Perinatal changes in the coupling of beta 1- and beta 3 adrenergic receptors to brown fat adenylyl cyclase.

The stimulation of adenylyl cyclase by catecholamines in neonatal brown adipose tissue (BAT) is markedly biphasic, suggesting the existence of receptors that have both high and low affinities for catecholamines. The identities of these receptors were examined by comparing responses in neonatal BAT membranes to those of Chinese hamster ovary cells which had been transfected to express the cloned rat beta 1 and beta 3 receptors. The results from these experiments indicate that high-affinity stimulation of adenylyl cyclase by catecholamines in BAT is mediated by beta 1 receptors, as evidenced by the potencies of norepinephrine and isoproterenol at this receptor and the potent blockade of the receptor by alprenolol. The low-affinity catecholamine receptor appears to be the beta 3 receptor, as indicated by the low potency of catecholamine agonists and the inability of low concentrations of alprenolol to block this activity. Furthermore, this receptor, like the cloned rat beta 3 receptor, was antagonized by (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP 12177) and was stimulated by (R',R')-4-(2-[(2[(3-chlorophenyl)-2- hydroxyethyl]amino)propyl]phenyl)phenoxyacetic acid (BRL 37344). These results indicate that both beta 1 and beta 3 receptors couple to adenylyl cyclase in BAT and that activation of adenylyl cyclase in neonatal BAT is mediated primarily by beta 3 receptors. Beta 3 receptors were also clearly detected in weanling BAT with the beta 3-selective agonist BRL 37344. However, when catecholamines were used to stimulate activity, the activation of adenylyl cyclase by beta 1 receptors, which occurred at low concentrations of catecholamines, obscured the activation of adenylyl cyclase by beta 3 receptors, which occurred only at high concentrations.     

Effects of agonist exposure on the coupling of beta 1 and beta 3 adrenergic receptors to adenylyl cyclase in isolated adipocytes.

The coupling of beta 1 and beta 3 adrenergic receptors to adenylyl cyclase was examined in membranes of isolated white adipocytes. The activation of adenylyl cyclase by isoproterenol (ISO) was biphasic. The high-affinity activation of adenylyl cyclase, which occurred at submicromolar concentrations of ISO, was mediated by beta 1 receptors, whereas low-affinity activation was mediated by beta 3 receptors. The relative activation of adenylyl cyclase by beta 3 receptors was less when ISO was used to stimulate activity, compared to when the beta 3-selective agonist BRL 37344 was used. These data indicate that both receptor subtypes can stimulate the same adenylyl cyclase in membranes of control cells, and that the activation of adenylyl cyclase by beta 1 receptors by low concentrations of catecholamines can obscure the activation by beta 3 receptors by high concentrations of catecholamines. Exposure of adipocytes to ISO at concentrations that either selectively stimulate beta 1 receptors or nonselectively stimulate both beta receptor subtypes greatly decreased the ability of beta 1, but not beta 3, receptors to activate adenylyl cyclase. In contrast, the exposure of cells to the beta 3-selective agonist BRL only slightly desensitized beta 1 receptors and did not affect beta 3 receptor activation of adenylyl cyclase. These data indicate that acute agonist exposure desensitizes beta 1, but not beta 3, receptors.     

Desensitization of adenylate cyclase and down regulation of beta adrenergic receptors after in vivo administration of beta agonist

In vitro incubation of cells with catecholamines leads to both down regulation of beta adrenergic receptor number and desensitization of agonist-stimulated adenylate cyclase activity. These same parameters, down regulation of beta adrenergic receptor number and desensitization of adenylate cyclase activity were assessed in rat lung membranes after in vivo administration of metaproterenol, a beta-2 selective agonist. In vivo treatment with metaproterenol leads to: 1) reduced beta adrenergic receptor number; 2) reduced isoproterenol-stimulated adenylate cyclase activity; 3) unaffected NaF or 5'-guanylylimidodiphosphate-stimulated adenylate cyclase activity; and 4) reduced affinity of the receptor for isoproterenol similar to the affinity observed in the presence of 5'-guanylylimidodiphosphate. The date suggest that in vivo metaproterenol administration results in an uncoupled receptor-adenylate cyclase complex. The effects of in vivo administration of the glucocorticoid, methylprednisolone, to metaproterenol-pretreated animals were also assessed. Glucocorticoid treatment was associated with 1) increased beta adrenergic receptor number in rats in which the receptors have been down regulated, 2) increased isoproterenol responsiveness in agonist-desensitized rats and 3) no effect on agonist affinity in desensitized animals. These data suggest that the restoration of agonist responsiveness by glucocorticoids in the catecholamine refractive state is not simply a reversal of receptor down regulation or adenylate cyclase desensitization.     

Comparison of beta adrenergic receptor binding characteristics and coupling to adenylate cyclase in rat pulmonary artery versus aorta

In an effort to define the mechanisms regulating pulmonary vasodilatation and explain the greater in vitro response to iso-proterenol in the pulmonary artery (PA) vs. aorta (AO), we compared beta adrenergic receptor binding characteristics and coupling to adenylate cyclase in PA and AO obtained from adult male rats. Beta adrenergic receptor binding characteristics and affinity for agonists were determined with [125I]-iodocyanopindolol. Agonist displacement studies were characteristic of a beta-2 adrenergic receptor subtype. Receptor density (44.7 +/- 7.3 vs. 39.6 +/- 0.8 fmol/mg of protein means +/- S.E.M., PA vs. AO) and the dissociation constant for the radioligand (10.3 +/- 2.6 vs. 13.4 +/- 3.5 pM) were similar in the two arteries. However, affinity for l-isoproterenol was greater (the inhibition constant was lower) in PA compared to AO (0.08 +/- 0.03 vs. 1.20 +/- 0.18 microM, P less than .05), as was affinity for l-epinephrine (0.89 +/- 0.20 vs. 3.87 +/- 0.62 microM, P less than .05). Affinity was similar for l-norepinephrine (18.93 +/- 3.63 vs. 13.49 +/- 3.12 microM). Base-line cyclic AMP (cAMP) content, basal adenylate cyclase activity and adenylate cyclase activity stimulated by GTP, isoproterenol plus GTP and forskolin were measured by radioimmunoassay for cAMP. Base-line cAMP content was greater in PA than in AO (513.5 +/- 46.9 vs. 125.5 +/- 19.1 pmol of cAMP per mg of protein, P less than .001), as was basal adenylate cyclase activity (10.8 +/- 1.2 vs. 5.7 +/- 1.3 pmol of cAMP per mg of protein per min, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

利培酮抑制3T3-L1前脂肪细胞的分化

目的研究利培酮对3T3-L1前脂肪细胞分化的影响。方法采用经典的激素鸡尾酒法诱导3T3-L1前脂肪细胞分化为成熟的脂肪细胞,油红O染色观察。向诱导培养基中加入利培酮研究其对3T3-L1前脂肪细胞分化的影响。结果用激素鸡尾酒法成功地将3T3-L1前脂肪细胞诱导为成熟的脂肪细胞。0.1、1、10μmol/L的利培酮均能够抑制3T3-L1前脂肪细胞的分化。结论利培酮能够抑制3T3-L1前脂肪细胞的分化。     

Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

3T3-L1前脂肪细胞与蚕丝支架的体外共培养

蚕丝具各较好的力学性能,生物相容性优于传统的人工合成的可降解高分子材料.实验拟观察蚕丝对3T3-L1前脂肪细胞吸附作用及对细胞形态和功能的影响.将原料蚕丝与经胰蛋白酶消化后的原料蚕丝制成蚕丝复合物三维支架:制备3T3-L1前脂肪细胞悬液,调整细胞浓度为6×1010 L1;将3T3-L1前脂肪细胞悬液接种在支架上,培养1～4周,倒置显微镜观察可见3T3-L1前脂肪细胞伸出细长的突起沿着蚕丝不断向前迁移延伸,细胞首尾相互融合,渐渐连成一片分布于蚕丝网眼内;扫描电镜观察可见细胞与支架紧密贴附,适度伸展,并有基质分泌.结果表明蚕丝复合物三维支架对3T3-L1前脂肪细胞具有良好的吸附作用,并能维持3T3-L1前脂肪细胞正常形态和功能.     

Pineal beta adrenergic receptor: correlation of binding of 3H-l-alprenolol with stimulation of adenylate cyclase.

3H-l-Alprenolol, a potent competitive beta adrenergic antagonist, binds to sites in rat pineal gland membranes. The properties of these binding sites were compared to those of the receptors which mediate the beta adrenergic activation of pineal adenylate cyclase. Both sites are highly stereospecific. The l-stereoisomers of alprenolol and propranolol were at least two orders of magnitude more potent than the d-stereoisomers in inhibiting isoproterenol-stimulated adenylate cyclase or 3H-l-alprenolol binding. The dissociation constants (Kd) of the l-stereoisomers of both alprenolol and propranolol were 10 to 22 nM as determined by competition for binding sites or by inhibition of isoproternol-stimulated adenylate cyclase. Beta adrenergic agonists which stimulated adenylate cyclase also competitively inhibited the binding of 3H-l-alprenolol. They showed the same order of potency (isoproterenol greater than norepinephrine greater than or equal to epinephrine) and the same individual affinities in the two systems. Alpha adrenergic blockers were ineffective in inhibiting either adenylate cyclase stimulation or 3H-l-alprenolol binding. Isoproternol stimulation of adenylate cyclase acrivity, and 3H-l-alprenolol binding, were rapid and rapidly reversible. The 3H-l-alprenolol binding sites were saturable and bound 0.6 pmol of ligand per mg of added protein. The data suggest that the binding of 3H-l-alprenolol occurs at sites indistinguishable from the pineal beta adrenergic receptor.     

Beta adrenergic receptor subtypes in the atria: evidence for close coupling of beta-1 and beta-2 adrenergic receptors to adenylate cyclase

The relationship between occupancy of beta adrenergic receptors and stimulation of adenylate cyclase in dog atrial tissue was examined by studying the binding of [125I]iodopindolol and the activation of adenylate cyclase. Computer-assisted nonlinear regression analysis was used to analyze the inhibition of isoproterenol-stimulated adenylate cyclase activity by beta-1- or beta-2-selective antagonists. The Ki values for each subtype of receptor for the selective antagonists resulting from studies of the inhibition of adenylate cyclase activity were similar to those determined in studies of the inhibition of the binding of [125I]iodopindolol. To compare further the occupancy of beta-1 or beta-2 adrenergic receptors with the activation of adenylate cyclase mediated by each class of receptor, computer modeling of the stimulation of adenylate cyclase by the beta-1-selective agonist norepinephrine was carried out. The EC50 values of norepinephrine for each receptor subtype, as measured in studies of norepinephrine-stimulated adenylate cyclase activity, were similar to the Ki values for the inhibition by norepinephrine of the binding of [125I]iodopindolol to each receptor subtype. The data led to the conclusion that beta-1 adrenergic receptors make up about 70% of the total number of beta adrenergic receptors and mediate 70% of the increase in adenylate cyclase activity produced by isoproterenol. These results suggest that the relationship between occupancy of each class of receptor and activation of adenylate cyclase is linear and that, when agonist-stimulated adenylate cyclase activity is used as a functional response, neither spare beta-1 nor spare beta-2 adrenergic receptors exist in the atrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

LYRM1基因过表达对3T3-L1脂肪细胞线粒体代谢的影响

目的观察LYRM1基因过表达对3T3-L1脂肪细胞线粒体代谢的影响。方法体外培养3T3-L1前体脂肪细胞,分别构建LYRM1基因过表达细胞株(LYRM1-pcDNA3.1/myc-His B)和空载对照细胞株(pcDNA3.1/myc-His B);实时定量RT-PCR验证转染情况;RT-PCR检测诱导分化成熟后的线粒体代谢酶HKI,ACC,CS,CPT1和Cyc-C基因表达水平。结果 RT-PCR结果证实LYRM1质粒转染成功;与空载对照组比较,LYRM1过表达组HKI,ACC,CS,CPT1和Cyc-C mRNA表达水平均显著降低(P均<0.05)。结论 LYRM1基因过表达下调了3T3-L1脂肪细胞中线粒体的代谢关键酶基因表达水平,可能参与调节脂肪细胞线粒体的代谢。     

Genetic influence on the regulation of beta adrenergic receptors in mice

The regulation of beta adrenergic receptors was investigated in inbred mouse strains in which previous studies revealed differences in the regulation of dopamine receptors. The density of beta adrenergic receptors in the cerebral cortex of BALB/J mice was about one-third of that in CBA/J and C57BL/6J mice. Strain differences in the binding of [125I]iodohydroxypindolol to beta adrenergic receptors were due to changes in the density of beta-1 adrenergic receptors. Chronic administration of propranolol did not result in an increase in the density of beta adrenergic receptors receptors in cortices of C57BL/6J and BALB/cJ mice were observed. In contrast, pretreatment with 6-hydroxydopamine resulted in increases in the density of beta adrenergic receptors in the cerebral cortex of all three strains. Analysis of the effects of these treatments on the subtypes of beta adrenergic receptors revealed that the changes were restricted to changes in the density of beta-1 receptors. The failure to observe a response to propranolol in CBA/J mice expands the extent of deficits reported previously in this strain for striatal dopamine receptor supersensitivity after chronic treatment with haloperidol (Severson et al., Brain Res. 210: 201-215, 1981). CBA/J mice may be a useful model for genetic analysis of mechanisms for the control of receptor sensitivity and to investigate the impairments of the regulation of catecholaminergic receptors observed in aged rodents.     

大黄素对3T3-L1前脂肪细胞诱导分化的作用

目的:观察大黄素(emodin)对3T3-L1前脂肪细胞的诱导分化作用,并探讨其可能机制。方法:采用油红O染色、三酰甘油(TG)含量测定及3-磷酸甘油脱氢酶(GPDH)活性测定等方法检测脂肪细胞的分化程度;用实时定量PCR法检测大黄素对脂肪细胞过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA、CCAAT增强子结合蛋白α(C/EBPα)mRNA和脂肪酸结合蛋白ap2mRNA表达的影响;用四甲基偶氮唑盐(MTT)比色法测定3T3-L1前脂肪细胞的增殖;运用表面等离子体共振技术(SPR)测定大黄素与PPARγ2的亲和力。结果:大黄素呈剂量依赖性促进3T3-L1前脂肪细胞的诱导分化,50μmol/L大黄素可使脂肪细胞内TG含量和GPDH活性分别增加约22%及60%。大黄素可显著升高脂肪细胞的PPARγ2mRNA、C/EBPαmRNA及脂肪酸结合蛋白ap2mRNA表达水平。同时,50μmol/L大黄素可使前脂肪细胞的细胞增殖率下降约17%。SPR检测结果显示,大黄素具与PPARγ2结合活性,提示其可能是PPARγ2的配体。结论:大黄素作为PPARγ2的配体,能促进脂肪细胞分化、抑制前脂肪细胞增殖,而该过程可能是通过上调PPARγ2、C/EBPα的表达来实现的。     

Rh-SAA对3T3-L1脂肪细胞胰岛素敏感性的影响

目的:通过检测不同浓度和不同时间点Rh-SAA干预的3T3-LI脂肪细胞对葡萄糖的转运情况,了解Rh-SAA对脂肪细胞胰岛素敏感性的影响.方法:采用不同浓度的Rh-SAA(1、10、20 μg/mL)干预3T3-LI脂肪细胞48 h及20 μg/mL Rh-SAA分别干预细胞6、12、24、48 h,采用3H-2-DG摄入法检测细胞对葡萄糖的转运率.结果:Rh-SAA显著减少3T3-L1脂肪细胞在胰岛素刺激下的葡萄糖摄取,呈剂量和时间依赖效应.结论:Rh-SAA能降低3T3-L1脂肪细胞对胰岛素的敏感性,提示炎症与胰岛素抵抗密切相关.     

胎球蛋白-a对3T3-L1脂肪细胞脂肪分解的影响

目的观察胎球蛋白-a(fetuin-a)对脂肪分解的影响并探讨其可能的机制。方法体外培养3T3-L1前脂肪细胞,分化成熟后用不同浓度的fetuin-a孵育。采用甘油检测试剂盒测定释放到上清液的甘油含量;采用Real-timePCR检测细胞中脂肪甘油三酯脂肪酶(Adipose triglyceride lipase,ATGL)的mRNA表达;采用western blot检测细胞内磷酸化激素敏感脂肪酶(Hormone Sensitive Lipase,HSL)和ATGL的蛋白表达;采用ELISA试剂盒检测细胞内环磷酸腺苷(cyclic adenosine monophosphate,cAMP)的含量。结果 Fetuin-a能够抑制成熟脂肪细胞的脂肪分解,具有剂量依赖关系。Fetuin-a能够降低细胞内cAMP及磷酸化的HSL含量,同时使脂肪细胞ATGL mRNA水平及蛋白表达降低。结论 Fetuin-a抑制3T3-L1脂肪细胞的脂肪分解,其机制可能是通过抑制cAMP-PKA-HSL通路以及降低细胞内ATGL的表达而发挥作用的。     

牛磺酸对3T3-L1前脂肪细胞分化的影响

目的:有研究发现,喂食牛磺睃的糖尿病鼠脂肪细胞摄糖的能力增加,但牛磺酸是否能影响脂肪细胞分化,目前还尚不清楚.本实验观察牛磺酸对3T3-L1前脂肪细胞分化的影响,并探讨牛磺酸影响3T3-L1前脂肪细胞分化的机制.方法:实验于2006-07/09在中南大学湘雅三医院实验中心完成.①实验材料:实验中应用的3T3-L1细胞由中国科学院上海细胞库提供.②实验方法:将3T3-L1细胞用含体积分数为0.10胎牛血清的高糖DMEM培养液培养,内含108 u/L青霉素,80×1010U/L链霉素.细胞达汇片后,按5×107L-1密度接种于培养瓶中,应用0.5mmol/L IBMX、0.5mg/L胰岛素和1μmol/L地塞米松诱导分化,实验组加入牛磺酸,对照组未实施牛磺酸干预.油红O染色观察脂肪细胞分化情况.10, 20 mmol/L牛磺酸分别干预C3T3-L1前脂肪细胞24,48 h,抽提实验组和对照组细胞RNA及蛋白.③实验评估:采用RT-PCR及Western-blot方法观察脂肪分化相关基因的表达变化.结果:①10 mmol/L牛磺酸干预后14 d,实验组油红O染色阳性脂肪细胞明显少于对照组.②10,20 mmol/L牛磺酸分别干预3T3-L1前脂肪细胞24.48 h后,对脂肪分化相关基因Insig-2蛋白、PPAR、Insing-2、脂联素、脂联素受体、GLUT-4、AP-2 mRNA表达无明显影响.结论:牛磺酸可抑制前脂肪细胞分化,但具体机制还需要进一步研究.     

3T3-L1脂肪细胞分化不同阶段抵抗素表达的变化

目的:抵抗素具有直接对抗胰岛素作用,观察3T3-L1脂肪细胞分化不同阶段过程中抵抗素mRNA和蛋白表达的变化。方法:实验于2006-03/10在郑州大学第一附属医院完成。3T3-L1前脂肪细胞由武汉同济医院儿科馈赠。①将3T3-L1前脂肪细胞接种于培养瓶,加入含体积分数为0.1小牛血清的高糖DMEM培养液,在37℃、体积分数为0.05的CO2条件下培养。待细胞融合2d后,加入体积分数为0.1小牛血清的高糖DMEM培养液培养(含0.5mmol/L3-异丁基-1-甲基黄嘌呤、5mg/L胰岛素、1μmol/L地塞米松),诱导3T3-L1前脂肪细胞分化成熟。②分别于诱导分化的第2,4,6,8天采集细胞,采用反转录-聚合酶链反应检测脂肪细胞抵抗素mRNA的表达,以免疫印迹分析方法检测脂肪细胞抵抗素蛋白的表达。结果:①3T3-L1前脂肪细胞分化的形态学变化:诱导分化前细胞呈梭形,胞浆中无脂滴,表现为成纤维细胞样的前脂肪细胞形态,待完全汇合后处于生长停滞期;诱导分化第2天细胞变圆;第4天时细胞变大,变圆,胞浆有少量脂滴出现;第6天脂滴明显增多;至第8天更多的脂滴积累,形成典型的“戒环”样结构。②抵抗素mRNA在3T3-L1脂肪细胞分化不同阶段表达的变化:3T3-L1脂肪细胞抵抗素mRNA在分化的第2,4,6,8天吸光度值分别为0.16±0.03,0.37±0.07,0.63±0.11和0.96±0.17,相邻两时间点比较抵抗素mRNA的表达差异均有显著性意义(P均<0.01)。③抵抗素蛋白在3T3-L1脂肪细胞分化不同阶段表达的变化:3T3-L1脂肪细胞抵抗素蛋白在分化的第2,4,6,8天吸光度值分别为28942.5±1392.7,59326.7±2954.3,107821.6±4325.1和173656.5±8356.7,相邻两时间点比较抵抗素蛋白的表达差异均有显著性意义(P均<0.01)。结论:抵抗素mRNA及蛋白在脂肪细胞分化成熟过程中表达逐渐升高。     

GEM基因在3T_3-L_1脂肪细胞诱导分化过程中的表达变化

目的观察3T3L1前体脂肪细胞诱导分化过程中GEM基因表达水平的变化,探讨GEM基因与肥胖发生的关系。方法体外培养3T3L1前体脂肪细胞,采用RTPCR技术检测分化不同时段的3T3L1脂肪细胞中GEM基因的mRNA表达水平。结果在3T3L1脂肪细胞的诱导分化过程中,GEM基因mRNA表达水平无显著变化(P>0.05)。结论GEM基因在脂肪细胞分化、脂质合成和积聚过程中的作用有待进一步研究。