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SECRETION OF TRIGLYCERIDE AND KETOGENESIS LIVERS FROM SPONTANEOUS DIABETIC BB WISTAR

1157-1162

BY RATS’

Ira Weinstein, Tarun B. Patel, and Murray Heimbergz Department of Pharmacology College of Medicine The University of Tennessee, Memphis The Health Science Center Memphis, Tennessee 38163 Received

March

14,

1991

SUMMARY: Livers from male and femaleBB Wistar spontaneouslydiabetic rats were perfusedin vitro to determinethe effects of spontaneouslyoccurring insulin-dependentdiabeteson the metabolismof fatty acid. The secretionof triglyceride and the incorporationof [l-l%] oleic acid into perfusateand hepatictriglyceride wasreducedby the diabetic state,whereasP-hydroxybutyrate production and output of total ketonebodieswere increased.The spontaneousdiabetic Wistar rat clearly is a suitablemodelto study the derangementsinducedin lipid/plasmalipoprotein metabolismby the insulin-dependentdiabetic state;the dataobtainedwith this modelconfirm our earlier observationson experimentalinsulin deficiency inducedwith alloxan, streptozotocin, and anti-insulin serum. 0 1991 Academic Press, Inc.

It hasbeenreportedpreviously from this laboratory that hepaticsecretionof triglyceride (i.e., very low density lipoprotein) is reducedandketogenesisis acceleratedin experimentaldiabetes inducedby alloxan (l-5), anti-insulin serum(6-7), or streptozotocin(8). The availability of a geneticmodel of insulin deficiency in the rat afforded us an opportunity to explore the previous observationsfurther. An animalmodel in which diabetesoccursspontaneously,suchasin the diabetic BB Wistar rat, may be a more physiologicalmodel than onein which the diabeteswas inducedchemically. Certain alterationsof serumlipoproteinsobservedin the spontaneousdiabetic BB Wistar rat were comparedto thoseobservedin humandiabetesmellitus (9). Thus, the objectives of the study reported herewere to determinewhether the changesin hepaticmetabolism of fatty acidsby livers from spontaneous diabetic BB Wistar rats were similarto thosealterations IThis researchwas supportedby grantsHL27850 (M.H.), andDK 35713 andDK 01742 (T.B.P.) from the National Institutes of Health, U.S. Public Health Service. Support for the BB Wistar rat colony wasobtainedfrom the American DiabetesAssociation,TennesseeAffiliate. 2To whom requestsfor reprints shouldbe addressed. Abbreviations used:VLDL, very low density lipoprotein; FFA, free fatty acids. 0006-291X/91

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observed earlier, and to determine whether it would validate those studies with livers from rats made diabetic by treatment with alloxan, streptozotocin or anti-insulin serum. METHODS Female rats (221-252 g) and male rats (250-455 g) were obtained from the colony of spontaneous diabetic BB Wistar rats maintained in the Department of Comparative Medicine at the University of Tennessee, Center for Health Sciences. The development of diabetes mellitus, as determined by appearance of glucose in the urine, was observed within 60-100 days of age (mean 84.5 days). This corresponds to approximately 150 and 190 g body weight for female and male rats, respectively. The diabetic animals were maintained with protamine zinc insulin (8 units/kg body wt/day). Litter mates which did not develop glucosuria were termed nondiabetic resistant animals (NDR). These animals served as the controls. Animals were fed laboratory chow and water ad Eibirum. Spontaneousdiabetic animalswere deprived of insulin 24 hr before the experiment. The livers were removed surgically (10) and perfusedin a recycling systemunderconditionsdescribed previously (11). A complex of oleic acid (NuChek Prep, Elysian, MN) and 10 t.tCi[l-t4C] oleic acid (New England Nuclear, Boston, MA) with bovine serumalbumin wasprepared(10) and was infusedat a rate of 166pmol/hr to give steadystateconcentrationsof 0.48 f 0.04 pmol/ml fatty acid in the perfusate,after removal of erythrocytes. Samplesof perfusateand liver were taken for the determinationof lipids asdescribedpreviously (11). Justprior to cannulationof the portal vein, 3-5 ml of blood wasobtainedfrom the abdominalaorta for estimationof serumlipids. aHydroxybutyrate wasdeterminedenzymatically (12). Incorporation of [ l-14C] oleic acid into triglyceride (11) andtotal ketonebodies(13) wasestimatedby liquid scintillation spectrometry. Statisticalevaluation of the differencesbetweenmeanswasdeterminedfrom a two-tailed unpaired Student’s“t” test. RESULTS The concentrationof triglyceride in serafrom femalerats was0.25 f 0.04 pruol/ml and 1.49 * 0.76 pmol/ml for the NDR and spontaneousdiabetic groups,respectively. In serafrom male animals,correspondingvalues were 0.46 f 0.15 and 1.96 f 0.65 pmol/ml for NDR and diabetic groups,respectively (n=3 for eachgroup). As observedpreviously with livers from rats in which insulin deficiency wasinducedchemically (l-8), livers from the BB-Wistar diabetic rats secretedlessVLDL triglyceride andincorporatedless[ 1-I%] oleic acid into perfusatetriglyceride than did livers from NDR animals(Table 1). The livers from malerats secretedlesstriglyceride 1. Secretion of triglycerideandincorporation of [I-t4C] oleicinto perfusate triglyceride

Table

by liversfrom maleandfemalespontaneous diabeticBB WistarRats Female(3)

Male(2)

GROUP pnol/g liver NDR

2.09 2.42

Diabetic

0.11 0.18

DPM/gliver 22947 210926

1

1737 1516

pmol/gliver f

4.99 0.12

1.89 zk0.46

DPM/gliver 301000 iz32729 33347 *8038

Datafor femalesareexpressed asmeans + SEandarecumulativeafter4-h of perfusion.Numberof observations areshownin parentheses. Differencesbetween NDR andDiabeticgroupsof liversfrom femaleratsarep < 0.05or better. 1158

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Table 2. Incorporation of [ 1-14C] oleic into total perfusate ketones bodies and production of B-hydroxybutyric (BOB) acid by livers from male andfemale spontaneous diabetic BB Wistar rats

Female(3)

Male (2) GROUP

BOB timol/g liver

DPM/g liver

NDR

8.80 17.70

70156 60817

20.84 zlz 3.39

111215 f 2326

Diabetic

63.04 36.18

24670 1 175632

110.93 + 24.19

441080 + 42713

BOB fimol/g liver

DPM/g liver

Data for females are expressed as means f SE and are cumulative after 4 h of perfusions. Number of observations are shown in parentheses. Radioactivity is incorporation of [l-t%] oleic into total ketone bodies. Differences between NDR and Diabetic groups of livers from female rats are p < 0.05 or better.

andincorporatedless[ 1-14C]oleic into triglyceride than did livers from femalerats, regardlessof the diabetic status. Sexual differencesin triglyceride secretionwere the subjectof previous reports from this laboratory (14). The incorporation of [ 1-14C]oleic acid into total ketone bodiesand biosynthesisof Bhydroxybutyrate was increasedin livers from spontaneouslydiabetic BB Wistar ratscomparedto the NDR controls (Table 2). Thesedata supportthosereportedearlier with alloxan, anti-insulin serum,and streptozotocin (l-8). It is of interestthat, in theseexperiments,livers from femalerats produced

more p-hydroxybutyrate

and incorporated

more [ l-14C] oleic acid into ketone bodies than

did livers from malerats. In contrast,ketogenesisby livers from normal malerats exceededthat from livers of normal female animals(14). Finally, asobservedpreviously in insulin deficiency inducedchemically, the spontaneous diabetic BB Wistar rats were alsocharacterizedby hepaticsteatosis,although,clearly, incorporation of [l-t4C] oleateinto hepatic triglyceride wasreducedin the diabetic state(Table 3). Table 3. Concentration of and incorporation of [l-l%] oleic acid into hepatic triglyceride of maleandfemale spontaneous diabetic BB Wistar Rats Male

GROUP pmol/g liver NDR Diabetic

5.26 8.75 66.4 28.2

Female (3)

(2)

DPM/g liver

j.unol/g liver

DPM/g liver

520412 480760

10.00 f 1.24

667600 IL 5027

35360 30120

20.27 3~ 4.68

215800 f 57717

The number of observations are indicated in parentheses. Values are expressed as means + SE and represent conditions in the liver after 4 hours of perfusion. Differences between NDR and Diabetic groups of livers from female rats are p < 0.05 or better.

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DISCUSSION The data reported here clearly support the conclusion that, in this genetic model of Type I insulin-dependent diabetes mellitus, hepatic oxidation of fatty acid is increased, while esterification of fatty acid to triglyceride and secretion in the VLDL is reduced, compared to the NDR control, when livers are exposed to equal concentrations of fatty acid. It has also been a constant reproducible observation that secretion of triglyceride (i.e., of VLDL)

by the isolated perfused liver

is reduced in insulin deficiency induced by any of the methods reported previously (l-8), in comparison to nondiabetic controls. Yet, it is equally obvious that the diabetic animals prepared by all of these methods are characterized by a severe hypertriglyceridemia

and fatty liver. Some of the

reasons for this apparent dichotomy were discussed earlier in a report from this laboratory (7). In studies with the perfused liver in vitro, comparisons of triglyceride synthesis and secretion were made at equal concentrations of perfusate fatty acid. In vivo, however, this is not the case. This is an important difference, since FFA are the primary stimulants for VLDL secretion. The ambient concentration of plasma FFA in the diabetic rat can be 1.5 - 2.0 mM, compared to approximately 0.5 mM in the normal fed animal (7,15). The diabetic liver in vivo is thus being deluged with elevated concentrations of substrate fatty acid, primarily derived from hydrolysis of adipose tissue triglycerides, in addition to those of dietary origin, while this is not so in normal controls in vivo; therefore, a direct comparison between the liver in vivo and in vitro is inappropriate under these conditions. Had the normal liver (i.e., in the presence of adequate insulin) been exposed to these same amounts of FFA, synthesis and secretion of triglyceride would have exceeded that of the diabetic by a wide margin while ketogenesis would have been much less. Most likely, as suggested earlier (7), the increased load of plasma FFA substrate in the diabetic animal is driving formation and secretion of triglyceride at rates exceeding that of the normal animal having lower ambient FFA levels, despite the fact that fatty acid oxidation is accelerated in the diabetic. Nevertheless, triglyceride (i.e., VLDL) formation and secretion is the preferred pathway in the insulin-primed animal, and not in the insulin-deficient diabetic. Therefore, increased synthesis and secretion of VLDL triglyceride may be contributing, in part, to the diabetic hypertriglyceridemia

of

insulin deficiency.

Clearly, the hypertriglyceridemia triglyceride.

may, in part, also result from impaired clearance of the

It is known that lipoprotein lipase activity is reduced by insulin deficiency (16). The 1160

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exact mechanism(s) responsible have not yet been resolved. Chen et al. suggested that the decreased lipoprotein lipase activity could not explain entirely the defect in clearance of triglyceride (17), as reported by others (18). Bar On er al. concluded that the defect in clearance may have resulted from quantitative changes in apolipoprotein CII concentration, an activator of lipoprotein lipase, and from possible structural alterations in the lipoproteins induced by the diabetes (19).

The triglyceride-rich

fatty liver occurs despite increased fatty acid oxidation, probably on the

same basis as discussed above for the hypertriglyceridemia

Although the triglyceride-rich

fatty

liver developed in the intact animal prior to perfusion of the liver in vitro (l), the incorporation of [ 1-14C] oleic acid into hepatic triglyceride was depressed in livers from the diabetic BB Wistar rats compared to the NDR controls. The reduced synthesis of triglyceride by the perfused liver in vitro in diabetic rats with concentration of fatty acid infused in these experiments may have been overcome, in part, the large supply of fatty acid to the liver from adipose tissue in vivo. Additionally, the relatively impaired secretion of triglyceride (i.e., VLDL)

by the livers from the

diabetic BB Wistar rats, despite increased concentrations of plasma fatty acids, may, indeed have contributed to the development of the fatty liver. Our current studies with the spontaneous diabetic BB Wistar rat confirm earlier observations on fatty acid metabolism and hepatic secretion of VLDL triglyceride in experimental insulin deficiency induced by alloxan, streptozotocin, and anti-insulin serum, and support the conclusion that the diabetic Wistar rat is an appropriate model to study the derangements of plasma lipid/lipoprotein metabolism in insulin-deficient, insulin-dependent diabetes mellitus. REFERENCES 1.

Heimberg, M., Dunkerley, A., and Brown, T. 0. (1966) B&him. Biophys. Acta 125:252264. 2. Heimberg, M., Van Harken, D.R., and Brown, T.O. (1967) B&him. Biophys. Acta 137:435-445. 3. Van Harken, D.R., Brown, T.O., and Heimberg, M. (1967) Lipids 2:231-238. 4. Wilcox, H.G., Dishmon, G., and Heimberg, M. (1968) J. Biol. Chem. 243:665-675. 5. Van Harken, D.R., Dixon, C.W., and Heimberg, M. (1969) J. Biol. Chem. 244:22782285. 6. Woodside, W. F., and Heimberg, M. (1976) J. Biol. Chem. 251:13-23. 7. Woodside, W.F., and Heimberg, M. (1978) Metabolism 27: 1763-1777. 8. Wasfi, I., Weinstein, I., and Heimberg, M. (1981) Proc. Sot. Exptl. Biol. and Med. 166:330-334. 9. Patel, S. T., Neuman, H. A. I., Yates, A. J., Thibert, P., and Folko, J. M. (1984) J. Lipid Res. 25:1072-1083. 10. Kohout, M., Kohoutova, B., and Heimberg, M. (1971) J. Biol. Chem. 246:5067-5074. 11. Weinstein, I., Wilcox, H. G., and Heimberg, M. (1986) B&him. Biophys. Acta 876:450459. 1161

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12. Williamson, D.H., and Mellanby, J. (1974) In: Methods of Enzymatic Analysis, edited by H.U. Bergmeyer. Vol. 4, pp. 1836-1839, Academic Press, New York. 13. Stakkestad, J. R., and Bremer, J. (1982) B&him. Biophys. Acta 711:90-100. 14. Soler-Argilaga, C., and Heimberg, M. (1976) J. Lipid Res. 17:605-615. 15. Fredrickson, D.S., and Gordon, R.S. Jr. (1958) Physiol. Rev. 38:585-630. 16. Bagdade, J. P., Porte, D. Jr., andBierman, E. L. (1968) Diabetes 17:127-132. 17. Chen, Y. I., Risser, T. R., Caully, M., and Reaven, G. R. (1979) Diabetes 28:893-898. 18. Nikkila, E. A., and Kekki, M. (1973) Metabolism 22:1-22. 19. Bar On, H., Levy, E., Oschry, Y., Ziv, E., and Shafrir, E. (1984) Biochim. Biophys. Acta 793:115-118.

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Secretion of triglyceride and ketogenesis by livers from spontaneous diabetic BB Wistar rats.

Livers from male and female BB Wistar spontaneously diabetic rats were perfused in vitro to determine the effects of spontaneously occurring insulin-d...
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