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Lipogenesis in aminonucleoside-induced nephrotic syndrome
241
Biochimica et Biophysics Acta, 360 (1974) 241-251 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56432
LIPOGENESIS SYNDROME
IN AMINONUCLEOSIDE-INDUCED
SOPHIA DiAMANT
and ELEAZAR
NEPHROTIC
SHAFRIR
Department of Biochemistry, Hebrew University-Hadassah Medical School and Hadassah University Hospital, Jerusalem (Israel) (Received
March 26th, 1974)
Summary The activity of rat liver lipogenic enzymes, acetyl-CoA carboxylase, fatty acid synthetase, ATP, citrate lyase and NADP-malate dehydrogenase and the in vitro . incorporation %of [l- ’ 4 C] acetyl-CoA to fatty acids became markedly reduced as a result of.se@al injections of aminonucleoside of puromycin, used to, mduce nephrosis. The activity of the lipogenic pathway returned to normal and even showed an adaptive increase in nephrotic rats 10-14 days after the termination of aminonucleoside injections. The initial decrease in the hepatic lipogenic capacity was attributed to the direct effect of aminonucleoside on enzyme synthesis since (1) it preceded the onset of proteinuria, (2) was observed in rats given doses of aminonucleoside insufficient to produce early nephrosis and (3) was also evident in adipose tissue. The rise in serum lipids closely followed the onset of nephrotic proteinuria and hypoproteinemia despite the coincident decrease in hepatic lipogenie capacity. The development of hyperlipidemia was associated with a marked increase in circulating free fatty acid levels which subsided later, coinciding with the rise in the activity of lipogenic enzymes. This observation, together with lack of change in liver triglyceride content, suggested an increased channeling of preformed free fatty acids into the lipoprotein-borne lipids. The findings indicate the need for careful dissociation of direct effects of aminonucleoside from those induced by nephrosis and demonstrate that the hepatic overproduction of apolipoproteins entails a complementary elaboration of lipid moieties either by de novo fatty acid synthesis or by the utilization of extrahepatic fatty acids.
Introduction Experimental aminonucleoside
nephrotic syndrome, caused by of puromycin [ 1,2], or antikidney
the administration of serum [3] to rats, is
242
accompanied by a marked rise in plasma triglyceride and cholesterol levels. Changes in the metabolic pattern in the liver aim toward compensation of the plasma protein loss resulting from the kidney lesion. There is an enhanced plasma albumin synthesis in antikidney serum induced nephrosis [ 3,4] in association with an increased production of apolipoproteins and increased incorporation of various precursors into the lipid moieties of the lipoproteins [5--S]. Similar observations were made in aminonucleoside-induced nephrotic syndrome [9,10]. To gain an insight into the cellular mechanisms leading to the hyperlipidemia in aminonucleoside-induced nephrosis, the function of the hepatic fatty acid synthesis pathway was presently investigated. The maximal activity of enzymes with regulatory implication for fatty acid synthesis was determined and a correlation was sought between changes in their activity and the nature of lipid precursors channeled into the lipoproteins. The possibility of direct influence of puromycin aminonucleoside on the enzyme activity pattern was also explored at different stages of the syndrome. Materials and Methods Male albino rats of Hebrew University strain, weighing 150 to 200 g were fed ad libitum a local pelleted diet containing 65% carbohydrate, 20% protein, 4% fat and 11% of salts, cellulose and inert material by weight. Nephrosis was induced by seven daily subcutaneous injections of aminonucleoside of puromycin [ 6dimethylamino-9-( 3’-amino-3’-deoxy-0-D -ribofuranosyl)-purine ] ,2 mg per 100 g rat weight or as otherwise indicated. The rats were sacrificed during the injection period and at various times within 3-14 days after the last injection. The rats were anesthetized by a short exposure to exposure to ether, and the blood was collected from the abdominal aorta. Liver and epidymal adipose tissue were homogenized (1:3, w/v), in 0.20 M sucrose solution containing 20 mM triethanolamine (pH 7.4), 1 mM disodium EDTA and 1 mM dithioerythreitol, or in other media as specified in the enzyme determination procedures. The homogenates were centrifuged at 4°C at 100 000 X g for 45 min, and the fatand particle-free fluids were used for enzyme assays. The activities of NADP-malate dehydrogenase (EC 1.1.1.40), ATP citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase, as well as the incorporation of [l-* 4 Clacetyl CoA into fatty acids were measured as outlined in a previous publication [ll]. The enzyme activities were expressed as nmoles of the respective substrate metabolized per min per mg protein at 37” C in the particle-free supernatant fluid. Tissue protein content was measured by a modified method of Lowry et al. [ 121. To avoid the chromogenic interference of some materials in the homogenizing solution, the protein was precipitated and washed with 5% trichloroacetic acid, and then solubilized in 5% NaOH prior to the determination. Urinary protein was determined by photometric measurement of sulphosalicylic acid turbidity using bovine albumin as a standard. Liver glycogen was measured enzymatically [13] after digestion of the tissue in 33% KOH, precipitation in 60% ethanol and resolubilization.
243
Cholesterol and triglycerides were determined ‘by an automated assay [14]. Extracts from serum were prepared in isopropanol in the ratio 0.3 : 9.7 (v/v); liver samples were first extracted with chloroform-methanol (2~1, v/v), the extracts evaporated and taken up into the water-isopropanol mixture (0.3:9:7; v/v). Serum free fatty acids were extracted and determined by a modification of the method of Dole and Meinertz [ 151. Auxilliary enzyme preparations and cofactors used in the assays were purchased from Sigma Chemical Co., (St. Louis, MO.) or Boehringer (Mannheim, Germany). Aminonucleoside was obtained from Nutritional Biochemicals Corp. (Cleveland, Ohio). Radioactive materials were purchased from the Radiochemical Centre, Amersham, U.K.
Results Preliminary determinations of the activity of enzymes associated with fatty acid synthesis in the liver of nephrotic rats indicated that the activity was decreased 3-5 days after the termination of a series of seven aminonucleoside injections [ 161. At this time, a marked proteinuria and hyperlipidemia were evident. To investigate whether the decrease in enzyme activity was secondary to the hyperlipidemia or directly due to aminonucleoside, a study of the time course of the changes in the activities of enzymes of lipogenesis was carried out during and after aminonucleoside administration. As shown in Fig. 1, aminonucleoside treatment induced an acute decrease in the activity of acetyl-CoA carboxylase and fatty acid synthetase, involved I
Fi& 1. Chenees in the activitv of enwmes coficemed with fatty acid synthesis during end after &onucleoside injections. L&t side (): changes duriug seven de& treatnwnta with 2 lsg/per 100 g amhonucleoside which induces nep&roeis. Right side (. - * -): changes in nemotie ntr reeovw from the extrerenel effects of eminonu@eoside. Vertical bars repreeent S.E. of the meen for S-10 ratr in wh group.
244
directly in fatty acid synthesis, as well as of NADP-malate dehydrogenase (“malate enzyme”), and ATP citrate lyase (“citrate cleavage enzyme”), enzymes auxiliary to fatty acid synthesis. The decrease in activity was significant already after two or three days of aminonucleoside injection and continued gradually to a nadir after seven days of injection. The drop in acitivity was approximately to 40% of the value in control animals in the case of NADPmalate dehydrogenase and to 55% of the value in control animals in case of fatty acid synthetase. The low level of the activity of the four enzymes of fatty acid synthesis pathway, reached after seven aminonucleoside injections, persisted for five days after cessation of aminonucleoside administration. A rise was evident after seven days of recovery from the treatment with aminonucleoside, whereas activities higher than initial ones were obtained after 10 or 14 days of recovery. The rise above the initial activity was statistically significant (p < 0.05) in the case of ATP citrate lyase, fatty acid synthetase and acetyl-CoA carboxylase. The rate of overall channeling of [l-l 4 C] acetyl-CoA to fatty acids, was about 40% of the control value, three days after the termination of aminonucleoside injections, but 10 days later it exceeded the control value (Table I). The relation of the changes in the activity of liver enzymes participating in fatty acid synthesis to the rise in serum triglyceride and cholesterol levels and to the onset of proteinuria and hypoproteinemia, was also examined during the induction of nephrosis. Fig. 2 shows that the changes in serum protein level and urinary protein excretion were small during the first 3-4 days of aminonucleoside injection. The hypoproteinemia, together with massive proteinuria, started between the fourth and fifth day of aminonucleoside injection. The abrupt rise in serum triglycerides and cholesterol also started between the fourth and sixth day of aminonucleoside injection. However, it should be stressed that the decrease in the activity of enzymes of lipogenesis (Fig. 1) preceded the onset of proteinuria and that the rise in serum lipid levels occurred despite the coincident decrease in the activity of enzymes of lipogenesis. Upon cessation of aminonucleoside injections the rise in serum triglyceride and cholesterol levels continued (Fig. 2). Highest levels, which were reached after 7-10 days of recovery were associated with a return of the activity of enzymes of lipogenesis to or above normal. At this time the proteinTABLE
I
INCORPORATION
OF [1-14Cl
ACETYL-CoA
TO FATTY
ACIDS
Fatty acid synthesis from [1-14Cl acetyl-CoA was measured in the 100000 X g liver supernatant fraction as described in ref. 11. Values in the table are means + S.E. for 6-8 rats in each group. The asterisk denotes significant difference from the control value (P < 0.01). Rats
Activity (nmolelmin per mg protein)
Control Four daily aminonucleoside injections 2 mg per 100 g Nephrotic, 3 days after termination of 7 aminonucleoside Nephrotic. 10 days after termination of 7 aminonucleoside
0.81 + 0.10 0.46 +_O.Ol* 0.24 + 0.02* 0.99 +- 0.08*
injections injections
245
Fig. 2. Time course of the hypoproteinemia and proteinuria in relation to the elevation of serum lipids, changes during seven daily treatments during and after aminonucleoside ink&ions. Left side ( -): with 2 mg per 100 g aminonucleoside. Right side (* -* -_): changes in nephrotic rats after cessation of aminonucleoside injections. Vertical bars represent S.E. of the mean for 12-20 rats in each group. FFA : free fatty acids: TG : triglycerides: TC : total cholesterol.
uria was at its peak, whereas the low levels of total serum protein, reached after seven days of recovery from aminonucleoside injections, remained without appreciable change. Fig. 2 also shows changes in levels of serum free fatty acids. A steep rise in serum free fatty acids occurred on the fifth day of treatment with aminonucleoside, coinciding with the onset of hypoproteinemia and hyperlipidemia. The rise in free fatty acids continued through the seven days of recovery. The levels then dropped almost to normal at the fourteenth day of recovery. The changes in hepatic enzymes concerned with fatty acid synthesis, which preceded the onset of nephrotic proteinuria and hyperlipidemia, suggested that loss in enzyme activity was induced directly by aminonucleoside and not by the changes associated with the nephrotic condition. To support this contention, aminonucleoside was administered to rats in doses insufficient to induce nephrosis within seven days. Table II shows that a significant decrease in the activity of enzymes participating in lipogenesis started on the fourth or fifth day of injection of the drug. There was no proteinuria at this time or even after seven days of injection when the enzyme activities were down to approximately one half of the initial value. These results extend those presented in Fig. 1 and accentuate the dissociation between the decrease in activity of lipogenic enzymes and the onset of nephrosis. The changes in the activity of liver enzymes participating in fatty acid
246 TABLE
II
EFFECT OF A LOW DOSE LIPOGENESIS AND LIVER Aminonucleoslde
OF AMINONUCLEOSIDE GLYCOGEN CONTENT
was injected
ON THE ACTIVITY
m the dose of 1 mg/lOO
g body
OF LIVER
ENZYMES
OF
weight per day instead of the usual dose of
2 mg/lOO g per day. used to obtain nephrosis in seven days. No proteinuria, hypoproteinemia or hyperlipidemia was observed in this series of rats. Liver enzyme activities and glycogen content were determined as described in the Methods. Values are means ?z S.E. for groups of four rats except the control group which comprised 12 rats. Significant differences from control rats (P < 0.05 at least) are indicated by an asterisk. ____
_ NADP-malate
Day of mjection
0
3 4 5 7
dehydrogenase (mnole /min per mg protein)
ATP citrate lyase (nmolel min per mg protein)
Acetyl-CoA carboxylase (nmolelmin per mg protein)
Fatty acid synthetase (nmolelmin per mg protein)
Liver glycogen
70.8 72.6 53.7 40.8 35.4
23.0 19.4 18.8 13.8 12.1
22.6 18.0 15.8 13.9 14.3
6.2 + 0.5
73.0 65.3 71.1 72.4 71.5
+ 6.9 +_9.0 + 6.0 + 2.9* + 2.6*
+ + f f t
2.4 0.8 3.6 2.6* 2.1*
f + + t +
2.3 2.7 1.8* 1.1* 2.0*
(mglg)
3.7 ? 1.0* 2.8 + 0.2’ 2.6 + 0.4*
f 5.6 t 2.2 + 2.0 + 2.5 +6.5
synthesis and in the levels of serum free fatty acids could have resulted from reduced food intake which might have been associated with the aminonucleoside treatment. Evidence that this is not the case was obtained from the determination of liver glycogen levels, as shown in Table II. Lack of significant change in liver glycogen during the seven days of aminonucleoside injections negates the possibility that the suppression of lipogenesis could have been caused by starvation. In experiments in which nephrosis-inducing doses of aminonucleoside were given, liver glycogen levels fell by 20-40’S on days 5-7 of aminonucleoside injections and returned to normal 3-5 days after cessation of injections. However, as shown in Fig. 1, the decrease in enzyme activities was evident earlier and was much more pronounced than in a glycogen-void liver of a 24 h fasted rat. The fact that hyperlipidemia was observed in spite of the decrease in the hepatic capacity of fatty acid synthesis raised the possibility of a shift of TABLE
III
LIVER TRIGLYCERIDE SIDE TREATMENT Values
in the Table
AND
are means
(P <0.05 at least) are indicated
CHOLESTEROL 2 S.E. for groups by an asterisk.
CONTENT of 6-8
DURING
rats. Significant
Days of treatment 0
3
Cholesterol
(mglg) (m&!/g)
AFTER
5
7
AMINONUCLEO-
differences
Days of recovery ___ ___~~~
__---___ Trlglycendes
AND
from
__~_
control
rats
_
3
5
7
10
7.9 * 0.7 5.9* i: 0.3
7.3 +_1.1 4.9 +_0.3
8.4 rt 0.8 5.0 to.5
8.3 + 0.5 5.4* ? 0.4
8.6 f 0.4 4.3 + 0.2
8.9 f 0.4 4.5 +_0.2
9.0 f 0.4 5.8* +0.4
7.7 f 0.7 5.2 f 0.2
247
DAYS
6
4
2
0
OF
TREATMENT
Fig. 3. Changes in the activity of enzymes concerned with fatty acid synthesis in rat epididymal adipose tissue during the course of seven aminonucleoside injections 2 mg per 100 g
preformed lipids from liver into the circulation. The results in Table III show that under the conditions of our experiments, the development of hyperlipidemia was not accompanied by a decrease in liver triglyceride content, neither during the course of aminonucleoside injections nor after four or ten days of recovery. On the contrary, there was a tendency toward an increase in liver cholesterol content. To investigate whether the effect of aminonucleoside is specific to the liver, as a site of compensatory plasma protein synthesis, or a general one, the activity of lipogenic enzymes was also determined in adipose tissue. Fig 3 documents a marked decrease in the activity of adipose tissue acetyl-CoA carboxylase, fatty acid synthetase, NADP-malate dehydrogenase and ATP citrate lyase two to three days before the onset of the massive protein&a and hyperlipidemia. These results indicate that adipose tissue lipogenesis was suppressed by aminonucleoside directly and independently of its nephrosis-inducing effect. Discussion Increased
hepatic
production
of lipoproteins
in the nephrotic
syndrome
is
248
documented by chemical and immunological measurements of lipoproteins released from liver slices or from perfused liver [5,6,16] and by electronmicroscopic studies [ 171. There is an augmented elaboration of both protein and lipid moieties of the lipoproteins in antikidney serum, as well as in aminonucleoside-induced nephrosis as inferred from the enhanced rates of incorporation of amino acids into apolipoproteins or of glucose, acetate, mevalonate or citrate into triglycerides or cholesterol [l-10,16,17] . The production of apolipoproteins seems to be stimulated as part of the overall compensatory hepatic effort to replace plasma proteins in response to hypoproteinemia, whereas the complementary synthesis of lipoprotein-borne lipids in the liver may result from: (1) an increased activity of rate-limiting enzymes of the pathway of lipogenesis, (2) an increased availability of lipogenic precursors which are channeled into fatty acid synthesis, (3) an enhanced flow of substrates through the lipogenic pathway by an accelerated removal of the end products, (4) an increased utilization of preformed fatty acids, either by a shift of liver lipids to the plasma or mobilization of adipose tissue fatty acids. Our results do not indicate that the primary cause for the increased lipoprotein lipid production in nephrosis was an increase in the activity of hepatic enzymes of lipogenesis. The activities of four regulatory enzymes of this pathway, determined under optimal in vitro assay conditions, showed a decrease during the acute phase of induction of nephrosis by aminonucleoside, which is seemingly incompatible with the increased liver triglyceride production. It could be conceived that the reason for the initial decline in lipogenic enzymes is feedback inhibition secondary to the hyperlipoproteinemia; this is unlikely, however, because the decline was already evident before the onset of extensive proteinuria and hyperlipoproteinemia. It is most probable that the loss of activity was the result of a selective inhibition of enzyme synthesis by aminonucleoside, in addition to the nephrotic lesion caused by this drug, since it was observed in the liver even at low doses of aminonucleoside, insufficient to induce proteinuria. The activity of enzymes of fatty acid synthesis was also reduced in adipose tissue, prior to the onset of nephrosis, indicating a direct effect of aminonucleoside on the enzymes of this tissue as well. Evidence has been presented that aminonucleoside may retain, toward certain systems, the protein-synthesis inhibition potency of its parent substance, puromycin, by selectively inhibiting the synthesis of certain cellular RNA species in the L-strain of mouse fibroblasts [18], of enzymes of gluconeogenesis in glucocorticoid-treated rat liver [ 191 and of hemin in rabbit bone marrow [ 201. The small but steady drop in serum protein level during the first five days of aminonucleoside administration (Fig. 2), preceding the onset of massive proteinuria indicates that liver protein synthesis, in general, was retarded. The marked increase in proteinuria without change in serum protein level, together with the increase in lipoproteinemia, which occurred seven days after cessation of aminonucleoside injections indicates that the compensatory hepatic synthesis of plasma proteins reached its capacity only at this time, after recovering from direct aminonucleoside effects.
249
A few days after the termination of aminonucleoside administration there was a return to normal values and then a significant and persistent rise in the activity of enzymes of fatty acid synthesis, over and above the levels in control animals, which appears to express a secondary adjustment to the increased flow of substrates or intermediates through the lipogenesis pathway. This seems to be a metabolic event characteristic of the nephrotic condition which became obscured during the acute stage of aminonucleoside presence. The observation that aminonucleoside may affect the enzymes of lipogenesis and, possibly, enzymes of other metabolic pathways in the liver or kidney [17,21-231 emphasizes the need for a long recovery period after aminonucleoside administration, in order to be able to dissociate the direct effects of the drug from the hepatic metabolic adjustments evoked by the nephrotic hypoproteinemia. With respect to the possibility of increased precursor supply, which might have presented a load on the pathway of fatty acids synthesis, previous observations point out that there is no increase in liver metabolites that may lead to enhanced acetyl-CoA production [ 231. Glycolysis, similarly to lipogenesis, was reduced a few days after the onset of the aminonucleoside-induced nephrotic syndrome [23]. Total hepatic levels of acetyl-CoA were not increased in the liver of nephrotic rats (Diamant, S. and Shafrir, E. unpublished observations). Amino acids represent poor precursors for fatty acid synthesis in nephrosis [16] even if the flow of amino acids to the liver is increased together with a rise in the activity of hepatic enzymes of amino acid catabolism [23,24]. The above considerations stress the fact that an enhanced triglyceride outflow from the liver of nephrotic rats does occur in the absence of accumulation of precursors of fatty acid synthesis, and even at low capacity of the enzymes of this pathway. The determining factor appears to be the excessive availability of specific protein carriers of the synthesized lipids, which draws upon the lipogenic pathway for the supply of lipids required for the completion of lipoprotein molecules. This requirement appears to be so large that it cannot be met solely by de novo fatty acid synthesis. It must be sustained by the utilization of preformed fatty acids taken up from the serum particularly during the acute stage of nephrosis induction, coinciding with the mobilization from adipose tissue as demonstrated here by the markedly increased levels of circulating free fatty acids during the acute stage of the development of the nephrotic hyperlipoproteinemia. Enhanced free fatty acid release and lipolytic activity were also observed by Gutman and Shafrir [25] in adipose tissue of aminonucleoside nephrotic rats and by Tashimo and Matsuda [26] in fat celis of antikidney serum nephrotic rats. Serum free fatty acid elevation was not observed by the latter workers, which was attributed to their rapid hepatic removal [ 271 for lipogenesis. The mechanism of nephrotic hyperlipidemia based on hepatic trapping of preformed fatty acids is supported by experiments showing augmented uptake of labeled oleate by the isolated, perfused liver from aminonucleoside nephrotic rats and its secretion as lipoprotein-borne lipids [16] and by in vivo demonstration of increased reappearance of esterified lipids, containing labeled palmitate in antikidney nephrosis [28]. It is also pertinent to mention that triglyceride synthesis from preformed fatty acids in a subcellular liver system is
250
greatly enhanced by the presence of appropriate protein acceptors for the synthesized products [ 291. The time course of the changes in serum free fatty acid levels, from the onset of nephrosis through the 14 days of recovery from aminonucleoside injections, shows that the gradual decrease in free fatty acid mobilization from the initial peak is accompanied by a rise in the capacity of the liver to synthesize fatty acids de novo. This is evident from the rise in the activity of enzymes of the lipogenic pathway. Thus the importance of the source of fatty acids for lipoprotein assembly appears to shift from depots, external to the liver, to fatty acids derived from de novo synthesis. Since the liver of our aminonucleoside-nephrotic rats was not depleted of triglycerides or cholesterol, it is unlikely that the lipid complement of the lipoproteins was taken from the structural or cytoplasmic liver lipids. However, there are reports that the hyperlipoproteinemia may be associated with a decrease [30,31], an increase [283, or lack of change [9,32] in liver lipid content. It is possible that exessive utilization of endogenous hepatic lipids for lipoprotein assembly may occur in some circumstances, in which the sources of extrahepatic fatty acids are restricted. It may be concluded that the hepatic overproduction of apolipoproteins, in association with the increased synthesis of other plasma proteins seems to be responsible for the demand for complementary lipid elaboration. The accelerated removal of end products may promote the channeling of substrates through the lipogenic pathway, and induce an adaptive rise in the activity of regulatory enzymes of lipogenesis. The incorporation of preformed fatty acids into the lipoprotein-borne lipids may also be increased. Thus nephrotic hyperlipoproteinemia may be of multifaceted origin as far as the supply of sources of the lipid moiety of the lipoproteins are concerned. Acknowledgement This research was supported by the United States-Israel Foundation, Research Grant Agreement No. 205.
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Biochimica et Biophysics Acta, 360 (1974) 241-251 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56432
LIPOGENESIS SYNDROME
IN AMINONUCLEOSIDE-INDUCED
SOPHIA DiAMANT
and ELEAZAR
NEPHROTIC
SHAFRIR
Department of Biochemistry, Hebrew University-Hadassah Medical School and Hadassah University Hospital, Jerusalem (Israel) (Received
March 26th, 1974)
Summary The activity of rat liver lipogenic enzymes, acetyl-CoA carboxylase, fatty acid synthetase, ATP, citrate lyase and NADP-malate dehydrogenase and the in vitro . incorporation %of [l- ’ 4 C] acetyl-CoA to fatty acids became markedly reduced as a result of.se@al injections of aminonucleoside of puromycin, used to, mduce nephrosis. The activity of the lipogenic pathway returned to normal and even showed an adaptive increase in nephrotic rats 10-14 days after the termination of aminonucleoside injections. The initial decrease in the hepatic lipogenic capacity was attributed to the direct effect of aminonucleoside on enzyme synthesis since (1) it preceded the onset of proteinuria, (2) was observed in rats given doses of aminonucleoside insufficient to produce early nephrosis and (3) was also evident in adipose tissue. The rise in serum lipids closely followed the onset of nephrotic proteinuria and hypoproteinemia despite the coincident decrease in hepatic lipogenie capacity. The development of hyperlipidemia was associated with a marked increase in circulating free fatty acid levels which subsided later, coinciding with the rise in the activity of lipogenic enzymes. This observation, together with lack of change in liver triglyceride content, suggested an increased channeling of preformed free fatty acids into the lipoprotein-borne lipids. The findings indicate the need for careful dissociation of direct effects of aminonucleoside from those induced by nephrosis and demonstrate that the hepatic overproduction of apolipoproteins entails a complementary elaboration of lipid moieties either by de novo fatty acid synthesis or by the utilization of extrahepatic fatty acids.
Introduction Experimental aminonucleoside
nephrotic syndrome, caused by of puromycin [ 1,2], or antikidney
the administration of serum [3] to rats, is
242
accompanied by a marked rise in plasma triglyceride and cholesterol levels. Changes in the metabolic pattern in the liver aim toward compensation of the plasma protein loss resulting from the kidney lesion. There is an enhanced plasma albumin synthesis in antikidney serum induced nephrosis [ 3,4] in association with an increased production of apolipoproteins and increased incorporation of various precursors into the lipid moieties of the lipoproteins [5--S]. Similar observations were made in aminonucleoside-induced nephrotic syndrome [9,10]. To gain an insight into the cellular mechanisms leading to the hyperlipidemia in aminonucleoside-induced nephrosis, the function of the hepatic fatty acid synthesis pathway was presently investigated. The maximal activity of enzymes with regulatory implication for fatty acid synthesis was determined and a correlation was sought between changes in their activity and the nature of lipid precursors channeled into the lipoproteins. The possibility of direct influence of puromycin aminonucleoside on the enzyme activity pattern was also explored at different stages of the syndrome. Materials and Methods Male albino rats of Hebrew University strain, weighing 150 to 200 g were fed ad libitum a local pelleted diet containing 65% carbohydrate, 20% protein, 4% fat and 11% of salts, cellulose and inert material by weight. Nephrosis was induced by seven daily subcutaneous injections of aminonucleoside of puromycin [ 6dimethylamino-9-( 3’-amino-3’-deoxy-0-D -ribofuranosyl)-purine ] ,2 mg per 100 g rat weight or as otherwise indicated. The rats were sacrificed during the injection period and at various times within 3-14 days after the last injection. The rats were anesthetized by a short exposure to exposure to ether, and the blood was collected from the abdominal aorta. Liver and epidymal adipose tissue were homogenized (1:3, w/v), in 0.20 M sucrose solution containing 20 mM triethanolamine (pH 7.4), 1 mM disodium EDTA and 1 mM dithioerythreitol, or in other media as specified in the enzyme determination procedures. The homogenates were centrifuged at 4°C at 100 000 X g for 45 min, and the fatand particle-free fluids were used for enzyme assays. The activities of NADP-malate dehydrogenase (EC 1.1.1.40), ATP citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase, as well as the incorporation of [l-* 4 Clacetyl CoA into fatty acids were measured as outlined in a previous publication [ll]. The enzyme activities were expressed as nmoles of the respective substrate metabolized per min per mg protein at 37” C in the particle-free supernatant fluid. Tissue protein content was measured by a modified method of Lowry et al. [ 121. To avoid the chromogenic interference of some materials in the homogenizing solution, the protein was precipitated and washed with 5% trichloroacetic acid, and then solubilized in 5% NaOH prior to the determination. Urinary protein was determined by photometric measurement of sulphosalicylic acid turbidity using bovine albumin as a standard. Liver glycogen was measured enzymatically [13] after digestion of the tissue in 33% KOH, precipitation in 60% ethanol and resolubilization.
243
Cholesterol and triglycerides were determined ‘by an automated assay [14]. Extracts from serum were prepared in isopropanol in the ratio 0.3 : 9.7 (v/v); liver samples were first extracted with chloroform-methanol (2~1, v/v), the extracts evaporated and taken up into the water-isopropanol mixture (0.3:9:7; v/v). Serum free fatty acids were extracted and determined by a modification of the method of Dole and Meinertz [ 151. Auxilliary enzyme preparations and cofactors used in the assays were purchased from Sigma Chemical Co., (St. Louis, MO.) or Boehringer (Mannheim, Germany). Aminonucleoside was obtained from Nutritional Biochemicals Corp. (Cleveland, Ohio). Radioactive materials were purchased from the Radiochemical Centre, Amersham, U.K.
Results Preliminary determinations of the activity of enzymes associated with fatty acid synthesis in the liver of nephrotic rats indicated that the activity was decreased 3-5 days after the termination of a series of seven aminonucleoside injections [ 161. At this time, a marked proteinuria and hyperlipidemia were evident. To investigate whether the decrease in enzyme activity was secondary to the hyperlipidemia or directly due to aminonucleoside, a study of the time course of the changes in the activities of enzymes of lipogenesis was carried out during and after aminonucleoside administration. As shown in Fig. 1, aminonucleoside treatment induced an acute decrease in the activity of acetyl-CoA carboxylase and fatty acid synthetase, involved I
Fi& 1. Chenees in the activitv of enwmes coficemed with fatty acid synthesis during end after &onucleoside injections. L&t side (): changes duriug seven de& treatnwnta with 2 lsg/per 100 g amhonucleoside which induces nep&roeis. Right side (. - * -): changes in nemotie ntr reeovw from the extrerenel effects of eminonu@eoside. Vertical bars repreeent S.E. of the meen for S-10 ratr in wh group.
244
directly in fatty acid synthesis, as well as of NADP-malate dehydrogenase (“malate enzyme”), and ATP citrate lyase (“citrate cleavage enzyme”), enzymes auxiliary to fatty acid synthesis. The decrease in activity was significant already after two or three days of aminonucleoside injection and continued gradually to a nadir after seven days of injection. The drop in acitivity was approximately to 40% of the value in control animals in the case of NADPmalate dehydrogenase and to 55% of the value in control animals in case of fatty acid synthetase. The low level of the activity of the four enzymes of fatty acid synthesis pathway, reached after seven aminonucleoside injections, persisted for five days after cessation of aminonucleoside administration. A rise was evident after seven days of recovery from the treatment with aminonucleoside, whereas activities higher than initial ones were obtained after 10 or 14 days of recovery. The rise above the initial activity was statistically significant (p < 0.05) in the case of ATP citrate lyase, fatty acid synthetase and acetyl-CoA carboxylase. The rate of overall channeling of [l-l 4 C] acetyl-CoA to fatty acids, was about 40% of the control value, three days after the termination of aminonucleoside injections, but 10 days later it exceeded the control value (Table I). The relation of the changes in the activity of liver enzymes participating in fatty acid synthesis to the rise in serum triglyceride and cholesterol levels and to the onset of proteinuria and hypoproteinemia, was also examined during the induction of nephrosis. Fig. 2 shows that the changes in serum protein level and urinary protein excretion were small during the first 3-4 days of aminonucleoside injection. The hypoproteinemia, together with massive proteinuria, started between the fourth and fifth day of aminonucleoside injection. The abrupt rise in serum triglycerides and cholesterol also started between the fourth and sixth day of aminonucleoside injection. However, it should be stressed that the decrease in the activity of enzymes of lipogenesis (Fig. 1) preceded the onset of proteinuria and that the rise in serum lipid levels occurred despite the coincident decrease in the activity of enzymes of lipogenesis. Upon cessation of aminonucleoside injections the rise in serum triglyceride and cholesterol levels continued (Fig. 2). Highest levels, which were reached after 7-10 days of recovery were associated with a return of the activity of enzymes of lipogenesis to or above normal. At this time the proteinTABLE
I
INCORPORATION
OF [1-14Cl
ACETYL-CoA
TO FATTY
ACIDS
Fatty acid synthesis from [1-14Cl acetyl-CoA was measured in the 100000 X g liver supernatant fraction as described in ref. 11. Values in the table are means + S.E. for 6-8 rats in each group. The asterisk denotes significant difference from the control value (P < 0.01). Rats
Activity (nmolelmin per mg protein)
Control Four daily aminonucleoside injections 2 mg per 100 g Nephrotic, 3 days after termination of 7 aminonucleoside Nephrotic. 10 days after termination of 7 aminonucleoside
0.81 + 0.10 0.46 +_O.Ol* 0.24 + 0.02* 0.99 +- 0.08*
injections injections
245
Fig. 2. Time course of the hypoproteinemia and proteinuria in relation to the elevation of serum lipids, changes during seven daily treatments during and after aminonucleoside ink&ions. Left side ( -): with 2 mg per 100 g aminonucleoside. Right side (* -* -_): changes in nephrotic rats after cessation of aminonucleoside injections. Vertical bars represent S.E. of the mean for 12-20 rats in each group. FFA : free fatty acids: TG : triglycerides: TC : total cholesterol.
uria was at its peak, whereas the low levels of total serum protein, reached after seven days of recovery from aminonucleoside injections, remained without appreciable change. Fig. 2 also shows changes in levels of serum free fatty acids. A steep rise in serum free fatty acids occurred on the fifth day of treatment with aminonucleoside, coinciding with the onset of hypoproteinemia and hyperlipidemia. The rise in free fatty acids continued through the seven days of recovery. The levels then dropped almost to normal at the fourteenth day of recovery. The changes in hepatic enzymes concerned with fatty acid synthesis, which preceded the onset of nephrotic proteinuria and hyperlipidemia, suggested that loss in enzyme activity was induced directly by aminonucleoside and not by the changes associated with the nephrotic condition. To support this contention, aminonucleoside was administered to rats in doses insufficient to induce nephrosis within seven days. Table II shows that a significant decrease in the activity of enzymes participating in lipogenesis started on the fourth or fifth day of injection of the drug. There was no proteinuria at this time or even after seven days of injection when the enzyme activities were down to approximately one half of the initial value. These results extend those presented in Fig. 1 and accentuate the dissociation between the decrease in activity of lipogenic enzymes and the onset of nephrosis. The changes in the activity of liver enzymes participating in fatty acid
246 TABLE
II
EFFECT OF A LOW DOSE LIPOGENESIS AND LIVER Aminonucleoslde
OF AMINONUCLEOSIDE GLYCOGEN CONTENT
was injected
ON THE ACTIVITY
m the dose of 1 mg/lOO
g body
OF LIVER
ENZYMES
OF
weight per day instead of the usual dose of
2 mg/lOO g per day. used to obtain nephrosis in seven days. No proteinuria, hypoproteinemia or hyperlipidemia was observed in this series of rats. Liver enzyme activities and glycogen content were determined as described in the Methods. Values are means ?z S.E. for groups of four rats except the control group which comprised 12 rats. Significant differences from control rats (P < 0.05 at least) are indicated by an asterisk. ____
_ NADP-malate
Day of mjection
0
3 4 5 7
dehydrogenase (mnole /min per mg protein)
ATP citrate lyase (nmolel min per mg protein)
Acetyl-CoA carboxylase (nmolelmin per mg protein)
Fatty acid synthetase (nmolelmin per mg protein)
Liver glycogen
70.8 72.6 53.7 40.8 35.4
23.0 19.4 18.8 13.8 12.1
22.6 18.0 15.8 13.9 14.3
6.2 + 0.5
73.0 65.3 71.1 72.4 71.5
+ 6.9 +_9.0 + 6.0 + 2.9* + 2.6*
+ + f f t
2.4 0.8 3.6 2.6* 2.1*
f + + t +
2.3 2.7 1.8* 1.1* 2.0*
(mglg)
3.7 ? 1.0* 2.8 + 0.2’ 2.6 + 0.4*
f 5.6 t 2.2 + 2.0 + 2.5 +6.5
synthesis and in the levels of serum free fatty acids could have resulted from reduced food intake which might have been associated with the aminonucleoside treatment. Evidence that this is not the case was obtained from the determination of liver glycogen levels, as shown in Table II. Lack of significant change in liver glycogen during the seven days of aminonucleoside injections negates the possibility that the suppression of lipogenesis could have been caused by starvation. In experiments in which nephrosis-inducing doses of aminonucleoside were given, liver glycogen levels fell by 20-40’S on days 5-7 of aminonucleoside injections and returned to normal 3-5 days after cessation of injections. However, as shown in Fig. 1, the decrease in enzyme activities was evident earlier and was much more pronounced than in a glycogen-void liver of a 24 h fasted rat. The fact that hyperlipidemia was observed in spite of the decrease in the hepatic capacity of fatty acid synthesis raised the possibility of a shift of TABLE
III
LIVER TRIGLYCERIDE SIDE TREATMENT Values
in the Table
AND
are means
(P <0.05 at least) are indicated
CHOLESTEROL 2 S.E. for groups by an asterisk.
CONTENT of 6-8
DURING
rats. Significant
Days of treatment 0
3
Cholesterol
(mglg) (m&!/g)
AFTER
5
7
AMINONUCLEO-
differences
Days of recovery ___ ___~~~
__---___ Trlglycendes
AND
from
__~_
control
rats
_
3
5
7
10
7.9 * 0.7 5.9* i: 0.3
7.3 +_1.1 4.9 +_0.3
8.4 rt 0.8 5.0 to.5
8.3 + 0.5 5.4* ? 0.4
8.6 f 0.4 4.3 + 0.2
8.9 f 0.4 4.5 +_0.2
9.0 f 0.4 5.8* +0.4
7.7 f 0.7 5.2 f 0.2
247
DAYS
6
4
2
0
OF
TREATMENT
Fig. 3. Changes in the activity of enzymes concerned with fatty acid synthesis in rat epididymal adipose tissue during the course of seven aminonucleoside injections 2 mg per 100 g
preformed lipids from liver into the circulation. The results in Table III show that under the conditions of our experiments, the development of hyperlipidemia was not accompanied by a decrease in liver triglyceride content, neither during the course of aminonucleoside injections nor after four or ten days of recovery. On the contrary, there was a tendency toward an increase in liver cholesterol content. To investigate whether the effect of aminonucleoside is specific to the liver, as a site of compensatory plasma protein synthesis, or a general one, the activity of lipogenic enzymes was also determined in adipose tissue. Fig 3 documents a marked decrease in the activity of adipose tissue acetyl-CoA carboxylase, fatty acid synthetase, NADP-malate dehydrogenase and ATP citrate lyase two to three days before the onset of the massive protein&a and hyperlipidemia. These results indicate that adipose tissue lipogenesis was suppressed by aminonucleoside directly and independently of its nephrosis-inducing effect. Discussion Increased
hepatic
production
of lipoproteins
in the nephrotic
syndrome
is
248
documented by chemical and immunological measurements of lipoproteins released from liver slices or from perfused liver [5,6,16] and by electronmicroscopic studies [ 171. There is an augmented elaboration of both protein and lipid moieties of the lipoproteins in antikidney serum, as well as in aminonucleoside-induced nephrosis as inferred from the enhanced rates of incorporation of amino acids into apolipoproteins or of glucose, acetate, mevalonate or citrate into triglycerides or cholesterol [l-10,16,17] . The production of apolipoproteins seems to be stimulated as part of the overall compensatory hepatic effort to replace plasma proteins in response to hypoproteinemia, whereas the complementary synthesis of lipoprotein-borne lipids in the liver may result from: (1) an increased activity of rate-limiting enzymes of the pathway of lipogenesis, (2) an increased availability of lipogenic precursors which are channeled into fatty acid synthesis, (3) an enhanced flow of substrates through the lipogenic pathway by an accelerated removal of the end products, (4) an increased utilization of preformed fatty acids, either by a shift of liver lipids to the plasma or mobilization of adipose tissue fatty acids. Our results do not indicate that the primary cause for the increased lipoprotein lipid production in nephrosis was an increase in the activity of hepatic enzymes of lipogenesis. The activities of four regulatory enzymes of this pathway, determined under optimal in vitro assay conditions, showed a decrease during the acute phase of induction of nephrosis by aminonucleoside, which is seemingly incompatible with the increased liver triglyceride production. It could be conceived that the reason for the initial decline in lipogenic enzymes is feedback inhibition secondary to the hyperlipoproteinemia; this is unlikely, however, because the decline was already evident before the onset of extensive proteinuria and hyperlipoproteinemia. It is most probable that the loss of activity was the result of a selective inhibition of enzyme synthesis by aminonucleoside, in addition to the nephrotic lesion caused by this drug, since it was observed in the liver even at low doses of aminonucleoside, insufficient to induce proteinuria. The activity of enzymes of fatty acid synthesis was also reduced in adipose tissue, prior to the onset of nephrosis, indicating a direct effect of aminonucleoside on the enzymes of this tissue as well. Evidence has been presented that aminonucleoside may retain, toward certain systems, the protein-synthesis inhibition potency of its parent substance, puromycin, by selectively inhibiting the synthesis of certain cellular RNA species in the L-strain of mouse fibroblasts [18], of enzymes of gluconeogenesis in glucocorticoid-treated rat liver [ 191 and of hemin in rabbit bone marrow [ 201. The small but steady drop in serum protein level during the first five days of aminonucleoside administration (Fig. 2), preceding the onset of massive proteinuria indicates that liver protein synthesis, in general, was retarded. The marked increase in proteinuria without change in serum protein level, together with the increase in lipoproteinemia, which occurred seven days after cessation of aminonucleoside injections indicates that the compensatory hepatic synthesis of plasma proteins reached its capacity only at this time, after recovering from direct aminonucleoside effects.
249
A few days after the termination of aminonucleoside administration there was a return to normal values and then a significant and persistent rise in the activity of enzymes of fatty acid synthesis, over and above the levels in control animals, which appears to express a secondary adjustment to the increased flow of substrates or intermediates through the lipogenesis pathway. This seems to be a metabolic event characteristic of the nephrotic condition which became obscured during the acute stage of aminonucleoside presence. The observation that aminonucleoside may affect the enzymes of lipogenesis and, possibly, enzymes of other metabolic pathways in the liver or kidney [17,21-231 emphasizes the need for a long recovery period after aminonucleoside administration, in order to be able to dissociate the direct effects of the drug from the hepatic metabolic adjustments evoked by the nephrotic hypoproteinemia. With respect to the possibility of increased precursor supply, which might have presented a load on the pathway of fatty acids synthesis, previous observations point out that there is no increase in liver metabolites that may lead to enhanced acetyl-CoA production [ 231. Glycolysis, similarly to lipogenesis, was reduced a few days after the onset of the aminonucleoside-induced nephrotic syndrome [23]. Total hepatic levels of acetyl-CoA were not increased in the liver of nephrotic rats (Diamant, S. and Shafrir, E. unpublished observations). Amino acids represent poor precursors for fatty acid synthesis in nephrosis [16] even if the flow of amino acids to the liver is increased together with a rise in the activity of hepatic enzymes of amino acid catabolism [23,24]. The above considerations stress the fact that an enhanced triglyceride outflow from the liver of nephrotic rats does occur in the absence of accumulation of precursors of fatty acid synthesis, and even at low capacity of the enzymes of this pathway. The determining factor appears to be the excessive availability of specific protein carriers of the synthesized lipids, which draws upon the lipogenic pathway for the supply of lipids required for the completion of lipoprotein molecules. This requirement appears to be so large that it cannot be met solely by de novo fatty acid synthesis. It must be sustained by the utilization of preformed fatty acids taken up from the serum particularly during the acute stage of nephrosis induction, coinciding with the mobilization from adipose tissue as demonstrated here by the markedly increased levels of circulating free fatty acids during the acute stage of the development of the nephrotic hyperlipoproteinemia. Enhanced free fatty acid release and lipolytic activity were also observed by Gutman and Shafrir [25] in adipose tissue of aminonucleoside nephrotic rats and by Tashimo and Matsuda [26] in fat celis of antikidney serum nephrotic rats. Serum free fatty acid elevation was not observed by the latter workers, which was attributed to their rapid hepatic removal [ 271 for lipogenesis. The mechanism of nephrotic hyperlipidemia based on hepatic trapping of preformed fatty acids is supported by experiments showing augmented uptake of labeled oleate by the isolated, perfused liver from aminonucleoside nephrotic rats and its secretion as lipoprotein-borne lipids [16] and by in vivo demonstration of increased reappearance of esterified lipids, containing labeled palmitate in antikidney nephrosis [28]. It is also pertinent to mention that triglyceride synthesis from preformed fatty acids in a subcellular liver system is
250
greatly enhanced by the presence of appropriate protein acceptors for the synthesized products [ 291. The time course of the changes in serum free fatty acid levels, from the onset of nephrosis through the 14 days of recovery from aminonucleoside injections, shows that the gradual decrease in free fatty acid mobilization from the initial peak is accompanied by a rise in the capacity of the liver to synthesize fatty acids de novo. This is evident from the rise in the activity of enzymes of the lipogenic pathway. Thus the importance of the source of fatty acids for lipoprotein assembly appears to shift from depots, external to the liver, to fatty acids derived from de novo synthesis. Since the liver of our aminonucleoside-nephrotic rats was not depleted of triglycerides or cholesterol, it is unlikely that the lipid complement of the lipoproteins was taken from the structural or cytoplasmic liver lipids. However, there are reports that the hyperlipoproteinemia may be associated with a decrease [30,31], an increase [283, or lack of change [9,32] in liver lipid content. It is possible that exessive utilization of endogenous hepatic lipids for lipoprotein assembly may occur in some circumstances, in which the sources of extrahepatic fatty acids are restricted. It may be concluded that the hepatic overproduction of apolipoproteins, in association with the increased synthesis of other plasma proteins seems to be responsible for the demand for complementary lipid elaboration. The accelerated removal of end products may promote the channeling of substrates through the lipogenic pathway, and induce an adaptive rise in the activity of regulatory enzymes of lipogenesis. The incorporation of preformed fatty acids into the lipoprotein-borne lipids may also be increased. Thus nephrotic hyperlipoproteinemia may be of multifaceted origin as far as the supply of sources of the lipid moiety of the lipoproteins are concerned. Acknowledgement This research was supported by the United States-Israel Foundation, Research Grant Agreement No. 205.
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