兔侧脑室内注射八肽胆囊收缩素对血浆自由脂肪酸及血糖浓度的影响

本工作通过脑内埋藏套管,向兔双侧侧脑室内注射微量八肽胆囊收缩素(CCK-8),观察对空腹血浆自由脂肪酸(FFA)和血糖浓度的影响,结果如下:(1)侧脑室内注射引起血浆FFA 浓度明显下降,但血糖浓度无明显改变,静脉注射,血浆 FFA 不仅未下降,反而明显升高,血糖浓度也无明显改变。(2)注射苄胺唑啉和心得安不能消除侧脑室注射 CCK-8所引起的血浆 FFA 下降。(3)注射阿托品和切断膈下迷走神经以阻断胆碱能神经的作用后,侧脑室注射 CCK-8不再引起血浆 FFA 浓度下降。从上述结果看来,侧脑室注射 CCK-8降低血浆 FFA 浓度的作用,不是由于 CCK-8吸收入血所致,而是脑内 CCK-8浓度升高所产生的特异作用。这个作用可能是通过迷走神经引起的,与交感神经活动的关系不大。     

兔侧脑室注射八肽胆囊收缩素抗血清对血浆自由脂肪酸浓度的影响

本工作根据抗原抗体间具有高度特异性的中和作用的原理,将微量 CCK-8抗血清注射入制备有埋藏套管的慢性实验兔的侧脑室内,观察在中和脑内外源性或内源性的 CCK-8后,血浆 FFA 浓度的变化,结果如下:1.侧脑室内注射正常兔血清,对血浆 FFA 浓度无明显影响;在注射 CCK-8同时注射兔正常血清,也不影响 CCK-8降低血浆 FFA 浓度的作用。2.侧脑室内注射 CCK-8抗血清能有效地阻断外源性脑室注射 CCK-8降低血浆 FFA 的作用,此阻断作用随 CCK-8抗血清剂量的增加而增强。3.侧脑室内单独注射 CCK-8抗血清,血浆 FFA 浓度明显升高,这一作用可能是由于中和了脑内内源性 CCK-8的作用所致。以上结果表明,CCK-8脑室注射引起的血浆 FFA 降低的作用具有一定的特异性;而在正常生理情况下,脑内释放的内源性 CCK-8可能参与血浆 FFA 代谢的调节。     

胆囊收缩素及其类似物对兔血浆自由脂肪酸浓度的降低作用

我们最近曾报道,向兔侧脑室内灌注八肽胆囊收缩素(CCK-8)有明显的降低血浆自由脂肪酸(FFA)的作用。本实验进一步观察不同分子形式的 CCK,以及与 CCK 结构相近似的雨蛙肽(Caerulein)和胃泌素(G-17)对血浆 FFA 浓度的影响,并在摩尔量基础上比较它们的相对作用强度。结果如下:(1)侧脑室灌注CCK-8(50pmol)可引起血浆 FFA 浓度明显降低。灌注后第45分钟时,平均降低47.8%,(2)等摩尔量的CCK-4也有降低血浆 FFA 的效应(-35.1%),但其作用比CCK-8弱;(3)灌注50pmol的CCK-33对血浆 FFA 浓度无明显作用,但随着灌注剂量的增加,作用也趋明显,在10倍量时(500pmol),其作用强度与 CCK-8(50 pmol)相似,(4)雨蛙肽具有与CCK-8相似的作用(-45.1%),但G-17的作用较弱(-24.5%)。以CCK-8的作用强度为1,上述肽类在等摩尔量基础上降低血浆 FFA 浓度的相对强度如下:CGK-8:CCK-4:CCK-33:雨蛙肽:G-171:0.76:0.09:0.94:0.51可以认为,脑内CCK-8作用的发挥与其化学结构具有一定的关系,羧基端八肽酰胺可能是脑内CCK-8降低血浆 FFA 浓度的重要结构。这一结果与哺乳动物脑内 CCK 的主要形式为CCK-8是一致的。     

八肽胆囊收缩素对链佐霉素引起的小鼠糖尿病的保护作用

用小剂量多次腹腔注射链佐霉素的方法破坏胰岛β细胞,造成小鼠糖尿病,其主要表现为血糖升高和血清胰岛素浓度下降。在每次注射链佐霉素前10—15min,由皮下注射 CCK-8(100μg/kg),可使链佐霉素的损伤作用減轻或不出现:小鼠血糖浓度由对照组的518.9±53.6mg%降低到267.1±16.8mg%,糖尿病发病率由83.3%降到30%;血清胰岛素浓度基本正常,表明 CCK-8对链佐霉素引起的糖尿病有一定的预防作用。用链佐霉素造成高血糖后,再用 CCK-8作治疗性注射,不能减轻高血糖的程度,表明 CCK-8无治疗作用。CCK-8对正常小鼠的血糖及胰岛素水平也无明显影响。以上结果提示,CCK-8对胰岛β细胞可能具有直接保护作用。     

新生犊牛胰腺内分泌机能的某些特点

６头装有暂时性颈静脉血管瘘的黑白花品种新生犊牛,在从出生至１５日龄的围产期内每天测定血浆葡萄糖（Ｇ）、胰岛素（Ｉｎｓ）和胰高血糖素（Ｇｃ）的基本浓度,可见１日龄喂初乳前Ｇ、Ｉｎｓ和Ｇｃ的浓度分别是１１９６．０±１５２．２ｍｇ／Ｌ、（７．３３±１．４３）×１Ｏ３Ｕ／Ｌ和６８５．３９±９５．４２ｎｇ／Ｌ。Ｇ于第２天明显下降,第３天回升,以后相对稳定;Ｉｎｓ第２天升高,以后下降,５日龄后相对稳定;Ｇｃ４日龄内逐日下降,５日龄后达到相对稳定水平。第１天喂初乳引起Ｉｎｓ显著升高,Ｇｃ下降,Ｇ无变化或者降低;以后喂初乳或常乳均引起Ｉｎｓ和Ｇ一致性升高,进食引起的Ｉｎｓ释放反应随日龄增长明显增加,Ｇｃ下降。不同日龄时灌注精氨酸均引起Ｉｎｓ显著增加,Ｇｃ早期无显著变化,７日龄后显著增加,同时Ｇ并无明显变化。灌注Ｇ引起Ｇ和Ｉｎｓ两者都增加,Ｇｃ下降。随日龄增长胰岛Ｂ细胞对精氨酸和Ｇ的反应明显加强。     

侧脑室注射胰高血糖素对兔血浆自由脂肪酸、血糖及胰岛素浓度的影响

胰高血糖素是一种脑-肠肽,但它在脑中的生理作用尚不清楚。本工作在家兔侧脑室埋藏慢性套管,并通过注射20μl含0.5—5μg的胰高血糖素溶液,在注射后的15、45、75、105分钟各取血样测定血糖、血浆自由脂肪酸(FFA)和胰岛素的浓度。结果发现:(1)对血糖及血浆胰岛素浓度无明显影响;(2)能引起血浆 FFA 浓度降低,且与剂量有依赖关系,在注射后的45分钟,FFA 降低最明显,以后逐渐恢复;(3)皮下注射阿托品(0.2mg/kg)或静脉注射酚妥拉明(5mg/kg)均不能消除侧脑室注射胰高血糖素降低 FFA 的作用;(4)静脉注射心得安(5mg/kg)能阻断侧脑室注射胰高血糖素的降低 FFA 的作用。这似表明脑中的胰高血糖素可能参与脂代谢的调节,并可能是通过肾上腺素β-受体起作用的。     

电损毁海马CA_3区及连合前穹窿对大鼠血浆胰岛素水平及胰岛细胞的影响

电损毁大鼠海马CA_3区(HCA_3-EL)或连合前穹窿纤维(ACHF-EL)后,其血浆胰岛素基础水平明显升高。HCA_3-EL两周后,大鼠空腹血糖浓度增高,且糖耐量降低。但胰岛B细胞对葡萄糖刺激之分泌反应却显著增强。HCA_3-EL或ACHF-EL大鼠经静脉糖耐量试验(IVGTT)后,其胰岛A细胞内胰高血糖素样及B细胞内胰岛素样免疫阳性物质的相对含量均明显低于对照组。HCA_3-EL组大鼠胰岛中生长抑素样免疫阳性物质的相对含量及阳性细胞数目均较对照组减少,而胰岛中胰多肽样免疫阳性物质却无明显变化。上述结果表明,海马CA_3区(HCA_3)及连台前穹窿(ACHF)对胰岛素的分泌有紧张性抑制作用。     

血浆游离脂肪酸浓度变化与消化间期综合肌电周期的关系

用狗进行慢性实验,在十二指肠浆膜缝植三对双极电极,记录空腹时的电活动。观察血浆游离脂肪酸(FFA)浓度变化与消化间期综合肌电(IDMEC)周期性活动的关系。实验结果提示:(1)血浆FFA浓度随IDMEC周期不同时相而变动,Ⅰ相时最低,Ⅲ相时最高(P<0.01);(2)静脉注射心得安后,血浆FFA浓度降低,随IDMEC的周期波动消失;(3)静脉注射酚妥拉明后,在IDMEC的各时相中血浆FFA浓度升高,且无周期性波动;(4)切除双侧迷走神经干后,血浆FFA浓度略有降低,随IDMEC的周期波动仍然存在;(5)静脉注射心得安、酚妥拉明和切除迷走神经后,对IDMEC的周期性活动没有明显的影响。以上结果证明,在空腹情况下血浆FFA浓度随IDMEC周期的不同时相而变动,交感神经系统在这些变动中具有一定的作用。     

饥饿状态大鼠胰腺高血糖素和胰岛素变化的定量分析

用免疫组织化学方法结合图象分析技术对饥饿状态大鼠胰岛Ａ、Ｂ细胞中胰因糖素和胰岛素的免疫反应强度进行定量分析。结果表明：与正常对照相比,饥饿大鼠胰岛细胞中的Ｇｌｕ含量明显下降,Ｂ细胞中Ｉｎｓ含量明显升高。提示饥饿可导致Ｇｌｕ释放增加,Ｉｎｓ和减少。与饥饿５天大鼠线要比较,饥饿５天后静脉注射葡萄糖组９０ｍｉｎ后胰岛内Ｇｌｕ含量明显升高,Ｉｎｓ含量无显著变化。提示：静脉注射葡萄糖要快速作用下胰岛Ａ细胞,     

消炎痛对四氧嘧啶引起的大鼠糖耐量和胰岛素分泌改变的影响

本实验以糖耐量为指标,观察了消炎痛抗四氧嘧啶引起的大鼠胰岛Ｂ细胞的损伤作用。皮下注射四氧嘧啶（１５０ｍｇ／ｋｇ）的大鼠控口灌注葡萄糖（５ｇ／ｋｇ以后,血糖浓度急剧升高并在灌胃后１８０ｍｉｎ仍继续升高。在葡萄糖刺激后血清胰岛素浓度无明显升高,未出现正常的分泌高峰。在注射四氧嘧啶前１２ｈ预先皮下注射消炎痛的大鼠则口服葡萄糖后的第６０ｍｉｎ血糖浓度迅速升高达到高峰,然后逐渐下降。在葡萄糖刺激后血清胰岛素浓度出现明显高峰,并具有类似正常的分泌高峰。基础血糖浓度和在口服葡萄糖后３０,６０,１２０,１８０ｍｉｎ血糖浓度及相应时相的胰岛素浓度在两组之间有显著性差异。消炎痛对正常大鼠糖耐量及胰岛素分泌没有明显的影响。本实验结果提示,消炎痛预处理可能对四氧嘧啶引起的胰岛Ｂ细胞损伤具有一定的预防作用。     

Mechanism of actions of cholecystokinin octapeptide on food intake and insulin and pancreatic polypeptide release in the dog

We investigated the mechanism by which CCK-8 injected into the third cerebral ventricle (ITV administration) inhibits food intake and stimulates insulin and pancreatic polypeptide (PP) secretion in the dog. ITV administration of CCK-8 (4.08 micrograms/5 min) resulted in a significant elevation of plasma insulin and PP concentrations. This effect was abolished by truncal vagotomy and promptly inhibited by ITV administration of atropine (20 micrograms) and proglumide (10 mg). CCK-8 was less effective in increasing insulin and PP concentrations than in reducing feeding. Thus, 1.36 micrograms of ITV CCK-8 markedly reduced food intake to 14, 15, 29 and 31% of control values at 10, 30, 60 and 120 min, respectively. Atropine and naloxone (50 micrograms) had no blocking effect on CCK-8-induced satiety, whereas proglumide antagonized it. These results indicate that ITV CCK-8 effects the endocrine pancreas and food intake through atropine-sensitive and atropine-insensitive mechanisms, respectively, both of which are likely to be mediated by CNS CCK receptors. Intravenous CCK-8 also stimulated PP and insulin release, through mechanisms that were atropine-sensitive and atropine-insensitive, respectively. However, its mode of action, especially on insulin secretion, was quite different from that of ITV CCK-8. Therefore, exogenous CCK appears to act in the brain and the periphery in concert with and independently from cholinergic systems.     

Role of Insulin and Free Fatty Acids in the Regulation of ob Gene Expression and Plasma Leptin in Normal Rats

Objective: It is under debate whether free fatty acids (FFAs) play an independent role in the regulation of adipose cell functions. In this study, we evaluated whether leptin secretion induced by FFA is due directly to an increased FFA availability or whether it is mediated by insulin levels. Research Methods and Procedures: To test this hypothesis, we compared the effects of six different experimental designs, with different FFA and insulin levels, on plasma leptin: euglycemic clamp, euglycemic clamp + FFA infusion, FFA infusion alone, FFA + somatostatin infusion, somatostatin infusion alone, and saline infusion. Results: Our results showed that euglycemic clamp, FFA infusion, or both in combination induced a similar increment of circulating leptin (3.31 ± 0.30, 3.40 ± 0.90, and 3.35 ± 0.80 ng/mL, respectively). Moreover, the inhibition of FFA‐induced insulin increase by means of somatostatin infusion completely abolished the rise of leptin in response to FFA (1.05 ± 0.30 vs. 3.40 ± 0.90 ng/mL, p < 0.001). Discussion: In conclusion, our data showed that the effects of high FFA levels on plasma leptin were mediated by the rise of insulin concentration. These data confirm a major role for insulin in the regulation of leptin secretion from rat adipose tissue and support the hypothesis that leptin secretion is coupled to net triglyceride synthesis in adipose tissue.     

Comparison of local and systemic effects of insulin on myocardial glucose extraction in ischemic heart disease

Physiological increases in circulating insulin level significantly increase myocardial glucose uptake in vivo. To what extent this represents a direct insulin action on the heart or results indirectly from reduction in circulating concentrations of free fatty acids (FFA) is uncertain. To examine this, we measured myocardial glucose, lactate, and FFA extraction in 10 fasting men (ages 49-76 yr) with stable coronary artery disease during sequential intracoronary (10 mU/min, coronary plasma insulin = 140 +/- 20 microU/ml) and intravenous (100 mU/min, systemic plasma insulin = 168 +/- 26 microU/ml) insulin infusion. Basally, hearts extracted 2 +/- 2% of arterial glucose and extracted 27 +/- 6% of FFA. Coronary insulin infusion increased glucose extraction to 5 +/- 3% (P < 0.01 vs. basal) without changing plasma FFA or heart FFA extraction. Conversion to intravenous infusion lowered plasma FFA by approximately 50% and heart FFA extraction by approximately 75%, increasing heart glucose extraction still further to 8 +/- 3% (P < 0. 01 vs. intracoronary). This suggests the increase in myocardial glucose extraction observed in response to an increment in systemic insulin concentration is mediated equally by a reduction in circulating FFA and by direct insulin action on the heart itself. Coronary insulin infusion increased myocardial lactate extraction as well (from 20 +/- 10% to 29 +/- 9%, P < 0.05), suggesting the local action may include stimulation of a metabolic step distal to glucose transport and glycolysis.     

N-terminal glycation of cholecystokinin-8 abolishes its insulinotropic action on clonal pancreatic B-cells.

Monoglycated cholecystokinin octapeptide (Asp(1)-glucitol CCK-8) was prepared under hyperglycaemic reducing conditions and purified by reverse phase-high performance liquid chromatography. Electrospray ionisation mass spectrometry and automated Edman degradation demonstrated that CCK-8 was glycated specifically at the amino-terminal Asp(1) residue. Effects of Asp(1)-glucitol CCK-8 and CCK-8 on insulin secretion were examined using glucose-responsive clonal BRIN-BD11 cells. In acute (20 min) incubations, 10(-10) mol/l CCK-8 enhanced insulin release by 1.2-1.5-fold at 5.6-11.1 mmol/l glucose. The stimulatory effect induced by 10(-10) mol/l CCK-8 was abolished following glycation. At 5.6 mmol/l glucose, CCK-8 at concentrations ranging from 10(-11) to 10(-7) mol/l induced a significant 1.6-1.9-fold increase in insulin secretion. Insulin output in the presence of Asp(1)-glucitol CCK-8 over the concentration range 10(-11)-10(-7) mol/l was decreased by 21-35% compared with CCK-8, and its insulinotropic action was effectively abolished. Asp(1)-glucitol CCK-8 at 10(-8) mol/l also completely blocked the stimulatory effects of 10(-11)-10(-8) mol/l CCK-8. These data indicate that structural modification by glycation at the amino-terminal Asp(1) residue effectively abolishes and/or antagonises the insulinotropic activity of CCK-8.     

Elevation of plasma CCK concentration after intestinal administration of a pancreatic enzyme secretion-stimulating peptide purified from rat bile-pancreatic juice: analysis with N-terminal region specific radioimmunoassay

The rat plasma cholecystokinin (CCK) concentration was measured after intestinal administration of a peptide purified from rat bile-pancreatic juice, which has a stimulatory effect on pancreatic enzyme secretion. The plasma CCK concentration was measured by means of a radioimmunoassay using CCK-8 N-terminal specific antibody, OAL-656. In experimental rats with protease-free intestines, intraduodenal infusion of 10 micrograms of the purified peptide, which stimulates pancreatic enzyme secretion 2.0-2.5 fold, induced a significant increase in the plasma CCK level. Furthermore, after removal of CCK from the plasma by immunoabsorption with an OAL-656-bound Sepharose 4B column, the stimulatory effect of the plasma on pancreatic enzyme secretion was abolished when it was injected intravenously into recipient rats. It was concluded that this peptide stimulates the release of CCK in the intestine and that this is responsible at least in part for the pancreatic enzyme secretion-stimulating activity of the peptide.     

Mechanism of rapid-phase insulin response to elevation of portal glucose concentration

The hepatoportal region is important for glucose sensing; however, the relationship between the hepatoportal glucose-sensing system and the postprandial rapid phase of the insulin response has been unclear. We examined whether a rapid-phase insulin response to low amounts of intraportal glucose infusion would occur, compared that with the response to intrajugular glucose infusion in conscious rats, and assessed whether this sensing system was associated with autonomic nerve activity. The increases in plasma glucose concentration did not differ between the two infusions at 3 min, but the rapid-phase insulin response was detected only in the intraportal infusion. A sharp and rapid insulin response was observed at 3 min after intraportal infusion of a small amount of glucose but not after intrajugular infusion. Furthermore, this insulin response was also induced by intraportal fructose infusion but not by nonmetabolizable sugars. The rapid-phase insulin response at 3 min during intraportal infusion did not differ between rats that had undergone hepatic vagotomy or chemical sympathectomy with 6-hydroxydopamine compared with control rats, but this response disappeared in rats that had undergone chemical vagotomy with atropine. We conclude that the elevation of glucose concentration in the hepatoportal region induced afferent signals from undetectable sensors and that these signals stimulate pancreas to induce the rapid-phase insulin response via cholinergic nerve action.     

Insulin suppresses PDK-4 expression in skeletal muscle independently of plasma FFA

Starvation and experimental diabetes induce a stable increase in pyruvate dehydrogenase kinase (PDK) activity in skeletal muscle, which is largely due to a selective upregulation of PDK-4 expression. Increased free fatty acid (FFA) level has been suggested to be responsible for the upregulation. Because these metabolic states are also characterized by insulin deficiency, the present study was designed to examine whether insulin has a significant effect to regulate PDK mRNA expression in rat skeletal muscle. In study 1, overnight-fasted rats received an infusion of saline or insulin for 5 h (n = 6 each). During the insulin infusion, plasma glucose was clamped at basal levels (euglycemic hyperinsulinemic clamp). A third group (n = 6) received Intralipid infusion during the clamp to prevent a fall in plasma FFA. PDK-2 mRNA level in gastrocnemius muscle was not altered by insulin or FFA (i.e., Intralipid infusion). In contrast, PDK-4 mRNA level was decreased 72% by insulin (P < 0.05), and Intralipid infusion prevented only 20% of the decrease. PDK-4 protein level was decreased approximately 20% by insulin (P < 0.05), but this effect was not altered by Intralipid infusion. In study 2, overnight-fasted rats were refed or received an infusion of saline or nicotinic acid (NA, 30 micromol/h) for 5 h (n = 5 each). During the refeeding and NA infusion, plasma FFA levels were similarly (i.e., 60-70% vs. saline control) lowered. Muscle PDK-4 mRNA level decreased 77% after the refeeding (P < 0.05) but not after the NA infusion. In conclusion, the present data indicate that insulin had a profound effect to suppress PDK-4 expression in skeletal muscle and that, contrary to previous suggestions, circulating FFA had little impact on PDK-4 mRNA expression, at least within 5 h.     

Continuous 24-h nicotinic acid infusion in rats causes FFA rebound and insulin resistance by altering gene expression and basal lipolysis in adipose tissue

Nicotinic acid (NA) has been used as a lipid drug for five decades. The lipid-lowering effects of NA are attributed to its ability to suppress lipolysis in adipocytes and lower plasma FFA levels. However, plasma FFA levels often rebound during NA treatment, offsetting some of the lipid-lowering effects of NA and/or causing insulin resistance, but the underlying mechanisms are unclear. The present study was designed to determine whether a prolonged, continuous NA infusion in rats produces a FFA rebound and/or insulin resistance. NA infusion rapidly lowered plasma FFA levels (>60%, P < 0.01), and this effect was maintained for ≥5 h. However, when this infusion was extended to 24 h, plasma FFA levels rebounded to the levels of saline-infused control rats. This was not due to a downregulation of NA action, because when the NA infusion was stopped, plasma FFA levels rapidly increased more than twofold (P < 0.01), indicating that basal lipolysis was increased. Microarray analysis revealed many changes in gene expression in adipose tissue, which would contribute to the increase in basal lipolysis. In particular, phosphodiesterase-3B gene expression decreased significantly, which would increase cAMP levels and thus lipolysis. Hyperinsulinemic glucose clamps showed that insulin's action on glucose metabolism was improved during 24-h NA infusion but became impaired with increased plasma FFA levels after cessation of NA infusion. In conclusion, a 24-h continuous NA infusion in rats resulted in an FFA rebound, which appeared to be due to altered gene expression and increased basal lipolysis in adipose tissue. In addition, our data support a previous suggestion that insulin resistance develops as a result of FFA rebound during NA treatment. Thus, the present study provides an animal model and potential molecular mechanisms of FFA rebound and insulin resistance, observed in clinical studies with chronic NA treatment.     

Intraportal glucose infusion and pancreatic islet blood flow in anesthetized rats

The aim of the study was to evaluate whether a selective increase in portal vein blood glucose concentration can affect pancreatic islet blood flow. Anesthetized rats were infused (0.1 ml/min for 3 min) directly into the portal vein with saline, glucose, or 3-O-methylglucose. The infused dose of glucose (1 mg. kg body wt(-1). min(-1)) was chosen so that the systemic blood glucose concentration was unaffected. Intraportal infusion of D-glucose increased insulin release and islet blood flow; the osmotic control substance 3-O-methylglucose had no such effect. A bilateral vagotomy performed 20 min before the infusions potentiated the islet blood flow response and also induced an increase in whole pancreatic blood flow, whereas the insulin response was abolished. Administration of atropine to vagotomized animals did not change the blood flow responses to intraportal glucose infusions. When the vagotomy was combined with a denervation of the hepatic artery, there was no stimulation of islet blood flow or insulin release after intraportal glucose infusion. We conclude that a selective increase in portal vein blood glucose concentration may participate in the islet blood flow increase in response to hyperglycemia. This effect is probably mediated via periarterial nerves and not through the vagus nerve. Furthermore, this blood flow increase can be dissociated from changes in insulin release.