Effects of Propionate on Lipid Biosynthesis in Isolated Rat Hepatocytes PATSY M. /Y/SH//YA1 AND RICHARD A. FREEDLAHD2 Department of Physiological Sciences, School of Veterinary Medicine, University of California, Dauis, CA 95616 terol synthesis from [14C]acetatewas inhibited. In this study, the effect of SCFA on lipid synthesis was assessed by measuring tritium incorporation from 3H2Ointo product so as to avoid problems in estimating endogenous metabolite pool size. In addition, the mech anisms by which propionate is able to depress fatty acid and cholesterol synthesis were studied by incubating isolated hepatocytes with substrates that merged into these biosynthetic pathways and bypassed key rate-lim iting enzymes.

ABSTRACT The effects of propionate, a product of intestinal fiber fermentation, on fatty acid and sterol synthesis were studied in isolated rat hepatocytes. Fatty acid synthesis, as measured by tritium incorpo ration from 'HO. was inhibited in the presence of 1 mmol/L propionate with no substrate additions or additions of acetate, butyrate, láclate or oleate. Incor poration of ( I-' 'CJacetate into fatty acids was also inhibited in the presence of propionate. Although pro pionate markedly depressed [1- 'Cjacetate incorpora tion into sterols in hepatocyte preparations, tritium incorporation from 'HO into sterols was not inhibited, indicating that overall sterol synthesis was not af fected. Thus, in vitro, the effect of propionate on lipid metabolism is apparently limited to inhibition of de novo fatty acid synthesis. J. Nutr. 120:668-673, 1990.

MATERIALS AND METHODS Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) weighing 300-400 g were housed in individual cages in a room illuminated from 1200 to 2400 h. The rats were allowed to eat a high carbohydrate, purified diet described in Table 1 from 2400 to 0800 h for a minimum of 2 wk prior to any experimental procedure. This design was used to mini mize variation between animals due to food intake and to ensure that the animals would be in a fully lipogenic state when hepatocytes were isolated. Hepatocytes were isolated from nonfasted rats at 0700 h by the method of Berry and Friend (12) with modifications described by Cornell et al. (13). Twenty mmol/L glucose was added to the perfusion medium to minimize glycogen shedding during the isolation pro cess. The final concentration of glucose in the incuba tion flasks was 0.5 mmol/L. Incubations were conducted in the presence or absence of 1 mmol/L propionate. Initially, concentrations of 1, 5 and 10 mmol/L propionate were tested in incubations. Because these concentrations all gave similar results, we chose


•fatty acid synthesis •sterol synthesis •propionate •fiber •hepatocytes

Serum cholesterol concentrations have been shown to decrease with the ingestion of soluble fibers such as pectin or guar gum. The decrease in cholesterol concen trations has been partially attributed to the ability of these dietary fibers to increase fecal bile acid excretion and to inhibit lipid absorption from the gut (1).Although adsorption of bile acids by dietary fiber may result in a twofold loss of cholesterol from the body pool (2), it is not sufficient to account for the observed decrease in serum cholesterol (3). It has been suggested that shortchain fatty acids (SCFA),particularly propionate, may be involved in lowering serum cholesterol concentra tions (4). SCFA arise from the fermentation of dietary fibers by colonie microflora and can be absorbed from the colon (5). Propionate, which is totally metabolized in the liver (6),has been shown to affect glycolysis (7,8), gluconeogenesis (9, 10) and ketogenesis (11).Anderson and Bridges (7)observed in isolated hepatocyte prepara tions containing propionate that fatty acid and choles

'Present address: Children's Hospital Oakland Research Insttute, 747 52nd Street, Oakland, CA 94609. 2To whom reprint requests should be addressed.

0022-3166/90 $3.00 ©1990 American Institute of Nutrition. Received 7 August 1989. Accepted 1 March 1990.

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Compositon of the purified diet

Fatty acid synthesis in isolated hepatocytes incubated with various substrates in the absence or presence of propionate1



g/kg Vitamin-free casein1 Glucose Corns tarch Corn oil2 Salt mix3'5 Vitamin mix4'5 Choline chloride

250 500 149 50 50 10 1

'Nutritional Biochemical, Cleveland, OH. 2Mazola corn oil, Best Food, Englewood Cliffs, NJ. 3Rogers-Harper salt mix (g/kg mix): ammonium molybdate-4H2O, 0.03; calcium carbonate, 292.9; calcium phosphate-2H2O, 4.3; cupric sulfate, 1.56; ferric citrate-6H2O, 6.23; mangesium sulfate-7H2O, 99.8; manganese sulfate-H2O, 1.21; potassium iodide, 0.005,- potassium phosphate, 343.1; sodium chloride, 250.6; sodium selenite-5H2O, 0.02; and zinc chloride, 0.2. 4AIN-76 vitamin mix (g/kg mix): thiamin-HCl, 0.6; riboflavin, 0.6; pyridoxine-HCl, 0.7; nicotinic acid, 3; D-calcium pantothenate, 1.6; folie acid, 0.2; D-biotin, 0.02; cyanocobalamin, 0.001; retinyl palmitate, 0.8 (500,000 iu/g); d7-a-tocopheryl acetate, 20 (250 iu/g); cholecalciferol, 0.00025; menaquionine, 0.005. 5Nutritional Biochemical, Cleveland, OH.

to use 1mmol/L because this would reflect more closely the propionate levels from the intestine to the liver via the portal vein. Also, there was an indication that pro pionate utilization plateaued at concentrations exceed ing 1 mmol/L, which was not true for butyrate and acetate. Two-hundred fifty microcuries of 3H2O(1 Ci/g), 1.0 uCi of [2-14C]lactate(14.5 Ci/mol), 0.1 uCi [l-14C]acetate (2.5 Ci/mol) or 0.1 uCi [U-14C]oleate(305 Ci/mol) was added without carrier to hepatocytes that had been preincubated at 37°Cfor 30 min with the substrates indicated in the tables. Cells were maintained in an atmosphere of 95% oxygen and 5% carbon dioxide at all stages of the incubations. Thirty minutes after the ad dition of the tracer amounts of radioisotopes, reactions were stopped with 60% perchloric acid (wt/v). Fatty acids and sterols synthesized were measured by the procedure of Newton and Freedland (14).The amount of acetyl units (AU) incorporated from 3H2O into fatty acids (15), 1 umol of 3H2Obeing equivalent to 1.15 umol of AU. In another set of flasks, hepatocytes were incubated with the indicated substrates and no radiolabeled com pounds. At 60 min, hepatocytes were separated from the incubation media by centrifuging the cells through sil icon oil into 14% perchloric acid (wt/v) (16).Neutralized aliquots were analyzed for pyruvate (17)and citrate (18). The [2-14C]lactateused in the incubations was obDownloaded from by Queen Mary University of London user on 26 March 2018

No propionate


Propionate (1 mmol/L)

p value

\imol acetyl units-g'1 30 min'1 EndogenousAcetate mmol/L)Butyrate (2 mmol/L)Láclate (2 mmol/L)Oleate (2 (0.5 mmol/L)1548843.2

0.4"6.7 ± 1.4b6.4 ± 0.9b11.1 ± 2.1C0.7 ± ±0.2d1.3

0.2"b1.7 ± 0.8'b2.9 ± 1.1a2.7 ± 0.8*0.2 ± ±

Effects of propionate on lipid biosynthesis in isolated rat hepatocytes.

The effects of propionate, a product of intestinal fiber fermentation, on fatty acid and sterol synthesis were studied in isolated rat hepatocytes. Fa...
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