Total Protein Biosynthesis Research Articles

DNAJB12 and Hsp70 Mediate Triage of Misfolded Membrane Proteins for Proteasomal versus Lysosomal Degradation.

The endoplasmic reticulum (ER) fills the cell with a continuous network of sealed membrane tubules and sheets. The ER is subdivided into microdomains mediating one-third of total protein biosynthesis, oxidative protein folding, secretion, protein quality control, calcium signaling, marcoautophagy/autophagy, stress sensing, and apoptosis. Defects in ER-calcium homeostasis underlie several diseases. Damage to the ER by misfolded membrane proteins is suppressed by specific HSPA/Hsp70 and DNAJ/Hsp40 chaperone pairs that select intermediates for ubiquitination and ER-associated degradation (ERAD) via the proteasome. The ER-transmembrane Hsp40 chaperone DNAJB12 and HSPA/Hsp70 also target toxic intermediates of misfolded membrane proteins for ER-associated autophagy (ERAA). DNAJB12-HSPA/Hsp70 maintain membrane protein degradation intermediates in detergent-soluble and degradation-competent states. DNAJB12-HSPA/Hsp70 also interact with the autophagy initiation kinase ULK1 on ER tubules containing ERAD-resistant misfolded membrane proteins (ERAD-RMPs). Omegasomes are ER microdomains where the autophagosome precursor or phagophore (PG) forms. ER tubules loaded with ERAD-RMPs enter omegasomes where they are converted into ER-connected PG (ER-PG). The Atg8 (autophagy related 8)-family member GABARAP (GABA type A receptor-associated protein) facilitates transfer of ERAD-RMPs from ER-PGs to autolysosomes (AL) that dock transiently with omegasomes. This article describes a model for DNAJB12-HSPA/Hsp70 action during the conformation-dependent triage in the ER of misfolded membrane proteins for folding versus proteasomal or AL degradation.

Overexpression of Prolidase Induces Autophagic Death in MCF-7 Breast Cancer Cells.

Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death.

The plant alkaloid conophylline inhibits matrix formation of fibroblasts

Conophylline is a Vinca alkaloid from leaves of the tropical plant Ervatamia microphylla and has been shown to mimic the effect of the growth and differentiation factor activin A on pancreatic progenitor cells. However, activin A stimulates fibrosis of pancreatic stellate cells, whereas conophylline inhibits it, suggesting that this compound may serve as an antifibrotic drug. Here we investigated the effects of conophylline on human foreskin fibroblasts, especially focusing on extracellular matrix (ECM) proteins. A gene microarray analysis revealed that conophylline remarkably suppressed expression of the gene for hyaluronan synthase 2 (HAS2) and of its antisense RNA, whereas the expression of collagen genes was unaffected. Of note, immunostaining experiments revealed that conophylline substantially inhibits incorporation of versican and collagens into the ECM in cells treated with transforming growth factor β (TGFβ), which promotes collagen synthesis, but not in cells not treated with TGFβ. Moreover, a protein biosynthesis assay disclosed that conophylline decreases collagen biosynthesis, concomitant with a decrease in total protein biosynthesis, indicating that conophylline-mediated inhibition of fibrosis is not specific to collagen synthesis. Conophylline affected neither TGFβ-induced nuclear translocation of SMAD family member 2/3 (SMAD2/3) nor phosphorylation of SMAD2. However, conophylline substantially inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that conophylline inhibits HAS2 expression via TGFβ-mediated activation of the ERK1/2 pathway. Taken together, our results indicate that conophylline may be a useful inhibitor of ECM formation in fibrosis.

The protein synthesis inhibitor brusatol normalizes high‐fat diet‐induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination

The naturally occurring quassinoid compound brusatol improves the survival of insulin-producing cells when exposed to the proinflammatory cytokines IL-1β and IFN-γ in vitro. The aim of the present study was to investigate whether brusatol also promotes beneficial effects in mice fed a high-fat diet (HFD), and if so, to study the mechanisms by which brusatol acts. In vivo, we observed that the impaired glucose tolerance of HFD-fed male C57BL/6 mice was counteracted by a 2 wk treatment with brusatol. Brusatol treatment improved both β-cell function and peripheral insulin sensitivity of HFD-fed mice. In vitro, brusatol inhibited β-cell total protein and proinsulin biosynthesis, with an ED50 of ∼40 nM. In line with this, brusatol blocked cytokine-induced iNOS protein expression via inhibition of iNOS mRNA translation. Brusatol may have affected protein synthesis, at least in part, via inhibition of eukaryotic initiation factor 5A (eIF5A) hypusination, as eIF5A spermidine association and hypusination in RIN-5AH cells was reduced in a dose- and time-dependent manner. The eIF5A hypusination inhibitor GC7 promoted a similar effect. Both brusatol and GC7 protected rat RIN-5AH cells against cytokine-induced cell death. Brusatol reduced eIF5A hypusination and cytokine-induced cell death in EndoC-βH1 cells as well. Finally, hypusinated eIF5A was reduced in vivo by brusatol in islet endocrine and endothelial islet cells of mice fed an HFD. The results of the present study suggest that brusatol improves glucose intolerance in mice fed an HFD, possibly by inhibiting protein biosynthesis and eIF5A hypusination.-Turpaev, K., Krizhanovskii, C., Wang, X., Sargsyan, E., Bergsten, P., Welsh, N. The protein synthesis inhibitor brusatol normalizes high-fat diet-induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination.

Omacetaxine: A Protein Translation Inhibitor for Treatment of Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is driven by the Bcr-Abl fusion protein, which is a result of a (9;22) chromosomal translocation. Imatinib, dasatinib, and nilotinib (tyrosine kinase inhibitors, TKI) have revolutionized how CML is treated. Although the majority of patients respond to these kinase inhibitors, a subset becomes resistant to these therapeutics. Synribo (omacetaxine mepesuccinate) was recently approved by the U.S. Food and Drug Administration for Philadelphia-positive CML either in the chronic or the accelerated phase whose disease failed two prior TKIs. With omacetaxine 1.25 mg/m(2) twice daily for 14 days during induction and for 7 days during maintenance, a major cytogenetic response occurred in 20% of patients in the chronic phase and major hematologic response in 27% of patients in the accelerated phase. Laboratory investigations unraveled the mechanism of action and effectiveness of this agent. Bcr-Abl protein is intrinsically programmed to turn over with a short half-life that makes it susceptible to protein translation inhibitors. Omacetaxine (homoharringtonine) inhibits total protein biosynthesis by binding to A-site cleft of ribosomes. As a corollary to this action, there is a diminution of short-lived proteins, such as Bcr-Abl, followed by cell death. Approval of this first-in-class protein translation inhibitor opens up new avenues for its use in other diseases as well as mechanism-based combinations.

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

PKCepsilon mediates glucose-regulated insulin production in pancreatic beta-cells

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCε in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCε) or expression of inactive PKCε (PKCε-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCε in the perinuclear region, an area known to be rich in ER–Golgi systems, associated with insulin-containing structures. ß’COP1 (RACK2) is the anchoring protein for PKCε. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCε-KD, and 60% in islets expressing RACK2 binding protein (εV1-2); total protein biosynthesis was not affected. In islets expressing εV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCε plays a specific role in mediating the glucose-signal into insulin production: binding to ß’COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.

An inhibitor of free radical processes decreases the rate of protein synthesis in gunshot wound tissues and decelerates the general adaptation syndrome

The dynamics of total protein and procollagen biosynthesis was studied in the muscle tissue in gunshot wound during the natural healing and after treatment with antioxidant (AO) 2,6-di-tert-butyl-4-methylphenol (BHT). AO treatment considerably decreased the rate of both total protein and procollagen synthesis in the muscle on the third day of healing. BHT notably decreased protein biosynthesis and inhibited corticosteroid production as measured from the electron spin resonance (ESR) signal of reduced adrenodoxin. AO treatment also decreased the rate of protein biosynthesis in bone marrow, thymus, and spleen. The decreased functional activity of the endocrine and immune systems suggests that AO considerably decelerates the systemic body response (general adaptation syndrome) to acute wound. Lipid peroxide radicals and the products of lipid hydroperoxide degradation are proposed as the primary mediators of the body response to stress.

Inhibition of glycosyl-phosphatidylinositol biosynthesis in Plasmodium falciparum by C-2 substituted mannose analogues.

Mannose analogues (2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose) have been used to study glycosylphosphatidylinositol (GPtdIns) biosynthesis and GPtdIns protein anchoring in protozoal and mammalian systems. The effects of these analogues on GPtdIns biosynthesis and GPtdIns-protein anchoring of the human malaria parasite Plasmodium falciparum were evaluated in this study. At lower concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D glucose (0.2 and 0.1 mm, respectively), GPtdIns biosynthesis is inhibited without significant effects on total protein biosynthesis. At higher concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-glucose (1.5 and 0.8 mm, respectively), the incorporation of [3H]glucosamine into glycolipids was inhibited by 90%, and the attachment of GPtdIns anchor to merozoite surface protein-1 (MSP-1) was prevented. However, at these concentrations, both sugar analogues inhibit MSP-1 synthesis and total protein biosynthesis. In contrast to 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose (mannosamine), the formation of new glycolipids was observed only in the presence of tritiated or nonradiolabelled 2-deoxy-D-glucose. Mannosamine inhibits GPtdIns biosynthesis at a concentration of 5 mm, but neither an accumulation of aberrant intermediates nor significant inhibition of total protein biosynthesis was observed in the presence of this analogue. Furthermore, the [3H]mannosamine-labelled glycolipid spectrum resembled the one described for [3H]glucosamine labelling. Total hydrolysis of mannosamine labelled glycolipids showed that half of the tritiated mannosamine incorporated into glycolipids was converted to glucosamine. This high rate of conversion led us to suggest that no actual inhibition from GPtdIns biosynthesis is achieved with the treatment with mannosamine, which is different to what has been observed for mammalian cells and other parasitic protozoa.

Biosynthetic response and mechanical properties of articular cartilage after injurious compression

Traumatic joint injury is known to produce osteoarthritic degeneration of articular cartilage. To study the effects of injurious compression on the degradation and repair of cartilage in vitro, we developed a model that allows strain and strain rate-controlled loading of cartilage explants. The influence of strain rate on both cartilage matrix biosynthesis and mechanical properties was assessed after single injurious compressions. Loading with a strain rate of 0.01 s −1 to a final strain of 50% resulted in no measured effect on the cells or on the extracellular matrix, although peak stresses reached levels of about 12 MPa. However, compression with strain rates of 0.1 and 1 s −1 caused peak stresses of approximately 18 and 24 MPa, respectively, and resulted in significant decreases in both proteoglycan and total protein biosynthesis. The mechanical properties of the explants (compressive and shear stiffness) were also reduced with increasing strain rate. Additionally, cell viability decreased with increasing strain rate, and the remaining viable cells lost their ability to exhibit an increase in biosynthesis in response to low-amplitude dynamic mechanical stimulation. This latter decrease in reparative response was most dramatic in the tissue compressed at the highest strain rates. We conclude that strain rate (like peak stress or strain) is an important parameter in defining mechanical injury, and that cartilage injuriously compressed at high strain rates can lose its characteristic anabolic response to low-amplitude cyclic mechanical loading.

Glucose uptake, utilization, and signaling in GLUT2-null islets.

We previously reported that pancreatic islet beta-cells from GLUT2-null mice lost the first phase but preserved the second phase of glucose-stimulated insulin secretion (GSIS). Furthermore, we showed that the remaining secretory activity required glucose uptake and metabolism because it can be blocked by inhibition of oxidative phosphorylation. Here, we extend these previous studies by analyzing, in GLUT2-null islets, glucose transporter isoforms and glucokinase expression and by measuring glucose usage, GSIS, and glucose-stimulated insulin mRNA biosynthesis. We show that in the absence of GLUT2, no compensatory expression of either GLUT1 or GLUT3 is observed and that glucokinase is expressed at normal levels. Glucose usage by isolated islets was increased between 1 and 6 mmol/l glucose but was not further increased between 6 and 20 mmol/l glucose. Parallel GSIS measurements showed that insulin secretion was not stimulated between 2.8 and 6 mmol/l glucose but was increased by >4-fold between 6 and 20 mmol/l glucose. Stimulation by glucose of total protein and insulin biosynthesis was also markedly impaired in the absence of GLUT2. Finally, we re-expressed GLUT2 in GLUT2-null beta-cells using recombinant lentiviruses and demonstrated a restoration of normal GSIS. Together, these data show that in the absence of GLUT2, glucose can still be taken up by beta-cells, albeit at a low rate, and that this transport activity is unlikely to be attributed to GLUT1 or GLUT3. This uptake activity, however, is limiting for normal glucose utilization and signaling to secretion and translation. These data further demonstrate the key role of GLUT2 in murine beta-cells for glucose signaling to insulin secretion and biosynthesis.

ADP-ribosylation Factor 1 and Its Activation of Phospholipase D Are Important for the Assembly of Very Low Density Lipoproteins

The role of ADP-ribosylation factor 1 (ARF-1) in the assembly of very low density lipoproteins (VLDL) was investigated by expressing dominant-negative mutants in McA-RH7777 cells. Transient expression of ARF-1(T31N), a GDP-restrictive mutant, significantly inhibited apolipoprotein B-100 (apoB-100) VLDL production without influencing the biosynthesis of apoB-100 low density lipoproteins or total apoB production (indicating that it inhibited the second step of VLDL assembly) and without altering total protein production or biosynthesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10-15% of the apoB-100 pool was converted to VLDL in a time- and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to N-terminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5'-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.

Effects of growth hormone in vitro on the glucose metabolism of fetal rat islet beta-cells.

This study was undertaken to investigate the effects of growth hormone (GH) on the in vitro maturation of the metabolism of fetal rat islets. For this purpose fetal islets were obtained from 21-day-old fetuses by mild collagenase digestion of the pancreas and cultured in RPMI 1640 supplemented with 10% fetal calf serum. After one day the medium was changed and supplemented with 1% fetal calf serum with or without GH (1 microgram/ml, human recombinant) and the islets cultured for another two days. Islets were then studied with regard to insulin secretion, (pro)insulin and total protein biosynthesis, glucose utilization and oxidation, thymidine incorporation, insulin and DNA contents and the contents of mRNAs for either insulin, adenine nucleotide translocator or cytochrome b. In addition, the activities of glucose phosphorylating enzymes and succinate-cytochrome c reductase were measured. Islets treated with GH showed increased insulin secretion in response to glucose, increased rates of glucose oxidation and utilization, increased thymidine incorporation and increased activities of succinate cytochrome c reductase and glucose phosphorylation at high glucose concentrations. There were, however, no changes in (pro)insulin and total protein biosynthesis, contents of insulin and DNA or the contents of any of the mRNAs. These combined data show that fetal beta-cells are sensitive to growth hormone with respect to glucose metabolism, insulin release and DNA replication. The increased rates of islet glucose phosphorylation may reflect glucokinase activity and explain part of the increased insulin responsiveness to glucose of the fetal rat beta-cell. These observations suggest that GH is of physiological significance for the maturation of the fetal beta-cell.

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.

Suppression of angiogenesis by the antitumor agent titanocene dichloride

Titanocene dichloride, which is an active antitumor agent against solid but not blood-borne tumors, suppresses angiogenesis and inhibits biosynthesis of collagenous proteins in the in vivo system of the chorioallantoic membrane of the chick embryo. The agent does not affect total protein biosynthesis in the same system. At non-toxic dose regimens titanocene dichloride retards the growth of Walker 256 carcinosarcoma transplants in rats and reduces the number of seeded implants in the mesenteric bed. At concentrations which suppress angiogenesis and inhibit biosynthesis of collagenous proteins, the agent does not affect the viability of Walker 256 carcinosarcoma cells, or the attachment and proliferation of human A549 lung adenocarcinoma or human umbilical vein endothelial cells in culture. It appears that the antitumor activity of titanocene dichloride may be attributed, at least in part, to its ability to suppress angiogenesis.

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.

Nicotinamide and dexamethasone inhibit interleukin-1-induced nitric oxide production by RINm5F cells without decreasing messenger ribonucleic acid expression for nitric oxide synthase.

Nitric oxide (NO) generation may be a final common pathway for beta-cell damage in early insulin-dependent diabetes mellitus. Insulin-producing cells express an inducible form of NO synthase (iNOS), which is similar to that observed in activated macrophages. Induction of iNOS mRNA in these cells depends on protein synthesis. To further characterize the regulation of iNOS induction in insulin-producing cells, RINm5F cells (RIN cells) were exposed for 6 h to human recombinant interleukin-1 beta (rIL-1 beta; 1 ng/ml) alone or in combination with either nicotinamide (10, 20, or 50 mM) or dexamethasone (1 or 5 microM). These agents have been previously shown to prevent activation of iNOS in macrophages, fibroblasts, and hepatocytes. rIL-1 beta induced the expression of iNOS mRNA in RIN cells and a 12- to 13-fold increase in medium nitrite accumulation, the latter indicating NO production. Nicotinamide decreased nitrite production in a dose-dependent way. Thus, 10 mM nicotinamide decreased rIL-1 beta-induced nitrite formation by 30%, 20 mM by 60%, and 50 mM by 90%. The highest concentration of nicotinamide also prevented rIL-1 beta-induced iNOS mRNA, an effect associated with inhibition of total protein biosynthesis. However, 10 or 20 mM nicotinamide did not modify rIL-1 beta-induced iNOS mRNA expression or inhibit protein biosynthesis. Dexamethasone also decreased rIL-1 beta-induced nitrite production without affecting iNOS mRNA expression. As a whole, these data suggest that both nicotinamide and dexamethasone may prevent NO accumulation in insulin-producing cells by posttranscriptional mechanisms. It is also possible that these drugs induce direct inhibition of iNOS enzymatic activity and/or scavenge NO. Higher concentrations of nicotinamide might also inhibit iNOS mRNA expression, possibly by blocking protein biosynthesis.

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.

Functional and metabolic perturbations in isolated pancreatic islets from the GK rat, a genetic model of noninsulin-dependent diabetes.

Spontaneously diabetic nonobese GK rats exhibit high basal plasma glucose and insulin levels and a poor insulin secretory response to glucose. We studied insulin biosynthesis, insulin release, and glucose metabolism in freshly isolated islets from GK rats and control Wistar rats. In GK rats, islet insulin content was decreased when expressed per islet but normal when related to DNA content. In the presence of a low concentration (2.8 mM) of glucose both (pro)insulin and total protein biosynthesis was doubled in islets from GK rats. As judged from the (pro)insulin/total protein synthesis ratio, (pro)insulin biosynthesis was normally stimulated by 16.7 mM glucose. In islets from diabetic rats both basal and glucose-stimulated insulin release were dramatically decreased. A reduced secretory response to 10 mM leucine or 10 mM leucine plus 10 mM glutamine and a lack of response to 10 mM monomethylsuccinate were observed. By contrast the insulinotropic capacity of nonnutrient secretagogues such as 62 microM gliclazide or the combination of 2 mM Ba2+ and 1.4 mM theophylline in the absence of extracellular Ca2+ remained normal. Glucose oxidation (estimated as the production of 14CO2 from D-[6-14C]glucose) was severely impaired, whereas no major alteration of glycolytic flux (as judged from the conversion of D-[5-3H]glucose to 3H2O) could be detected. Accordingly, the D-[6-14C]glucose oxidation/D-[5-3H]glucose use ratio was less markedly increased in response to a rise in glucose concentration in islets from GK rats than in islets from control rats. Thus, in islets from diabetic GK rats, glucose-induced insulin release but not insulin biosynthesis was impaired. This defect is associated with, and probably at least in part due to, a deficient oxidative metabolism of glucose in islet mitochondria.