Octavi Viñas

Adenine nucleotide translocase 3 (ANT3) overexpression induces apoptosis in cultured cells

Mitochondrial adenine nucleotide translocase 1 (ANT1), but not ANT2, can dominantly induce apoptosis [Bauer et al. (1999) J. Cell Biol. 439, 258–262]. Nothing is known, however, about the apoptotic activity of ANT3. We have transfected HeLa cells with the three human ANT isoforms to compare their potential as inducers of apoptosis. Transient overexpression of ANT3 resulted, like ANT1, in apoptosis as shown by an increase in the sub‐G1 fraction, annexin V staining, low ΔΨ m, and activation of caspases 9 and 3. Moreover, the apoptosis produced by ANT3 was inhibited by bongkrekic acid and by cyclosporin A. The pro‐apoptotic activities of the ANT1 and ANT3 isoforms contrast with the lack of apoptotic activity of ANT2. This finding may help to identify the specific factors associated with the pro‐apoptotic activities of ANT isoforms.

Effects of chronic ethanol consumption on lactational performance in rat: Mammary gland and milk composition and pups' growth and metabolism ☆

The effects of chronic ethanol consumption on lactational performance were studied in the rat on day 15 after delivery by determining mammary gland and milk composition, while growth rate and metabolic parameters were studied in pups coming from untreated mothers but being suckled by ethanol-treated mothers. Alcohol treatment increases the dry weight and lipoprotein lipase activity in the mammary gland, and decreases both absolute and relative mammary gland weight and mammary tissue protein content. The triacylglycerol concentration of milk from treated dams is increased, whereas lactose concentration is decreased in comparison to milk from controls, although the total energy content of milk from alcohol-treated dams is higher than that from controls. Ethanol treatment produces a reduction of daily milk production. Pups nursed by alcoholic mothers show a retarded growth with respect to pups nursed by untreated mothers. Furthermore, they present a reduction in the levels of circulating glucose, insulin, glycerol and free fatty acids, whereas an increase in acetoacetate and in urea levels is observed. Pups from alcoholic mothers show reduced glycogen concentration in the liver while the protein content is increased. Plasma free amino acids in pups nursed by alcoholic mothers are lower than in control pups, the differences in Ala, Glu+Gln, Gly, Pro, 4-OH-Pro, citrulline, Cys, Tyr, Phe and the combined total values being statistically significant. We may therefore draw the conclusion that chronic ethanol treatment impairs lactational performance affecting mammary gland function as shown by the decline in milk production and altered milk composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Opposite regulation of PPAR-α and -γ gene expression by both their ligands and retinoic acid in brown adipocytes

Abstract The peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors involved in the regulation of lipid metabolism and adipocyte differentiation. Little is known, however, about the control of the expression of the genes encoding each of all three receptor subtypes: α, δ, and γ. We have addressed this question in the brown adipocyte, the only cell type that co-expresses high levels of the three PPAR subtypes. Differentiation of brown adipocytes is associated with enhanced expression of PPAR genes. However, whereas PPARγ and PPARδ genes are already expressed in preadipocytes, the mRNA for PPARα appears suddenly in association with the acquisition of the terminally differentiated phenotype. Both retinoic acid isomers and PPAR agonists, specific for either PPARα or PPARγ, regulate expression of each PPAR subtype gene in the opposite way: they up-regulate PPARα and down-regulate PPARγ. The effects on PPARα mRNA are independent of protein synthesis, whereas inhibition of PPARγ mRNA expression depends on protein synthesis, except when its specific ligand prostaglandin J 2 is used. Our results indicate a strictly opposite autoregulation of PPAR subtypes, which supports specific physiological roles for them in controlling brown fat differentiation and thermogenic activity.

Uncoupling protein‐3 gene expression in skeletal muscle during development is regulated by nutritional factors that alter circulating non‐esterified fatty acids

Uncoupling protein‐3 gene expression in skeletal muscle is up‐regulated during postnatal development of mice. A high‐carbohydrate diet at weaning induces a decrease in uncoupling protein‐3 mRNA levels that does not occur when mice were weaned onto a high‐fat diet. Uncoupling protein‐3 mRNA levels do not increase in response to fasting in young pups. Only after day 15 of life, when fasting increases serum non‐esterified fatty acids, uncoupling protein‐3 mRNA is up‐regulated by starvation. Over‐nutrition or under‐nutrition during lactation increases or decreases, respectively, uncoupling protein‐3 mRNA expression in skeletal muscle. Regulation of uncoupling protein‐3 gene expression in skeletal muscle during development is mediated by ontogenic and nutritional factors determining changes in circulating non‐esterified fatty acids.

Both retinoic-acid-receptor- and retinoid-X-receptor-dependent signalling pathways mediate the induction of the brown-adipose-tissue-uncoupling-protein-1 gene by retinoids.

The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte.

9-cis Retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes

The expression of uncoupling protein‐2 (UCP2) mRNA is up‐regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down‐regulated. 9‐cis Retinoic acid causes a dose‐dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all‐trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9‐cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.

Effects of chronic ethanol treatment on amino acid uptake and enzyme activities in the lactating rat mammary gland

The effects of chronic ethanol consumption on mammary gland amino acid uptake at the 15th day of lactation in the rat have been studied. Ethanol treatment decreased the arterial levels of Ala, Asp, Gly, Pro, Lys and Met, and increased those of Gln and alpha-amino-butyrate. Chronic ethanol treatment produced a decrease in the arteriovenous differences of Asp, Thr, Arg, Met and Phe, and increased those of Ala, Gln, Gly, Pro and Tyr. The combination of the calculated values of relative extraction and the arteriovenous differences indicate that these alterations in amino acid uptake are related to changes in the transport process for Ala, Asp, Thr, Pro, Arg, Asn, Gly, Tyr, and Phe, and that the alterations in the arteriovenous differences of Gln, Lys and Met are due to the affected arterial levels of these amino acids. Measurements of enzymatic activities in the mammary gland show that these alterations in the amino acid transport process cannot be ascribed to changes in the gamma-glutamyl cycle.

Lipoprotein lipase mRNA expression in brown adipose tissue: translational and/or posttranslational events are involved in the modulation of enzyme activity.

Lipoprotein lipase mRNA abundance in rat brown adipose tissue increases during the first 24 h of cold exposure. Lipoprotein lipase mRNA levels do not change in brown fat throughout pregnancy and lactation, whereas enzyme activity is significantly lowered. After 5 h of acute cold or noradrenaline administration there is a 2-fold increase in lipoprotein lipase mRNA abundance, whereas lipoprotein lipase activity is stimulated to more than 6-fold the basal values. It is concluded that translational and/or posttranslational mechanisms are involved in the noradrenergic modulation of lipoprotein lipase activity in brown fat.

Cationic and anionic amino acid transport studies in rat red blood cells

The transport of L-proline, L-lysine and L-glutamate in rat red blood cells has been studied. L-proline and L-lysine uptake were Na+-independent. When the concentration dependence was studied both showed a non-saturable uptake assimilable to a difussion-like process, with high Kd values (0.718 and 0.191 min−1 for L-proline and L-lysine respectively). Rat red blood cells showed high impermeability to L-glutamate. No sodium dependence was observed and the Kd value was low (0.067 min−1). Our results show firstly, that rat red blood cells do not have amino acid transport systems for anionic and cationic amino acids and secondly that erythrocytes show no sodium-dependent L-proline transport, and that these cells are very permeable to this amino acid.

Changes in Glycine and Leucine Transport During Red Cell Maturation in the Rat

Both glycine and leucine transport in rat red blood cells have been studied. The glycine uptake showed two different components, one sodium-dependent and another diffusion-like process. In contrast, leucine uptake was sodium independent. Both, Na+-dependent glycine and the overall leucine uptake in red blood cells showed a saturable pattern. Kinetic parameters in reticulocytes were: i) glycine: apparent Km 0.16 mM; Vmax 100.2 nmol/ml ICW/min; ii) leucine: apparent Km 2.11 mM; Vmax 3.88 μmol/ml ICW/min. The erythrocytes kinetic parameters were: i) glycine: apparent Km 0.17 mM; Vmax 9.47 nmol/ml ICW/min; leucine; apparent Km 4.77 mM; Vmax 7.42 μmol/ml ICW/min. The Kd values (sodium independent glycine uptake) were similar in both kind of cells, but the importance of this component in total glycine uptake in erythrocytes was much higher than in reticulocytes. Our results confirm that rat red blood cells have both saturable leucine and Na+-dependent glycine uptake, but some important changes occur during cell maturation.