Fish Oil-induced Yellow Fat Disease in Rats

Transcription

1 Vet. Pathol. 15: (1978) Fish Oil-induced Yellow Fat Disease in Rats I. Histological Changes L. H. J. C. Danse and P. M. Verschuren Department of Veterinary Pathology, University of Utrecht, Utrecht Abstract. Yellow fat disease was induced in young rats given a vitamin E-deficient diet supplemented with 15% fish oil. The changes in adipose tissue of this oil-induced disorder were different from those of natural yellow fat disease in horse, pig and mink. In the natural disease all fat depots had the early stage of yellow fat disease with interstitial lipofuscin-laden macrophages exclusively. In the rat, however, this change was seen only in the subcutaneous fat depot. Moreover, affected adipose tissue of animals with natural disease had extensive fibrosis, but in the rat fibrosis was always absent. Rats with fish oil-induced yellow fat disease had degenerative changes in various fat depots that occurred at various times but in the horse, pig and mink fat depots were affected simultaneously. Lipofuscin accumulated in the reticuloendothelial system in rats. Accumulation in spleen and liver was dependent on vitamin E deficiency, but only the accumulation in the Kupffer cells was correlated with yellow fat disease. Lipofuscin accumulation in the mesenteric lymph node did not depend on vitamin E deficiency. Yellow fat disease primarily is a generalized disorder of fat depots. It usually is characterized by extensive adipose-cell degeneration (steatosis), inflammation and fibrosis of adipose tissue (steatitis) and accumulation of lipofuscin pigment. Yellow fat disease is considered an expression of vitamin E deficiency. Moreover the intake of polyunsaturated fatty acids, especially those with three or more double bonds, from dietary plant and fish oil is important [13]. The natural disorder has been seen in the horse [30], pig [19], mink [ll], cat [12] and rabbit [17]. The horse, pig and mink are considered to be especially sensitive. Experimentally induced yellow fat disease has been described in the rat [22], but there are differences in the development of the disease between rat and sensitive species. These differences could be important for the pathogenesis of yellow fat disease, as they may indicate factors that determine species and tissuedependent sensitivity. In the pig [19], mink [7] and horse [20], both in natural and experimental disease, all fat depots are affected simultaneously and in the same degree. This may not be so for the rat [22]. 114

2 Fish Oil-induced Yellow Fat Disease 115 Table 1. Composition of basal vitamin-e deficient semisynthetic diet Ingredient Casein Soy-protein Corn starch Glucose Cell u lose DL-methionine Minerals and vitamin mixture Percent Morphogenetic studies in mink [7], horse [29] and pig [8] proved that the early changes of yellow fat disease were characterised by interstitial lipofuscin-laden macrophages without adipose-cell degeneration. In the rat this initial stage of yellow fat disease has never been reported but early changes such as adipose-cell degeneration and the induction of an inflammatory reaction have been described [221. In the pig [6] and horse [29] extensive fibrosis of adipose tissue has been seen during the development of yellow fat disease, but in rat this fibrosis of degenerated adipose tissue might be absent [22]. In several species [8, 16, 221 intensive accumulation of lipofuscin in the reticuloendothelial system has been seen next to changes in adipose tissue during yellow fat disease development. The significance of this accumulation for the function of the reticuloendothelial system and for the development of yellow fat disease is unknown. First, however, the changes in various parts of the reticuloendothelial system must be differentiated on the basis of their vitamin E dependency, since previous reports were inconclusive. Materials and Methods Yellow fat disease was induced with a vitamin E deficient diet containing a large amount of fish oil. Female Wistar rats weighing grams were bred for feeding experiments. Forty to 50 rats were born within a 3-day period. From these eight to 10 were selected. The rats were kept in constant tcmpcraturc (22" C). humidity (65% rclativc) and artificial light (12 hours light). Females and their young in each group received the same diet. Rats fed a fish-oil supplemented diet received a basal vitamin E deficient semisynthetic diet (Muracon S. S. E., Trouw & C., the Netherlands) (table I). Control rats were fed the same basal diet supplemented with 9% lard, 6% soy oil and 30 milligrams vitamin E per kilogram food. Dietary fish oil was given by gastric intubation at 15% by weight of the daily feed intake, except during the first week after. Young rats were fed 15% fish oil mixed with the basal diet the first week after. During gestation and, fish oil was fed only to the females. Feed consumption was determined daily and refused feed was removed before refeeding. In experiment rats on vitamin E supplemented conditions 5 milligrams dl-a-tocopherol acetate, (Duphrafalw, Philips-Duphar B. V., the Netherlands) weekly was injected intramuscularly. Young rats were weighed weekly, starting at. The cod liver oil (Unimills, the Netherlands) used in the fish oil supplemented groups had a fatty acid pattern (table II) and the peroxide value was The oil was stored in

3 116 Danse and Verschuren I Table 11. Fatty acid composition of cod liver oil (% by weight) Cm:n' % :O 16:l 17:O 18:O 18:l 18:2w6' 18:3O3 20:o 20: 1 20:5w3 22: 1 22:6w3 others I Cm = number of carbon atoms; n = number of double bonds. o = position of first double bond starting at methyl ending of fatty acid. dark at 4" C to prevent oxidative changes. The peroxide value was determined weekly according to the method of Wheeler [14]. When food mixed with fish oil was used, the peroxide value was controlled after lipid extraction with petroleum ether in a Soxhlet extraction apparatus at 45" C. Experiment 1. One control group (group 1) and two groups (group 2 and 3) fed a diet supplemented with fish oil were used; each group consisted of 40 young rats and their mothers. Group 3 was reared vitamin E deficient. The young rats were identified by group and by dam. At, and 4 and 8 weeks after five female and five male young rats from each group were killed. Dams were killed with the last group. Experiment 2. Two groups, identical to groups 2 and 3 of experiment I, each consisting of 30 pups and their mothers were used. Two and 4 weeks after five female and five male young rats from each group were killed to study fat depots. Adipose tissue samples from the subcutaneous (inguinal), mesenteric, gonadal fat depots and brown fat (interscapular) (experiments 1, 2) and samples of liver, kidney, myocardial and skeletal muscle (M. psoas), spleen and mesenteric lymph nodes (experiment 1) were fixed in 10% buffered formalin and embedded in paraplast for microscopical and histochemical study. Sections were cut at 7 micrometers and stained with hematoxylin and eosin (HE), periodic acid-schiff (PAS), carbol fuchsin for acid fast lipofuscin [24], and azan. Unstained sections were embedded in fluormount and studied with a fluorescence microscope (Zeiss, exciting filter BG 12, barrier filters 53 and 44, Osram super pressure lamp HBO 200 W) [24]. In the reticuloendothelial system of liver, spleen and mesenteric lymph node, the intensity of fluorescence caused by lipofuscin accumulation was evaluated. For identification of macrophages in adipose tissue, one young rat of group 3 (experiment 1) was anaesthetised at 4 weeks after. Colloidal carbon was injected in the subcutaneous and paraepididymal fat depots. After 1 hour, tissue samples were taken from the injected region and processed for histological study. Results Rats in experiment 1 fed a fish-oil supplemented diet grew faster than control animals (fig. 1). This growth-promoting effect of fish oil is known [18]. In our

4 Fish Oil-induced Yellow Fat Disease 117, m n meksoleqe x m 11 rreeksalape Fig. 1: Bodyweight gain of young rats in cxpcrimcnt I. Fish oil diet with ( ) or without (-.-.-.) vitamin E and control (-); differences bctwccn curves are significant (p < 0.02). study, however, there was growth retardation in the control group during the fourth week. Body weight gain of group 3 fed a fish oil diet deficient in vitamin E was lower than when the vitamin was supplemented. Differences between these two groups were not always significant when tested at several intervals, but when the growth curves were paired tested (Student s t-test for paired observation) [9] there was a significant difference. Body weight gains of the different groups in experiments 1 and 2 were the same. Gonadal fat depot was yellow in young rats of group 3 at 4 and 8 weeks after. Mesenteric fat depot also was discoloured 8 weeks after. There was no difference in consistency of the adipose tissue. No other macroscopical changes were seen. Yellow fat disease development was restricted to young rats in group 3 of both experiments. There was no difference in disease development between sexes nor between the young of different dams. The fat depots of dams were normal. The initial change in adipose tissue consisted of interstitial lipofuscin-laden macrophages ( stage M ). Lipofuscin pigment was acid-fast, PAS-positive and had bright yellow autofluorescence (fig. 2) [7]. Macrophages could be identified because these cells had phagocytized injected carbon particles (fig. 3). Adipose cells looked normal at this stage of the disease. In a more advanced stage of the disorder ( stage S ) some degenerated fat cells and an inflammatory reaction surrounding these cells were seen next to interstitial macrophages (fig. 4). The inflammatory cells were polynucleated granulocytes, lymphocytes and monocytes. In the final stage ( stage E ) more than 30% of all fat cells were affected and the inflammatory reaction had increased greatly. The composition of the inflammatory infiltrate was the same as in stage S, but fibrosis of adipose tissue was never seen. Areas of stcatosis with sparse inflammatory activity, as described in porcinc yellow fat disease [6], were seen only in stage E (fig. 5).

5 118 Danse and Verschuren Fig. 2: Autofluorescence of lipofuscin in interstitial macrophages. Fig. 3: Phagocytosis of carbon particles (arrow) by interstitial lipofuscin-laden macrophages. Fig. 4: Degenerated fat ccll (D) surrounded by inflammatory cells (steatitis) in "stage S" yellow fat disease. HE. Degenerative changes occurred in sequence in the fat depots (table 111). All fat depots were the same as those in control animals at. Two weeks after waning (experimcnt 2) eight rats had "stage S" changes in the gonadal and two rats had them in mesenteric fat depots. *'Stage M" changes were seen in eight rats in the subcutaneous fat depot. At 4 weeks after (experiments 1, 2) all rats had "stage E" and "stage S" changes in gonadal and mesenteric fat depots. These rats had "stage M" changes in the subcutaneous fat depots. Eight weeks after two rats had "stage E" yellow fat disease in their mesenteric fat depots and four rats had "stage S" changes in subcutaneous fat. Brown fat was unchanged. Histological changes and accumulation of lipofuscin (table IV) occurred in several parts of the reticuloendothelial system. There were lipofuscin-laden Kupffer cells in the liver of group 3 vitamin E-deficient young rats at and 8 weeks after (fig. 6). The disappearance of lipofuscin in the Kupffer cells of these rats 4 weeks after, which also was noticed by Mason cannot be explained. Changes in the spleen also were restricted to group 3. Eight weeks after HE sections of the spleen showed proliferation of foamy macrophages in the marginal zone surrounding the white pulp. There was intensive accumulation of lipofuscin in these macrophages and also in the reticuloendothelial system of the red pulp (fig. 7, 8). From 4 weeks after the pigment accumulation in group 3 was significant, but at this age foamy macrophages were not yet visible. Changes in the mesenteric lymph node did not depend on the vitamin E deficiency but on the type of dietary fat. Eight weeks after there was extensive proliferation of foamy macrophages in the subcapsular sinus and in the cortical and paracortical regions (fig. 9) in lymph nodes of young rats

6 Fish Oil-induced Yellow Fat Disease 119 Fig. 5: Degenerated fat cells (D) with sparse inflammatory activity (steatosis) in "stage E" ycllow fat discasc. HE. Fig. 6: Kupffcr cells of young rat (group 3) 8 wccks after. Autofluorescencc of lipofuscin (). Fig. 7: Spleen of young rat (group 3) 8 weeks after. Strong fluorescence of lipofuscin () in macrophages of white pulp (W), marginal zone (M) and red pulp (R). Fig. 8: Spleen of young rat (group 2) 8 weeks aftcr wcaning. Fluorcsccnce in few cells (2) of red pulp (R). fed a fish oil diet. These changes were present to a lesser degree 4 weeks earlier. Lipofuscin accumulation was primarily associated with macrophage proliferation (fig I). Lipofuscin was in macrophages in medullary cords and sinuses in control rats and in rats fed fish oil. Therefore in control rats 8 weeks after, the fluorescence in lymph nodes of this group was positive (table IV). Changes in the spleens and lymph nodes were the same in dams as in their young 8-weeks aftcr. There was no lipofuscin. however, in Kupffer cells of the liver of dams.

7 120 Danse and Verschuren Table 111. Development of yellow fat disease in various fat depots of young rats (experiments I, 2) Age taneous Inci- Mesen- Inci- Gona- Inci- Brown Incidence teric dence dal dence fat dence Weaning N 10/10 N 10/10 N 10/10 N 10/10 2 weeks after M 8/10 s 2/10 s 8/10 N 10/10 4 weeks after M 10/10 S 10/10 E 10/10 N 10/10 8 weeks after S 4/10 E 2/10 E 10/10 N 10/10 I N = normal adipose tissue; M = stage M yellow fat; S = stage S yellow fat; E = stage E yellow fat. Skeletal muscle degeneration characterized by disintegrated and necrotic fibers and an inflammatory reaction was seen in young rats of group 3 (experiment 1) at 4 and 8 weeks after. The same, but much less severe lesions were seen in group 3 dams. Degenerative changes were seen in kidneys of all groups both in dams and young 4 weeks after and later. The lesions were wedge-shaped areas of atrophy of cortical tubules and nephrocalcinosis in the outer medulla. Females had four times as many lesions as did males. No changes were seen in myocardial muscle and liver parenchyma. Discussion The development of fish oil-induced yellow fat disease in the rat differed from that of natural yellow fat disease in species considered to be sensitive (horse, pig, mink). There were differences in the character of the changes and their progression in several fat depots. In yellow fat disease in horse. pig and mink the initial change in adipose tissue with interstitial lipofuscin-laden macrophages without adipose cell degeneration was seen in all fat depots 17, 8, 291. In the rat this change was seen only in subcutaneous fat, whilst other fat had adipose cell degeneration and interstitial macrophage reaction from the beginning. In contrast to the horse 1291 and pig (61. no fibrosis was seen in rat adipose tissue during yellow fat disease development. These different inflammatory reactions may result from various lipids released during lipolysis of fat cell vacuoles in the degenerating adipose tissue. These specific inflammatory reactions also were seen when various lipids were injected subcutaneously I I]. Differences in fatty acid composition may exist between different fat depots. Fatty acid composition of fat depots varies with the dietary fat composition 15, 271. In natural cases of porcine yellow fat disease a high percentage of linolenic acid (18:3w3, table 11) was found in food and in animal fat depots I 191. Moreover, linolenic acid is also the most important polyunsaturated fatty acid in fat depots of the normal horse 121). In our study,

8 Fish Oil-induced Yellow Fat Disease 121 Table 1V. lntcnsity of fluorescence caused by lipofuscin accumulation in the reticuloendothelial system of liver, spleen and mesenteric lymph node of young rats (exmriment I Organ Age Groups Liver Spleen Lymph node 4 weeks after 8 weeks after 4 weeks after wcaning 8 weeks after 4 weeks after 8 weeks after wcaning -2? / - I 10 rats in each group at each age were evaluated. - = fluorescence ncgativc ; = fluorescencc in a few cells; = fluorescencc in many cells; = strong fluorescence in many cells. however, the other members of the w3 family (table 11), eicosapentaenoate (20:5w3) and docosahexaenoate (22:6w3) may be important also. In species susceptible to yellow fat disease all fat depots were affected simultaneously and to the same degree [7, 19, 201. In the rat, however, degenerative changes were seen at different times in subcutaneous, mesenteric and gonadal fat depots. No changes were seen in the brown adipose tissue. The different adipogenetic and metabolic rates of the fat depots may be the reason why these depots were not affected simultaneously. In the rat these differences were seen between gonadal and subcutaneous fat depot [lo, 261. Fat in the gonadal fat depot forms much faster and is metabolically more active than any other fat. The next most active is mesenteric fat. In the rat, subcutaneous adipose tissue is the most sluggish. In the pig these differences may be equalized by the very high metabolic activity of subcutaneous fat depots [2]. Moreover, adipogenesis is rapid in these subcutaneous fat depots [23]. Therefore research should be done to examine possible similarities of fat depot characteristics between species sensitive to yellow fat disease. Horses [30] and pigs [16] develop yellow fat disease in fetus and young during early. In the rat, however, we found no sign of yellow fat disease occurred in offspring until 2 weeks after even though mothers were fed a vitamin E-deficient diet supplemented with a high amount of fish oil beginning at mating. Our study indicates that lipofuscin accumulation in the reticuloendothelial system of liver and spleen depended on the vitamin E-deficient diet. In the mesenteric lymph node, however, equal accumulations were seen in both groups on a fish oil diet. This proves that in this part of the reticuloendothelial system accumulation was dependent on the type of dietary fat and not on the vitamin E deficiency. In agreement with previous reports [7, 151 pigment accumulation in macrophages of liver and spleen is regarded as an expression of vitamin E

9 122 Danse and Verschuren Fig. 9: Mesenteric lymph node of young rat (group 3) 8 weeks after. Proliferation of foamy macrophages in subcapsular sinus and cortical and paracortical region. HE. Fig. 10: Mesenteric lymph node of young rat (group 3) 8 weeks after. Strong fluorescence ( ) in macrophages. Fig. 11: Mesenteric lymph nodc of young control rat 8 weeks after. Fluoresccnce () in medullary region (M). deficiency and moreover, it may be related to dietary polyunsaturated fatty acids with three or more double bonds. Lipofuscin accumulation in spleen macrophages also was seen in dams on a vitamin E-deficient diet. Since fat depots of these dams were normal, the changes in spleen and the lesions of adipose tissue will not be discussed. Lipofuscin accumulation in Kupffer cells of the liver, however, was seen only in young rats with degenerative changes in adipose tissue. This suggests there is a connection between these two lesions and yellow fat disease. The same observation was made in porcine yellow fat disease [8]. The consequences of lipofuscin accumulation in the reticuloendothelial system is unknown. Being an undigestible final product of oxidised unsaturated fatty acids, lipofuscin accumulation may cause overloading of macrophages and a decreased digestive function of these cells [4]. The effects of dietary plant oil and unsaturated fatty acids on reticuloendothelial phagocytosis were reported. Depending on the type of fatty acid the effect might be suppressive [3, 281 or stimulating [25]. Possible pigment accumulation in the reticuloendothelial system was not studied. Because skeletal muscle degeneration was seen both in young and their dams there seems no more specific cause than vitamin E deficiency for the relationship between this change and yellow fat disease. Both changes affected young rats. From this work we conclude that two things may be important in the etiology and pathogenesis of yellow fat disease. Release of various lipids in degenerating adipose tissue may cause the difference in inflammatory reactions between natural and fish oil-induced yellow fat disease. *Also, adipogenetic and metabolic characteristics of fat depots may be important for the progression of degenerative

10 Fish Oil-induced Yellow Fat Disease 123 changes. The sensitivity of some species for developing yellow fat disease may be based on these factors. In a preceding study [7] we stressed the significance of the reticuloendothelial system in the pathogenesis of yellow fat disease but in this study we found that only changes in the Kupffer cells were correlated with the disease. Acknowledgements We thank Mrs. C. Mcnsinga for histological preparations. References 1 ABDULLA, Y. H.; ADAMS, C. W. M.; MORGAN, R. S.: Connective-tissue reactions to implantation of purified sterol, sterol esters, phosphoglycerides, glycerides and free fatty acids. J Pathol Bact 94:63-7 I, I968 2 ANDERSON, D. B.; KAUFMAN, R. G.; KASTENSCHMIDT, L. L.: Lipogenic enzyme activities and cellularity of porcine adipose tissue from various anatomical locations. J Lipid Res 13: , BERKEN, A,; SHERMAN, A. A.: Rcticuloendothelial system depression in man after olive oil ingestion. Proc Soc Exp Biol Med 141: , DAEMS, W.TH.; WISSE, E.; BREDEROO, P.: Electron microscopy of the vacuolar apparatus in Lysosomes, ed. Dingle; 1st cd., pp , North-Holland Publishing Co., Amsterdam, DAHL, 0.; PERSSON, K. A.: Properties of animal depot fat in relation to dietary fat. J Sci Food Agric 16: , DANSE, L. H. J. C.: STEENBERGEN-BOII ERWEG, W. A,: Enzyme histochemical studies of adipose tissue in porcine yellow fat disease. Vet Pathol 11: , DANSE, L. H. J. C.; STEENBERGEN-BOTTERWEG, W. A.: Early changes of yellow fat disease in mink fed a vitamin E deficient diet supplemented with fresh or oxidised fish oil. Zentrdlbl Veterinaermed [A] 23: , DAVIS, C. L.; GORHAM, J. R.: The pathology of experimental and natural cases of yellow fat disease in swine. Am J Vet Rcs 15:55-59, DE JONGE, H.: lnleiding tot de medische statistiek. Verh Ned lnst Preventieve Geneesk 48:XLVIII, DIGIROLAMO, M.; THURMAN, L.; CULLEN, J.: Observations on adipose tissue cellularity and development in rats and rabbits fed ud lib. in The Regulation of the Adipose Tissue Mass, ed. Vague and Boyer; pp , American Elsevier Publishing Co., New York, 1974 I 1 ENDER, F.; HELGEBOSTAD, A.: Yellow fat disease in furbearing animals. XV Int Vet Congr Stockholm, Proc , I GASKELL, C. J.; LEEDALE, A. H.; DOUGLAS, S. W.: Pansteatitis in the cat: a report of four cases. J Small Anim Pract 16: I, GREEN, J.; BUNYAN, J.: Vitamin E and the biological antioxidant theory. Nutr Abstr Rev 39~ , I HADORN, H.; BUFER, K.; SUTER, H.: Bemerkungen uber die jodometrischen Verfahren zur Bestimmung der Peroxydzahl in Speiseblen. Zschr fur Lebensmittelunt und Forschung 104: , I HAYES, K. C.: Pathophysiology of vitamin E deficiency in monkeys. Am J Clin Nutr 27:l , HERMANS, P. G.: Steatosis as a congenital disease in pigs. Tijdschr Diergeneeskd 98~ , 1973

11 124 Danse and Vcrschuren 17 JONES, D.; HOWARD, A. N.; GRESHAM, G. A.: Aetiology of yellow fat disease (Pansteatitis) in the wild rabbit. J Comp Pathol 79: , KARRICK, N. L.: Nutritional value as animal feed in Fish Oils, ed. Stansby; pp , A.V.I. Publishing Co., Westport, Conn., KERK, P. VAN DE; DANSE, L. H. J. C.: Yellow fat disease in piglets and fattening pigs. Tijdschr Diergeneeskd 98:1I , KRONEMAN, J.; WENSVOORT, P.: Muscular dystrophy and yellow fat disease in Shetland pony foals. Neth J Vet Sci 1:42-48, LANGNER, H. J.: Die Erkennbarkeit der Verfalschung von Rinderfett mit Pferdefett. Fette, Seifen, Anstrichm 71: , MASON, K. E.; DAM, H.; GRANADOS, H.: Histological changes in adipose tissue of rats fed a vitamin E deficient diet high in cod liver oil. Anat Rec 94: , MERSMANN, H. J.; GOODMAN, J. R.; BROWN, L. J.: Development of swine adipose tissue: morphology and chemical composition. J Lipid Res 16: , PEARSE, A. G. E.: Histochemistry: Theoretical and Applied; 3rd ed., vol. 11, Churchill, London PIPY, B.; GAILLARD, D.; Derache, R.: Influence de regimes contenant des huiles de colza et de canbra sur le systeme reticuloendothelial du rat. J Physiol (Paris) 64: , SHAFRIR, E.; WERTHEIMER, E.: Comparative physiology of adipose tissue in different sites and in different species in Handbook of Physiology, Adipose Tissue, ed. Renold and Cahill; pp , American Physiological SOC, Washington, D.C., SHIER, P. D.; SCHEMMEL, R.: Effects of diet, age, strain and anatomical site on fat depot triglyceride and fatty acid content in rats. Proc SOC Exp Biol Med 149: , SPRATT, M. G.; KRATZING, C. C.: Oleic acid as a depressant of reticuloendothelial activity in rats and mice. J Reticuloendothel SOC 17: , WENSVOORT, P.: Morphogenesis of the altered adipose tissues in generalised steatitis in equidae. Neth J Vet Sci 99: , WENSVOORT, P.: Age-linked fcatures of generalised steatitis in equidae. Neth J Vet Sci 99: I 974 Request reprints from L. H. J. C. Danse. Department of Veterinary Pathology. University of Utrccht. Biltstraat 172. Utrecht (The Netherlands).