Gerald B. Storer

Hypocholesterolaemic Effects of Dietary Propionate: Studies in Whole Animals and Perfused Rat Liver

In adult male rats fed a non-purified diet supplemented with 5% sodium propionate, plasma cholesterol concentrations were significantly depressed. Although liver cholesterol was increased by feeding propionate, rates of hepatic cholesterol and fatty acid synthesis were unchanged. Tissue concentrations and rates of synthesis of cholesterol were also unaffected by dietary propionate in stomach, small intestine and caecum. Concentrations of propionate in hepatic portal venous plasma were raised by feeding the supplemented diet but the increase was low in comparison to the dietary intake. Examination of the gut contents revealed concentrations of total volatile fatty acids (VFA) of 19 mumol/ml in the stomach contents of control rats and 148 mumol/ml (of which propionate contributed 116 mumol/ml) in those fed the supplemented diet. Duodenal and ileal concentrations of VFA were very low and were only slightly raised in the propionate-fed rats while caecal VFA were the same in both groups with a combined mean of 159 mumol/ml. These data indicate that in the rat, the absorption of dietary propionate appears to occur in the stomach. In pigs fed a standard ration hepatic portal venous VFA remained low for the first 4 h after feeding but then rose with the onset of large bowel fermentation. Feeding the diet supplemented with propionate caused hepatic portal venous plasma concentrations to rise by approximately 0.4 mumol/ml. This increase was apparent 30 min after feeding and was sustained for 3 h but subsequently there was no difference to controls. As in the rat, the absorption of dietary propionate appeared to occur in the upper gastrointestinal tract. The transport of propionate via the porcine hepatic portal vein also appeared insufficient to account for the dietary intake and suggests metabolism of the acid by the upper gastrointestinal tract. Further studies with perfused livers from fed rats indicated that propionate at a concentration of 1 mumol/ml did not alter cholesterol synthesis but that inhibition occurred at 18 mumol of propionate/ml. It appears that a redistribution of cholesterol from the plasma to the liver, rather than inhibition of hepatic and intestinal cholesterol synthesis, is responsible for the hypocholesterolaemic effects of dietary propionate. Because the absorption and transport of dietary propionate appears to follow a time course which differs considerably to that of the acid produced by the large bowel microflora, we conclude also that VFA produced by such fermentation would not seem to be responsible for the hypocholesterolaemic effects of certain water-soluble plant fibres.

Effects of saponins on bile acids and plasma lipids in the rat.

1. The effects of feeding isolated saponins on plasma lipid concentrations and on concentrations of biliary and faecal bile acids and neutral sterols were studied in the rat. 2. The animals were given one of four diets, i.e. a standard low-cholesterol synthetic diet, the diet + 10 g saponins/kg, the diet + 10 g cholesterol/kg, the diet + 10 g cholesterol + 10 g saponins/kg. 3. Saponins partially reversed the hypercholesterolaemia caused by the high-cholesterol diet and increased both the rate of bile acid secretion and the faecal excretion of bile acids and neutral sterols. The proportionate contribution of the primary bile acids (particularly chenodeoxycholic) to faecal excretion was also increased by saponins. 4. The results are discussed in relation to the hypothesis that saponins act by inducing the adsorption of bile acids by dietary fibre.

Failure of insulin to stimulate lipogenesis and triacylglycerol secretion in perfused livers from rats adapted to dietary fish oil

Livers from male rats fed a standard commercial diet supplemented with 8% (w/w) marine fish or safflower oils were perfused for 70 min with undiluted blood in the presence and absence of insulin. Lipogenesis, as measured by the incorporation of 3H2O into liver and perfusate fatty acids, was inhibited by the feeding of fish oil. Net triacylglycerol secretion was also depressed by this dietary treatment. Infusion of insulin stimulated triacylglycerol secretion and the incorporation of newly synthesised fatty acids into liver and perfusate lipids with dietary safflower oil but not with fish oil. Hepatic cholesterol synthesis was also depressed by feeding fish oil. Net ketogenesis was raised by feeding fish oil and was depressed by insulin with both safflower and fish oil. Blood glucose was raised in the fish oil group but with both dietary oils the hormone exerted a significant hypoglycaemic effect. The data are discussed with respect to the observations that in vivo dietary fish oil (but not safflower oil) opposes the hypertriglyceridaemia arising from the hepatic overproduction of very-low-density lipoproteins.

Effects of dietary oat bran and diabetes on plasma and caecal volatile fatty acids in the rat

Abstract Diets high in plant fibre, particularly viscous gums, are of potential benefit in the control of diabetes in man. This study compares the effects of oat bran, one such fibre preparation, with purified cellulose on volatile fatty acid metabolism in rats fed a high carbohydrate diet. Concentrations of total volatile fatty acids were significantly higher both in hepatic portal venous plasma and caecal contents of rats fed oat bran. Major differences were also found in the relative contributions of individual acids with proportionately less acetate and more propionate and butyrate in these animals compared with rats fed cellulose. In rats fed a standard laboratory diet, diabetes induced by streptozotocin produced a sustained increase in caecal and hepatic portal venous concentrations of acetate, propionate and butyrate. Because of the high simple carbohydrate content of the experimental diet, the tolerance of diabetic rats for the bran and cellulose diets was poor and no differences in VFA metabolism were observed after 24 hr of diabetes. It is concluded that volatile fatty acid concentrations were in proportion to the probable digestibility of the source of dietary fibre and are significant metabolic fuels liable to altered metabolism by insulin insufficiency.

Metabolic effects of acetate in perfused rat liver. Studies on ketogenesis, glucose output, lactate uptake and lipogenesis.

Abstract 1. Livers from fed male rats were perfused in situ in a non-recirculating system with whole rat blood containing acetate at six concentrations, from 0.04 to 1.5 μmol/ml, to cover the physiological range encountered in the hapatic portal venous blood in vivo. 2. Below a concentration of 0.25 μmol/ml there was net production of acetate by the liver, while above it there was ner uptake with a fractional extraction of 40%. 3.No relationship was observed between blood [acetate] and hepatic ketogenesis, the ration [3-hydroxybutyrate]/[acetoacetate] or glucose output, either at low fatty acid concentration s or during oleate infusion. 4. Following the increase in serum fatty acid concentration, induced by oleate infusion, there were suquential incresase in ketogenesis and the ratio of [3-hydroxybutyrate]/[acetoacetate] while glucose output rose and lactate uptake fell significantly after in redox state. 5. There was a highly significant negative correlation between blood [acetate] and hepatic lactate uptake during oleate infusion. At the highest acetate concentration of 1.5 μmol/ml there was a small net hepatic lactate output. After oleate infusion ceased, lactate uptake increased, but the negative correlation between blood [acetate] and hepatic lactate uptake persisted. 6. Livers were also perfused with iether [1-14C]acetate or [U-14C]lactate at a concentration of acetate of either 0.3 or 1.3 μmol/ml of blood. With [1-14C]acetate, most of the radioactivity was recovered as fatty acids at the lower concentration of blood acetate. At the higher blood [acetate] a considerably smaller proportion of the radioactivity was recovered in lipids. With [U-14C]lactate the reverse pattern obtained i.e., recovery was greater at the high concentration of acetate and fell at the low concentration. Fatty acid biosynthesis, measured with 3H2O, was stimulated from 2.4 to 6.6 μmol of fatty acid/g of liver per h by high blood [acetate] although the contribution of (acetate+lactate) to synthesis remained constant at 33–38% of the total. 7. These results emphasize the important role of the liver in regulating blood acetate concentrations and indicate that it can be major hepatic substrate. Acetate taken up by the liver appeared to compete directly with lactate, for lipogenesis and metabolism and acetate uptake was inhibited by raised bloodd [lactate].

Time-Course of Changes in Plasma Lipids in Diabetic Rats Fed Diets High in Fish or Safflower Oils

Adult male rats were maintained for 10 days on a standard chow diet or that diet supplemented with either safflower or marine fish oils, and then rendered diabetic with streptozotocin (40 mg/kg of body weight) and circulating metabolites determined over the next 3 days. Pre-diabetic concentrations of glucose and insulin did not differ between groups, and the severity of hyperglycaemia and lowering of insulin in streptozotocin-treated animals were also similar. Pre-diabetic concentrations of plasma free fatty acids and triacylglycerols were lower, and blood ketone bodies were higher in non-diabetic rats fed fish oil than in both other groups. However, following streptozotocin treatment, plasma free fatty acids rose significantly more in both groups of oil-fed animals than in chow-fed ones. Plasma triacylglycerols were unaltered from pre-treatment levels in rats fed chow, but rose considerably in both groups fed oil-supplemented diets. In a subsequent experiment it was shown that the increase in triacylglycerols persisted for up to 11 days after streptozotocin and the hypertriglyceridaemia was greatest in the fish oil group. The rise would seem to result from defective clearance of lipoproteins of dietary origin. It appears that fish oil-supplemented diets should be avoided in diabetics until the possibility of increased hypertriglyceridaemia has been excluded by controlled studies.

Effects of solvent extraction on the hypocholesterolaemic action of oat bran in the rat

In adult male rats fed on a cholesterol-free synthetic diet, plasma cholesterol concentrations were lowest with oat bran, intermediate with cellulose and highest with wheat bran. Plasma triacylglycerols (TAG) were similar with wheat bran and cellulose but higher with oat bran. The concentrations and pools of caecal volatile fatty acids (VFA) were lowest with cellulose and equally higher with oat bran and wheat bran. Plasma VFA concentrations in the hepatic portal vein reflected those in caecal digesta and were unrelated to plasma cholesterol. Feeding oat bran after extraction with n-pentane gave plasma cholesterol concentrations similar to that found with wheat bran. Reconstitution of oat bran with extracted lipids did not restore the cholesterol-lowering effect. Addition of the extracted material to a wheat-bran diet had no effect on plasma cholesterol. Plasma TAG were higher with the oat bran and reconstituted-oat-bran diets than with wheat-bran or cellulose diets. However, extracted oat bran + safflower oil gave similar TAG concentrations to that with wheat bran. These extractions and additions did not change caecal bile acid or neutral sterol concentrations. Effects of these diets on plasma cholesterol were unrelated to their tocotrienol or tocopherol content. Addition of n-pentane to oat bran followed by evaporation of solvent gave plasma cholesterol concentrations that were significantly higher than untreated oat bran but lower than similarly treated wheat bran. It is concluded that oat bran affects cholesterol metabolism through a pentane-soluble component as well as non-starch polysaccharides. It appears that the activity of this lipid is not transferable by simple addition of the solvent extract to the whole diet.

PLASMA AND CAECAL VOLATILE FATTY ACIDS IN MALE AND FEMALE RATS: EFFECTS OF DIETARY GUM ARABIC AND CELLULOSE

Abstract Adult male and female rats were fed synthetic diets containing 10% gum arabic or cellulose. Concentrations of total volatile fatty acids in caecal fluid and hepatic portal venous plasma of both sexes were higher on the gum arabic diet. Total and individual acids in caecal fluid did not differ between male and female rats fed gum arabic, but with cellulose concentrations of acetate and propionate were significantly lower in females than in males. Caecal pH correlated negatively with volatile fatty acid concentration in both sexes, but with gum arabic, pH was significantly lower in males than in females with the same caecal volatile fatty acid concentration. The concentration and distribution of plasma volatile fatty acids in the hepatic portal vein generally reflected that of caecal fluid although acetate concentrations were lower in male than in female rats fed gum arabic. Blood glucose and plasma triacylglycerols were unrelated to hepatic portal venous plasma volatile fatty acids but in males liver glycogen in each dietary group was positively correlated with volatile fatty acid concentration. This relationship was absent in females. It is concluded that there are differences in the production and absorption of volatile fatty acids between male and female rats and that these are affected by the source of dietary fibre.

Effects of varying the content and proportions of gum arabic and cellulose on caecal volatile fatty acid concentrations in the rat

Abstract Adult male rats were fed a synthetic diet containing 5, 10 or 15% by weight of gum arabic (GA) or cellulose (C). Food consumption over the 10 d experimental period was similar in rats fed the 5C and 15C and all the GA diets but was raised in the 10C group. Final body weights were similar in the 5C, 10C and 15C groups and were equally lower in rats fed the GA diets. The wet weight of caecal digesta was the same with all three C diets while digesta wet weight rose with increasing dietary GA content and diarrhoea was observed with the 15GA diet. Concentrations of total caecal VFA were similar in the 5C and 5GA groups and were depressed equally by the 10C and 15C diets. Total VFA did not change in the 10GA rats but were depressed in the 15GA group. Calculation of the mass of caecal VFA showed that this was increased with the 10GA diet but substantially reduced with that containing 15GA, suggesting that VFA were not causative of diarrhoea. In animals fed diets containing mixtures of both fibres to a level of 15% by weight, caecal digesta was the same with 10C+5GA and 7.5C+7.5 GA diets but increased with 5C+10GA. Total VFA concentrations and pool size were unaffected by changing from 10C+5GA to 7.5C+7.5GA but the concentration and mass of propionate was increased. In rats fed 5C+10GA VFA concentration was greatly depressed although their mass was unchanged from the 10C+5GA group. We conclude that VFA concentrations are influenced by the type and concentration of dietary fibre and that fibre mixtures favour production of propionate. Below a certain level of fermentable fibre digesta mass seems to reflect enhanced fermentation but above that level, the presence of unfermented fibre. Differences between the present experiments and previous ones may be due to changes in the large bowel microflora.

Direct stimulation by glucose and insulin of glycogen synthesis in perfused rat liver

The central role of the liver in blood glucose regulation was first demonstrated in the intact animal [I] with inhibition of glucose production under a glucose load. Insulin plays a key role in this regulatory process, diminishing hepatic glucose output and accelerating glycogen deposition [2]. While this situation appears in the intact animal, studies on isolated liver preparations have given inconclusive results. For example, in perfused liver and isolated hepatocytes inhibition of net glucose output and stimulation of glycogen synthesis occurs only at unphysiologically high concentrations of glucose [3-S] or gluconeogenic substrates [3,5] and is insulin-insensitive [6]. A defect in isolated liver preparations has been proposed [2]. added to maintain 100 ng/ml of serum [7] while glucose was infused initially at 200 I.tmol/min for 1.5 min and subsequently at 20 ~mol/min [IO]. Both infusions were in 0.15 M NaCl and control livers were infused with 0.15 M NaCl only. Blood samples were taken at zero time and then at 15 min intervals and assayed for glucose and lactate [7]. At the end of the experiment a portion of liver (1.0-l .3 g) was quickly taken, blotted dry and weighed for glycogen estimation [IO]. The remainder of the liver was similarly weighed for calculation of metabolic activity/g liver. Bile was collected at 15 min intervals [71. All data are shown as the mean f SEM of the numbers of observations in parentheses. Statistical significance was determined by analysis of variance. Insulin insensitivity of the perfused rat liver depends upon the use of whole blood as perfusate and that the hormone responsiveness was lost following dilution of blood with buffer [7]. It was of interest to determine whether insulin enhanced hepatic glucose uptake and glycogen synthesis in livers perfused with whole blood. Here, we report that there was a substantial insulindependent uptake of added glucose by the perfused liver which was, additionally, accelerated by the hormone. Glycogen synthesis paralleled the rate of glucose uptake and was increased by insulin. 3. Results and discussion