小檗碱对糖尿病db/db小鼠骨骼肌蛋白质代谢的影响及机制研究

目的 探讨小檗碱对糖尿病/胰岛素抵抗模型db/db小鼠骨骼肌蛋白质代谢及肌萎缩蛋白Fbox-1(Atrogin-1)表达的影响.方法 db/db小鼠为研究对象,以野生型为对照组.予小檗碱(5 mg·kg-1·d-1)腹腔注射3周,观察体重和食量;3周末处死,分离胫骨前肌和腓肠肌.胫骨前肌冰冻切片行层黏连蛋白免疫组化染色,荧光显微镜下观察并拍照,测量肌纤维横截面积并计算肌纤维面积分布图;同位素[14C]-苯丙氨酸掺入法检测肌肉蛋白合成,[3H]-酪氨酸释放率分析检测肌肉分解代谢;Northern印迹检测腓肠肌Atrogin-1和肌环指蛋白1(MurF-1)转录水平,Western印迹检测蛋白翻译水平.结果 小檗碱能明显降低db/db小鼠血糖水平[(18.55---3.79对26.32±4.02)mmol/L,P＜0.01],降低脂肪重量[(2.75±0.30对3.77±0.52)g,P＜0.05],使胫骨前肌的肌重/胫骨长度比值及肌纤维横截面积下降,肌纤维面积分布图明显左移;小檗碱使野生型j、鼠和db/db小鼠的蛋白质合成率下降18％ ～ 22％;蛋白质分解率升高24％～ 26％,使肌肉特异性的Atrogin-1、MurF-1转录和翻译水平增高,同时使真核转录因子3调节亚基(eIF3-f)蛋白水平降低.结论 小檗碱具有降糖降脂作用,但可引起骨骼肌蛋白质合成下降,促进蛋白质分解代谢,加剧糖尿病的骨骼肌消耗,其机制与上调Atrogin-1和MurF-1表达,同时下调elF3-f有关.     

肌肉特异性糖皮质激素受体基因敲除对慢性肾脏病骨骼肌消耗的影响

目的:内源性糖皮质激素水平异常升高可引起和加重慢性肾脏病(CKD)引起的骨骼肌消耗。本研究观察肌肉特异性糖皮质激素受体基因敲除(MGRKO)对CKD状态下小鼠骨骼肌消耗的影响。方法:采用5/6肾切除制作CKD小鼠模型,以假手术组(CTL组)为对照组。组织病理学检测胫骨前肌横截面积并计算肌纤维面积分布图;荧光定量PCR检测肌肉萎缩蛋白Fbox-1(Atrogin-1)和肌环指蛋白1(MuRF-1)mRNA水平;Western Blot检测其蛋白表达及Akt/FoxO1信号通路;ELISA方法检测血清皮质酮水平。结果:造模1月后,lox/lox-CKD和MGRKO-CKD小鼠的血尿素氮和皮质酮水平均明显升高;lox/lox-CKD的体重明显下降胫骨前肌湿重明显降低。而MGRKO-CTL和MGRKO-CKD两组间体重无明显差异,胫骨前肌湿重仅轻微降低。组织学观察到lox/lox-CKD胫骨前肌肌纤维明显萎缩,横截面积减少,肌纤维面积分布图明显左移。而MGRKO-CKD组的体重下降不明显,胫骨前肌仍较饱满,肌肉湿重略降低,横截面积略降低,与MGRKO-CTL组比较,肌纤维面积分布图仅轻度左移。荧光定量PCR和Western Blot结果显示lox/lox-CKD组的腓肠肌Atrogin-1和MuRF-1的mRNA和蛋白表达水平显著升高,同时其Akt磷酸化水平(pAkt/Akt)较lox/lox-CTL组显著降低3.1倍,FoxO1磷酸化水平(pFoxO1/FoxO1)较lox/lox-CTL组降低2.3倍;而MGRKO-CKD组的Atrogin-1表达仅轻微上调,MuRF-1则无明显变化;MGRKO-CKD组Atrogin-1蛋白表达水平仅比MGRKO-CTL组升高,同时pAkt/Akt、pFoxO1/FoxO1水平较对照组仅轻微降低。结论:内源性糖皮质激素水平升高在CKD肌肉消耗中发挥重要作用;MGRKO可减轻/阻止CKD状态下的体重下降和肌肉萎缩,下调肌萎缩关键基因Atrogin-1和MuRF-1表达水平;该效应与阻断糖皮质激素影响Akt/FoxO1信号途径有关,抑制糖皮质激素信号通路可能改善CKD肌肉消耗。     

心脏营养素-1延缓失神经骨骼肌萎缩作用机制研究

目的探讨外源性心脏营养素-1（CT-1）延缓失神经骨骼肌萎缩的作用机制。方法将120只小鼠随机分为两组各60只，制作腓肠肌失神经模型后实验组腹腔注射CT-1 100μg／（kg·d），对照组注射等量CT-1溶媒，分别于给药后2、4、6周完整切取腓肠肌，检测骨骼肌细胞凋亡、蛋白代谢、兴奋一收缩耦联等相关指标。结果与对照组比较，观察组Fas mRNA表达降低、Bcl-2 mRNA表达增加、骨骼肌细胞凋亡率下降；肌细胞收缩蛋白中α-肌动蛋白、肌球蛋白重链Ⅱa mRNA表达增加；肌质网Ca^2＋-ATP酶水平升高；但泛素、RC2 mRNA表达水平及MeHis释放量无明显差异。结论外源性CT-1延缓失神经骨骼肌萎缩的机制为促进失神经骨骼肌结构性蛋白合成、抑制肌细胞凋亡、提高Ca^2＋-ATP酶水平等。     

不同频率神经肌肉电刺激对ARDS相关性ICU-AW小鼠肌肉萎缩防治及临床意义

目的不同频率下神经肌肉电刺激(neuromuscular electrical stimulation, NMES)在ARDS相关性ICU获得性衰弱(ICU-acquired weakness, ICU-AW)中的作用及机制。 方法健康雄性C57BL/6小鼠88只随机分为11组,每组8只。分为：空白对照组(C1)、气管内注入无菌水组(C2)、ICU-AW模型组(ICU-AW)、ICU-AW+AMPK激动剂A-769662组(ICU-AW-A)、ICU-AW+A-769662溶剂对照组(ICU-AW-V)、NMES 20 Hz组(ICU-AW-20)、NMES 40 Hz组(ICU-AW-40)、NMES 60 Hz组(ICU-AW-60)、NMES 80 Hz组(ICU-AW-80)、ICU-AW-40+AMPK抑制剂Compound C组(ICU-AW-40-C)、ICU-AW-40+Compound C溶剂对照组(ICU-AW-40-V)。检测小鼠四肢抓力和存活状态,7 d后收集小鼠肺组织和腓肠肌标本,采用HE染色观察肺和肌肉病理学变化,采用western blot以及qRT-PCR的方法检测小鼠腓肠肌中Atrogin-1、MuRF-1 mRNA和蛋白表达。 结果与C1、C2组相比,ICU-AW小鼠出现肺损伤及腓肠肌萎缩,四肢抓力及存活率显著降低(P<0.05),腓肠肌组织中MuRF-1、Atrogin-1基因及蛋白表达显著降低(P值分别为<0.001和<0.05);和C2组相比,ICU-AW组p-AMPK蛋白水平显著降低(P<0.01);与ICU-AW-V组相比,ICU-AW-A组小鼠腓肠肌肌肉萎缩改善,四肢抓力显著提高,Atrogin-1和MuRF-1蛋白及基因表达量显著降低(P<0.01);相比于ICU-AW组,ICU-AW-20组、ICU-AW-60组、ICU-AW-40组四肢抓力均显著提升(P<0.05),其中,ICU-AW-40提升最为显著(P<0.05);ICU-AW-40组Atrogin-1和MuRF-1蛋白及基因表达量也较其它四组显著降低(P<0.01);而AMPK抑制剂Compound C干预后能够显著逆转NMES 40 Hz方案的ICU-AW肌无力的保护作用(P<0.05,ICU-AW-40-V vs. ICU-AW-40-C)。 结论早期应用40 Hz NMES能够显著改善ARDS相关性ICU-AW小鼠肌肉萎缩无力,其保护机制可能是通过激活AMPK发挥作用。     

补肾健脾方对地塞米松诱导大鼠骨骼肌萎缩的影响

目的探讨补肾健脾方对地塞米松诱导的骨骼肌萎缩的保护作用机制。方法将24只SD大鼠随机分为正常组、模型组、中药组、美雄酮组。在预设时间节点进行体质量称量,第14天测试大鼠跑台能力,第15天采用多功能生理仪检测大鼠肌张力,光镜检测肌纤维横截面;采用Western印迹方法检测骨骼肌肌球蛋白重链(Myhc)、肌肉环状指基因(MuRF)-1蛋白表达变化。结果与正常组比较,模型组的体重、有氧运动能力、肌张力、Myhc表达显著下降,MuRF-1表达显著升高(P<0.05);与模型组比较,中药组、美雄酮组的体重、有氧运动能力、肌张力、Myhc表达显著升高,MuRF-1表达显著降低(P<0.05)。结论补肾健脾方能有效抑制蛋白质的分解代谢,从而延缓地塞米松诱导的骨骼肌萎缩。     

AKT/FOXO1信号通路在心力衰竭小鼠骨骼肌萎缩中的作用

目的:探讨AKT/FOXO1信号通路在心力衰竭(HF)小鼠骨骼肌萎缩中的作用。方法:采用主动脉弓横向结扎8周,复制小鼠HF动物模型。采用实时定量-聚合酶链式反应和Western blot技术检测HF小鼠胫骨前肌内,E3连接酶的2个肌肉萎缩特异性指标Atrogin-1和MuRF1,对胫骨前肌中转录因子FOXO1和激酶AKT的磷酸化水平和总蛋白水平进行测定,并比较磷酸化蛋白和总蛋白的比率。结果:与对照组小鼠相比,TAC 8周诱导HF小鼠的胫骨前肌肌纤维变小,质量减轻。RT-PCR分析结果显示,HF小鼠胫骨前肌内的Atrogin-1和MuRF1的mRNA表达明显上调(P0.01)。Western blot分析结果显示,HF小鼠胫骨前肌组织中Atrogin-1和MuRF1的蛋白相对表达量HF组较对照组明显增加(P0.01)。HF组小鼠胫骨前肌中p-FOXO1的表达水平较对照组增加,FOXO1的总蛋白水平显著下降;p-AKT的蛋白表达较对照组增加(P0.05),AKT的总蛋白水平差异无统计学意义。p-FOXO1/FOXO1和p-AKT/AKT比率HF组明显高于对照组(P0.01)。结论:AKT/FOXOs信号通路参与HF后骨骼肌萎缩的过程并发挥重要作用。     

肺癌组织中Atrogin-1基因mRNA表达及其临床意义

目的 研究肺癌组织中Atrogin-1基因mRNA表达及其与临床病理参数的关系.方法 用PCR技术和免疫组化技术检测60例肺癌患者癌组织和癌旁正常肺组织的Atrogin-1基因mRNA及其蛋白表达.结果 肺癌组织中Atrogin-1基因mRNA表达及Atrogin-1蛋白表达观显高于癌旁正常肺组织(P均<0.01).临床病理参数分析显示,Atrogin-1基因mRNA表达与结核病史、淋巴结转移、肿瘤体积、肿瘤分期、体质量下降和血清白蛋白有关(P均<0.05);Atrogin-1蛋白表达与淋巴结转移、家族史、肿瘤体积、肿瘤分期、体质量下降和血清白蛋白有关(P均<0.05).结论 Atrogin-1基因在肺癌发生发展中起重要作用,其对监测患者病情进展、转移潜能及估计预后有临床价值.     

AG490对心肌营养素-1致心肌细胞肥大的影响

目的:研究AG490(一种酪氨酸酶抑制剂)对心肌营养素-1(cardiotrophin-1,CT-1)诱导的心肌细胞肥大的影响。方法:利用CT-1刺激心肌细胞造成细胞肥大,同时用AG490进行干预;检测心肌细胞大小、3H-亮氨酸掺入率和β-肌球蛋白重链(β-MHC)mRNA的表达。结果:经CT-1刺激后心肌细胞形态变大(q=16.47,P<0.01),3H-亮氨酸掺入率和β-MHCmRNA表达增加(P<0.01);AG490能抑制CT-1引起的心肌细胞肥大(q=10.37,P<0.01)、3H-亮氨酸掺入率和β-MHCmRNA表达的增高(P<0.01)。结论:AG490能够部分抑制CT-1所诱导的心肌细胞肥大。     

慢性心力衰竭合并骨骼肌萎缩大鼠模型的建立与评价

目的:探讨慢性心力衰竭合并骨骼肌萎缩模型建立方法,通过观察骨骼肌组织形态学和涉及萎缩相关的指标,探讨慢性心力衰竭合并骨骼肌萎缩涉及的分子靶点。方法:通过结扎大鼠左侧冠状动脉前降支的方法来建立大鼠慢性心力衰竭模型。术后3 d进行心电图检测。术后饲养12周,检测大鼠心脏射血分数(EF)和血清N末端脑钠肽前体(NT-proBNP)浓度,观察心脏大体结构和苏木精-伊红(HE)染色后组织形态,确定心力衰竭模型是否复制成功。同时测量大鼠抓力、腓肠肌重量,观察腓肠肌的大体结构和HE染色后组织形态,确定大鼠慢性心力衰竭合并骨骼肌萎缩模型成功建立。应用蛋白免疫印迹法(Western Blot)检测腓肠肌组织中肌球蛋白重链(MyHC)、肌萎缩Fbox-1蛋白(Atrogin-1)、肌肉环指蛋白-1(MuRF-1)、叉头转录因子1(FOXO1)蛋白表达。结果:与假手术组相比,模型组心电图出现病理性Q波,心脏EF明显下降(P<0.01),血清NT-proBNP浓度明显升高(P<0.01),心脏体积变大而且水肿,左心室前壁明显凹陷,HE染色观察到心肌组织的排列很不规则;大鼠腓肠肌组织疏松,颜色偏暗淡...     

Foxo1及泛素溶酶体系统在慢性肾脏病致骨骼肌萎缩作用中的初步观察

 目的 观察终末期肾病患者骨骼肌萎缩表现,并初步探讨腹直肌中转录因子Foxo1及泛素溶酶体系统活性变化与骨骼肌萎缩的关系。方法 对慢性肾脏病(CKD)5期的22例尿毒症患者在腹膜透析置管术中行腹直肌活检。并将8例诊断为子宫腺肌病并拟行经腹全子宫切除术患者及6例确诊为腹壁疝并拟行腹壁疝修补术患者设为对照组,于手术时留取少许腹直肌标本。观察肌纤维形态、计算肌纤维横截面积,并检测Foxo1及泛素溶酶体系统标志物Atrogin-1及MuRF1的mRNA和蛋白的表达情况。结果 CKD组患者腹直肌肌纤维的横截面面积较对照组明显缩小(P<0.01),CKD组Foxo1、Atrogin-1及MuRF1的mRNA含量及蛋白表达量均较对照组明显增加(P<0.05),而p-Foxo1蛋白表达量较对照组降低(P<0.05)。结论 CKD患者存在肌萎缩现象,推测其与Foxo1表达上调、磷酸化程度降低以及泛素溶酶体系统活性亢进有关。     

Reactive oxygen species play an essential role in IGF-I signaling and IGF-I-induced myocyte hypertrophy in C2C12 myocytes

IGF-I induces skeletal muscle hypertrophy by stimulating protein synthesis and suppressing the protein degradation pathway; the downstream signaling pathways Akt-mammalian target of rapamycin (mTOR)-p70-kDA-S6-kinase (p70S6K), and Forkhead box O1 (FoxO1) play essential roles in this regulation. Reactive oxygen species (ROS) modulate the signaling of various growth factors via redox regulation. However, the role of ROS in IGF-I signaling is not fully understood. In this study, we investigated whether ROS regulate the signaling and biological action of IGF-I in C2C12 myocytes. We found that IGF-I induces ROS in C2C12 myocytes. While treatment with H(2)O(2) significantly enhanced IGF-I-induced phosphorylation of the IGF-I receptor (IGF-IR), IGF-IR phosphorylation was markedly attenuated when cells were treated with antioxidants. The downstream signaling pathway, Akt-mTOR-p70S6K was subsequently down-regulated. Furthermore, the phosphorylation of FoxO1 by IGF-I decreased concomitantly with the restoration of the expression of its target genes, Atrogin-1 and muscle RING finger 1, which are related to muscle atrophy. Nox4 knockdown, which is reportedly to produce ROS in insulin signaling, attenuated IGF-I-induced IGF-IR phosphorylation, indicating that Nox4 is involved in the regulation of IGF-I signaling. Importantly, antioxidant treatments inhibited IGF-I-induced myocyte hypertrophy, demonstrating that ROS are necessary for IGF-I-induced myocyte hypertrophy in vitro. These results indicate that ROS play an essential role in the signaling and biological action of IGF-I in C2C12 myocytes.     

Alcohol-induced modulation of rictor and mTORC2 activity in C2C12 myoblasts

Background: The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1. Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real‐time PCR, respectively, while protein–protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by ³⁵S‐methionine/cysteine incorporation into cellular protein. Results: EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline‐rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14‐3‐3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline‐rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts . Conclusions: EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1‐dependent feedback mechanisms, functioning in addition to the insulin receptor substrate‐I/PI3K signaling pathway to regulate protein synthesis.     

Alcohol and indinavir adversely affect protein synthesis and phosphorylation of MAPK and mTOR signaling pathways in C2C12 myocytes

BACKGROUND: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. METHODS: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S]methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. RESULTS: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. CONCLUSIONS: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.     

1,25-Dihydroxyvitamin D improved the free fatty-acid-induced insulin resistance in cultured C2C12 cells

BACKGROUND: Epidemiological evidence has indicated that vitamin D deficiency increased the risk of insulin resistance in metabolic syndrome. The present study was conducted to test the hypothesis that 1,25-dihydroxyvitamin D may improve the free fatty-acid (FFA)-induced insulin resistance in muscle cells. METHOD: The insulin resistance of muscle cell model was established by treatment of FFA in differentiated C2C12 cells. Glucose uptake of C2C12 myotubes was analysed by the 3H-labelled 2-deoxyglucose uptake assay. The diameter of myotubes was measured under the condition of glutaraldehyde-induced autofluorescense. Tyrosine phosphorylated insulin receptor substrate 1 (IRS-1) was measured by immunoprecipitation. Serine phosphorylated IRS-1 and protein kinase B (Akt), extracellular signal-related kinase (ERK), c-Jun amino-terminal kinases (JNK) as well as their phosphorylated form were analysed by Western blots. RESULTS: Compared with a vehicle-treated group, FFA treatment in myotubes was associated with 70.6% reduction in insulin-mediated uptake of glucose, a five-fold increase in serine phosphorylation of IRS-1, 76.9% decrease in tyrosine phosphorylation of IRS-1 and 81.8% decrease in phosphorylation of Akt. Supplement of 1,25-dihydroxyvitamin D improved the FFA-induced inhibition of glucose uptake in a dose- dependent (p < 0.001) and time-dependent manner (p < 0.01). This was accompanied by increase in tyrosine phosphorylation of IRS-1 and phosphorylated Akt and decrease in serine phosphorylation of IRS-1 (p < 0.001). 1,25-Dihydroxyvitamin D also inhibited the FFA-induced reduction in myotube diameter by 35.3% (p < 0.001). JNK phosphorylation was reduced by 126.7% with treatment of 1,25-dihydroxyvitamin D (p < 0.001). 1,25-Dihydroxyvitamin D had no effect on FFA-induced ERK phosphorylation (p = 0.84). CONCLUSION: 1,25-Dihydroxyvitamin D improved the FFA-induced insulin resistance in muscle cells. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Induction of Ca signal mediated apoptosis and alteration of IP3R1 and SERCA1 expression levels by stress hormone in differentiating C2C12 myoblasts

Glucocorticoid (GC) are stress hormones, whose cytotoxicity has been shown in various cells. The imbalance of calcium homeostasis is believed to be associated with the dexamethasone (DEX, a synthetic GC)-induced apoptosis. Here we show that in C2C12 myoblasts, DEX markedly up-regulated the expression of inositol 1,4,5-triphosphate receptor 1 (IP3R1) and down-regulated the expression of SERCA1 (sarcoendoplasmic reticulum Ca²⁺-ATPase 1), leading to calcium overload. Furthermore, the imbalance of calcium homeostasis increased the level of BAX, decreased the level of Bcl-2, induced cytochrome c release and activated caspase-3, leading to intranucleosomal DNA fragmentation and plasma membrane damage, eventually resulting in cell apoptosis. Taken together, by using C2C12 myoblasts as a model system, we demonstrated a novel mechanism for stress hormone-induced apoptosis: it is dependent on the induction of intracellular calcium overload via the alterations of IP3R1 and SERCA1 expressions.     

Dexamethasone increases 1,25-dihydroxyvitamin D3 receptor levels and augments bioresponses in rat osteoblast-like cells

Glucocorticoids increase the level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors in primary cultures of rat calvarial osteoblast-like (OB) cells. The present study investigated how this dexamethasone (DEX) up-regulation of 1,25-(OH)2D3 receptors modulates three 1,25-(OH)2D3 bioresponses: inhibition of collagen synthesis, stimulation of osteocalcin synthesis, and induction of 25-hydroxyvitamin D3-24-hydroxylase activity. Pretreatment of OB cells with 13 nM DEX for 24 h doubled the 1,25-(OH)2D3 receptor level without changing receptor affinity for 1,25-(OH)2D3 to study bioresponses. After DEX treatment to increase the 1,25-(OH)2D3 receptor level, the magnitude and sensitivity of all three 1,25-(OH)2D3 bioresponses were enhanced. The maximal 1,25-(OH)2D3 inhibition of collagen synthesis was increased by DEX pretreatment compound to control values: 30 to 50% (1 day treatment) and 50 to 70% (2 day treatment). The sensitivity to 1,25-(OH)2D3, as measured by reduction of the half-maximal inhibitory dose (ED50), was increased 50%. This potentiation of 1,25-(OH)2D3 inhibitory action on collagen synthesis was still evident after correction for the inhibitory effect on collagen synthesis by DEX alone. The maximal stimulation of osteocalcin by 1,25-(OH)2D3 was also enhanced from 2- to 3-fold in controls to over 4- to 5-fold by DEX pretreatment. Similarly, the ED50 of the response was reduced 50%. For the induction of 25-hydroxyvitamin D3-24-hydroxylase activity, DEX doubled the enzyme activity over that seen with 1,25-(OH)2D3 alone, but only slightly affected the sensitivity of the enzyme induction. In conclusion, after DEX up-regulation of 1,25-(OH)2D3 receptor levels, there was a general potentiation of 1,25-(OH)2D3 bioresponses in rat OB cells. However, the detailed patterns of the augmented responses were different for each of the three biological functions we studied.