单羧酸转运蛋白基因对肿瘤细胞内pH值及生长特性的调节作用

目的 以反义技术抑制肿瘤细胞的单羧酸转运蛋白基因第一亚型(MCT1)表达,观察其对肿瘤细胞内pH值(pHi)调节、乳酸转运及生长性质的影响.方法 (1)应用逆转录-聚合酶链式反应(RT-PCR)从人肺癌细胞系A549中扩增MCT1目的 基因片段,将克隆的基因片段反向插入逆转录病毒载体pLXSN,构建反义表达重组载体pLXSN-MCT1,并对其进行DNA测序验证.(2)通过电穿孔法将pLXSN、重组载体pLXSN-MCT1转染于肺腺癌细胞系A549中,经G418筛选转染细胞阳性克隆.用PCR方法 鉴定pLXSN-MCT1重组载体在基因组的转染整合及表达;以分光光度法测定细胞内pH及乳酸含量变化,并以细胞生长曲线研究细胞的生长情况.结果 (1)对所构建的重组载体进行双酶切电泳分析及DNA序列分析证明反义载体构建成功.(2)与未转染的A549细胞比较,转染MCT1反义表达重组体的细胞pHi降低、乳酸显著升高(P＜0.001);且细胞的生长受到明显抑制.结论 单羧酸转运蛋白MCT1基因在肿瘤细胞pHi调节、乳酸转运及细胞的生长中起着重要的调节作用.     

罗格列酮对老年胰岛素抵抗小鼠骨骼肌细胞葡萄糖转运体4转位的影响

目的 探讨罗格列酮对胰岛素刺激的老年胰岛素抵抗（IR）小鼠骨骼肌细胞葡萄糖转运体4（GLUT4）转位的影响.方法 18月龄雌性C57BL/6J老年小鼠30只,随机平均分成三组：老年普通膳食组,老年IR组喂养高脂肪膳食（58%脂肪,21%蛋白,21%碳水化合物）,罗格列酮组给予高脂肪膳食的同时服用罗格列酮,同时设立青年组.16 w后提取各组小鼠完整的骨骼肌束,在胰岛素浓度为10-7的缓冲液中孵育30min.用超速蔗糖梯度离心方法分离细胞内膜和外膜的匀浆,用Western blot方法检测各组小鼠骨骼肌细胞内、外膜GLUT4蛋白水平.结果 老年对照组小鼠胰岛素刺激的GLUT4转位功能较青年组明显下降（P＜0.05）,老年IR组的内、外膜GLUT4蛋白总量及GLUT4转位均较老年对照组降低（P＜0.05）,而罗格列酮组的GLUT4转位较IR组有明显的上升（P＜0.05）.结论 罗格列酮增加了细胞内膜GLUT4向细胞外膜的转位.     

Possible mechanism for the regulation of glucose on proliferation, inhibition and apoptosis of colon cancer cells induced by sodium butyrate

AIM： To study the effect of glucose on sodium butyrate- induced proliferation inhibition and apoptosis in HT-29 cell line, and explored its possible mechanisms.
METHODS： HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, and were allowed to adhere for 24 h, and then replaced with experimental medium. Cell survival rates were detected by MTr assay. Apoptosis was detected by TUNEL assay. Glucose transport protein 1 （GLUT1） and monocarboxylate transporter 1 （MCT1） mRNA expression was detected by RT-PCR. RESULTS： Low concentration of glucose induced apoptosis and regulated proliferation in HT-29 cell line, and glucose can obviously inhibit the effect of proliferation inhibition and apoptosis induced by sodium butyrate. Glucose also down-regulated the expression of MCT1mRNA （0.28 ± 0.07 vs 0.19± 0.10, P 〈 0.05）, and decreased the expression of GLUTlmRNA slightly （0.18 ± 0.04 vs 0.13 ± 0.03, P 〈 0.05）.
CONCLUSION： Glucose can regulate the effect of proliferation inhibition and apoptosis induced by sodium butyrate and this influence may be associated with the intracellular concentration of glucose and sodium butyrate.     

罗格列酮上调高脂饮食老年大鼠骨骼肌GLUT4和PKB的表达

目的 观察高脂饮食对老年大鼠骨骼肌葡萄糖转运蛋白4(GLUT4)和蛋白激酶B(PKB)的表达变化及罗格列酮的干预效果.方法 老年大鼠随机分为对照组、高脂组(HF)、高脂+罗格列酮干预组(RSG),每组20只.应用清醒状态下正常葡萄糖高胰岛素钳夹技术的葡萄糖输注率评价胰岛素抵抗,用荧光定量PCR法和Western印迹技术检测骨骼肌GLUT4和PKB的表达.结果 高脂组骨骼肌长链脂酰辅酶A(LCACoA)升高而葡萄糖输注率明显下降(P＜0.01),骨骼肌GLUT4和PKB的表达明显降低(P＜0.05,P＜0.01),罗格列酮干预组显著缓解高脂组上述变化(P＜0.05,P＜0.01).结论 罗格列酮上调高脂饮食老年大鼠骨骼肌GLUT4和PKB的表达,是改善老年胰岛素抵抗的机制之一.     

Chronic caffeine intake reverses age-induced insulin resistance in the rat: effect on skeletal muscle Glut4 transporters and AMPK activity

The role of caffeine consumption on insulin action is still under debate. The hypothesis that chronic caffeine intake reverses aging-induced insulin resistance in the rat was tested in this work. The mechanism by which caffeine restores insulin sensitivity was also investigated. Six groups of rats were used: 3 months old (3 M), 3 months old caffeine-treated (3MCaf), 12 months old (12 M), 12 months old caffeine-treated (12MCaf), 24 months old (24 M), and 24 months old caffeine-treated (24MCaf). Caffeine was administered in drinking water (1 g/l) during 15 days. Insulin sensitivity was assessed by means of the insulin tolerance test. Blood pressure, body weight, visceral and total fat, fasting glycemia and insulinemia, plasma nonesterified fatty acids (NEFA), total antioxidant capacity (TAC), cortisol, nitric oxide, and catecholamines were monitored. Skeletal muscle Glut4 and 5′-AMP activated protein kinase (AMPK) protein expression and activity were also assessed. Aged rats exhibited diminished insulin sensitivity accompanied by hyperinsulinemia and normoglycemia, increased visceral and total fat, decreased TAC and plasma catecholamines, and also decreased skeletal muscle Glut4 and AMPK protein expression. Chronic caffeine intake restored insulin sensitivity and regularized circulating insulin and NEFA in both aging models. Caffeine neither modified skeletal muscle AMPK expression nor activity in aged rats; however, it decreased visceral and total fat in 12 M rats and it restored skeletal muscle Glut4 expression to control values in 24 M rats. We concluded that chronic caffeine intake reverses aging-induced insulin resistance in rats by decreasing NEFA production and also by increasing Glut4 expression in skeletal muscle.     

Reduced expression of sarcalumenin and related Ca2+ -regulatory proteins in aged rat skeletal muscle

In skeletal muscle, Ca(2+)-cycling through the sarcoplasm regulates the excitation-contraction-relaxation cycle. Since uncoupling between sarcolemmal excitation and fibre contraction may play a key role in the functional decline of aged muscle, this study has evaluated the expression levels of key Ca(2+)-handling proteins in senescent preparations using immunoblotting and confocal microscopy. Sarcalumenin, a major luminal Ca(2+)-binding protein that mediates ion shuttling in the longitudinal sarcoplasmic reticulum, was found to be greatly reduced in aged rat tibialis anterior, gastrocnemius and soleus muscle as compared to adult specimens. Minor sarcolemmal components of Ca(2+)-extrusion, such as the surface Ca(2+)-ATPase and the Na(+)-Ca(2+)-exchanger, were also diminished in senescent fibres. No major changes were observed for calsequestrin, sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor Ca(2+)-release channel. In contrast, the age-dependent reduction in the alpha(1S)-subunit of the dihydropryridine receptor was confirmed. Hence, this report has shown that downstream from the well-established defect in coupling between the t-tubular voltage sensor and the junctional Ca(2+)-release channel complex, additional age-related alterations exist in the expression of essential Ca(2+)-handling proteins. This may trigger abnormal luminal Ca(2+)-buffering and/or decreased plasmalemmal Ca(2+)-removal, which could exacerbate impaired signaling or disturbed intracellular ion balance in aged fibres, thereby causing contractile weakness.     

Effects of treadmill exercise and training frequency on anabolic signaling pathways in the skeletal muscle of aged rats

Physical exercise is the most effective intervention against sarcopenia of aging; however, the cellular and molecular mechanisms mediating training-induced adaptations are not yet completely understood. Furthermore, it is unclear whether exercise training initiated late in life affects myocyte anabolic signaling in a dose-dependent manner. Hence, we sought to investigate the effects of treadmill exercise and training frequency on anabolic pathways, including insulin signaling, in the skeletal muscle of old rats. Aged (14-16-month-old) male Wistar rats were trained on a treadmill for 3 (EX3) or 5 days/week (EX5) during 8 weeks and compared with age-matched sedentary controls (SED). Four-month-old rats were used as young controls (YC). Protein expression levels of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), activated (phosphorylated) mammalian target of rapamycin (p-mTOR) and glucose transporter GLUT4 were determined in quadriceps muscle extracts via immunoblotting. Mitochondrial cytochrome c oxidase (COX) activity was assessed by histochemical staining, while electron microscopy was employed to quantify the sarcomere volume (V_src). Body weight (BW) increased, whereas muscle weight (MW) and V_src decreased with age. EX5, but not EX3 increased MW and V_src, without affecting BW. The expression of IR and GLUT4 was higher in SED rats relative to the YC group. Conversely, protein levels of IRS-1 and p-mTOR as well as COX activity were reduced in advanced age. Compared with SED rats, EX3 animals displayed reduced IR expression and increased IRS-1 levels and COX activity. The expression of GLUT 4 and p-mTOR was unaffected by EX3. EX5 up-regulated IRS-1 and p-mTOR expression and COX activity, while decreasing GLUT4 levels, with no effect on IR expression. In summary, substantial impairments in muscle anabolic pathways, including insulin signaling, were detected in aged sedentary rats. These changes were ameliorated by exercise training, concomitant with improvements in muscle trophism. Benefits were more evident in rats trained for 5 days/week, suggesting that physical exercise initiated late in life affects anabolic signaling in a dose-dependent manner.     

Computer simulation of entry into glycolysis and lactate output in the ischemic rat heart.

A computer model of glucose uptake, glycogenolysis, and export of pyruvate and lactate in the ischemic, isolated rat heart was constructed. This model predicts that, after an initial burst of glycogenolysis which consumes the outer glycogen branches, glycolysis is limited by glucose uptake. Reduced glucose uptake in this model then results from decreased rate of substrate delivery despite increased affinity of the cell membrane carrier for glucose. Lactate accumulation in this model is due partially to its incomplete washout from the interstitium at low coronary flow rates and partially to a dramatic reduction in the activity of a cell membrane lactate permease.     

低出生体重大鼠发育期骨骼肌形态结构变化及胰岛素抵抗情况

观察低出生体重大鼠不同发育时期骨骼肌形态结构的变化和胰岛素抵抗情况.采用孕期低蛋白饮食法建立低出生体重仔鼠模型.于出生后7 d、21 d、2月龄测定仔鼠空腹血糖、血清胰岛素值,并检测骨骼肌形态结构.自7 d至2月龄,低出生体重仔鼠骨骼肌纤维均有明显萎缩、排列稀疏紊乱;2月龄时超微结构明显异常.2月龄内低出生体重仔鼠血糖和血清胰岛素与对照组比较无差异.     

低出生体重大鼠发育期骨骼肌形态结构变化及胰岛素抵抗情况

观察低出生体重大鼠不同发育时期骨骼肌形态结构的变化和胰岛素抵抗情况.采用孕期低蛋白饮食法建立低出生体重仔鼠模型.于出生后7 d、21 d、2月龄测定仔鼠空腹血糖、血清胰岛素值,并检测骨骼肌形态结构.自7 d至2月龄,低出生体重仔鼠骨骼肌纤维均有明显萎缩、排列稀疏紊乱;2月龄时超微结构明显异常.2月龄内低出生体重仔鼠血糖和血清胰岛素与对照组比较无差异.     

Melatonin prevents mitochondrial dysfunction and insulin resistance in rat skeletal muscle

Melatonin has a number of beneficial metabolic actions and reduced levels of melatonin may contribute to type 2 diabetes. The present study investigated the metabolic pathways involved in the effects of melatonin on mitochondrial function and insulin resistance in rat skeletal muscle. The effect of melatonin was tested both in vitro in isolated rats skeletal muscle cells and in vivo using pinealectomized rats (PNX). Insulin resistance was induced in vitro by treating primary rat skeletal muscle cells with palmitic acid for 24 hr. Insulin‐stimulated glucose uptake was reduced by palmitic acid followed by decreased phosphorylation of AKT which was prevented my melatonin. Palmitic acid reduced mitochondrial respiration, genes involved in mitochondrial biogenesis and the levels of tricarboxylic acid cycle intermediates whereas melatonin counteracted all these parameters in insulin‐resistant cells. Melatonin treatment increases CAMKII and p‐CREB but had no effect on p‐AMPK. Silencing of CREB protein by siRNA reduced mitochondrial respiration mimicking the effect of palmitic acid and prevented melatonin‐induced increase in p‐AKT in palmitic acid‐treated cells. PNX rats exhibited mild glucose intolerance, decreased energy expenditure and decreased p‐AKT, mitochondrial respiration, and p‐CREB and PGC‐1 alpha levels in skeletal muscle which were restored by melatonin treatment in PNX rats. In summary, we showed that melatonin could prevent mitochondrial dysfunction and insulin resistance via activation of CREB‐PGC‐1 alpha pathway. Thus, the present work shows that melatonin play an important role in skeletal muscle mitochondrial function which could explain some of the beneficial effects of melatonin in insulin resistance states.     

Catecholamine regulation of local lactate production in vivo in skeletal muscle and adipose tissue: role of -adrenoreceptor subtypes

CONTEXT: The regulation of lactate production in skeletal muscle (SM) and adipose tissue (AT) is not fully elucidated. OBJECTIVE: Our objective was to investigate the catecholamine-mediated regulation of lactate production and blood flow in SM and AT in healthy, normal-weight subjects by using microdialysis. METHODS: First, lactate levels in SM and AT were measured during an iv norepinephrine infusion (n = 11). Local blood flow was determined with the 133Xe-clearance technique. Second, muscle lactate was measured during hypoglycemia and endogenous epinephrine stimulation (n = 12). Third, SM was perfused with selective beta(1-3)-adrenoreceptor agonists in situ (n = 8). Local blood flow was measured with the ethanol perfusion technique. RESULTS: In response to iv norepinephrine, the fractional release of lactate (difference between tissue and arterial lactate) increased by 40% in SM (P = 0.001), whereas remaining unchanged in AT. Blood flow decreased by 40% in SM (P < 0.005) and increased by 50% in AT (P < 0.05). In response to hypoglycemia, epinephrine increased 10-fold, and the fractional release of lactate in SM doubled (P < 0.0001). The blood flow remained unchanged. The beta2-agonist, terbutaline, caused a marked concentration-dependent increase of muscle lactate and blood flow (P < 0.0001). The beta(1)-agonist, dobutamine, induced a discrete increase of muscle lactate (P < 0.0001), and the blood flow remained unchanged. The beta3-agonist, CPG 12177, did not affect muscle lactate or blood flow. CONCLUSIONS: Catecholamines stimulate lactate production in SM, but not in AT. In SM, the beta2-adrenoreceptor is the most important beta-adrenergic receptor subtype in the regulation of lactate production.     

Insulin-induced translocation of GLUT 4 in skeletal muscle of insulin-resistant Zucker rats

Summary The genetically obese Zucker rat (fa/fa) is an animal model with severe insulin resistance of the skeletal muscle. We investigated whether a defect of insulin-dependent glucose transporter (GLUT 4) translocation might contribute to the pathogenesis of the insulin-resistant state. fa/fa rats, lean controls (Fa/Fa) as well as normal Wistar rats were injected intraperitoneally with insulin and were killed after 2 or 20 min, respectively. Subcellular fractions were prepared from-hind-limb skeletal muscle and were characterized by determination of marker-enzyme activities and immunoblotting applying antibodies against 1 Na⁺/K⁺ AT Pase. The relative amounts of GLUT 1 and GLUT 4 were determined in the fractions by immunoblotting with the respective antibodies. Insulin induced an approximately two-fold increase of GLUT 4 in a plasma membrane and transverse tubule enriched fraction and a decrease in the low density enriched membrane fraction in all three groups of rats. There was a high individual variation in GLUT 4 translocation efficiency within the groups. However, no statistically significant difference was noted between the groups. No effect of insulin was detectable on the distribution of GLUT 1 or 1 Na⁺K⁺ ATPase. The data suggest that skeletal muscle insulin resistance of obese Zucker rats is not associated with a lack of GLUT 4 translocation.     

间歇低氧对大鼠胰岛素抵抗及骨骼肌细胞GLUT4、Akt2的影响

目的 检测不同间歇低氧暴露时间对骨骼肌葡萄糖转运蛋白(GLUT)4与蛋白激酶B(PKB/Akt)2表达的影响,探讨二者在间歇低氧导致胰岛素抵抗中的作用.方法 选取健康雄性Sprague-Dawley大鼠40只,按照随机数字表法分为5组:常氧对照组(NC组),间歇低氧2周组(IH2组),间歇低氧4周组(IH4组),间歇低氧6周组(IH6组),间歇低氧8周组(IH8组),每组8只.IH2组、IH4组、IH6组、IH8组每天给予8h间歇低氧暴露(9:00～17:00),NC组室内环境正常饲养.检测各组空腹血糖和空腹胰岛素水平,计算稳态模型评估-胰岛素抵抗指数(HOMA-IR).采用免疫组织化学法检测大鼠骨骼肌GLUT4及Akt2蛋白的表达,蛋白表达量用平均灰度值表示,并分析GLUT4与Akt2的相关性.结果 与NC组相比,IH2组、IH4组、IH6组、IH8组空腹血糖、HOMA-IR升高,骨骼肌GLUT4与Akt2灰度值升高,并且随间歇低氧暴露时间的延长而升高明显(F =87.67～288.63,P均＜0.05);与NC组相比,IH2组、IH4组、IH6组、IH8组空腹胰岛素升高,其中IH2组、IH4组、IH6组,随间歇低氧暴露时间的延长而升高明显,IH8组较IH6组下降(F=86.04,P＜0.01).Pearson相关分析显示GLUT4与Akt2的表达呈正相关(r=0.895,P ＜0.05).结论 随着间歇低氧暴露时间的延长胰岛素抵抗程度增加,GLUT4与Akt2蛋白表达水平下降,二者在间歇低氧导致胰岛素抵抗的过程中起协同作用.     

Effect of exercise training on antioxidant and metabolic functions in senescent rat skeletal muscle.

Aging is known to be associated with alterations of both oxidative capacity and antioxidant status in skeletal muscle. In the present investigation we compared the activity of enzymes involved in both metabolic functions and antioxidant defense capacity in young adult (5 months old) and senescent (27.5 months old) Fischer 344 rats. In addition, we studied the effects of chronic exercise training on these enzymes in the senescent skeletal muscle. Old sedentary rats had significantly lower glutathione peroxidase (GPX) activity (-22%, p less than 0.05) in the deep portion of vastus lateralis muscle (DVL) than young sedentary rats, but after a progressive 10-week treadmill training program GPX activity in DVL was significantly increased in old rats to a level higher than that seen in young sedentary rats. Superoxide dismutase and catalase activities in the DVL were not altered significantly with aging or by training. Glutathione S-transferase activity in the same muscle was elevated (p less than 0.05) with aging but unaffected by training. Citrate synthase and malate dehydrogenase activities in the DVL muscle were significantly decreased in senescence, whereas training increased these two enzyme activities by 71 and 48%, respectively (both p less than 0.05). Lactate dehydrogenase activity in the same muscle decreased with age but increased 23% (p less than 0.05) in old rats. These data indicate that while aging may significantly affect antioxidant and metabolic capacities in skeletal muscle, regular exercise can preserve functions of these enzyme systems at old age.     

低出生体重大鼠发育期骨骼肌形态结构变化及胰岛素抵抗情况

观察低出生体重大鼠不同发育时期骨骼肌形态结构的变化和胰岛素抵抗情况.采用孕期低蛋白饮食法建立低出生体重仔鼠模型.于出生后7 d、21 d、2月龄测定仔鼠空腹血糖、血清胰岛素值,并检测骨骼肌形态结构.自7 d至2月龄,低出生体重仔鼠骨骼肌纤维均有明显萎缩、排列稀疏紊乱;2月龄时超微结构明显异常.2月龄内低出生体重仔鼠血糖和血清胰岛素与对照组比较无差异.     

Direct metabolic regulation in skeletal muscle and fat tissue by leptin: implications for glucose and fatty acids homeostasis

In recent years, the adipose tissue has emerged as an important endocrine organ. It is now recognized that besides storing energy the adipocytes also secrete several bioactive peptides, collectively called adipocytokines. Among these adipocytokines, leptin, the product of the ob gene, has been extensively investigated over the last decade. Skeletal muscle and adipose tissue, two major tissues involved in the regulation of glucose and fatty acids metabolism, have been consistently demonstrated to be directly affected by leptin. By binding to its receptors located in skeletal muscle and fat cells, leptin promotes energy dissipation and prevents fatty acid accumulation and 'lipotoxicity' in these tissues. On the other hand, under conditions of peripheral leptin resistance, such as observed in obese humans, the activation of pathways involved in fatty acid oxidation may be impaired. This leads to intracellular accumulation of lipid intermediates and causes insulin resistance. This review examines the metabolic pathways that are directly activated by leptin and how it regulates glucose and fatty acids metabolism in skeletal muscle and fat tissue. Furthermore, the impact of peripheral leptin resistance in these tissues leading to dysfunctional metabolic adaptations is also discussed.