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The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells. Through its interaction with the 5¢ cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome. The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns. Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates. Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis. Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen.Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure. However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities. eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs. Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs. Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast. It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled.

SummaryIn vertebrates and invertebrates, relationships between diet and health are controlled by a conserved signalling pathway responsive to insulin-like ligands. In invertebrate models for example, forkhead transcription factor family O (FOXO) transcription factors in this pathway regulate the rate of aging in response to dietary cues, and in vertebrates, obesity and age-induced deficits in the same pathway are thought to contribute to dysregulation of hepatic gluconeogenesis through genes such as phosphoenolpyruvate carboxykinase (PEPCK). Recently, we have begun to screen for dietary constituents capable of regulating this pathway in our cell culture model. Here, we identify three black tea theaflavins, theaflavin 3-Ogallate, theaflavin 3′ ′ ′ ′ -O -gallate, theaflavin 3,3′ ′ ′ ′ di-O -gallate and thearubigins as novel mimics of insulin/IGF-1 action on mammalian FOXO1a, PEPCK and moreover we provide evidence that the effects on this pathway of the green tea constituent (-)-epigallocatechin gallate depend on its ability to be converted into these larger structures. With the exception of water, tea is the most popular drink globally, but despite this, little is known about the biological availability of black tea polyphenols in vivo or the molecular target(s) mediating the effects presented here. Further investigation in these two areas might provide insight into how age-related metabolic disease may be deferred.

Metal-binding compounds have recently been reported to have anti-hyperglycaemic properties in vivo. In the current study, we have investigated the ability of these compounds and related structures to induce insulin-like signal transduction to downstream effectors such as the transcription factor FOXO1a and the key gluconeogenic regulatory enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). Our results indicate that β-thujaplicin, diethyldithiocarbamate (DEDTC) and its clinically-used dimer disulfiram, induce insulin-like dose-dependent effects on signalling to FOXO1a in a manner that is strictly dependent on the presence of zinc ions, as other ions including aluminium, cobalt, copper, lithium and manganese cannot substitute. The most potent compound tested on gluconeogenesis is disulfiram, which in the presence of 10 μM zinc, inhibited both PEPCK and G6Pase with an IC50 of 4 μM. Our results demonstrate that metal-binding compounds with diverse structures can induce zinc-dependent insulin-like effects on signal transduction and gene expression.

Anti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK. Salicylate also reduced mTOR signalling, but this property was observed widely among the analogues. In a sub-panel of analogues, salicylate alone reduced promoter activity of the key gluconeogenic enzyme glucose 6-phosphatase and suppressed basal glucose production in mouse primary hepatocytes. Both salicylate and 2,6 dihydroxybenzoic acid suppressed TNFα-induced IκB degradation, and in genetic knockout experiments, we found that the effect of salicylate on IκB degradation was AMPK-independent. Previous data also identified AMPK-independent regulation of glucose but we found that direct inhibition of neither NF-κB nor mTOR signalling suppressed glucose production, suggesting that other factors besides these cell signalling pathways may need to be considered to account for this response to salicylate. We found, for example, that H4IIE cells were exquisitely sensitive to uncoupling with modest doses of salicylate, which occurred on a similar time course to another anti-hyperglycaemic uncoupling agent 2,4-dinitrophenol, while there was no discernible effect at all of two salicylate analogues which are not anti-hyperglycaemic. This finding supports much earlier literature suggesting that salicylates exert anti-hyperglycaemic effects at least in part through uncoupling.

Many diseases of aging including AD (Alzheimer's disease) and T2D (Type 2 diabetes) are strongly associated with common risk factors, suggesting that there may be shared aging mechanisms underlying these diseases, with the scope to identify common cellular targets for therapy. In the present study we have examined the insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as CQ (clioquinol). The IIS [insulin/IGF-1 (insulin-like growth factor-1) signalling] kinase Akt/PKB (protein kinase B) inhibits the transcription factor FOXO1a (forkhead box O1a) by phosphorylating it on residues that trigger its exit from the nucleus. In HEK (human embryonic kidney)-293 cells, we found that CQ treatment induces similar responses. A key transcriptional response to IIS is the inhibition of hepatic gluconeogenic gene expression, and, in rat liver cells, CQ represses expression of the key gluconeogenic regulatory enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). The effects on FOXO1a and gluconeogenic gene expression require the presence of Zn2+ ions, reminiscent of much earlier studies examining diabetogenic properties of 8-hydroxyquinolines. Comparative investigation of the signalling properties of a panel of these compounds demonstrates that CQ alone exhibits FOXO1a regulation without diabetogenicity. Our results suggest that Zn2+-dependent regulation of FOXOs and gluconeogenesis may contribute to the therapeutic properties of this drug. Further investigation of this signalling response might illuminate novel pharmacological strategies for the treatment of age-related diseases.

Out of 1610 children's hearts with congenital malformations there were 27 specimens showing double outlet right ventricle. Cases with dextrocardia, situs inversus, or l-venticular loop were excluded. Anatomical examination was performed with particular reference to the infundibular region, the great vessels, and the ventricular septum. The commonest associated malformations were ventricular septal defect and pulmonary stenosis. Aortic stenosis was the predominant finding in those cases dying in the neonatal period. An aortic conus was associated with pulmonary stenosis, ventricular septal defect, and d-transposition, a pulmonary conus with ventricular septal defect and a double conus with stenosis of either great vessel. The anterior vessel always had a muscular conus and the posterior vessel was commonly stenotic.