Proc. Nati. Acad. Sc. USA Vol. 74, No. 8, pp. 3550-3554 August 1977
Correction. This article, which originally appeared in the July 1977 issue, is reprinted here in its entirety because the pictures were not reproduced well in the first printing.
Medical Sciences
Fatty liver induced in Zucker "fatty" (ff) rats by a semisynthetic diet rich in
sucrose
(hyperlipidemia/obesity/congenital disorders/Golgi apparatus/GERL)
PHYLLIS M. NOVIKOFF Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461
Communicated by Alex B. Nomlkoff, May 6,1977
producing a hyperlipidemia in Holtzman-Sprague-Dawley rats. More recently, we used the same diet in studies demonstrating: (a) that clofibrate will neutralize the orotic acid effect if fed simultaneously with the orotic acid (10); and (b) that a clofibrate + orotic acid diet will reverse an already established orotic acid-induced fatty liver (11). I was interested in studying the effects of the hyperlipidemia-inducing semisynthetic diet on the liver of a rat with congenital hyperlipidemia. This led to the development of the fatty liver model described here.
ABSTRACT A new fatty liver model is described in which heredity plays a major role. A marked fatty liver develops when the homoz gous mutant Zucker rat (ff or "fatty") is fe a semisynthetic Jiet enriched in sucrose. Lean littermates do not develop fatty livers on this experimental diet. Even old ff rats do not develop fatty livers on chow; the experimental diet is required. The fatty liver is described, including light microscopic and preliminary electron microscopic observations. The clinical importance of understanding the pathogenesis of fatty liver is obvious, and a variety of animal models have been intensively studied. In the new model described here, genetic constitution exerts a major influence. We have induced a fatty liver in a mutant rat widely used in studies of obesity since 1961, when Zucker and Zucker reported that its obesity is inherited as an autosomal Mendelian recessive trait (1). Much is known about hormonal imbalances and lipogenesis in the Zucker rat (ff or "fatty"); these features have been reviewed most recently by Bray, who compared this type of obesity with a hypothalamic model of obesity (2). In contrast, little is known about the livers of Zucker rats, either lean or ff, and we have been unable to find reports on either their light microscopic histology or their ultrastructure. The ff rats develop enormous numbers of large adipocytes but their livers do not become fatty. The fatty liver we are describing was produced in theff rats by dietary means. The diet is a semisynthetic diet to which we added 1% orotic acid to produce a fatty liver in Holtzman-Sprague-Dawley rats (3-7) and to study the. effects of the hypolipidemic agent ethyl pchlorophenoxyisobutyrate (clofibrate) (8). The diet contains 60% sucrose, wt/wt, and it will be referred to as the "experimental diet." This diet was recognized by Shiff et al. (9) as
MATERIALS AND METHODS The Zucker rats were purchased from Harriet G. Bird Memorial Laboratory, Stow, MA. A total of 6 ff and 8 lean littermate rats, 2-4 months of age and of both sexes, were fed on Purina rat chow. Five ff and 7 lean littermate rats, of the same age and of both sexes, were fed the experimental diet, for 1 week or 3 weeks. As additional controls, four 7-month-old fatties and 4 littermate leans maintained on chow were studied. The experimental diet consisted of 60% sucrose, 20% vitamin-free casein, 4% corn oil, 1% vitamin mixture (12), 4% salt mixture (U.S. Pharmacopeia XIV), and 10% cellulose. This diet and the Purina rat chow were purchased from Teklad Mills, Madison, WI. While the animal was under ether anesthesia, a small piece of liver from the left lobe was quickly placed into cold 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, and diced. After a 2-hr fixation, the pieces were processed for electron microscopy. A larger piece, cut into ca 1.5-mm slices, was fixed in cold formaldehyde/glutaraldehyde/CaCI2 fixative (13) for 3 hr. Such tissue was used for preparing frozen sections to be studied by light microscopy, in which the bile canaliculi were visualized, using the Wachstein-Meisel "ATPase" medium, and then the lipid was stained, using oil red 0. Also the 3-hr alde-
Abbreviations: ATPase, adenosine triphosphatase; ER, endoplasmic reticulum; VLDL, very low density lipoprotein.
FIG. 1 (on following page). Gross appearance of livers from rats fed the experimental diet 3 weeks: on the left from the lean rat; on the right, from the ff rat. The livers were photographed following removal of 10-15 ml of blood via the abdominal aorta. Note the marked enlargement (it was about three times larger by weight) and the beige color of the fatty liver in the ff rat. The gross appearance of the fatty liver in the ff rat was essentially the same at 1 week of feeding the experimental diet. FIGS. 2 and 3. Frozen sections of liver, 10 ,gm thick, incubated in "ATPase" medium, then stained in oil red 0. (X150.) Fig. 2 is from the ff rat fed chow. Tiny red-stained lipid droplets are barely evident at this low magnification. As Fig. 4 shows, they are located close to the sinusoidal surface of the hepatocytes, with a greater number in the hepatocytes nearer the portal tracts (P). A central vein is present at C. Note the darkly stained bile canaliculi, thicker in the periportal region. Fig. 3 is from the ff rat fed the experimental diet 1 week. Large red-stained lipid spheres occupy the cytoplasm of the hepatocytes; the spheres are fewer and smaller in the centrolobular region. A central vein is present at C. Note that the bile canaliculi are attentuated and of variable thickness. The few empty vacuoles in the figure have resulted from loss of lipid spheres during preparation of the slide. FIGS. 4-6. Epon sections 1 um thick, stained in toluidine blue. (X400.) The sections for Figs. 4 and 5 were fixed in 1% OS04. The section for Fig. 6 was fixed initially in aldehyde and post-fixed in 1% 0804. Portal veins are indicated by P. Fig. 4, ff rat on chow; compare Fig. 7. At the arrows, tiny electron-lucent lipid droplets are barely evident; they are close to the sinusoidal aspect of the hepatocytes. Note nuclei (arrowheads); the irregular clear areas are sites of glycogen. Figs. 5 and 6 are from ff rats fed the experimental diet, 1 week and 3 weeks, respectively. Large electron-lucent spheres occupy much of the hepatocyte cytoplasm. Note the enormous increase in size and number of lipid spheres. Although the cell boundaries are indistinct in these illustrations, the size of the heptocytes is markedly increased and they have rounded out. Note that many nuclei (arrowheads) have been displaced from their positions, producing "signet ring" appearances. At the middle and lower arrowheads of Fig. 5 and at all three arrowheads of Fig. 6, the nuclei are distorted to "half-moon" shapes. The lipid spheres are larger in Fig. 6 than in Fig. 5. Fatty cysts (19) are not observed.
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Medical Sciences: Novikoff
Proc. Natl. Acad. Sci. USA 74 (1977)
FIGS. 1-6. (Legends appear at the bottom of the preceding page.)
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Medical Sciences: Novikoff
Proc. Nad. Acad. Sci. -USA 74 (1977)
FIGS. 7 and 8. (Legends appear at the bottom of the following page.)
l eSn o i
Medical Sciences: Novikoff
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o. Nt Ad S - Proc. Natl. Acad. Sci. USA 74 (1977)
3553
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Correction. This article, which originally appeared in the July 1977 issue, is reprinted here in its entirety because the pictures were not reproduced well in the first printing.
Medical Sciences
Fatty liver induced in Zucker "fatty" (ff) rats by a semisynthetic diet rich in
sucrose
(hyperlipidemia/obesity/congenital disorders/Golgi apparatus/GERL)
PHYLLIS M. NOVIKOFF Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461
Communicated by Alex B. Nomlkoff, May 6,1977
producing a hyperlipidemia in Holtzman-Sprague-Dawley rats. More recently, we used the same diet in studies demonstrating: (a) that clofibrate will neutralize the orotic acid effect if fed simultaneously with the orotic acid (10); and (b) that a clofibrate + orotic acid diet will reverse an already established orotic acid-induced fatty liver (11). I was interested in studying the effects of the hyperlipidemia-inducing semisynthetic diet on the liver of a rat with congenital hyperlipidemia. This led to the development of the fatty liver model described here.
ABSTRACT A new fatty liver model is described in which heredity plays a major role. A marked fatty liver develops when the homoz gous mutant Zucker rat (ff or "fatty") is fe a semisynthetic Jiet enriched in sucrose. Lean littermates do not develop fatty livers on this experimental diet. Even old ff rats do not develop fatty livers on chow; the experimental diet is required. The fatty liver is described, including light microscopic and preliminary electron microscopic observations. The clinical importance of understanding the pathogenesis of fatty liver is obvious, and a variety of animal models have been intensively studied. In the new model described here, genetic constitution exerts a major influence. We have induced a fatty liver in a mutant rat widely used in studies of obesity since 1961, when Zucker and Zucker reported that its obesity is inherited as an autosomal Mendelian recessive trait (1). Much is known about hormonal imbalances and lipogenesis in the Zucker rat (ff or "fatty"); these features have been reviewed most recently by Bray, who compared this type of obesity with a hypothalamic model of obesity (2). In contrast, little is known about the livers of Zucker rats, either lean or ff, and we have been unable to find reports on either their light microscopic histology or their ultrastructure. The ff rats develop enormous numbers of large adipocytes but their livers do not become fatty. The fatty liver we are describing was produced in theff rats by dietary means. The diet is a semisynthetic diet to which we added 1% orotic acid to produce a fatty liver in Holtzman-Sprague-Dawley rats (3-7) and to study the. effects of the hypolipidemic agent ethyl pchlorophenoxyisobutyrate (clofibrate) (8). The diet contains 60% sucrose, wt/wt, and it will be referred to as the "experimental diet." This diet was recognized by Shiff et al. (9) as
MATERIALS AND METHODS The Zucker rats were purchased from Harriet G. Bird Memorial Laboratory, Stow, MA. A total of 6 ff and 8 lean littermate rats, 2-4 months of age and of both sexes, were fed on Purina rat chow. Five ff and 7 lean littermate rats, of the same age and of both sexes, were fed the experimental diet, for 1 week or 3 weeks. As additional controls, four 7-month-old fatties and 4 littermate leans maintained on chow were studied. The experimental diet consisted of 60% sucrose, 20% vitamin-free casein, 4% corn oil, 1% vitamin mixture (12), 4% salt mixture (U.S. Pharmacopeia XIV), and 10% cellulose. This diet and the Purina rat chow were purchased from Teklad Mills, Madison, WI. While the animal was under ether anesthesia, a small piece of liver from the left lobe was quickly placed into cold 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, and diced. After a 2-hr fixation, the pieces were processed for electron microscopy. A larger piece, cut into ca 1.5-mm slices, was fixed in cold formaldehyde/glutaraldehyde/CaCI2 fixative (13) for 3 hr. Such tissue was used for preparing frozen sections to be studied by light microscopy, in which the bile canaliculi were visualized, using the Wachstein-Meisel "ATPase" medium, and then the lipid was stained, using oil red 0. Also the 3-hr alde-
Abbreviations: ATPase, adenosine triphosphatase; ER, endoplasmic reticulum; VLDL, very low density lipoprotein.
FIG. 1 (on following page). Gross appearance of livers from rats fed the experimental diet 3 weeks: on the left from the lean rat; on the right, from the ff rat. The livers were photographed following removal of 10-15 ml of blood via the abdominal aorta. Note the marked enlargement (it was about three times larger by weight) and the beige color of the fatty liver in the ff rat. The gross appearance of the fatty liver in the ff rat was essentially the same at 1 week of feeding the experimental diet. FIGS. 2 and 3. Frozen sections of liver, 10 ,gm thick, incubated in "ATPase" medium, then stained in oil red 0. (X150.) Fig. 2 is from the ff rat fed chow. Tiny red-stained lipid droplets are barely evident at this low magnification. As Fig. 4 shows, they are located close to the sinusoidal surface of the hepatocytes, with a greater number in the hepatocytes nearer the portal tracts (P). A central vein is present at C. Note the darkly stained bile canaliculi, thicker in the periportal region. Fig. 3 is from the ff rat fed the experimental diet 1 week. Large red-stained lipid spheres occupy the cytoplasm of the hepatocytes; the spheres are fewer and smaller in the centrolobular region. A central vein is present at C. Note that the bile canaliculi are attentuated and of variable thickness. The few empty vacuoles in the figure have resulted from loss of lipid spheres during preparation of the slide. FIGS. 4-6. Epon sections 1 um thick, stained in toluidine blue. (X400.) The sections for Figs. 4 and 5 were fixed in 1% OS04. The section for Fig. 6 was fixed initially in aldehyde and post-fixed in 1% 0804. Portal veins are indicated by P. Fig. 4, ff rat on chow; compare Fig. 7. At the arrows, tiny electron-lucent lipid droplets are barely evident; they are close to the sinusoidal aspect of the hepatocytes. Note nuclei (arrowheads); the irregular clear areas are sites of glycogen. Figs. 5 and 6 are from ff rats fed the experimental diet, 1 week and 3 weeks, respectively. Large electron-lucent spheres occupy much of the hepatocyte cytoplasm. Note the enormous increase in size and number of lipid spheres. Although the cell boundaries are indistinct in these illustrations, the size of the heptocytes is markedly increased and they have rounded out. Note that many nuclei (arrowheads) have been displaced from their positions, producing "signet ring" appearances. At the middle and lower arrowheads of Fig. 5 and at all three arrowheads of Fig. 6, the nuclei are distorted to "half-moon" shapes. The lipid spheres are larger in Fig. 6 than in Fig. 5. Fatty cysts (19) are not observed.
3550
Medical Sciences: Novikoff
Proc. Natl. Acad. Sci. USA 74 (1977)
FIGS. 1-6. (Legends appear at the bottom of the preceding page.)
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Proc. Nad. Acad. Sci. -USA 74 (1977)
FIGS. 7 and 8. (Legends appear at the bottom of the following page.)
l eSn o i
Medical Sciences: Novikoff
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o. Nt Ad S - Proc. Natl. Acad. Sci. USA 74 (1977)
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At
