Biosynthesis of fatty acids in cell-free homogenates of lactating gerbil mammary gland

Cell-free homogenates of lactating mammary gland of gerbils maintained on a diet of sunflower seed, guinea pig chow and oats (Diet 1) or a diet of guinea pig chow and oats (Diet 2) and of rats maintained on laboratory chow (Diet 3) were incubated with¹⁴C-labeled acetate, acetyl CoA or malonyl CoA aerobically. A large proportion of the¹⁴C from¹⁴C-acetate and¹⁴C-acetyl CoA incorporated into fatty acids by homogenates from gerbils on Diet 1 was in unsaturated compounds, particularly in 18-carbon and 20-carbon dienoic acids, compared to preparations from animals on Diets 2 or 3. The two radioactive dienoic acids were proven to be Δ^9,12 18∶2 and Δ^11,14 20∶2, and the latter was shown to be a direct elongation product of Δ^9,12 18∶2 by the substrate¹⁴C-acetyl CoA. In all experiments¹⁴C from¹⁴C-malonyl CoA was incorporated predominantly into 14∶0 and 16∶0, and very little incorporation occurred into unsaturated fatty acids in homogenates made either from gerbil or rat. Total fatty acids isolated from homogenates and from milk fat (fat floating on the centrifuged homogenates) of gerbils on Diet 1 had a higher proportion of 18∶2 than animals on the other two diets, a reflection of the large dietary intake of linoleic acid by gerbils on Diet 1. Under these conditions the amount of 18∶2 in the mammary gland had a significant effect on the products of the incubation.     

The effect of dietary fat on fatty acid synthesis in cell-free preparations of lactating mammary gland

Cell-free preparations of lactating mammary gland of rats maintained during lactation on a fat-free diet incorporated C¹⁴-acetate into fatty acids to a greater degree than preparations made from rats fed a similar diet containing 20% fat. The type of fat used did not affect the degree of inhibition of synthesis. C¹⁴-acetate was incorporated mainly into dodecanoic and tetradecanoic acids although labeling was observed in fatty acids from 8–18 carbons. The pattern of labeling was not significantly different in the various groups except for slightly decreased amounts of C¹⁴ in the shorter chain fatty acids of preparations made from glands of rats on the fat-free or coconut oilcontaining diet. The fatty acids characteristic of the fed fat became prominent components of the microsomes and mitochondria as well as of the fat floating on the centrifuged homogenates (presumably milk fat). Presented at the AOCS Meeting, Chicago, October 1964.     

Asymmetric distribution of decanoate in triacylglycerol synthesized in vitro by mammary glands of lactating mice

Slices, prepared from the mammary glands of lactating mice, were incubated with either [1-¹⁴C]acetate, [U-¹⁴C]glucose, or [1-¹⁴C]decanoate. From all 3 substrates, radioactivity in the synthesized lipids was found mainly in triacylglycerols (TG). When acetate or glucose served as substrate, decanoate (C₁₀) accounted for 24% of the fatty acids in TG. Hydrolysis of the TG by pancreatic lipase yielded [¹⁴C] fatty acids which had relatively more C₁₀ (38%) than did either of the other hydrolysis products mono- or diacylglycerol (14–17%). However, when TG produced by slices from C₁₀ were hydrolyzed, the acid was found to be esterified equally at the C-1, C-2 and C-3 of glycerol. Thus, when fatty acids are synthesized de novo and are converted to TG by gland slices, C₁₀ is predominantly located in the C-3 position, a finding in accord with the situation in milk TG, although such preferential incorporation does not occur when the free acid is presented to the tissue slices.     

Triglyceride synthesis from dihydroxyacetone phosphate and palmitate by microsomes from mammary glands of lactating mice

Both di- and triglycerides were synthesized when microsomes isolated from mammary glands of lactating mice were incubated with dihydroxyacetone phosphate (DHAP), (1-¹⁴C)palmitate, ATP, CoASH, GSH, KF, MgCl₂, and NADPH. When NADH replaced NADPH, glyceride synthesis was very low. In the absence of either NADPH or NADH, DHAP was acylated to palmityl-DHAP. Since microsomes do not have glycerol 3-phosphate NAD:oxidoreductase activity, we inferred that glycerol 3-phosphate (GP) is not an intermediate in triglyceride biosynthesis from DHAP. This reductase, present in the cytosol, was active only with NADH. With the same concentration of either GP or DHAP, microsomes yielded essentially similar amounts of di- and triglycerides. Mitochondria, while capable of synthesizing palmityl-DHAP, did not produce di- and triglycerides.     

Effect of human mammary MX-1 tumor on plasma free fatty acids in fasted and fasted-refed nude mice

We hypothesized that tumor-bearing (TB) nude mice, because they are reported to have no detectable, circulating tumor necrosis factor (TNF)/cachectin, would regulate their plasma free fatty acid (FFA) levels normally when fasted and refed. We compared levels of individual plasma FFA in response to fasting (24 hr) and to refeeding (for 20 hr after fasting) a fat-free, 65% sucrose diet in control, nude mice and in mice bearing 1.3±0.4 g MX-1 tumors. Total plasma FFA levels were typically high in 24-hr fasted mice [mean concentrations in μM±SE (n); controls 515±63 (6); TB, 625±63 (6)]. FFA levels were reduced by 65% in each group in response to refeeding. Each major plasma FFA species fell in response to refeeding, except arachidonate, which did not change significantly (fastedvs. fasted-refed concentrations) in either controls or TB mice. In refed mice, the molar FFA ratio of oleate to linoleate rose; however, that of oleate to arachidonate fell markedly. TB nude mice had normal appetites. We conclude that all species of FFA were mobilized from adipose tissue in a normal manner in TB nude mice; therefore, regulation of adipose triacylglycerol fatty acid mobilization (as plasma FFA) by dietary sugar is probably not affected by MX-1 tumor growth in these mice. Our findings are consistent with the hypothesis that nude mice may be unable to secrete TNF/cachectin in response to tumor growth, but they do not establish whether or not endogenous, circulating TNF/cachectin increases FFA mobilization in any TB animal.     

Effect of polyunsaturated fatty acids in obese mice

Genetically obese (ob/ob) mice display a variety of metabolic differences from lean litter mates. In the obese state, fatty acid desaturation-elongation in brown adipose tissue mitochondria is apparently altered, resulting in differences in membrane fatty acid composition. This change in membrane lipid environment appears to influence GDP binding and there-fore the activity of the proton conductance pathway associated with regulation of energy expenditure in these animals. In liver, binding of insulin to the nuclear membrane is increased by feeding a high polyunsaturated/saturated (P/S) diet fat. Consumption of a high P/S diet decreased mRNA levels for fatty acid synthase, acetyl-CoA carboxylase, malic enzyme, and pyruvate kinase in obese and lean animals. Expression of mRNA for these lipogenic enzymes was higher in obese animals and suggests that obese mice may be resistant to polyunsaturated fatty acid feedback control of gene expression.     

Effect of dietary fatty acid composition on the biosynthesis of unsaturated fatty acids in rat liver microsomes

A study was made of the influence of semisynthetic diets of low and high unsaturation on the fatty acid composition and desaturation-chain elongation enzymatic activity of the liver microsomal fractions of male Sprague-Dawley rats of different ages. Groups of rats were fed 5 or 20% coconut oil (CO), or a 5 or 20% mixture of corn and menhaden oils (3∶7) (CME) from weaning to 100 wk of age. Growth rate and food consumption were measured during this period in which animals were sacrificed at 36, 57, 77 and 100 wk of age. Both the level and composition of the dietary fat supplements produced marked effects on the fatty acid composition of the liver microsomal lipids. In general, the fatty acid composition of the microsomal fractions reflected that of the dietary fat and was more unsaturated with the higher level of fat fed. The rate of conversion of linoleic to arachidonic acid in assays performed in vitro with liver microsomal preparations from animals of the different groups also showed marked differences. The 6-desaturase-chain elongation activity was higher in the 5% than 20% group and corresponded to the essential fatty acid (EFA) status of the animals in these groups as represented by the triene-tetraene ratio of the microsomal lipid. The relationship of the 6-desaturase activity to fatty acid composition of the microsomal lipid indicated that if varied directly with the level of 20∶3ω9, 18∶1 and 16∶1 and was inhibited by arachidonic acid. The activity of the 6-desaturase enzyme system was lowest in the liver microsomal fraction obtained from the animals fed the CME diets and appeared to be suppressed by the high levels of 20∶5 and 22∶6 that accumulated in the microsomal lipid. Accordingly, the levels of arachidonic acid were lower in the microsomal lipid of these groups than those of the corresponding CO groups in spite of a greater abundance of linoleic acid in the diet. The data suggest that the activity of the 6-desaturase-chain elongation system is regulated by the fatty acid composition of the microsomal lipid as influenced by the composition of the dietary fat.     

Effect of dietary fats on desaturase activities and the biosynthesis of fatty acids in rat-liver microsomes

Four groups of rats were fed diets containing 15% (w/w) high-oleic safflower oil (SFO, rich incis-18∶1 acids), a mixture of 80% partially hydrogenated soybean oil plus 20% corn oil (H+CO, rich intrans-18∶1 acids), lard (L, rich in saturated fatty acids) and corn oil (Co, rich in 18∶2ω6). Fatty acid composition of liver microsomes and activities of the Δ⁵, Δ⁶ and Δ⁹ desaturases were determined. Microsomal Δ⁶ desaturase activity and arachidonic acid were lower in the H+CO group compared with SFO of L. No difference was found in the Δ⁵ or Δ⁶ desaturase activity of CO and SFO groups. Thus, the oleic-acid level of the SFO diet had no effect on the metabolism of 18∶2ω6. Fluorescent polarization studies, usingtrans-parinaric acid as a probe, showed no differences between the physical states of phospholipid vesicles made from lipids isolated from each group. We concluded that thetrans-18∶1 acids in partially hydrogenated soybean oil have a more inhibitory effect than saturated acids on EFA metabolism, even in the presence of adequate amounts of essential fatty acid.     

Effect of dietary organic selenium on fatty acid composition in nutria (Myocastor coypus Mol.) livers

Young, female nutrias (n = 13) were fed a diet supplemented with 0.36 mg/kg selenium in selenium‐enriched yeast (SeY; Sel‐Plex; Alltech, Inc., Nicholasville, KY) for 60 days (total Se concentration in the basal diet was ～0.1 mg/kg). Concentrations of fatty acids (FA) in the liver were compared to those of nutrias on a control diet (n = 11). Animals were sacrificed at 8 months of age and liver samples (approximately 30 g) were collected. The gas‐chromatographic analysis of tissue samples from the experimental group revealed a significant decrease in saturated fatty acids (p<0.001), monounsaturated fatty acid (p = 0.006), and polyunsaturated fatty acid (PUFA) (p = 0.02) compared to controls. The linoleic and linolenic acids, which are precursors of n‐6 and n‐3 PUFA, respectively, were significantly lower (p = 0.01 and p<0.001, respectively) in the supplemented group. The n‐6 to n‐3 PUFA ratio was significantly affected (p = 0.001) by the SeY dietary supplement (13.17 vs. 8.93, respectively).     

Inhibition of phenytoin bioactivation and teratogenicity by dietary n−3 fatty acids in mice

Evidence suggests that the teratogenicity of the anticonvulsant drug phenytoin (DPH) can result from its bioactivationvia embryonic prostaglandin synthase and/or maternal cytochromes P450. This study examined whether DPH bioactivation and teratogenicity could be reduced by dietary n−3 fatty acids. Female CD-1 mice were fed diets containing 2 wt% safflower oil and 10 wt% of either hydrogenated coconut oil, safflower oil, or a cod liver oil/linseed oil mixture (CLO/LO) for three weeks prior to impregnation and throughout gestation. DPH (55 or 65 mg/kg) was administeredvia intraperitoneal injections to pregnant mice at 0900 on gestational days 12 and 13, and on day 19 fetuses were given teratologic assessments. A similar dietary study evaluatedin vivo covalent binding of radiolabeled DPH administered on day 12, and dams were killed 24 h later. A reduction in DPH-induced cleft palates and a decrease in DPH covalent binding to embryonic protein was observed in the CLO/LO group. Feeding CLO/LO enhanced incorporation of n−3 fatty acids into embryos and inhibited embryonic prostaglandin synthase activity. No differences in maternal hepatic cytochromes P450 activities were observed among dietary treatments. These data indicate that dietary n−3 fatty acids could reduce DPH teratogenicityvia inhibition of embryonic prostaglandin synthase bioactivation of DPH. Presented in part at the annual meeting of the Federation of the American Societies for Experimental Biology, New Orleans, LA, May 1989 [Kubow, S. (1989)FASEB J. 3, A726] and at the annual meeting of the Canadian Federation of Biological Societies, Kingston, Ontario, Canada, June 1991 [Kubow, S. (1991)Proc. Can. Biol. Soc. 34, 117].     

Interaction of dietary fatty acids and cyclosporine a in the borderline hypertensive rat: Tissue fatty acids

In this study we examined (i) the effects of cyclosporine A (CS) on tissue lipid composition and (ii) the effect of changes in dietary n−6 fatty acids on tissue responses to CS. Fatty acid composition of liver, kidney, heart and brain were determined after 4 wk of treatment with CS (10 mg/kg·d p.o.) in male borderline hypertensive rats (BHR, n=4/group), whose diet was supplemented with either safflower oil or evening primrose oil (EPO). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine/phosphatidylinositol, triglyceride and cholesteryl ester fatty acids were measured in kidney, heart, brain and liver. The same parameters were also measured in safflower-fed BHR (n=4) receiving placebo. The effects of CS on liver microsomal Δ9, Δ6 and Δ5 desaturasesin vitro were also followed. CS affected the fatty acid composition of all tissues examined, with the greatest changes seen in the renal phosphatidylcholine and phosphatidylserine/phosphatidylinositol fractions. All CS-induced changes that occurred in the liver, brain and renal fatty acids were reversed by EPO. CS elevated Δ9 desaturase but had no effect on Δ6 and Δ5 desaturase. In light of (i) the observation that EPO normalizes renal function and blood pressure in CS-treated BHR, and (ii) the importance of the kidney in blood pressure regulation, the data suggest that the beneficial effects of EPO on CS toxicity may involve changes in renal phospholipid fatty acid profiles.     

n-3 polyunsaturated fatty acids endogenously synthesized in fat-1 mice are enriched in the mammary gland

In this study, we determined the phospholipid FA composition in the mammary gland of the transgenic Fat-1 mouse. This is the first animal model developed that can endogenously synthesize n−3 PUFA. The synthesis of n−3 PUFA is achieved through the expression of the fat-1 transgene encoding for an n−3 desaturase from Caenorhabditis elegans, which utilizes n−6 PUFA as substrate. Wild-type and Fat-1 female mice were terminated at 7 wk of age and the fifth mammary gland was removed. Lipids were extracted and phospholipids were separated by TLC and converted to FAME for analysis by GC. There was no significant change in total saturated, monounsaturated, and PUFA composition. However, there was a significant increase in total n−3 PUFA and a corresponding decrease in n−6 PUFA. The major n−3 PUFA that were enriched included 20:5n−3 and 22:6n−3. The n−6 PUFA that were reduced included 20:4n−6, 22:4n−6, and 22:5n−6. Overall, these findings demonstrate that female Fat-1 mice have elevated levels of n−3 PUFA in the mammary gland. Moreover, the n−3 desaturase products are the same n−3 PUFA found in fish oil, which have been shown to have chemoprotective properties against breast cancer. Therefore, this newly developed mouse model may be highly useful for investigating molecular and cellular mechanisms by which n−3 PUFA prevents and inhibits breast cancer growth.     

Placental and mammary transfer of chlorinated fatty acids in rats

Oleic acid was chlorinated with ³⁶Cl and administered orally along with [³H]oleic acid to both pregnant and lactating rats. There was about one-half as much ³⁶Cl as ³H in foetal lipids on the day after dosing of the dams and the level of ³⁶Cl declined more rapidly than ³H, indicating that the foetus may have the ability to dechlorinate fatty acids or selectively to eliminate them from the body. Greater amounts of chlorinated lipid crossed the placenta into the foetus as gestation advanced, reaching an average of 0·42% of the dose at day 19. Mammary transfer of ³⁶Cl from chlorinated oleic acid into the body lipids of 2-wk-old suckling rats was 15·9% in 24 hr compared with 34·0% for [³H]oleic acid, while the corresponding transfer of ³⁶Cl-chlorinated linoleic and linolenic acids accounted for only 2·43 and 2·73% of the dose, respectively.     

Effect of dietary safflower oil upon lipogenesis in neonatal lamb

The effect of dietary safflower oil upon lipogenesis has been investigated in neonatal lambs. Preliminary experiments with lambs suckled by their mothers showed that there was a 10-fold increase in the rate of incorporation of [¹⁴C] from acetate into fatty acids in adipose tissue slices during the first 10 days post partum. Barely detectable rates of [¹⁴C] acetate incorporation into fatty acids were found in liver slices from lambs during the same period. In lambs given cows' milk from birth until 11 days of age, there was also a 10-fold increase in the rate of lipogenesis in adipose tissue slices. Supplementing the diet of cows' milk with safflower oil (5 ml/lamb/day) resulted in significantly lower rates of lipogenesis in adipose tissue slices from 11 day old lambs. Administration of safflower oil had no effect upon the concentration of unesterified fatty acids, including linoleic acid, in the lamb adipose tissue slices. The data show that lipogenesis in ovine adipose tissue, like that in rodent liver and adipose tissue, is sensitive to dietary polyunsaturated fatty acids, and that, for the neonatal lamb, the effect of polyunsaturated fatty acids upon lipogenesis is not dependent upon an increase in the tissue concentration of polyunsaturated fatty acids.     

Effects of dietary polyunsaturated fatty acids on neuronal function

Diets deficient in linoleic acid (18∶2n−6), or that have unusual ratios of linoleic acid to α-linolenic acid (18∶3n−3) induce changes in the polyunsaturated fatty acid (PUFA) composition of neuronal and glial membranes. Such changes have been linked to alterations in retina and brain function. These functional effects are presumed to follow from the biochemical consequences of modifying membrane PUFA content; known effects include modifications in membrane fludity, in the activities of membrane-associated, functional proteins (transporters, receptors, enzymes), and in the production of important signaling molecules from oxygenated linoleic and α-linolenic acid derivatives. However, despite the demonstration that central nervous system function changes when dietary PUFA intake is altered, and that in general, membrane PUFA content influences membrane functions, little work has focused specifically on brain and retina to reveal the underlying biochemical bases for such effects. This review examines this issue, looking at known effects of dietary PUFA on neurons in both the central and peripheral nervous systems, and attempts to identify some approaches that might promote productive investigation into the underlying mechanisms relating changes in dietary PUFA intake to alterations in neuronal and overall nervous system functioning.     

Effect of low levels of dietary fish oil on fatty acid desaturation and tissue fatty acids in obese and lean rats

The effect of very low levels of dietary long-chain n−3 fatty acids on Δ6 desaturation of linoleic acid (18∶2n−6) and α-linolenic acid (18∶3n−3), and on Δ5 desaturation of dihomo-γ-linolenic acid (20∶3n−6), in liver microsomes and its influence on tissue fatty acids were examined in obese and lean Zucker rats and in Wistar rats. Animals fed for 12 wk a balanced diet containing ca. 200 mg of long-chain polyunsaturated n−3 fatty acids per 100 g of diet were compared to those fed the same amount of α-linoleic acid. Low amounts of long-chain n−3 fatty acids greatly inhibited Δ6 desaturation of 18∶2n−6 and Δ5 desaturation of 20∶3n−6, while Δ6 desaturation of 18∶3n−3 was not inhibited in Zucker rats and was even stimulated in Wistar rats. Inhibition of the biosynthesis of long-chain n−6 fatty acids was reflected in a decrease in arachidonic acid (20∶4n−6) content of serum lipids when fasting, and also in the phospholipid fatty acids of liver microsomes. On the contrary, heart and kidney phospholipids did not develop any decrease in 20∶4n−6 during fish oil ingestion. Docosahexaenoic acid (22∶6n−3), present in the dietary fish oil, was increased in serum lipids and in liver microsome, heart, and kidney phospholipids.     

Effect of subacute toxic levels of dietary cyclopropenoid fatty acids upon membrane function and fatty acid composition in the rat

The effect of subacute toxicity levels of dietary cyclopropenoid fatty acids upon several physiological parameters was determined in the rat. Diets containing 2% corn oil, 2%Sterculia foetida oil or 2% hydrogenatedSterculia foetida oil were fed.Sterculia foetida oil (50% cyclopropenoid fatty acids) fed rats exhibited retarded growth, elevated organ to body wt ratios, increased saturation of tissue lipid, and abnormal histopathology when compared to corn oil and hydrogenatedSterculia foetida oil fed rats. Growth was retarded 50%, liver/body wt doubled, and the percentage of saturated fatty acids in adipose tissue increased 2.5-fold forSterculia foetida oil vs. corn oil comparisons. Three membrane systems were examined in corn oil andSterculia foetida oil fed rats. Erythrocyte hemolysis rate in 0.3 M glycerol was increased by 30%; induction of mitochondrial swelling by reduced glutathione was inhibited completely and microsomal codeine demethylase activity was depressed nearly 50% inSterculia foetida oil fed rats. The ability of cyclopropenoid fatty acids to inhibit fatty acyl desaturase and influence tissue and membrane lipid composition is discussed. Most of the detrimental effects observed in cyclopropenoid fatty acids fed rats may be associated with alteration of normal lipid metabolism and membrane function.     

Effect of dietary fat on the lipid composition and utilization of short-chain fatty acids by rat colonocytes

The objective of the present studies was to examine the effect of dietary fat on the lipid composition of rat colonocytes and their utilization of short-chain fatty acids (SCFA). Rats were fed 14% beef fat, fish oil or safflower oil plus 2% corn oil in a semi-synthetic base diet for 4 wk. Colonocytes were isolated and their lipid composition was examined. Feeding beef fat and fish oil resulted in an increase in monounsaturated fatty acids and a reduction in ω-6 fatty acids. Feeding fish oil resulted in an enrichment with ω-3 fatty acids. These was no dietary influence on the amount of either cholesterol or phospholipids of colonocytes. Fish oil feeding resulted in significant increase in colonocyte free fatty acids (FFA) as compared to other diets. Dietary fat was found to have no effect on SCFA utilization by colonocytes. Colonocytes were found to utilize SCFA in the order of butyrate ≥acetate ≥propionate. The presence of acetate and propionate in the medium had no effect on the rate of butyrate utilization.