Glucose Regulates Rat Beta Cell Number through Age-Dependent Effects on Beta Cell Survival and Proliferation

Background

Glucose effects on beta cell survival and DNA-synthesis suggest a role as regulator of beta cell mass but data on beta cell numbers are lacking. We examined outcome of these influences on the number of beta cells isolated at different growth stages in their population.

Methods

Beta cells from neonatal, young-adult and old rats were cultured serum-free for 15 days. Their number was counted by automated whole-well imaging distinguishing influences on cell survival and on proliferative activity.

Results

Elevated glucose (10–20 versus 5 mmol/l) increased the number of living beta cells from 8-week rats to 30%, following a time- and concentration-dependent recruitment of quiescent cells into DNA-synthesis; a glucokinase-activator lowered the threshold but did not raise total numbers of glucose-recruitable cells. No glucose-induced increase occurred in beta cells from 40-week rats. Neonatal beta cells doubled in number at 5 mmol/l involving a larger activated fraction that did not increase at higher concentrations; however, their higher susceptibility to glucose toxicity at 20 mmol/l resulted in 20% lower living cell numbers than at start. None of the age groups exhibited a repetitively proliferating subpopulation.

Conclusions

Chronically elevated glucose levels increased the number of beta cells from young-adult but not from old rats; they interfered with expansion of neonatal beta cells and reduced their number. These effects are attributed to age-dependent differences in basal and glucose-induced proliferative activity and in cellular susceptibility to glucose toxicity. They also reflect age-dependent variations in the functional heterogeneity of the rat beta cell population.     

孕期地塞米松暴露对大鼠子代海马神经元增殖以及突触可塑性的影响

地塞米松是一种糖皮质激素药物,具有抗炎、抑制免疫等多种药理作用,广泛应用于治疗多种疾病。临床上常使用地塞米松来促进早产儿的肺成熟以及预防胎儿呼吸窘迫综合征。目前的流行病学以及试验研究表明,地塞米松孕期暴露会增加子代患软骨病、肾脏损伤等疾病的风险。为了探究孕期地塞米松暴露(prenatal dexamethasone exposure,PDE)对大鼠子代胎鼠海马神经元增殖发育以及胎鼠海马突触可塑性形成的影响,对孕中晚期Wistar大鼠皮下注射地塞米松(0.2 mg·kg^-1·d^-1),对照组注射等剂量0.9%氯化钠溶液。收集GD20子代海马,采用实时荧光定量PCR以及Western blot法对海马神经增殖、突触可塑性形成和APPL1(adaptor protein containing pH domain,PTB domain and leucine zipper motif 1)进行相关功能检测,并进一步使用投射电镜观察海马突触超微结构。结果显示,与空白对照组相比,PDE胎海马Ki67、增殖细胞核抗原(proliferating cell ...     

Myocilin Regulates Cell Proliferation and Survival

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.     

NGF 对大鼠胚胎中脑神经细胞体外增殖和分化的影响

目的:探讨神经生长因子(nerve growth factor,NGF)对大鼠胚胎中脑神经细胞体外增殖和分化的影响。方法:在体外分离培养大鼠胚胎中脑神经细胞的培养液中加入不同浓度(10、50、100、200ng/ml)的NGF,培养不同时间,以不加神经营养因子的细胞为对照组,通过MTT法检测细胞活性,神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经细胞,光镜下形态学观察各组大鼠中脑神经细胞体外增殖和分化情况。结果:胚胎中脑神经细胞胞体增大、突起延长且有丰富的神经纤维连结成网络状,细胞集落数增加,显示出剂量-效应关系。结论:一定剂量的NGF能促进大鼠中脑神经细胞分化和增殖,增强其活性。     

Effects of Adrenalectomy and Replacement Therapy of Corticosterone on Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

Newly generated neurons in the dentate gyrus differentiate into mature granule cells. In the present study, we observed the effects of adrenalectomy (ADX) and corticosterone replacement therapy (CRT) on cell death, cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG). For this, the animals received vehicle or CRT after ADX, and were sacrificed 5 or 42 days later. Plasma corticosterone levels were very low in the adrenalectomized groups, whereas CRT after ADX significant increased serum corticosterone levels at 42 days, not 5 days, after ADX. ADX induced some neuronal damage in the dentate gyrus at 5 days post-ADX. CRT did not significantly reduce the neuronal damage at 5 days post-ADX; however, neuronal damage was not shown at 42 post-ADX with CRT. Ki67 (a marker for cell proliferation) and doublecortin (DCX, a marker for neuronal differentiation) immunoreaction was detected in the SZDG. ADX transiently increased cell proliferation and neuroblast differentiation 5 days after ADX, not 42 days, after ADX, and the CRT 42 days after ADX prominently decreased cell proliferation and neuroblast differentiation in the dentate gyrus. These results suggest that adrenal corticosteroid hormone is not essential for cell proliferation and neuroblast differentiation in long-term period after ADX.     

The Dual Role of HIV-1 gp120 V3 Loop-Induced Autophagy in the Survival and Apoptosis of the Primary Rat Hippocampal Neurons

Neurochemical Research - HIV-1 gp120, an important subunit of the envelope spikes that decorate the surface of virions, is known to play a vital role in neuronal injury during HIV-1-associated...     

The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity

Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer?5E and overexpression of synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.     

c-ski对大鼠皮肤成纤维细胞增殖的调节作用及机制

c-ski是成纤维细胞增殖的复杂调节子,它对中胚层来源的皮肤成纤维细胞增殖的作用还不清楚。在观察正常成纤维细胞周期c-ski表达的时相特点的基础上,通过体外转染c-ski,观察它对细胞增殖活性、细胞周期进展以及周期蛋白表达的影响。结果显示：c-ski mRNA表达在加入血清后开始升高,在细胞周期G,期的高峰期达到峰值,S期显著下降,在G2／M期维持在较低的水平：转染的c-ski可以以剂量依赖的方式增加细胞的增殖活性,并且可以逆转Smad3对细胞增殖活性的抑制作用;C-ski使成纤维细胞提前达到G0／G1期的最低点,进入S期：同时细胞G1期周期蛋白cyclinD的表达增加。这些结果表明：C-ski是皮肤成纤维细胞G1期的调节子,通过加快G1期进展促进增殖,抑制Smad3活性,促进cyclinD的表达可能与这一作用的分子机制有关。     

微清蛋白中间神经元在氯胺酮抗抑郁中的作用

目的:观察微清蛋白(PV)中间神经元在氯胺酮抗抑郁中的作用。方法:32只Wistar雄性大鼠随机均分为4组(n=8),包括生理盐水组(S组)、氯胺酮组(K组)、夹竹桃麻素预处理+生理盐水组(AS组)、夹竹桃麻素预处理+氯胺酮组(AK组)。夹竹桃麻素预处理组将药物溶于大鼠饮水中,共喂养1周,于第8 d制备模型。大鼠强迫游泳15 min制备急性应激抑郁模型,24 h后给大鼠分别腹腔注射1 mL生理盐水或氯胺酮10 mg/kg,给药后0.5 h行敞箱实验记录大鼠水平运动及垂直运动得分,行强迫游泳6 min记录后5 min内不动时间。行为学测试结束后,取大鼠前额皮层,Western印迹检测PV中间神经元中PV及谷氨酸脱羧酶67(GAD67)的表达。结果:与S组相比,K组大鼠强迫游泳不动时间减少,PV及GAD67的表达下降(P0.05),AS组则无显著变化(P0.05);与K组相比,AK组大鼠强迫游泳不动时间增加,PV及GAD67的表达增加(P0.05)。生理盐水、氯胺酮、夹竹桃麻素均未显著影响大鼠自主活动(P0.05)。结论:氯胺酮通过下调大鼠前额皮层PV中间神经元功能发挥快速有效的抗抑郁作用。     

Effects of Antidepressant Drug Imipramine on Gene Expression in Rat Prefrontal Cortex

We have investigated gene expression changes produced by acute and chronic daily treatment with a prototypical antidepressant, imipramine, using DNA microarrays. The analysis of similarities in gene expression patterns among functionally related genes revealed four expression profile cluster areas that showed a highly significant overrepresentation of several functional classes. Genes encoding for proteins involved in cAMP metabolism, postsynaptic membrane proteins, and proto-oncogenes were overrepresented in different cluster areas. Furthermore, we found that serine proteases as a group were similarly regulated by chronic antidepressant treatment. Our data suggest that cAMP metabolism, synaptic function, and protein processing by serine proteases may be important targets of antidepressant treatment and potential objects for antidepressant drug development.     

pERK, pAkt and pBad: A Possible Role in Cell Proliferation and Sustained Cellular Survival During Tumorigenesis and Tumor Progression in ENU Induced Transplacental Glioma Rat Model

Gliomas remain to be an unresolved medical problem. Better understanding of complex regulation and key molecules involved in glioma pathology are needed for designing new and effective treatment modalities. Activation of mitogen-activated protein kinase/extracellular signal regulated kinase (ERK) pathway is known to be having a critical role in cell proliferation and differentiation during the invasion and metastasis of the tumor cells. In the present study, N-ethyl N-nitrosourea induced glioma rat model was used to understand the role of ERK1/2 and Akt pathways in the progression of tumor malignancy. Twenty-four glioma rat brains of early (P90) and progressive (P180) stages were used for histological and immunoblot analysis. Results have shown increased levels of activated ERK1/2, activated Akt or protein kinase B, Bcl-2 and pBad in the glioma rats. This study may indicate increased cell proliferation and angiogenesis, mediated through activation of both ERK and Akt pathways along with increased levels of pBad. Further, pAkt and Bcl-2 levels in the progressive stage glioma rats may indicate existence of sustained tumor cell survival signals. Moreover, enhanced pBad levels in tumor may indicate that there are anti-apoptotic mechanisms, further making the malignant cells resistant to apoptosis.     

Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, ³H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.     

APP5肽模拟物P165对体外培养大鼠海马神经干细胞增殖和分化的影响

目的研究一种小分子多肽─APP5肽的模拟物P165对体外培养的大鼠胚胎海马神经干细胞（neuralstem cells,NSCs）增殖和分化的影响,以期能找到一种可代替神经营养因子的小分子物质,能够促进NSCs的增殖或分化,为将来的临床应用提供理论依据。方法（1）原代培养SD大鼠胚胎脑海马NSCs;（2）利用5-溴脱氧尿嘧啶核苷（BrdU）和神经元、星型胶质细胞、少突胶质细胞的特异性标记物微管相关蛋白2（MAP2）、胶质纤维酸性蛋白（GFAP）、2,3-环核苷酸-3磷酸二酯酶（CNPase）对培养的NSCs进行鉴定;（3）将培养的NSCs分为对照组、血清组、APP5肽反序列组和P165组,观察各组细胞形态的变化;（4）将培养的NSCs分为对照组、APP5肽反序列组和P165组,利用细胞计数,测定干细胞克隆形成率、干细胞克隆形成大小的方法分析P165对海马NSCs增殖的影响。结果（1）海马神经干细胞呈神经球聚集生长,BrdU染色阳性;加入血清后神经球周围有细胞呈放射状向四周生长,并带有突起。染色呈MAP2、GFAP或CNPase阳性;（2）海马NSCs加入P165及其反序列后细胞形态上与对照组相比没有明显改变;（3）与对照组相比,加P165后海马NSCs数量明显增加,克隆形成率和克隆形成的直径均有明显的增加,并有统计学差异。结论P165能够促进海马NSCs的增殖,但并不促进其分化。     

二十二碳六烯酸对大鼠脂肪细胞增殖分化的影响

体外培养大鼠脂肪细胞,分别以0 μmol/L（对照组）、40 μmol/L（低剂量组）和160 μmol/L（高剂量组）的二十二碳六烯酸（DHA）处理细胞。采用台盼蓝排斥试验和MTT比色法检测细胞活性及增殖状况;油红O染色化学比色法定量分析细胞内脂肪生成及细胞分化程度;逆转录聚合酶链反应 (RT-PCR) 分析过氧化物酶增殖物激活受体γ2（PPARγ2）mRNA表达情况,探讨DHA对前体脂肪细胞增殖分化的影响及其可能机制。结果显示,各组细胞活力及MTT测得的光密度值(OD值)均低于对照组,160μmol/L组在60～72h作用显著（P<0.05）;脂肪细胞经DHA处理后, 160μmol/L组细胞油红O染色的OD值及PPARγ2 mRNA表达量均显著下降(P<0.01)。以上结果说明,DHA对脂肪细胞增殖分化均有一定抑制作用,高剂量DHA（160μmol/L）可显著减少细胞内脂肪的合成、抑制脂肪细胞分化,PPARγ2 mRNA表达量的下降可能是DHA抑制细胞分化的部分原因。     

Effects of the Antidepressant/Antipanic Drug Phenelzine on Alanine and Alanine Transaminase in Rat Brain

1. Phenelzine (PLZ) is an antidepressant with anxiolytic properties. Acute and chronic PLZ administration increase brain GABA levels, an effect due, at least in part, to an inhibition of the activity of the GABA metabolizing enzyme, GABA transaminase (GABA-T).2. Previous preliminary reports have indicated that acute PLZ treatment also elevates brain alanine levels. As with GABA, the metabolism of alanine involves a pyridoxal phosphate-dependent transaminase.3. In the study reported here, the effects of acute PLZ treatment on the levels of various amino acids, some of which are also metabolized by pyridoxal phosphate-dependent transaminases were compared in rat whole brain. Of the 6 amino acids investigated, only GABA and alanine levels were elevated (in a time- and dose-dependent manner).4. The elevation in brain alanine levels could be explained, at least in part, by a time- and dose-dependent inhibitory effect of PLZ on alanine transaminase (ALA-T), although as with GABA the increases are higher than expected from the degree of enzyme inhibition produced. In addition, we also showed that the elevation in alanine levels and the inhibition of alanine transaminase in the brain are retained after 14 days of PLZ treatment, and that PLZ produces a marked increase in extracellular levels of alanine.5. These results are discussed in terms of their relevance to synaptic function and to the pharmacological profile of PLZ.     

骨桥蛋白对细胞生存的多重作用

骨桥蛋白(osteopontin, OPN)作为一种分泌性蛋白质广泛存在于多种组织细胞中,不仅调控肿瘤细胞的转移、侵袭和增殖,也在炎症反应、血管再生等生理过程中发挥作用。OPN通过与受体结合直接或间接地激活细胞内信号途径,介导细胞与细胞、细胞与细胞外基质之间的相互作用,从而参与调控细胞的生存。大量实验证实,OPN能够促进细胞的增殖,抑制细胞凋亡,尤其是对于肿瘤细胞。但在有些细胞中,OPN对细胞命运的影响却恰恰相反。本文综述了OPN在不同条件下对细胞存活、活化和增殖,细胞自噬和细胞凋亡的多重作用及其作用途径,为进一步研究OPN对不同细胞作用的受体及其信号网络机制提供重要理论基础。     

Evaluation of the Effects of Pentagastrin, Gastrin and Pancreatic Glucagon On Cell Proliferation In the Rat Gastrointestinal Tract

ABSTRACT The effects of six injections of a range of doses (100–1000 μg/kg bodyweight) of pentagrastrin and single injection of a range of doses of porcine gastrin (10–40 μg/kg bodyweight) and pancreatic glucagon (25–100 μg/kg bodyweight) on cell proliferation in the intestine of fasted rats has been investigated. the end-point employed included the measurement of ¹⁴C leucine incorporation and thymidine-derived tritium content of the body of the stomach, duodenum, jejunum, ileum and colon. the carbon 14 and tritium content per μg of tissue in triplicate samples of fifty individually dissected crypts of glands were determined. From these data and the wet weight of the washed, blotted, intestinal segments, values for crypts/μg tissue and crypts/segment were calculated. the results demonstrated that pentagastrin at physiological doses decreased cell proliferation slightly in stomach, while gastrin and glucagon were without effect. In the small intestine, pentagastrin and gastrin were without significant effect with the exception that they increased the weight of the duodenum. In contrast, a high physiological dose of glucagon increased DNA and protein synthesis throughout the small bowel, but particularly in the ileum. Pharmacological doses of pentagastrin and all doses of gastrin appeared to increase cell proliferation in the colon although the possibility could not be excluded that this was due to stimulation of precursor uptake. Gastrin also increased colonic weight. Glucagon had no effects in the colon. These observations are compatible with the hypothesis that (i) the primary effects of gastrin and pentagastrin on the proximal intestine are as secretogogues and effects on cell proliferation may be secondary, (ii) gastrin and pentagastrin at physiological levels do not stimulate small intestinal cell proliferation, however glucagon does, and (iii) gastrin at physiological levels and pentagastrin at pharmacological levels may stimulate cell proliferation in the colon.     

Long-Term Effects of Peripubertal Binge EtOH Exposure on Hippocampal microRNA Expression in the Rat

Adolescent binge alcohol abuse induces long-term changes in gene expression, which impacts the physiological stress response and memory formation, two functions mediated in part by the ventral (VH) and dorsal (DH) hippocampus. microRNAs (miRs) are small RNAs that play an important role in gene regulation and are potential mediators of long-term changes in gene expression. Two genes important for regulating hippocampal functions include brain-derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT1), which we identified as putative gene targets of miR-10a-5p, miR-26a, miR-103, miR-495. The purpose of this study was to quantify miR-10a-5p, miR-26a, miR-103, miR-495 expression levels in the dorsal and ventral hippocampus of male Wistar rats during normal pubertal development and then assess the effects of repeated binge-EtOH exposure. In addition, we measured the effects of binge EtOH-exposure on hippocampal Drosha and Dicer mRNA levels, as well as the putative miR target genes, BDNF and SIRT1. Overall, mid/peri-pubertal binge EtOH exposure altered the normal expression patterns of all miRs tested in an age- and brain region-dependent manner and this effect persisted for up to 30 days post-EtOH exposure. Moreover, our data revealed that mid/peri-pubertal binge EtOH exposure significantly affected miR biosynthetic processing enzymes, Drosha and Dicer. Finally, EtOH-induced significant changes in the expression of a subset of miRs, which correlated with changes in the expression of their predicted target genes. Taken together, these data demonstrate that EtOH exposure during pubertal development has long-term effects on miRNA expression in the rat hippocampus.     

Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation

Cleavage and polyadenylation specific factor 4 (CPSF4), a member of CPSF complex, plays a key role in mRNA polyadenylation and mRNA 3′ ends maturation. However, its possible role in lung cancer pathogenesis is unknown. In this study, we investigated the biological role and clinical significance of CPSF4 in lung cancer growth and survival and elucidated its underlying molecular mechanisms. We found that CPSF4 was highly expressed in lung adenocarcinoma cell lines and tumor tissue but was undetectable in 8 normal human tissues. We also found that CPSF4 overexpression was correlated with poor overall survival in patients with lung adenocarcinomas (P<0.001). Multivariate survival analyses revealed that higher CPSF4 expression was an independent prognostic factor for overall survival of the patients with lung adenocarcinomas. Suppression of CPSF4 by siRNA inhibited lung cancer cells proliferation, colony formation, and induced apoptosis. Mechanism studies revealed that these effects were achieved through simultaneous modulation of multiple signaling pathways. Knockdown of CPSF4 expression by siRNA markedly inhibited the phosphorylation of PI3K, AKT and ERK1/2 and JNK proteins. In contrast, the ectopic expression of CPSF4 had the opposite effects. Moreover, CPSF4 knockdown also induced the cleavage of caspase-3 and caspse-9 proteins. Collectively, these results demonstrate that CPSF4 plays a critical role in regulating lung cancer cell proliferation and survival and may be a potential prognostic biomarker and therapeutic target for lung adenocarcinoma.