Synaptotagmins family affect glucose transport in retinal pigment epithelial cells through their ubiquitination-mediated degradation and glucose transporter-1 regulation

BACKGROUND Synaptotagmins(SYTs)are a family of 17 membrane transporters that function as calcium ion sensors during the release of Ca2+-dependent neurotransmitters and hormones.However,few studies have reported whether members of the SYT family play a role in glucose uptake in diabetic retinopathy(DR)through Ca2+/glucose transporter-1(GLUT1)and the possible regulatory mechanism of SYTs.AIM To elucidate the role of the SYT family in the regulation of glucose transport in retinal pigment epithelial cells and explore its potential as a therapeutic target for the clinical management of DR.METHODS DR was induced by streptozotocin in C57BL/6J mice and by high glucose medium in human retinal pigment epithelial cells(ARPE-19).Bioinformatics analysis,reverse transcriptase-polymerase chain reaction,Western blot,flow cytometry,ELISA,HE staining,and TUNEL staining were used for analysis.RESULTS Six differentially expressed proteins(SYT2,SYT3,SYT4,SYT7,SYT11,and SYT13)were found between the DR and control groups,and SYT4 was highly expressed.Hyperglycemia induces SYT4 overexpression,manipulates Ca2+influx to induce GLUT1 fusion with the plasma membrane,promotes abnormal expression of the glucose transporter GLUT1 and excessive glucose uptake,induces ARPE-19 cell apoptosis,and promotes DR progression.Parkin deficiency inhibits the proteasomal degradation of SYT4 in DR,resulting in SYT4 accumulation and enhanced GLUT1 fusion with the plasma membrane,and these effects were blocked by oe-Parkin treatment.Moreover,dysregulation of the myelin transcription factor 1(Myt1)-induced transcription of SYT4 in DR further activated the SYT4-mediated stimulus-secretion coupling process,and this process was inhibited in the oe-MYT1-treated group.CONCLUSION Our study reveals the key role of SYT4 in regulating glucose transport in retinal pigment epithelial cells during the pathogenesis of DR and the underlying mechanism and suggests potential therapeutic targets for clinical DR.

Effects of suppressing glucose transporter-1 by an antisense oligodeoxynucleotide on the growth of human hepatocellular carcinoma cells

BACKGROUND:The glucose transporter-1(Glut-1),a key ratelimiting factor in the transport and metabolism of glucose in cancer cells,is over-expressed in many human cancer cells and this overexpression is correlated with poor biological behavior. The increased levels of Glut-1 expression in hepatocellular carcinoma(HCC)cells functionally affect tumorigenicity.This study was undertaken to investigate effects of suppressing Glut-1 by an antisense oligodeoxynucleotide(AS-ODN)on the growth of human hepatocellular carcinoma(HepG-2)cells. METHODS:We used AS-ODN targeting against the Glut-1 gene in a HepG-2 cell line.There were four experimental groups: empty pcDNA3.1 vector(mock transfection),pcDNA3.1-anti-Glut(+),pcDNA3.1-Glut(+),and non-transfected HepG-2 cells. The Glut-1 mRNA expression was detected by RT-PCR and the Glut-1 protein expression by Western blotting after cell culture, and the glucose uptake was detected after glucose stimulation in each group. RESULTS:Compared with non-transfected HepG-2 or Glut-1 pcDNA3.1,a down-regulation of Glut-1 mRNA in HepG-2 cells transfected with anti-Glut-1 pcDNA3.1 was noted(P<0.05).Glut-1 protein in HepG-2 cells transfected with Glut-1 AS-ODN was decreased compared with non-transfected HepG-2,Glut-1 pcDNA3.1,or empty vectors. Glucose uptake by the HepG-2 cells transfected with AS-ODN was decreased at 1 hour after glucose stimulation.CONCLUSIONS:The application of Glut-1 AS-ODN can down-regulate the expression of Glut-1 at mRNA and protein,and inhibit glucose uptake partially in HepG-2 cells.The Glut-1 gene maybe a potential therapeutic target for HCC.

Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） in rats, OBJECTIVE： This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-X^TM system. DESIGN： An in vitro cell-based experiment. SETTING： This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS： Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5 a and human embryonic kidney 293 cells （HEK293 cells） used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody （Chemicon, U.S.A.） and primers （Shanghai Boya Bioengineering Co., Ltd） were also used. METHODS： E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES： Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively. RESULTS： Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION： We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.

Current and Future Clinical Applications of Zinc Transporter-8 in Type 1 Diabetes Mellitus

Objective： To evaluate the utility of zinc transporter-8 （ZnT8） in the improvement of type 1 diabetes mellitus （T1DM） diagnosis and prediction, and to explore whether ZnT8 is a potential therapeutic target in TI DM. Data Sources： A search was conducted within the medical database PubMed for relevant articles published from 2001 to 2015. The search terms are as follows： ＂ZnTS,＂ ＂type 1 diabetes,＂ ＂latent autoimmune diabetes in adults,＂ ＂type 2 diabetes,＂ ＂islet autoantibodies,＂ ＂zinc supplement,＂ ＂T cells,＂ ＂[3 cell,＂ ＂immune therapy.＂ We also searched the reference lists of selected articles. Study Selection： English-language original articles and critical reviews concerning ZnT8 and the clinical applications of islet autoantibodies in diabetes were reviewed. Results： The basic function of ZnT8 is maintaining intracellular zinc homeostasis, which modulates the process of insulin biosynthesis, storage, and secretion. Autoantibodies against ZnT8 （ZnTSA） and ZnTS-specific T cells are the reliable biomarkers for the identification, stratification, and characterization of T1DM. Additionally, the results from the animal models and clinical trials have shown that ZnT8 is a diabetogenic antigen, suggesting the possibility of ZnT8-specific immunotherapy as an alternative for T1DM therapy. Conclusions： ZnT8 is a novel islet autoantigen with a widely potential for clinical applications in T1DM. However, before the large-scale clinical applications, there are still many problems to be solved.

Regulation of the expression and function of glucose transporter-1 by TGF- β1 and high glucose in mesangial cells

Objective To study the effects of high glucose and transforming growth factor β1 (TGF β1) on the expression and function of glucose transporter 1 (GLUT1) in mouse mesangial cells Methods Cultured mouse mesangial cells were used The expression of GLUT1 mRNA was detected by Northern Blot; glucose uptake and its kinetics were determined with a 2 Deoxy [ 3H] D glucose uptake assay Results Mesangial cells exposed to enriched glucose medium (20?mmol/L) for 72 hours demonstrated a decrease in both GLUT1 mRNA and V max for uptake of the glucose analog, 2 deoxy D glucose (2DOG), as compared to mesangial cells cultured in physiologic glucose concentrations(5 5?mmol/L) In contrast, hypertonic mannitol had no effect on GLUT1 mRNA levels TGF β1 treatment for 10 hours stimulated 2DOG uptake, both in 5 5?mmol/L and 20?mmol/L glucose medium, by approximately 4 28 fold in a dose dependent manner (2?ng/ml maximum) Kinetic analysis of 2DOG uptake revealed an increase in V max and a decrease in K m in the presence of TGF β1 TGF β1 also up regulated the expression of GLUT1 mRNA in mesangial cells The addition of anti TGF β neutralizing antibody (30?μg/ml) in mesangial cells cultured in enriched glucose medium (20?mmol/L) led to a 40% decrease in 2DOG uptake Conclusions The expression of GLUT1 can be suppressed by exposure of mesangial cells to high glucose medium, which may serve as a protective mechanism against possible adverse effects of excessive glucose flux into cells TGF β1 stimulates glucose uptake by enhancing the expression and function of GLUT1 in mesangial cells This effect is independent of the glucose milieu in the cultured medium

乳腺浸润性导管癌血氧水平依赖功能磁共振成像与葡萄糖转运蛋白1表达的关系

目的 探讨血氧水平依赖功能磁共振成像（BOLD-fMRI）的R2＊值与乳腺浸润性导管癌组织葡萄糖转运蛋白1 （GLUT-1）表达的相关性.方法 回顾性分析2011年12月至2013年6月本院接受乳腺癌手术治疗且术后病理证实为乳腺浸润性导管癌患者77例的临床资料,所有患者均于术前行乳腺BOLD-fMRI检查并测定肿瘤组织的R2＊值.免疫组化染色检测肿瘤组织GLUT-1的表达.Spearman相关分析肿瘤组织BOLD-fMRI R2＊值与GLUT-1表达的关系.结果 病理学检查显示77例患者均为乳腺浸润性导管癌,肿瘤分级为Ⅰ级3例、Ⅱ级48例、Ⅲ级26例,其中伴腋窝淋巴结转移40例.免疫组化显示肿瘤组织GLUT-1表达阴性6例、弱阳性11例、中度阳性25例、强阳性35例,GLUT-1表达阳性率为92.2％（71/77）.腋窝淋巴结转移组GLUT-1表达显著高于无腋窝淋巴结转移组（x2=5.718,P=0.016）.BOLD-fMRI检查显示乳腺浸润性导管癌呈混杂信号,R2＊值为26.4～90.5 Hz,平均（54.9±16.9）Hz.Spearman相关分析表明,乳腺浸润性导管癌肿瘤组织R2＊值与GLUT-1的表达呈正相关（r=0.587,P=0.000）.结论 BOLD-fMRI可用于无创性评价乳腺浸润性导管癌组织慢性乏氧及葡萄糖代谢水平.

Hepatitis C virus infection and insulin resistance

Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.

Dynamic expression of cerebral cortex and hippocampal glutamate transporters in a rat model of chest compression-induced global cerebral ischemia

The present study established a rat model of global cerebral ischemia induced by chest compression for six minutes to dynamically observe expressional changes of three glutamate transporters in the cerebral cortex and hippocampus. After 24 hours of ischemia, expression of glutamate transporter-1 significantly decreased in the cerebral cortex and hippocampus, which was accompanied by neuronal necrosis. At 7 days post-ischemia, expression of excitatory amino acid carrier 1 decreased in the hippocampal CA1 region and cortex, and was accompanied by apoptosis Expression of glutamate-aspartate transporter remained unchanged at 6 hours 7 days after ischemia. These results suggested that glutamate transporter levels were altered at different periods of cerebral ischemia.

Corticosterone induced D-serine release deficit play an important role in long-term potentiation impairment by corticosterone in perforant path-dentate gyrus pathway

OBJECTIVE Previous studies showed that over activation of NMDA receptors may be a crucial cause of long-term potentiation(LTP)and cognitive impairment induced by stress or corticosterone.However,other studies showed that the function of NMDA receptors is insufficient since the NMDA receptors co-agonist D-serine could improve stress-induced cognitive impairment.The purpose of this study is to clarify whether over activation of NMDA receptors or hypofunction of NMDA receptors is involved in hippocampal impairment of LTP by corticosterone and the underlying mechanisms.METHODS Cort was injected subcutaneously 1 h before the high-frequency stimulation(HFS)to induce LTP impairment.NMDA receptor antagonists and agonists were administrated by icv.RESULTS Hippocampal LTP and object location recognition memory were impaired in corticosterone-treated mice.Corticosterone increased the glutamate level in hippocampal tissues,neither NMDA receptors antagonist nor its subtype antagonists alleviated impairment of LTP,while enhancing the function of NMDA receptors by D-serine did alleviate impairment of LTP by corticosterone,suggesting that hypofunction of NMDA receptors might be one of the main reasons for impairment of LTP by corticosterone.Further results showed that the level of D-serine and its precursor L-serine did not change.D-serine release-related protein Na+-independent alanine-serine-cysteine transporter-1(ASC-1)in the cell membrane was decreased and increasing D-serine release by the selective activator of ASC-1 antiporter activity alleviated impairment of LTP by corticosterone.CONCLUSION Taken together,this study demonstrates that hypofunction of NMDA receptors may be involved in impairment of LTP by corticosterone and reduced D-serine release may be an important reason for its hypofunction,which is an important complement to existing mechanisms of corticosterone-induced LTP and cognitive impairment.

New and emerging therapies in gout

Gout is a disease that has been pestering humankind for centuries.Hence,gout therapeutics has always been a field of ongoing research.Beginning from colchicine,which was the first drug to come into use,to the latest inflammasome inhibitors and monoclonal antibodies,therapies for gout have evolved remarkably over the years.Although gout-related research came to a standstill in the late 20th century,the 21st century witnessed a renaissance in gout therapeutics with the advent of new xanthine oxidase inhibitors.Our present armamentarium against gout includes drugs that exploit the newfound knowledge about the various mechanisms involved in the pathogenesis of gout as well as drugs used in other conditions,which are effective in gout as well.Many more drugs that promise better management of gout in the future are in the pipeline.

Molecular characterization of circulating tumor cells in pancreatic ductal adenocarcinoma:potential diagnostic and prognostic significance in clinical practice

Background:The clinical value of heterogeneous sub-populations of circulating tumor cells(CTCs)in pancreatic ductal adenocarcinoma(PDAC)remains unclear.Methods:Peripheral blood samples were obtained from 67 PDAC patients.CTCs were isolated by employing CD45 negative enrichment technique and further characterized for epithelial to mesenchymal transition(EMT)or human equilibrative nucleoside transporter-1(hENT-1).The relationships between CTCs sub-phenotypes with clinicopathological factors or post-operative recurrence in PDAC patients were analyzed.Results:EMT related CTCs could be isolated and identified from the 81%of patients(54/67),and both the total count(median:5 vs.17/mL,P<0.0001)and M-CTC percentage(median:0.2 vs.0.345,P=0.0244)of CTCs could differentiate local/regional with metastatic disease.Multivariate analysis showed that both AJCC stage(P=0.025)and M-CTC percentage(P=0.001)were independent prognostic indicators of recurrence free survival(RFS)in resected patients.Moreover,Kaplan-Meier survival analysis showed that M-CTC after 2 courses of chemotherapy was significantly associated with inferior RFS(49.5 weeks vs.undefined,P=0.0288).No significant correlation in hENT-1 expression was found between CTCs and matched tumor tissues,and further multivariate analysis suggested hENT-1 expression in CTCs as independent prognostic factor for RFS(P=0.016).Patients with low hENT-1 expression in CTCs had decreased RFS(32 weeks vs.undefined,P=0.0337).Conclusions:CTCs could be the promising diagnostic biomarkers in PDAC patients,and phenotypic profiling of CTCs based on EMT or hENT-1 could help establish novel prognostic biomarkers in resected patients undergoing adjuvant gemcitabine-based chemotherapy.

Adolescent stress increases depression-like behaviors and alters the excitatory-inhibitory balance in aged mice

Background:Depression affects approximately 5% of elderly people and its etiology might be related to chronic stress exposure during neurodevelopmental periods.In this study,we examined the effects of adolescent chronic social stress in aged mice on depressive behaviors and the excitatory-inhibitory (E/I) balance in stress-sensitive regions of the brain.Methods:Sixty-four adolescent,male C57BL/6 mice were randomly assigned to either the 7-week (from post-natal days 29 to 77) social instability stress (stress group,n =32) or normal housing conditions (control group,n =32).At 15 months of age,16 mice were randomly selected from each group for a series of behavioral tests,including two depression-related tasks (the sucrose preference test and the tail suspension test).Three days following the last behavioral test,eight mice were randomly selected from each group for immunohistochemical analyses to measure the cell density of parvalbumin (PV+)-and calretinin (CR+)-positive gamma-aminobutyric-acid (GABA)ergic inhibitory inter-neurons,and the expression levels of vesicular transporters of glutamate-1 (VGIuT1) and vesicular GABA transporter (VGAT) in three stress-sensitive regions of the brain (the medial pre-frontal cortex [mPFC],hippocampus,and amygdala).Results:Behaviorally,compared with the control group,adolescent chronic stress increased depression-like behaviors as shown in decreased sucrose preference (54.96 ± 1.97% vs.43.11 ± 2.85%,t(22)=3.417,P =0.003) and reduced latency to immobility in the tail suspension test (92.77 ± 25.08 s vs.33.14 ± 5.95 s,t(25)=2.394,P =0.025),but did not affect anxiety-like behaviors and pre-pulse inhibition.At the neurobiologic level,adolescent stress down-regulated PV+,not CR+,inter-neuron density in the mPFC (F(1,39)=19.30,P < 0.001),and hippocampus (F(1,42)=5.823,P =0.020) and altered the CR+,not PV+,inter-neuron density in the amygdala (F(1,28)=23.16,P < 0.001).The VGluT1/VGAT ratio was decreased in all three regions (all F > 10.09,all P < 0.004),which suggests stress-induced hypoexcitability in these regions.Conclusions:Chronic stress during adolescence increased depression-like behaviors in aged mice,which may be associated with the F/I imbalance in stress-sensitive brain regions.