effect of glycerol and dihydroxyacetone on hepatic lipogenesis

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

Effect of Glycerol and Dihydroxyacetone on Hepatic Lipogenesis

ANDRl?S CARMONA’ AND R. A. FREEDLAND

Department 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 en- ergy 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 y Humanistico, Universidad Central de Venezuela.

2 To whom correspondence should be addressed.

accumulation of glycerol-P, does not de- plete 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 sup- porting these hypotheses is lacking, the effect of glycerol on lipogenesis was reeval- uated 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

0003-9861/89 $3.00 130 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

EFFECT OF GLYCEROL ON LIPOGENESIS 131

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 non- purified diet (Ralston Purina, St. Louis, MO) or a semipurified diet containing 60R sucrose, fructose, or glucose (8).

Cell preparation. Isolated hepatocytes were pre- pared as described by Berry and Friend (9) with the modifications of Cornell et ul. (10). In order to mini- mize glycogen shedding during cell isolation, 20 mM

glucose was added to the perfusion medium. The final glucose concentration in the reconstituted hepato- cgtes 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 Sys- tem 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 Krebs- Ringer 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

PM), 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-IuCif- erin, and luciferase from P. pyrulis were from Boer- inger-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.

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 theoret- ically, 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 AND FREEDLAND

TABLE I

EFFECT OF 20 mu GLYCEROL ON LIPOGENESIS IN ISOLATED HEPATOCYTES FROM RATS

FED VARIOUS DIETS

Fatty acid synthesis” Y”

Diet -Glycerol +Glycerol Inhibition

Commercial (chow) 4.38 f 1.13 0.40 f 0.10 91

60% Glucose 7.61 -t 1.83 2.10 f 0.39 72 60% Fructose 11.77 k 0.99 6.82 f 0.89 42 60% Sucrose 12.99 + 0.94 6.22 f 1.15 52

Anova table

Source df F values P

Diet (A) Glycerol (B) AXB

3 18.14 to.01 1 52.69 <O.Ol 3 9.35 10.01

Note. Isolated hepatocytes from female rats were preincubated for 30 min with or without 20 mM glycerol. At the end of this period 0.25 mCi of ‘Hz0 was added and the incubation continued for 30 more min. Results are means + SEM for four experiments performed in duplicate. Results were analyzed using a two-way ANOVA.

” The rate of fatty acid synthesis was expressed as micromoles acetyl units per gram cells incorporated after 30 min of incubation.

Addition of both glycerol and dihydroxyacetone, at either 1 or 10 mM, increased glucose output (Table II). At 10 mM, both substrates were better precursors of glucose than either lactate or pyruvate. Addi- tion of the secondary substrates, in combination with either glycerol or dihydroxyacetone, resulted in additional increases in glucose output.

Dihydroxyacetone, fructose, lactate, and pyruvate stimulated fatty acid synthesis, while 10 mM glycerol significantly inhibited lipogenesis by more than 50% (Table II). Although the addition of fructose or lactate significantly increased lipogenesis above the values observed with 10 mM glycerol alone, they did not restore fatty acid synthesis to the rates observed with the secondary substrates alone. On the other hand, only pyruvate was able to overcome the effect of glycerol, increasing the rate of

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.

TABLE II

RATES OF LIPOCENESIS AND GLUCOSE OUTPUT IN ISOLATED HEPATOCYTES INC~JBATED

WITH VARIO~JS SUBSTRATES

Lipogenesis Glucose (wmol A.U.1 output

Substrate addition g/30 min) (pmol/g/min)

None 10 mM Lactate 10 mM Pyruvate 1 mM Fructose

1 mM Glycerol (1 GUY)

1 Gly + lactate 1 Gly + pyruvate 1 Gly + fructose

10 mM Glycerol (10 GUY)

10 Gly + lactate 10 Gly + pyruvate 10 Gly + fructose

1 mM Dihydroxy- acetone (1 DHA)

1 DHA + lactate 1 DHA + pyruvate 1 DHA + fructose 10 InM Dihydroxy-

acetone (10 DHA)

10 DHA + lactate 10 DHA + pyruvate 10 DHA + fructose

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

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

5.3 i 0.8” 10.4 rt 2.2” 36.6 + 5.3”*

7.0 rt 0.9’“‘”

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

18.1 t 2.2” 30.8 k 3.6’& 43.9 t 4.3& 25.9 -+ 6.7”

0.06 t 0.16 0.72 -t 0.08” 0.78 f 0.11” 0.51 k 0.07”

0.46 t 0.12” 0.92 * 0.05& 1.06 i O.lOh 0.70 * o.oP

1.12 k 0.10” 1.64 f 0.11”“’ 1.86 f 0.16”” 1.50 + 0.06&

0.37 + 0.07” 1.25 + 0.17& 1.39 + 0.18& 0.99 f 0.16”k

1.48 + 0.17” 1.95 f 0.20& 2.32 f 0.21 a6e 1.84 * 0.17””

Note. Isolated hepatocytes were incubated as described in the text. For lipogenesis the results are means + SEM for four experiments performed in duplicate. The values of glucose output are the means -t SEM for five experiments. A.U., acetyl units.

(L P < 0.05 (paired t test) significantly different from the value obtained in the absence of any added substrate.

‘Significantly different from the value obtained with the primary substrate alone at the respective concentration.

‘Significantly different from the value obtained with the respectively secondary substrate alone.


The combination of 10 mM dihydroxyacetone and pyruvate resulted in a rate of fatty acid synthesis significantly higher than that obtained with pyruvate alone (see Table II).

.% Eflect of Glycerol and Dihydroxyacetone on the Accumulation sf Lactate and Pyruvate and La~ctate/Pyruvate Ratio

Addition of 1 or 10 mM glycerol to isolated hepatocytes significantly increased the accumulation of lactate by 31 and 75%) respectively (Table III). In contrast, at both concentrations glycerol significantly decreased the rate of pyruvate accumulation by 20 and 77%, respectively. The same trend was observed in cells incubated with glycerol in combination with fructose.

In sharp contrast, dihydroxyacetone (10 mM) significantly increased the production of both lactate and pyruvate. The addition of fructose caused significant increases in lactate and pyruvate production only when dihydroxyacetone was present at 1 mM (Table III).

In isolated liver cells the accumulation of lactate plus pyruvate can be used as an index of the rate of glycolysis. Addition of 10 mM glycerol increased the accumulation of lactate plus pyruvate by almost 30% as compared to that of cells incubated without exogenous substrates (see Table III). This increment could be accounted for almost exclusively by the increase in lactate. In addition, in the presence of fructose, glycerol (1 or 10 mM) did not affect lactate plus pyruvate accumulation.

The redox state of the NADH/NAD cou- ple in the cytosol has been indirectly as- sessed by measuring the lactate/pyruvate concentration ratio. In hepatocytes incubated without exogenous substrates the lactate/pyruvate ratio was 2.29 (Table III). Addition of fructose or dihydroxyacetone (1 or 10 mM), alone or in combination with fructose, caused only minor changes in the lactate/pyruvate ratio. One millimolar glycerol increased the lactate/pyruvate ratio by 60%, while the addition of 10 mM glycerol, alone or in combination with fructose, caused a sevenfold elevation in the lactate/pyruvate ratio. The rise in the

TABLE III

LACTATE AND PYRUVATE PRODUCTION AND LACTATE/ PYRUVATE RATIOS IN ISOLATED HEPATOCYTES

INCUBATED WITH VARIOUS SUBSTRATES

Lactate Pyruvate Substrate accumulation accumulaion additions (Fmol/g/min) (pmol/g/min) L/P

None 0.80 i 0.06 0.35 + 0.02 2.29 Lactate (L) 0.89 +- 0.02” - Pyruvate (P) 1.04 + 0.11” - Fructose (F) 1.27 t 0.10” 0.45 t 0.03” 2.82

1 mM Gly 1.05 t 0.07” 0.28 i 0.03” 3.75 1 mM Gly + L - 0.77 k o.02”k - 1 mM Gly + P 1.60 ir 0.19”h’ - - 1 mM Gly + F 1.54 t O.OYb 0.35 -+ 0.03’” 4.52

10 mM Gly 1.40 i- 0.12” 0.08 k 0.02” 17.50 lOmMGlg+L 0.42 k 0.05’” - 10 mM Gly + P 2.34 i-0.27”” - - 10 mM Gly + F 1.84 k 0.11” 0.11 k 0.03”” 16.70

1 mM DHA 1.02 + 0.14 0.39 f 0.02”k 2.61 lmMDHA+L - 0.91 F o.ol”* - lmMDHA+P 1.40f o.18”k - - lmMDHA+F 1.33 +- 0.16”” 0.45 i 0.03”* 2.95

10 mM DHA 2.03 t 0.13” 0.61 i 0.05” 3.32 lOmMDHA+L - 0.96 f 0.03”‘” - 10 mM DHA + P 1.83 f 0.29”’ - - 10 mM DHA + F 1.84 k 0.21” 0.66 f 0.04”” 2.83

Nofe. Results (wmol/g cells/min) are means k SEM for five experiments. Gly, glycerol; DHA, dihydroxyacetone; L, lactate; P, pyruvate; F, fructose; L/P, lactate/pyruvate ratio.

“P < 0.05 (paired t test) significantly different from the value obtained in the absence of any added substrate.


“Significantly different from the value obtained with the respective secondary substrate alone.

lactate/pyruvate ratio in cells incubated with glycerol was caused by a large decrease in pyruvate rather than by an increase in lactate.

4. Efect oj’Glycero1 a,nd Dihydroxyacetone on Total Ketone Body Production and fi- Hydroxybutyrate/Acetoacetate Ratio

The rate of ketogenesis in hepatocytes from fed rats incubated without exogenous


TABLE IV

TOTAL KETONE BODY PRODUCTION, p- HYDROXYBUTYRATE/ACETOA~ETATE RATIO, AND

ATP CONTENT OF ISOLATED HEPATOCYTES INCUBATED WITH VARIOUS SUBSTRATES

Total ketone body

Substrate production BHWAcAc ATP addition (nmol/g/min) ratio (mM)

NOSY? 100.6? 4.2 0.83 2.60 + 0.22 Lactate(L) 95.3 * 15.4 0.93 2.50 + 0.22 Pyruvate (P) 180.3 + 19.5” 0.95 2.72 * 0.28 Fructose (F) 86.8 2 13.5 0.79 2.28 t 0.25”

1 mM Gly 64.8~ 6.3” 0.84 2.62 k 0.19 lmMGty+L 80.6 k 12.9” 1.40 2.66 2 0.26 1 mM Gly + P 173.8 + 21.7”b 0.90 2.57 f 0.27 1 mM Gly + F 71.7? 11.4 0.82 2.39 + 0.29

10 nlM Gly 40.4i 6.3” 1.03 2.16 + 0.22” 10 mM Gly + L 69.1 + 8.4& 1.42 2.12 ? 0.26” 10 mM Gly + P 153.3 i- 19.9& 0.89 2.30 + 0.32 lOm~Gly+F 49.5 k 11.4” 0.95 2.09 2 0.22”

1 mM DHA 94.1 5 11.1 0.77 2.73 + 0.25 1 mMDHA+L 112.7 f 23.2 1.12 2.37 + 0.27* I~MDHA+P 183.6 + 22.6& 0.91 2.62 t 0.26 ~IIIMDHA+F 92.1 + 19.5 0.88 2.33 k 0.38

10 l,,M DHA 90.1 + 3.6 0.70 2.54 + 0.24 10 mM DHA + L 108.3 + 19.3’ 0.99 2.34 310.30 10 mM DHA + P 202.2 + 17.8& 0.97 2.41 + 0.32 10 mM DHA + F 119.7 + 24.9 0.88 2.43 + 0.35

Note. Isolated hepatocytes were incubated for 60 min in the presence of the indicated substrates. Ke- tone bodies and ATP were measured as described in the text. The results are means t SEM for five experiments (ATP) or four experiments (ketone bodies). Gly, glycerol; DHA, dihydroxyacetone; L, lactate; P, pyruvate; F, fructose; BHB, /j’-hydroxybutyrate; AcAc, acetoacetate.

” P < 0.05 (paired t test) significantly different from the value obtained in the absence of any added substrate.


‘Significantly different from the value obtained with the respective secondary substrate alone.

fatty acids is very small. Glycerol (10 mM) significantly decreased the production of ketone bodies by 60%, while the effect of dihydroxyacetone was not significant (Ta- ble IV). Only the addition of pyruvate caused a significant increase in ketone body output (80%).

Addition of lactate, but not fructose, to cells incubated with 10 mM glycerol increased the production of ketone bodies by 50% above the rate observed with glycerol alone, although the ketone body output was still significantly lower than in cells incubated without exogenous substrates or with lactate alone (Table IV). In contrast, cells incubated with pyruvate and glycerol had a rate of ketogenesis that was only slightly lower than in cells incubated with pyruvate alone, but it was almost four times higher than that in cells incubated with glycerol alone. In the case of hepatocytes incubated with dihydroxyacetone, addition of pyruvate also increased the rate of ketogenesis.

Under the conditions of our incubations we did not observe large changes in the p- hydroxybutyrate/acetoacetate ratio. In general, the addition of glycerol rendered the mitochondrial compartment slightly more reduced while dihydroxyacetone tended to make it more oxidized (Table IV).

5. Efect of Glycerol und Dih,ydroxyacetone on Total A TP Content

The total cellular ATP of isolated hepatocytes incubated without exogenous substrates was 2.6 mM (Table IV). This value is within the normal range of liver ATP concentrations. Only in the presence of fructose or 10 mM glycerol (alone or in combination with fructose or lactate) did we observe a small but significant decrease in ATP levels as compared to that of cells incubated without exogenous substrates. All other combinations, including those with dihydroxyacetone, failed to significantly change the ATP concentration.

6. Efect of TMPD on Lipogenesis, Lactate/Pyruvate Ratio and ATP Content of Hepatocytes Incubated with Glycerol

Since pyruvate, but not lactate, was able to overcome the inhibition of lipogenesis caused by glycerol it appeared that the increase in the NADH/NAD ratio in the cytosolic compartment was a major factor in the inhibition of lipogenesis by glycerol.

135 EFFECT OF GLYCEROL ON LIPOGENESIS

TABLE V

EFFECT OF TMPD ON LIPOGENESIS, LACTATE/~YRUVATE RATIOS, AND ATP CONTENT IN ISOLATED HEPATOCYTES

-

Lipogenesis L/P ATP (mM) TMPD level

(PM) Substrate addition -TMPD +TMPD -TMPD +TMPD -TMPD +TMPD

50 None 9.93 - 2.20 2.80 Glycerol 6.70 6.86 12.28 9.8 2.40 2.43 Lactate 25.57 23.55 2.74 2.64 Glycerol + lactate 15.00 19.97 - 2.40 2.40

100 None 11.75 - 2.31 - 3.04 -

Glycerol 8.34 12.04 12.73 7.3 2.66 2.71 Lactate 47.90 51.29 - 3.02 3.03 GLycerol + lactate 38.98 54.54 - - 2.74 2.92

Note. Isolated hepatocytes were incubated with or without TMPD as described in the text. The concentrations of glycerol and lactate were 10 mM. Results are averages of two experiments. Lipogenesis is expressed as pm01 acetyl units/g cells/30 min. L/P, lactate/pyruvate ratio.

Considering that TMPD is a hydrogen acceptor that favors the reoxidation of cytosolic NADH, we tested the effect of this dye on lipogenesis, lactate/pyruvate ratio, and ATP content in hepatocytes incubated with glycerol.

In the presence of 10 mM glycerol, 50 pM TMPD decreased the lactate/pyruvate ratio by 20% without changing the rate of lipogenesis as compared to cells incubated with glycerol alone (Table V). At 100 PM, TMPD decreased the lactate/pyruvate ratio by 57% in cells incubated with glycerol and caused a 44% increase in lipogenesis. In the presence of lactate and glycerol, lipogenesis was increased by 40% to a level slightly above that observed with lactate alone. Under the conditions tested, TMPD did not alter the ATP content of the hepatocytes (Table V).

7. Multiple Correlation Analysis

Table VI presents the results of a multiple correlation analysis performed to as- sess the relationships among some variables considered in this study. There was a highly significant correlation between pyruvate levels and lipogenesis (P < 0.001); the change in this variable accounted for 78% of the explained variability of lipo-

genesis. On the other hand, no relationship between ATP levels and lipogenesis (P > 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 Sequential F variable df R” values P

Pyruvate 1 0.7792 68.42 <O.OOl P/L ratio 1 0.8204 3.62 <0.08 ATP 1 0.8205 0.00 NS Ketone bodies 1 0.8292 0.76 NS

Note. For the multiple correlations analysis, use was made of the data corresponding to cells incubated with glycerol or dihydroxyacetone (1 and 10 mM) alone or in combination with 1 mM fructose, for which a complete set of values was available.


level. Glycerol enters glycolysis only after oxidation with the production of NADH. After conversion to dihydroxyacetone-P, either of these substrates can be converted to glucose or to pyruvate and eventually to fatty acids. In the perfused liver, dihydroxyacetone is metabolized more rapidly than glycerol (4) with the phosphorylation apparently being the rate-limiting step (20). In contrast, glycerol in perfused livers (3, 4) and isolated hepatocytes (5) causes an accumulation of glycerol-P with the translocation of reducing equivalence from a cytosol into the mitochondria being the rate-limiting step of glycerol metabolism (5).

Gluconeogenesis appears to be the major pathway of utilization of these two substrates; in perfused livers from starved rats the rates of gluconeogenesis from 10 mM dihydroxyacetone was four times higher than that from 10 mM glycerol (21). The same trend was observed in isolated hepatocytes from fed rats (Table II). The observation that additional substrates such as fructose, pyruvate or lactate added to cells incubated with either glycerol or dihydroxyacetone significantly increased glucose production suggests that the ca- pacity for gluconeogenesis was not satu- rated in cells incubated with glycerol or dihydroxyacetone at 10 mM.

In hepatocytes from fed rats, additions of substrates that merge into the glycolytic pathway such as dihydroxyacetone, fructose (below 5 mM), lactate, and pyruvate resulted in significant increases in the rate of lipogenesis (Table II). In contrast, glycerol inhibited lipogenesis in hepatocytes from rats fed various diets (Tables I and II), these results are in agreement with those of previous reports (6, 7). The degree of inhibition with glycerol was less severe when hepatocytes were obtained from rats fed fructose containing diets (Table I).

Clark et al. (6) speculated that the antilipogenic effect of glycerol could be attributed to depletion of the ATP pool. Several authors (4,20,22,23) have shown that glycerol caused a 30-50s decrease of hepatic ATP in rats either fed a commercial diet or starved. In hepatocytes from sucrose fed

rats, we observed only a 15-20s reduction in the ATP level of the hepatocytes after 1 h of incubation with 10 mM glycerol (Table IV). Our results do not support the pro- posal of Clark et al. (6) since no significant correlation between ATP levels and lipogenesis was found (Table VI). In addition, 100 mM TMPD increased lipogenesis in hepatocytes incubated with glycerol without affecting the ATP content of the cells (Ta- ble V). Therefore, under our conditions, the depletion of the ATP pool could not ac- count for the inhibition of lipogenesis.

Since acute administration of fructose also causes depletion of the ATP pool (23, 24), it has been suggested that fructose feeding may induce a metabolic adaptation such that the ATP concentration can be maintained close to its normal levels (25). Such an adaptation may explain why hepatocytes from rats fed fructose-containing diets were more resistant to the effect of glycerol on hepatic lipogenesis.

An alternative explanation for the effect of glycerol on fatty acid synthesis has been put forward by Lin et al. (7). According to these authors the antilipogenic effect of glycerol is due to the increased cytosolic NADH/NAD ratio which inhibits the conversion of glucose (or glycogen) to pyruvate at glyceraldehyde-3-P dehydrogenase (7, 26).

Addition of 10 mM glycerol to cells caused a fivefold increase in a lactate/pyruvate ratio which resulted from a large decrease in pyruvate and a small increase in lactate (Table III), which is indicative of a more reduced cytosolic compartment. Despite the increase in NADH/NAD ratio it is unlikely that glycolysis was impaired to a large extent as suggested by Lin et al. (7), since there was a marked accumulation of lactate plus pyruvate after incubation with glycerol or glycerol plus fructose (Ta- ble III). This is in agreement with other ob- servations (3-5, 20) which showed lactate as one of the major products of glycerol metabolism.

Comparison of the data presented in Ta- bles II and III suggests an inverse relationship between the rate of lipogenesis and the lactate/pyruvate ratio determined under various experimental conditions. Gu-


maa et al. (26) have shown that under a variety of metabolic and hormonal conditions there existed an inverse relationship between the rate of lipogenesis and the cytosolic NADH/NAD ratio. Although this was true for the extreme cases (Table II and III), the multiple correlation analysis revealed that the ratio between pyruvate and lactate was not significantly corre- lated with the rate of fatty acid synthesis (see Table VI). The lack of significant correlation between these two variables may reflect the fact that a given pyruvate/lactate ratio may result from a large number of combinations between pyruvate and lactate levels.

Pyruvate, but not lactate or fructose, was able to overcome the inhibitory effect of glycerol and lipogenesis (Table II). There are two possibilities for this observation. Pyruvate may act as a cytosolic hydrogen acceptor rendering the cytosol compartment more oxidized, or it may act as a direct precursor of acetyl-CoA inside the mitochondrial compartment.

In rat adipose tissue, pyruvate is a better lipogenic substrate than lactate, and it has been suggested (27) that lactate metabolism is limited by the rate of removal of cytosolic NADH. TMPD, which transports reducing equivalents from the cytosol into the mitochondria without affecting the ATP pool (27), caused a twofold increase in lipogenesis in adipocytes incubated with 5 mM lactate (27, 28). A similar effect of TMPD was observed in hepatocytes incubated with glycerol or glycerol plus lactate (Table V), where a 40-50% stimulation of lipogenesis was observed. It was also found (Table V) that the lactate/pyruvate ratio decreased by 60%.

The effect of TMPD on the lactate/pyruvate ratio could be accounted for almost completely by an increase in pyruvate (results not shown; (27)). These findings suggest that in the presence of glycerol, the concentration of pyruvate may limit the provision of acetyl-CoA for lipogenesis. This situation is similar to the effect of ethanol on gluconeogenesis from lactate (29).

The acetyl-CoA generated from pyruvate in the mitochondria can be converted

to citrate, the major precursor of cytosolic acetyl-CoA (30), or to ketone bodies. Tables II and IV show that glycerol had the same effect on lipogenesis and ketogenesis. In our incubations, glycerol caused a 60% decrease in ketone output which may be caused by the low levels of pyruvate found in these cells. The addition of pyruvate, but not lactate, caused a large increase in ketogenesis (see Table IV). It appears that at high pyruvate levels, pyruvate dehydrogenase is activated (31, 32), increasing the availability of acetyl-CoA in the mitochondria. Our finding that pyruvate was the best predictor variable (see Table VI), ac- counting for 78% of the variability of lipogenesis, supports the theory that the concentration of pyruvate, when present at low levels, determines the rate of lipogenesis.

The results presented in this paper strongly suggest that the antilipogenic effect of glycerol is caused by the large decrease in cytosolic pyruvate levels which may result from an increase in the NADH/ NAD ratio. The decreased pyruvate may limit the production of citrate, and conse- quently, of acetyl units for fatty acid synthesis. In this regard, our results may indirectly support the suggestion of Lin et al. (7), who considered the fall in citrate levels as one of the possible factors responsible for the impairment of fatty acid synthesis.

In hepatocytes from sucrose fed rats, the change in ATP concentration does not seem to play a role in the inhibition of lipogenesis. However, the possibility is not ex- cluded that, in hepatocytes from rats fed diets of low-fructose content, depletion of the ATP pool might contribute to the inhibition of this metabolic process by glycerol.

ACKNOWLEDGMENTS

The authors express their appreciation to Ernest Avery for his excellent technical assistance and to Steve Pimentel and Jerry McMurphy for their help with the processing of the samples. Our appreciation is also extended to Barbara Washburn for her suyges- tions and critical reading of the manuscript.

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effect of glycerol and dihydroxyacetone on hepatic lipogenesis

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