Vet. Pathol. 15: 544-548 (1978) Fish Oil-induced Yellow Fat Disease in Rats 111. Lipolysis in Affected Adipose Tissue L. H. J. C. DANSE and P. M. VERSCHUREN Department of Veterinary Pathology, University of Utrecht, Utrecht Abstract. Basal and hormone-stimulated lipolysis of adipose tissue was measured at successive stages during the development of fish oil-induced yellow fat disease in rats. Changes of lipolytic activity at an early stage of yellow fat disease were not seen. There was a significant increase of basal lipolysis and a decrease of stimulated lipolysis when many fat cells were affected (stage E). Since the increased basal lipolysis probably originates from degenerated fat cells, the mechanism of enzyme activation is not clear. The decreased stimulated lipolysis was proportional to the number of affected fat cells and resulted from membrane damage of these cells. Increased 5-nucleotidase activity, seen in affected fat cells, may be important, to this reduced stimulated lipolysis. Animals with yellow fat disease often have poor appetite, lassitude and loss of weight [7, 8, 121. Sometimes a cachexia, leading to the death of the animals, will develop [9]. Despite this period of illness and malnutrition, atrophy of adipose tissue has not been seen. On the contrary, the animals look fat [9, 12, 131. It has often been reported, therefore, that the diseased animals were the best of the group. This apparent inavailability of depot fat as an energy reserve may result either from an early functional blockade or from a very acute and extensive progression of the degenerative processes in adipose tissue. These signs mainly were seen in natural cases. In order to facilitate causative and pathogenetic investigation, yellow fat disease was experimentally induced in the rat [4]. Lipolysis is the hydrolytic cleavage of triglycerides in free fatty acids and glycerol, which is catalysed by a hormone-sensitive lipase. This lipase is activated through the cyclic AMP system (fig. 1) [2]. Since the hormone receptors are localised in the plasma membrane, the intactness of this structure is essential for lipolysis in adipose tissue [ 161. When adipose tissue was induced to release its lipid stores, there was increased basal and hormone-stimulated lipolytic activity and cyclic AMP concentration [l]. This membrane dependency of lipolysis in adipose 544
Fish Oil-induced Yellow Fat Disease 545 Extracellular lntracellular AT\ 5-nucleotidase 57P-Adenosine Hormone + "1 phosphodiesterase /' activated lipase ps001 Normal Stage S Stage E Fig. 1: Activation of hormone-sensitive lipase in the fat cell. Fig. 2: Basal and adrenalin-stimulated (1 7 pg/ml) lipolytic activity in epididymal adipose tissue at 1 (normal), 2 (stage S) and 6 (stage E) weeks development of yellow fat disease. Values given are means of triplicate incubations. Statistically significant differences are indicated by their P value. tissue and the necessity of this process for fatty acid mobilisation from adipose tissue in energy deficit, makes measurement of lipolysis suitable to assess the present problem. Membrane damage after lipid oxidation should be an important factor in the pathogenesis of yellow fat disease [3]. Materials and Methods One hundred and twenty young male Wistar rats of weaning age and weighing 40? 5 grams were divided into two equal groups. In the experiment group yellow fat disease was induced with a vitamin E-deficient, cod liver oil-containing diet. The control group was kept on a vitamin E-supplemented fish oil diet. The experimental conditioning, which started at weaning age, and the qualification and treatment of the dietary cod liver oil has been described [4]. Rats were killed at 1, 2 and 6 weeks after weaning. Both epididymal fat pads were immediately removed and put in a Krebs-Ringer bicarbonate buffer, ph 7.35, with 5% bovine serum albumine (Bovine albumin Fraktion V@, Fluka, Germany) at 37" C. Within each group the adipose tissue of the rats was pooled (5 to 20 rats, depending on their age). The incubations for measuring lipolysis were done according to Metz [14]. After incubation of 25 minutes the main incubation was started by adding an aqueous solution of adrenalin (L-adrenalin-D-hydrogenetartrate, Fluka, Germany) at a final concentration of 17 pg/ml. With this concentration of adrenalin, lipolysis was maximally stimulated. Main
546 Danse and Verschuren incubation time was 2 hours. Basal lipolytic activity was determined by adding saline instead of adrenalin. Lipolytic activity was measured as glycerol release. Glycerol was determined enzymatically according to Eggstein and Kreutz [6] with a test set (Boehringer, Germany). Epididymal adipose tissue from each rat was fixed in 10% buffered formalin and embedded in paraplast for microscopical and histochemical determination of the stage in the development of yellow fat disease [4]. Sections were cut at 7 micrometers and stained with hematoxylin and eosin (HE) and with carbol fuchsin for acid-fast lipofuscin. Unstained sections were studied with a fluorescence microscope [15]. Statistical calculations were done by Student s t-test. Results In the first week after weaning adipose tissue of the experiment group looked normal. Two weeks after weaning this group had stage S yellow fat disease with a small number of degenerated fat cells and the interstitial macrophage reaction. During the 6th week after weaning these lesions had progressed extensively to stage E yellow fat disease. At that time the number of affected fat cells was between 10 and 20%. In stage S yellow fat disease there was no significant difference in basal and adrenalin-stimulated lipolysis of adipose tissue between experiment rats and controls (fig. 2). At stage E adipose tissue of experiment rats had a small but significant increase of basal lipolysis and a significant decrease of stimulated lipolysis. Discussion In our study the extent of degenerative changes in adipose tissue 6 weeks after weaning was the same as in rats 4 weeks after weaning in previous experiments [4]. This retarded progression of the disease was because experimental conditioning was started at different times in the experiments. In this study young rats were treated from weaning age, whereas in the previous study mother rats were treated during gestation and the weaning period. Blockade of lipolysis at an early stage of yellow fat disease did not occur. In stage S yellow fat disease in which few but distinct morphological changes were seen in adipose tissue, basal and stimulated lipolysis had not changed significantly. When a considerable number of fat cells was affected ( stage E77), however, there were significant changes in lipolytic activity. Basal lipolysis was increased, whilst adrenalin-stimulated lipolysis was reduced. The increased basal lipolytic activity during stage E yellow fat disease may result from activation of fat cells, that are still undamaged. Neither microscopical [4] nor enzyme histochemical [5] changes indicating activation were seen, however, in these fat cells. On the contrary, degenerative fat cells at this stage had an increased esterase activity (lipolytic activity). The origin of this increased esterase is not clear. There were few esterase-positive inflammatory cells surrounding these affected fat cells. Considering the damaged membranes of the affected fat cells and the decreased hormone-stimulated lipolysis during this stage, activation of the fat cell-associated lipase in these cells seems improbable. During the development of
Fish Oil-induced Yellow Fat Disease 547 adipose tissue necrosis after acute experimental pancreatitis, increased basal lipolysis also was seen both histochemically [ll] and biochemically [18]. In this disorder, however, leakage of pancreatic lipase was suggested to be the cause of increased enzyme activity in adipose tissue. In yellow fat disease identical observations were made but pancreatic lesions were absent. In stage E yellow fat disease there was about 30% reduction of stimulated lipolysis. Membrane damage in the affected fat cells probably would prevent the activation of lipase. The increased 5-nucleotidase activity in affected fat cells in rats [5] and pigs [3], however, also may be important. This enzyme plays a part in the catabolism of cyclic AMP (fig. 1). An increase of 5-nucleotidase activity, which also was seen in the liver of vitamin E deficient ducklings and which was considered to be a primary effect of membrane injury [lo], may result secondarily in a decreased activity of cyclic AMP regulated metabolic processes [ 171. Our study indicates the absence of a functional blockade in adipose tissue during the slow development of yellow fat disease in the rat. The decrease of hormonestimulated lipolysis was proportional to the number of degenerated fat cells. Therefore in species in which yellow fat disease was associated with poor appetite, body weight loss and cachexia without atrophy of fat depots, rapid progression of lesions in adipose tissue seems probable. Acknowledgements We thank Dr. S. H. M. Metz, Institute for Animal Feeding and Nutrition Research, Lelystad, for help with the manuscript; and Mrs. C. Mensinga for the histological preparations. References 1 BUTCHER, R.W.; BAIRD, C.E.; SUTHERLAND, E.W.: Effects of lipolytic and antilipolytic substances on adenosine 3,5 -monophosphate levels in isolated fat cells. J Biol Chem 243~1705-1712,1968 2 BUTCHER, R.W.: The role of cyclic AMP in the actions of some lipolytic and antilipolytic agents in Hormone and Metabolic Research, Suppl. 2. Adipose Tissue, Regulation and Metabolic Function, ed. Levine and Pfeiffer; pp. 5-10; Georg Thieme Verlag, Stuttgart, 1970 3 DANSE, L.H.J.C.; STEENBERGEN-BOTTERWEG, W.A.: Enzyme histochemical studies of adipose tissue in porcine yellow fat disease. Vet Pathol 11:465-476, 1974 4 DANSE, L.H.J.C.; VERSCHUREN, P.M.: Fish oil-induced yellow fat disease in rats. I. Histological changes. Vet Pathol 15114-124, 1978 5 DANSE, L.H.J.C.; STEENBERGEN-BOTTERWEG, W.A.: Fish oil-induced yellow fat disease in rats. 11. Enzyme histochemistry of adipose tissue. Vet Pathol 15125-132, 1978 6 EGGSTEIN, M.; KREUTZ, F.H.: Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe. Klin Wochenschr 44:262-267, 1966 7 GASKELL, C.J.; LEEDALE, A.H.; DOUGLAS, S.W.: Pansteatitis in the cat: a report of four cases. J Small Anim Prac 16:117-121, 1975 8 GORHAM, J.R.; BOE, N.; BAKER, G.A.: Experimental yellow fat disease in pigs. Cornell Vet 41:332-338, 1951 9 HARDEMAN, Y.H.P.; WENSVOORT, P.: Steatitis in piglets. Tijdschr Diergeneeskd. 98:199-201, 1973
548 Danse and Verschuren 10 HULSTEART, C.E.; GIJZEL, W.P.; HARDONK, M.J.; KROON, A.M.; MOLENAAR, I.: Cellular membranes and membrane-bound enzymes in vitamin E deficiency: a histochemical, cytochemical, biochemical, and morphologic study of the liver of the Pekin duckling. Lab Invest 33:176-186, 1975 11 ITO, T.: A pathological study on fat necrosis in swine. Jpn J Vet Sci 35229-310, 1973 12 KRONEMAN, J.; WENSVOORT, P.: Muscular dystrophy and yellow fat disease in Shetland pony foals. Neth J Vet Sci 1:42-48, 1968 13 MASON, K.E.; HARTSOUGH, G.R.: Steatitis or yellow fat in mink, and its relation to dietary fats and inadequacy of vitamin E. J Am Vet Med Assoc 119:72-75, 1951 14 METZ, S.H.M.; MULDER, I.; VAN DEN BERGH, S.G.: Regulation of lipolysis in bovine adipose tissue by the degree of saturation of plasma albumin with fatty acids. Biochim Biophys Acta 306:42-50, 1973 15 PEARSE, A.G.E.: Histochemistry: Theoretical and Applied, 3rd ed., vol. 11; Churchill, London, 1972 16 RODBELL, M.: The fat cell in mid-term: its past and future in Hormone and Metabolic Research, suppl. 2. Adipose Tissue, Regulation and Metabolic Functions, ed. Levine and Pfeiffer; pp. 1-4, Georg Thieme Verlag, Stuttgart, 1970 17 SURAN, A.A.: 5 Nucleotidase and an acid phosphatase of spinal cord: comparative histochemistry and specificity of the enzymes in mouse and cat spinal cords. Cytologic localisation in mouse substantia gelatinosa. J Histochem Cytochem 22:802-811, 1974 18 THEVE, N.O.; HALBERG, D.; CARLSTROM, A.: Studies in fat necrosis. I. Lipolysis and calcium content in adipose tissue from rats with experimentally induced fat necrosis. Acta Chir Scand 139:131-133, 1973 Request reprints from L.H.J.C. Danse, Department of Veterinary Pathology, University of Utrecht, Biltstraat 172, Utrecht (The Netherlands).