Virchows Archiv B Cell Pathol (1992) 63:43-49

VirchowsArch&B CellPathology IncladingMolecular

9 Springer-Verlag 1992

Proliferation of myocardial peroxisomes in experimental rat diabetes: a biochemical and immunocytochemical study S a d a k i Yokota ~ and Kohtaro A s a y a m a 2

Departments of ~Anatomy and 2 Pediatrics, Yamanashi Medical University, Yamanashi 409-38, Japan ReceivedJune 16 / AcceptedAugust 21, 1992

S u m m a r y . Myocardial peroxisomes were investigated in

normal and diabetic rats. Catalase and acyl-CoA oxidase activities were increased in the diabetic rat heart and immunoblot analysis showed that both enzyme proteins were markedly enhanced in diabetic heart homogenates. After immunoenzyme staining, catalase and acyl-CoA oxidase were localized in fine granules in the myocardium, which were increased in number in diabetic rats. The numerical density of the granules stained for catalase was increased 1.7 times and that for acyl-CoA oxidase 1.8 times, compared with controls. Protein A-gold labeling for catalase and acyl-CoA oxidase was present in myocardial peroxisomes. The labeling density for both enzymes was increased in diabetic rats by 1.6 times for catalase and 1.5 times for acyl-CoA oxidase, compared with controls. The results indicate that myocardial peroxisomes are increased in the diabetic rat and that this proliferation is accompanied by an increase in catalase and acyl-CoA oxidase activities. Key words: D i a b e t e s - Heart - P e r o x i s o m e s - Catalase

- Acyl-CoA oxidase

Introduction

It has been established that /3-oxidation of fatty acids occurs in both peroxisomes and the mitochondria (Mannaerts et al. 1979). In peroxisomal/3-oxidation, the initial key enzyme is acyl-CoA oxidase, whereas in mitochondrial oxidation it is acyl-CoA dehydrogenase (Osumi et al. 1980). The former enzyme oxidizes long-chain fatty acid and produced H 2 0 2 that is immediately degraded by catalase. In addition, extensive studies on patients with the Zellweger syndrome, in which peroxisomes are lacking in the liver and other tissues (Goldfischer et al. 1973; Mooi etal. 1983; Mfiller-H6cker etal. 1981; Pfeifer and Sandhage 1979) have clarified several physioCorrespondence to: S. Yokota

logical functions of peroxisomes, such as the formation of bile acid (Kase et al. 1983), the synthesis of plasmalogen (Hajra and Bishop 1982), the catabolism of phytanic acid (Poulos et al. 1984), the oxidation of pipecoplic acid (Trijbels et al. 1979), and the catabolism of amino acids (Noguchi 1987). Thus, there is increasing attention being paid to peroxisomes as their various important functions are clarified (Schutgens et al. 1986). Although liver peroxisomes have been extensively studied (for reviews, see B6ck et al. 1980; de Duve and Baudhuin 1969; Masters and Holmes 1977), investigations of myocardial peroxisomes are few (Asano and Ashraf 1980; Herzog and Fahimi 1974, 1976; Hicks and Fahimi 1977). In a previous study, we have shown that peroxisomal proliferation in the rat heart and soleus muscles is induced by starvation (Yokota and Asayama 1990). This proliferation was accompanied by an increase in catalase and acyl-CoA oxidase activities. In addition, it has been shown that catalase and acyl-CoA oxidase activities are increased in the diabetic rat heart (Asayama et al. 1991). Since these enzyme are contained in peroxisomes, it would be expected that the increased enzyme activities would be associated with proliferation of myocardial peroxisomes. The purpose of this study was to examine this suggestion by analyzing normal and diabetic rat myocardium using biochemical and immunocytochemical methods.

Materials and methods Animals. Sprague Dawley rats (Japan SLC, Shizuoka, Japan) aged

6 weeks were fed on standard chow and water ad libitum. They were divided into weight-matched experimental and control groups. The experimental groups were given 65 mg/kg streptozotocin (Sigma, St. Louis, Mo., USA) to induce diabetes, which was confirmed by demonstrating hyperglycemiaafter 3 days (Asayama et al. 1991). The diabetic rats were killed under pentobarbital anesthesia (50 mg/kg i.p.) 2 weeks after the induction of diabetes and controls were sacrificedon the same day. The hearts were removed, rinsed with cold phosphate-buffered saline (PBS), blotted with gauze and then stored frozen at -80 ~ C.

44

Enzyme assay. To assay the acyl-CoA oxidase activity, the tissues were homogenized with 10mM potassium phosphate buffer (pH 7.4) containing 30 mM sucrose. The homogenate was centrifuged at 800 g for 10 min, then at 5200 g for 10 min. The resulting supernatant was used for the assay. The acyl-CoA oxidase activity was assayed according to the method described by Small and coworkers (1980). Immunoblot analysis. A 10% homogenate (g/vol) was prepared as follows. The tissues were homogenized with 10 mM potassium phosphate buffer containing 0.1% Triton X-100, 4 mM leupeptin (Peptide Institute, Osaka, Japan), 4 mM chymostatin (Sigma), 4 mM antipain (Sigma) and 4 mM pepstatin (Sigma). The homogenates were centrifuged at 10000g for 10 min and the resulting supernatants were mixed with the same volume of sample buffer consisting of 2% sodium dodecyl sulfate (SDS), 0.2% mercaptoethanol, 8 M urea, 0.002% bromphenol blue and 0.2 M TRIS-HC1 buffer (pH 6.8). The mixture was heated in boiling water for 2 min and then electrophoresed on gradient polyacrylamide gel (7%20%) prepared in 0.56 M TRIS-HC1 buffer (pH 8.8) and 0.1% SDS. After electrophoresis, proteins were transferred to nitrocellulose membrane (Towbin et al. 1979). The membrane was incubated with anti-catalase antiserum or with anti-acyl-CoA oxidase antiserum (diluted • 4000). After rinsing with 0.3% Tween 20-PBS, the membrane was incubated with horseradish peroxidase (HRP)-labeled Fab' fragments of goat anti-rabbit IgG (Yokota and Asayama 1990) and HRP activity was stained by diaminobenzidine (DAB) reaction.

Tissue preparation. The hearts were perfused through the left ventricle with fixative for 5 min. The fixative contained 4% paraformaldehyde, 0.25% glutaraldehyde, 5% sucrose and 0.05 M cacodylate buffer (pH 7.4). The tissue was cut into 100-~tm-thick slices with a Vibratome (Oxford Labs, Los Angeles, Calif., USA) and the slices were divided into four groups. The first group was postosmicated and embedded in Epon. The second and third groups were refixed with 2% glutaraldehyde for 30 min. The former was then stained for catalase by the alkaline DAB technique (LeHir et al. 1979) and the latter was embedded in Epon without postosmication. The fourth group was embedded in LR-White at - 2 0 ~ C.

Quantification of the immunolabeling A total of seven light micrographs were taken from the longitudinal sections of each tissue block at magnification of x 500 and the numerical density of the cytoplasmic granules stained by the immunoenzyme technique was determined. The myocardial cell area in the photographs was determined by a semi-computing system and the number of the stained granules was counted. The numerical density was expressed as the stained granules per 100 Ixm2. The labeling of peroxisomes with gold particles was analyzed quantitatively. Electron micrographs (n =7) including the peroxisomes were taken for each tissue block at magnification of x 20 000. The positive pictures were analyzed by a semi-computing system (Yokota et al. 1988). The labeling density was expressed as gold particles per square micrometer (Bendayan et al. 1980). The data are presented as the mean_+ SEM and statistical significance was determined by the unpaired t-test.

Table 1. Catalase and acyl-CoA oxidase activities in heart homogenates from control and diabetic rats Catalase a

Control (C) Diabetes (D) D/C

1.09 + 0.04 3.92 _ 0.19 3.6

Acyl-CoA oxidase b 7.1 + 0.2 10.5 + 0.4

1.5

a VKIS-1)/gtissue

b nmol/min per gram of tissue

Immunoeytochemical procedures Light microscopy. Semi-thin sections (1 pm thick) from the third group were stained for catalase and acyl-CoA oxidase by the immunoenzyme technique (Litwin et al. 1984). Briefly, the sections were treated with 10% sodium ethoxide to remove the epoxy resin. After 3 min digestion with 0.5% trypsin (Difco, Detroit, Mich., USA), the sections were incubated with specific antibodies for 2 h. After rinsing with PBS, the sections were treated with HRP-labeled Fab' fragments of goat anti-rabbit IgG and the HRP activity was visualized by the DAB reaction. Anti-catalase and anti-acyl-CoA oxidase antibodies used were from the batches described previously (Yokota and Asayama 1990, 1992). The monospecificity of the antibodies was demonstrated by immunoblot analysis as shown in the results. For immunocytochemical control the sections were incubated with antisera absorbed by the corresponding antigens. This was followed by treatment with the secondary antibody and the DAB reaction. Electron microscopy. For a single labeling, both surfaces of ultrathin sections of the fourth group were treated with anti-catalase or anti-acyl-CoA oxidase, and then 15 nm protein A-gold probe was applied (Roth 1982). For double labeling, each surface of the sections was treated separately with anti-catalase antibody and anti-acyl-CoA oxidase antibody, respectively, followed by 15 nm and 5 nm protein A-gold probes (Bendayan 1982). For immunocytochemical control, sections were incubated with x 1000 diluted antisera absorbed by the corresponding antigens instead of the specific antibodies.

Fig. 1. Immunoblot analysis of catalase and acyl-CoA oxidase in cardiac muscle homogenates. Acyl-CoA oxidase is shown in lanes 1-3, and catalase in lanes 4-6. Lanes I and 4, Rat liver (8 p.g protein); lanes 2 and 5, control rat heart (22 p.g); lanes 3 and 6, diabetic rat heart (22 ~tg). The numbers on the left indicate the molecular masses of subunits

45

Fig. 2A, B. Longitudinal sections of rat heart showing immunoenzyme staining for catalase. A Control rat heart. Discrete granular staining is observed in myocardium. B Diabetic rat heart. Stained granules are increased in number and size. • 2000. Bar: 10 Ixm

Fig. 3 A, B. Longitudinal sections of rat heart showing immunoenzyme staining for acyl-CoA oxidase. A Control rat heart. Cytoplasmic granules are stained for the enzyme. B Diabetic rat heart. Reaction positive granules are increased in number and size. x 2000. Bar: 10 ~tm

Results

applied for electrophoresis (Fig. 1, lane 4). After incubation with anti-acyl-CoA oxidase, three bands (molecular masses 75 kDa, 53 kDa and 23 kDa) appeared in the homogenate of the control rat liver (Fig. 1, lane 1). The 53 kDa band was intensely stained, whereas the other two bands were intermediately stained. In the homogenate of control rat heart, an additional 45 kDa band was developed. These four bands were also observed in homogenate of diabetic rat heart (Fig. 1, lane 3); the 75 kDa, 53 kDa and 23 kDa bands were increased, whereas the 45 kDa band was unchanged (Fig. 1, lanes 2 and 3). The polypeptide component of 53 kDa was the most markedly increased.

Biochemical assay Catalase activity in the diabetic rat heart was increased by 3.6 times over control and that of acyl-CoA oxidase by 1.4 times (Table 1). These increases were statistically significant (P < 0.001).

Immunoblot analysis of catalase and acyl-CoA oxidase After treatment with anti-catalase antibody, a single band (molecular mass, approximately 60 kDa) was developed in the homogenate of normal rat heart (Fig. 1, lanes 4-6). In the diabetic rat heart, a single signal was noted. The staining intensity of which was markedly enhanced (Fig. 1, lane 6) but was still lower than the normal rat liver homogenate, a lesser amount of which was

Immunocytochemical staining Light microscopy. After incubation with the anti-catalase antibody, staining of scattered fine cytoplasmic granules

46

Fig. 4 A, B. Myocardium stained for catalase by the protein A-gold technique. Both surfaces of a section were stained. A Control rat heart; B diabetic rat heart. Peroxisomes (P) are labeled with gold particles but mitochondria (M) are not, Labeling intensity in the diabetic rat is higher than that in the control, x 49000. Bar: 0.5 I~m

Fig. 5A, B. Heart muscle stained for acyl-CoA oxidase by the protein A-gold technique. Both surfaces of a section were stained. A Control rat; B diabetic rat heart. Gold particles are observed in peroxisomes (P) but not in mitochondria (M). Labeling intensity is increased in the diabetic rat. • 49900. Bar: 0.5 ~tm

was noted in the normal rat myocardium (Fig. 2A). Similar granules were markedly increased in size and number in the diabetic rat myocardium (Fig. 2B). Similar scattered cytoplasmic granules were stained after incubation with anti-acyl-CoA oxidase in the normal rat myocardium (Fig. 3A) and their number was markedly increased in the diabetic rat myocardium (Fig. 3B) in which they frequently formed small clusters. No granular staining was observed after incubation with absorbed antisera (not shown).

oxisomes (Fig. 5A, B) with more intense staining in the diabetic animals (Fig. 5 B). Myofibrils and mitochondria were consistently unstained by these techniques.

Electron microscopy. Gold particles showing the antigenic sites for catalase were present exclusively in the peroxisomes in both normal and diabetic rat myocardial cells (Fig. 4A, B). The labeling density was increased in the latter (Fig. 4B). Gold particles marking acyl-CoA oxidase antigenic sites were also located in myocardial per-

Double labeling. The peroxisomes of normal and diabetic rat myocardial cells were doubly labeled with differently sized gold particles. Catalase and acyl-CoA oxidase were colocalized in the same peroxisomes (Fig. 6A, B). The labeling intensity for both enzymes was lower than that in the preceding figures, since only one side of the sections was used for each enzyme. Quantitative analysis of staining intensity. The results for the numerical densities of peroxisomes in the normal and diabetic rat myocardium are shown in Table 2. The numerical density of peroxisomes stained for catalase was similar to that stained for acyl-CoA oxidase and

47

Fig. 6A, B. Double labeling of peroxisomes for catalase and acylCoA oxidase. One surface of the section was labeled with a 15-nm protein A-gold probe and the other surface with a 5-nm protein A-gold probe. Large and small (arrows) gold particles represent catalase and acyl-CoA oxidase, respectively. A Control rat heart; B diabetic rat heart, x 64000. Bar: 0.25 ~tm

Table 2. Numerical density of myocardial peroxisomes in control

and diabetic rats (peroxisomes/100 ~tm2)

Control (C) Diabetes (D) D/C

Catalase

Acyl-CoA oxidase

14.82 _+1.47 25.56 +_4.38 1.72

10.94_+ 1.49 19.36 + 2.52 1.77

Table 3. Labeling density for catalase and acyl-CoA oxidase in myocardial peroxisomes of control and diabetic rats (gold particles/

~tmz)

Control (C) Diabetes (D) D/C

Catalase

Acyl-CoA oxidase

392 + 98 620_ 122 1.58

253_ 55 383 + 56 1.51

both were increased in the diabetic, compared with the normal rat myocardium (1.7-fold for catalase and 1.8fold for acyl-CoA oxidase). The results for labeling density are shown in Table 3. In myocardium of both the normal and diabetic rats, the labeling density for catalase was higher than that for acyl-CoA oxidase. The labeling density for catalase in the diabetic rat heart was 1.6-fold higher, and that for acyl-CoA oxidase in diabetic rats was 1.5-fold higher than in controls.

Discussion

In recent years peroxisomal function and its modification in nutritional disorders have excited increasing attention. A significant production of cellular fatty acid

oxidation is attributed to the peroxisomal fl-oxidation (Veekamp and Van Moerlerk 1986). The activities of catalase and peroxisomal oxidases are known to alter in diabetic tissues (Asayama et al. 1989, 1991 ; Horie et al. 1981; Kawashima et al. 1983), but the fine structural aspects of these changes have not been elucidated previously. In the present study, the immunoblot analysis of rat myocardial homogenates showed three polypeptide subunits of 75 kDa, 53 kDa and 23 kDa with an additional polypeptide component of 45 kDa that is not detected in the rat liver. Similar results were obtained in a previous study (Yokota and Asayama 1992). In the diabetic rat heart, the 53 kDa and 23 kDa subunits were clearly increased. Recently, Schepers et al. (1990) reported that the 52 kDa and 22.5 kDa subunits are increased following the administration of the peroxisome proliferator, clofibrate. Since these two subunits seem to correspond to our 53 kDa and 23 kDa subunits, these results are consistent with our own. If the 45 kDa polypeptide observed here is a dimer of the 23 kDa subunit, it should be increased in the diabetic rat heart, although this was not demonstrated in the present study. Alternatively, the 45 kDa polypeptide may be another unknown polypeptide reacting with anti-acyl-CoA oxidase and present in rat heart but not in the liver. The 23 kDa subunit found in the diabetic rat heart was somewhat smaller than that in the normal rat liver and migrated to the same position as the corresponding band in the normal heart. The same slight difference was shown previously in the starved rat heart (Yokota and Asayama 1992). In the present study, immunostaining for both catalase and acyl-CoA oxidase revealed that an almost twofold myocardial peroxisomal proliferation occurred in the diabetic rat heart, and that protein A-gold labeling in peroxisomes for both enzymes increased in a parallel manner. We have previously demonstrated similar changes in hearts of starved rats (Yokota and Asayama 1990, 1992). In the diabetic state, impaired glucose utilization due to insulin deficiency is reported to render tissues more dependent on lipid oxidation for energy metabolism (Fritz 1961) and mitochondrial fl-oxidation is known to be activated in diabetic tissues (Debeer and Mannaerts 1983), although there is no overall increase in mitochondrial oxidative metabolism in the heart (Asayama et al. 1989). The heart in streptozotocin-diabetic rats is known to oxidize less glucose and more fatty acids than that of non-diabetic controls (Roesen and Reinauer 1984). It has been postulated that the peroxisomal fl-oxidation system plays a permissive role for the mitochondrial system (Berry et al. 1983). The peroxisomes produce acetylCoA and fatty acids with chain lengths shorter than C18, that is independent of mitochondrial oxidative metabolism, and supply these components to mitochondria for oxidation. There is also the suggestion that the peroxisomal fl-oxidation system takes over from the mitochondrial system when the latter is saturated (Brady and Hoppel 1983). Thus, both systems appear to work in concert for fatty acid oxidation, and the activation of

48 myocardial acyl-CoA oxidase activity observed here may reflect the overall increase in lipid oxidation in the diabetic heart. During prolonged fasting, a shortage o f glucose resuits in hypoinsulinism and the tissues become more dependent on endogenous fatty acids released from fat tissue as an energy source. Mitochondrial oxidative metabolism is suppressed by prolonged fasting (Asayama et al. 1989) and it is conceivable that it becomes saturated and its fl-oxidation function taken over by the peroxisomal system as postulated in the diabetic state. This would explain the similar ultrastructural changes observed here in the diabetic rat heart and in the previous series in the starved animals (Yokota and Asayama 1990, 1992). The present results have provided solid evidence that a parallel increase in acyl-CoA oxidase and catalase activities occur in diabetic rat heart. Erucic acid (Norseth and Thomassen 1983) and ciprofibrate (Nemali et al. 1988) are known to be specific inducers of myocardial acyl-CoA oxidase activity. These substances are reported to increase acyl-CoA oxidase more markedly than catalase. Thus, the mechanism by which increased acyl-CoA oxidase activity is induced in the starved and diabetic rat heart appears to be different from that occurring as a response to these chemicals. The elucidation of this difference will require further study.

Acknowledgements. We wish to acknowledge the technical assistance of Mrs. H. Horiuchi and Mr. Y. Futumata. This study was supported in part by Grants-in-aid 03833013 (to S.Y.) and 63570431 (to K.A.) from the Ministry of Education, Science and Culture of Japan.

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