Lipoprotein lipase-mediated sequestration of long-chain polyunsaturated triacylglycerols in serum LDL from normal and hypothyroid rats

Rat serum VLDL, unlike human, contains significant proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids. Hypothyroidism in this species is characterized by low levels of serum VLDL, the accumulation of LDL, elevated levels of lipoprotein lipase and depressed hepatic lipase activity. The hypothyroid rat thus represents an interesting model in which to study hepatic VLDL metabolism and the substrate specificity of lipoprotein lipase. This report shows that serum IDL and LDL in both euthyroid and hypothyroid rats contain progressively enhanced proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids when compared to VLDL. Hypothyroidism resulted in a decrease in the proportion of 22:6 fatty acid within the serum VLDL triacylglycerols when compared to euthyroid VLDL. Lipolysis of VLDL from euthyroid rats in vitro using the perfused rat heart system resulted in increases or sequestration of triacylglycerols containing long-chain polyunsaturated fatty acids within the IDL fraction similar to those seen in vivo. It is concluded that lipoprotein lipase-mediated hydrolysis of VLDL triacylglycerols and the conversion of VLDL to IDL and LDL in the rat results in a progressive sequestration of the longer-chain polyunsaturated triacylglycerol molecular species with the IDL and LDL.     

Cyclic di-GMP is essential for the survival of the lyme disease spirochete in ticks

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host.     

Some kinetic properties of phosphorylated ATPase of sarcoplasmic reticulum formed in the absence of added alkali metal salts.

In the rat both hypothyroidism and diabetes decrease heparin-releasable liver lipase activity. This defect may be reversed by feeding a diet rich in polyunsaturated fatty acids. It is suggested that a diet-induced increase of membrane fluidity restores liver lipase activity, which contributes to the hypolipidemic effect of polyunsaturated fatty acids.     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Post-heparin triacylglycerol lipases in ovine plasma

Lipoprotein lipase and hepatic lipase have been shown to be present in the post-heparin plasma of sheep. Intravenous injection of heparin into sheep produced a rapid increase in the free fatty acid concentration and lipolytic enzyme activity of the plasma, both peaking within 5-15 min and then falling to pre-heparin levels within 30-60 min. Lipolytic activity was not detected in plasma before heparin treatment. Two distinct lipolytic activities were separated from the plasma by chromatography on heparin-Sepharose 6B. Lipoprotein lipase was identified on the basis that the lipolytic activity was dependent upon the addition of plasma, inhibited by 1M NaCl, and inhibited by a specific antiserum against lipoprotein lipase. The second lipolytic activity of plasma was identified as hepatic lipase, as it was not dependent upon plasma for activity, nor was it inhibited by 1M NaCl or antiserum against lipoprotein lipase. Its properties were identical to the lipase extracted from the liver of sheep. Lipoprotein-lipase activity, but not hepatic-lipase activity, was dependent upon the nutritional state of the sheep at the time of heparin injection. However, hepatic lipase comprised a significant proportion of the total lipolytic activity.     

Genetics and Regulation of Chitobiose Utilization in Borrelia burgdorferi

Borrelia burgdorferi spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of N-acetylglucosamine (GlcNAc). The B. burgdorferi celA, celB, and celC genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the celB gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes chbA, chbB, and chbC, since they probably encode a chitobiose transporter. We also found that the chbC gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.     

Importance of lipolysis in oral cavity for orosensory detection of fat

Lingual lipase is usually secreted from von Ebner's glands, although there is great variation between species. Lingual lipase is thought to be an auxiliary enzyme for fat digestion and absorption in mammals; however, the reason for lipolysis in the oral cavity is not known. We focused on the gustatory sense and investigated the significance of lingual lipase in the perception of a fat taste by using orlistat, a potent lipase inhibitor. Five-minute two-bottle preference tests demonstrated that the addition of orlistat diminished the preference for triacylglycerides but not for free fatty acids. Radioactive triolein applied on rats' circumvallate papilla revealed that lingual lipase was released continuously to generate significant amounts of fatty acids and other lipolytic products within 1-5 s, which was enough time to taste fat. These findings suggest that lingual lipase is released to perceive the taste of triacylglycerides and to find nutritive lipids in food.     

Regeneration of renal proximal tubules after mercuric chloride injury is accompanied by increased binding of aminoacyl-transfer ribonucleic acid.

The liver of the foetal guinea pig accumulates a large quantity of triacyglycerol late in gestation at the same time that adipose-tissue mass grows at its maximum rate and foetal adipose-tissue lipoprotein lipase activity and sensitivity to lipolytic hormones has substantially declined. The fatty acid for triacyglycerol synthesis is not synthesized in the foetal liver and it is unlikely that it originates from any of the foetal tissues. Before the accumulation of hepatic triacyglycerol the concentration of free fatty acids increases in both the umbilical vein and the maternal inferior vena cava. This occurs at a time when the triacyglycerol lipase activity in maternal adipose tissue is elevated and the rate of lipolysis, but not of fatty acid esterification, is higher than earlier in gestation or than in the non-pregnant state. It is proposed that the increase in lipolysis in maternal adipose tissue, brought about by an increase in circulating lipolytic hormones, mobilizes fatty acid, which passes to the foetus and is partly stored as hepatic triacylglycerol. The foetal liver effectively removes both long-and short-chain fatty acids from umbilical-vein blood. The rate of placental fatty acid transfer is more than adequate to account for the triacylglycerol accumulation.     

Degradation of triglycerides by a pseudomonad isolated from milk: the roles of lipase and esterase studied using recombinant strains over-producing, or specifically deficient in these enzymes

The roles of lipase and esterase in causing hydrolytic spoilage of milk by a highly lipolytic psychrotrophic strain of Pseudomonas fluorescens , LS107d2, has been studied. Strains of LS107d2 have been constructed that over-produce, or are specifically deficient in, a lipase (encoded by lip A ) and an esterase (encoded by est A ). Southern blot analysis reveals that LS107d2 contains only one esterase and one lipase (encoded by est A and lip A ) and this was confirmed by the phenotypes of mutants on triolein and tributyrin-containing agar. Analysis of broth cultures showed that the lipase is secreted into the culture medium; in contrast, the esterase is not secreted. Free fatty acid (FFA) levels in whole milk cultures of wild-type, over-producing and the mutant strains of LS107d2 have been examined. From these studies it is concluded that esterase is not involved in the accumulation of FFA by hydrolysing short chain fatty acid esters; that the highly lipolytic phenotype of LS107d2 is due solely to a single secreted lipase; and that the main FFA accumulated in milk cultures of LS107d2 are C4, C16, C18 and C18: 1. Evidence is also presented demonstrating that FFA degradation, as well as production, determines the level of FFA in milk contaminated with lipolytic organisms.     

Efficient targeted mutagenesis in Borrelia burgdorferi

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1). The utility of the coumermycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.     

Borrelia burgdorferi bb0426 encodes a 2'-deoxyribosyltransferase that plays a central role in purine salvage

Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5'-diphosphates to deoxynucleosides-5'-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2'-deoxyribosyltransferase (DRTase, encoded by bb0426 ), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues.     

Molecular characterisation of the haemolytic isolates of Bacillus anthracis originated in Poland

Bacillus anthracis is generally considered non-haemolytic, when cultured on the solid media. However, strains capable to lyse sheep erythrocytes have been reported. Anthrolysin O, an orthologue of cereolysin was proposed as a putative haemolysin of B. anthracis. AIM: to determine whether anthrolysin O, haemolytic enterotoxin HBL and the pleiotropic regulator PlcR that activates antrholysin O production are associated with a haemolytic activity of B. anthracis strains isolated in Poland. MATERIAL: in total 8 B. anthracis strains - the fully virulent BL1 and seven the pXO2 lacking strains including: a vaccine strain Sterne 34F2 together with three haemolytic and three non-haemolytic strains isolated from different samples of the same animal died from anthrax in Poland. METHODS: The haemolytic activity was detected using Columbia agar plates supplemented with 5% of sheep blood. Anthrolvsin O, cereolysin and gene hblA encoding the key subunit of the HBL were detected by PCR. In addition, the plcR gene fragment containing the B. anthracis specific non-sense mutation was analysed by the DNA sequencing. Ten marker loci based MLVA genotyping was performed to distinguish tested strains. RESULTS: The alo gene encoding anthrolysin O was detected in both the haemolytic and non-haemolytic strains while hblA was absent. The B. anthracis specific plcR non-sense mutation was detected in both the groups of tested strains, suggesting that the haemolysis in tested strains may rather be conferred by the PlcR-independent factors. Moreover, haemolytic and non-haemolytic strains were indistinguishable by the MLVA. Obtained results may argue the haemolytic and non-haemolytic strains are isogenic and most probably a single mutational event is responsible for the haemolytic phenotype induction.     

Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala,a fermented African locust bean condiment

AIMS: To investigate predominant isolates of Bacillus subtilis and B. pumilus in soumbala, a fermented African locust bean condiment, for their ability to degrade African locust bean oil (ALBO). METHODS AND RESULTS: Agar diffusion test in tributyrin and ALBO agar was used for screening of the isolates for esterase and lipase activity, respectively. The quantity and the profile of free fatty acids (FFA) during 72 h of degradation of ALBO by the Bacillus isolates were studied by titration and gas chromatography. The degradation of tributyrin and ALBO was variable among the isolates. Two strains of B. subtilis and two strains of B. pumilus showed significantly higher esterase and lipolytic activities than the others. The degradation ALBO was most pronounced in enriched nutrient agar except for one isolate of B. pumilus degrading ALBO to the same extent regardless of the enrichment. The quantity of FFA released from ALBO by the most lipolytic strains of Bacillus increased mainly between 0 and 24 h and differed among the isolates. The profile of FFA was similar for the Bacillus isolates with oleic acid (C18:2) occurring as the major FFA in all the samples except in samples incubated with B. subtilis B9 where stearic acid (C18) was dominant. CONCLUSION: Bacillus isolates from soumbala showed high strain dependent lipolytic activity against ALBO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of soumbala.     

Generation of polyunsaturated fatty acids from vegetable oils using the lipase from ground oat (Avena sativa L.) seeds as A Catalyst

Summary Ground oat seeds can serve as a source of lipase in both aqueous-oil emulsions and in organic solvents. In organic solvents the lipase lost selectivity for monounsaturated fatty acids. Because of this, polyunsaturated vegetable oils were rapidly split. The lipase source is very inexpensive and is recyclable, to a certain extent.     

Fate of fat: The role of adipose triglyceride lipase in lipolysis

Lipolysis, the coordinated catabolism of triacylglycerol (TG) stored in cellular lipid droplets, provides fatty acids, di-, and monoglycerides. These products are important energy substrates, precursors for other lipids, or lipid signaling molecules. Following their discovery by Hollenberg, C.H., Raben, M.S., and Astwood, E.B.(1961) and Vaughan, M., Berger, J.E., and Steinberg, D. (1964), hormone-sensitive lipase (HSL) and monoacylglycerol lipase stayed in the focus of research for three decades. Within the last decade, however, it became evident that the lipolytic pathway is incompletely understood. Studies on the regulation of lipolysis and the characterization of HSL-deficient mice indicated that additional previously unrecognized factors that contribute to fat catabolism must exist. This led to the discovery of the perilipin, adipophilin, Tip47 (PAT) family of lipid droplet binding proteins and the identification of a novel TG hydrolase named adipose triglyceride lipase (ATGL). This review focuses on the importance of ATGL as TG lipase within the “lipolytic machinery” and the current knowledge of molecular mechanisms that regulate ATGL activity.     

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.     

Lipolysis: pathway under construction

PURPOSE OF REVIEW: The lipolytic catabolism of stored fat in adipose tissue supplies tissues with fatty acids as metabolites and energy substrates during times of food deprivation. This review focuses on the function of recently discovered enzymes in adipose tissue lipolysis and fatty acid mobilization. RECENT FINDINGS: The characterization of hormone-sensitive lipase-deficient mice provided compelling evidence that hormone-sensitive lipase is not uniquely responsible for the hydrolysis of triacylglycerols and diacylglycerols of stored fat. Recently, three different laboratories independently discovered a novel enzyme that also acts in this capacity. We named the enzyme 'adipose triglyceride lipase' in accordance with its predominant expression in adipose tissue, its high substrate specificity for triacylglycerols, and its function in the lipolytic mobilization of fatty acids. Two other research groups showed that adipose triglyceride lipase (named desnutrin and Ca-independent phospholipase A2zeta, respectively) is regulated by the nutritional status and that it might exert acyl-transacylase activity in addition to its activity as triacylglycerol hydrolase. Adipose triglyceride lipase represents a novel type of 'patatin domain-containing' triacylglycerol hydrolase that is more closely related to plant lipases than to other known mammalian metabolic triacylglycerol hydrolases. SUMMARY: Although the regulation of adipose triglyceride lipase and its physiological function remain to be determined in mouse lines that lack or overexpress the enzyme, present data permit the conclusion that adipose triglyceride lipase is involved in the cellular mobilization of fatty acids, and they require a revision of the concept that hormone-sensitive lipase is the only enzyme involved in the lipolysis of adipose tissue triglycerides.     

Borrelia burgdorferi membranes are the primary targets of reactive oxygen species

Spirochetes living in an oxygen-rich environment or when challenged by host immune cells are exposed to reactive oxygen species (ROS). These species can harm/destroy cysteinyl residues, iron-sulphur clusters, DNA and polyunsaturated lipids, leading to inhibition of growth or cell death. Because Borrelia burgdorferi contains no intracellular iron, DNA is most likely not a major target for ROS via Fenton reaction. In support of this, growth of B. burgdorferi in the presence of 5 mM H₂O₂ had no effect on the DNA mutation rate (spontaneous coumermycin A1 resistance), and cells treated with 10 mM t -butyl hydroperoxide or 10 mM H₂O₂ show no increase in DNA damage. Unlike most bacteria, B. burgdorferi incorporates ROS-susceptible polyunsaturated fatty acids from the environment into their membranes. Analysis of lipoxidase-treated B. burgdorferi cells by Electron Microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (50-fold) in these cells, with a corresponding increase in the levels of malondialdehyde by-product (fourfold). These data suggest that B. burgdorferi membrane lipids are targets for attack by ROS encountered in the various stages of the infective cycle.