Interleukin-6 affects insulin secretion and glucose metabolism of rat pancreatic islets in vitro

Recently it has been postulated that interleukin-1 (IL-1) locally released by infiltrating mononuclear cells may destroy the pancreatic B cells during the development of insulin-dependent diabetes mellitus. Since IL-1 is a potent inducer of interleukin-6 (IL-6) in various cells, it is conceivable that IL-6 is a second mediator of the IL-1 action. In the present study the effects of IL-6 alone or in combination with IL-1 were studied on pancreatic islet function in vitro after tissue culture and compared with the effects observed after exposure to IL-1 only. Rat pancreatic islets were cultured in medium RPMI 1640 + 10% calf serum with or without the addition of human recombinant IL-6 (500-5000 pg/ml) for 48 h. The medium insulin accumulation was increased by 40-50% after culture with 500-2000 pg/ml IL-6, but was similar to the controls at 5000 pg/ml. When islets were cultured for 18 h only, also 5000 pg/ml IL-6 stimulated the medium insulin accumulation. IL-6 did not affect the islet insulin content and the rates of islet (pro)insulin and total protein biosynthesis. It inconsistently decreased the islet DNA content. In short-term experiments after 48-h culture with IL-6, there was a dose-dependent inhibition of the glucose-stimulated insulin release. On the other hand, islets cultured with IL-6 (5000 pg/ml) exhibited an elevated glucose oxidation and oxygen uptake, but a lower ATP content at 16.7 mM glucose and an unaffected glucose utilization and glutamine oxidation compared to the controls. This raises the possibility that IL-6 had induced a condition with an increased energy expenditure, resulting in an enhanced mitochondrial metabolism of glucose. Islets cultured with human recombinant IL-1 beta (25 units/ml) showed a strong inhibition of the insulin accumulation in the culture medium and of glucose-stimulated insulin release and a marked decrease in the islet DNA and insulin content. A combination of IL-1 (25 U/ml) + IL-6 (1000 pg/ml) did not alter the inhibitory action of IL-1 alone. The present findings thus show that IL-6 induces a dissociation between insulin secretion and glucose oxidation in islets in vitro. This has not been observed in islets exposed to IL-1, which suggests that IL-6 does not solely mediate the inhibitory effects of IL-1 on islet function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of in vitro maturation of stimulus-secretion coupling in fetal rat islet beta-cells

We have studied the maturation of a glucose-responsive insulin release from fetal rat islets, and specifically investigated the impact of nutrients, α-adrenoceptors, imidazoline receptors, and cyclic adenosine monophosphate (cAMP). Islets were isolated from 21-d-old fetal rats and maintained for 7 d in tissue culture at 3.3 or 11.1 mM glucose and various supplements. Culture in the presence of the nonglucidic nutrient α-ketoisocaproic acid (KIC), markedly enhanced both basal and stimulated insulin release from islets cultured at either low or high glucose. Additionally, KIC significantly elevated the insulin content of islets maintained in low glucose, whereas it slightly lowered it is islets cultured at high glucose. Culture with phentolamine, an antagonist of α-adrenergic and imidazoline receptors, markedly amplified both basal and glucose-stimulated insulin secretion when added with islets cultured in either low or high glucose. By contrast, the pure α₂-adrenoreceptor antagonist benextramine had no such effects. Addition to culture media of a membrane-permeant agonist (Sp-cAMP[S]) or antagonist (Rp-cAMP[S]) of cAMP-dependent protein kinases types I and II failed to influence basal or glucose-responsive insulin secretory rates at either glucose concentration during culture as well as islet insulin content. In conclusion, islet β-cell differentiation and functional maturation of the stimulus-secretion coupling can be accelerated in vitro in fetal rat pancreatic tissue by nutrient stimulation, and by interference with imidazoline receptors, whereas cAMP seems virtually ineffective in this respect. These effectors may be of regulatory significance in the vivo development of glucose-sensitive β-cells.     

Erythropoietin affects the maturation pattern of fetal G-CSF-responsive progenitors.

A transient hyporegenerative neutropenia has been reported in neonates, but not in older children or adults, undergoing treatment with recombinant erythropoietin (epo). Monocytopenia has not been reported. We postulated that epo might selectively reduce the responsiveness of neonatal progenitors to Granulocyte Colony-Stimulating Factor (G-CSF), while not similarly affecting their responsiveness to Macrophage Colony-Stimulating Factor (M-CSF). To test this hypothesis two types of experiments were performed. First, progenitors of adult or fetal origin were pre-incubated with epo (or control), then washed, and their responsiveness to G-CSF and M-CSF evaluated in clonogenic culture assays. Second, clonogenic maturation was initiated using either G-CSF or M-CSF, after which the effect of a late addition of epo to the developing clones was evaluated. Indeed, pre-incubation with epo resulted in production of fewer neutrophils from fetal progenitors grown in G-CSF (P less than 0.001), but it did not reduce the number of macrophages generated from progenitors grown in M-CSF. Adding epo to the already-developing G-CSF-responsive and M-CSF-responsive adult and fetal clones did not alter colony development. Thus, epo appears to have an action on G-CSF-responsive, but not-M-CSF-responsive fetal progenitors, resulting in reduced production of neutrophils. This effect is no longer apparent, however, when progenitors have matured to the 8-cell clone stage.     

Androgens delay human fetal lung maturation in vitro

The mature lung produces pulmonary surfactant, a lipid-protein complex that prevents lung alveoli from becoming atelectatic. The prenatal surge of surfactant production in preparation for birth occurs between 28-34 weeks gestation and is triggered by glucocorticoids, which stimulate surfactant synthesis by alveolar epithelial cells through a series of biochemical steps mediated by mesenchymal-epithelial interactions. In contrast to this, when explanted midgestation human fetal lung tissue is maintained in serum-free medium, there is a spontaneous increase (40%) in de novo saturated phosphatidylcholine (SPC) synthesis on the fifth day in culture. Addition of dexamethasone (DEX; 1 x 10(-8) M) to the culture medium causes the increased synthesis in SPC to occur earlier (day 4) and to a greater extent (87%). Addition of an equimolar concentration of dihydrotestosterone (DHT) to the medium delays both the spontaneous and DEX-stimulated increases in SPC synthesis by 24 h. Weak fetal adrenal androgens are also able to block both spontaneous and DEX-stimulated SPC synthesis. Addition of DHT to the explant cultures at daily intervals reveals that inhibition of the DEX effect occurs within the first 24 h after exposure. The antiandrogen flutamide neutralizes the effect of DHT, indicating that it acts through the androgen receptor to block the glucocorticoid. Therefore, the human fetal adrenal cortex may time lung development through both inhibitory and stimulatory mechanisms.     

胰岛与Sertoli细胞体外共同培养

目的 研究胰岛体外长期培养的新方法。方法 采用绿色荧光蛋白（GFP）标记成年SD大鼠胰岛，与Sertoli细胞共同培养20周，应用形态学方法和透射电镜观察胰岛细胞单独培养和共同培养的生长特性；放射免疫法测定胰岛素分泌量。结果 胰岛单独培养3周。细胞存活率显著下降，胰岛阳性细胞数目减少，胰岛素24h累积分泌量和对葡萄糖刺激的反应性下降，大部分胰岛β细胞超微结构破坏，分泌颗粒减少；与Sertoli细胞共同培养的胰岛存活时间显著延长，细胞存活率和胰岛素阳性细胞数目较单独培养显著增加，胰岛素分泌量始终处于高水平状态，培养20周胰岛β细胞超微结构基本正常，结论 大鼠胰岛与Sertoli细胞共同培养可以促进胰岛生长，显著延长存活时间，是一种新的体外长期培养胰岛的方法。     

β细胞素对体外培养大鼠胰岛的影响

研究β细胞素(BTC)对体外培养大鼠胰岛的作用。BTC无促胰岛急性分泌作用,但对长期培养大鼠胰岛的葡萄糖刺激的胰岛素分泌(GSIS)具有一定保护效力;实时PCR及免疫荧光检测胰高血糖素、胰岛素、PDX-1、葡萄糖转运子2的表达未随培养时间延长而丰度下降,BTC可能不是通过调节上述基因的表达来维护胰岛GSIS功能的。     

Glucose decreases extracellular adenosine levels in isolated mouse and rat pancreatic islets

The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca²⁺ suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.     

Glucose decreases extracellular adenosine levels in isolated mouse and rat pancreatic islets

The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca²⁺ suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.     

Glucose utilization in islets of hyperglycemic rat models with impaired glucose-induced insulin secretion

Under most experimental conditions islet glucose metabolism is well-correlated with short-term glucose-induced insulin secretion. Two hyperglycemic rat models (neonatal streptozotocin and glucose infusion) have been previously found to have markedly impaired insulin responses to glucose, and the glucose utilization of islets isolated from these models was therefore studied to see if reduced glucose metabolism might be related to the secretory abnormalities. It was found that glucose utilization in the islets of the two models was similar or higher than in comparable control islets. These data suggest that the secretory defect of these models, which is presumably induced by chronic hyperglycemia, is at a step in the secretion process distal to glucose metabolism.     

大鼠胚胎垂体生长激素细胞的体外诱导研究

目的探讨地塞米松和生长激素释放激素(GHRH)对大鼠胚胎垂体生长激素(GH)细胞分化的作用,为将来选择性细胞移植治疗生长激素缺乏症(GHD)奠定基础。方法利用免疫组织化学方法和放射免疫分析方法,在大鼠胚胎垂体组织的原代培养中,对糖皮质激素诱导GH细胞分化的最佳浓度、诱导GH细胞发生分化所需要的最短时间以及GHRH在大鼠胚胎垂体细胞分化中的作用进行实验研究。结果地塞米松诱导GH细胞分化的最佳浓度为50nmol/L(P<0.01)。体外培养的胚胎垂体细胞经地塞米松诱导16h,GH细胞开始分化,诱导48h,分化更明显,上清液中GH水平也明显增加(P<0.01);当GHRH的浓度达到10-7mol/L时,可以增强地塞米松对GH细胞的诱导分化作用,明显提高GH细胞的百分比和GH的分泌量(P<0.01)。结论地塞米松可以诱导生长激素前体细胞最终分化为具有功能活性的GH细胞,GHRH与地塞米松发挥协同作用,调节GH细胞的分化与GH分泌。     

Ontogeny of four cell types in fetal rat islets using histochemical techniques

Summary The pancreas of the fetal rat was collected from the first appearance of the pancreatic bud at 11 days of gestation, and every day thereafter until birth. After birth the neonatal pancreas was collected every day for one week, and at intervals thereafter. Fetal B-cells were stained with Gomori’s aldehyde fuchsin at 16 μ days, and with the immunofluorescent technique for insulin at 14 days. The A-cells were stained as early as 13 days using the fluorescent antibody technique for glucagon. The D-cells first stained at 17 days with pseudoisocyanin. A 4th cell type was found which stained black with silver nitrate, using a method derived from the Grimelius technique for A-cells. This 4th cell type appeared at 15 days in the fetus, reaching its greatest abundance around 19 days, and then declined in numbers after birth until adulthood, when occasionally one or two cells were found.     

Glucose enhances adenylyl cyclase responses in normal but not diabetic GK rat islets

In the GK rat model of type 2 diabetes, adenylyl cyclase (AC) expression and stimulation are increased. Whether the prevalent glucose level has any effects on AC responses is, however, unclear. We have studied concurrent insulin release and cyclic adenosine monophosphate (cAMP) generation in response to 5 microM forskolin in islets cultured for 48 hours in 5.5 or 11 mM glucose. Insulin release was impaired in GK rat islets, irrespective of culture condition, in response to 3.3 and 16.7 mM glucose and was fully restored by forskolin through exaggerated insulin responses. Stimulation of normal islets with 5 microM forskolin elicited different islet cAMP responses, which were dependent on the dose of glucose in the culture medium. Thus in normal islets cultured in 11 mM glucose, forskolin increased cAMP levels fivefold to sixfold at 3.3 and 16.7 mM glucose, whereas forskolin increased cAMP levels only twofold in islets cultured at 5.5 mM glucose. In GK islets, forskolin induced a consistently exaggerated approximately eightfold increase in cAMP generation irrespective of glucose concentration in the culture medium. In conclusion, culturing normal islets at hyperglycemic glucose levels (11 mM) primed and markedly enhanced cAMP generation in response to forskolin.     

Glucose metabolism by rat pancreatic eslets in vitro

[U-¹⁴C]-glucose was incubated with rat islets in a Krebs-Ringer buffer. The uptake of glucose and its oxidation to CO₂ were in accord with linear increases over the range of medium concentration of glucose from 0.6–3.0 mg/ml. Uptake was similar at 3.0 and 5.0 mg/ml indicating saturation of uptake in the range of these concentrations. These values are in agreement with reports of insulin release as a function of increasing glucose concentration and hence of insulin release as a function of uptake. Between 62% and 76% of glucose uptake was accounted for in CO₂ and lactate. The amount of [¹⁴C]-lactate formed was similar to the amount of ¹⁴CO₂ formed at 0.6 mg/ml, and it was about one-half as much or less at 3.0 and 5.0 mg/ml. Therefore, lactate is not the major product of glucose metabolism by rat islets in vitro. The formation of [3-¹⁴C]-lactate from [1-¹⁴C]-glucose indicates that lactate formation from glucose proceeds via pyruvate without intermediate randomization of the pyruvate carbons in the Krebs cycle.     

Glucose affects the release of thyrotropin-releasing hormone from the rat hypothalamus

We have attempted to elucidate the effect of glucose concentrations on the release of thyrotropin-releasing hormone (TRH), a brain neuropeptide possessing glucoregulatory function, from rat hypothalamic slices in vitro. Rat hypothalamic slices were preincubated for 60 min at 37 degrees C in Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) containing varying concentrations of glucose (10, 5, 2.5 and 1 mM), and then tissues were incubated in KRB buffer, followed by stimulation with 60 mM K+ or 1 mM ouabain. In addition, after inducing hypoglycemia by insulin administration in the rat, hypothalamic tissues were dissected out and preincubated in KRB buffer (10 mM glucose) and then incubated in fresh KRB buffer containing 1 mM ouabain. A decrease in the glucose concentration of incubation medium caused a dose-dependent decrease in both K(+)- and ouabain-stimulated TRH release from rat hypothalamic slices. Furthermore, the ouabain-stimulated TRH release from the hypothalamus of rats with insulin-induced hypoglycemia was significantly reduced (45% of control values; p less than 0.01). The present results indicate that in vitro and in vivo hypoglycemia resulted in a significant decrease in the release of TRH from the hypothalamus, suggesting that circulating glucose levels affect TRH release which, in turn, might be responsible for peripheral glucoregulation.     

Glucose increases the synthesis of lipoxygenase-mediated metabolites of arachidonic acid in intact rat islets.

Previous studies suggested that products of a 12-lipoxygenase pathway in the pancreatic islet may promote insulin release. To determine whether glucose augments the production of such metabolites, intact rat islets prelabeled with [3H]arachidonate were stimulated with glucose, and 12-hydroxy-5,8,10,14-icosatetraenoic acid (12-HETE) release was measured by using HPLC. D-Glucose (16.7 mM) augmented the enzymatic synthesis of 12-HETE by 271% above that seen with 0-1.7 mM glucose. The glucose effect was stereospecific and preferential for the alpha anomer; it was modestly potentiated by the cyclo-oxygenase inhibitor ibuprofen. Glucose-stimulated 12-HETE accumulation was abrogated by mannoheptulose and was reproduced by the trioses glyceraldehyde or dihydroxyacetone, suggesting that the metabolism of glucose to glucose 6-phosphate or triose phosphates (or both) is critical. Glucose also augmented [3H]arachidonate labeling of islets, suggesting an action at the level of substrate release or re-uptake (or both). These features of islet 12-HETE synthesis accord well with other known effects of glucose on beta cell function and suggest that lipoxygenase-mediated metabolites of arachidonate may be suitable candidates to mediate or amplify glucose's effects on insulin release.     

The effect of beta-endorphin on rat oocyte maturation in vitro

Cumulus enclosed or denuded oocytes obtained from ovaries of 25- to 27-day Sprague-Dawley rats underwent spontaneous germinal vesicle breakdown (GVBD) when cultured for 6 h in Krebs-Ringer's buffered solution (KRBS). This spontaneous division was found to be inhibited by adding beta-endorphin to the culture system and the inhibition was dose dependent, ranging from 200 to 800 pg/ml KRBS. Naloxone, a potent opioid antagonist without any agonistic action, did not stimulate spontaneous GVBD when added to the KRBS at doses ranging from 80 to 120 pg/ml. However, by adding 80 pg/ml naloxone to the culture system containing 600 pg/ml beta-endorphin, the inhibitory effect of beta-endorphin on spontaneous GVBD could be reversed completely.     

瘦素对离体大鼠胰岛分泌胰岛素的双向影响

目的 了解不同浓度的瘦素，在不同条件下对离体大鼠胰岛分泌胰岛素的影响。方法 利用细胞培养技术，在不同的葡萄糖浓度（5.6mmol/L或16.7mmol/L）和不同作用时间（10min或2h)下，以0,1,5,10,15,50ak 100μg/L瘦素作用大鼠胰岛，观察其对胰岛素分泌的影响；用放免法检测培养物上清液胰岛素浓度。结果 培养液葡萄糖浓度为5.6mmol/L，培养10min，1μg/L、5μg/L瘦素促进被孵育的胰岛的胰岛素分泌；培养2h,5μg/L瘦素促进基础胰岛素分泌，≥50μg/L瘦素抑制胰岛素分泌。葡萄糖浓度为16.7mmol/L，培养10min，≥50μg/L瘦素抑制胰岛素分泌；培养2h，≥5μg/L瘦素抑制胰岛素分泌。结论 重组瘦素对离体大鼠胰岛分泌胰岛素具有双向作用，并受瘦素浓度、作用时间和环境葡萄糖浓度等因素变化的影响。     

Steroid production in vitro by rat ovaries during sexual maturation

To determine changes in steroidogenesis by rat ovaries during sexual maturation, ovaries obtained at various ages (days 10-35) and at the first pro-oestrus were incubated in the absence or presence of LH and the accumulation of steroids in the medium was measured. Basal and LH-stimulated oestradiol-17 beta and testosterone release into the medium, expressed in pmol/4 h per mg ovary, was high at day 10 of age and at first pro-oestrus. Between days 20 and 35 basal oestradiol and testosterone release was low and could not be stimulated by LH. Addition of testosterone to the culture medium increased oestradiol production at all ages studied. Release of progesterone occurred at all ages even in LH-free medium. Incubation in the presence of LH resulted in a dose-dependent increase in progesterone with a maximal response at pro-oestrus. Androsterone and 5 alpha-androstane-3 alpha,17 beta-diol production in the absence or presence of LH was high during the entire prepuberal period. Production of 5 alpha-reduced androgens in response to LH increased from days 10 to 20 but decreased thereafter. Similarly, 5 alpha-reductase activity, measured in ovarian homogenates, increased from days 10 to 20 but was decreased again by first pro-oestrus. A further decrease in basal and LH-stimulated 5 alpha-reduced androgen production occurred after first ovulation. These results demonstrated age-related changes in steroid release after in-vitro incubation. At day 10 progesterone can be converted to aromatizable androgens allowing production of oestrogens, while after day 10 progesterone is converted to 5 alpha-reduced C19 steroids.(ABSTRACT TRUNCATED AT 250 WORDS)