Data on lipid metabolism in the genetically obese Zucker rat

Pergamon Press

Lifa Sciaacea Vol . 20, pp . 541550, 1117 . Printed in the II .S .A.

DATA ON LIPID METABOLISM IN THE GENETICALLY OBESE ZUCKER RAT André Bach, Marthe Bauer and Henri Schirardin Laboratoire de la Clinique Médicale A HBpital Civil

67000

Strasbourg, France

(kecaived in final form January 5, 1977) Sume~ary

We assayed various lipid fractions as well as some intermediate metabolites and adenine nucleotides in the liver and the blood of the genetically obese Zucker rat (fa/fa) and compared their values with the corresponding ones obtained for nonobese controls and for Sprague-Dawley rats . Results for the two latter groups were similar, and differed greatly from those for the obese rats . The obese rats had hepaton~egaly due mainly to high levels of triglycerides . The plasma of the obese anie~als contained high levels of triglycerides, phospholipids, cholesterol, and nonesterified fatty acids . Obese rats had slightly higher levels of ketone bodies than nonobese rats . In the liver of obese rats, we observed a decreased level of citrate/g and an elevated malate level . Levels of adenine nucleotides were similar in all rats . In 1929, the Danish physiologist August Krogh (1) wrote : "For a large nwaber of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied . . . I have no doubt that there is quite a number of animals which are similarly "created' for special physiology purposes, . . ." . And Hans A . Krebs (2) added in 1975 : "A general lesson to be learned . . . is the importance of looj~ing out for a good experimental material when trying to tackle a specific biological problem ." Such "experimental material" now exists for the study of same aspects of obesity : it is the genetically obese Zucker rat (3), in which homozygotes for the recessive "fatty" mutant gene (fa) are obese . Before undertaking a study of the particular metabolism of these animals, we felt it would be useful to establish a baseline pattern of lipids in the blood and the liver, and also of some intermediate metabolites that can enter into lipid metabolism . We report here the results of this study and compare them with the results obtained for two other groups of rats : nonobese control rats and a strain often used in animal experimentation, the Sprague-Dawley rat . Materials and Methods We used three types of 9-week-old male rats (CSEAL-CNRS) : genetically obese Zucker rats (fa/fa) ; their nonobese litter mates (Fa/fa or Fa/Fa, here represented Fa/-) ; and Sprague-Dawley (S-D) strain rats . All animals were allowed water and laboratory chow for a week and then, still not fasting, were stunned and decapitated between 8 :00 and 8 :30 a .m .

541

542

Lipide 1n Geaetically Obese Zucker Rat

Vol . 20, No . 3, 1977

In three groups of eight animals each, about 1 .5 ml of blood was immediately collected from each animal into 3 ml 0 .5 M ice-cold HC10q and shaken . Enough 0 .5 M ice-cold HC10q was then added to bring the final HC10q/blood (v/w) ratio to 3/1 . Yarious substances were assayed after neutralization of the supernatant with 7N KOH followed by addition of powdered HKCOg and removal of the precipitated KC104 . The remaining blood from each rat was collected in a hemolysis tube containing EDTA . After gentle shaking, the blood was centrifuged and the plasma removed . Plasma lipoproteins were electrophoresed the same day on cellulose acetate (4) . Finally, we assayed plasma tri lycerides (5), total and esterified cholesterol (6), nonesterified fatty acids ~7), and phospholipids (8) . The liver was then removed and weighed . A fragment weighing about 1 g was used to determine water content . Another was used for extraction of lipids with a methanol/chloroform mixture (2/1, v/v) . About 2 g of liver were ground in a mortar under liquid nitrogen, then put into the solvent and homogenized . This operation was repeated twice . The extraction and washing liquids were pooled, the solvent evaporated at 40° C under a partial vacuum, and lipids removed with benzene . After evaporation, the dry extract was weighed and its total cholesterol, triglyceride, and phospholipid levels were determined, as above . Another piece of the liver, 0 .2 g, was used for total protein determination (9) . In other animals (three groups of eight rats each), a piece of liver weighing about 2 g was freeze-clamed immediately after the rat had been killed . It was then ground in a mortar under liquid nitrogen, weighed, transferred to 0 .5 M ice-cold HC10q, and homogenized . The precipitate was washed once, to give a final HC10q/liver (v/w) ratio of 3/1 . The supernatant was neutralized with 7N KOH and then with powdered KHC03 . The KC104 precipitate was removed by centrifugation, and various substrates and adenine nucleotides were assayed the same day . Using enzymatic methods, we determined plasma or liver levels, or both, of ketone bodies (10), lactate (11) , pyruvate (12) , citrate (13), glucose 14 , malate (15), acetyl-CoA (16), glycogen (17), ATP (18), and ADP and AMP ~19 ; . Inorganic phosphorus was assayed colorimetrically (20) ._ All results are expressed as X t ô

(n - 8) . Means were compared using

Student's t-test for paired differences . The difference between two means was considered significant (HS) when p < 0 .05 . Percentages of variation indicated in the text were calculated from the means of the results obtained in the two groups of Zucker rats . Results and Discussion Nonobese controls weighed less than S-D rats of the same age (Table I) . Obese rats weighed on the average 52X more than their Fa/- litter mates . This difference was mainly due to the very high lipid accumulation in the whole body of fa/fa rats (21, 22) . Table I also shows that the liver was much heavier (+ 86X) (23, 24, 25~ in obese rats than in nonobese controls, and that its relative size was signif cantly greater . Thus, liver weight/100g of body weight was 4 .2 for Fa/- rats compared with 5 .2, or 24X (HS) more, for fa/fa rats .

Vol . 20, No . 3, 1977

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Lipids in Genetically Obese Zucker Rat

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Lipide in Genetically Obese Zucker Rat

544

Vol . 20, No . 3, 1977

TABLE II Lipid Composition of Liver Rats w ~T °'°' â~ NO

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otal cholesterol Total cholesterol phospholipids (mg/g wet liver) m liver 2 .4 29 .8

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The sum of the three lipid fractions represents about 80% (Fa/-) and 90% (S-D and fa/fa) of the total lipids obtained directly after extraction . This fact can explain at least in part why the three categories .of lipids were less abundant in nonobese controls . For further details, see Table I . The water and the protein content per g of wet liver (24) seemed lower in obese rats than in their nonobese litter mates, but these differences are at the threshold of significance . However, the livers of obese rats were richer in glycogen (+ 19% HS) than were livers from the two other groups of animals (26) . But for the whole] aver, the amounts of water, protein, and glycogen for obese rats were nearly double those for Fa/- and S-D rats ; this finding accounts in part for the observed difference in organ weight . Lipids The main cause of greater liver weight in obese rats was higher lipid content - as much as 69% (HS) or 223% higher, according to whether it expressed per g of liver or for the whole organ (Table I) (24) . Even on visual inspection, the yellowish but nevertheless smooth-looking liver suggests this accumulation of fat .

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We determined the various categories of lipids extracted from the liver . Table II shows that 1 g of liver from an obese rat contained roughly as much cholesterol (- 14%, NS) and phospholipids (+ 5% HS) as an equivalent piece from a nonobese litter mate . However, the liver of genetically obese rats was mainly characterized by a huge elevation of-triglyceride levels (+ 1348%) (24) . Wahle (27) found greater incorporation of long-chain fatty acids in hepatic triglycerides for obese rats than for controls . A similar but less marked elevation of trigl.yceride level was observed in plasma : the plasma triglyceride content was 377% higher in obese rats than in their litter mates (Table III) (28, 29, 30) .

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545

546

Lipide in Genetically Obeae tucker Rat

Vol . 20, No . 3, 1977

The plasma ohospholipid level in fa/fa rats was twice (+ 111%, HS) that in S-D or Fa/-rats (28, 31) . In plasma, unlike the liver, levels of cholesterol were significantly higher (+ 52%, HS) in obese rats than in controls (24, 28, 29, 31) . This was true of both the esterified (+ 62%, HS) and the free (+ 19%, NS) form of cholesterol . Thus, the ratio of esterified to total cholesterol was about the same in all animals, suggesting that cholesterol esterification pathways are not changed in obese rats . Furthermore, it is worth noting that the ratio of total cholesterol to phospholipids was slightly dower in obese than in nonobese rats, in both liver and plasma . In view of the clinical significance attached to these two ratios, one might guess that obese rats are no more predisposed to atheroma than are normal rats . Finally, the level of nonesterified fatty acids was higher (+ 95%)i~5 .obese rats than in controls (23, 26, 31, 32, 33) . Lypolysis is known to take place at a higher rate in the adipose tissue of obese than of normal rats (31, 32, 33), and the excess of mobilized fat returns to storage tissue via lipoproteins(26) . Such increases in the levels of all these various lipid fractions must of course be reflected in a rise in plasma lipid levels . Table III, while revealing that there is no significant differénce between S-D and Fa/-rats, confirms the very high lipemia already reported in obese rats (28) : 2 .5 times as high in our 9-week-old obese rats as in the two other groups of animals . This hyperlipemia in obese rats has to be correlated with their very high plasma triglyceride/phospholipid ratio (+ 140%, HS) . Although migration on cellulose acetate is not very well suited to analysis of rat plasma lipoproteins, the results obtained by this technique are nevertheless worth reporting . The electrophoretic pattern was very similar for the S-D and the Fa/- rats (Fig . 1) . For obese rats the peaks were higher and sharper, revealing an increase in lipoproteins (their relative surface was greater by some 77%, HS) ; an additional peak also appeared, raising the question of a possible correlation with the abundance of very low-density lipoproteins(28,35) . In any case, it is clear from the electrophoregram that 9-week-old obese rats did not show any sign of the hyperchylomicronemia found by Zucker (23) in 12-month-old animals .

FIG . 1 Densitometric Tracings (limits) of Plasma Lipoproteins . Values are expressed as the absolute peak area ( ) t standard error of the mean (n = 8) .

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Lipide in Genetically Obese Zucker Rat

Vol. 20, No . 3, 1977

547

TABLE IV

Hepatic Levels of Intermediate Metabolites and Adenine Nucleotides Lactate

Pyruvate

1 .4 15 .8

182 .4 1 19 . 047 1 224

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40 .4 452 .2

1 1 .2 1 18 .7

54 .3 1 4.2 34 .61 0.9 603.4 1 45 .E 387 .7115 .E

24 .9 ± 1 .8 ~ 259.1 ± 19 .4

194.8 1 19 . 1 178 004

7 .0 1 0 .9 69 .0 ± 7 .4

51 .1 524.5

1 6.8 1 65 .8

61 .4 1 6 .3 34 .7± 1 .5 639.E ± 67 .4 362 .3120.9

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Lipide in Genetically Obesa Zucker Rat

548

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Vol . 20, No . 3, 1977

Vol. 20, No . 3, 1977

Lipids is Genetically Obeaa Zucker Rat

Finally, in agreement with the (36), and York et al . (32, 33, 37), and insulinemia n o se rats (1 .04 vely) than in their nonobese litter ml) .

549

results of Lemonier (25), Lemonier et al . we observed significantjy higher glycen~ïa t 0 .02 m9/m1 and 6 .4 t 0 .8 ng/ml, respectimates (0 .96 ± 0.02 mg/ml and 1 .3 t 0 .1 ng/

Intermediate metabolites and adenine nucleotides . Considering the very high incorporation of fatty acids in hepatic lipids, one might expect the rate of fatty acid catabolism to be depressed . However, Table IV shows that the level of acetyl-CoA - the first step in the oxidation process - is roughly the same per g of wet liver in the three groups of animals . This is also true for the ketone body levels expressed per g of the organ, although when measured for the total liver, they are twice as abundant in obese as in nonobese animals . This observation can also account for the higher (+ 42X HS) ketonemia in obese rats (Table V) . This increase in ketonemia is true for acetoacetate as well as ß-hydroxybutyrate . This result contrasts with the results of Malewiak and Griglio (38), who reported a slight hypoketonemia in 5-month-old obese Zucker rats compared with controls . The ratio of 9-hydroxybutyrate to acetoacetate seemed slightly higher in Zucker than in S-D rats, but this difference is not significant . Citrate levels were higher (+ 32X, HS) in the blood and lower (- 5016, HS) in the liver of the obese than of the other rats . On the other hand, considered for the liver as a whole, total citrate was the same in all cases . This observa tion correlates with that of Taketomi et al . (39), who showed that thie .activities of ATP citrate-lyase and acetyl-CoA carl~xylase were greatly increased in the livers of obese rats . On the other hand, malate levels were markedly higher (+ 79X) in livers of obese than of nonobese rats . While we could not show whether the observed 20X increase in plasma lactate levels of obese rats was significant, we did show that this substance is definitely more abundant (+ 76X) in the livers of these animals . As for pyru vate, its levels are hi her in obese rats, both in the blood (+ 31X, HS) and in the liver (+ 83X, HSjg . Finally, there was almost no difference between the three types of rats for adenine nucleotides (Table IV) except of course when amounts were reported for the whole liver; in that case obese rats had more . Our results show that there is no significant difference between 5-D and the nonobese Zucker rats . However, the ôbesity of fa/fa rats induces striking changes affecting both the liver and the blood and consisting mainly in higher levels of lipids, particularly triglycerides. For these animals, in which obesity is already well established by 8 weeks of age, there are relatively few differences in intermediate metabolites and adenine nucleotides .

The authors are grateful to Prof . J . WARTER for the interest he has taken in this work .

550

Lipids in Genetically Obese Zucker Rat

Vol . 20, No . 3, 1977

References 1 . A . KROGH, Amer . J . Ph siol . 90, 243-251 (1929) . 2 . H .A . KREBS, ~~~ x~~ o0~ 9~ 221-226 (1975) . 3 . L .M~. ZUCKER,, .heredity 52, 275-278 (1961) . J 4 . J . CANAL and . RIQUOIS, Feuil . biol . 12, 53-62 (1971) . 5 . H . SCHIRARDIN and M . B UER, Feuil . ~, 63-68 (1976) . 6 . H . SCHIRARDIN, M . BAUER and J . NEY, to be published . .F 7. :G . SOLONI nd L .C . SARDINA, Clin . them . 19, N° 4, 419-424 (1973) . 8 . H . WACHTER, ~rztl . Lab . 11, 9 . G . BEISENHERT,~ B~L~, T . RUCHER, R . CZOK, K .H . GARBADE, E . MEYER ARENDT and G . PFLEIDERER, Z . Naturforsch . 8b, 555-577 (1953) . 10 . D .H . WILLIAMSON, J . MELLANBY and H . . KREBS, Biochem . J . 82, 90-96 (1962 . 11 . H .J . HOHORST, in Methoden der enzymatischen Ana yTse, e~ . ~y H .U . BERGMEY~R (P . 1425-1429), Verlag Chemie, Weinheim, (1970) . 12 . R . CZOK and W . LAMPRECHT, in Methoden der enz,rnatischen Analyse, ed . by H .U . BERGMEYER (P . 1407-1411), Verlag Ch~oie, Weinheim (1970) . 13 . S . DAGLEY, in Methoden der enzymatischen Analyse, ed . by H .U . BERGMEYER (P . 1520-1523), Verlas Chemie, Weinheim (1970) . 14 . H .U . BERGMEYER, E . BERNT, F . SCHMIDT and H . STORK, in Methoden der enzymatischen Analyse, ed . by H .U . BERGMEYER (P . 1163-1168), Verlag Chemie, Weinheim (1970) . 15 . H .J . HOHORST, in Methoden der enzymatischen Analyse, ed . by H .U . BERGMEYER (P . 1544-1548), Verlag Chemie, Weinheim (1970) . 16 . K . DECKER, in Methoden der enzymatischen Analyse, ed . by H .U . BERGMEYER (P . 1922-1927), Verlag Chemie, Weinheim (1970) . 17 . D . KEPPLER, K . DECKER, in Methoden der enzymatischen Analyse, ed . by H :U . BERGMEYER (P . 1089-1094), Verlag Chemie Weinheim (1970) . 18 . D . JAWOREK, W . GRUBER and H .U . BERGMEYER, in Methoden der enzymatischen Analyse, ed . by H .U . BERGMEYER (P . 2020-2024), Verlag Chemie, Weinheim (1970) . 19 . D . JAWOREK, W . GRUBER and H .U . BERGMEYER, in Methoden der enzymatischen Analyse, ed . by H .U . BERGMEYER (P . 2051-2055), Verlag Chemie, Weinheim (1970) . , 20 . E .S . BAGINSKI, P . P . FOA and B . ZAK, Clin . Chim . Acta 15, 156-158 (1967) . 21 . L .M . ZUCKER and H .N . ANTONIADES, Endocr~no o , 3f6-1330 (1972) 22 . G .A . BRAY, D .A . YORK and R .S . SW , eta o ~sm 22, 435-442 (1973) . 23 . L .M . ZUCKER, Ann . N .Y . Acad . Sci . 131, 24 . L .C . FILLIOS an a ~sm 14, 734-745 (1965) . 25 . D . LEMONIER, Horm . metab . res~~87-258 (1971) . 26 . L .M . ZUCKER, ~ . _~~r_e_ s. , 234-243 (1972) . 27 . K .W .J . WAHLE, om~p:6iôchem. siol . 48 B, 565-574 (1974) . 28 . G . SCHONFELD ana~ßFCÉ~ , r . J . ~siol . 220, 1178-1181 (1971) . 29 . W .S . BARRY and G .A . BRAY, Mete sm - 3 (1969) . 30 . ~A . SHINO, T . MATSUO, H . IWATSUf~ana T. SUZUODI, Diabetologia 9, 413-421 (1973) . 31 . T .F . ZUCKER and L .M . ZUCKER, Proc . Soc . exp . biol . med . 110, 165-171 (1962) . 32 . D .A . YORK and G .A . BRAY, Horm . metab . res . 5, 355-350 (1973) . 33 . D .A . YORR and G .A . BRAY, eta o sm 2, 43-454 (1973) . 34 . N . ZÖLLNER and K . KIRSCH,~xér . Med . 135, 545 (1962) . 35 . G . SCHONFELD, C . FELSKI an ~ i ~ res . 15, 457-464 (1974) . 36 . D . LEMONIER, R . AUBERT, J .P . SUQUET and G ~tOSSELIFr D~iäbetologia 10, 697-701 (1974) . 37 . D .A . YORK, J . STEINKE and G .A . BRAY, Metabolism _21, 277-284 (1972) . 38 . M . I . MALEWIAK and S . GRIGLIO, to be pu s e . 39 . S . TAKETOMI, E . ISHIKAWA and H . IWATSUKA, Horm . metab . res . 1, 242-246 (1975) .

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