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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 271, No. 1, May 15, pp. 130-138, 1989

Effect of Glycerol and Dihydroxyacetone ANDRl?S CARMONA’ Department

AND

on Hepatic Lipogenesis

R. A. FREEDLAND

of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, Califwnia 95616

Received September 12,1988, and in revised form January

9,1989

Glycerol is a dietary component which is metabolized primarily by the liver and kidney where it is used mainly for glucose synthesis. The metabolism of glycerol is very similar to that of dihydroxyacetone which can be considered its more oxidized counterpart. The effects of these substrates on hepatic lipogenesis and gluconeogenesis were examined. In isolated hepatocytes, 10 mM dihydroxyacetone caused a large increase in glucose output and stimulated lipogenesis without affecting the lactate/pyruvate ratio or the total ATP content of the cells. (As compared to dihydroxyacetone, 10 mM glycerol was less effective as a gluconeogenic substrate, increased the lactate/pyruvate ratio, caused a slight decrease in the total ATP content, and inhibited lipogenesis by at least 40% depending on the type of diet fed to the rats.) The fall in ATP levels was very small and did not correlate with the changes in fatty acid synthesis. The immediate cause of the inhibition of lipogenesis, brought about by glycerol in hepatocytes from sucrose fed rats, seemed to be a large decrease in pyruvate levels. This did not result from impairment of glycolysis but from a rise in the cytosolic NADH/NAD ratio. o 1989 Academic press, I~~.

Glycerol is a normal dietary component which is well utilized by the rat as an energy source, when administered as the main dietary component (1). In mammals, glycerol is metabolized primarily by the liver and kidney (see (2) for review) where it is mainly used for glucose synthesis. In perfused liver preparations (3,4) and in isolated hepatocytes (5) glycerol administration causes a large increase in glycerol-P, which is accompanied by an elevation in the cytosolic NADH/NAD ratio, and depletion of the ATP pool (4). In addition, when present at concentrations above 1 mM, glycerol inhibits hepatic fatty acid synthesis (6,7). In contrast, dihydroxyacetone, a more oxidized counterpart of glycerol, is utilized by the liver at a faster rate, does not cause 1 Sponsored by Consejo de Desarrollo Cientifico Humanistico, Universidad Central de Venezuela. 2 To whom correspondence should be addressed. 0003-9861/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

y

130

accumulation of glycerol-P, does not deplete the ATP pool, and does not increase the cytosolic NADH/NAD ratio (3,4). Clark et al. (6) suggested that the inhibition of lipogenesis brought about by glycerol was due to the accumulation of glycerol-P and depletion of the ATP pool. Lin et al. (7) considered that the effect of glycerol was due to both an increase in the cytosolic NADH/NAD ratio, which may impair glycolysis at the level of glyceraldehyde-P dehydrogenase, and cause/bring about a decrease in citrate levels. Since direct experimental evidence supporting these hypotheses is lacking, the effect of glycerol on lipogenesis was reevaluated using isolated hepatocytes incubated with glycerol or dihydroxyacetone, under a variety of conditions set to contrast their effects on several metabolic parameters. The results reported in this paper suggest that the inhibition of lipogenesis could not be attributed to the decrease in ATP levels

EFFECT

OF GLYCEROL

mediate cause of the depression of fatty acid synthesis was the large decrease in pyruvate concentration. MATERIALS

AND

METHODS

Artinruls. Female Sprague-Dawley rats weighing 200-300 g (Charles River Breeding Laboratories, Inc., Wilmington, MA) were fed either a commercial nonpurified diet (Ralston Purina, St. Louis, MO) or a semipurified diet containing 60R sucrose, fructose, or glucose (8). Cell preparation. Isolated hepatocytes were prepared as described by Berry and Friend (9) with the modifications of Cornell et ul. (10). In order to minimize glycogen shedding during cell isolation, 20 mM glucose was added to the perfusion medium. The final glucose concentration in the reconstituted hepatocgtes was between 1 and 2 mM. Eqerimental desiyn. Isolated hepatocytes were incubated as described below in the absence of any added substrates (endogenous) or in the presence of 1 or 10 mM glycerol or dihydroxyacetone (primary substrates), alone or in combination with fructose, lactate, or pyruvate (secondary substrates). For the statistical analysis we used the two-way ANOVA, multiple correlation analysis, and paired t test routines contained in the Number Cruncher Statistical System for IBM computers (version 4.20, Kaysville, UT). For reasons of clarity only the results of the following comparisons are reported: (i) all experimental flasks versus endogenous and (ii) the means obtained in the presence of both a primary plus a secondary substrate versus the value obtained with the primary substrate alone and versus the secondary substrate alone. Erc.perimentnl procedures. Lipogenesis was measured using a modification of the procedure of Newton and Freedland (11) in which the cells (50-60 mg wet wt) were preincubated for 30 min at 37°C in the presence of the indicated substrates before the addition of 0.25 mCi of [“Hjwater and the incubation was continued for 30 more min. The amount of acetyl units incorporated into fatty acids was calculated using the conversion factor derived by Jungas (12). For the measurement of cellular metabolites 40-50 mg of cells (wet wt) was incubated for 1 h in KrebsRinger buffer with 1% albumin and the indicated substrates in a final volume of 3 ml. The incubations were terminated with 0.23 ml of 60% perchloric acid, Samples of the neutralized perchloric acid extract were used for the determination of glucose (13), pyruvate (14), lactate (15). B-hydroxybutyrate, and acetoacetate (16). ATP was measured as follows: The incubation mixture contained, in a final volume of 0.45 ml, Tris-acetate buffer (50 mM, pH 7.75), EDTA (1.5 mM), dithiothreitol (40 GM), bovine serum albumin (0.0X%), magnesium acetate (10 mM), D-luciferin (35

131

ON LIPOGENESIS

and luciferase from Photinus pyrulis (2400 units). The bioluminescence determinations were carried out in a luminescence photometer (Picolite Model 6100, Packard Instruments, Co., Downers Grove, IL). Materials. Glucose oxidase, peroxidase, lactate dehydrogenase, /3-hydroxybutyrate dehydrogenase, NAD, NADH, and o-dianisidine were from Sigma Chemical Co. (St. Louis, MO). Collagenase, D-IuCiferin, and luciferase from P. pyrulis were from Boeringer-Mannhein Biochemicals (Indianapolis, IN). N,N,N’,N’-Tetramethyl-p-phenylenediamine dichloride (TMPD)” was purchased from Eastman Kodak Co. (Rochester, NY). “HI,0 (25 mCi/g) was from New England Nuclear (Boston, MA). All other material and reagents were of analytical grade. PM),

RESULTS

1. Eflect of Glycerol on Lipogenesis in Hepatocytes from Rats Fed Various Diets The effect of diet on lipogenesis in isolated hepatocytes incubated with or without 20 rnhl glycerol is presented in Table I. As compared to the endogenous rate, addition of 20 mM glycerol significantly impaired lipogenesis regardless of the diet fed to the animals. However, the extent of the inhibition was dependent upon the type of diet fed to the animals, being significantly more intense (90% inhibition) in hepatocytes from rats fed chow and less severe (40% ) in those from rats fed fructose. Inhibition of lipogenesis with hepatocytes from sucrose fed rats was close to 50%) and the rates of lipogenesis, measured both in the presence or absence of glycerol, were higher than those for glucose or chow fed rats. In the subsequent experiments only cells from sucrose fed rats were used. 2. Effect of Glycerol and Dihydrozyacetone on Lipogenesis and Glucose Output in Isolated Hepatocytes Both glycerol and dihydroxyacetone merge into the glycolytic pathway at the triosephosphate level and, at least theoretically, the incoming carbons can be used for either glucose or fatty acid synthesis. a Abbreviation used: TMPD, N,i\‘,N’,N’-tetramethyl-p-phenylenediamine dichloride.

132

CARMONA TABLE

AND

I

EFFECT OF 20 mu GLYCEROL ON LIPOGENESIS IN ISOLATED HEPATOCYTES FROM RATS FED VARIOUS DIETS Fatty acid synthesis” Diet Commercial (chow) 60% Glucose 60% Fructose 60% Sucrose

-Glycerol

4.38 7.61 11.77 12.99

f -t k +

1.13 1.83 0.99 0.94

+Glycerol

0.40 2.10 6.82 6.22

f f f f

0.10 0.39 0.89 1.15

FREEDLAND

lipogenesis slightly above that of pyruvate alone. In contrast, in cells incubated with dihydroxyacetone (1 or 10 mM), addition of lactate or pyruvate, but not fructose, significantly increased the rate of lipogenesis.

Y” Inhibition

91 72 42 52

TABLE

RATES OF LIPOCENESIS AND GLUCOSE OUTPUT IN ISOLATED HEPATOCYTES INC~JBATED WITH VARIO~JS SUBSTRATES Lipogenesis (wmol A.U.1 g/30 min)

Glucose output (pmol/g/min)

None 10 mM Lactate 10 mM Pyruvate 1 mM Fructose

12.4 k 1.5 29.7 f 2.6” 30.0 2 2.6” 16.9 +- 2.4”

0.06 0.72 0.78 0.51

t -t f k

0.16 0.08” 0.11” 0.07”

1 mM Glycerol (1 GUY) 1 Gly + lactate 1 Gly + pyruvate 1 Gly + fructose

10.7 +- 1.1 16.7 + 2.9’ 29.6 f 3.1ub 12.3 + 2.5”

0.46 0.92 1.06 0.70

t * i *

0.12” 0.05& O.lOh o.oP

10 mM Glycerol (10 GUY) 10 Gly + lactate 10 Gly + pyruvate 10 Gly + fructose

5.3 i 10.4 rt 36.6 + 7.0 rt

0.8” 2.2” 5.3”* 0.9’“‘”

1.12 k 1.64 f 1.86 f 1.50 +

0.10” 0.11”“’ 0.16”” 0.06&

15.2 + 3.0 24.6 3~3.4”“” 29.5 + 2.3”b 18.4 it 3.6”

0.37 + 1.25 + 1.39 + 0.99 f

0.07” 0.17& 0.18& 0.16”k

18.1 30.8 43.9 25.9

1.48 + 1.95 f 2.32 f 1.84 *

0.17” 0.20& 0.21 a6e 0.17””

Substrate

Anova table Source

df

F values

P

Diet (A) Glycerol (B) AXB

3 1 3

18.14 52.69 9.35

to.01 0.98) was found. DISCUSSION

The metabolism of glycerol is similar to that of dihydroxyacetone. Both are first phosphorylated (2, 17-19) and dihydroxyacetone phosphate enters directly into the glycolytic pathway at the triosephosphate

TABLE

VI

MULTIPLE CORRELATION ANALYSIS AMONG LIPOGENESIS RATE, PYRUVATE AND KETONE BODY PRODUCTION RATES, ATP LEVEL, AND PYRTJVATE/LACTATE RATIO Independent variable

df

Sequential R”

F values

P

Pyruvate P/L ratio ATP Ketone bodies

1 1 1 1

0.7792 0.8204 0.8205 0.8292

68.42 3.62 0.00 0.76