Effects of dietary methionine, cystine, and glycine on endogenous hypercholesterolemia in hepatoma-bearing rats

The effects on hypercholesterolemia of dietary additions of cystine (Cys), methionine (Met), glycine (Gly), and a combination of Met and Gly to a 20% casein diet were studied in male Donryu rats subcutaneously implanted with an ascites hepatoma line of AH109A cells. The hepatoma-bearing rats fed the 20% casein diet lapsed into both endogenous hypertriglyceridemia and hypercholesterolemia when compared to hepatoma-free (normal) rats fed the same diet. The hypercholesterolemia was due to an elevation (3.2 fold) in the very low-density lipoprotein plus low-density lipoprotein (VLDL + LDL)-cholesterol (Ch) level. The high-density lipoprotein (HDL)-Ch level was slightly but significantly decreased. These lipoprotein changes in hepatoma-bearing rats resulted in a marked (4.5 fold) increase in the atherogenic index (AI, (VLDL + LDL)-Ch/HDL-Ch) in comparison with that of tumor-free rats. The dietary additions of 1.2% Met, 1.2% Cys, and a combination of 1.2% Met and 2.5% Gly significantly suppressed the hepatoma-induced increase in (VLDL + LDL)-Ch with no influence on the hepatoma-induced decrease in HDL-Ch, leading to a noticeable fall in AI. These results indicate that hepatoma-bearing rats are useful as an endogenously hyperlipidemic model and that some dietary amino acids are capable of improving hepatoma-induced hypercholesterolemia and abnormal serum lipoprotein profiles.     

Effects of dietary triglycerides on serum and liver lipids and sterol excretion of rats

The effects of several highly purified simple and mixed dietary triglycerides (TGs) on serum and liver cholesterol and on sterol excretion were studied in rats. The TGs contained 4- to 18-carbon fatty acids with melting points of -75 to 63.5 degrees C. Ratios of polyunsaturated to saturated fatty acids ranged from 0.1 to 105. Ratios of total unsaturated to saturated fatty acids ranged from 0.1 to 115. All diets contained 8% TG plus 0.82% safflower oil. Sterols were quantified directly by a new and improved high resolution gas chromatographic method and were identified by mass spectrometry. TG digestibilities correlated negatively with melting points above 30 degrees C (R = -0.9). Serum cholesterol was lower in rats fed tributyrin, tricaproin, tricaprylin, tricaprin, trielaidin, trilinolein or partially hydrogenated soybean oil (43-49 mg/dl) than in those fed trilaurin, trimyristin, tripalmitin, tristearin, triolein or corn oil (54-59 mg/dl). Liver lipid levels correlated (R = 0.65) with the degree of unsaturation of dietary TGs. Liver cholesterol levels correlated negatively with fecal excretion of coprostanol plus cholesterol (R = -0.4). Coprostanol plus cholesterol excreted in feces correlated weakly (R = 0.3) with intake of total sterol and of polyunsaturated TGs (R greater than or equal to 0.4 are at least 80% significant). The results demonstrate that consumption of polyunsaturated TGs was associated with higher hepatic lipid levels. Also, greater fecal excretion of coprostanol plus cholesterol was associated with lower hepatic cholesterol levels.     

Effect of ethanol on lipoprotein synthesis and fecal sterol excretion

The effect of variable doses of ethanol on plasma lipoprotein composition, lipoprotein synthesis and fecal sterol excretion was examined in male, atherosclerosis susceptible squirrel monkeys. Primates were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys given liquid diet with vodka substituted isocalorically for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. Circulating high density lipoprotein (HDL) free cholesterol and phospholipid, very low density-low density lipoprotein (VLDL-LDL) total cholesterol, and total plasma cholesterol and triglyceride were significantly elevated in High Ethanol primates compared to the other treatments. However, the percent distribution of cholesterol among the lipoprotein fractions was identical for the three groups. There were no significant differences in serum glutamate oxalo-acetate transaminase. High Ethanol primates also had significantly greater HDL free cholesterol specific activity following intravenous injection of ³H mevalonolactone compared to the other groups while radioactive VLDL-LDL free cholesterol was elevated in both High and Low Ethanol animals. Although, total fecal bile acid mass was significantly greater in both alcohol treatment groups compared to Controls, fecal neutral sterol specific activity was only higher in monkeys fed the high ethanol diet. This study provides evidence that ethanol at 24% of calories: 1) raises HDL cholesterol levels by enhancing lipoprotein synthesis; 2) increases the fecal output of newly synthesized cholesterol without causing liver dysfunction; and 3) maintains a constant relative distribution of cholesterol among lipoprotein classes.     

Effect of dietary glycine on methionine metabolism in rats fed a high-methionine diet

The alleviation mechanism of methionine toxicity by dietary glycine was investigated in weanling rats fed a high-methionine diet. When rats were fed a 10% casein diet containing 2% methionine, the activities of methionine adenosyltransferase, cystathionine beta-synthase, and cystathionine gamma-lyase, which participate in the methionine metabolism in the transsulfuration pathway, were significantly enhanced. But the addition of 2% glycine to the high methionine diet did not cause further increase in these enzyme activities; the activities of methionine adenosyltransferase and cystathionine beta-synthase were rather decreased while cystathionine gamma-lyase activity was not altered. Methionine transaminase activity was essentially insensitive to the dietary addition of methionine and glycine. In rats fed a high methionine diet, the hepatic methionine level was significantly increased with a concomitant decrease in the levels of glycine, serine, and threonine. The addition of glycine to the high methionine diet effectively suppressed the enhancement of the hepatic methionine level and almost completely restored the glycine level, but it only partially restored the serine level and further decreased the threonine level. From these results, it is suggested that the alleviating effect of dietary glycine on methionine toxicity is primarily elicited by the restoration of the hepatic glycine level rather than by an increase in hepatic enzyme activity.     

Hepatic glycine N-methyltransferase is up-regulated by excess dietary methionine in rats

Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAM) levels and the ratio of SAM:S-adenosylhomocysteine (SAH). In liver, methionine availability, both from the diet and via the folate-dependent one-carbon pool, modulates GNMT activity to maintain an optimal SAM:SAH ratio. The regulation of GNMT activity is accomplished via posttranslational and allosteric mechanisms. We more closely examined GNMT regulation in various tissues as a function of excess dietary methyl groups. Sprague Dawley rats were fed either a control diet (10% casein plus 0.3% L-methionine) or the control diet supplemented with graded levels (0.5-2%) of L-methionine. Pair-fed control groups of rats were included due to the toxicity associated with high methionine consumption. As expected, the hepatic activity of GNMT was significantly elevated in a dose-dependent fashion after 10 d of feeding the diets containing excess methionine. Moreover, the abundance of hepatic GNMT protein was similarly increased. The kidney had a significant increase in GNMT as a function of dietary methionine, but to a much lesser extent than in the liver. For pancreatic tissue, neither the activity of GNMT nor the abundance of the protein was responsive to excess dietary methionine. These data suggest that additional mechanisms contribute to regulation of GNMT such that synthesis of the protein is greater than its degradation. In addition, methionine-induced regulation of GNMT is dose dependent and appears to be tissue specific, the latter suggesting that the role it plays in the kidney and pancreas may in part differ from its hepatic function.     

Brewer's spent grain, serum lipids and fecal sterol excretion in human subjects with ileostomies

A crossover design studying lipid and apoprotein levels in serum and excretion of sterol, nitrogen and fat in ileostomy effluent was performed in 10 subjects fed diets with or without supplementation with brewer's spent grain, which is the residue of barley after the brewing of beer. More cholesterol, nitrogen, fat and energy were excreted in the ileostomy effluents when the subjects consumed a brewer's spent grain supplemented, high fiber diet than when they consumed a low fiber diet. No significant change was found in the daily net sterol excretion. The six subjects with low daily excretion of bile acids (less than 1000 mg/24 h) had increased cholesterol and net cholesterol and decreased bile acid excretion per day, and lowered serum LDL-cholesterol and apoprotein B levels after supplementation with brewer's spent grain. We propose that subjects with low daily bile acid excretion are suitable models for studying the effect of dietary changes on sterol excretion and serum lipid levels. Increased fecal cholesterol excretion is suggested to be the primary mechanism for the serum LDL-cholesterol lowering effect of brewer's spent grain.     

Effects of dietary fiber on fecal mucinase and beta-glucuronidase activity in rats

Mucinase and beta-glucuronidase enable colon bacteria to degrade protective mucins and recycle glucuronide conjugates of toxins and carcinogens. The response of these bacterial enzymes to dietary fiber was studied in the laboratory rat. Fiber-free basal diet was mixed with guar gum, pectin, carrageenan, or cellulose at levels of 5 and 15%. These diets were fed for 21 days to groups of six male Fischer-344 rats having an average weight of 150 g. Mucinase and beta-glucuronidase activities were assayed in fresh rat feces. Rats fed 15% guar gum or pectin gained significantly (P less than 0.05) less weight than the other rats. Mucinase specific activity was highest in the fiber-free diet group and lowest in the 15% guar gum group. Total daily output of mucinase was highest in rats fed fiber-free diet or cellulose and lower in rats fed more readily fermentable fiber. Specific activity and total output of beta-glucuronidase were highest in rats fed fiber-free diet and significantly lower in those fed 15% fiber diets. These data are consistent with the hypothesis that some kinds of dietary fiber may play a role in the etiology of intestinal disease.     

Effects on liver and serum lipids of dietary supplements of methionine and excess lysine given to previously-starved rats

The addition of lysine hydrochloride (50 g/kg) to a diet supplemented with methionine (3 g/kg) had no effect on liver lipids when rats (Wistar and Sprague-Dawley) were fed ad lib. for 14 d. However, refeeding a diet supplemented with methionine and excess lysine hydrochloride to previously-starved rats (Wistar) for 6, 7 or 9 d, but not 3 or 4 d, resulted in liver lipid accumulation, mainly triglycerides, which was prevented by the addition of arginine, citrulline, ornithine, adenine, allopurinol or maize oil, but not by guanine, cytosine, thymine or uracil. The addition of lysine hydrochloride to a methionine-supplemented diet resulted in reduced serum triglyceride levels after refeeding for 3 or 4 d and reduced serum cholesterol levels after refeeding for 3, 4 or 6 d. Serum lipids were unaffected by refeeding the diets for 7 or 9 d. Since hepatic lipid accumulation was preceded by the decline in the level of serum triglycerides, one of the factors responsible for lipid accumulation in the liver might be reduced transport of triglycerides from the liver into the blood. When adenine and allopurinol were added to the diet supplemented with methionine and lysine hydrochloride refed for 4 d, there was an increase in serum triglycerides. However, when this diet was refed for 7 d, with the addition of arginine, citrulline, guanine, pyrimidine bases, allopurinol or maize oil, no effects on serum cholesterol and triglyceride levels were observed.     

Inhibitory effect of coffee on hepatoma proliferation and invasion in culture and on tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats

We have already reported that instant coffee powder (ICP) and ICP-loaded rat sera could suppress proliferation and invasion of rat ascites hepatoma cell line of AH109A in vitro. In this report, we examined the mechanisms for suppression of tumor cell proliferation and invasion by ICP, and the effect of ICP on in vivo tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats. ICP, when directly added to the culture media, induced cell cycle arrest (elongation of S phase) at a lower concentration (0.3 mg/mL) and apoptosis at a higher concentration (0.6-1.2 mg/mL). ICP and ICP-loaded rat sera showed reactive oxygen species (ROS)-scavenging property and canceled the enhancement of invasive activity of hepatoma cells induced by ROS in vitro. These results suggest that ICP suppresses the proliferation by inducing cell cycle arrest and apoptosis, and the invasion by scavenging ROS and that ICP could retain these properties after their gastrointestinal absorption. The hepatoma-bearing rats were fed with a 20% casein diet (20C) or 20C supplemented with 0.1%, ICP for 14 d. Dietary ICP significantly reduced solid tumor growth and tended to reduce hepatoma metastases to lung and lymphatic nodes, suggesting that ICP could suppress tumor cell proliferation and invasion in vivo. In addition, dietary ICP significantly increased serum high-density lipoprotein (HDL)-cholesterol and tended to reduce very low-density and low-density lipoprotein (VLDL+LDL)-cholesterol, resulting in amelioration of abnormal lipoprotein profiles occurred in hepatoma-bearing rats. In conclusion, ICP has the ability to induce cell cycle arrest and apoptosis in hepatoma cells and to suppress tumor cell invasion by reducing oxidative stresses in vitro, and it could also exhibit these effects in vivo, leading to the inhibition of tumor growth and metastases.     

Effect of dietary cholesterol and degree of fat unsaturation on plasma lipid levels, lipoprotein composition, and fecal steroid excretion in normal young adult men

Effect of four test diets differing in the ratio of polyunsaturated to saturated fats (P/S, 1.8 vs 0.28) and two cholesterol levels (1,000 vs 300 mg/day) for each level of the P/S ratio was determined on plasma lipid levels, lipoprotein compositions and concentrations, and fecal steroid excretion in a controlled diet study with 11 normal young men using a crossover design. Plasma cholesterol levels were significantly decreased by the diets high in P/S ratio regardless of the dietary cholesterol levels (14% decrease by high cholesterol and 20% in low cholesterol) while the diets low in P/S ratio increased cholesterol by an average of 24 and 22% in presence of high and low cholesterol, respectively. Lipids and apoproteins of lower-density lipoproteins were changed in accordance with those of plasma cholesterol but changes in high-density lipoprotein (HDL) appear to depend on both cholesterol content and P/S ratio. Dietary cholesterol level profoundly influenced the excretion of neutral sterols and diets high in P/S ratio significantly (p less than 0.05) increased fecal bile acid extraction. The present study demonstrated that dietary polyunsaturated fats, when a moderate amount was consumed, were effective and beneficial hypocholesterolemic nutrients without reducing HDL-cholesterol.     

Dietary soluble fiber and cholesterol affect serum cholesterol concentration, hepatic portal venous short-chain fatty acid concentrations and fecal sterol excretion in rats.

Sprague-Dawley rats were fed diets containing 7.5% dietary fiber as cellulose (control), pectin, psyllium or oat bran with or without 0.3% added cholesterol for 3 wk. Among rats fed cholesterol, liver total lipid and cholesterol concentrations were significantly lower in groups fed pectin, psyllium and oat bran compared with cellulose-fed controls. Cholesterol feeding resulted in significantly greater liver cholesterol in rats fed cellulose, psyllium and oat bran but not in those fed pectin. Among rats fed cholesterol, total serum cholesterol levels were significantly lower in those fed pectin than in those fed psyllium, oat bran or cellulose. When cholesterol was fed, the oat bran-fed group had significantly higher butyrate and the pectin-fed group had significantly higher propionate concentrations in the hepatic portal vein than did cellulose-fed controls. The groups fed psyllium, oat bran and pectin all had significantly higher fecal neutral sterols than did the cellulose-fed group when cholesterol was fed. Without dietary cholesterol only pectin-fed rats had significantly higher fecal excretion of neutral sterols than those fed cellulose. Dietary fiber did not influence fecal acidic sterol excretion. However, the addition of cholesterol to these fiber diets was accompanied by a significantly higher bile acid excretion than that of animals fed cellulose without cholesterol. The results of this study indicate that soluble dietary fibers may exert their hypocholesterolemic effect by increasing excretion of fecal neutral sterols.     

Effects of coingestion of different fibers on fecal excretion and cecal fermentation in rats

The effects of the concurrent ingestion of 2 dietary fibers of different fermentabilities on fecal excretion and cecal fermentation were examined in rats. Poorly fermentable, low-molecular-weight sodium alginate supplementation of a fiber-free diet increased fecal excretion and the water content dose-dependently but did not affect cecal pH. Highly fermentable guar gum hydrolysate supplementation decreased cecal pH dose-dependently, whereas there was only a slight increase in fecal excretion. The concurrent ingestion of both fibers increased fecal excretion, lowered cecal pH, and increased the fecal water content and cecal short-chain fatty acids, especially butyrate. The increases in both fecal excretion and cecal fermentation were not achieved by excessive ingestion of the respective fibers alone and were also observed in loperamide-induced constipation. In conclusion, the concurrent ingestion of low-molecular-weight sodium alginate and guar gum hydrolysate increases both fecal excretion and cecal fermentation as a consequence of each fiber compensating for their respective dietary disadvantages in a complementary manner.     

Effects of dietary Angelica keiskei on serum and liver lipid profiles, and body fat accumulations in rats

Angelica keiskei (Ashitaba) is a perennial plant belonging to the Umbelliferae family. Recently, much attention has been focused on Ashitaba products as a so-called health food for the breakdown of cellulite among various physiological benefits of Ashitaba. The current study was carried out to investigate the physiological efficacy of dietary Ashitaba on serum and liver lipid profiles and body fat accumulation in rats. Rats were fed a high-fat diet with various amounts of Ashitaba for 28 d. Perirenal adipose tissue weights of rats fed the x 10 (170 mg/100 g BW) Ashitaba diet were significantly higher (p < 0.05) than those of the control group. Serum triacylglycerol concentrations of rats fed the x 100 (1,700 mg/100 g BW) Ashitaba diet were significantly higher (p < 0.05) than those of the x 1 (17 mg/100 g BW) group. Fecal weights and bile acid excretions of rats fed the x 10 or x 100 Ashitaba diet were significantly higher (p < 0.05) than those of the control group. However, there were no significant differences in the body weight gain, epididymal adipose tissue weight, serum cholesterol or liver lipid concentrations or other biochemical profiles in the serum. Furthermore, even the excessive ingestion of Ashitaba had no significant pathological impact on the liver or kidney. These results indicate that the large intake of Ashitaba products may supply dietary fiber and thus improve gastrointestinal condition through the increased excretion of feces containing high level of bile acids, although even excessive intake of Ashitaba for a short period of 28 d did not show any impact on the decrease in body fat or modification of lipid profiles in this study.     

Effects of dietary methionine, cystine and potassium sulfate on serum cholesterol and urinary ascorbic acid in rats fed PCB

Liver weight, liver and urinary ascorbic acid levels and serum cholesterol concentration were higher in rats fed polychlorinated biphenyls (PCB) than in controls. The influences of methionine, cystine and potassium sulfate on these metabolic responses were studied. Methionine or equivalent moles of cystine or potassium sulfate were added to a basal diet containing 10% soy protein isolate. The basal diet contained 0.3% of total sulfur-containing amino acids (S-AAs). When methionine was added to the basal diet, maximum gain in body weight was obtained with 0.5% of dietary S-AAs, while the highest values in serum cholesterol and urinary ascorbic acid were obtained with 0.8% of dietary S-AAs in rats fed PCB. Dietary addition of cystine had little effect on body weight gain. Nevertheless, in rat fed PCB, urinary ascorbic acid and serum cholesterol were significantly higher in rats fed the cystine-supplemented diet than in those fed the unsupplemented diet. Addition of potassium sulfate had no effect on body weight gain, urinary ascorbic acid or serum cholesterol. These results suggest that more S-AAs are required for the highest metabolic response to PCB than for maximum growth, and the higher requirement for S-AAs cannot be replaced by inorganic sulfate.     

Influence of a fiber mixture on serum and liver lipids and on fecal fat excretion in rats

Male Sprague-Dawley rats (8 per group) were fed a modified AIN-76A diet containing either 10% cellulose or 10% of a proprietary fiber mixture for 21 days. Weight gain and liver weight were similar in the 2 groups but feed efficiency was higher in the test group. Serum cholesterol and triglycerides were lowered by 11% and 15%, respectively, by the test diet. Liver cholesterol was lowered significantly in the fiber-fed rats whereas liver triglycerides were elevated. Twenty-four–hour fecal collections were made on days 12 and 19. The weight of feces was unaffected by the test diet, but water content and fecal fat were increased by 27% and 44% and by 99% and 100%, respectively, on days 12 and 19. The amount of Liebermann-Burchard–positive material in the feces was significantly higher in rats fed the test material. The increases on days 12 and 19 were 139% and 105%, respectively.