Shamus P. Burns

Epigenetic Modification of the Renin-Angiotensin System in the Fetal Programming of Hypertension

Hypertension is a major risk factor for cardiovascular and cerebrovascular disease. Lifelong environmental factors (eg, salt intake, obesity, alcohol) and genetic factors clearly contribute to the development of hypertension, but it has also been established that stress in utero may program the later development of the disease. This phenomenon, known as fetal programming can be modeled in a range of experimental animal models. In maternal low protein diet rat models of programming, administration of angiotensin converting enzyme inhibitors or angiotensin receptor antagonists in early life can prevent development of hypertension, thus implicating the renin-angiotensin system in this process. Here we show that in this model, expression of the AT1b angiotensin receptor gene in the adrenal gland is upregulated by the first week of life resulting in increased receptor protein expression consistent with the increased adrenal angiotensin responsiveness observed by others. Furthermore, we show that the proximal promoter of the AT1b gene in the adrenal is significantly undermethylated, and that in vitro, AT1b gene expression is highly dependent on promoter methylation. These data suggest a link between fetal insults to epigenetic modification of genes and the resultant alteration of gene expression in adult life leading ultimately to the development of hypertension. It seems highly probable that similar influences may be involved in the development of human hypertension.

Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation.

Maternal protein restriction is a model of fetal programming of adult glucose intolerance. Perfused livers of 48-h- starved adult offspring of rat dams fed 8% protein diets during pregnancy and lactation produced more glucose from 6 mM lactate than did control livers from rats whose dams were fed 20% protein. In control livers, a mean of 24% of the glucose formed from lactate in the periportal region of the lobule was taken up by the most distal perivenous cells; this distal perivenous uptake was greatly diminished in maternal low protein (MLP) livers, accounting for a major fraction of the increased glucose output of MLP livers. In control livers, the distal perivenous cells contained 40% of the total glucokinase of the liver; this perivenous concentration of glucokinase was greatly reduced in MLP livers. Intralobular distribution of phosphenolpyruvate carboxykinase was unaltered, though overall increased activity could have contributed to the elevated glucose output. Hepatic lobular volume in MLP livers was twice that in control livers, indicating that MLP livers had half the normal number of lobules. Fetal programming of adult glucose metabolism may operate partly through structural alterations and changes in glucokinase expression in the immediate perivenous region.

Investigation of urea cycle enzyme disorders by 1H-NMR spectroscopy

High resolution proton nuclear magnetic resonance spectroscopy (1H-NMR) has been used to study patients with inborn errors of the urea cycle to evaluate further the diagnostic potential of this technique. The 1H-NMR metabolic profile from the urine of patients with citrullinaemia and argininosuccinic aciduria consistently demonstrated the presence of the diagnostic metabolites citrulline, N-acetylcitrulline and argininosuccinate, respectively. The profile from the urine of patients with ornithine carbamoyl transferase deficiency, is potentially diagnostic, but orotate was only detected in samples from three out of four patients. The characteristic fingerprint that each of the metabolites produces is unlike that of any other we have seen, including analogues of the metabolites which are structurally very similar such as arginine, ornithine and aspartate. The level of excretion of the metabolites from the patients with citrullinaemia and argininosuccinic aciduria has been well within the range of NMR detection.

PROTON NMR SPECTROSCOPIC ANALYSIS OF MULTIPLE ACYL-COA DEHYDROGENASE DEFICIENCY CAPACITY OF THE CHOLINE OXIDATION PATHWAY FOR METHYLATION IN VIVO

Proton NMR spectra of urine from subjects with multiple acyl-CoA dehydrogenase deficiency, caused by defects in either the electron transport flavoprotein or electron transport flavoprotein ubiquinone oxidoreductase, provide a characteristic and possibly diagnostic metabolite profile. The detection of dimethylglycine and sarcosine, intermediates in the oxidative degradation of choline, should discriminate between multiple acyl-CoA dehydrogenase deficiency and related disorders involving fatty acid oxidation. The excretion rates of betaine, dimethylglycine (and sarcosine) in these subjects give an estimate of the minimum rates of both choline oxidation and methyl group release from betaine and reveal that the latter is comparable with the calculated total body methyl requirement in the human infant even when choline intake is very low. Our results provide a new insight into the rates of in vivo methylation in early human development.

Propionylcarnitine excretion is not affected by metronidazole administration to patients with disorders of propionate metabolism

Propionylcarnitine (PC) excretion has been measured during a clinical trial of metronidazole therapy in two patients with propionic acidaemia and two patients with methylmalonic aciduria. All patients were in good metabolic control and were receivingl-carnitine. While total propionate excretion was reduced by up to 40% in all four patients during metronidazole therapy, the excretion of propionylcarnitine remained largely unchanged. PC comprised up to 80% of total propionate excretion in patients with propionic acidaemia.

An investigation of argininosuccinic acid anhydrides in argininosuccinic acid lyase deficiency by 1H-NMR spectroscopy.

Argininosuccinate (ASA) is excreted in large amounts in the urine of patients with argininosuccinate lyase (EC 4.3.2.1) deficiency (ASLD). Previous workers have reported the presence of anhydrides of ASA in the urine of patients with this disorder. We have used 1H-NMR spectroscopy to investigate the presence of these compounds in untreated urine. Our results indicate that the anhydrides were absent from urine and are artifacts produced by the conditions prevailing in the methods of analysis previously used to investigate the urine from patients with ASLD. We have also attempted to reproduce the conditions that might promote the transition to the anhydrides in the urine from a patient with ASLD and in solutions of argininosuccinate. Resolution of ASA and the two anhydrides is possible and a spectrum of the urine from a patient with ASLD shows that the anhydrides are present at below 1% of the total ASA under normal physiological conditions.

Lactate supply as a determinant of the distribution of intracellular pH within the hepatic lobule.

When isolated livers from starved rats are perfused with lactate at constant perfusate pH and P(co(2)), there is a marked gradient of cell pH (pH(i)) along the length of the lobular radius, with periportal cells being substantially more alkaline than perivenous cells. In the present studies, the perivenous 21% of the lobular volume was destroyed by retrograde digitonin perfusion, and antegrade perfusion restored. pH(i) was determined by (31)P-NMR. The remaining periportal cells, the site of gluconeogenesis from lactate, had a substantially higher mean pH(i) (7.42) than did the intact liver (7.23). When lactate was removed from the perfusate, mean pH(i) decreased to 7.25. The corresponding concentration of cell bicarbonate fell with a half-time of approximately 5 min. When lactate was re-introduced mean pH(i) rose to 7.34. We conclude that a major contributor to periportal alkalinity under these conditions is proton consumption during gluconeogenesis from lactate ions.

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.

Fetal programming of hepatic lobular architecture in the rat demonstrated ex vivo with magnetic resonance imaging.

We demonstrate that MRI imaging at sub‐millimetre resolution can distinguish between periportal and perivenous zones of the rat liver lobule. This made it possible to measure the hepatic lobular radius in ex‐vivo perfused fixed livers using MRI. Comparisons of histomorphometric and MRI measurements of lobular radius were in good agreement, although MRI measurements were significantly smaller (P < 0.001). Male rats whose mothers were fed 40% of the protein of controls during gestation and lactation, had a significantly larger hepatic lobular radius than that of controls [449 ± 11 µm vs 373 ± 9 µm (mean ± SEM), respectively, p < 0.001, n = 12; histomorphometry data]. The proton T2 in periportal and perivenous zones was mapped both before and after antegrade or retrograde perfusion of 10 ml of digitonin (4 mg ml−1). Only the T2 of the hypointense regions increased significantly following antegrade perfusion of digitonin and conversely only that of the intense regions following retrograde perfusion. Digitonin causes permeabilization of cells in specific hepatic zones, determined by the direction of perfusion. The intense and hypointense regions of the hepatic MR images thus arise from the perivenous and periportal zones of the hepatic lobule, respectively. Copyright