Charlotte E. Borgeson

Effects of dietary fish oil on human mammary carcinoma and on lipid-metabolizing enzymes

The growth rate of a human mammary carcinoma, MX-1, was significantly reduced in athymic “nude” mice fed fish oil. Tumors from the fish oil-fed animals also showed a greater sensitivity to two anti-neoplastic agents, mitomycin C and doxorubicin. Mitochondria were isolated from control livers, host livers and tumors from fish oil-and corn oil-fed animals, and increased levels of 20∶5n−3 and 22∶6n−3 were found in mitochondrial lipids in all three tissues from the fish oil-fed animals. To investigate the effect of dietary n−3 fatty acids on lipid metabolism, the activity of the acyl-CoA:carnitine acyltransferase and three acyl-CoA desaturases were measured. Carnitine acyltransferase activity toward all four acyl-CoA substrates tested was markedly increased in mitochondria from liver by feeding fish oil. In mitochondria from tumors, feeding fish oil resulted in an increased activity toward only 18∶3n−3. These data suggest that fish oil may induce an increase in the oxidation of fatty acids. The Δ9-desaturase activity was decreased in microsomes from liver and tumor from fish oil-fed animals. However, both the Δ6 and Δ5 desaturases were increased in tumor and in control liver as a result of feeding fish oil. The Δ5 desaturase was not altered in microsomes from the host animals. The effect of fish oil on the Δ5 and Δ6 desaturases may involve alterations to metabolism of specific polyunsaturated fatty acids especially in the tumor tissue.

Polyunsaturated fatty acids and eicosanoids in insects

Abstract Novel aspects of the metabolism of polyunsaturated fatty acids (PUFAs), precursors of prostaglandins and other eicosanoids, in insects are reviewed. A number of insect species are able to produce linoleic acid, 18:2 ( n-6 ), de novo , a fatty acid that was previously believed to be essential for all animals. These insect species are unique among animals in that they possess a Δ12 desaturase which converts oleic acid, 18:1 ( n-9 ) to 18:2 ( n-6 ) and makes them nutritionally independent of plant derived polyunsaturated fatty acids (PUFA). The potential role of microorganisms in linoleate production was ruled out by studies using isolated tissue under axenic conditions from the house cricket, Acheta domesticus , an insect which does not contain intracellular microorganisms. The results showed that it is insect tissue that contains the Δ12 desaturase. This enzyme has been charactized from the house cricket and the American cockroach. In contrast to the plant Δ12 desaturase, which converts 18:1 esterified in a phospholipid as substrate to 18:2 ( n-6 ), the insect Δ12 desaturase uses oleoyl-CoA as substrate. A number of insect species, including representatives of both those that do and do not produce linoleate, elongate and desaturate 18:2 ( n-6 ) and 18:3 ( n-3 ) to 20:4 ( n-6 ) and 20:5 ( n-3 ), respectively. A conspicuous exception to this are mosquitoes, which require 20:4 ( n-6 ) or structurally related fatty acids in their diet. A number of insect species have been shown to metabolize 20:4 to prostaglandins and other eicosanoids.

Juvenile hormone regulation of HMG-R gene expression in the bark beetle Ips paraconfusus (Coleoptera: Scolytidae): implications for male aggregation pheromone biosynthesis

Abstract. Juvenile hormone III (JH III) induces acyclic isoprenoid pheromone production in male Ips paraconfusus. A likely regulatory enzyme in this process is 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). To begin molecular studies on pheromone production, a 1.16-kb complementary DNA representing approximately one-third of I. paraconfususHMG-R was isolated by polymerase chain reaction and sequenced. The predicted translation product is 59% and 75% identical to the corresponding portion of HMG-R from the fruit fly, Drosophila melanogaster, and the German cockroach, Blattella germanica, respectively. Northern blots show that topical application of JH III increases HMG-R transcript levels in male thoraces in an apparent dose- and time-dependent manner. These data support the model that JH III raises HMG-R transcript levels, resulting in increased activity of the isoprenoid pathway and de novo pheromone production.

The Δ12 desaturase from the house cricket, Acheta domesticus (Orthoptera: Gryllidae): characterization and form of the substrate.

A novel delta 12-desaturase from animals, which converts oleic acid (18:1n-9) to linoleic acid (18:2n-6), was characterized in the house cricket, Acheta domesticus. The delta 12-desaturase product, linoleic acid, was determined by silver nitrate thin-layer chromatography, radio-gas-liquid chromatography and radio-high-performance liquid chromatography with the latter being used for routine analyses. Enzyme activity was located in the microsomal fraction of whole insect homogenates. NADPH or NADH was required for activity, with NADPH being the more efficient electron donor. In short incubation times with oleoyl-CoA as substrate, the highest amount of product, linoleic acid, was found as linoleoyl-CoA. With longer incubation periods, most of the linoleic acid was recovered in the polar lipid fraction containing phospholipid. Preincubation of the microsomal preparation in the absence of NADPH, which allowed 90% of the oleoyl moiety to be transacylated into complex lipid, resulted in no detectable desaturation upon addition of NADPH. These data indicate that the oleic acid moiety used as substrate was in the form of a CoA derivative and not in the form of a phospholipid, as it is for the plant delta 12-desaturase. This is the first characterization of a delta 12-desaturase from an animal system and the first report of a delta 12-desaturase that uses oleoyl-CoA as substrate.

Pheromone precursor synthesis is localized in the metathorax of Ips paraconfusus Lanier (Coleoptera : Scolytidae)

Pheromone precursor synthesis is localized in the metathorax of Ips paraconfusus Lanier (Coleoptera : Scolytidae)

De novo biosynthesis of linoleic acid in two non-insect invertebrates: The land slug and the garden snail

The de novo biosynthesis of linoleic acid [18:2(n-6)], a fatty acid considered to be essential for most animals, was demonstrated in the land snail,Bulimulis alternatus mariae, and the garden slug,Arion circumscriptus. Radiolabeled acetate injected into the animals was incorporated into both 18:2 and 20:2, as demonstrated by radio-high performance liquid chromatography (radio-HPLC) and radio-gas chromatography (radio-GC). GC-mass spectrometry (GC-MS) of the methoxy derivatives of the 18:2 and 20:2 isolated from the snail showed that major isomers had the double bonds in the n-6,9 positions. Radio-gas-liquid chromatography (radio-GLC) of the ozonolysis products from the labeled dienoic fatty acid methyl esters showed that both ends of the molecules were labeled, confirming de novo synthesis. The production of linoleic acid by these animals suggests the capability to produce linoleic acid may be widespread in invertebrates.

Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences.

We have isolated fatty acyl‐CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two ∼1.66 kb cDNAs were recovered. They had identical coding regions and 3′ untranslated regions (UTRs), but differed in their 5′ UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta‐9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a Δ9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more Δ9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl‐CoA desaturases are a dynamically evolving gene family.

Characterization of the Δ12 desaturase in the American cockroach, Periplaneta americana: The nature of the substrate

The cockroach, Periplaneta americana, can convert oleic acid (18:1(n - 9], to linoleic acid, (18:2(n - 6], by a microsomal delta 12 desaturase. Most of the desaturase activity was present in the fat body tissue, with lower activity in the epidermis and no detectable activity in the thorax or gut tissue. In incubations of microsomal preparations from fat body tissues with [1-14C]18:1-CoA, increased amounts of [1-14C]18:2 were found with increasing time and protein concentration. The form of the substrate for the delta 12 desaturase was determined to be 18:1-CoA by comparing activity towards [1-14C]18:1-CoA and [1-14C]18:1 transesterified to phospholipid. Ozonolysis of the 18:2 formed from [1-14C]oleoyl-CoA followed by radio-gas-liquid chromatography gave one labeled peak, 9-oxononanoate, which showed that the product of the delta 12 desaturase is the physiologically important isomer, 18:2(n - 6).

Biochemical and molecular characterizaton of house cricket (Acheta domesticus, Orthoptera: Gryllidae) Δ9 desaturase ☆

Studies of insect fatty acyl-CoA desaturases have heretofore concentrated on the Diptera and Lepidoptera. We report here the isolation and characterization of a fatty acyl-CoA Δ9 desaturase from the house cricket, Acheta domesticus (Orthoptera). Two desaturase cDNAs were isolated from a library, one of which contained two intron sequences. The clones were identical in their respective coding regions, but had divergent 5′ and 3′ untranslated regions. The cDNAs encode a 359 amino acid desaturase enzyme that could rescue a fatty acyl-CoA desaturase auxotrophic phenotype when expressed in yeast. Biochemical analysis of lipids from transformed yeast cells confirmed that the enzyme is a Δ9 desaturase with activity on both palmitic and stearic acid substrates. Southern blotting indicated multiple Δ9 desaturase genes within the genome. A single message that was up-regulated in fed insects vs. starved insects was observed on northern blots, indicating transcriptional regulation in response to diet.

Insect tissues, not microorganisms, produce linoleic acid in the house cricket and the American cockroach

Biosynthesis of linoleic acid, 18∶2(n−6), was unambiguously demonstrated to occur in the cockroach,Periplaneta americana, and the cricket,Acheta domesticus. Axenic tissue from both of these insect species was demonstrated by radio-gas-liquid chromatography (radio-GLC) and radio-high-performance liquid chromatography (radio-HPLC) to incorporate [1-14C]acetate and [1-14C]oleate into this essential fatty acid.