Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections

In situ cDNA:mRNA hybridization is a technique that has been developed for the visualization of cDNA:mRNA hybrids in individual cells. To use this technique to answer questions of regulation in heterogeneous populations of cells in the brain, it must be combined with other procedures allowing for the identification of functional subgroups of neurons. We report here a procedure by which in situ cDNA:mRNA hybridization may be combined with retrograde axonal tracing using the fluorescent tracer fast blue. Using this technique, it now becomes possible to measure mRNA regulation in functional subsets of cells defined by their axonal projections.     

Dysregulation of the Hypothalamus-Pituitary-Adrenal Axis in Male and Female, Genetically Obese (ob/ob) Mice

The autosomal, recessive obesity of ob/ob mice is associated with hypercorticosteronemia and amelioration of most symptoms of obesity following adrenalectomy. Increased adrenocorticotropic hormone (ACTH) secretion has been hypothesized on the basis of several reports of higher pituitary ACTH content in ob/ob mice compared to lean littermates. However, the only measurement of ACTH blood concentration found lower levels in ob/ob mice than in leans suggesting that hypercorticosteronemia might result solely from an enhanced adrenal response to ACTH and also suggesting that the ob/ob's elevated pituitary ACTH content might be due to decreased ACTH secretion rather than increased ACTH synthesis. In our study, basal serum ACTH levels were higher in ob/ob males and females compared to sex-matched lean littermates. Anterior pituitary ACTH synthesis was also elevated as indicated by increased content of ACTH and proopiomelanocortin mRNA in obese mice of both sexes; however hypothalamic corticotropin-releasing factor content was not different in lean and obese mice. Basal serum ACTH and corticosterone (CS) levels showed normal circadian rhythm in both phenotypes and sexes, but the circadian increase in CS level was much greater in obese mice than in leans despite equal serum ACTH increases in the two phenotypes. Ether stress at both peak and trough of the circadian rhythm also stimulated much larger serum CS increases in obese mice even though ACTH increases were again equal in the two phenotypes. Taken together, these results strongly indicate that ob/ob mice have increased synthesis and secretion of pituitary ACTH despite the presence of chronically elevated serum CS. This hyperactivity of the hypothalamo-pituitary-adrenal axis appears to be most pronounced in ob/ob females since pituitary ACTH content was equal in obese males and females despite much higher circulating CS levels in the females. Furthermore, the results also indicate an enhanced response to ACTH by the adrenal cortex of the obese mouse. Thus, ob/ob mice exhibit abnormal hypothalamo-pituitary-adrenal axis function with hyperactivity occurring at the level of pituitary ACTH synthesis/secretion as well as at the level of adrenocortical response to ACTH.     

体复康对运动性疲劳大鼠脑组织中阿黑皮素原基因表达动态变化的影响

目的 :从基因转录水平阐明运动性疲劳产生及药物调节的机理。方法 :采用生物素标记阿黑皮素原 (POMC)cRNA探针 (Bio cRNA)原位杂交法结合图像分析 ,观察中药复方体复康对运动性疲劳大鼠不同脑区POMCmRNA表达。结果 :对于慢性运动应激 ,体复康能增强POMC在海马CA1 区、下丘脑部位的基因表达 ,而能降低POMC在额叶皮层的基因表达。长时期 (共 7周 )大强度的跑台运动 (速度由 1 5m/min递增至 3 5m/min ,运动时间为每天 2 0～2 5min)的大鼠 ,末次运动后POMC基因表达在额叶皮层、海马CA1 下降 (前者至运动结束后 3h仍未恢复 ,后者至运动结束后 3 0min即恢复 ) ,在下丘脑则增强 ;体复康能增强末次运动后POMC在额叶皮层、海马CA1 区、下丘脑等脑区的基因表达 ,尤其是在运动结束后 3h最为显著。结论 :POMCmRNA表达在运动性疲劳大鼠不同脑区呈现不同的动态变化趋势 ,体复康对其POMCmRNA表达有重要动态调节作用。     

Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.     

胆汁酸通过p-STAT3-SOCS3途径调节下丘脑神经元食欲及抑制信号肽表达

目的 探索胆汁酸牛磺石胆酸或鹅去氧胆酸对小鼠下丘脑GT1-7细胞食欲抑制信号肽表达的影响及可能的作用途径。方法 将实验分为3组,每组3个平行样本。空白对照组：仅给予含10%胎牛血清高糖培养基培养GT1-7细胞。胆汁酸牛磺石胆酸处理组：分别采用10 nmol/L,100 nmol/L,1 μmol/L和10 μmol/L浓度的胆汁酸牛磺石胆酸处理GT1-7细胞12、24和48 h。鹅去氧胆酸处理组：分别采用10 nmol/L,100 nmol/L,1 μmol/L和10 μmol/L浓度的鹅去氧胆酸处理GT1-7细胞12、24和48 h。采用Real-time PCR 检测GT1-7细胞食欲抑制信号肽阿黑皮素原（POMC）mRNA表达水平;ELISA法检测POMC剪切产物抑制食欲肽α-促黑素细胞激素水平。采用Western blot检测G蛋白偶联胆汁酸受体（TGR5）和法呢醇X受体表达以及磷酸化信号转导转录激活因子3（p-STAT3）,磷酸化蛋白激酶（p-AKT）,细胞因子信号传导抑制因子3（SOCS3）蛋白表达量。结果 Western blot结果显示,下丘脑神经元GT1-7细胞能表达TGR5和FXR两种胆汁酸受体,且表达量受胆汁酸调节。Real-time PCR检测结果显示,10 μmol/L tLCA或鹅去氧胆酸处理GT1-7细胞24 h后POMC mRNA表达水平上升,ElISA结果也发现10 μmol/L 胆汁酸牛磺石胆酸或鹅去氧胆酸处理GT1-7细胞24 h后具有抑制食欲作用的POMC衍生肽[α]-MSH释放增多。Western blot结果显示,胆汁酸处理可上调GT1-7细胞p-STAT3和p-AKT蛋白表达,且调控STAT3信号通路的SOCS3蛋白表达也有增加。结论 胆汁酸牛磺石胆酸或鹅去氧胆酸通过GT1-7细胞胆汁酸受体TGR5和FXR促进POMC mRNA表达,增加[α]-促黑素细胞激素水平,提示胆汁酸具有通过抑制食欲信号肽表达调节食欲的潜在作用,该作用是通过p-AKT和p-STAT3 -SOCS3 信号通路实现。     

Endothelin in coronary artery disease

Abstract

Objectives. The aim of the present study was to compare changes in circulating levels of proopiomelanocortin (POMC) derivates and lactate after remote ischemic preconditioning (IPC) and physical exercise. Introduction. Remote IPC (rIPC) is cardioprotective following acute myocardial infarction and major cardiac surgery. A blood-borne, transferable factor, released following not only rIPC but also vigorous exercise, mediates protection that is abolished by naloxone suggesting involvement of an opioid-receptor-dependent pathway. Design. Eight healthy volunteers underwent rIPC by four cycles of 5-min inflation of a pneumatic tourniquet to 200 mmHg interrupted by 5 min of deflation. Subsequently, circulating plasma levels of POMC derivates, cortisol, and lactate were measured. After 3 days, the volunteers completed a vigorous exercise program, after which the same compounds were measured. Results. While rIPC was not associated with any significant increase in circulating POMC derivates or lactate, exercise induced significant elevation of both compared with baseline. Conclusions. We were not able to demonstrate a detectable increase in circulating POMC derivates by a standard rIPC stimulus, suggesting that rIPC effect is not mediated by local or detectable central release of these derivates.     

Role of AMPK in pancreatic beta cell function

Pharmacological activation of AMP activated kinase (AMPK) by metformin has proven to be a beneficial therapeutic approach for the treatment of type II diabetes. Despite improved glucose regulation achieved by administration of small molecule activators of AMPK, the potential negative impact of enhanced AMPK activity on insulin secretion by the pancreatic beta cell is an important consideration. In this review, we discuss our current understanding of the role of AMPK in central functions of the pancreatic beta cell, including glucose-stimulated insulin secretion (GSIS), proliferation, and survival. In addition we discuss the controversy surrounding the role of AMPK in insulin secretion, underscoring the merits and caveats of methods used to date.     

肾衰养真胶囊对慢性肾衰竭营养不良大鼠下丘脑神经肽Y mRNA及原阿片黑皮素mRNA表达的影响

目的 观察肾衰养真胶囊对慢性肾衰竭(CRF)营养不良大鼠下丘脑神经肽Y(NPY)mRNA及原阿片黑皮素(POMC)mRNA的表达及其营养状态的影响.方法 采用含0.5%腺嘌呤及4%酪蛋白饲料联合喂养的方法制作CRF营养不良大鼠模型,观察其营养不良发生时间,符合CRF营养不良模型的随机分为模型组、肾衰养真胶囊组和开同组.治疗4周后,检测下丘脑NPY mRNA和POMC mRNA表达水平.结果 肾衰养真胶囊组大鼠营养状况明显改善,下丘脑的NPY mRNA表达上调及POMC mRNA表达下调.结论 肾衰养真胶囊可改善CRF营养不良大鼠的营养状况,其机制可能是通过上调下丘脑NPY mRNA表达,下调下丘脑POMC mRNA表达,从而促进大鼠摄食.     

The different time courses of reading different levels of Chinese characters: an ERP study

AMP-activated protein kinase (AMPK) is an energy sensor that is activated by the increase of intracellular AMP:ATP ratio. AMPK in the hypothalamic arcuate nucleus (ARC) is activated during fasting and the activation of AMPK stimulates food intake. To clarify the pathway underlying AMPK-induced feeding, we monitored the activity of single ARC neurons by measuring cytosolic Ca(2+) concentration ([Ca(2+)](i)) with fura-2 fluorescence imaging. An AMPK activator, AICA-riboside (AICAR), at 200 μM increased [Ca(2+)](i) in 24% of ARC neurons. AMPK and acetyl CoA carboxylase were phosphorylated in the neurons with [Ca(2+)](i) responses to AICAR. AICAR-induced [Ca(2+)](i) increases were inhibited by Ca(2+)-free condition but not by thapsigargin, suggesting that AICAR increases [Ca(2+)](i) through Ca(2+) influx from extracellular space. Among AICAR-responding ARC neurons, 38% were neuropeptide Y (NPY)-immunoreactive neurons while no proopiomelanocortin (POMC)-immunoreactive neuron was observed. Intracerebroventricular administration of AICAR increased food intake, and the AICAR-induced food intake was abolished by the co-administration of NPY Y1 receptor antagonist, 1229U91. These results indicate that the activation of AMPK leads to the activation of ARC NPY neurons through Ca(2+) influx, thereby causing NPY-dependent food intake. These mechanisms could be implicated in the stimulation of food intake by physiological orexigenic substances.