJB can induce remarkable alteration of ileal lncRNA and mRNA expression. The role of the ileum in DJB tends to re-establish the energy homeostasis by regulating the lipid and amino acid metabolism.

A Rodent Model of Jejunal-Ileal Loop Bipartition (JILB): a Novel Malabsorptive Operation.

BACKGROUND: We designed a novel malabsorptive procedure named as jejunal-ileal loop bipartition (JILB), in which a jejunal-ileal loop is created to reduce the effective length of food chyme passage in the small bowel, but without exclusion of any segment of the intestine. This study is to investigate the feasibility and efficacy of JILB on weight loss and glycemic control in obese diabetic mouse model. METHODS: High-fat diet-induced C57BL/6 mice with typical obese and diabetic phenotypes were randomly divided into two groups according to the surgical procedure performed, including JILB (n = 8) and sham group (n = 8). Age-matched naïve C57BL/6 mice fed with rodent chow diet were adopted as normal controls. Body weight, food intake, fasting plasma glucose (FPG), fasting plasma insulin (FPI), and oral glucose tolerance test (OGTT) were measured in vivo before and 2, 4, and 8 weeks after surgery. Plasma glucagon-like peptide 1 (GLP-1) was assayed before and 15 min after oral glucose challenge at the 8th week postoperatively. RESULTS: Comparing to the sham animals, JILB group consumed similar amount of food, but had lower body weight after surgery (P < 0.01). It led to significant lower FPG (p < 0.05) and improved glucose tolerance with lower FPI (p < 0.001). And GLP-1 secretion at 15 min after oral glucose challenge was higher than shams (P < 0.05). No intestinal obstruction was identified. CONCLUSIONS: JILB is potentially a metabolic and bariatric procedure that leads to effective weight loss and diabetes remission in obese diabetic subjects.

miR-1-3p suppresses proliferation of hepatocellular carcinoma through targeting SOX9.

BACKGROUND: Liver cancer was the fourth leading cause of cancer-related death in 2015. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. miR-1-3p plays important roles in cancer, including prostate, bladder, lung cancer, and colorectal carcinoma. The function of miR-1-3p in HCC remains poorly understood. METHODS: qRT-PCR was performed to detect the miR-1-3p expression in HCC cell lines (HCCLM3, Hep3B, Bel-7404, SMMC-7721) and the normal human hepatic cell line (LO2). HCCLM3 and Bel-7404 cells were transfected with miR-1-3p mimic or scramble control followed by water-soluble tetrazolium salt (WST-1) assay. Western bolt analysis was performed to determine the protein levels. TargetScan7.1 (http://www.targetscan.org/vert_71/) was used to predict the potential targets of miR-1-3p. SRY (sex determining region Y)-box 9 (SOX9), which has been previously shown to play an important role in HCC, was found to be a target of miR-1-3p. Luciferase reporter assay was used to explore the targeting of miR-1-3p on SOX9. For in vivo tumorigenesis assay, HCCLM3 cells with stable overexpression of miR-1-3p or control plasmid were injected subcutaneously into the flank of the SCID mice and animals were monitored for tumor growth. RESULTS: miR-1-3p was significantly downregulated in HCC cell lines (HCCLM3, Hep3B, Bel-7404, and SMMC-7721) compared to normal human hepatic cell line (LO2). Overexpression of miR-1-3p significantly inhibited the proliferation and induced apoptosis in HCCLM3 and Bel-7474 cells. SOX9 was a direct target of miR-1-3p in HCC cells. Inhibition of SOX9 significantly inhibited the proliferation of HCCLM3 and Bel-7474 cells. In vivo, overexpression of miR-1-3p decreased tumor volume in a xenograft model. CONCLUSION: These results highlight the role of miR-1-3p in HCC. Overexpression of miR-1-3P inhibited the proliferation of HCC at least partly due to the regulation of SOX9. miR-1-3p may be a promising therapeutic candidate for HCC.

Comparison of Diabetes Remission and Micronutrient Deficiency in a Mildly Obese Diabetic Rat Model Undergoing SADI-S Versus RYGB.

BACKGROUND: Single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has launched a huge challenge to classic Roux-en-Y gastric bypass (RYGB). Our objective was to compare diabetes remission and micronutrient deficiency in a mildly obese diabetic rat model undergoing SADI-S versus RYGB. METHODS: Thirty adult male mildly obese diabetic rats were randomly assigned to sham (S), SADI-S, and RYGB groups. Body weight, food intake, fasting plasma glucose (FPG), oral glucose tolerance test (OGTT), plasma insulin, GLP-1, and ghrelin levels were measured at indicated time points. Meanwhile, insulin sensitivity and pancreatic ß cell function were assessed during OGTT. Finally, plasma micronutrient evaluation and islet ß cell mass analysis were performed after all animals were sacrificed. RESULTS: As compared to sham, the SADI-S and RYGB groups achieved almost equivalent efficacy in caloric restriction and FPG control without excessive weight loss. During OGTT, the SADI-S and RYGB groups also provided comparable effects on glycemic excursion, insulin sensitivity, and ß cell function; however, only rats in the RYGB group showed significant changes in gut hormones, whereas the three groups were found to exhibit no significant difference in ß cell mass. In addition, only vitamin E in the RYGB group was deficient as compared with the SADI-S and S groups. CONCLUSION: In mildly obese diabetic rat, SADI-S and RYGB procedures have comparable efficacy in diabetes remission and risk of micronutrient deficiency. These data show that each of the surgery accomplishes diabetes improvements through both overlapping and distinct mechanisms requiring further investigation.

Loss of thioredoxin 2 alters mitochondrial respiratory function and induces cardiomyocyte hypertrophy.

Thioredoxin 2 (Trx2), as a member of the thioredoxin system in mitochondria, is involved in controlling mitochondrial redox state. However, the role of Trx2 in cardiac biology is not fully understood. In the present study, the expression of Trx2 is silenced in quiescent neonatal rat ventricular cardiomyocytes (NRVCs) and mitochondrial respiratory function and cardiomyocyte hypertrophy are assessed. The results show that Trx2 depletion does not induce significant cytotoxicity in quiescent NRVCs. Remarkably, Trx2 depletion results in cardiomyocyte hypertrophy as determined by increased cell size and protein synthesis. Furthermore, Trx2 depletion inhibits AMPK activity and AMPK activator reversed cellular hypertrophy. Trx2 depletion enhances mitochondrial ROS generation without impact on cellular ROS level. Trx2 depletion has no effect on mitochondrial biogenesis. Specifically, Trx2 depletion increases mitochondrial respiration flux and total ATP concentration under quiescent conditions. To decipher the relationship between ROS generation, mitochondrial respiration flux, and AMPK signaling, mitochondrial metabolism and ROS was specifically inhibited, and the results show that AMPK inactivation and hypertrophic response in Trx2-silenced cells is reversed by respiration blockers but not ROS scavenger. In conclusion, these results show that beyond mitochondrial ROS scavenging, Trx2 controls mitochondrial respiratory function in quiescent cardiomyocytes and is implicated in cardiomyocyte hypertrophy via AMPK signaling.

Jejunal long noncoding RNAs are associated with glycemic control via gut-brain axis after bariatric surgery in diabetic mice.

BACKGROUND: Metabolic and bariatric surgery is effective in ameliorating type 2 diabetes, although its underlying mechanisms are largely unknown. Our previous study indicated that the distinctly expressed duodenal long noncoding RNAs (lncRNAs) induced by the duodenal-jejunal bypass (DJB) might play a role in improving glycemic control via the enteropancreatic axis. Therefore, the physiologic role of the jejunum in metabolic regulation after DJB requires investigation. OBJECTIVES: To investigate the alterations in the jejunal Roux limb lncRNA expression signatures after DJB and analyze the functional pathways associated with metabolic improvement on a genome-wide scale in high-fat diet-induced diabetic mice. SETTING: University medical center. METHODS: Diabetic mice induced by high-fat diet were randomly assigned into 2 groups undergoing either DJB or sham surgery. The lncRNA and messenger (m)RNA expression profiles of the Roux limb segment of the jejunum in both groups were investigated using microarray. To identify the functional characteristics of the distinctly expressed lncRNAs, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted. The lncRNA-mRNA and lncRNA-transcription factor interaction networks were constructed based on Pearson correlation analysis. RESULTS: Compared with the sham group, 827 dysregulated (fold change ≥2.0) jejunal lncRNAs were identified in the DJB group. Both Kyoto Encyclopedia of Genes and Genomes pathway and gene ontology enrichment analysis revealed that 601 lncRNA-co-expressed mRNAs (fold change ≥2.0) were associated with neuromodulation-related pathways or biological processes, including serotonergic, glutamatergic, and dopaminergic synapses. In addition, hormonal regulation-related pathways, especially steroid biosynthesis, were also enriched. The results were further confirmed by bioinformatic analysis of target genes or transcription factors predicted on the basis of dysregulated jejunal lncRNAs. Furthermore, the NONMMUT023781 lncRNA may simultaneously target the Adcy8 mRNA both in cis and in trans and participate in neuromodulation and hormonal regulation. CONCLUSION: Alterations of jejunal Roux limb lncRNA and mRNA expression profiles trigger both neuromodulation and endocrine-related pathways, which play a critical role in type 2 diabetes remission after metabolic and bariatric surgery via the gut-brain axis. NONMMTU023781 and Adcy8 were identified as potential targets, which warrant further research.

Duodenal-Jejunal Bypass Surgery Reverses Diabetic Phenotype and Reduces Obesity in db/db Mice.

Type 2 diabetes mellitus (T2DM), a complex metabolic disorder typically accompanying weight gain, is associated with progressive ß-cell failure and insulin resistance. Bariatric surgery ameliorates glucose tolerance and provides a near-perfect treatment. Duodenal-jejunal bypass (DJB) is an experimental procedure and has been studied in several rat models, but its influence in db/db mice, a transgenic model of T2DM, remains unclear. To investigate the effectiveness of DJB in db/db mice, we performed the surgery and evaluated metabolism improvement. Results showed that mice in DJB group weighed remarkably less than sham group two weeks after surgery. Compared to the preoperative level, postoperative fasting blood glucose (FBG) was dramatically reduced. Statistical analysis revealed that changes in body weight and FBG were significantly correlated. Besides, DJB surgery altered plasma insulin level with approximate 40% reduction. Thus, for the first time we proved that DJB can achieve rapid therapeutic effect in transgenic db/db mice with severe T2DM as well as obesity. In addition, decreased insulin level reflected better insulin sensitivity induced by DJB. In conclusion, our study demonstrates that DJB surgery may be a potentially effective way to treat obesity-associated T2DM.

MiR-9 Promotes Apoptosis Via Suppressing SMC1A Expression in GBM Cell Lines.

OBJECTIVE: Glioblastomas multiforme (GBM) is the most malignant brain cancer, which presented vast genomic variation with complicated pathologic mechanism. METHOD: MicroRNA is a delicate post-transcriptional tuner of gene expression in the organisms by targeting and regulating protein coding genes. MiR-9 was reported as a significant biomarker for GBM patient prognosis and a key factor in regulation of GBM cancer stem cells. To explore the effect of miR-9 on GBM cell growth, we over expressed miR-9 in U87 and U251 cells. The cell viability decreased and apoptosis increased after miR-9 overexpression in these cells. To identify the target of miR-9, we scanned miR-9 binding site in the 3'UTRs region of expression SMC1A (structural maintenance of chromosomes 1A) genes and designed a fluorescent reporter assay to measure miR-9 binding to this region. Our results revealed that miR-9 binds to the 3'sUTR region of SMC1A and down-regulated SMC1A expression. RESULT: Our results indicated that miR-9 was a potential therapeutic target for GBM through triggering apoptosis of cancer cells.

Duodenal long noncoding RNAs are associated with glycemic control after bariatric surgery in high-fat diet-induced diabetic mice.

BACKGROUND: The duodenum plays a role in the mechanism of type 2 diabetes remission after bariatric surgery. Roux-en-Y gastric bypass (RYGB) may change gene expression in the duodenum and metabolism. Long noncoding RNAs (lncRNAs) constitute a novel class of RNAs that regulate gene expression. Little is known about how duodenal lncRNAs respond to RYGB. Logically, studies on the changes of duodenal lncRNAs potentially can lead to an understanding of the mechanisms of bariatric surgery, as well as discovery of antidiabetic drug targets and biomarkers predicting postoperative outcome. OBJECTIVES: To investigate the expression signature of duodenal lncRNAs associated with glycemic improvement by duodenal-jejunal bypass (DJB), a component of RYGB, on a genome-wide scale in high-fat diet-induced diabetic mice. SETTING: University medical center. METHODS: High fat diet-induced diabetic mice were randomized into 2 groups receiving either the DJB or a sham procedure. Microarray was applied to screen the differentially expressed lncRNAs and messenger RNAs (mRNAs) in the duodenum between the DJB and sham groups, and the result was validated by quantitative real-time polymerase chain reaction in another cohort of animals. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the potential lncRNA functions. Based on Pearson correlation analysis, the lncRNA-mRNA and lncRNA-transcription factor (TF) interaction networks were constructed to identify and rank core regulatory lncRNAs and transcription factors. RESULTS: A total of 301 lncRNAs, including 232 that were upregulated and 69 downregulated (fold change≥2.0), were differentially expressed in the duodenum between the DJB and sham groups. GO enrichment indicated that these lncRNA-coexpressed mRNAs were correlated with biological processes including cell proliferation, digestion, and catabolic and biosynthetic processes. KEGG pathway analysis revealed that in addition to the digestion and absorption signaling pathways, pancreatic secretion- and inflammatory process-related signaling pathways were mostly enriched in the DJB group. In addition, the lncRNA-mRNA interaction network combined with GO and KEGG pathway analysis suggested that as a top-ranked gene, NONMMUG021726 may play an important role in the mechanism of type 2 diabetes remission after DJB. CONCLUSION: DJB leads to drastic changes in lncRNA and mRNA expressions in the duodenum. The majority of top-ranked lncRNAs and mRNAs have roles in pancreatic secretion and inflammatory processes, implying that bypass of the duodenum may initiate insulin secretion and attenuate inflammation. In addition, modulators of such lncRNAs, most likely NONMMUG021726, have potential to become therapeutic targets or biomarkers for prediction of the outcomes of bariatric surgery.

Gastric volume reduction is essential for the remission of type 2 diabetes mellitus after bariatric surgery in nonobese rats.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) has shown positive outcomes in the remission of type 2 diabetes (T2D) and weight loss in obese patients by inhibiting food intake and nutrient absorption as well as inducing favorable hormonal changes. The purpose of the present study was to investigate whether gastric volume reduction is still required in addition to intestinal bypass for the purpose of T2D remission in nonobese patients. SETTING: University Medical Center. METHODS: Nonobese T2D Goto-Kakizaki rats were employed in the study. All rats were randomized into 3 groups according to the surgical procedure performed, including (1) RYGB, (2) duodeno-jejunal bypass (DJB) without gastric volume reduction, and (3) sham surgery (control). In addition, age-matched Wistar rats were adopted as normal nondiabetic controls. Weight, food intake, fasting plasma glucose, and intraperitoneal glucose tolerance test were measured in vivo before and 2, 4, and 8 weeks after the treatment. Whole body metabolic parameters including respiratory exchange ratio, energy expenditure, and activities were also recorded in all animals at the third week postoperatively. RESULTS: Compared with DJB and sham animals, the RYGB group had lower weight, less food intake, lower fasting plasma glucose, and improved glucose tolerance at all measuring time points postoperatively. By measuring whole body metabolic parameters, we found that RYGB, but not DJB, increased metabolic rate manifested by increased energy expenditure but less activity at night. In the meantime, respiratory exchange ratio was lower in RYGB group than in the other 3 groups at daytime, meaning adipose tissue became the main source of internal energy production during the resting phase in the group. CONCLUSION: For nonobese T2D patients, adding gastric volume reduction to intestine bypass gave better efficacy in remission of T2D by increasing metabolic rate and adipolysis, especially during the resting period.

SalA attenuates ischemia/reperfusion-induced endothelial barrier dysfunction via down-regulation of VLDL receptor expression.

BACKGROUND: Salvianolic acid A (SalA) has been shown to confer robust protection against endothelial injury. VLDL receptor is expressed at high levels on the endothelial surface, however its biological effect on endothelial cells has not yet been completely elucidated. Here, we investigated molecular effects of SalA on endothelial VLDL expression and barrier dysfunction under conditions of ischemia/reperfusion (IS/RP). METHODS: Human umbilical vein endothelial cells (HUVECs) treated with SalA were subjected to IS/RP stimulation. Endothelial permeability, ZO-1 distribution, actin cytoskeleton reorganization, and intracellular reactive oxygen species (ROS) generation were examined. The mRNA levels were tested by real-time RT-PCR and the protein levels were determined by immunoblot analysis. RESULTS: Pretreatment of HUVECs with SalA markedly attenuated IS/RP-induced endothelial hyperpermeability, discontinuous ZO-1 staining, actin stress fiber formation, and intracellular ROS generation. IS/RP activated p38 MAPK signaling and enhanced VLDL receptor expression, and inactivation of p38 MAPK abolished increase of VLDL receptor expression. Furthermore, siRNA experiments showed that VLDL receptor was a crucial mediator of endothelial barrier dysfunction and intracellular ROS generation induced by IS/RP. Importantly, SalA effectively suppressed IS/RP-induced activation of p38 MAPK signaling and increase of VLDL receptor expression. CONCLUSION: These results for the first time demonstrated that SalA protected against IS/RP-induced endothelial barrier dysfunction through suppression of VLDL receptor expression.