Inosine-5'-monophosphate dehydrogenase is required for mitogenic competence of transformed pancreatic beta cells.

The relation of inosine-5'-monophosphate dehydrogenase (IMPDH; the rate-limiting enzyme in GTP synthesis) to mitogenesis was studied by enzymatic assay, immunoblots, and RT-PCR in several dissimilar transformed pancreatic ss-cell lines, using intact cells. Both of the two isoforms of IMPDH (constitutive type 1 and inducible type 2) were identified using RT-PCR in transformed beta cells or in intact islets. IMPDH 2 messenger RNA (mRNA) and IMPDH protein were both regulated reciprocally by changes in levels of their end-products. Flux through IMPDH was greatest in rapidly growing cells, due mostly to increased uptake of precursor. Glucose (but not 3-0-methylglucose, L-glucose, or fructose) further augmented substrate uptake and also increased IMPDH enzymatic activity after either 4 or 21 h of stimulation. Serum or ketoisocaproate also increased IMPDH activity (but not uptake). Two selective IMPDH inhibitors (mycophenolic acid and mizoribine) reduced IMPDH activity in all cell lines, and, with virtually identical concentration-response curves, inhibited DNA synthesis (assessed as bromodeoxyuridine incorporation) in response to glucose, serum, or ketoisocaproate. Inhibition of DNA synthesis was reversible, completely prevented by repletion of cellular guanine (but not adenine) nucleotides, and could not be attributed to toxic effects. Despite the fact that modulation of IMPDH expression by guanine nucleotides was readily detectable, glucose and/or serum failed to alter IMPDH mRNA or protein, indicating that their effects on IMPDH activity were largely at the enzyme level. Precursors of guanine nucleotides failed, by themselves, to induce mitogenesis. Thus, adequate IMPDH activity (and thereby, availability of GTP) is a critical requirement for beta-cell proliferation. Although it is unlikely that further increases in GTP can, by themselves, initiate DNA synthesis, such increments may be needed to sustain mitogenesis.

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.

Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants.

Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes adverse alterations in beta cell function, a phenomenon termed glucose toxicity and one that may play a secondary pathogenic role in type 2 diabetes. However, no mechanism of action has been definitively identified for glucose toxicity in beta cells. To ascertain whether chronic oxidative stress might play a role, we chronically cultured the beta cell line, HIT-T15, in medium containing 11.1 mM glucose with and without the antioxidants, N-acetyl-L-cysteine (NAC) or aminoguanidine (AG). Addition of NAC or AG to the culture medium at least partially prevented decreases in insulin mRNA, insulin gene promoter activity, DNA binding of two important insulin promoter transcription factors (PDX-1/STF-1 and RIPE-3b1 activator), insulin content, and glucose-induced insulin secretion. These findings suggested that one mechanism of glucose toxicity in the beta cell may be chronic exposure to reactive oxygen species, i.e., chronic oxidative stress. To ascertain the effects of these drugs on diabetes, NAC or AG was given to Zucker diabetic fatty rats, a laboratory model of type 2 diabetes, from 6 through 12 weeks of age. Both drugs prevented a rise in blood oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde + 4-hydroxy-2-nonenal), and partially prevented hyperglycemia, glucose intolerance, defective insulin secretion as well as decrements in beta cell insulin content, insulin gene expression, and PDX-1 (STF-1) binding to the insulin gene promoter. We conclude that chronic oxidative stress may play a role in glucose toxicity, which in turn may worsen the severity of type 2 diabetes.

A wound-induced [Ca2+]i increase and its transcriptional activation of immediate early genes is important in the regulation of motility.

Upon mechanical wounding of a confluent quiescent monolayer, cells move into the denuded zone. However, it is not well known what signaling cascade connects release from contact inhibition to cell movement at the wound edge. Mechanical wounding induced an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) in endothelial cells at the wound edge. The [Ca2+]i signal was required for the transcriptional activation of two immediate early genes (IEGs), c-fos and c-jun, since blocking Ca2+ influx with Gd3+ or EGTA reduced IEG transcription, while augmenting Ca2+ influx increased IEG transcription. The transcriptional activation of the IEGs depended on protein kinase C and calmodulin-dependent protein kinase since treatment with the inhibitors Calphostin C and KN-62 significantly reduced IEG expression. Briefly blocking Ca2+ influx also produced a long-term reduction of cell motility, while augmenting Ca2+ influx increased cell motility. To evaluate whether expression of IEGs might control cell movement, we microinjected sense or antisense cDNA to c-fos into cells after wounding. Antisense c-fos cDNA inhibited motility, while sense cDNA increased motility rates. These results suggested that the [Ca2+]i rise, induced by wounding, regulated the initiation of subsequent motility through the transcriptional activation of IEGs during wounding.

Co-ordination between localized wound-induced Ca2+ signals and pre-wound serum signals is required for proliferation after mechanical injury.

The signals which initiate proliferation of endothelial cells after injury are important for selective blood vessel growth during wound healing or tumour growth. Upon mechanically wounding quiescent cells, a transient [Ca2+]i increase was induced in cells at the wound edge. These same cells proliferated 18-24 h post wounding, as measured by bromodeoxyuridine incorporation. The localized Ca2+ signal was required specifically during wounding since blocking Ca2+ influx reduced proliferation by 40-50%. Proliferation also required serum since starvation reduced proliferation by 80%. Serum-starved cells proliferated if briefly primed with serum prior to wounding. The signals derived from serum and [Ca2+]i combined at least additively to induce proliferation. Therefore, serum priming followed by a single, transient Ca2+ signal induced by mechanical injury must occur in a temporally and spatially regulated manner for normal proliferation. Co-ordination between signalling cascades induced by growth factors and release from contact inhibition might be obligatory for localized re-endothelialization after injury.

How do injured cells communicate with the surviving cell monolayer?

Mechanically scratching cell monolayers relieves contact inhibition and induces surviving cells near the wound edge to move and proliferate. The present work was designed to test whether surviving cells passively respond to newly available space, or whether cells are actively stimulated by signals from injured cells nearby. We monitored intracellular free Ca2+ ([Ca2+]i) while scratching confluent monolayers of bovine pulmonary endothelial cells and mouse mammary epithelial cells. Within seconds after wounding, a transient elevation of [Ca2+]i was observed in surviving cells. In endothelial cells, the [Ca2+]i elevation propagated into the monolayer for a distance of 10 to 12 cell rows at a speed of 20 to 28 microm/second. The amplitude of the wave of [Ca2+]i was reduced as it propagated into the monolayer, but the velocity of the wave was nearly constant. Cells that experienced the [Ca2+]i elevation had intact plasma membranes, and survived for over 24 hours post wounding. Removing extracellular Ca2+ decreased the amplitude by two-thirds and reduced the propagation rate by half, suggesting that Ca2+ influx contributed to the increased [Ca2+]i. To determine how [Ca2+]i waves were stimulated, we blocked extracellular communication by fluid perfusion or intercellular communication by breaks in the monolayer. In bovine pulmonary artery endothelial cultures, the [Ca2+]i wave passed over breaks in the monolayer, and was prevented from traveling upstream in a perfusion chamber. Conditioned media from injured cells also elevated [Ca2+]i in unwounded reporter cultures. In mouse mammary epithelial monolayers with established cell-cell contacts, the [Ca2+]i wave passed over breaks in the monolayer, but was only partially prevented from traveling upstream during perfusion. These experiments showed that mechanical wounds lead to long distance, [Ca2+]i-dependent communication between the injured cells and the surviving cell monolayer through at least two mechanisms: first, extracellular release of a chemical stimulus from wounded cells that diffused to neighboring cells (present in both monolayers); second, transmission of an intercellular signal through cell-cell junctions (present in the mammary epithelial monolayers). Thus, mechanical injury provided a direct, chemical stimulus to nearby cells which have not themselves been damaged.

The presence of M protein in nontypeable group A streptococcal upper respiratory tract isolates from Southeast Asia.

Previous studies have suggested that group A streptococcal strains from southeast Asia, serotypically different from temporally related North American and European isolates, may represent unrecognized M serotypes. Sixty non-M typeable group A streptococcal upper respiratory tract isolates from Thailand were evaluated for the presence of M protein using a modification of the direct serum bactericidal test. Of them, 59 (98%) grew rapidly in human blood. Typeability by T agglutination and opacity factor production did not influence their growth in blood. It was concluded that these isolates produce M protein and likely represent previously uncharacterized M serotypes. Identification of such non-M typeable strains is important in understanding the epidemiology and pathogenesis of group A streptococcal infections and their sequelae in areas of the world where they remain a significant health problem and will also be necessary in the development of a vaccine with global efficacy.