Patrick Brachet

Age-associated B vitamin deficiency as a determinant of chronic diseases

The number of elderly individuals is growing rapidly worldwide and degenerative diseases constitute an increasing problem in terms of both public health and cost. Nutrition plays a role in the ageing process and there has been intensive research during the last decade on B vitamin-related risk factors in vascular and neurological diseases and cancers. Data from epidemiological studies indicate that subclinical deficiency in most water-soluble B vitamins may occur gradually during ageing, possibly due to environmental, metabolic, genetic, nutritional and pathological determinants, as well as to lifestyle, gender and drug consumption. Older adults have distinct absorption, cell transport and metabolism characteristics that may alter B vitamin bioavailability. Case-control and longitudinal studies have shown that, concurrent with an insufficient status of certain B vitamins, hyperhomocysteinaemia and impaired methylation reactions may be some of the mechanisms involved before a degenerative pathology becomes evident. The question that arises is whether B vitamin inadequacies contribute to the development of degenerative diseases or result from ageing and disease. The present paper aims to give an overview of these issues at the epidemiological, clinical and molecular levels and to discuss possible strategies to prevent B vitamin deficiency during ageing.

Alterations in intestinal uptake of putrescine and tissue polyamine concentrations in tumor-bearing rats

Intestinal absorption of putrescine and tissue metabolism of polyamines were investigated in rats grafted with the rapidly growing Mat-Lylu prostatic tumor. These animals exhibited a dramatic 21% decrease in weight and protein, but not DNA, content of their intestinal mucosa, relative to healthy rats reared under similarly controlled nutritional conditions. No significant variation in the specific activities of intestinal brush-border membrane enzymes was observed, however, suggesting a comparable differentiation state of intestinal cells exists in both groups. Putrescine uptake by brush-border membrane vesicles prepared from cancerous or healthy rat intestine was a time dependent process at 25 degrees C. Equilibrium uptake was much greater than could be explained by equilibration of the vesicle space with putrescine, indicating that the diamine was bound to membrane sites. Kinetics of putrescine uptake at 2 min revealed that the process involves two components, a saturable Michaelis-Menten carrier and passive diffusion. With respect to the kinetic parameters of putrescine transport, no significant changes were observed between the tumor-bearing and the control rats. After correction for nonspecific binding to the membranes, putrescine accumulation at equilibrium (75 min) was concentration-dependent and fit a single-site saturable model. Maximum accumulation of the diamine at equilibrium (Bmax) was increased by more than 46% in the cancerous rats relative to the controls, but the dissociation constant (Kd) was unchanged. Efflux of putrescine from the vesicles was slightly slower in the tumor-bearing group, but the differences were generally not significant. No change was observed with respect to the specific activity of ornithine decarboxylase and the concentration of polyamines in the intestinal mucosa. In Mat-Lylu grafted rats fed a standard diet supplemented with [14C]putrescine, about 19% of body radioactivity was recovered in the tumor within 24 h. This was concomitant with a decrease in the percentage of radioactivity retained in the intestinal, renal and hepatic tissues, relative to that retained in the same tissues of healthy rats. Our findings indicate that the presence of the tumor evolves an adaptive response in the small intestine of the rat, involving an increased capacity of the brush-border membrane to accumulate putrescine.

Increased translation efficiency and antizyme-dependent stabilization of ornithine decarboxylase in amino acid-supplemented human colon adenocarcinoma cells, Caco-2.

The mechanisms of the response of ornithine decarboxylase(ODC), the rate-limiting enzyme in polyamine biosynthesis, to amino acid supplementation were studied in the human colon adenocarcinoma cell line, Caco-2. Supplementation of serum-deprived, subconfluent Caco-2 cells with any one of a series of amino acids (10 mM) resultedin increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with iso-osmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effectors and acted in a concentration-dependent manner; millimolar concentrations of most of these amino acids being sufficient to significantly increase ODC activity. In contrast, supplementation with D-methionine, L-lysine, L-aspartate or L-glutamate had little or no effect on ODC activity, whereas supplemental L-methionine, L-arginine, L-ornithine or L-cysteine was inhibitory. Polyamine assays showed that the putrescine content of cells varied in accordance with the changes in ODC activity. Western-blot and Northern-blot analyses revealed specifically increased levels of ODC protein but not mRNA,respectively, in response to supplementation with an ODC-inducing amino acid. Suppression of the increase in cycloheximide-treated cellsconfirmed a requirement for protein synthesis. Pulse-labelling of cellswith [(35)S]methionine showed a 3-fold increase in thesynthesis of ODC protein after 4 h of supplementation with glycineor L-serine. Supplemental glycine also augmented, reversibly, the half-life of ODC by almost 4-fold and simultaneously decreased the activity of putrescine-induced free antizyme. These results suggest that translational, but not transcriptional, regulation of ODC takes part in ODC induction by amino acids in Caco-2 cells. However, it also appears to occur in concert with decreased enzyme in activation and/or degradation.

Myocardium proteome remodelling after nutritional deprivation of methyl donors

Methyl donor (MD: folate, vitamin B12 and choline) deficiency causes hyperhomocysteinemia, a risk factor for cardiovascular diseases. However, the mechanisms of the association between MD deficiency, hyperhomocysteinemia, and cardiomyopathy remain unclear. Therefore, we performed a proteomic analysis of myocardium of pups from rat dams fed a MD-depleted diet to understand the impact of MD deficiency on heart at the protein level. Two-dimension gel electrophoresis and mass spectrometry-based analyses allowed us to identify 39 proteins with significantly altered abundance in MD-deficient myocardium. Ingenuity Pathway Analysis showed that 87% of them fitted to a single protein network associated with developmental disorder, cellular compromise and lipid metabolism. Concurrently increased protein carbonylation, the major oxidative post-translational protein modification, could contribute to the decreased abundance of many myocardial proteins after MD deficiency. To decipher the effect of MD deficiency on the abundance of specific proteins identified in vivo, we developed an in vitro model using the cardiomyoblast cell line H9c2. After a 4-day exposure to a MD-deprived (vs. complete) medium, cells were deficient of folate and vitamin B12, and released abnormal amounts of homocysteine. Western blot analyses of pup myocardium and H9c2 cells yielded similar findings for several proteins. Of specific interest is the result showing increased and decreased abundances of prohibitin and α-crystallin B, respectively, which underlines mitochondrial injury and endoplasmic reticulum stress within MD deficiency. The in vitro findings validate the MD-deficient H9c2 cells as a relevant model for studying mechanisms of the early metabolic changes occurring in cardiac cells after MD deprivation.

Regional differences for the D-amino acid oxidase-catalysed oxidation of D-methionine in chicken small intestine.

1. Enzymic oxidation of D-[1-14C]methionine (D-met) to 2-keto-4-methylthiobutanoate (KMB) has been determined using 100,000 g supernatants prepared from chicken tissue homogenates. 2. The small intestinal mucosa contains substantial oxidative activity towards D-met, which represents about one-half and one-tenth the hepatic and renal activity, respectively. 3. KMB is poorly decarboxylated and rather transaminated to L-met. 4. The specific activity for D-met oxidation in the duodenal mucosa is 1.5- and 4.0-fold than in the jejunal and ileal mucosa, respectively. 5. The intestinal D-met-oxidizing activity is dramatically altered by the D-amino acid oxidase specific inhibitor benzoate.

Selective sites for polyamine binding to rabbit intestinal brush-border membranes

The intestinal polyamine transporters have not yet been identified. Our aim was to characterize specific polyamine binding sites in rabbit intestinal brush-border membranes (IBBM) as a starting step for identification of polyamine transporters. This was investigated at 4 degrees and at low membrane concentration. Saturation isotherms for [3H]putrescine (PUT) binding indicated a single population of sites (puT) with a dissociation equilibrium constant Kd of 3.8 microM and a density of sites Bmax of 58 pmol/mg of protein. [3H]spermidine (SPD) binding also involved only one class of sites (spD), albeit with a lower affinity (Kd = 106 microM) and higher abundance (Bmax = 1240 pmol/mg of protein) than puT. On the contrary, [14C]spermine (SPM) bound two classes of sites (spM1 and spM2) differing in their affinity (Kd = 2.5 and 31.4 microM) and abundance (Bmax = 467 and 1617 pmol/mg of protein, respectively). Membrane association of SPM at 4 degrees was much faster than that of SPD and PUT, both of which proceeded at a similar rate. In contrast to PUT and SPD dissociation, SPM dissociation at 23 degrees did not follow a first-order reaction. Specifically bound [3H]PUT, unlike [3H]SPD and [14C]SPM, dissociated at 23 degrees independently of the addition of nonradioactive polyamine. Methylglyoxal-bis-(guanylhydrazone) was an extremely potent inhibitor of PUT binding (Ki = 3.2 +/- 1.5 nM), but as with PUT and cadaverine (CAD), it did not alter [3H]SPD and [14C]SPM binding substantially. The intestinal brush-border membrane may contain at least three sites specific for polyamine binding and exhibiting different ligand selectivity. Site puT might be associated with the transport system already described for intestinal uptake of PUT.

Decreased plasma folate concentration in young and elderly healthy subjects after a short-term supplementation with isotretinoin

Background  In the last two decades, there has been an increasing use of isotretinoin (13‐cis‐retinoic acid or 13‐CRA) for treatment of severe, and recently mild and moderate, acne in Westernized populations. Recent human and animal studies emphasized alterations caused by 13‐CRA administration on folate‐dependent, one‐carbon metabolism. Folate deficiency and subsequent hyperhomocysteinemia increase the risk of degenerative diseases.

Expression of spermidine/spermine N1-acetyltransferase in HeLa cells is regulated by amino acid sufficiency.

The effect of amino acids on the regulation of the expression of spermidine/spermine N(1)-acetyltransferase (SSAT), the key enzyme of polyamine catabolism, was studied in HeLa cells. When compared with similar exposure to complete medium, deprivation of arginine, methionine or leucine gave rise to a time-dependent, slowly reversible increase in the cellular level of SSAT mRNA that started to be significant after 8, 12 or 16h and reached four-, five- and two-fold after 16h, respectively. Experiments utilizing (i) constructs containing fragments of the SSAT promoter linked to a luciferase reporter gene or (ii) actinomycin D (Act-D)-treated cells indicated that the increase in the SSAT mRNA level was due to an augmentation in gene transcription and message stability after omission of one of the polyamine precursor amino acids. By contrast, SSAT mRNA stabilisation was only observed when leucine was the omitted amino acid. Amino acid deprivation was also found to cause increased intracellular activity of SSAT concurrent with changes in the cell polyamine content, namely increased putrescine but decreased spermine levels. Furthermore, stable expression of a dominant negative mutant of stress-activated protein kinase/extracellular signal-regulated protein kinase (SAPK/ERK) kinase 1 in HeLa cells was found to inhibit the increase in SSAT mRNA by amino acid deprivation. The data suggest that c-Jun N-terminal kinase/SAPK (JNK/SAPK) may be involved in the amino acid-dependent regulation of SSAT expression.

Activation of c-Jun N-terminal kinase 1 (JNK-1) after amino acid deficiency in HeLa cells.

Long-term amino acid starvation represents a form of metabolic stress which stimulates gene expression. Here we report that depriving HeLa cells for any one of a series of amino acids activates c-Jun N-terminal kinase-1 (JNK-1). In contrast, the other mitogen-activated protein kinases (MAPKs) ERK-1 and, to a lesser extent, p38 activities decreased under such conditions. In methionine- or leucine-deprived cells, JNK-1 activation occurred after 4 or 6 h, respectively, and reached a steady maximum of 5- to 7-fold over control cells afterwards. This activation was dependent on the amino acid concentration and it could be reversed by resupplying the complete medium. Limitation for all amino acids also augmented JNK-1 activity, whereas increased amino acid concentrations had an opposite effect. The free radical scavenging thiol antioxidant N-acetylcysteine (NAC) alleviated partially JNK-1 activation in amino acid-deprived cells. The data indicate that activation of JNK-1 by long-term amino acid deprivation may be mediated in part by oxidative stress.

Transport of N-α(or N-ϵ)-L-methionyl-L-lysine and acetylated derivatives across the rabbit intestinal epithelium

Abstract Intestinal absorption constitutes an important step in the biological utilization of L -Methionine and N-Acetyl- L -methionine covalently bound to ϵ-NH2 groups of food protein lysyl residues. The transport of synthesized and purified N-α-(or N-ϵ-) L -Methionyl- L -lysine and N-α-(or N-ϵ-)Acetyl- L -methionyl- L -lysine was studied in vitro in rabbit ileum mounted in the Ussing chamber and compared to that of both free L -Methionine and L -Lysine or both free N-Acetyl- L -methionine and L -Lysine. Addition of all solutes to the mucosal reservoir, except N-ϵ-Acetyl- L -methionyl- L -lysine, led to an increase in the short-circuit current, the lowest response obtained by N-α-Acetyl- L -methionyl- L -lysine. In all cases, only the constitutive amino acids were recovered in the serosal chamber. When free L -Methionine and L -Lysine were added together to the mucosal reservoir, comparable fluxes were obtained: when any of the dipeptides were added, transport of lysine was the highest. Although the mucosal reservoir disappearance of N-α- L -Methionyl- L -lysine was faster than that of N-ϵ- L -Methionyl- L -lysine, the mucosal to serosal fluxes of their constitutive amino acids were not significantly different. However, the transepithelial flux of L -Methionine originating from the dipeptides was 40–50% less than that of the free amino acid, whereas L -Lysine flux was unchanged. Substantial tissue oxidation of L -Methionine unlike L -Lysine, which are slowly released from peptide hydrolysis by the brush border membrane aminopeptidases, likely altered the amino acid transport. Moreover, the observed higher transepithelial flux for L -Lysine relative to L -Methionine when both were derived from the dipeptides may have resulted from intracellular L -Methionine stimulation of L -Lysine absorption. As compared to the normal di- and isodipeptides, lower net fluxes of L -Methionine, and to a lesser extent of L -Lysine, were observed from the acetylated peptides, the former being zero even in the case of N-ϵ-Acetyl- L -methionyl- L -lysine. The results demonstrate the significant contribution of brush border aminopeptidases to the intestinal absorption of N-ϵ- L -Methionyl- L -lysine in the rabbit. Our findings also suggest that the availability of amino acids from acetylated peptides is limited by their transport rates and/ or their hydrolysis from cytosolic N-acylpeptide hydrolase and N-acylase.