A novel mammalian glucokinase exhibiting exclusive inorganic polyphosphate dependence in the cell nucleus.

BACKGROUND: Hexokinase and glucokinase enzymes are ubiquitously expressed and use ATP and ADP as substrates in mammalian systems and a variety of polyphosphate substrates and/or ATP in some eukaryotic and microbial systems. Polyphosphate synthesising or utilizing enzymes are widely expressed in microbial systems but have not been reported in mammalian systems, despite the presence of polyphosphate in mammalian cells. Only two micro-organisms have previously been shown to express an enzyme that uses polyphosphate exclusively. METHODS: A variety of experimental approaches, including NMR and NAD-linked assay systems were used to conduct a biochemical investigation of polyphosphate dependent glucokinase activity in mammalian tissues. RESULTS: A novel mammalian glucokinase, highly responsive to hexametaphosphate (HMP) but not ATP or ADP as a phosphoryl donor is present in the nuclei of mammalian hepatocytes. The liver enzyme exhibited sigmoidal kinetics with respect to glucose with a S0.5 of 12 mM, similar to the known kinetics of mammalian ATP-glucokinase. The Km for HMP (0.5 mM) was also similar to that of phosphoryl donors for mammalian ATP-glucokinases. The new enzyme was inhibited by several nucleotide phosphates. CONCLUSIONS: We report the discovery of a polyphosphate-dependent enzyme system in mammalian cells with kinetics similar to established ATP-dependent glucokinase, also known to have a nuclear location. The kinetics suggest possible regulatory or redox protective roles. GENERAL SIGNIFICANCE: The role of polyphosphate in mammalian systems has remained an enigma for decades, and the present report describes progress on the significance of this compound in intracellular metabolism in mammals.

The effect of maternal undernutrition on the rat placental transcriptome: protein restriction up-regulates cholesterol transport.

BACKGROUND: Fetal exposure to a maternal low protein diet during rat pregnancy is associated with hypertension, renal dysfunction and metabolic disturbance in adult life. These effects are present when dietary manipulations target only the first half of pregnancy. It was hypothesised that early gestation protein restriction would impact upon placental gene expression and that this may give clues to the mechanism which links maternal diet to later consequences. METHODS: Pregnant rats were fed control or a low protein diet from conception to day 13 gestation. Placentas were collected and RNA sequencing performed using the Illumina platform. RESULTS: Protein restriction down-regulated 67 genes and up-regulated 24 genes in the placenta. Ingenuity pathway analysis showed significant enrichment in pathways related to cholesterol and lipoprotein transport and metabolism, including atherosclerosis signalling, clathrin-mediated endocytosis, LXR/RXR and FXR/RXR activation. Genes at the centre of these processes included the apolipoproteins ApoB, ApoA2 and ApoC2, microsomal triglyceride transfer protein (Mttp), the clathrin-endocytosis receptor cubilin, the transcription factor retinol binding protein 4 (Rbp4) and transerythrin (Ttr; a retinol and thyroid hormone transporter). Real-time PCR measurements largely confirmed the findings of RNASeq and indicated that the impact of protein restriction was often striking (cubilin up-regulated 32-fold, apoC2 up-regulated 17.6-fold). The findings show that gene expression in specific pathways is modulated by maternal protein restriction in the day-13 rat placenta. CONCLUSIONS: Changes in cholesterol transport may contribute to altered tissue development in the fetus and hence programme risk of disease in later life.

Processes underlying the nutritional programming of embryonic development by iron deficiency in the rat.

Poor iron status is a global health issue, affecting two thirds of the world population to some degree. It is a particular problem among pregnant women, in both developed and developing countries. Feeding pregnant rats a diet deficient in iron is associated with both hypertension and reduced nephron endowment in adult male offspring. However, the mechanistic pathway leading from iron deficiency to fetal kidney development remains elusive. This study aimed to establish the underlying processes associated with iron deficiency by assessing gene and protein expression changes in the rat embryo, focussing on the responses occurring at the time of the nutritional insult. Analysis of microarray data showed that iron deficiency in utero resulted in the significant up-regulation of 979 genes and down-regulation of 1545 genes in male rat embryos (d13). Affected processes associated with these genes included the initiation of mitosis, BAD-mediated apoptosis, the assembly of RNA polymerase II preinitiation complexes and WNT signalling. Proteomic analyses highlighted 7 proteins demonstrating significant up-regulation with iron deficiency and the down-regulation of 11 proteins. The main functions of these key proteins included cell proliferation, protein transport and folding, cytoskeletal remodelling and the proteasome complex. In line with our recent work, which identified the perturbation of the proteasome complex as a generalised response to in utero malnutrition, we propose that iron deficiency alone leads to a more specific failure in correct protein folding and transport. Such an imbalance in this delicate quality-control system can lead to cellular dysfunction and apoptosis. Therefore these findings offer an insight into the underlying mechanisms associated with the development of the embryo during conditions of poor iron status, and its health in adult life.

Cell cycle regulation and cytoskeletal remodelling are critical processes in the nutritional programming of embryonic development.

Many mechanisms purport to explain how nutritional signals during early development are manifested as disease in the adult offspring. While these describe processes leading from nutritional insult to development of the actual pathology, the initial underlying cause of the programming effect remains elusive. To establish the primary drivers of programming, this study aimed to capture embryonic gene and protein changes in the whole embryo at the time of nutritional insult rather than downstream phenotypic effects. By using a cross-over design of two well established models of maternal protein and iron restriction we aimed to identify putative common "gatekeepers" which may drive nutritional programming.Both protein and iron deficiency in utero reduced the nephron complement in adult male Wistar and Rowett Hooded Lister rats (P<0.05). This occurred in the absence of damage to the glomerular ultrastructure. Microarray, proteomic and pathway analyses identified diet-specific and strain-specific gatekeeper genes, proteins and processes which shared a common association with the regulation of the cell cycle, especially the G1/S and G2/M checkpoints, and cytoskeletal remodelling. A cell cycle-specific PCR array confirmed the down-regulation of cyclins with protein restriction and the up-regulation of apoptotic genes with iron deficiency.The timing and experimental design of this study have been carefully controlled to isolate the common molecular mechanisms which may initiate the sequelae of events involved in nutritional programming of embryonic development. We propose that despite differences in the individual genes and proteins affected in each strain and with each diet, the general response to nutrient deficiency in utero is perturbation of the cell cycle, at the level of interaction with the cytoskeleton and the mitotic checkpoints, thereby diminishing control over the integrity of DNA which is allowed to replicate. These findings offer novel insight into the primary causes and mechanisms leading to the pathologies which have been identified by previous programming studies.

Prenatal protein restriction leads to a disparity between aortic and peripheral blood pressure in Wistar male offspring.

A host of animal studies have been used to model the effects of exposure to a low protein diet in utero on adult blood pressure. Collection of systolic blood pressure data by the indirect tail-cuff plethysmography method consistently shows increased pressures in low protein exposed rodent offspring compared to controls, but this technique has been criticised as the associated stress artefacts may confound the observed effects. Conversely, radiotelemetry systems allow unrestrained and continuous monitoring of blood pressure through the awake and sleep phases of the diurnal cycle. In this novel study, we directly compared blood pressure parameters in male offspring from low protein and control-fed dams measured simultaneously using tail-cuff and radiotelemetry systems. Control rats showed a good correlation between tail-cuff and radiotelemetry derived blood pressure data. Conversely, low protein males were relatively hypertensive at 8 weeks of age when measured by tail-cuff, but had significantly lower blood pressure than controls at 12 weeks of age when measured by telemetry. Heart rate and length of systole did not differ between the two groups. Individual stress protocols mimicking those imposed by tail-cuff plethysmography (novel environment, heat, restraint, inflation), caused similar increases in blood pressure and heart rate in control and low protein animals, ruling out an effect of enhanced pressor response to stress following prenatal protein restriction. Instead, an increase in peripheral vascular resistance in these animals is considered possible. Such a disparity between central and peripheral blood pressure measurements could have important clinical implications regarding cardiovascular risk assessment and treatment.