Mechanisms for the effects of ethanol on hepatic phosphatidate phosphohydrolase.

1. The effects of the intramuscular administration of glycerol and dihydroxyacetone (40mmol per kg body wt.), sorbitol and glucose (20mmol per kg body wt.) or NaCl (1.5mmol per kg body wt. in 10ml of water per kg body wt.) were investigated on soluble phosphatidate phosphohydrolase and certain metabolites in rat liver. 2. The effects of ethanol and glycerol on phosphatidate phosphohydrolase were also studied in isolated perfused livers. 3. The administration of glycerol, sorbitol and dihydroxyacetone in vivo increased hepatic phosphatidate phosphohydrolase activity by 137, 63 and 32% respectively in 4h. 4. A significant positive correlation was found between the hepatic sn-glycerol 3-phosphate concentration and phosphatidate phosphohydrolase after the administration of various substrates in vivo. 5. The soluble phosphatidate phosphohydrolase activity tended to increase during perfusions of isolated rat livers without added substrates, and neither ethanol nor glycerol produced additional effects. 6. The activity of soluble phosphatidate phosphohydrolase was 2.5 times higher in the livers of hyperthyroid rats than in normal rats. This activity was not influenced by intragastric ethanol or glycerol administration, nor was the concentration of sn-glycerol 3-phosphate changed by these compounds. 7. It is concluded that the ethanol-induced increase in hepatic phosphatidate phosphohydrolase may at least in part be mediated by the hepatic concentration of metabolites, probably by the concentration of sn-glycerol 3-phosphate.     

Regulation of cellular respiration in myoglobin-deficient mouse heart

The regulation of cardiac O2 consumption according to energy demand is best studied in the intact organ by non-destructive methods, using probes detectable by their fluorescence or light absorption. However, myoglobin is normally present in high concentrations and swamps the cytochrome spectra, thereby bringing about an oxygen-dependent internal filter effect which quenches the fluorescence of probes. A viable myoglobin-deficient mouse strain (Myo(-/-)) has been generated previously and isolated perfused Myo(-/-) hearts are used here as an ideal model for studying mitochondrial metabolism by non-destructive optical methods. In this model we monitored the redox state of cytochrome aa3 and flavoprotein (Fp) during perturbations of myocardial work output upon changes in extracellular [Ca2+], KCl-induced arrest and pacing. Increased consumption of energy and O2 led to a concomitant reduction of cytochrome aa3 and oxidation of Fp. Administration of a medium chain-length fatty acid caused a marked reduction of Fp, but even then an increase in energy consumption caused Fp oxidation. The results show that cell respiration in the intact myocardium is regulated at the site of the respiratory chain. Our findings do not support the NMR-based hypothesis that O2 consumption is mainly regulated at the level of intermediary metabolism and by the pressure of reducing equivalents to the mitochondrial respiratory chain.     

Oxygen and substrate dependence of hepatic cellular respiration: sinusoidal oxygen gradient and effects of ethanol in isolated perfused liver and hepatocytes

The oxygen dependence of hepatic cellular respiration was studied by employing simultaneous organ spectrophotometry of cytochromes and hemoglobin, the latter used as an intrasinusoidal optical oxygen probe. The Km of cytochrome aa3 for oxygen was found to be 6.8 microM in the isolated perfused liver and 0.3 microM in suspensions of isolated hepatocytes. The results indicate that the sinusoid-to-cell pO2 gradient is about 5 torr. Optical determination of the average effective pO2 indicates that the axial sinusoidal O2 profile does not conform to zero-order O2 uptake in the liver. Because of extensive NAD+ reduction, ethanol increases the thermodynamic driving force of oxidative phosphorylation, and it also increased the oxygen consumption in both the perfused liver and the hepatocyte suspension, but had no effect on the grade of steady-state cytochrome aa3 reduction, the cellular energy state [ATP]/[ADP].[Pi], or the Km of cytochrome aa3 for oxygen. The results indicate that hepatic energy metabolism is oxygen independent at very low O2 concentrations, but that the sinusoidal axial O2 concentration is anomalous, probably due to the spatial arrangement of the metabolizing systems.     

Beta-oxidation of polyunsaturated fatty acids in peroxisomes. Subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase activity in rat liver

The metabolism of the double bonds at the delta 3 position in fatty acids was studied in rat liver. Infusion of delta 3-trans-dodecenoic acid into isolated perfused liver and subcellular fractionation studies showed the presence of both peroxisomal and mitochondrial delta 3,delta 2-enoyl-CoA isomerase activity (EC 5.3.3.8). These findings together with the previous demonstration of peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34) [(1981) J. Biol. Chem. 256, 8259-8262] and D-3-OH-acyl-CoA epimerase (EC 5.1.2.3) [(1985) FEBS Lett. 185, 129-134] activities show that peroxisomes possess all the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids.     

Effect of acetate and octanoate on tricarboxylic acid cycle metabolite disposal during propionate oxidation in the perfused rat heart

Tricarboxylic acid cycle pool size is determined by anaplerosis and metabolite disposal. The regulation of the latter during propionate metabolism was studied in isolated perfused rat hearts in the light of the characteristics of NADP-linked malic enzyme, which is inhibited by acetyl-CoA. The acetyl-CoA concentration was varied by infusions of acetate and octanoate, and the rate of metabolite disposal was calculated from a metabolic balance sheet compiled from the relevant metabolic fluxes. Propionate addition increased the tricarboxylic acid cycle pool size 4-fold and co-infusion of acetate or octanoate did not change it further. Propionate caused a decrease in the CoA-SH concentration and a 10-fold increase in the propionyl-CoA concentration. A paradoxical increase in the CoA-SH concentration was observed upon co-infusion of acetate in the presence of propionate, an effect probably caused by competitive inhibition of propionate activation. A more pronounced decline in the propionyl-CoA concentration was observed upon the co-infusion of octanoate. In a metabolic steady state, acetate and octanoate reduced propionate disposal only slightly, but did not increase the tricarboxylic acid cycle pool size. The results are in accord with the notion that the tricarboxylic acid pool size is mainly regulated by the anaplerotic mechanisms.     

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

To study the role of metallothioneins (MTs) in Zn accumulation,the expression of TcMT2a, TcMT2b, and TcMT3 was analysed inthree accessions and 15 F₃ families of two inter-accession crossesof the Cd/Zn hyperaccumulator Thlaspi caerulescens, with differentdegrees of Zn accumulation. The highest expression levels werefound in the shoots of a superior metal-accumulating calamineaccession from St Laurent le Minier, with >10-fold TcMT3expression compared with another calamine accession and a non-metallicolousaccession. Moreover, F₃ sibling lines from the inter-accessioncrosses that harboured the MT2a or MT3 allele from St Laurentle Minier had higher expression levels. However, there was noco-segregation of TcMT2a or TcMT3 expression and Zn accumulation.To examine the functions of TcMTs in plants, TcMT2a and TcMT3were ectopically expressed in Arabidopsis. The transformantlines had reduced root length in control medium but not at highmetal concentrations, suggesting that the ectopically expressedproteins interfered with the physiological availability of essentialmetals under limited supply. The Arabidopsis transformant linesdid not show increased tolerance to Cd, Cu, or Zn, nor increasedCd or Zn accumulation. Immunohistochemical analysis indicatedthat in roots, MT2 protein is localized in the epidermis androot hairs of both T. caerulescens and Arabidopsis thaliana.The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarilyinvolved in Zn accumulation as such. However, the elevated expressionlevels in the metallicolous accessions suggests that they docontribute to the metal-adapted phenotype, possibly throughimproving Cu homeostasis at high Zn and Cd body burdens. Alternatively,they might function as hypostatic enhancers of Zn or Cd tolerance. Key words: Cd, crosses, metallothionein, protein, quantitative real-time PCR, Thlaspi caerulescens, Zn Received 14 August 2008; Revised 14 October 2008 Accepted 15 October 2008     

Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells

Glycosylation is one of the most common modifications of proteins and lipids and also a major source of biological diversity in eukaryotes. It is critical for many basic cellular functions and recognition events that range from protein folding to cell signaling, immunological defense, and the development of multicellular organisms. Glycosylation takes place mainly in the endoplasmic reticulum and Golgi apparatus and involves dozens of functionally distinct glycosidases and glycosyltransferases. How the functions of these enzymes, which act sequentially and often competitively, are coordinated to faithfully synthesize a vast array of different glycan structures is currently unclear. Here, we investigate the supramolecular organization of the Golgi N- and O-glycosylation pathways in live cells using a FRET flow cytometric quantification approach. We show that the enzymes form enzymatically active homo- and/or heteromeric complexes within each pathway. However, no complexes composed of enzymes that operate in different pathways, were detected, which suggests that the pathways are physically distinct. In addition, we show that complex formation is mediated almost exclusively by the catalytic domains of the interacting enzymes. Our data also suggest that the heteromeric complexes are functionally more important than enzyme homomers. Heteromeric complex formation was found to be dependent on Golgi acidity, markedly impaired in acidification-defective cancer cells, and required for the efficient synthesis of cell surface glycans. Collectively, the results emphasize that the Golgi glycosylation pathways are functionally organized into complexes that are important for glycan synthesis.     

Temporal variation in abundance, return rate and life histories of previously spawned Atlantic salmon in a large subarctic river

The 30 year time series analyses revealed large temporal variation in the return rates and a recent increase in abundance of previous spawning Atlantic salmon Salmo salar in the River Teno, northern Scandinavia. The mean proportion of repeat spawners was 7 and 4% in the total Atlantic salmon catch and 9 and 22% in multi‐sea‐winter (MSW) catch component for females and males, respectively. Previous spawners constituted on the average 7% of the catch in mass but up to 20%(31 t) and 30%(19 t) in 2003 and in 2004, respectively. In 1975–2000, the proportion of previous spawners varied between 1 and 6%(3–12% of MSW Atlantic salmon), whereas in 2001–2004, they accounted for 8–21%(16–35% of MSW Atlantic salmon) of the total Atlantic salmon catch. The number of previous spawners in the catch correlated significantly with the preceding numbers of respective 1–3 sea‐winter (SW) maiden Atlantic salmon 2 years earlier. The recent increase in the numbers of 1S1 and 2S1 (1 or 2 years at sea followed by first spawning and 1 year reconditioning period at sea) alternate spawning Atlantic salmon was a consequence of higher numbers of maiden 1SW and 2SW Atlantic salmon in the catches and increased sea temperatures. Similarly, the return rate of 1SW Atlantic salmon to second spawning has improved in recent years. Most previous spawners ascended and were captured early in the fishing season. The smolt and sea‐age combinations of repeat spawners comprised 68 age groups contributing with the annual mean of 15 age groups to the great diversity of the River Teno Atlantic salmon population complex.     

Myocardial Overexpression of Mecr,a Gene of Mitochondrial FAS II Leads to Cardiac Dysfunction in Mouse

It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy.