Rate Of Protein Biosynthesis Research Articles

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA.

The binding of small molecules to distinctive three-dimensional structures in mRNA provides a new dimension in RNA control, previously limited to the targeting of secondary structures with antisense and RNA interference; such targeting can modulate mRNA function and rates of protein biosynthesis. Small molecules that selectively bind the iron-responsive element (IRE), a specific three-dimensional structure in the noncoding region of the ferritin mRNA model that is recognized by the iron-regulatory protein repressor, were identified by using chemical footprinting. The assay used involved an oxoruthenium(IV) complex that oxidizes guanine bases in RNA sequences. Small molecules that blocked oxidation of guanines in the internal loop region were expected to selectively increase the rate of ferritin synthesis, because the internal loop region of the ferritin IRE is distinctive from those of other IREs. The natural product yohimbine was found (based on gel mobility shifts) to block cleavage of the internal loop RNA site by >50% and seemed to inhibit protein binding. In the presence of yohimbine, the rate of biosynthesis of ferritin in a cell-free expression system (rabbit reticulocyte lysate) increased by 40%. Assignment of the IRE-yohimbine interaction as the origin of this effect was supported by a similar increase in synthesis of luciferase protein in a chimera of the IRE and luciferase gene. The identification of a small, drug-like molecule that recognizes a naturally occurring three-dimensional mRNA structure and regulates protein biosynthesis rates raises the possibility that small molecules can regulate protein biosynthesis by selectively binding to mRNA.

Role of apolipoprotein A-I in the anabolic effect of steroid hormones

As early shown, a portion of steroid hormones binds to blood lipoproteins, primarily to high-density lipoproteins (HDL) [Panin et al. 1988]. Steroid hormones together with HDL are captured by resident macrophages of the liver where in secondary lysosomes HDL are degraded to form apoA-I and steroid hormones restore a ∆4, 3-keto group with the participation of 5-α and 5β- reductases to give rise to tetrahydro compounds. In this study, an attempt was undertaken to show a role of a complex of some steroid hormones with apo A-I in realization of the anabolic action of these steroid hormones by using the cultured hepatocytes and concurrently cultured hepatocytes and Kupffer’s cells isolated from the liver of male Wistar rats weighing 180-200 g.
 Steroid hormones having an anabolic action, such as androsterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and tetrahydrocortisol as ingredients of a complex with apolipoprotein A-I (apoA-I), increased the rate of protein biosynthesis and dehydroepiandrosterone sulfate and tetrahydrocortisol also did the rate of DNA synthesis in the cultured hepatocytes. All the hormones had a restored ∆4,3-keto group in the A ring structure. Restoration of this group of steroid hormones and formation of their complex with apoA-I are associated with the action of resident macrophages (Kupffer’s cells). That is the reason that addition of HDL (a source of apoA-I) and cortisol (a source of the restored form - tetrahydrocortisol) to the coculture of hepatocytes and macrophages, by concurrently stimulating the latter by lipopolysaccharide led to a significant increase in the rate of protein and DNA biosynthesis. The findings show an important role of a ∆4,3-keto group of the A ring of steroid hormones and their complex with apo A-I in realizing the anabolic action of steroids.

Up-regulation of tRNA biosynthesis affects translational readthrough in maf1-delta mutant of Saccharomyces cerevisiae.

Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-delta mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ- luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-delta compared with the parental strain. The maf1-delta mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1- Delta using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11or by [PSI(+)], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1- Delta increase translational fidelity and, independently, affect growth under special conditions.

Effect of tetrahydrocortisol-apolipoprotein A-I complex on RNA polymerase interaction with eukaryotic DNA and the rate of protein biosynthesis in hepatocytes

A mechanism of activation of protein biosynthesis in hepatocytes was proposed as effected by the conditioned medium of nonparenchymal liver cells incubated in the presence of high density lypoproteins, cortisol, and lypopolysaccharides. It was found that the increase in the biosynthesis rate was associated with the formation of the tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) complex in macrophages, which display 5 alpha- and 5 beta-reductase activity and are constituents of nonparenchymal liver hepatocytes. Using the small-angle X-ray scattering technique, it was shown that the THC-apoA-I-eukaryotic DNA interaction may break hydrogen bonds between pairs of complementary nucleic bases and cause the formation of single-stranded DNA fragments capable of binding to DNA-dependent RNA polymerase. The interaction is highly cooperative and has a saturating mode, up to six enzyme molecules being bound per DNA molecule.

Effect of tetrahydrocortisol on protein biosynthesis in hepatocytes

Cooperative effect of high-density lipoproteins and glucocorticoids on protein biosynthesis in hepatocytes was associated with the formation of biologically active hormone in Kupffer cells. Apolipoprotein A-I, a constituent of high-density lipoproteins, transported the active hormone from liver macrophages to hepatocytes. Apolipoprotein A-I-induced stimulation of protein biosynthesis was observed only in Kupffer cells. Apolipoprotein A-I alone or in combination with hormones did not change the rate of protein biosynthesis in liver endotheliocytes.

Effect of mild hypothermia during and after transient in vitro ischemia on metabolic disturbances in hippocampal slices at different stages of development

In the present study the neuroprotective effect of mild hypothermia (decrease of temperature from 37°C to 33°C) during and after transient ischemia in brain tissue at different stages of development was tested in vitro by measuring energy metabolism, glutamate release and protein biosynthesis rate (PSR) in hippocampal slices. Slices were taken from immature (E40) and mature (E60) guinea pig fetuses and adult guinea pigs. The slices were exposed to ischemia-like conditions (oxygen/glucose deprivation, OGD) for periods of between 10 to 40 min followed by a 2-h or 12-h recovery phase. During OGD, mild hypothermia slowed down the depletion of energy stores only in slices from immature fetuses, but had no effect on slices prepared from mature fetuses and adult animals. Hypothermia also reduced glutamate release significantly during oxygen/glucose deprivation. Lowering temperature to 33°C had no effect on energy metabolism and only a minor effect on PSR of slices from mature fetuses and adult animals subjected to 2 h of recovery. However, 12 h after OGD PSR was markedly improved by mild hypothermia in slices from mature animals and in slices from adults that had been exposed to OGD for only 20 or 30 min. The inhibition of PSR was more severe in the slices from adults than in those from mature fetuses subjected to the same duration of OGD. Age- and temperature-related differences in glutamate release during OGD did not fully agree with corresponding disparities in the values for PSR obtained 12 h after OGD. These results indicate that the neuroprotective effect of mild hypothermia was not mediated by a temperature-dependent retardation of the depletion of energy stores during OGD. Age-related disparities in the vulnerability of the brain to ischemia and the neuroprotective efficiency of mild hypothermia appear to be only partially reflected by the varying levels of glutamate release during ischemia but best reflected by the extent of PSR inhibition. It is concluded that mild hypothermia may be a suitable therapeutical intervention for the suppression of hypoxic-ischemic cell damage during birth.

Non-Selective Effects of Adenosine A 1 Receptor Ligands on Energy Metabolism and Macromolecular Biosynthesis in Cultured Central Neurons

To investigate the effects of adenosine A 1 receptor activation on energy metabolism and RNA and protein biosynthesis in central neurons, cultured neurons from the rat forebrain were exposed for 1 hr to 72 hr to various concentrations (10 nM–100 μM) of the selective A 1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA) or the A 1 receptor antagonist 8-cyclopentyltheophylline (CPT). At all concentrations tested, the adenosinergic compounds did not affect cell viability within 72 hr of treatment, except for CPT, which reduced viability by 19.7% when used at the concentration of 100 μM. Energy metabolism was analysed by studying the specific uptake of 2-D-[ 3H]deoxyglucose ([ 3H]2DG). Rates of RNA and protein biosynthesis were assessed by the measurement of [ 3H]uridine and [ 3H]leucine incorporation, respectively. Neuronal [ 3H]2DG uptake was increased by 16% ( P < 0.01) after 8 hr in the presence of 100 μM CCPA, whereas 100 μM CPT for 24 hr also increased [ 3H]2DG uptake (8%, P < 0.01). At these concentrations, both ligands inhibited [ 3H]uridine incorporation after a 3-hr treatment by 92% and 30%, respectively. CCPA never altered [ 3H]leucine incorporation when compared to controls, and CPT significantly inhibited protein synthesis only at 10–100 μM. Additional experiments to analyse the influence of A 1 ligands on the transport of [ 3H]2DG, [ 3H]leucine and [ 3H]uridine suggested that CCPA and CPT, which interact functionally with adenosine receptors by regulating cyclic AMP production in this model, are able to alter energy metabolism and RNA synthesis in central neurons in a nonspecific manner by interacting with glucose and uridine transporters.

Effect of the ATP level on the overall protein biosynthesis rate in a wheat germ cell-free system

A sensitive assay which examines the effects of ATP level on the overall activity of a cell-free translation system in a protein synthesis is described. The translational activity of cell-free system was measured in terms of a rate of protein synthesis directed by the ‘test’ template. The test template encodes a photoluminescent protein, obelin. The rate of obelin accumulation was determined from the kinetic curves of obelin mRNA translation. Time-dependent nucleotide level measurements were conducted throughout the translation processes. It has been shown that the rate of translation decreases exponentially with the decrease of the ATP level. This fall in the overall translation rate is due in part to the mRNA becoming inactive in the translation process. This is not caused by degradation, this mRNA can be restored for translation in a fresh cell-free system by phenol treatment. The reported results provide evidence that the level of ATP unambiguously determines the translational activity of the system.

Long-term effects of nicotinamide-induced inhibition of poly(adenosine diphosphate-ribose) polymerase activity in rat pancreatic islets exposed to interleukin-1 beta.

Nicotinamide (NIC) is presently extensively studied as a potential agent that might prevent the development of insulin-dependent diabetes mellitus. This study aimed to examine the consequence of exposing isolated rat pancreatic islets to various concentrations of NIC (0, 0.5, 1.0, 5.0, 10, and 25 mM) over a prolonged period (6 days) in tissue culture and also to assess the efficacy of NIC to counteract interleukin-1 beta (IL-1; 25 U/ml)-induced beta-cell dysfunction. Except for a 30-40% increase at 5.0 mM NIC, the insulin content of islets was not affected by NIC. Also, the islet DNA content remained essentially unchanged. The insulin accumulation in the culture medium declined at 5-25 mM NIC between days 4-6. The insulin release in response to 16.7 mM glucose on day 6 was enhanced after culture with the addition of 0.5 mM NIC, but 25 mM NIC caused a strong inhibition of insulin secretion. However, neither the (pro)insulin nor the total protein biosynthesis rate of the islets was affected by NIC. A significant inhibition of the islet poly(ADP-ribose) polymerase activity by 40-80% was observed at 5-25 mM NIC. IL-1 was then tested together with 1.0 and 10 mM NIC. The islet DNA content was markedly reduced in all groups treated with IL-1, as was the medium insulin accumulation. Moreover, NIC failed to prevent IL-1 induced impairment of islet insulin release on day 6. The cytokine induced a very pronounced and sustained increase in the medium nitrite accumulation, and NIC could not influence this elevation. Thus, these data show that prolonged exposure to elevated NIC levels impaired the function of rat beta-cells, and NIC failed to counteract IL-1 actions. Whether these events are mimicked in the ongoing clinical trials with NIC remain to be established.

Chronic exposure of cultured rat pancreatic islets to elevated concentrations of islet amyloid polypeptide (IAPP) causes a decrease in islet DNA content and medium insulin accumulation

The biological action of islet amyloid polypeptide (IAPP) remains to be established, although a role for IAPP in causing β-cell failure in diabetes has been proposed. Acute in vitro experiments with IAPP have given controversial results as to β-cell insulin secretion. We have studied the effect of a long-term exposure of pancreatic islets to elevated IAPP concentrations. Thus, rat pancreatic islets were cultured for 4 days in medium supplemented with 0, 0.1, 1, 10, 100 or 1000 nM of synthetic amidated rat IAPP. Islets exposed to the two highest IAPP concentrations contained about 20% less DNA, whereas the rate of DNA synthesis was unchanged. Culture with 1000 nM IAPP, but not the lower concentrations of the peptide, slightly decreased the glucose oxidation rate. There was a correlation between increasing IAPP concentrations and and a decline in the medium insulin accumulation. The preceding IAPP exposure did, however, neither significantly affect basal and glucose-stimulated insulin secretion nor (pro)insulin and total protein biosynthesis rates, when tested after the culture. The finding of a decreased islet cell DNA content after culture with elevated IAPP concentrations suggests a toxic action to islet cells, whereas a putative inhibition of the islet insulin production appears to be transient.

Fibronectin gene expression differs in normal and abnormal human wound healing

The overproduction of fibronectin and type I collagen in keloids and hypertrophic scars implicates altered regulation of extracellular matrix components as an important aspect of these wound healing pathologies. However, little is known about the similarities and differences in extracellular matrix gene expression during normal and abnormal wound healing. This study compared the content of fibronectin messenger RNA and rates of fibronectin protein biosynthesis in fibroblasts derived from normal skin, normal scar, keloid, and hypertrophic scar. Fibronectin expression was enhanced in cells from both normal and abnormal wounds relative to cells from quiescent normal skin. Matched pairs of normal and keloid fibroblasts from the same individuals were also compared, and three of the four pairs showed higher fibronectin expression by the keloid cells at the levels of messenger RNA and protein synthesis. This was consistent with previous studies showing elevated steady state content of fibronectin in keloid cells relative to normal cells from the same individual. Fibronectin messenger RNA and protein content in the tissues from which these cells were derived was examined by in situ hybridization and immunohistochemistry. These studies revealed that in vivo, the steady state content of fibronectin messenger RNA and protein was highest in abnormal wounds, less in most normal scars, and lowest in normal skin. Thus, fibroblasts from keloids and hypertrophic scars overexpressed fibronectin in vivo relative to normal skin and normal scar and retain this characteristic in vitro relative to normal skin. Although normal scars contained little fibronectin protein and messenger RNA, cultured fibroblasts derived from these scars had contents of fibronectin messenger RNA and rates of biosynthesis in vitro similar to those of keloid fibroblasts. This indicates that the fibronectin regulatory pathway in scar fibroblasts is influenced by the tissue environment. These results are discussed with respect to the relationship of fibronectin expression in keloids, hypertrophic scars, and normal wounds in human beings.

Effects of nicotinamide supplementation on human pancreatic islet function in tissue culture.

Nicotinamide currently attracts considerable interest as a compound that might prevent the development of human insulin-dependent diabetes mellitus. The present study investigated the direct actions of nicotinamide on human pancreatic beta-cells. For this purpose, islets were isolated from 11 adult cadaveric donors. The human islets were subsequently precultured in RPMI-1640 (5.6 mmol/L glucose) and 10% fetal calf serum for 3-4 days. The islets were cultured for another 6 days in the same medium in either the presence or absence of 10 mmol/L nicotinamide, and subsequently, islet function was examined. After culture, both groups of islets contained similar amounts of DNA, and DNA synthesis, determined by the tritiated thymidine incorporation rate, was unchanged. The insulin content both on a per islet basis and per DNA was similar in both groups as was the islet glucose oxidation rate. Insulin accumulation into the culture medium was sustained over the entire culture period and did not differ at any time point between the nicotinamide and the control group. Nicotinamide affected neither basal and glucose-stimulated insulin secretion nor (pro)insulin and total protein biosynthesis rates after culture. The insulin response to glucose of the human islets was about 5-fold. In conclusion, the present study shows that nicotinamide does not directly affect human beta-cell function or the cell replicatory rate. This would suggest that any potential beneficial effects observed after treatment with nicotinamide in patients with insulin-dependent diabetes mellitus may not necessarily reflect an action at the beta-cell level.

Interleukin-10 Stimulates Rat Pancreatic Islets in Vitro, but Fails to Protect against Interleukin-1

Rat pancreatic islets were cultured for 42 h in medium supplemented without or with 0.1 - 10 ng/ml of human interleukin-10 (IL-10). Medium insulin accumulation was increased by 50% after culture with 0.1 ng/ml IL-10. All concentrations of IL-10 increased the islet insulin content, but did not affect the islet DNA content. IL-10 did neither affect basal and glucose-stimulated insulin secretion nor (pro)insulin and total protein biosynthesis rates. Addition of 25 U/ml of interleukin-1β during the last 24 h of culture, led to a marked inhibition of glucose-stimulated insulin release and a 5-6 fold increase in nitrite production by rat insulinoma cells. These events could not be counteracted by any concentration of IL-10. The present data suggest that IL-10 might in some aspects stimulate pancreatic β-cell function, but it fails to block the inhbitory action of IL-1β.

Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function.

The aim of the present study was to clarify whether prolonged in vitro exposure of human pancreatic islets to high glucose concentrations impairs the function of these cells. For this purpose, islets isolated from adult cadaveric organ donors were cultured for seven days in RPMI 1640 medium supplemented with 10% fetal calf serum and containing either 5.6, 11, or 28 mM glucose. There was no glucose-induced decrease in islet DNA content or signs of morphological damage. However, islets cultured at 11 or 28 mM glucose showed a 45 or 60% decrease in insulin content, as compared to islets cultured at 5.6 mM glucose. Moreover, when such islets were submitted to a 60-min stimulation with a low (1.7 mM) followed by a high (16.7 mM) concentration of glucose, the islets cultured at 5.6 mM glucose showed a higher insulin response to glucose than those of the two other groups. Islets cultured at the two higher glucose concentrations showed increased rates of insulin release in the presence of low glucose, and a failure to enhance further the release in response to an elevated glucose level. Islets cultured at 28 mM glucose showed an absolute decrease in insulin release after stimulation with 16.7 mM glucose, as compared to islets cultured at 5.6 mM glucose. The rates of glucose oxidation, proinsulin biosynthesis, and total protein biosynthesis were similar in islets cultured at 5.6 or 11 mM glucose, but they were decreased in islets cultured at 28 mM glucose. These combined results suggest that lasting exposure to high glucose concentrations impairs the function of human pancreatic islets.

Morphological Characterization of Immortalized Hypothalamic Neurons Synthesizing Luteinizing Hormone-Releasing Hormone

An immortalized LHRH cell line has recently been developed by genetically targeting these neurons for tumorigenesis. One of the subclones, the GT1-7 cells, was characterized at both the light and electron microscopic levels to study the cellular and subcellular organization of these cells, particularly as they relate to biosynthesis, processing, and secretion. The cells were fixed onto slides 18-36 h after plating. LHRH and GnRH-associated peptide (GAP) immunoreactivities (IR) were detected by immunocytochemistry using colloidal gold labeling. These cultured cells exhibited the classical neuronal appearance of LHRH neurons, and they established numerous interconnections. Neighboring neurons were coupled by tight junctions, while more distant cells were interconnected with neural axon-like processes and collaterals. This cellular organization is suggestive of a neural network where neuronal activity is coordinated. At the ultrastructural level, the nondividing cells possessed indented nuclei, well developed Golgi complexes, and abundant numbers of ribosomes and secretory granules. Clathrin-coated vesicles were found in fusion with the plasma membrane. The ribosomes and secretory vesicles were particularly prominent, suggestive of high rates of protein biosynthesis and secretion. All of the cells immunostained for both LHRH and GAP; however, GAP IR was always more pronounced than that for LHRH. This finding was corroborated by biochemical data reported in a companion paper. The GAP IR was associated with ribosomes and secretory vesicles. By comparison, LHRH IR was restricted mainly to the secretory vesicles. Using colloidal gold particles of different sizes to denote LHRH or GAP IR, it was determined that both GAP and LHRH IR were colocalized within the same secretory vesicle. Taken together, these data suggest that pro-LHRH is biosynthesized on the ribosomes, packaged as an intact protein into the secretory vesicles, processed to LHRH and GAP-(1-56) within these vesicles, and transported to the periphery of the cell in preparation for secretion. These morphological data emphasize the utility of using these immortalized LHRH neuronal cells to dissect the cellular and subcellular architecture involved in biosynthesis, processing, and secretion. In addition, our results provide the first detailed evidence for the intracellular pathway involved in pro-LHRH biosynthesis, processing, and secretion in these cultured neuronal cells.

Regulation of protein synthesis at the elongation stage New insights into the control of gene expression in eukaryotes

There are many reports which demonstrate that the rate of protein biosynthesis at the elongation stage is actively regulated in eukaryotic cells. Possible physiological roles for this type of regulation are: the coordination of translation of mRNA with different initiation rate constants; regulation of transition between different physiological states of a cell, such as transition between stages or the cell cycle; and in general, any situation where the maintenance of a particular physiological state is dependent on continuous protein synthesis. A number of covalent modifications of elongation factors offer potential mechanisms for such regulation. Among the various modifications of elongation factors, phosphorylation of eEF-2 by the specific Ca 2+/calmodulin-dependent eEF-2 kinase is the best studied and perhaps the most important mechanism for regulation of elongation rate. Since this phosphorylation is strictly Ca 2+-dependent, and makes eEF-2 inactive in translation, this mechanism could explain how changes in the intracellular free Ca 2+ concentration may regulate elongation rate. We also discuss some recent findings concerning elongation factors, such as the discovery of developmental stage-specific elongation factors and the regulated binding of eEF-1α to cytoskeletal elements. Together, these observations underline the importance of the elongation stage of translation in the regulation of the cellular processes essential for normal cell life.

Effects of a diabetogenic strain of encephalomyocarditis (EMC) virus on protein synthesis in mouse islets of Langerhans

The effects of a diabetogenic strain of encephalomyocarditis (EMC) virus on total protein and insulin biosynthesis in mouse islets of Langerhans have been studied in tissue culture. In dispersed mouse islets, the rates of protein biosynthesis were assessed by measuring the incorporation of [3H]leucine into proteins. In infected dispersed islets incubated in 20 mM-glucose, both insulin and total protein biosynthesis were decreased at 6 h; only insulin biosynthesis was significantly decreased at 3 h. In whole islets, EMC virus brought about a decrease in glucose-stimulated protein and insulin biosynthesis as early as 2 h after infection without concomitant effects on insulin release. This inhibition of protein biosynthesis was still apparent at 20 h post-infection, at which time insulin release was found to be markedly elevated, and the islet insulin content was moderately decreased. At 44 h post-infection, glucose-induced insulin biosynthesis was preferentially inhibited. Infected islets at this later time point also displayed elevated levels of insulin release, and a marked loss of islet insulin content. When insulin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were assessed by dot-blot hybridization using appropriate cDNA probes, levels of insulin mRNA were shown to decrease steadily during the first 20 h of infection, in contrast with the levels of GAPDH mRNA. At 44 h post-infection, both types of mRNA were markedly decreased. It is suggested that there is an initial early 'shut-off' of protein synthesis without other detectable changes in islet function. This is followed by a phase where both insulin mRNA levels and insulin synthesis are dramatically decreased.

Role of aminoacyl-tRNA-synthetases in changes in rate of protein biosynthesis in rabbit liver during myocardial ischemia

Composition of high-molecular-weight aminoacyl-tRNA synthetases complexes from rabbit liver both in norm and after 12 h experimental myocardial ischemia (EMI) has been investigated. Partial redistribution of aminoacyl-tRNA synthetases activity from 1820 kD complex into 840 kD complex was observed in case of EMI which resulted in changes of protein biosynthesis rate in cell-free system.

Interleukin-6 Affects Insulin Secretion and Glucose Metabolism of Rat Pancreatic Isletsin 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)

Tissue Culture of Human Fetal Pancreas; Effects of Nicotinamide on Insulin Production and Formation of Isletlike Cell Clusters

Human fetal pancreas (HFP) is a potential source of beta-cells for transplantation to insulin-dependent diabetic patients. We have previously described a method for tissue culture of HFP that results in the in vitro development of isletlike cell clusters (ICCs) containing a minority of insulin-positive cells. Recently we found that nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, induces an increased islet cell DNA replication both in vivo and in vitro. In this study, this culture technique was used to evaluate the effects of addition of 10 mM nicotinamide on HFP explants cultured in RPMI-1640 medium plus 10% human serum. ICCs developed in 11 of 19 consecutive cultures with nicotinamide increased the yield of ICCs by 40%. Also, the insulin content of ICCs increased approximately 50% with nicotinamide supplementation, although measurements of DNA indicated an unchanged number of cells in each ICC. Neither the rates of insulin release in response to 16.7 mM glucose plus 5 mM theophylline nor the (pro)insulin or total protein biosynthesis rates were affected by nicotinamide addition. The combined results of this study suggest that nicotinamide is useful for stimulating the formation of ICCs from HFP.