Salivary alpha amylase and salivary cortisol response to fluid consumption in exercising athletes.

The objective of the study was to examine salivary biomarker response to fluid consumption in exercising athletes. Exercise induces stress on the body and salivary alpha amylase (sAA) and salivary cortisol are useful biomarkers for activity in the sympathoadrenal medullary system and the hypothalamic pituitary adrenal axis which are involved in the stress response. Fifteen college students were given 150 ml and 500 ml of water on different days and blinded to fluid condition. The exercise protocol was identical for both fluid conditions using absolute exercise intensities ranging from moderate to high. Saliva was collected prior to exercise, post moderate and post high intensities and analyzed by Salimetrics assays. Exercise was significant for sAA with values different between pre-exercise (85 ± 10 U · ml(-1)) and high intensity (284 ± 30 U · ml(-1)) as well as between moderate intensity (204 ± 32 U · ml(-1)) and high intensity. There was no difference in sAA values between fluid conditions at either intensity. Exercise intensity and fluid condition were each significant for cortisol. Cortisol values were different between pre-exercise (0.30 ± 0.03 ug · dL(-1)) and high intensity (0.45 ± 0.05 ug · dL(-1)) as well as between moderate intensity (0.33 ± 0.04 ug · dL(-1)) and high intensity. Moderate exercise intensity cortisol was lower in the 500 ml condition (0.33 ± 0.03 ug · dL(-1)) compared with the 150 ml condition (0.38 ± 0.03 ug · dL(-1)). This altered physiological response due to fluid consumption could influence sport performance and should be considered. In addition, future sport and exercise studies should control for fluid consumption.

The effects of varying dietary fat on the nutrient intake in male and female runners.

OBJECTIVE: The present study examined the effects of varying dietary fat levels on nutrients in female and male endurance runners. METHODS: Three diets (low, medium and high fat) were designed for each subject using their food preferences and three-day food records. Each diet was eaten for 28 to 31 days. The diets were self-selected from seven-day sample menus. Twelve male and 13 female runners between 18 and 55 years of age who averaged 42 miles/week participated in the study. Daily food intakes, activity records and weekly palatability/hunger scales were completed. RESULTS: Dietary fat intakes, as a percent of total energy intake (%E), averaged 17%E, 31%E, and 44%E on the low, medium and high fat diets, respectively. Energy consumption was less than their estimated energy expenditure (EEE) on all diets. On the low fat diet, the female runners were consuming approximately 60% of their EEE. As dietary fat increased, the difference between calorie intake and estimated energy expenditure became less and the subjects were less hungry on the two higher fat diets. For all subjects, as energy intakes increased, so did carbohydrate intake. Therefore, carbohydrate intake was not different on the two lower fat diets. Irrespective of gender, calcium and zinc intakes, which were below 1989 RDAs, increased with increasing fat intakes, between the low and medium fat diets. Zinc intake was also higher on the highest fat diet. Essential fatty acid intakes for females on the low fat diet were less than 2.5%E. Half of the female runners ate less than the RDA of calcium and zinc on the low fat diet and Fe on the medium fat diet. CONCLUSION: This study suggests that endurance runners may not be consuming enough calories on a low fat diet and that increasing dietary fat increased energy consumption. On the low fat diet, essential fatty acids and some minerals (especially zinc) may be too low. A low fat diet could compromise health and performance.

The effects of varying dietary fat on performance and metabolism in trained male and female runners.

OBJECTIVES: Low dietary fat intake has become the diet of choice for many athletes. Recent studies in animals and humans suggest that a high fat diet may increase VO2max and endurance. We studied the effects of a low, medium and high fat diet on performance and metabolism in runners. METHODS: Twelve male and 13 female runners (42 miles/week) ate diets of 16% and 31% fat for four weeks. Six males and six females increased their fat intakes to 44%. All diets were designed to be isocaloric. Endurance and VO2max were tested at the end of each diet. Plasma levels of lactate, pyruvate, glucose, glycerol, and triglycerides were measured before and after the VO2max and endurance runs. Free fatty acids were measured during the VO2max and endurance runs. RESULTS: Runners on the low fat diet ate 19% fewer calories than on the medium or high fat diets. Body weight, percent body fat (males=71 kg and 16%; females=57 kg and 19%), VO2max and anaerobic power were not affected by the level of dietary fat. Endurance time increased from the low fat to medium fat diet by 14%. No differences were seen in plasma lactate, glucose, glycerol, triglycerides and fatty acids when comparing the low versus the medium fat diet. Subjects who increased dietary fat to 44% had higher plasma pyruvate (46%) and lower lactate levels (39%) after the endurance run. CONCLUSION: These results suggest that runners on a low fat diet consume fewer calories and have reduced endurance performance than on a medium or high fat diet. A high fat diet, providing sufficient total calories, does not compromise anaerobic power.

Muscle structure with low- and high-fat diets in well-trained male runners.

Endurance capacity, maximal oxygen uptake capacity (VO2max) and quantitative muscle ultrastructural composition was analyzed in 7 well-trained male runners (mean age 37.1 years, mean VO2max 60 ml/min/kg) after a one month period of a low-fat diet (dietary fat intake 18.4% and a similar period of a high-fat diet (dietary fat intake 40.6%). Between these two interventional periods a washout period of one month was interspersed in which the nutritional fat content was approx. 32%; close to the average American Diet. During all three periods protein content of the nutrition was kept nearly constant at 15%. After the high-fat diet time to exhaustion in the endurance test increased significantly by 21% while VO2max remained unchanged. Muscle mitochondrial volume density remained unchanged while the intramyocellular fat content increased by 60%. Due to large interindividual differences in this variable this difference did not become statistically significant. While some 20% of the mitochondria are located in a subsarcolemmal location, only 10% of the lipid stores are associated with these mitochondria. Less than 2% of the mitochondrial outer surface are in contact with lipid droplets whereas 25-35% of the lipid surface is in contact with mitochondria. None of these variables is significantly altered after a high-fat diet. It is concluded that the change in endurance capacity of the subjects cannot be explained based on the structural changes observed in skeletal muscle tissue. This may be related to methodological problems associated with the determination of intramyocellular fat content.

Influence of the level of dietary lipid intake and maximal exercise on the immune status in runners.

Chronic exercise and high fat diets are associated with immune suppression. This study compares cellular immune responses at rest and after maximal exercise in runners after eating diets comprised of 17% low fat (LF), 32% medium fat (MF), and 41% high fat (HF) (4 wk each). VO2max increased significantly from the 17% to 41% fat diet. The leukocyte cell counts were significantly increased after exercise. In men, significantly higher proliferative response to phytohemagglutinin (PHA) (P < 0.004) was observed with MF diet, while response to pokeweed mitogen (PWM) was significantly decreased by MF and HF diets. The number of CD8+ (suppressor) T cells was significantly higher in men and exercise increased it significantly, while CD4+ (helper) T cells were not affected. Natural killer cells number was significantly increased 2.5 fold by exercise and with increase in dietary fat. The production of IL-2 by peripheral blood mononuclear cells was significantly higher in men (P < 0.0001) and increasing dietary fat significantly increased IL-2 production (P < 0.001). In men, exercise decreased the level of the proinflammatory cytokines (IL-1, IL-6 and TNF-alpha), whereas in women, with the exception of MF diet for IL-6, exercise had no effect. This study indicates that short, intense bouts of exercise in runners training 40 miles.wk-1 have mixed effects on the immune system. A high percentage of fat intake (41%) did not have any deleterious effects on the immune system of the well-trained runners.

Type of dietary fiber, not fat, alters phospholipase D and ornithine decarboxylase activities in the rat large intestine.

We investigated the effect of dietary fatty acid composition (n-6 vs. n-3) and fiber (highly fermentable vs. less fermentable) on the activities of phospholipase D (PLD) and ornithine decarboxylase (ODC) in the rat large intestine (cecum and proximal and distal colon). Twenty-four Sprague-Dawley rats (215-270 g) ate synthetic diets with 2% safflower oil plus 21.5% safflower or fish oil and 10% cellulose or guar gum for four weeks. Cecal bile acids and free fatty acids were higher in rats fed guar gum than in rats fed cellulose. Rats fed fish oil had more proximal colonic mucosal and cecal bile acids than those fed safflower oil. PLD activity was 23% lower in the proximal colon of rats fed guar gum than in those fed cellulose, but the mucosal weight was not different. ODC activity was lower but cecal mucosal wet weight was higher in the cecum of the rats fed guar gum than in the cecum of the rats fed cellulose. The activities of PLD and ODC are affected by dietary fiber and may not be accurate markers for tissue growth in the colonic mucosa.

Effect of membrane lipid alteration on the growth, phospholipase C activity and G protein of HT-29 tumor cells.

The objective of the present study was to examine the effect of modifying the fatty acid composition of membranes on cell growth and phosphoinositide specific phospholipase C (PLC) activity in HT-29 colon cancer cells. Cells were seeded at a density of 12 x 10(3) cells/cm2 and supplemented with 30 microM of either 18:0, 18:2 (n6) or 18:3 (n3) complexed to bovine serum albumin (BSA) in DMEM medium. Cell growth was followed for 12 days. The 18:0 supplemented cells (control) reached maximum growth at day nine which was greater than either 18:2 (n6) or 18:3 (n3) supplemented cells. There was no difference between the latter two groups in their growth. To investigate the fatty acid incorporation of the supplemented fatty acid and how they may influence composition in the cell membrane, we examined the fatty acid composition of each phospholipid (PL) species. Both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly influenced by the type of fatty acid supplemented. Supplementation with 18:0 resulted in HT-29 cell membranes having more monounsaturated fatty acids than the cells grown in the other fatty acids. Polyunsaturated fatty acid (PUFA) supplementation (both 18:2 and 18:3) resulted in the enrichment of PUFA in the PL fractions. Cells supplemented with 18:3 (n3) had the highest unsaturation index in membrane PE as compared to the other phospholipid species. PLC activity of the membranes was measured using PIP2 as a substrate in the presence of 15 micrograms alamethicin and 42 microM free calcium. The contribution of G protein to the activity of the enzyme was assessed using GTP gamma(S). PLC activity of HT-29 cells was 16% higher in the presence of GTP gamma(S) response. GTP gamma(S)-activated PLC activity of 18:3 (n3) supplemented cells was 81% of those supplemented with either 18:0 or 18:2 (n6) cells. It is concluded that the decrease in cell proliferation with supplementation with 18:3 (n3) may be mediated through its inhibitory effect on PLC, which provides the second messengers for protein kinase C (PKC) activation. PLC may be influenced by an increased unsaturation index of the PE fraction of the HT-29 tumor cell membranes.

The role of dietary fat on performance, metabolism, and health.

This paper presents a model to evaluate the nutritional status of trained athletes based on work in our laboratory as well as others. The model proposes that substrate use is set by the muscle fibers recruited, based on the exercise intensity. Second, the substrate available is primarily determined by the intramuscular stores. In trained athletes, intramuscular fat plays an important role in metabolism at exercise intensities as high as 80% of maximal aerobic power. Based on these factors, increasing the fat in the diet (while maintaining adequate intramuscular glycogen) increases VO2max and intramuscular stores of fat (presumably due to increased mitochondrial volume). These two factors result in a significant increase in the time to exhaustion at set levels of exercise (endurance). It also appears that fatigue is associated with depletion of either glycogen or fat. These conclusions hold true for athletes on diets where sufficient calories are taken in to meet demands and for exercise levels below 80% of VO2max, where primarily slow-twitch oxidative fibers are used. These data may not apply in exercise where predominantly fast-twitch fibers are used. Also, these data do not apply to runners eating a hypocaloric diet, where reducing the percentage of carbohydrates may compromise their glycogen stores. It would appear that the fat in the diet can be increased to a very high level without compromising the cardiovascular or immune systems of athletes. Moreover, it can be proposed that these data could be applied to sedentary persons, as long as they are isocaloric. This would imply that the fat consumed in the diet would be used in the muscle, as in the runners, although at a lower level. Thus, the dietary intake should be matched in both total calories and percentage of fats and carbohydrates to calories consumed by daily activity. It should be cautioned that if glycogen and fat stores are compromised, protein resynthesis is inhibited and loss of muscle mass may result. This has a negative effect on the athlete's ability to perform at high levels.

Prostaglandin synthesis in human cancer cells: influence of fatty acids and butyrate.

Previous research has suggested that prostaglandins (PGs) may play a role in the development of colon cancer since tumor cells produce more PGs than normal cells. However, the exact mechanism by which PGs play a role in the development of cancer is not known. In addition, factors that influence PG synthesis are not known since they are complicated by the presence of homeostatic mechanisms. To avoid the homeostatic mechanisms, the present research was designed to examine factors that may influence PG synthesis in an in vitro system, i.e., a tissue culture. We have chosen two human colon cancer cell lines that differ in their ability to metabolize long-chain fatty acids (LCFAs), LS174T cells and HT-29 cells. We examined the effect of LCFAs on their membrane fatty acid composition, growth, and ability to release the main PGs (PGE2 and PGI). The LCFAs used were those most common in the colonic lumen [18:0, 18:2 (n-6), and 18:3 (n-3)]. In addition, we examined the effect of butyrate on the above mentioned parameters. Butyrate is produced in the colon through fermentation of dietary fibers. The data obtained suggest that although both of these tumor cell lines are of human colonic origin, they differ in their response to LCFAs and butyrate in some of the characteristics studied, such as growth, composition of membranes, and the relationship between membrane FA composition and PG synthesis. Polyunsaturated fatty acid supplementation stimulated the growth of HT-29 cells but not of LS174T cells when compared with growth in media supplemented with 18:0.(ABSTRACT TRUNCATED AT 250 WORDS)

18:1 n7 fatty acids inhibit growth and decrease inositol phosphate release in HT-29 cells compared to n9 fatty acids.

Studies have shown that trans fatty acids may play a role in the development of chronic diseases such as heart disease and cancer. The objective of the present project was to examine the effect of supplementation with 18:1 isomers, both positional and geometrical, as compared to 18:0 on the growth, membrane fatty acid composition and the phosphoinositide cycle of HT-29 human colon cancer cells. Cells were supplemented with 30 microM stearic acid (18:0), elaidic acid (18:1, n9, trans), oleic acid (18:1, n9, cis), vaccenic acid (18:1, n7, cis) or trans-vaccenic acid (18:1, n7, trans) as sodium salts complexed to fatty acid-free bovine serum. Cells were grown in these media for 9 days. Cell growth was examined by counting the number of cells and expressed as percentage of control (18:0 supplemented cells). The phosphoinositide (PI) cycle was examined by measuring the inositol phosphate (IP) released from phosphoinositides in the absence (basal) or presence of stimuli (0.1 mM carbachol, 0.1 mM A23187 or 20 mM NaF). The results obtained indicated that cis and trans n7 fatty acids inhibited the growth of HT-29 cells by 11% and 23%, respectively, as compared to 18:0 supplementation. 18:1, n9 had no effect on tumor growth. Supplementation with all forms of 18:1 resulted in an increase in IP and IP2 production as compared to 18:0 supplemented cells without influencing IP3. The presence of the double bond at the 9 position in the supplemented fatty acid increases total IP production by 59% and in the cis form by 37% above the control. The breakdown of phosphoinositides in the absence and presence of several stimuli supports the observed finding on IP. Trans fatty acid supplementation resulted in lower hydrolysis of PI as compared to cis fatty acids. It is concluded that the observed inhibition of tumor growth by the vaccenic acids may be mediated by their effect(s) on the PI cycle which may be associated with their incorporation into membrane lipids.

In vitro [U-14C]glucose utilization by tissues of weanling rats with lateral hypothalamic area lesions one month after lesion production.

The role of the lateral hypothalamic area (LHA) in intermediary metabolism was investigated by quantitation of [U-14C]glucose oxidation to 14CO2 and 14C incorporation into the glycogen and lipid fraction of the liver, epididymal fat pad, and diaphragm. Weanling male Sprague-Dawley rats received bilateral electrolytic lesions in the LHA (LHAL rats). Sham operated rats were either fed ad libitum (CON-ADLIB) or pair-gained to the LHAL rats (CON-PG). The experiment was terminated 1 month after lesion production. LHAL rats were significantly (SIG) lighter and shorter and ate less than CON-ADLIB; LHAL rats were also SIG shorter than CON-PG, pointing to a food intake-independent lesion effect. Both LHAL and CON-PG rats had SIG less percent carcass fat than CON-ADLIB, but there was no SIG difference between LHAL and CON-PG rats. Also, LHAL rats had a SIG higher percentage of carcass protein than both CON-ADLIB and CON-PG. Furthermore, LHAL rats incorporated SIG less glucose into liver glycogen than CON-ADLIB but SIG more into CON-PG, whereas CON-PG rats incorporated SIG less into liver glycogen than CON-ADLIB, again suggesting a food intake-independent effect. There was no difference among the groups in glucose oxidation and incorporation into lipids and glycogen in both diaphragm and epididymal fat pads and liver total lipid. However, livers of CON-PG metabolized SIG more [U-14C]glucose to CO2 than did livers of CON-ADLIB, suggesting a food intake-dependent effect. There was no difference between LHAL and CON-PG rats in this parameter.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro activation and inhibition of rat colonic phospholipase D by fatty acids.

The present study was designed to examine the effect of long-chain fatty acids on phospholipase D (PLD) of the proximal colon mucosa of the rat. These fatty acids are present in the colon, in close contact with the membranes which contain the enzyme. The results indicate that unsaturated fatty acids activate the enzyme. At 4 mM, trienes are more potent activators as compared to 18:2 and 18:1. The number of double bonds and the configuration around the double bond were found to be important factors in activation of the enzyme. The metabolism of polyunsaturated fatty acids may be involved in the activation since the presence of antioxidants and cyclooxygenase inhibitors were found to influence the activation of PLD by polyunsaturated fatty acids.

Influence of dietary fat and feeding period on phosphoinositide metabolism in rat colonocytes.

The objective of the present study was to examine the effect of dietary fat content on phosphoinositide (PI) metabolism, fatty acid composition in colonocytes, and colonic luminal content of bile acids (BA) and free fatty acids (FFA) in rats. Male Sprague-Dawley rats weighing approximately 166 g were fed to semipurified diet containing 3% or 21.5% beef fat and 2% corn oil. The nonfat ingredients were adjusted to correct for differences in food consumption of these diets. Animals were fed these diets ad libitum for one or four weeks. The isolated colonocytes had a viability of 88.9% in all groups. PI metabolism was examined in the absence (basal) or presence of agonists, 2 mM deoxycholic acid or 10 microM A23187. Dietary fat concentration had no effect on PI metabolism, but the length of feeding had a significant effect on basal and stimulated PI metabolism. Colonocytes of animals fed the diets for four weeks were less sensitive to stimulation of PI cycle by agonists than those of animals fed for one week. Colonocyte fatty acid composition was influenced by dietary fat and feeding period. Only the relative percentage of 20:3(n-6) was significantly lower in rats fed the high-fat diet for one week; 18:0 was lower and 18:3(n-6) was higher in colonocytes of animals fed the diets for one week than in those fed for four weeks. Several colonic fatty acids, namely, 16:0, 20:3(n-6), and 22:5(n-6), also exhibited diet-by-feeding period interaction. Intracolonic luminal contents from rats fed the high-fat diet contained elevated concentrations of BA and FFA (44% and 62%, respectively). It was concluded that despite the effects of dietary fat concentration on increased colonic BA and FFA and on altered membrane fatty acid composition, dietary fat had no effect on PI metabolism in colonocytes under the conditions in the present experiment. A difference in components between the purified diet and the commercial rat chow and/or an aging effect of the rats may alter the PI cycle of colonocytes.

Differential response of glucose utilization by three rat muscle tissues to dietary fatty acid composition.

Rats were fed a semisynthetic diet containing 14% of either beef fat, safflower oil, or menhaden oil plus 2% corn oil for 7 weeks, and three tissues, diaphragm, heart, and skeletal muscle, were examined for fatty acid composition in their phospholipids and triglycerides. In addition, the lipid concentrations in these tissues were examined. The in vitro oxidation and incorporation of glucose into lipids of these tissues were also examined. Skeletal muscle showed the greatest change in phospholipid composition with diet. All tissues were responsive to changes in diet in regards to the triglyceride fraction. Dietary alteration of tissue phospholipid composition did not alter lipid concentration in these tissues. However, in diaphragm tissue, rats fed the beef fat diet had lower phospholipid and higher triglyceride synthesis compared with those fed either menhaden oil or safflower oil. In addition, triglyceride synthesis was higher in the diaphragm of animals fed the menhaden oil diet as compared with the safflower oil diet. Therefore, dietary fatty acid composition may play a role in the triglyceride and phospholipid metabolism of rat diaphragm.

Effect of dietary fat on metabolic adjustments to maximal VO2 and endurance in runners.

The present study examined the effects of dietary manipulations on six trained runners. The percent energy contributions from carbohydrate, fat, and protein were 61/24/14, 50/38/12, and 73/15/12 for the normal (N), fat (F), and carbohydrate (C) diets, respectively. Expiratory gases and blood responses to a maximum (VO2max) and a prolonged treadmill run were determined following 7 d on each diet. Free fatty acids (FFA), triglycerides, glycerol, glucose, and lactate were measured. Dietary assessment of subjects' N diet indicated that they were consuming approximately 700 kcal.d-1 less than estimated daily expenditures. Running time to exhaustion was greatest after the F diet (91.2 +/- 9.5 min, P < 0.05) as compared with the C (75.8 +/- 7.6 min, P < 0.05) and N (69.3 +/- 7.2 min, P < 0.05) diets. VO2max was also higher on the F diet (66.4 +/- 2.7 ml.kg-1 x min-1, P < 0.05) as compared with the C (59.6 +/- 2.8 ml.kg-1 x min-1, P < 0.05) and N (63.7 +/- 2.6 ml.kg-1 x min-1, P < 0.05) diets. Plasma FFA levels were higher (P < 0.05) and glycerol levels were lower (P < 0.05) during the F diet than during the C and N diets. Other biochemical measures did not differ significantly among diets. These data suggest that increased availability of FFA, consequent to the F diet, may provide for enhanced oxidative potential as evidenced by an increase in VO2max and running time. This implies that restriction of dietary fat may be detrimental to endurance performance.

Alteration of membrane fatty acid composition and inositol phosphate metabolism in HT-29 human colon cancer cells.

The present study was designed to investigate the role of membrane fatty acid (FA) composition on inositol phosphate (InsP) release by a human colon tumor cell line. Cells were supplemented for five days in culture with 0, 10, 30, or 100 microM sodium stearate (18:0), linoleate [18:2(omega-6)], or linolineate [18:3(omega-3)]. These FAs were supplied as a complex with FA-free bovine serum albumin. InsP release was examined in these cells with or without stimulation with deoxycholic acid (DCA) after they were labeled with [3H]myoinositol. FA enrichment was found to influence inositol incorporation into membrane lipids. Although 18:0 had no effect, 18:2(omega-6) decreased the incorporation. On the other hand, 18:3(omega-3) increased the incorporation of inositol compared with the cells supplemented with the other FAs, but they were not different from control. Basal release of total InsP was elevated only with supplementation of 10 and 30 microM 18:3(omega-3). FA supplementation with 18:0 at 30 microM and 18:2 at 30 and 100 microM resulted in downregulation of bsal release of InsP. Enrichment of HT-29 cell membranes with polyunsaturated FAs resulted in a significant increase in stimulated release of InsP, but this was not seen with saturated FA supplementation. At 10 microM supplementation, 18:2 had the greatest effect on stimulated InsP release. This effect of 18:2 disappeared at 30 microM. However, the increase in the stimulated InsP release caused by 18:3 occurred at 10 and 30 microM. DCA-stimulated release of InsP was not downregulated by any FA supplementation. This study showed that enrichment of the membranes with polyunsaturated FAs increases the response of the phosphatidylinositol cycle to DCA stimulation. In addition, enrichment with 18:3(omega-3) increases the basal turnover of InsP. It is concluded that alteration of membrane FAs has a profound effect on the phosphatidylinositol cycle.

Differential effect of butyrate on lipids of human colon cancer cells.

Recently, we demonstrated that treatment of LS174T cells with 2 mM butyrate for one day had a significant effect on the composition of cellular fatty acids. In an attempt to further explore this phenomenon, we investigated the effect of long-term butyrate treatment in the presence of different fatty acids in the medium on cellular phospholipids (PLs) and triacylglycerol (TG). Cells were supplemented with 100 microM sodium salts of 18:2 (n-6), 20:4 (n-6), 20:5 (n-3), or 22:6 (n-3) as a fatty acid-free-albumin complex. The molar ratio of the albumin and these long-chain fatty acids (LCFAs) was 3:1. One-half of these cultures were supplied with 2 mM butyrate, and the pH was adjusted to 7.4. The supplementation of the LCFAs and butyrate was maintained for eight days. The present study indicates that butyrate had a differential effect on the fatty acid composition of PLs and TG of LS174T cells. This includes an increase in monounsaturates and elongation of the supplemented LCFA, and this effect was more pronounced on TG than PL fatty acids. Butyrate resulted in a significant reduction in polyunsaturated fatty acid concentration only in PLs. In general, butyrate decreased the unsaturation index (UI) of the PLs but increased that of TG. The present study also confirmed our previous observation regarding the effect of LCFAs on cellular lipids. PL and TG fatty acid chain lengths reflect those of supplemented fatty acids. The UI of these two lipid fractions increased more with supplementation of n-3 than n-6 fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

n-butyrate reduces the expression of beta-galactoside alpha 2,6-sialyltransferase in Hep G2 cells.

n-Butyrate, a short chain fatty acid that is produced by colonic bacterial fermentation, is detectable in portal blood and induces differentiation in various human neoplastic cell lines. Earlier reports indicated approximately 20-fold induction in vitro by n-butyrate of the sialyltransferase that catalyzes terminal glycosylation of GM3 ganglioside in HeLa and colon cancer cells. We previously isolated a 1.3-kilobase cDNA for a human beta-galactoside alpha 2,6-sialyltransferase, for which N-linked glycoproteins are the acceptors. We report here that treatment of Hep G2 cells with 5 mM n-butyrate for 24 h reduced beta-galactoside alpha 2,6-sialyltransferase mRNA levels by approximately 90%. Reductions in mRNA level were followed by approximately 75 and approximately 90% reductions, respectively, in specific beta-galactoside alpha 2,6-sialyltransferase enzyme activity after treatment for 24 and 36 h with 5 mM n-butyrate. However, in contrast with earlier reports of enhanced ganglioside synthesis in response to n-butyrate treatment, incubation of Hep G2 cells with n-butyrate did not alter the ganglioside pattern as assessed by thin layer chromatography of lipids extracted from treated cells. Nuclear run-on reactions indicated that the rate of transcription of beta-galactoside, alpha 2,6-sialyltransferase was not altered by treatment with 5 mM n-butyrate for 24 h, but the effects of this treatment on cytoplasmic levels of beta-galactoside alpha 2,6-sialyltransferase mRNA were largely negated by co-treatment with actinomycin D or cycloheximide. Therefore, our results show that n-butyrate reduces expression of mature beta-galactoside alpha 2,6-sialyltransferase mRNA by post-transcriptional mechanisms.

Dietary fat and phospholipase A2 activity of Sprague-Dawley rat large intestine.

The present studies were conducted to examine the effect of dietary lipid content and composition [(n-6) vs. (n-3) fatty acids] on the activity of mucosal phospholipase (PL)A2 of the large intestinal tract of rats. Three segments of the large intestinal tract were examined: cecum, proximal colon and distal colon. Weanling male Sprague-Dawley rats were fed diets containing either 5% (LS) or 16% safflower (HS) or 14% menhaden oil plus 2% safflower oil (HM) for 3 wk with the oil replacing starch in the HS and HM diets on a weight basis. The lipid extracts of microsomal fractions from mucosal scrapings were examined for phospholipid and cholesterol content and fatty acid composition. Phospholipase A2 was assayed using a fluorescent substrate. Rats fed the high fat diets had lower PLA2 specific activities. The (n-3) or (n-6) fatty acid enrichment of the membranes had no effect of the activity of the enzyme. The activity of the enzyme decreased aborally from the cecum to the distal colon; the proximal colon had an intermediate specific activity.

Influence of butyrate on lipid metabolism, survival, and differentiation of colon cancer cells.

The present work was designed to study the differentiating effect of butyrate on LS174T cells after modification of their lipids with long-chain fatty acid (LCFA) supplementation. The LCFAs 18:1(n-9), 18:2(n-6), 20:4(n-6), 20:5(n-3), and 22:6(n-3) bound to added to the media of confluent cells for eight days. The fatty acid-to-albumin ratio was 3:1. The concentration of fatty acids in the media was 100 microM. On the last day, half of the flasks were treated with 2 mM butyrate. The data indicate that supplementation with polyunsaturated LCFAs having 20-22 carbon atoms resulted in a significant reduction in cell density and viability, whereas all LCFA supplementation reduced differentiation as measured by alkaline phosphatase activity. Butyrate treatment increased the density, viability, and differentiation of the tumor cells. The effect of butyrate on differentiation was mainly with cells supplemented with 18:1, 20:5, and 22:6. In the absence of LCFA supplementation, butyrate reduced the concentration of 22:5(n-6) in the cellular lipids. Also, butyrate modified the LCFAs incorporated in cells supplemented with 18:2 and 20:5, with changes occurring in 20:5(n-3), 22:5(n-3), and 22:5(n-6). Thus the present study suggests an interaction between butyrate and LCFA on differentiation and LCFA metabolism of human colon cancer cells.