Uncoupling protein 2 deficiency of non-cancerous tissues inhibits the progression of pancreatic cancer in mice

Background: Pancreatic ductal adenocarcinoma(PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. Methods: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient( Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type(WT) PDAC cells(cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. Results: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ m RNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. Conclusions: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.

Uncoupling protein 2 deficiency reduces proliferative capacity of murine pancreatic stellate cells

BACKGROUND： Uncoupling protein 2 （UCP2） has been suggested to inhibit mitochondrial production of reactive oxygen species （ROS） by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deficiency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells （PSCs）, which are involved in pancreatic wound repair and fibrogenesis. METHODS： PSCs were isolated from 12 months old （aged） UCP2^-/- mice and animals of the wild-type （WT） strain C57BL/6. Proliferation and cell death were assessed by em- ploying trypan blue staining and a 5-bromo-2＇-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of mRNA were determined by RT-PCR, while protein expression was analyzed by immunoblotting and immunofluorescence analysis. Intracellular ROS levels were measured with 2＇,7＇-dichlorofluorescin diacetate. Expression of senescence-associated β-galactosidase （SA β-Gal） was used as a surrogate marker of cellular senescence. RESULTS： PSCs derived from UCP2^-/- mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dose- dependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2^-/- mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2^-/- mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, expression of the activation marker α-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleukin-6 and transforming growth factor-I~ 1. CONCLUSIONS： A reduced proliferative capacity of PSC from aged UCP2^-/- mice may contribute to the retarded regeneration after acute pancreatitis. Apart from their slower growth, PSC of UCP2^-/- mice displayed no functional abnormalities. The antifibrotic potential of UCP2 inhibitors deserves further attention.

Effect of Target-directed Regulation of Uncoupling Protein-2 Gene Expression on Ischemia-reperfusion Injury of Hepatocytes

The effect of target-directed regulation of the uncoupling protein-2 （UCP-2） gene expression on the ischemia-reperfusion injury of hepatocytes under different conditions was investigated. The expression plasmid and RNAi plasmid targeting UCP-2 gene were constructed and trans- fected into normal hepatocytes and fatty liver cells, respectively. The expression of UCP-2 mRNA was detected by real time PCR. The cells were divided into normal cell group （NCG）, group of normal cells transfected with empty vector （EVNCG）, group of normal cells transfected with expression plasmid （EPNCG）, fatty liver cell group （FCG） and group of fatty liver cells transfected with RNAi plasmid （RPFCG）. The ischemia-reperfusion model in vitro was established. One, 6, 12 and 24 h after reperfusion, Annexin V/PI flow cytometry was used to measure cell necrosis rate, apoptosis rate and survival rate. Simultaneously, the intracellular ATP, ROS and MDA levels were determined. The re- sults showed that 1, 6, 12 and 24 h after ischemia-reperfusion, the intracellular ROS, MDA and ATP levels and cell survival rate in EPNCG were significantly lower, and cell necrosis rate significantly higher than in NCG and EVNCG, but there was no significant difference in apoptosis rate among NCG, EVNCG and EPNCG （P〉005）. Six, 12 and 24 h after reperfusion there was no significant dif- ference in ROS, MDA levels and apoptosis rate between FCG and RPFCG （P〉0.05）, but the ATP level and survival rate of cells in RPFCG were higher than in FCG （P〈0.05）. It was concluded that down-regulation of the UCP-2 gene expression in steatotic hepatocytes could alleviate the ische- mia-reperfusion injury of liver cells.

Role and mechanism of uncoupling protein 2 on the fatty acid-induced dysfunction of pancreatic alpha cells in vitro

Background Uncoupling protein （UCP） 2 is related to the dysfunction of beta cells induced by fatty acids. However,whether UCP2 has similar effects on alpha cell is still not clear. This study aimed to investigate the effects of UCP2 and its possible mechanisms in lipotoxicity-induced dysfunction of pancreatic alpha cells.Methods The alpha TC1-6 cells were used in this study to evaluate the effects of palmitate and/or UCP2 inhibit factors on the glucagon secretory function, glucagon content, the glucagon mRNA level and the nitrotyrosine level in the supernatant. Meantime, the expression levels of UCP2 and peroxisome proliferator-activated receptor-γ coactivator-1 alpha （PGC-1 alpha） were measured by real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting. Furthermore, the possible relationship between UCP2 and insulin signal transduction pathway was analyzed.Results Palmitate stimulated alpha cell glucagon secretion and the expression of UCP2 and PGC-1 alpha, which could be partially decreased by the inhibition of UCP2. Palmitate increased nitrotyrosine level and suppressed insulin signal transduction pathway in alpha cells. Inhibition of UCP2 influenced the effects of free fatty acid on alpha cells and may relate to glucagon secretion.Conclusion UCP2 played an important role on alpha cell dysfunction induced by free fatty acid in vitro, which may be related to its effects on oxidative stress and insulin signal transduction pathway.

Effects of Berberine on Hepatic Sirtuin 1-uncoupling Protein 2 Pathway in Non-alcoholic Fatty Liver Disease Rats Induced by High-fat Diet

Objective To investigate the involvement of sirtuin 1（SIRT1）-uncoupling protein 2（UCP2） pathway in the development of non-alcoholic fatty liver disease and whether berberine exerts its effects by regulating this pathway. Methods Male SD rats were divided into three groups： normal control group, high-fat diet group, and berberine supplement group. The rats in the normal control group were given normal diet while the rats in the other two groups were fed with high-fat diet. Rats in the berberine supplement group were concurrently given berberine（100 mg/kg body weight） once daily. After 16 weeks, the levels of serum, liver lipids, and serum aminotransferase were measured using an automatic biochemical analyzer. Superoxide dismutase（SOD） activity and malondialdehyde（MDA） content in the liver were measured using commercial kits. Histopathological changes of liver tissues were observed by hematoxylin and eosin（HE） staining and Oil Red O staining. The hepatic m RNA and protein levels of SIRT1 and UCP2 were assayed by reverse transcription polymerase chain reaction（RT-PCR） or Western blotting. Results Berberine supplement could significantly decrease the serum and liver lipid contents in rats fed with high-fat diet. Meanwhile, SOD level was significantly elevated, but MDA level was reduced in the liver. The results of HE and Oil Red O staining showed that the hepatic steatosis was alleviated in berberine supplement group. Furthermore, berberine induced an increase in SIRT1 expression but a decrease in UCP2 expression. Conclusion The regulation of hepatic SIRT1-UCP2 pathway may be an important mechanism by which berberine exerts the beneficial effects in NAFLD rats.

An epidemiologic study of mitochondrial membrane transporter protein gene polymorphism and risk factors for neural tube defects in Shanxi, China

The present study involved a questionnaire survey of 156 mothers that gave birth to children with neural tube defects or had a history of pregnancy resulting in children with neural tube defects （case group） and 156 control mothers with concurrent healthy children （control group） as well as detection of mitochondrial membrane transporter protein gene [uncoupling protein 2 （UCP2）] polymorphism. The maternal UCP2 3＇ untranslated region （UTR） D/D genotype and D allele frequency were significantly higher in the case group compared with the control group （odds ratio （OR） 3.233; 95% confidence interval （C/） 1.103 9.476; P= 0.040; OR： 3.484; 95% CI： for neural tube defects 2.109 5.753; P 〈 0.001）. Univariate and multivariate logistic regression analysis of risk factors for neural tube defects showed that a matemal UCP2 3＇ UTR D/D genotype was negatively interacted with the mothers＇ consumption of frequent fresh fruit and vegetables （S = 0.007）, positively interacted with the mothers＇ frequency of germinated potato consumption （S = 2.15） and positively interacted with the mothers＇ body mass index （S = 3.50）. These findings suggest that maternal UCP2 3＇ UTR gene polymorphism, pregnancy time, consumption of germinated potatoes and body mass index are associated with an increased risk for neural tube defects in children from mothers living in Shanxi province, China. Moreover, there is an apparent gene-environment interaction involved in the development of neural tube defects in offspring.

NADPH Oxidase-dependent Oxidative Stress and Mitochondrial Damage in Hippocampus of D-galactose-induced Aging Rats

Mitochondrial DNA(mtDNA) common deletion(CD) plays a significant role in aging and age-related diseases.In this study,we used D-galactose(D-gal) to generate an animal model of aging and the involvement and causative mechanisms of mitochondrial damage in such a model were investigated.Twenty 5-week-old male Sprague-Dawley rats were randomly divided into two groups:D-gal group(n=10) and control group(n=10).The quantity of the mtDNA CD in the hippocampus was determined using a TaqMan real-time PCR assay.Transmission electron microscopy was used to observe the mitochondrial ultrastructure in the hippocampus.Western blot was used to detect the protein levels of NADPH oxidase(NOX) and uncoupling protein 2(UCP2).We found that the level of mtDNA CD was significantly higher in the hippocampus of D-gal-induced aging rats than in control rats.In comparison with the control group,the mitochondrial ultrastructure in the hippocampus of D-gal-treated rats was damaged,and the protein levels of NOX and UCP2 were significantly increased in the hippocampus of D-gal-induced aging rats.This study demonstrated that the levels of mtDNA CD and NOX protein expression were significantly increased in the hippocampus of D-gal-induced aging rats.These findings indicate that NOX-dependent reactive oxygen species generation may contribute to D-gal-induced mitochondrial damage.

Antifibrotic Effect of Total Flavonoids of Astmgali Radix on Dimethylnitrosamine-lnduced Liver Cirrhosis in Rats

Objective：To study the effect of total flavonoids of Astmgali Radix（TFA）on liver cirrhosis induced with dimethylnitrosamine（DMN）in rats,and the effect on peroxisome proliferator-activated receptorγ（PPARγ）,uncoupling protein 2（UCP2）and farnesoid X receptor（FXR）.Methods：Fifty-three Sprague-Dawley rats were randomly divided into a control group（10 rats）and a DMN group（43 rats）.Rats in the DMN group were given DMN for 4 weeks and divided randomly into a model group（14 rats）,a low-dosage TFA group（14 rats）and a high-dosage TFA group（15 rats）in the 3rd week.Rats were given TFA for 4 weeks at the dosage of 15 and 30 mg/kg in the low-and high-TFA groups,respectively.At the end of the experiment blood and liver samples were collected.Serum liver function and liver tissue hydroxyproline content were determined.hematoxylin-eosin（HE）,Sirus red and immunohistochemical stainings of collagenⅠ,smooth muscle actin（α-SMA）was conducted in paraffinembedded liver tissue slices.Real time polymerase chain reaction（PCR）was adopted to determine PPARγ,UCP2 and FXR m RNA levels.Western blot was adopted to determine protein levels of collagenⅠ,α-SMA,PPARγ,UCP2 and FXR.Results：Compared with the model group,TFA increased the ratio of liver/body weight（low-TFA group P〈0.05,high-TFA group P〈0.01）,improved liver biochemical indices（P〈0.01 for ALT,AST,GGT in both groups,P〈0.05 for albumin and TBil in the high-TFA group）and reduced liver tissue hydroxproline content（P〈0.01 in both groups）in treatment groups significantly.HE staining showed that TFA alleviated liver pathological changes markedly and Sirus red staining showed that TFA reduced collagen deposition,alleviated formation and extent of liver pseudolobule.CollagenⅠandα-SMA immunohistochemical staining showed that staining area and extent markedly decreased in TFA groups compared with the model group.TFA could increase PPARγ,it regulated target UCP2,and FXR levels significantly compared with the model group（in the low-TFA group all P〈0.05,in the high group all P〈0.01）.Conclusions：TFA could improve liver function,alleviate liver pathological changes,and reduce collagen deposition and formation of liver pseudolobule in rats with liver cirrhosis.The antifibrotic effect of TFA was through regulating PPARγsignal pathway and the interaction with FXR.

Role and mechanism of rosiglitazone on the impairment of insulin secretion induced by free fatty acids on isolated rat islets

Background Prolonged exposure of pancreatic β-cells to fatty acids increases basal insulin secretion but inhibits glucose-stimulated insulin secretion. Rosiglitazone is a new antidiabetic agent of the thiazolidinediones. However, the relationship between thiazolidinediones and insulin secretion is highly controversial. The aim of this study is to explore the effect and mechanism of rosiglitazone on insulin secretion of islets under chronic exposure to free fatty acids （FFA）.Methods Pancreatic islets were isolated from the pancreata of male Sprague-Dawley rats by the collagenase digestion and by the dextran gradient centrifugation method. The purified islets were cultured in the presence or absence of rosiglitazone and palmitate for 48 hours. The insulin secretion was measured by radioimmunoassay. The mRNA level of peroxisome proliferator-activated receptor y, uncoupling protein 2 0dCP-2） and insulin were determined by real-time polymerase chain reaction （PCR）. The cell cytotoxicity assay was measured by cell counting kit-8.Results Islets exposed to elevated palmitate for 48 hours showed an increased basal and a decreased glucose-stimulated insulin secretion （P〈0.01）. The mRNA level of UCP-2 was increased by 3.7 fold in the 0.5 mmol/L concentration of palmitate. When islets were cultured with palmitate （0.5 mmol/L） in the presence of rosiglitazone （1.0 pmol/L）, both basal and glucose-stimulated insulin secretion reversed to a pattern of control islets （P〈0.05, P〈0.0 1）. The addition of rosiglitazone in the culture medium decreased the mRNA level of UCP-2 by 2.2 fold, having a statistically significant difference （P〈0.05） as compared with islets cultured with palmitate alone. The cell viability was not affected.Conclusion The protective effects of rosiglitazone on insulin secretion of isolated pancreatic islets under chronic exposure to palmitate might be mediated through the downregulation of UCP-2 expression.

Obesity phenotype in relation to gene polymorphism among samples of Egyptian children and their mothers

Obesity is complex heterogeneous disease controlled by genes,environmental factors,and their interaction.Genetic factors account for 40e90%of the body mass index variations.Body mass index(BMI)of children correlates more closely with maternal than paternal BMI.So,this studu was aimed to investigate the role of leptin receptor LEPR Gln223Arg,the uncoupling protein 2(UCP2 G 866 A)and insulin receptor gene(INSR exon 17)polymorphisms in the pathogenesis of obesity.A cross-sectional study executed on 130 children and their obese mothers;classified into 2 groups according to their BMI.The 2 groups were evaluated regarding the anthropometry.Restriction fragment length analysis for LEPR Gln223Arg,UCP2-866 G/A and INSR exon 17 polymorphisms were applied.It was reported that increased risk of obesity was found in LEPR AG t AA genotype and the A allele.Significant statistical difference was detected only in female children.Concerning UCP2,the AG followed by the GG genotype was the most frequent in all groups and the G allele was the mostly present in obese mothers and obese male children but with no statistical significance.There was difference in the INSR genotype and alleles between groups,but this difference was not statistically significant.This study concluded that the LEPR Gln223Arg,UCP2 G 866 A and INSR exon 17 polymorphisms are related to obesity in Egyptian population.Further researches on larger population are recommended to ascertain the implications of LEPR,UCP2 and INSR polymorphisms in obesity.