Using gene knockouts to investigate plant metabolism

Arabidopsis functional genomics resources now make the isolation of knockout mutants in any gene of choice both realistic and increasingly straightforward. Coupled with the completion of the genome sequence, this reverse genetics approach provides a platform facilitating dramatic progress in our understanding of fundamental aspects of plant metabolism. Recent experience shows that knockouts of genes encoding enzymes of primary metabolism can produce mutants with clear and sometimes unexpected phenotypes. They can provide new information about old pathways. Specific functions for individual members of multigene families can be revealed. Knockouts of enzymes of undefined function can lead to the discovery of those functions, and the analysis of enzymes which have previously never been studied at the biochemical level offers the potential to reveal new pathways of plant metabolism. Furthermore, the mutants isolated provide the starting point for genetic modification experiments to determine exactly how metabolism fuels growth and development, so providing a rational basis for the future modification of plant productivity.     

Microscopy approaches to investigate protein dynamics and lipid organization

The structure of cell membranes has been intensively investigated and many models and concepts have been proposed for the lateral organization of the plasma membrane. While proteomics and lipidomics have identified many if not all membrane components, how lipids and proteins interactions are coordinated in a specific cell function remains poorly understood. It is generally accepted that the organization of the plasma membrane is likely to play a critical role in the regulation of cell function such as receptor signalling by governing molecular interactions and dynamics. In this review we present different plasma membrane models and discuss microscopy approaches used for investigating protein behaviour, distribution and lipid organization.     

Paradoxical role of lipid metabolism in heart function and dysfunction

The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effecive than quercetin in causing DNA breakage. In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(1)-sequestering reagent, bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.     

Intraperitoneal leptin regulates lipid metabolism in ethanol supplemented Mus musculas heart

Diseases of the heart and blood vessels are a major cause of illness and disability worldwide. The relationship between ethanol consumption and cardiovascular disease are both complex and interconnected. Our aim of this study was to explore the effect of leptin on lipid metabolism in ethanol supplemented mice. Male Swiss mice (Mus musculas) weighing 25+/-2 g were administered ethanol (6.32 g kg(-1) body weight) for the first 30 days. Subsequently, ethanol fed mice were given intraperitoneal injections of exogenous mouse recombinant leptin (230 microg kg(-1) body weight) every alternate day for 15 days. Food and water intake and total body weight were measured every day and at the end of the experimental period of 45 days, plasma and cardiac lipids were analyzed. Exogenous leptin injections to ethanol fed mice significantly (P < 0.05) prevented the accumulation of total cholesterol, phospholipids (PL), triglycerides (TG) and free fatty acids (FFA) in the mouse heart and blood as compared to the untreated ethanol fed mice whereas, the plasma concentration of free cholesterol was significantly increased on leptin administration as compared to normal untreated mice. Moreover leptin administration significantly elevated the activities of cardiac lipoprotein lipase (LPL) and plasma lecithin cholesterol acyl transferase (LCAT) and significantly reduced the activities of cardiac HMG CoA reductase and cholesterol ester synthase (CES) on leptin administration to ethanol fed mice. Thus we could postulate that an increase in systemic leptin level prevents the accumulation of lipids in the plasma and heart of ethanol treated mice.     

Dietary modulation of lipid metabolism and mechanical performance of the heart

Sudden Cardiac Death resulting from sustained ventricular fibrillation or malignant cardiac arrhythmia has been linked to the type of dietary fat intake in several economically well developed countries where high levels of saturated fatty acids are common. Experimental studies with the small non-human primate marmoset monkey have clearly demonstrated the health benefit of substituting polyunsaturated fatty acids (PUFA's) for dietary saturated fatty acids. Heart rate and blood pressure are lowered, while the left ventricular ejection fraction and the electrical threshold for the induction of ventricular fibrillation are both increased after prolonged feeding of PUFA enriched diets. All these changes in heart function reduce the risk of developing malignant cardiac arrhythmias.The fatty acid composition of cardiac membrane phospholipids is profoundly altered by these changes in dietary lipid intake. In particular the proportions of arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexae noic acid (DHA) are altered in such a way that the production of myocardial eicosanoids is affected. Although the changes in proportion of these long-chain PUFA's in cardiac phosphatidyl ethanolamine and phosphatidyl inositol are not identical, the shift in balance between these substrates or inhibitors of cyclo-oxygenase activity leads to relatively greater production of prostacyclin (PGI₂) than thromboxane (TXA₂).The effect of the omega-3 PUFA's of fish oil is proportionally greater than that of linoleic acid (LA; 18:2, 6) rich sunflower seed oil, particularly during ischaemia, and probably reflects the different nutritionally induced changes in cardiac membrane fatty acid composition by these different types of dietary PUFA's. (Mol Cell Biochem 116: 19–25, 1992).     

Restriction polymorphism in patients with lipid metabolism disorders and ischemic heart disease

Using the RELP analysis we studied the frequency of X2 allele of apoB gene in three groups of patients: 1) men at the age of 20-59 with lipid metabolism disorders revealed in population inspection of Oktyabrsky district in Moscow; 2) men with ischaemic heart disease and 3) healthy men. It was established that in individuals suffering from type IIa hyperlipidemia the frequency of X2 allele was significantly higher than in healthy donors from Moscow population. Homozygotes for X2 allele of XbaI RELP had 7-9% higher serum cholesterol levels, than homozygotes for X1 allele. The study suggests the X2 allele of the apoB gene to be associated with the development of high plasma cholesterol level. No significant difference in X2 allele frequencies was found between patients with ischaemic heart disease and healthy donors. There was also no association found between cholesterol and triglyceride levels and the presence of X2 allele in this group of patients.     

Impact of fasting time on hepatic lipid metabolism in nutritional animal studies

Many animal studies on improvement of lipid metabolism, using dietary components, fast the animals on the final day of the feeding. Although fasting has a significant impact on lipid metabolism, its time-dependent influence is not fully understood. We examined the effects of several fasting times on lipid metabolism. Rats fed with a semisynthetic diet for 2 wk were killed after 0 (9:00?am), 6 (7:00?am–1:00?pm), 9 (0:00?am–9:00?am), and 13?h (8:00?pm–9:00?am) of fasting. Compared to the 0?h group, marked reduction of liver weight and hepatic triacylglycerol content was observed in the 9 and 13?h groups. Activities of hepatic enzymes involved in fatty acid synthesis gradually decreased during fasting. In contrast, drastic time-dependent reduction of gene expression, of the enzymes, was observed. Expression of carnitine palmitoyltransferase mRNA was higher in the fasting groups than in the 0?h group. Our study showed that fasting has a significant impact on several parameters related to lipid metabolism in rat liver.     

Maternal lipid metabolism and placental lipid transfer

During early pregnancy, long-chain polyunsaturated fatty acids (LC-PUFA) may accumulate in maternal fat depots and become available for placental transfer during late pregnancy, when the fetal growth rate is maximal and fetal requirements for LC-PUFAs are greatly enhanced. During this late part of gestation, enhanced lipolytic activity in adipose tissue contributes to the development of maternal hyperlipidaemia; there is an increase in plasma triacylglycerol concentrations, with smaller rises in phospholipid and cholesterol concentrations. Besides the increase in plasma very-low-density lipoprotein, there is a proportional enrichment of triacylglycerols in both low-density lipoproteins and high-density lipoproteins. These lipoproteins transport LC-PUFA in the maternal circulation. The presence of lipoprotein receptors in the placenta allows their placental uptake, where they are hydrolysed by lipoprotein lipase, phospholipase A(2) and intracellular lipase. The fatty acids that are released can be metabolized and diffuse into the fetal plasma. Although present in smaller proportions, maternal plasma non-esterified fatty acids are also a source of LC-PUFA for the fetus, their placental transfer being facilitated by the presence of a membrane fatty acid-binding protein. There is very little placental transfer of glycerol, whereas the transfer of ketone bodies may become quantitatively important under conditions of maternal hyperketonaemia, such as during fasting, a high-fat diet or diabetes. The demands for cholesterol in the fetus are high, but whereas maternal cholesterol substantially contributes to fetal cholesterol during early pregnancy, fetal cholesterol biosynthesis rather than cholesterol transfer from maternal lipoproteins seems to be the main mechanism for satisfying fetal requirements during late pregnancy.     

Cytochemical studies of lipid metabolism: immunogold probes for lipoprotein lipase and cholesterol

In this article, cytochemical methods are presented for the study of lipid metabolism both in normal cells and in mutant cells with genetic disorders characterized by abnormal lipid metabolism. The benefit of using an immunocytochemical approach to the study of lipase in tissues is discussed, and a review is presented of the results on immunolocalization of lipoprotein lipase in cardiac tissue of normal mice. Immunocytochemical techniques are applied to the study of lysosomal proliferation in hepatocytes from liver of mutant mice with a genetic defect responsible for the lack of hepatic lipase and lipoprotein lipase activity in these animals. Localization of lipids in tissues with structural techniques has been an area of great interest to our laboratory for many years. Attention is called to the development of a technique for the visualization of fatty acids as a function of their ionization state and the production of fatty-acid myelin figures in membranes. Results on the use of filipin to detect unesterified cholesterol in membranes are reviewed. Filipin produces fluorescent filipin-cholesterol complexes but also perturbs cell membranes. Application of this cytochemical probe, in combination with immunocytochemistry of lysosomes, produced useful information on defects in low-density lipoprotein-derived cholesterol translocation in mutant human fibroblasts. Initial results on the application of immunological techniques to the study of cholesterol in lipid model systems indicate a novel approach, which may be applicable to specialized cell systems. Recent advances in cryoultramicrotomy and development of immunoprobes present valuable opportunities for the structural assessment of lipids and lipases in cell organelles and cell membranes.     

Copper intake affects rat heart performance during ischemia-reperfusion: possible relation to altered lipid and fatty acid metabolism

Hearts from rats fed low copper (1.3 mg copper/kg diet) or a copper-supplemented diet (243 mg copper/kg diet) were perfused for 90 min according to the Langendorff method. The perfusion protocol included 30 min normoxia, 30 min ischemia and 30 min reperfusion. After 90 min perfusion, hearts from the low copper group had gained more weight, had lower coronary perfusion pressure, developed less force of contraction and secreted less 6-keto PGF1 alpha into the perfusate than hearts from the copper-supplemented group. After perfusion, the major lipid change in the hearts from both groups was a 85-90% decrease in total triacylglycerol. In both groups, stearic acid and arachidonic acid (mg%) were increased in the triacylglycerol fraction after heart perfusion. The quantitative (mg/g) decrease in the triacylglycerol content of stearic acid and arachidonic acid was significantly less in the copper-supplemented group. After perfusion, dihomo-gamma-linolenic acid (mg/g) was lower in heart phospholipids from the low copper group. Dihomo-gamma-linolenic/arachidonic acid (microgram/mg) was significantly decreased after perfusion only in the hearts from the low copper group. Lipid and fatty acid changes in the hearts of the rats fed low dietary copper may contribute to abnormal heart function in this group.     

Use of the isolated perfused rat lung in studies on lung lipid metabolism

A procedure for the use of the isolated perfused rat lung in studies on metabolic regulation has been developed. The procedure, reasonably uncomplicated, yet physiological, maintains the lung so that edema is not observed. The phospholipid content remains normal, and incorporation of [1-(14)C]-palmitate, [2-(14)C]acetate, and [U-(14)C]glucose is linear with time for a minimum of 2 hr. The incorporation of [1-(14)C]-palmitate and [2-(14)C]acetate into the total lung phospholipid fraction and into the phosphatidylcholine and phospatidylethanolamine fractions has been studied. Increasing the concentration of palmitate in the medium from 0.14 to 0.51 mm increased by 60% the incorporation of [1-(14)C]palmitate into the total lung phospholipid fraction at 2 hr. When the palmitate concentration of the medium was 0.14 mm, addition of 0.11 and 0.79 mm oleate to the medium decreased [1-(14)C]palmitate incorporation into the total lung phospholipid fraction at 2 hr by 37 and 49%, respectively. The results suggest that the incorporation of exogenous fatty acids, present in the medium perfusing the lung, into lung phospholipids may depend upon the fatty acid composition of the medium. Known specific acyltransferase activities may be responsible for the ordered incorporation of available fatty acids into lung phospholipids.