Fat gain with physical detraining is correlated with increased glucose transport and oxidation in periepididymal white adipose tissue in rats

As it is a common observation that obesity tends to occur after discontinuation of exercise, we investigated how white adipocytes isolated from the periepididymal fat of animals with interrupted physical training transport and oxidize glucose, and whether these adaptations support the weight regain seen after 4 weeks of physical detraining. Male Wistar rats (45 days old, weighing 200 g) were divided into two groups (n=10): group D (detrained), trained for 8 weeks and detrained for 4 weeks; and group S (sedentary). The physical exercise was carried out on a treadmill for 60 min/day, 5 days/week for 8 weeks, at 50-60% of the maximum running capacity. After the training protocol, adipocytes isolated from the periepididymal adipose tissue were submitted to glucose uptake and oxidation tests. Adipocytes from detrained animals increased their glucose uptake capacity by 18.5% compared with those from sedentary animals (P<0.05). The same cells also showed a greater glucose oxidation capacity in response to insulin stimulation (34.55%) compared with those from the S group (P<0.05). We hypothesize that, owing to the more intense glucose entrance into adipose cells from detrained rats, more substrate became available for triacylglycerol synthesis. Furthermore, this increased glucose oxidation rate allowed an increase in energy supply for triacylglycerol synthesis. Thus, physical detraining might play a role as a possible obesogenic factor for increasing glucose uptake and oxidation by adipocytes.     

Apelin-13对高尿酸诱导的脂肪细胞氧化应激的作用

目的:探讨apelin-13对高尿酸诱导的3T3-L1脂肪细胞氧化应激的作用及其机制。方法:3T3-L1脂肪细胞予以10 mg/dL尿酸刺激,部分细胞予以1μmol/L apelin-13预处理,以100μmol/L H₂O₂刺激的细胞为阳性对照。48 h后,流式细胞术检测活性氧族(ROS)含量,生化试剂盒检测细胞及培养液上清抗氧化酶[超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)]和促氧化酶NADPH氧化酶(NOX)的活性以及丙二醛(MDA)含量,real-time PCR法检测细胞局部肾素-血管紧张素系统(RAS)各组份血管紧张素原(AGT)、血管紧张素转化酶I(ACE1)、血管紧张素II 1型受体(AT1R)和AT2R及血管紧张素II 1型受体相关蛋(APJ)的mRNA表达水平,ELISA法检测细胞及培养液中血管紧张素II(AngⅡ)浓度。结果:10 mg/dL的尿酸明显降低3T3-L1脂肪细胞SOD、GSH-Px和CAT的活性,升高NOX的活性,增加MDA的含量,细胞内ROS的含量相应升高;apel...     

人骨髓基质干细胞体外诱导和鉴定

目的建立人自体骨髓基质干细胞(human menchymal stem cells,hMSCs)体外分离、鉴定体系,建立经诱导表达成骨、软骨、脂肪细胞表型的体外培养体系,探讨作为骨组织工程种子细胞的可能性,为组织工程技术应用打下基础。方法抽取患者骨髓5ml,以密度梯度法分离hMSCs,使用流式细胞仪鉴定细胞表型;应用免疫组化和分子生物学技术,对hMSCs进行成骨、软骨、脂肪细胞的诱导培养和表型鉴定。结果第2代hMSCs阳性细胞表达率CD105(78±6)%,CD166(43±7)%,CD29(69±12)%;hMSCs成骨诱导培养第3代平均扩增(163.4±13.4)倍,形成钙结节,骨细胞转录因子、骨钙素、骨桥蛋白和I型胶原免疫荧光阳性,RT-PCR证实有I型胶原、骨钙素、骨桥蛋白和骨结合素mRNA表达,对照组阴性;成软骨细胞诱导培养后II型胶原、SOX9(SRY-type HMGbox9,是在哺乳动物性别决定和软骨生成中起着关键调控作用的一个基因)。免疫荧光阳性,RT-PCR证实II型胶原、软骨聚集蛋白聚糖mRNA表达,对照组阴性;成脂肪细胞诱导培养后,油红-O染色阳性,RT-PCR证实PPAR2 mRNA...     

Differences in functional cyclic AMP compartments mediating lepolysis by isoprenaline and BRL 37344 in four adipocyte types

Triglyceride mobilization and adenylyl cyclase activation in adipocytes from Wistar rats, lean Zucker (Fa/?) rats, obese Zucker (fa/fa) rats and humans were investigated in concentration-response studies with (−)-isoprenaline and the atypical β₃-adrenoceptor selective agonist BRL 37344. Maximum FFA production by both agonists was identical in Wistar rat and lean Zucker rat adipocytes, while obese Zucker rat adipocytes and human adipocytes produced significantly less FFA, especially with BRL 37344. Maximum adenylyl cyclase activation by (−)-isoprenaline was similar for all types of adipocyte ghosts, whereas BRL 37344 was a partial agonist in all cases with the lowest intrinsic activity in human adipocytes. For (−)-isoprenaline the relationship between cAMP and lipolysis was steepest with Wistar rat adipocytes, followed by human and lean Zucker rat adipocytes, while obese Zucker rat cells showed a shallow relationship. For BRL 37344, the relationship was very steep and similar for all four adipocyte types, despite the marked differences in maximal lipolysis and cyclic AMP production. The results strongly argue in favour of cyclic AMP compartmentalization, the activity ratio between the functional and the non-functional compartment being least favourable in obese Zucker rat adipocytes. The atypical β₃-adrenoceptor agonist BRL 37344 very efficiently directs the generated cyclic AMP into the functional compartment in all four adipocytes types investigated.     

Effect of maturation and senescence on carbohydrate utilization and insulin responsiveness of rat adipose tissue

Summary The aim of the present investigation was to study the effect of aging upon the metabolism and the responsiveness to insulin of epididymal adipose tissue from rats (6 weeks, 6 and 24 months). Basal glucose metabolism by these tissues and adipocytes was positively related to cell size, for each age group. But age per se plays an important role: for the same diameter, the U-¹⁴C-glucose oxidation to CO₂ and its incorporation into triglycerides decreased markedly between 6 weeks and 6 months, as reported previously, and decreased still further between 6 months and 24 months, for any diameter. In contrast, insulin responsiveness of adipose tissue fragments and fat cells was negatively correlated to adipose cell size, when we analyzed the role of cell diameter for a group of given age. When comparing the sensitivity to hormone for a given cell volume but at different ages, it appeared that insulin resistance increased considerably between 6 weeks and 6 months and was still more marked in old age. The mechanisms underlying these facts have been discussed.     

A mixed meal potentiates the insulin sensitivity of glucose transport and metabolism in adipocytes from patients with type 2 diabetes mellitus

Post-glucose enhancement of insulin action may represent a physiological mechanism for the acute regulation of insulin sensitivity of target tissues. To clarify whether a similar mechanism is operative in the insulin-resistant diabetic state we have investigated the effects of a mixed meal on adipocyte insulin action in eight patients with Type 2 diabetes mellitus. Ninety minutes after ingestion of breakfast insulin binding to fat cells increased by 21% (p less than 0.05). In the fasting state 6 patients had a significant response of glucose transport and lipogenesis to insulin whereas two exhibited non-responsiveness. In the 6 responders insulin sensitivity, as estimated by the insulin concentration at which half-maximal effect was achieved, increased for glucose transport (before, 260 +/- 46 pmoll-1; after, 105 +/- 21 pmol l-1; p less than 0.05) and for lipogenesis (before, 36 +/- 9 pmol l-1; after, 9 +/- 2 pmol l-1; p less than 0.05). No significant changes occurred in basal or maximal glucose transport or lipogenesis. In the two primary non-responders intake of the meal was associated with average increase in maximal insulin responsiveness of 52% for glucose transport and 28% for lipogenesis. Intake of a mixed meal is associated with a slight increase of insulin binding to adipocytes from patients with Type 2 diabetes mellitus but a marked increase of adipocyte insulin sensitivity at the post-binding levels of glucose transport and metabolism.     

母体来源人胎盘间充质细胞的分离培养及其分化能力的研究

目的探讨胎盘母体来源间充质细胞的分离、培养方法及其向脂肪和成骨诱导分化潜能。方法采用酶消化获得母体来源胎盘间充质干细胞体外扩增后,利用流式细胞仪检测其表面标志物;进行诱导分化后,采用油红O及茜素红染色鉴定其向脂肪和成骨诱导分化潜能。结果体外培养的母体来源胎盘间充质干细胞呈长梭形,细胞形态均一;细胞表面标志鉴定：CD73、CD90和CD105呈阳性表达,而CD14、CD34、CD45,和HLA—DR呈阴性表达;细胞诱导分化后,经油红O、茜素红染色证实其可分化为脂肪细胞和成骨细胞。结论建立了母体来源胎盘间充质干细胞的分离培养方法;证实其具有成脂、成骨分化潜能,有望成为细胞治疗及组织工程更为理想的种子细胞。     

Fatty acid metabolism in adipose tissue of aging mice after direct tracer injection into fat pads

We have examined previously reported age-related defects in triglyceride synthesis from [1-¹⁴C]palmitate in adipose tissue of mice. Three techniques were used: in vitro, using adipocytes isolated from epididymal fat pads of young and old mice; and in vivo, using two new methods to measure free fatty acid (FFA) esterification by adipose tissue (direct injection of labeled palmitate-albumin complexes in large or small volumes into the extracellular spaces of the epididymal or inguinal fat pads of young and old mice). When the entire fat pad was filled with tracer we no longer observed heterogeneous labeling of adipocytes in epididymal fat pads that occurred in an earlier study in which an in vivo-in vitro method has been used. Free fatty acids were converted to triacylglycerol faster by adipocytes of large cells from older animal than by those of small cells from young mice; when the cell sizes of young and old mice were approximately equal, then the rates of FFA esterification were the same in young and old adipocytes. When FFA was injected as a small bolus the fractional rates of FFA disappearance and of FFA incorporation into triacylglycerol in the different fat pads, observed during a 60-min period, were the same (about 5 min or less) regardless of the region of the fat pad studied (distal or proximal epididymal fat pad), the type of fat pad (epididymal or inguinal), or the age of the mice (12–92 weeks). Other potential applications of the direct injection technique for studying FFA metabolism and structure-function in adipose tissue in vivo are discussed. Our findings, coupled with the earlier study in which labeled FFA was added to the outside of fat pads, indicate that, in adipose tissue of old mice, there exist barriers comprising mesothelial cells, collagenous structures, and/or the outer layer of adipocytes in fat pads, that interfere in the transport of FFA to the interior adipocytes when FFA is added outside the fat pad. This age-related defect may be circumvented by injecting tracer directly into the interstitial fluid compartment.     

孕酮对脂肪细胞促酰化蛋白受体和下游信号蛋白表达的作用

目的 观察孕酮对3T3-L1(前)脂肪细胞促酰化蛋白(ASP)受体C5L2 mRNA和细胞表面C5L2蛋白表达的影响,以及孕酮对ASP下游信号蛋白的作用.方法 体外培养3T3-L1细胞,诱导细胞分化,不同浓度孕酮作用于3T3-L1(前)脂肪细胞,孵育过夜后收获细胞,分别采用RT-PCR和流式细胞仪检测ASP受体mRNA和蛋白表达情况;采用Western印迹法检测基础状态和ASP激发后Gαq/11,Gβ,p-PKCα和p-PKCζ蛋白表达.结果 孕酮最大抑制成熟脂肪细胞14% C5L2 mRNA (P＞0.05)和蛋白表达22%(36%±15%vs 46%±12%,P＜0.01),高浓度孕酮(1 × 10-6 mol/L)能显著性抑制前脂肪细胞66%C5L2 mRNA(0.17±0.11 vs 0.50±0.18,P＜0.01)和29%C5L2蛋白表达(36%±16%vs 51%±20%,P＜0.05).高浓度孕酮在一定程度上抑制ASP激发后成熟脂肪细胞Gαq/11,Gβ,p-PKCα和p-PKCζ的表达,各蛋白表达分别减少了41%(0.71±0.21 vs 1.20 ±0.24,P＜0.05),63%(0.55±0.32 vs 1.48±0.40,P＜0.05),49%(0.53±0.20 vs 1.04 ±0.19,P＜0.01)和32%(0.36 ±0.10 vs 0.53 ±0.20,P＞0.05).在前脂肪细胞,高浓度孕酮显著性抑制ASP刺激的59%Gαq/11(0.42 ±0.18 vs 1.04±0.28,P＜0.01),43%Gβ(0.77 ±0.09 vs 1.35 ±0.27,P＜0.05),51%p-PKCα(0.44 ±0.15 vs 0.90 ±0.25,P＜0.05)和30%p-PKCζ(0.27±0.08vs 0.39±0.12,P＜0.05)蛋白表达.结论 孕酮诱导ASP抵抗的发生,ASP抵抗参与了高浓度孕酮引起的脂肪细胞胰岛素抵抗状态的病理生理过程.     

高灵敏酶联免疫分析法测定人脂肪细胞瘦素分泌

目的 建立高灵敏、特异的酶联免疫分析法测定人脂肪细胞瘦素的分泌.方法 制备兔抗人瘦素多克隆抗体和鼠抗人瘦素单克隆抗体,建立生物素一亲和素放大酶联免疫分析方法(BAELISA),观测原代培养的人网膜前脂肪细胞分化过程中瘦素的分泌及曲格列酮对其影响.结果 BA-ELISA方法灵敏度可达0.03 ng/ml,曲线工作范围0.05～5 ng/ml,批内批间变异系数分别小于7.4%和9.3%,平均回收率97.8%.测定经柱层析分离的混合人血清或脂肪细胞培养上清,仅显示单峰游离瘦素.观测到人网膜前脂肪细胞随诱导分化成熟而瘦素分泌量递增;10 umol/L曲格列酮可使瘦素的分泌峰值增加1倍.健康成人空腹血清瘦素水平女性显著高于男性(均值:7.6 ng/ml vs 3.2 ng/ml,P<0.001),与体重指数均有较强的相关性(r=0.61、0.67;P<0.001).结论 建立了高灵敏的BA-ELISA测定游离瘦素水平,尤其适于瘦素浓度低的临床血样本测定及基础研究之用.