Value of glucose transport protein 1 expression in detecting lymph node metastasis in patients with colorectal cancer

BACKGROUND There are limited data on the use of glucose transport protein 1(GLUT-1)expre-ssion as a biomarker for predicting lymph node metastasis in patients with colorectal cancer.GLUT-1 and GLUT-3,hexokinase(HK)-II,and hypoxia-induced factor(HIF)-1 expressions may be useful biomarkers for detecting primary tumors and lymph node metastasis when combined with fluorodeoxyglucose(FDG)uptake on positron emission tomography/computed tomography(PET/CT).AIM To evaluate GLUT-1,GLUT-3,HK-II,and HIF-1 expressions as biomarkers for detecting primary tumors and lymph node metastasis with 18F-FDG-PET/CT.METHODS This retrospective study included 169 patients with colorectal cancer who underwent colectomy and preoperative 18F-FDG-PET/CT at Chungbuk National University Hospital between January 2009 and May 2012.Two tissue cores from the central and peripheral areas of the tumors were obtained and were examined by a dedicated pathologist,and the expressions of GLUT-1,GLUT-3,HK-II,and HIF-1 were determined using immunohisto-chemical staining.We analyzed the correlations among their expressions,various clinicopathological factors,and the maximum standardized uptake value(SUVmax)of PET/CT.RESULTS GLUT-1 was found at the center or periphery of the tumors in 109(64.5%)of the 169 patients.GLUT-1 positivity was significantly correlated with the SUVmax of the primary tumor and lymph nodes,regardless of the biopsy site(tumor center,P<0.001 and P=0.012;tumor periphery,P=0.030 and P=0.010,respectively).GLUT-1 positivity and negativity were associated with higher and lower sensitivities of PET/CT,respectively,for the detection of lymph node metastasis,regardless of the biopsy site.GLUT3,HK-II,and HIF-1 expressions were not significantly correlated with the SUVmax of the primary tumor and lymph nodes.CONCLUSION GLUT-1 expression was significantly correlated with the SUVmax of 18F-FDG-PET/CT for primary tumors and lymph nodes.Clinicians should consider GLUT-1 expression in preoperative endoscopic biopsy in interpreting PET/CT findings.

Transforming growth factor beta-1 upregulates glucose transporter 1 and glycolysis through canonical and noncanonical pathways in hepatic stellate cells

BACKGROUND Hepatic stellate cells(HSCs)are the key effector cells mediating the occurrence and development of liver fibrosis,while aerobic glycolysis is an important metabolic characteristic of HSC activation.Transforming growth factor-β1(TGF-β1)induces aerobic glycolysis and is a driving factor for metabolic reprogramming.The occurrence of glycolysis depends on a high glucose uptake level.Glucose transporter 1(GLUT1)is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism,thus affecting cell proliferation and growth.However,little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs.AIM To investigate the mechanisms of action of GLUT1,TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis.METHODS Immunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue.A Seahorse extracellular flux(XF)analyzer was used to examine changes in aerobic glycolytic flux,lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation.The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothersagainst-decapentaplegic-homolog 2/3(Smad2/3)signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase(PI3K)/AKT signaling pathways.In addition,GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs.Finally,a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis.RESULTS GLUT1 protein expression was increased in both mouse and human fibrotic liver tissues.In addition,immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins,indicating that GLUT1 expression was related to the development of liver fibrosis.TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad,p38 MAPK and P13K/AKT signaling pathways.The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression.GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration.A GLUT1 inhibitor was administered in a mouse model of liver fibrosis,and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis.CONCLUSION TGF-β1 induces GLUT1 expression in HSCs,a process related to liver fibrosis progression.In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs.In addition,in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.

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.

Transcriptional activation of glucose transporter 1 in orthodontic tooth movement-associated mechanical response

The interplay between mechanoresponses and a broad range of fundamental biological processes, such as cell cycle progression,growth and differentiation, has been extensively investigated. However, metabolic regulation in mechanobiology remains largely unexplored. Here, we identified glucose transporter 1(GLUT1)—the primary glucose transporter in various cells—as a novel mechanosensitive gene in orthodontic tooth movement(OTM). Using an in vivo rat OTM model, we demonstrated the specific induction of Glut1 proteins on the compressive side of a physically strained periodontal ligament. This transcriptional activation could be recapitulated in in vitro cultured human periodontal ligament cells(PDLCs), showing a time-and dose-dependent mechanoresponse. Importantly, application of GLUT1 specific inhibitor WZB117 greatly suppressed the efficiency of orthodontic tooth movement in a mouse OTM model, and this reduction was associated with a decline in osteoclastic activities. A mechanistic study suggested that GLUT1 inhibition affected the receptor activator for nuclear factor-κ B Ligand(RANKL)/osteoprotegerin(OPG)system by impairing compressive force-mediated RANKL upregulation. Consistently, pretreatment of PDLCs with WZB117 severely impeded the osteoclastic differentiation of co-cultured RAW264.7 cells. Further biochemical analysis indicated mutual regulation between GLUT1 and the MEK/ERK cascade to relay potential communication between glucose uptake and mechanical stress response. Together, these cross-species experiments revealed the transcriptional activation of GLUT1 as a novel and conserved linkage between metabolism and bone remodelling.

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.

Effects of electroacupuncture on microcirculatory blood flow and glucose transporter function in the hippocampus

Nerve cell metabolism in post brain ischemia depends on increased microcirculation perfusion and transport function of microvascular endothelial cells. In the present study, a rat model of middle cerebral artery occlusion was established to investigate the influence of electroacupuncture （EA） on hippocampal CA1 cerebral blood flow and glucose transporter 1 （GLUT1） expression in the microvascular endothelial cells. Following EA at Neiguan （PC 6）, the cerebral blood flow in the ischemic hippocampal CA1 region was significantly elevated, the number and microvascular integrated absorbance of the GLUTl-positive cells were significantly increased, nerve cell damage was ameliorated, and GLUT1 protein expression in the ischemic hippocampus was significantly increased. Results demonstrate that EA increased the cerebral blood flow of the hippocampal CA1 region and improved the glucose transport function, thereby attenuating neuronal injuries.

Glucose transporter expression in the human colon

AIM To investigate by immunostaining glucose transporter expression in human colorectal mucosa in controls and patients with inflammatory bowel disease(IBD). METHODS Colorectal samples were obtained from patients undergoing lower endoscopic colonoscopy or rectosigmoidoscopy. Patients diagnosed with ulcerativecolitis(n = 18) or Crohn's disease(n = 10) and scheduled for diagnostic colonoscopy were enrolled. Patients who underwent colonoscopy for prevention screening of colorectal cancer or were followed-up after polypectomy or had a history of lower gastrointestinal symptoms were designated as the control group(CTRL, n = 16). Inflammatory status of the mucosa at the sampling site was evaluated histologically and/or endoscopically. A total of 147 biopsies of colorectal mucosa were collected and processed for immunohistochemistry analysis. The expression of GLUT2, SGLT1, and GLUT5 glucose transporters was investigated using immunoperoxidase labeling. To compare immunoreactivity of GLUT5 and LYVE-1, which is a marker for lymphatic vessel endothelium, doublelabeled confocal microscopy was used. RESULTS Immunohistochemical analysis revealed that GLUT2, SGLT1, and GLUT5 were expressed only in short epithelial portions of the large intestinal mucosa. No important differences were observed in glucose transporter expression between the samples obtained from the different portions of the colorectal tract and between the different patient groups. Unexpectedly, GLUT5 expression was also identified in vessels, mainly concentrated in specific areas where the vessels were clustered. Immunostaining with LYVE-1 and GLUT5 antibodies revealed that GLUT5-immunoreactive(-IR) clusters of vessels were concentrated in areas internal to those that were LYVE-1 positive. GLUT5 and LYVE-1 did not appear to be colocalized but rather showed a close topographical relationship on the endothelium. Based on their LYVE-1 expression, GLUT5-IR vessels were identified as lymphatic. Both inflamed and noninflamed mucosal colorectal tissue biopsies from the IBD and CTRL patients showed GLUT5-IR clusters of lymphatic vessels. CONCLUSION Glucose transporter immunoreactivity is present in colorectal mucosa in controls and IBD patients. GLUT5 expression is also associated with lymphatic vessels. This novel finding aids in the characterization of lymphatic vasculature in IBD patients.

BcSDR1 is involved in regulation of glucose transport and cAMP and MAPK signaling pathways in Botrytis cinerea

Botrytis cinerea is a typical necrotrophic pathogenic fungus that causes severe diseases in a wide range of plant species, leading to significant economic losses. Our previous study showed that BcSDR1 positively regulates growth,development, and pathogenicity of B. cinerea. However, the regulation mechanism of BcSDR1 and the relationship between BcSDR1 and cAMP and MAPK signaling pathways are not well understood. In this study, transcriptome data showed that BcSDR1 is involved in glucose transmembrane transport, signal transduction, secondary metabolism, and other biological processes. BcSDR1 mutant(BCt41) showed remarkably weak sensitivity to cAMP and MAPK signaling pathways specific inhibitors, SQ22536 and U0126, and significantly decreased cAMP content. The key genes of cAMP and MAPK signaling pathways, BcGB1, BcBTP1, BcBOS1, BcRAS1, and BcBMP3 were significantly upregulated,whereas BcPLC1, BcBCG1, BcCDC4, BcSAK1, BcATF1, and BcBAP1 were significantly downregulated(P<0.05).BcSDR1 was obviously upregulated in BcBCG2, BcBCG3, BcPKA1, and BcPKAR RNA interference(RNAi) mutants, but significantly downregulated in BcPKA2, BcBMP1, and BcBMP3 RNAi mutants. Thus, BcBCG2, BcBCG3, BcPKA1, and BcPKAR negatively regulate BcSDR1 expression, whereas BcPKA2, BcBMP1, and BcBMP3 positively regulate BcSDR1expression.

Icariin ameliorates learning and memory function via improving cerebral glucose metabolism disorder in APP/PS1/Tau triple transgenic Alzheimer disease mice

OBJECTIVE To investigate the protective effect of icariin(ICA) on learning and memory function in APP/PS1/Tau triple transgenic Alzheimer disease mice(3×Tg-AD mice),and then to explore whether its mechanism is related to the improvement of brain glucose metabolism disorder.METHODS Three-month-old male 3 ×Tg-AD mice were randomly divided into three groups(n=10):3×Tg group,3×Tg+ICA low-dose group(30 mg·kg-1) and 3×Tg + ICA high-dose group(60 mg·kg-1).Age-matched male wild type(WT) mice were randomly divided into two groups(n=10):WT control group and WT+ICA60 mg·kg-1 group.ICA in vehicle(0.5% Tween-80 in distilled water) was given orally once a day for five months in the 3×Tg+ICA groups.3×Tg and WT control group were given an equal volume vehicle.Morris water maze was used to detect the learning and memory function of mice.Brain glucose metabolism in 3×Tg mice was observed by 18 F-FDG microPET imaging technique.Nissl staining and HE staining were used to evaluate the survival neurons in hippocampus of mice.Glucose oxidase assay was used to detect glucose contents in cortex of mice.The protein expression of APP,Aβ1-40,Aβ1-42 and glucose transporter 1(GLUT1),and the phosphorylation level of tau protein at multiple sites in hippocampus were detected by Western blotting.RESULTS Behavioral examination revealed a profound decrease learning and memory function,accompanied by a decrease in number of neuronal cells in 3×Tg-AD mice.Moreover,the cerebral18 F-FDG uptake rate per gram tissue was reduced and the glucose contents in the cortex were increased in 3×Tg-AD mice.In addition,Western blotting analysis showed that the expression of APP,Aβ1-40,Aβ1-42 proteins and the levels of tau protein phosphorylation at Ser199/202 and PHF-1(Ser396/404) sites were increased significantly,followed by a decrease of GLUT1 expression in hippocampus of 3×Tg-AD mice.All of these changes in behavioral functions,neuronal loss and related protein expression were reversed when mice were treated with ICA.CONCLUSION ICA can improve the learning and memory ability of AD model mice,the mechanism may be related to the improvement of cerebral glucose metabolism dysfunction by increasing the expression of GLUT1.

Effect of exercise during pregnancy on offspring development through ameliorating high glucose and hypoxia in gestational diabetes mellitus

BACKGROUND Gestational diabetes mellitus(GDM)women require prenatal care to minimize short-and long-term complications.The mechanism by which exercise during pregnancy affects organ development and whether glucose transporter(GLUT)1 plays a role in GDM offspring organ development remains unknown.AIM To determine the effect of exercise during pregnancy on the cardiac,hepatic and renal development of GDM mother’s offspring.METHODS Placenta samples were collected from humans and mice.GDM mouse models were created using streptozotocin along with a GDM with exercise group.The hearts,livers and kidneys of 3-and 8-week-old offspring were collected for body composition analysis and staining.The effects of high glucose levels and hypoxia were investigated using HTR8/SVneo.Transwell and wound-healing assays were performed to assess cell migration.Immunofluorescence accompanied with TUNEL and Ki67 staining was used to explore apoptosis and proliferation.RESULTS Exercise during pregnancy downregulated the GLUT1 and hypoxia inducible factor-1αexpression in placenta from individuals with GDM.Cobalt chloride induced hypoxia and high glucose levels also significantly decreased migration and apoptosis of HTR8/SVneo cells.In addition,exercise reduced inflammatory cell infiltration in the liver and decreased the tubular vacuolar area in the kidneys of offspring.CONCLUSION GDM affects the growth and development of organs in offspring.Exercise during pregnancy can reverse adverse effects of GDM on the development of the heart,liver,and kidney in offspring.

Photoactivation of GLUT4 translocation promotes glucose uptake via PI3-K/Akt2 signaling in 3T3-L1 adipocytes

Insulin resistance is a hallmark of the metabolic syndrome and type 2 diabetes.Dysfunction of PI-3K/Akt signaling was involved in insulin resistance.Glucose transporter 4(GLUT4)is a keyfactor for glucose uptake in muscle and adipose tissues,which is closely regulated by Pi-3K/Aktsignaling in response to insulin treatment.Low-power laser irradiation(LPLI)has been shown toregulate various physiological processes and induce the synthesis or release of multiple moleculessuch as growth factors,which(especially red and near infrared light)is mainly through theactivation of mitochondrial respiratory chain and the initiation of intracellular signaling path-ways.Nevertheless,it is unclear whether LPLI could promote glucose uptake through activationof PI-3K/Akt/GLUT4 signaling in 3T3L-1 adipocytes.In this study,we investigated how LPLIpromoted glucose uptake through activation of PI-3K/Akt/GLUT4 signaling path way.Here,we showed that GLUT4 was localized to the Golgi apparatus and translocated from cytoplasm tocytomembrane upon LPLI treatment in 3T3L-1 adipocytes,which enhanced glucose uptake.Moreover,we found that glucose uptake was mediated by the PI3-K/Akt2 signaling,but notAkt1 upon LPLI treatment with Akt isoforms gene silence and PI3-K/Akt inhibitors.Collec-tively,our results indicate that PI3-K/Akt2/GLUT4 signaling act as the key regulators forimprovement of glucose uptake under LPLI treatment in 3T3L-i adipocytes.More importantly,our findings suggest that activation of PI3-K/Akt2/GLUT4 signaling by LPLI may provideguidance in practical applications for promotion of glucose uptake in insulin-resistant adiposetissue.

The influence of OCT1 gene polymorphisms on the metformin response in Uygur patients with glucose metabolism disorder

Objective To determine the effects of genetic variation in the organic cation transporter 1(OCT1)on the short-term responses of the antidiabetic drug,metformin.Method A total of 22 patients recruited with type 2 diabetes or IFG were treated with metformin(2 000 mg/day)for 1 week.The patients were screened from Second Jikun hospital and Kashidonglu community medicine service,Urumqi,China and their surrounding districts.To examine the effects of metformin on plasma glucose,total cholesterol,low-density lipoprotein-cholesterol,high-density lipoprotein-cholesterol and triglyceride in relation with R61C,G465R and 420 del variants of OCT1(gene encoding organic cation transporter 1,mainly locating in liver,which is metformin's major target)in subjects.In all,R61C,G465R and 420del of OCT1 gene were examined using DNA extracted from whole blood and PCR-RFLP.Data concerning with gene and metformin treatment were handled by t-test.Result After metformin treatment,there were increases both in FPG and LDL(P=0.011and P=0.013 respectively).To divide all participants into mutant and wild groups,according to the polymorphisms of R61C,G465R and 420 del respectively,as well as carriers with one of the mutant genotypes at least and carriers with none of the mutant sites.Analysis was made to compared FPG,Chol,TG,and LDL and HDL between carriers of wild genotypes and carriers of other genotypes showed no statistic significance both before the metformin treatment and after the treatment.The same is the case with changes of FPG,Chol,TG,and LDL and HDL of wild genotype carriers and variant genotype carriers,except of LDL changes(P=0.05)in patients grouped by G465R polymorphisms and TG changes(P=0.03)in subjects differed by 420del genotypes.Conclusion In this study,it is suggested that OCT1 gene polymorphisms have little contribution to the clinical efficacy of blood glucose control by metformin among Uygur people with type 2 diabetes or IFG,but it may have possible relationship with the clinical efficacy on fat metabolism by metformin.

Cinnamon free phenolic extract regulates glucose absorption in intestinal cells by inhibiting glucose transporters

Cinnamon is a traditional herbal medicine that is a valuable source of bioactive phenolic compounds.In this study,we determined the effects of cinnamon free phenolic extract(CFPE)on glucose transport in Caco-2 cells and its possible mechanisms of action.First,the contents of phenolic compounds and flavonoids in bark were compared among four cinnamon cultivars.The‘Taiwan’cultivar had the highest contents of these compounds,and the CFPE was prepared from it.A Caco-2 monolayer was established,and the effect of CFPE on glucose transport across the monolayer was determined.The results showed that CFPE(at 150-300μg/mL)suppressed glucose transport across the monolayer in a dose-dependent manner.Procyanidin C1,procyanidin B2,procyanidin B1,procyanidin B3,procyanidin A2,and(􀀀)-epicatechin derivatives in CFPE may have contributed to its suppressive effect.Treatment with CFPE decreased the transcript levels of SGK1 encoding serum and glucocorticoid-inducible kinase and PLC/PKC encoding phospholipase C/protein kinase C,which are involved in the signaling pathway associated with glucose transport;and the transcript levels of SGLT1 encoding sodium/glucose cotransporter 1 and GLUT2 encoding glucose transporter 2,the key glucose transporters in this pathway.These findings suggest that CFPE inhibits glucose transport via its effects on the signaling pathway and glucose transporters involved in glucose absorption in intestinal cells.Thus,CFPE has potential applications in preventing postprandial hyperglycemia.

肾上腺嗜铬细胞瘤患者组织葡萄糖转运蛋白1和拓扑异构酶ⅡA表达与临床特征及预后的相关性分析

目的探讨葡萄糖转运蛋白1(GLUT1)和拓扑异构酶ⅡA(TOP2A)在肾上腺嗜铬细胞瘤(PPGL)患者组织中的表达与临床特征及预后的相关性。方法选取2014年3月至2020年6月于新疆维吾尔自治区人民医院行手术切除的120例PPGL组织作为实验组。另收集同期行手术切除的100例无功能肾上腺腺瘤的肾上腺组织作为对照组。采用免疫组织化学染色法检测组织中GLUT1和TOP2A表达情况。通过Cox回归分析PPGL患者预后的影响因素。结果实验组中GLUT1的低表达率和TOP2A的高表达率均高于对照组[51.7%(62/120)比14.0%(14/100)、52.5%(63/120)比19.0%(19/100)],差异均有统计学意义(χ^(2)=34.225、31.829,均P<0.001)。GLUT1、TOP2A表达水平与镜下组织浸润和Ki-67有关(均P<0.05)。预后良好组中GLUT1高表达和TOP2A低表达比例均高于预后不良组,差异均有统计学意义(均P<0.001)。单因素分析结果显示,患者镜下组织浸润、Ki-67、GLUT1、TOP2A均是影响预后的因素(均P<0.001)。Cox多因素回归分析显示TOP2A、镜下组织浸润及Ki-67均是导致PPGL患者预后不良的危险因素,GLUT1为保护因素(均P<0.001)。结论GLUT1在PPGL组织中呈低表达,TOP2A呈高表达,与患者预后不良有关。

Low glucose metabolism in hepatocellular carcinoma with GPC3 expression

AIM To investigate the relationship between glucose metabolism and glypican-3(GPc3)expression in hepatocellular carcinoma(Hcc).METHODSImmunohistochemical staining of pathological samples for GPc3 and glucose transporter 1(GLUT1),and whole-body ^(18)F-FDG PET/c T for measuring tumour glucose uptake were performed in 55 newly diagnosed Hcc patients.The maximum standard uptake value(s UVmax)and tumour-to-non-tumourous liver uptake(T/NT)ratio were used to quantify ^(18)F-FDG uptake.In vitro ^(18)F-FDG uptake assay of GPc3-expressing Hep G2 and non-GPc3-expressing RH7777 cel ls was used to examine the effect of GPc3 in cellular glucose metabolism.The relationships between GPc3 expression and ^(18)F-FDG uptake,GLUT1 expression,tumour differentiation,and other clinical indicators were analysed using spearman rank correlation,univariateand multiple logistic regression analyses.RESULTSPositive GPc3 expression was observed in 67.3%of Hcc patients,including 75.0%of those with well or moderately differentiated Hcc and 36.4%of those with poorly differentiated Hcc.There was an inverse relationship between GPc3 expression and s UVmax(Spearman correlation coefficient=-0.281,P=0.038)and a positive relationship between GLUT1 expression and sU Vmax(Spearman correlation coefficient=0.681,P<0.001)in patients with Hcc.Univariate analysis showed that two glucose metabolic parameters(sU Vmax and T/NT ratio),tumour differentiation,lymph node metastasis,and TNM stage were all significantly associated with GPc3 expression(P<0.05),whereas GLUT1 expression,sex,age,tumour size,intrahepatic lesion number,and distant metastasis showed no statistical association(P>0.05).Further multivariate analysis revealed that only the T/N ratio was significantly correlated with GPC3 expression in patients with Hcc(P<0.05).In vitro assay revealed that the uptake of ^(18)F-FDG in GPc3-expressing HepG2 cells was significantly lower than that of non-GPc3-expressing RH7777 cells(t=-20.352,P<0.001).CONCLUSIONThe present study demonstrated that GPc3 expression is inversely associated with glucose metabolism,suggesting that GPc3 may play a role in regulating glucose metabolism in Hcc.

Characterization of monocarboxylate transporter activity in hepatocellular carcinoma

AIM: To assess the immunoexpression of hypoxia-related markers in samples from cirrhosis and primary and metastatic hepatocellular carcinoma (HCC).

Possible mechanism for the regulation of glucose on proliferation, inhibition and apoptosis of colon cancer cells induced by sodium butyrate

AIM： To study the effect of glucose on sodium butyrate- induced proliferation inhibition and apoptosis in HT-29 cell line, and explored its possible mechanisms. METHODS： HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, and were allowed to adhere for 24 h, and then replaced with experimental medium. Cell survival rates were detected by MTr assay. Apoptosis was detected by TUNEL assay. Glucose transport protein 1 （GLUT1） and monocarboxylate transporter 1 （MCT1） mRNA expression was detected by RT-PCR. RESULTS： Low concentration of glucose induced apoptosis and regulated proliferation in HT-29 cell line, and glucose can obviously inhibit the effect of proliferation inhibition and apoptosis induced by sodium butyrate. Glucose also down-regulated the expression of MCT1mRNA （0.28 ± 0.07 vs 0.19± 0.10, P 〈 0.05）, and decreased the expression of GLUTlmRNA slightly （0.18 ± 0.04 vs 0.13 ± 0.03, P 〈 0.05）. CONCLUSION： Glucose can regulate the effect of proliferation inhibition and apoptosis induced by sodium butyrate and this influence may be associated with the intracellular concentration of glucose and sodium butyrate.

Free fatty acids receptors 2 and 3 control cell proliferation by regulating cellular glucose uptake

BACKGROUND Colorectal cancer(CRC)is a worldwide problem,which has been associated with changes in diet and lifestyle pattern.As a result of colonic fermentation of dietary fibres,short chain free fatty acids are generated which activate free fatty acid receptors(FFAR)2 and 3.FFAR2 and FFAR3 genes are abundantly expressed in colonic epithelium and play an important role in the metabolic homeostasis of colonic epithelial cells.Earlier studies point to the involvement of FFAR2 in colorectal carcinogenesis.AIM To understand the role of short chain FFARs in CRC.METHODS Transcriptome analysis console software was used to analyse microarray data from CRC patients and cell lines.We employed short-hairpin RNA mediated down regulation of FFAR2 and FFAR3 genes,which was validated using quantitative real time polymerase chain reaction.Assays for glucose uptake and cyclic adenosine monophosphate(cAMP)generation was done along with immunofluorescence studies to study the effects of FFAR2/FFAR3 knockdown.For measuring cell proliferation,we employed real time electrical impedancebased assay available from xCELLigence.RESULTS Microarray data analysis of CRC patient samples showed a significant down regulation of FFAR2 gene expression.This prompted us to study the FFAR2 in CRC.Since,FFAR3 shares significant structural and functional homology with FFAR2,we knocked down both these receptors in CRC cell line HCT 116.These modified cell lines exhibited higher proliferation rate and were found to have increased glucose uptake as well as increased level of glucose transporter 1.Since,FFAR2 and FFAR3 signal through G protein subunit(Gαi),knockdown of these receptors was associated with increased cAMP.Inhibition of protein kinase A(PKA)did not alter the growth and proliferation of these cells indicating a mechanism independent of cAMP/PKA pathway.CONCLUSION Our results suggest role of FFAR2/FFAR3 genes in increased proliferation of colon cancer cells via enhanced glucose uptake and exclude the role of PKA mediated cAMP signalling.Alternate pathways could be involved that would ultimately result in increased cell proliferation as a result of down regulated FFAR2/FFAR3 genes.This study paves the way to understand the mechanism of action of short chain FFARs in CRC.

Decarboxylated osteocalcin,a possible drug for type 2 diabetes,triggers glucose uptake in MG63 cells

BACKGROUND Uncarboxylated osteocalcin(GluOC)has been reported to improve glucose metabolism,prevent type 2 diabetes,and decrease the severity of obesity in mice with type 2 diabetes.GluOC can increase glucose uptake in a variety of cells.Glucose metabolism is the main source of energy for osteoblast proliferation and differentiation.We hypothesized that decarboxylated osteocalcin(dcOC),a kind of GluOC,can increase glucose uptake in MG63 cells(osteoblast-like osteosarcoma cells)and influence their proliferation and differentiation.AIM To investigate the effects of dcOC on glucose uptake in human osteoblast-like osteosarcoma cells and the possible signaling pathways involved.METHODS MG63 cells(human osteoblast-like osteosarcoma cells)were treated with dcOC(0,0.3,3,10,or 30 ng/mL)for 1 and 72 h,and glucose uptake was measured by flow cytometry.The effect of dcOC on cell proliferation was measured with a CCK-8 assay,and alkaline phosphatase(ALP)enzyme activity was measured.PI3K was inhibited with LY294002,and hypoxia-inducible factor 1 alpha(HIF-1α)was silenced with siRNA.Then,GPRC6A(G protein-coupled receptor family C group 6 subtype A),total Akt,phosphorylated Akt,HIF-1α,and glucose transporter 1(GLUT1)levels were measured by Western blot to elucidate the possible pathways by which dcOC modulates glucose uptake.RESULTS The glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after short-term(1 h)treatment with dcOC at different concentrations(0.3,3,and 10 ng/mL groups,P<0.01;30 ng/mL group,P<0.05).Glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after long-term(72 h)treatment with dcOC at different concentrations(0.3,3,and 10 ng/mL groups,P<0.01;30 ng/mL group,P<0.05).DcOC triggered Akt phosphorylation in a dose-dependent manner,and the most effective stimulatory concentration of dcOC for short-term(1 h)was 3 ng/mL(P<0.01).LY294002 abolished the dcOC-mediated(1 h)promotion of Akt phosphorylation and glucose uptake without affecting GLUT1 protein expression.Long-term dcOC stimulation triggered Akt phosphorylation and increased the protein levels of HIF-1α,GLUT1,and Runx2 in a dose-dependent manner.Inhibition of HIF-1αwith siRNA abolished the dcOC-mediated glucose uptake and substantially decreased GLUT1 protein expression.DcOC interven-tion promoted cell proliferation in a time-and dose-dependent manner as determined by the CCK-8 assay.Treatment with both 3 ng/mL and 10 ng/mL dcOC affected the ALP activity in MG63 cells after 72 h(P<0.01).CONCLUSION Short-and long-term dcOC treatment can increase glucose uptake and affect proliferation and ALP activity in MG63 cells.This effect may occur through the PI3K/Akt,HIF-1α,and GLUT1 signaling factors.

GLUT1、GLUT2介导的葡萄糖摄取对小鼠牙齿早期发育的影响

目的:探究葡萄糖转运蛋白(glucose transporter,GLUT)1、GLUT2介导的葡萄糖摄取对小鼠牙齿早期发育的影响。方法:收集胚胎13.5 d(embryonic day 0.5,E13.5)、E14.5、E16.5、E18.5和出生后1 d(postnatal day 1,P1)时期的下颌磨牙牙胚、上颌切牙牙胚;实时荧光定量聚合酶链反应(real time-quantitative polymerase chain reaction,RT-qPCR)和Western blot检测牙胚中Glut1、Glut2 mRNA和蛋白水平;免疫组织化学染色检测牙胚中GLUT1、GLUT2、Ki67、糖原水平。将下颌磨牙牙胚在无糖DMEM培养基、高糖且含不同浓度的根皮素(0、0.25、0.5 mmol/L)的DMEM培养基中培养9 d;并对其进行苏木精-伊红(hematoxylin-eosin,HE)染色。结果:(1)在E13.5时期,GLUT1在成釉器中高表达,细胞增殖活跃,糖原在牙板和牙囊中大量沉积;在E14.5、E18.5时期,GLUT1在成釉器中表达逐渐降低,细胞增殖减少,糖原在成釉器和牙乳头中大量沉积;在P1时期,GLUT1在中间层和内釉上皮中表达较多,细胞增殖增多,糖原在牙乳头中少量沉积。在整个牙胚发育阶段,GLUT2表达相对较少。(2)GLUT1在前成釉细胞、前成牙本质细胞中高表达,而在分化后的成釉细胞、成牙本质细胞中表达较少;GLUT2与GLUT1呈现相反的表达趋势。(3)0.5 mmol/L根皮素能够抑制E13.5时期外植体牙胚发育,0.25 mmol/L根皮素不抑制E13.5、E14.5时期外植体牙胚发育,但能够导致牙胚变小,且具有根皮素浓度依赖性。无糖培养基能够抑制E13.5、E14.5时期外植体牙胚发育。结论:GLUT1、GLUT2在牙齿早期发育中的表达受到精确的时空调控,由GLUT1、GLUT2介导的葡萄糖摄取在小鼠牙齿早期发育中发挥重要作用。