重组屋尘螨2类变应原疫苗免疫治疗小鼠过敏性气道炎症的研究

目的 观察以聚乳酸-羟基乙酸共聚物（PLGA）材料为佐剂制备的重组屋尘螨2类变应原（rDer p 2）纳米微粒疫苗（DEPN）对小鼠过敏性气道炎症的影响，并探讨其免疫治疗机 制。 方法 制备PLGA-rDer p 2纳米粒子并鉴定其特性。40只BALB/c小鼠随机分为5组，A组（对照组）均给予生理盐水（100 μl）。B、C、D和E组腹部皮下注射屋尘螨粗浸液（10 μg）免疫小鼠致敏，然后分别用PBS（100 μl）、2 mg 空白PLGA粒子（empty PLGA，EP）、100 μg rDer p 2、2 mg的DEPN纳米疫苗（载有100 μg rDer p 2）皮下注射进行免疫治疗，连续免疫治疗3 d，1次/d，各组用rDer p 2（50 μg）滴鼻激发，激发后第2天剖杀，收集支气管肺泡灌洗液（BALF）并对细胞进行总计数和分类计数；HE染色和PAS染色（Periodic Acid?鄄Schiff Stain）观察小鼠肺部组织炎症和支气管黏液分泌；用ELISA检测BALF和脾细胞培养上清的细胞因子（IL-4、 IFN-γ）和血清中变应原特异性IgG2a和IgE抗体浓度。 结果 B、C 组肺部呈明显的变态反应性炎症，D、E组变应原诱导的肺部嗜酸粒细胞浸润和黏液分泌比B、C 组显著减轻。BALF中的细胞总数B组比A组明显增多，分类细胞以中性和嗜酸粒细胞为主，超过50%。rDer p 2特异性IgE抗体水平，D组（0.93±0.04）和E组（0.77±0.10）均低于B组（1.14±0.10）（P＜0.01）；特异性IgG2a抗体水平，D组（1.02±0.01）和E组（1.17±0.46）均高于B组（0.14±0.01）（P＜0.01）。在BALF中，D组[（55.60±3.79） pg/ml]和E组[（48.60±4.50） pg/ml]IL-4水平均低于B组[（78.90±6.07） pg/ml]（P＜0.01）；IFN-γ水平E组[（68.50±2.87） pg/ml]显著高于B组[（27.30±3.51） pg/ml] （P＜0.01）。脾细胞上清的IL-4水平，D组[（56.3±4.85） pg/ml]和E组[（40.2±4.36） pg/ml]显著低于B组[（81.20±6.84） pg/ml] （P＜0.01）；IFN-γ水平，E组[（70.20±3.85） pg/ml]显著高于B组[（34.60±2.25） pg/ml] 。 结论 DEPN免疫治疗可抑制小鼠肺部过敏炎症，其机制可能与调节Th1/Th2平衡有关。     

ANXA2敲除的A549细胞系的构建及其对H1N1和H9N2流感病毒复制的影响北大核心CSCD

目的旨在利用CRISPR/Cas9基因编辑技术构建ANXA2敲除的A549细胞,并探索ANXA2敲除对流感病毒复制的影响。方法本研究设计了3对靶向ANXA2基因外显子的特异性sgRNAs,分别将sgRNAs构建到LentiCRISPRv2载体上获得重组质粒,与辅助质粒共转染293T细胞包装成慢病毒后感染A549细胞,通过嘌呤霉素压力及有限稀释法筛选ANXA2基因敲除的单克隆细胞株,用靶基因测序及Western-blot验证ANXA2的敲除效果,并通过CCK-8试验比较ANXA2敲除细胞和野生型A549细胞的细胞活力。再分别用人流感病毒WSN(H1N1)和禽流感病毒SD98(H9N2)感染ANXA2敲除和野生型A549细胞,经TCID_(50)检测ANXA2敲除对流感病毒复制的影响。结果成功获得了ANXA2敲除的A549细胞系,且与野生型A549细胞相比,细胞活力无显著差异;ANXA2敲除后WSN(H1N1)和SD98(H9N2)流感病毒的病毒滴度均升高,其中ANXA2对SD98(H9N2)流感病毒的影响要大于WSN(H1N1)。结论本研究利用CRISPR/Cas9基因编辑技术构建了ANXA2敲除的A549细胞,并发现ANXA2敲除促进了流感病毒的复制,本研究为流感病毒复制和致病机制的研究奠定了基础。     

急性肺损伤大鼠肺水通道蛋白1和5的表达及功能的实验研究

目的观察脂多糖(LPS)、肿瘤坏死因子α(TNFα)、白细胞介素1β(IL1β)对大鼠肺微血管内皮细胞(LMECs)水通道蛋白1(AQP1)表达和功能的影响;同时在LPS诱发的大鼠急性肺损伤(ALI)模型观察AQP1、AQP5表达的变化。方法(1)体外实验:将第3代LMECs随机分为LPS组、TNFα组、IL1β组和DMEM对照组,实验组分别给予LPS、TNFα、IL1β刺激,应用逆转录聚合酶链反应(RTPCR)测定AQP1mRNA的表达以及免疫组化测定AQP1蛋白表达,同时采用放射性核素示踪法测定LMECs内氚水(3H2O)的放射强度。(2)体内实验:40只雄性Wistar大鼠随机分为LPS2h组、LPS4h组、LPS6h组、LPS8h组和对照组,LPS各组制成ALI模型,采用RTPCR测定ALI大鼠AQP1、AQP5mRNA表达以及免疫组化观察AQP1、AQP5蛋白表达。结果(1)体外实验:LPS、TNFα、IL1β组LMECsAQP1mRNA和蛋白表达显著低于DMEM对照组(mRNA表达分别为0.428±0.026、0.446±0.029、0.454±0.023和0.793±0.035,蛋白表达分别为0.366±0.009、0.374±0.014、0.377±0.007和0.660±0.013,P均<0.01);且LMECs内3H2O掺入量[(726±58)、(738±45)、(774±44)脉冲数/min]也显著低于DMEM对照组[(1148±70)脉冲数/min,P均<0.01]。(2)体内实验:ALI大鼠肺组织AQP1、AQP5mRNA表达(LPS2h组0.409±0.018、0.421±0.020,LPS4h组0.421±0.023、0.412±0.023,LPS6h组0.435±0.020、0.388±0.031,LPS8h组0.438±0.016、0.386±0.019)也显著低于对照组(0.794±0.015、0.787±0.022,P均<0.01)。结论AQP1、AQP5可能参与ALI/ARDS液体的异常转运,可能与肺水肿的发病机制有关。     

弓形虫GRA4和SAG2基因重组BCG疫苗免疫保护性的比较研究

目的 比较弓形虫致密颗粒蛋白4（GRA4）基因和表面抗原2（SAG2）基因的重组卡介苗（BCG）对小鼠的免疫保护效果。 方法 108只SPF级雌性BALB/c小鼠随机分成6组：PBS组、BCG空白菌组、BCG?鄄空白载体组、BCG?鄄SAG2组、BCG-GRA4组和BCG-SAG2+GRA4组,每组18只。每鼠分别注射对应液体/疫苗100 μl,共2次,间隔2周。接种前尾静脉采血,接种后4、6、8周每组分别剖杀3只,取脾和眼眶血检测细胞因子、IgG与IgM抗体,T淋巴细胞亚群计数,淋巴细胞转化率等。末次免疫后3周,每组剩余小鼠分别腹腔接种RH株弓形虫速殖子50个进行攻击感染,观察各组小鼠存活时间。 结果 弓形虫SAG2和GRA4重组BCG疫苗均能诱导小鼠产生免疫应答。第4周时,BCG-GRA4+SAG2免疫组小鼠的CD3⁺CD4⁺/CD3⁺CD8⁺ 的比值最高,为14.06%±1.17%。第6周时,BCG-GRA4+SAG2免疫组小鼠的IgG抗体水平最高, 为0.18±0.02。第8周时,BCG-SAG2免疫组小鼠的IgM抗体水平最高,为0.82±0.05;弓形虫速殖子攻击后,BCG-SAG2组平均存活8.61 d,PBS对照组平均存活7.33 d,3个免疫组小鼠比其他3组的平均存活时间长1 d。 结论 弓形虫重组BCG疫苗具有一定的免疫保护性。     

低剂量辐射对荷人胶质瘤(U251)裸小鼠移植瘤细胞凋亡相关基因蛋白Bcl-2表达的影响

目的研究低剂量辐射对荷人胶质瘤（U251）裸小鼠移植瘤细胞凋亡相关基因蛋白Bcl-2表达的影响。方法对荷人胶质瘤裸小鼠进行全身深部X射线照射，采用免疫组化技术检测荷人胶质瘤裸小鼠移植瘤组织的Bcl-2蛋白表达水平。结果D1（75mGy）照射后，胶质瘤细胞的Bcl-2蛋白表达呈下降趋势，与假照组（0mGy）比，无显著差异（P〉0．05）；D2（4Gy）组、D1＋D2组的Bcl-2蛋白表达明显减少，与假照组（0mGy）比有统计学差异（P〈0．05）；而D1＋D2组与D2组比较，有统计学差异（P〈0．05），以D1＋D2组Bcl-2蛋白的表达减少更为显著。结论低剂量辐射在一定程度上可能下调了胶质瘤细胞的Bcl-2蛋白的表达，同时对其后的大剂量辐射有协同作用。     

人血管生成素相关蛋白2重组腺病毒体外转染对小鼠毛细血管内皮细胞分化的作用

目的研究血管生成素相关蛋白2重组腺病毒(Ad．ARP2)体外的转染效率、产物表达和其表达产物对血管内皮细胞分化的作用。方法体外培养扩增ICR小鼠心脏冠状动脉毛细血管内皮细胞(CMECs),通过免疫荧光染色评价人腺病毒转染CMECs的转染效率;应用Western blot和ELISA方法检测ARP2在细胞的内表达和分泌,并分别与对照组[空病毒载体(Ad．Null)组和PBS组]比较。观察种植在Matrigel凝胶上分别转染Ad．ARP2和Ad．Null的CMECs的生长状态。结果腺病毒转染倍数为200时,转染效率为93．5％,且对细胞生长无影响。Ad．ARP2转染CMECs后有ARP2蛋白表达,ELISA检测第1、2、3、5、8 d的ARP2分泌量分别为76．0±1．7、490．1±9．5、389.6±2．5、240．1±1．1、73．4±1．3 pg／ml,而对照组未检测到有ARP2蛋白表达和分泌。含有ARP2的培养上清能促进血管内皮细胞分化。结论腺病毒可以安全、有效地转染CMECs,其携带ARP2基因可获得高水平表达,并能促进血管内皮细胞分化。     

德国小蠊重组变应原（rBla g 2）治疗过敏性哮喘小鼠的实验研究

目的 探讨重组德国小蠊变应原（rBla g 2）治疗过敏性哮喘小鼠的效果及机制。 方法 18只BALB/c小鼠随机均分为阴性对照组（A组）、哮喘模型组（B组）和重组蛋白rBla g 2治疗组（C组）。B、C两组分别腹腔注射经Al（OH）3佐剂乳化的重组蛋白rBla g 2,剂量为50 mg/（只?次）,共3次,每次间隔1周。A组以50 ml生理盐水代替变应原。末次致敏后2周,进行免疫治疗,C组腹腔注射rBla g 2,100 mg/（只?次）,每两天1次,连续8次。A、B两组以PBS代替变应原。末次治疗后1周,将小鼠麻醉,B、C两组每鼠每天滴鼻50 mg rBla g 2,连续7 d。A组以PBS代替变应原滴鼻。于最后1次滴鼻后24 h检测各项指标：各组小鼠气道高反应性,支气管肺泡灌洗液（BALF）中细胞总数和细胞种类以及血清中rBla g 2抗原特异性IgE和IgG2a的变化;HE染色观察小鼠肺组织的改变,免疫组化检测肺嗜酸粒细胞（EOS）B淋巴细胞瘤/白血病-2（Bcl-2）的表达。 结果 与B组相比,C组气道高反应检测中扩大间隙（Penh）值下降（P＜0.05）;血清中rBla g 2特异性IgE降低,IgG2a增加（P＜0.01）;C组EOS细胞阳性数量明显减少,且Bcl-2蛋白阳性表达较弱。B组BALF的细胞总数和EOS数分别为（24.60±15.08）×105个/ml和（22.20±3.76）×105个/ml,而C组细胞总数［（14.30±4.95）×105个/ml］和EOS数［（5.20±1.56）×105个/ml］显著减少（P＜0.01）。B组肺部气管周围炎性细胞聚集,肺组织上皮损伤,组织水肿,而C组肺部变应性炎症明显减轻。与A组相比,C组各项检测指标接近A组。 结论 rBla g 2对小鼠过敏性哮喘有治疗作用,可能是EOS细胞凋亡在其中起重要作用。     

RNA干扰技术阻断小鼠巨噬细胞受体相互作用蛋白2表达的实验研究

目的观察阻断受体相互作用蛋白2（Rip2）对巨噬细胞产生炎症细胞因子的影响,以及对内毒素血症小鼠的保护作用。方法构建Rip2小干扰RNA（siRNh）重组表达质粒,转染细胞后RT．PCR和Western blot检测Rip2的mRNA和蛋白表达,四甲基偶氮唑盐（M1Tr）法检测细胞增殖水平,脂多糖（LPS）刺激后,测定TNFa和高迁移率组蛋白1（HMGB1）的水平。Rip2 siRNA质粒转染小鼠后,观察小鼠病死率,测定血清TNFct水平和肝组织Rip2和HMGB1表达。结果Rip2 siRNA表达质粒可阻断Rip2 mRNA和蛋白表达。Rip2阻断的细胞增殖明显,LPS刺激后产生TNFα、HMGB1减少;Rip2阻断的小鼠生存率较其他组高（P〈0．05）,肝组织中HMGB1[（40．21±11．03）Pg／g]表达和血清TNFα[（300．43±59．26）ng／L]水平均较其他组低（P〈0．05）。结论Rip2 siRNA表达质粒可阻断Rip2的表达,从而减少TNFα、HMGB1等炎症细胞因子的产生,降低小鼠内毒素血症的病死率。     

丝裂原活化蛋白激酶选择性抑制剂对葡聚糖硫酸钠结肠炎的影响

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）选择性抑制剂SB203580对葡聚糖硫酸钠（DSS）诱导的小鼠结肠炎的影响。方法24只雌性BALB／C小鼠均分为三组：正常对照组、DSS结肠炎组和SB203580干预组。结肠炎组小鼠每日自由摄取5％DSS溶液．干预组小鼠在摄取5％DSS溶液72h后，每日腹腔注射SB2035801mg／kg体质量。各组小鼠每日记录疾病活动指数（DAI）评分，H-E染色观察小鼠结肠黏膜组织学改变。ELISA法测定结肠组织巾肿瘤坏死因子（TNF）-a含量。免疫组化染色观察磷酸化的活化转录因子2（P-ATF2）在结肠组织炎性细胞内的表达情况。结果正常对照组和DSS结肠炎组小鼠的DAI评分为0．224-0．09和4．81±1．08（P〈0．05），组织学评分为1．03±0．38和11．0±0．75（P〈0．05），经SB203580干预后分别为1．96±0．89和7．16±1．46，与结肠炎组小鼠比较差异有统计学意义（P00．05）。正常对照组与DSS结肠炎组小鼠的结肠匀浆TNF-a含量分别为（42．98±17．31）和（128．34±33．76）pg／ml（P〈0．05），SB203580于预后下降为（81．86±25．11）pg／ml，与结肠炎组小鼠比较差异有统计学意义（P〈0．05）。正常对照组与结肠炎组小鼠结肠组织炎性细胞P-ATF2的表达分别为（6．91±1．83）％和（81．02±12．35）％（P〈0．05），经SB203580处理后为（38．59±8．12）％，与结肠炎组小鼠比较差异有统计学意义（P00．05）。结论阻断p38MAPK信号转导通路可明显缓解DSS诱导的结肠炎。预示此信号转导通路可为溃疡性结肠炎新药研究提供有价值的靶标。     

甘舒霖强化治疗初诊2型糖尿病37例研究

75例初诊T2DM患者，按单、双日随机分为两组，对照组瑞格列奈1～3mg及二甲双胍0．25～0．5，每日各三次口服；强化组给予甘舒霖R（三餐前30分钟），甘舒霖N（睡前）皮下注射。4周后观察治疗前后空腹血糖（FBG）、餐后2小时血糖（2HBG）、糖化血红蛋白A1c（HbA1c）、胰岛β细胞功能指数（HOMA—β）、胰岛素抵抗指数（HOMA—IR）、血糖达标时间等。结果：两组病人治疗后，上述指标均有明显下降，强化组下降更明显分别为（15．8±3．2）mmol／Lvs（5．9±0．5）mmol／L，（20．2±3．8）mmol／L（7．5±0．5）mmool／L，（10．4±1．36）％vs（6．96±0．74）％。均P〈0．01，（3．49±0．82）vs（5．32±2．20），（2．20±0．81）vs（1．02±0．41），均P〈0．05。结论：甘舒霖强化治疗初诊T2DM，能明显增强胰岛β细胞的功能，减轻胰岛素抵抗，使患者血糖良好控制。     

Insulin Deficiency and Increased Plasma Concentration of Intact and 32/33 Split Proinsulin in Subjects with Impaired Glucose Tolerance

In order to determine insulin status and beta cell function during the oral glucose tolerance test (OGTT), in impaired glucose tolerance (IGT), 51 such subjects and matched controls, identified during a population survey for diabetes, underwent a 75 g OGTT. Fasting, 30 min and 2 h insulin and intact proinsulin, and fasting and 2 h 32/33 split proinsulin, were measured by specific two-site immunoradiometric assays. The subjects with IGT had higher fasting (geometric mean ± SD, 5.0 ± 4.0 pmol^?1 vs 2.9 ± 1.7, p < 0.02) and 2 h intact proinsulin (23 ± 14 vs 14 ± 12, p < 0.0001), and fasting (3.2 ± 3 pmol^?1 vs 1.8 ± 1.8, p < 0.0007) and 2 h 32/33 split proinsulin (18.3 ± 19 pmol^?1 vs 6.6 ± 15, p < 0.0001). Despite higher plasma glucose concentrations, the IGT group had similar fasting insulin, lower 30 min insulin (216 ± 124 pmol^?1 vs 278 ± 130, p < 0.02), and a lower 30 min insulin/glucose ratio (23.7 ± 2.1 vs 34.8 ± 2.3, p < 0.002). The percentage of fasting proinsulin-like to total insulin-like molecules was higher in those with IGT (15.3 ± 8% vs 11.6 ± 8, p < 0.04). After 6 months, at repeat OGTT, the same subjects with IGT were classified as ‘persisters’ or ‘reverters’. The persister (24/51 47.1%), at initial OGTT, had a higher 2 h glucose level, a greater BMI and higher systolic blood pressure, but other parameters were similar to the reverters. In the reverters, when baseline variables were compared to those recorded at six month follow-up, there was a reduction in 2 h intact (23.8 ± 13 pmol^?1 vs 19.4 = 10, p < 0.02) and 32/33 split proinsulin (20.4 ± 18 pmol^?1 vs 13.8 ± 13, p < 0.006), and an increase in fasting insulin (41 ± 30 pmol^?1 vs 54 ± 35, p < 0.02), respectively, despite no change in fasting glucose. These findings show that IGT is associated with beta cell dysfunction and reduced early insulin secretion during the OGTT. In some subjects with IGT these abnormalities show improvement in the short term.     

急性冠状动脉综合征患者血液凝固性加强

目的通过研究急性冠状动脉综合征患者凝血状态的变化,探讨急性冠状动脉综合征患者的发病与血栓前状态的关系,以期对危重冠心病患者及早作出诊断和治疗。方法选择急性冠状动脉综合征患者86例,对照组为稳定型心绞痛患者75例,以酶联免疫吸附法测定两组患者血浆凝血酶原片段1和2、可溶性纤维蛋白单体复合物等凝血分子标志物的含量并进行比较。结果急性冠状动脉综合征患者血浆凝血酶原片段1和2及可溶性纤维蛋白单体复合物较稳定型心绞痛患者均显著升高(1.21±0.23nmolL比0.76±0.20nmolL;85.4±12.4mgL比68.7±13.8mgL,P均<0.001)。急性冠状动脉综合征合并2型糖尿病时血浆凝血酶原片段1和2及可溶性纤维蛋白单体复合物较不伴有2型糖尿病时显著升高(1.28±0.19nmolL比1.16±0.20nmolL;89.8±12.4mgL比82.7±13.7mgL,P均<0.05)。急性冠状动脉综合征合并原发性高血压时血浆凝血酶原片段1和2及可溶性纤维蛋白单体复合物较不伴有原发性高血压时显著升高(1.26±0.24nmolL比1.16±0.20nmolL;90.0±12.8mgL比82.7±13.7mgL,P均<0.05)。结论稳定型心绞痛患者的凝血系统处于稳定状态,而急性冠状动脉综合征患者处于高凝状态,合并2型糖尿病或原发性高血压的急性冠状动脉综合征患者高凝状态更显著,提示高凝状态与急性冠状动脉综合征的发病密切相关。     

Expression and externalization of annexin 1 in the adrenal gland: structure and function of the adrenal gland in annexin 1-null mutant mice

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins with a well demonstrated role in early delayed (30 min to 3 h) inhibitory feedback of glucocorticoids in the hypothalamus and pituitary gland. This study used adrenal gland tissue from ANXA1-null transgenic mice, in which a beta-galactosidase (beta-Gal) reporter gene was controlled by the ANXA1 promoter, and wild-type control mice to explore the potential role of ANXA1 in adrenal function. RT-PCR and Western blotting revealed strong expression of ANXA1 mRNA and protein in the adrenal gland. Immunofluorescence labeling of ANXA1 in wild-type and beta-Gal expression in ANXA1-null adrenals localized intense staining in the outer perimeter cell layers. Immunogold electron microscopy identified cytoplasmic and nuclear ANXA1 labeling in outer cortical cells and capsular cells. Exposure of adrenal segments in vitro to dexamethasone (0.1 mum, 3 h) caused an increase in the amount of ANXA1 in the intracellular compartment and attached to the surface of the cells. The N-terminal peptide ANXA1(Ac2-26) inhibited corticosterone release. Corticosterone release was significantly greater from ANXA1-null adrenal cells compared with wild type in response to ACTH (10 pm to 5 nm). In contrast, basal and ACTH-stimulated aldosterone release from ANXA1-null adrenal cells was not different from wild type. Morphometry studies demonstrated that ANXA1 null adrenal glands were smaller than wild-type, and the cortical/medullary area ratio was significantly reduced. These results suggest ANXA1 is a regulator of adrenocortical size and corticosterone secretion.     

Hyperlipidaemia is associated with increased insulin-mediated glucose metabolism, reduced fatty acid metabolism and normal blood pressure in transgenic mice overexpressing human apolipoprotein C1

Aims/hypothesis. Insulin resistance for glucose metabolism is associated with hyperlipidaemia and high blood pressure. In this study we investigated the effect of primary hyperlipidaemia on basal and insulin-mediated glucose and on non-esterified fatty acid (NEFA) metabolism and mean arterial pressure in hyperlipidaemic transgenic mice overexpressing apolipoprotein C1 (APOC1). Previous studies have shown that APOC1 transgenic mice develop hyperlipidaemia primarily because of an impaired hepatic uptake of very low density lipoprotein (VLDL). Methods. Basal and hyperinsulinaemic (6 mU · kg^–1· min^–1), euglycaemic (7 mmol/l) clamps with 3-³H-glucose or 9,10-³H-palmitic acid infusions and in situ freeze clamped tissue collection were carried out. Results. The APOC1 mice showed increased basal plasma cholesterol, triglyceride, NEFA and decreased glucose concentrations compared with wild-type mice (7.0 ± 1.2 vs 1.6 ± 0.1, 9.1 ± 2.3 vs 0.6 ± 0.1, 1.9 ± 0.2 vs 0.9 ± 0.1 and 7.0 ± 1.0 vs 10.0 ± 1.1 mmol/l, respectively, p < 0.05). Basal whole body glucose clearance was increased twofold in APOC1 mice compared with wild-type mice (18 ± 2 vs 10 ± 1 ml · kg^–1· min^–1, p < 0.05). Insulin-mediated whole body glucose uptake, glycolysis (generation of ³H₂O) and glucose storage increased in APOC1 mice compared with wild-type mice (339 ± 28 vs 200 ± 11; 183 ± 39 vs 128 ± 17 and 156 ± 44 vs 72 ± 17 μmol · kg^–1· min^–1, p < 0.05, respectively), corresponding with a twofold to threefold increase in skeletal muscle glycogenesis and de novo lipogenesis from 3-³H-glucose in skeletal muscle and adipose tissue (p < 0.05). Basal whole body NEFA clearance was decreased threefold in APOC1 mice compared with wild-type mice (98 ± 21 vs 314 ± 88 ml · kg^–1· min^–1, p < 0.05). Insulin-mediated whole body NEFA uptake, NEFA oxidation (generation of ³H₂O) and NEFA storage were lower in APOC1 mice than in wild-type mice (15 ± 3 vs 33 ± 6; 3 ± 2 vs 11 ± 4 and 12 ± 2 vs 22 ± 4 μmol · kg^–1· min^–1, p < 0.05) in the face of higher plasma NEFA concentrations (1.3 ± 0.3 vs 0.5 ± 0.1 mmol/l, p < 0.05), respectively. Mean arterial pressure and heart rate were similar in APOC1 vs wild-type mice (82 ± 4 vs 85 ± 3 mm Hg and 459 ± 14 vs 484 ± 11 beats · min^–1). Conclusions/interpretation. 1) Hyperlipidaemic APOC1 mice show reduced NEFA and increased glucose metabolism under both basal and insulin-mediated conditions, suggesting an intrinsic defect in NEFA metabolism. Primary hyperlipidaemia alone in APOC1 mice does not lead to insulin resistance for glucose metabolism and high blood pressure. [Diabetologia (2001) 44: 437–443] Received: 14 September 2000 and in revised form: 23 November 2000     

The set point for maternal glucose homeostasis is lowered during late pregnancy in the rat: the role of the islet beta-cell and liver

Summary The aim of this study was to determine the effects of late pregnancy on the ability of insulin to suppress maternal hepatic glucose production in the rat. Unlike in most previous studies, suppression of hepatic glucose production was measured at levels of glycaemia above the relatively hypoglycaemic basal pregnant level. Glucose kinetics were measured using steady-state tracer methodology in chronically catheterised, conscious virgin control and pregnant rats, firstly, during basal and low-dose hyperinsulinaemic euglycaemic clamp conditions and secondly, during a three-step glucose infusion protocol (glucose infusion rates of 0, 60 and 150 μmol · kg⁻¹· min⁻¹). During the clamps, plasma glucose levels were not different (6.1 ± 0.4 vs 6.5 ± 0.3 mmol/l, pregnant vs virgin; N. S.), but plasma insulin levels were higher in the pregnant rats (242 ± 30 vs 154 ± 18 pmol/l, pregnant vs virgin; p < 0.05) most probably due to stimulated endogenous insulin release in this group. Hepatic glucose production was suppressed from basal levels by 41 % in virgin and 90 % in pregnant rats. During the glucose infusion studies, at matched insulin levels (147 ± 10 vs 152 ± 14 pmol/l), but at plasma glucose levels which were much lower in the pregnant rats (5.5 ± 0.2 vs 8.4 ± 0.6 mmol/l, pregnant vs virgin; p < 0.0001), hepatic glucose production was shown to be suppressed by a similar degree in both groups (41 ± 5 vs 51 ± 5 % from basal, pregnant vs virgin; N. S.). Both the plasma insulin and percentage suppression of hepatic glucose production dose responses to plasma glucose were markedly shifted to the left indicating that the plasma glucose set point is lowered in pregnancy. In conclusion, suppression of hepatic glucose production by insulin is not impaired and the set point for plasma glucose homeostasis is lowered during late pregnancy in the rat. [Diabetologia (1996) 39: 785–792] Received: 2 October 1995 and in final revised form: 1 February 1996     

Forearm Substrate Exchange during Hyperinsulinaemic Hypoglycaemia in Normal Man

To assess muscle substrate exchange during hypoglycaemia, 8 healthy young male subjects were studied twice during 2 h of hyperinsulinaemic euglycaemia followed by 4 h of (1) hypoglycaemia (plasma glucose < 2.8 mmol l^?1), and (2) euglycaemia. Insulin was infused at a rate of 1.5 mU kg^?1 min^?1 throughout. When compared to euglycaemia, hypoglycaemia was associated with: (1) increment in circulating glucagon (65 ± 8 vs 23 ± 4 ng l^?1, p < 0.05), growth hormone (19.9 ± 3.6 vs 2.6 ± 1.3 μg l^?1, p < 0.05), adrenaline (410 ± 88 vs 126 ± 32 ng l^?1, p < 0.05) and increased suppression of C-peptide (0.5 ± 0.1 vs 1.0 ± 0.1 μg l^?1, p < 0.05) along with a modest lowering of insulin (103 ± 10 vs 130 ± 13 mU l^?1, p < 0.05); (b) decrease in plasma glucose level (3.0 ± 0.07 vs 5.0 ± 0.12 mmol l^?1 p < 0.05), forearm glucose uptake (0.21 ± 0.09 vs 1.21 ± 0.21 mmol l^?1, p < 0.05) and requirement for exogenous glucose (5.6 ± 1.1 vs 13.2 ± 0.9 mg kg^?1 min^?1 p < 0.005) together with an impaired suppression of isotopically determined endogenous glucose production (0.34 ± 0.5 vs ?2.3 ± 0.3 mg kg^?1 min^?1, p < 0.05); (3) exaggerated increase in blood lactate (1680 ± 171 vs 1315 ± 108 μmol l^?1, p < 0.05) and a decrease in alanine (215 ± 18 vs 262 ± 19 μmol l^?1, p < 0.05). Forearm release of lactate (130 ± 43 vs 12 ± 31 μmol l^?1, p = 0.09) tended to be increased, whereas alanine balance (18 ± 6 vs 17 ± 5 μmol l^?1) was unchanged. (4) Total forearm blood flow increased similarly during both studies (4.4 ± 0.6 vs 4.2 ± 0.5 ml 100 ml^?1 min^?1). These data suggest that the human forearm is not a major site for glucose uptake nor for lactate production during protracted hypoglycaemia; the fact that forearm glucose uptake is reduced sixfold during hypoglycaemia further suggests that restriction of glucose uptake in muscles plays a frontline role in the defence against hypoglycaemia.     

Evidence for Impaired Pancreatic Beta Cell Function in South African Indians with Impaired Glucose Tolerance

In a prospective study of South African Indians with impaired glucose tolerance (IGT), the serum insulin response during a 75 g oral glucose tolerance test (OGTT) was examined in 128 subjects who were classified as IGT 1 year previously (year 0) and in 60 matched control subjects. Based on the results at year 1, study subjects were divided into three groups, using World Health Organization criteria for glucose tolerance: IGT (n = 47), diabetes (n = 41), and transient IGT (normal glucose tolerance) (n = 40). When compared with the control group, despite higher plasma glucose concentrations, the IGT group showed similar fasting insulin, but lower 30-min insulin response (57.4 ± 1.9 mUI^?1 vs 86.5 ± 1.8, p<0.001) and lower 30-min insulin/glucose ratio (7.4 ± 5.2 vs 13.3 ± 8.7, p < 0.001). The insulinogenic index was lower in the IGT group than in the control group at 30, 60, 90, and 120 min (p < 0.01, p < 0.001, p < 0.001, p < 0.001, respectively). The 2-h insulin response was higher in the IGT group (106.7 ± 1.9 mUI^?1 vs 59.2 ± 1.9, p < 0.01). The IGT group displayed a delayed pattern of insulin response with maximum levels only at 2-h. Insulin area was similar in the two groups. In the transient IGT group, despite similar plasma glucose levels, the insulin responses at 0, 15, 30, and 60 min (p < 0.01, p < 0.001, p < 0.001, p < 0.001, respectively) were lower than in the control group; the 30-min insulin/glucose ratio (7.1 ± 5.1 vs 13.3 ± 8.7, p < 0.001) and 60-min insulinogenic index (46.9 ± 86.3 vs 123.4 ± 206.3, p < 0.001) were also lower in the transient IGT group. This study has shown that IGT in South African Indians is characterized by a diminished early phase insulin response and delayed (2-h) hyperinsulinaemia during OGTT. Such findings would suggest that in this population group impaired early beta cell function is an important pathophysiological abnormality underlying IGT.     

Glucagon receptor antagonism improves islet function in mice with insulin resistance induced by a high-fat diet

Aims/hypothesis Increased glucagon secretion predicts deterioration of glucose tolerance, and high glucagon levels contribute to hyperglycaemia in type 2 diabetes. Inhibition of glucagon action may therefore be a potential novel target to reduce hyperglycaemia. Here, we investigated whether chronic treatment with a glucagon receptor antagonist (GRA) improves islet dysfunction in female mice on a high-fat diet (HFD). Materials and methods After 8 weeks of HFD, mice were treated with a small molecule GRA (300 mg/kg, gavage once daily) for up to 30 days. Insulin secretion was studied after oral and intravenous administration of glucose and glucagon secretion after intravenous arginine. Islet morphology was examined and insulin secretion and glucose oxidation were measured in isolated islets. Results Fasting plasma glucose levels were reduced by GRA (6.0 ± 0.2 vs 7.4 ± 0.5 mmol/l; p = 0.017). The acute insulin response to intravenous glucose was augmented (1,300 ± 110 vs 790 ± 64 pmol/l; p < 0.001). The early insulin response to oral glucose was reduced in mice on HFD + GRA (1,890 ± 160 vs 3,040 ± 420 pmol/l; p = 0.012), but glucose excursions were improved. Intravenous arginine significantly increased the acute glucagon response (129 ± 12 vs 36 ± 6 ng/l in controls; p < 0.01), notably without affecting plasma glucose. GRA caused a modest increase in alpha cell mass, while beta cell mass was similar to that in mice on HFD + vehicle. Isolated islets displayed improved glucose-stimulated insulin secretion after GRA treatment (0.061 ± 0.007 vs 0.030 ± 0.004 pmol islet⁻¹ h⁻¹ at 16.7 mmol/l glucose; p < 0.001), without affecting islet glucose oxidation. Conclusions/interpretation Chronic glucagon receptor antagonism in HFD-fed mice improves islet sensitivity to glucose and increases insulin secretion, suggesting improvement of key defects underlying impaired glucose tolerance and type 2 diabetes.     

Modulation of Glucose and Growth Hormone Responses to Meals and Exercise in Type 1 Diabetes by Cholinergic Muscarinic Blockade

Anticholinergic drugs suppress nocturnal and exercise-related growth hormone (GH) secretion in Type 1 diabetes; nocturnal GH suppression is associated with a fall in fasting plasma glucose levels. The aim of this study was to assess the effect of GH suppression on glucose levels following a period of meals and exercise in physiological pattern. Six Type 1 diabetic men recruited from the outpatient clinic were studied in random order at least 1 week apart. After an overnight fast subjects received two-thirds of their usual subcutaneous insulin and either 200 mg oral pirenzepine or placebo at time 0 min. Between 90 and 120 min subjects exercised continuously on an ergometric cycle. Standard meals or snacks were eaten at 30, 150, 270, and 390 min. Venous blood was collected from an indwelling cannula between 0 and 570 min. The mean incremental rise in plasma glucose after breakfast (δ peak/_{90 min}) was 2.6 ± 0.5 (mean ±SEM mmol l^?1 (pirenzepine) vs 4.5 ± 0.8 (placebo)), p < 0.05. Following exercise the fall in plasma glucose (δ gluc_{90–240 min}) was 6.4 ± 1.9 (pirenzepine) vs 2.0 ± 1.3 (placebo), p < 0.005. The exercise-related peak rise in GH was 12.6 ± 3.3 (pirenzepine) vs 28.5 ± 6.0 mU l^?1 (placebo), p = 0.08. Excluding one outlying result there was an inverse correlation between the integrated exercise-related increase in GH between 90 and 240 min and the fall in glucose over the corresponding time period (n = 11, r = ?0.75, p = 0.008). In conclusion suppression of exercise-related GH secretion by pirenzepine is associated with a subsequent lowering of plasma glucose levels. The smaller post-prandial glucose rise pre-exercise implies also a direct effect of pirenzepine on meal-related glucose tolerance in Type 1 diabetes.