Carbohydrates, lipids and lipoproteins and islet ... - Springer Link

J. Biosci., Vol. 7, Numbers 3 & 4, June 1985, pp. 387-397. © Printed in India.

Carbohydrates, lipids and lipoproteins and islet changes in diabetes with superimposed myocardial infarction S. D. BHATT†, P. S. BORA and L. M. SRIVASTAVA* Department of Zoology, Kumaun University Campus, Almora 263601, India * Department of Biochemistry, All India Institute of Medical Sciences, New Delhi 110029, India MS received 11 October 1984; revised 23 January 1984 Abstract. Short-term metabolic and concomitant morphologic effects of streptozotocin diabetes on isoproterenol-induced myocardial infarction was studied in Wistar rats, Of particular significance was the observation that myocardial infarction in concert with diabetes brought about a distinctive exacerbation of the severity and complexity of the histopathological lesions. Of all the biochemical parameters, serum glucose and free fatty acids registered maximum elevation and serum lactate and cardiac glycogen levels a maximum reduction. Among the lipoproteins, an inverse relationship was found between high density lipoproteins and low density and very low density lipoproteins; while high density lipoproteins, ratio of high density lipoprotein to low density lipoprotein and the percentage of high density lipoprotein were decreased, there was a significant increase in low density lipoprotein concentration and percentage values of low density and very low density lipoproteins. In diabetes, the Β cell of the endocrine pancreas depicted selective necrosis. Loss of insulin granules and wide-spread necrobiosis of cellular elements of the pancreatic islets were observed, respectively, in myocardial infarction and in diabetes plus myocardial infarction combinations. Pathological evidence of chemical-induced mild toxicity was present in the exocrine parenchyma. Mitotic features and the presence of centroacinar cells in the damaged Langerhans’ islets supposedly formed the basis of regeneration of the tissue in diabetes, with or without vascular complications. Keywords. Experimental diabetes; myocardial infarction; pathophysiologic relationship.

Introduction The multiple systemic disturbances in diabetes result from defective utilization of glucose by cells and the extensive use of alternate energy-generating metabolic pathways. Further, several considerations explain the well recognized association of diabetes with atherosclerosis and myocardial infarction (MI) (Opie et al., 1979; Bajaj, 1984). Experimentally, the changes in hormonal and metabolite levels, enzyme profiles and myocardial histopathology brought about by the administration of isoproterenol, a potent β -adrenergic stimulating agent, are known to mimic those observed in humans † To whom correspondence should be addressed. Abbreviations used: AF, Aldehyde fuchsin; CAHP, chromalum haematoxylin phloxine; FFA, free fatty acids; HDL, high density lipoproteins; Η and Ε, haematoxylin and eosine; ISO, isoproterenol; LDL, low density lipoproteins; LH, lead haematoxylin; MI, myocardial infarction; PTAH, phosphotungstic acid haematoxylin; SZ, streptozotocin; TG, triglycerides; VLDL, very low density lipoproteins.

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during massive myocardial ischaemia (Wexler, 1975; Bora, 1984). Though considerable evidence to justify the β-cytotoxic property of the diabetogenic drugs such as streptozotocin has accumulated (Fox et al., 1978; Srivastava et al., 1982; Tancrede et al., 1983), virtually no information in respect to islet cell changes as the basis for understanding biochemical and morphologic patterns in diabetes with vascular disease is available thus far. The study of cholesterol metabolism, until quite recently, has been neglected perhaps because the more common and obvious abnormality of plasma lipids in diabetes is hypertriglyceridemia (Schnatz et al., 1972; Christopher and Young, 1981). In addition to their function in the transport of the nonpolar lipids, the lipoproteins have recently been shown to impart an important metabolic function (Brown et al., 1981). Of the four major categories of lipoproteins separable on the basis of their flotation at densities ranging between 1·063 and 1·21 g/ml, the triglyceride-rich very low density lipoproteins (VLDL) are known to be associated with lipolysis, loss of TG, and conversion to a smaller cholesterol-rich lipoprotein class – the low density lipoprotein (LDL). Another class of lipoprotein, the smallest of the plasma lipoproteins consisting approximately 50% protein by mass, the high density lipoproteins (HDL), is known not only to exchange certain proteins and phospholipids with VLDL but also favour the conversion of VLDL to LDL (Tall and Small, 1978). Following the epidemiological demonstration that the HDL and LDL levels are associated, negatively or positively, with risk of clinical sequelae of atherosclerosis (Wolinsky, 1980) interest in the study of interrelationships within the circulating lipoprotein classes has markedly increased. The present study was intended to obtain an insight into some of the biochemical and histopathological alterations in experimental diabetes with superimposed myocardial infarction. Materials and methods Adult Wistar rats of either sex with an average weight of 195 g (150–240 g) used in the present study were obtained from All India Institute of Medical Sciences’s animal facility. The animals were maintained under controlled conditions of ambient temperature (25 ± 0·5°C) and photoperiod (13 h light: 11 h dark) and were fed on chow pellets (Hindustan Lever) and water ad libitum. The rats were arranged in four groups of 5–7 individuals each and treated as follows: Group I: Group II:

Rats administered vehicle solutions served as controls. Rats received a single dose of streptozotocin (SZ: Upjohn, Kalamazoo, Michigan, USA) (85 mg/kg body wt) intraperitonially in phosphatecitrate buffer (pH 4·0). Group III: Rats were treated subcutaneously (s.c.) with isoproterenol (ISO: CIPLA, Bombay, India) (85 mg/kg body wt) for 2 days at 24 h interval. Group IV: 1 h after SZ treatment as in group II, the rats received 2 s.c. injections, spaced 24 h apart, of ISO at the same dose level as in group III. The animals were sacrificed using light anasthesia 48 h after the final treatment. Blood collected at autopsy was subjected to various estimations, i.e., glucose (Feteris,

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1965), lactate (Hohorst, 1974), triglyceride (Schettler and Nussel, 1975), cholesterol (Chiamori and Henry, 1959), and FFA (Novak, 1965). The method of Montgomery (1957) was employed for the analysis of cardiac glycogen. The lipoprotein cholesterol sub-fractions in the serum, viz, HDL, LDL, VLDL were estimated by precipitation with sodium phosphotungstate-magnesium chloride reagent and sodium dodecyle sulphate (Lopez-Virella et al., 1977). Based on the cholesterol content, the HDL assay was performed by the technique described by Burstein et al. (1970), using polyanions. For the estimation of LDL, VLDL was precipitated first by the addition of 10 % sodium dodecyle sulphate in 0·15 Μ sodium chloride solution. After centrifugation, the HDL and LDL in the subnatant were estimated. The differences in the LDL and VLDL concentrations and the total cholesterol values were calculated. Further, in order to determine the percentage values of HDL, LDL and VLDL, the gels obtained by the method of Christopher et al. (1971) were scanned in Gilford scanner at the absorbance range of 580–640 nm. In order to observe the nuclear and cytoplasmic changes in the various cell types in the pancreatic islets and the exocrine acini after the administration of the drugs used in the present study, the serially-cut 4–6 μm thick sections of the paraffin-embedded and Bouin's or Helly’s -fixed tissue were subjected to differential haematoxylin staining procedures such as haematoxylin and eosine (H and E), chromalum haematoxylin phloxine (CAHP), lead haematoxylin (LH), phosphotungstic acid haematoxylin (PTAH) and to aldehyde fuchsin (AF) using different counterstains. Student's ‘t’ test was used for comparisions between the metabolic data of control and experimental animals. Results General observations Diabetic rats, in general., showed classical symptoms of overt diabetes, with signs such as polydipsia, polyphagia and polyurea. Rats receiving ISO alone were beset with, within minutes, extremely rapid respiration. The severity of the symptoms became prominent in D plus MI rats but for no mortality. Blood chemistry Carbohydrates and lipids: SZ treatment caused significant elevation in serum glucose level (P < 0·001) (table 1). The MI rats while showing only a meagre hyperglycemic response, MI in concert with diabetes brought about significant worsening of the hyperglycemia with the sugar level increased markedly. The lactate in diabetes was slightly elevated, remained unchanged in MI while in D-ΜΙ combination, a highly significant decrease (P < 0·001) was evident. The cardiac glycogen reached levels of equal significance (P < 0·001) in all the experimental groups; howbeit, in contradistinction to the rising trend in diabetes, the glycogen levels in the other two groups showed a decline. As expected, the induction of diabetes led to a significant hypertriglyceridemia (Ρ < 0·001). An exacerbation of the situation was clearly evident in rats subjected to the B— 11

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Table 1. Blood and tissue metabolites in different groups of rats.

NS: Nonsignificant Values are mean ± S.D. of 5-7 observations.

combined treatments of SZ and ISO, than to ISO alone. A similar response was exhibited by FFA whose levels increased strikingly in the combined treatment group. However, the magnitude of hypercholesterolemia was similar in diabetic and D plus MI rats (P < 0·001) while in the MI animals alone, a less significant increase (P < 0·01) was observed in the serum cholesterol. Lipoproteins HDL and LDL concentrations: The induction of diabetes did not alter HDL content (table 2). The SZ-ISO combination dampened the depression in HDL levels brought about significantly (P < 0·001) by the treatment of ISO. In contrast to HDL, the LDL rose significantly in all the experimental groups with the values in combined treatment being maximum followed, in decreasing chronological order, by MI and diabetes. HDL/LDL ratio: The HDL to LDL ratio in MI and in D plus MI groups demonstrated a fall of equal significance (P < 0·001); in diabetes alone, the reduction in the ratio was less significant (table 2). Table 2.

Lipoprotein cholesterol sub-fractions in rats under different experimental conditions.

Designations the same as in table 1.


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Percentage values of HDL, LDL and VLDL: While diabetes was not associated with abnormal HDL, its percentage value in MI was significantly lowered (P < 0·001). The combined treatment led to a slight recuperation. The values of LDL displayed an inverse relationship with H D L, i. e., a decrease and. an increase respectively in diabetes (P < 0·01) and MI (P < 0·005). Further, diabetes invoked a supernormal increase (P < 0·01) in the percentage of VLDL while in other groups the response was found insignificant (table 2). Light microscopic histopathology With their density being greatest in the splenic portion, the pancreatic islets from the control rats comprised a homogeneously normal Β cell configuration (figure 1). The extent of Β cell necrosis varied in different islets from almost total to no damage at all in one and the same pancreas. In general., the A and D cells and the acinar parenchyma were not affected by the drugs. In diabetes, the pancreatic islets were predominately devoid of active Β cells in the central parts of the islets and the peripherily deployed A and D cells did not show any change (figure 2). Such islets also depicted cytoplasmic vacuolation and lymphocytic infiltration. In MI, diminution of AF + ve insulin granules and indistinctness of the cellular borders were seen in some Β cells (figure 3). The superimposition of MI in diabetes caused a definite aggravation of the severity of histopathological lesions. In some islets of such rats there was no trace of survival of any of the cellular elements with the cytoplasm appearing amorphous (figure 4). In other islets, the Β cell became necrotic with some remaining cellular ‘debris’ dispersed throughout the hyalinized islets (figure 5). Presence of large oedematous spaces in the exocrine pancreata (figure 6) was noticed in majority of the D plus MI group of rats. Mitotic features in the islets of rats treated with SZ, alone or in combination with ISO, were seen rather seldom (figure 7). Further, signs of transformation of the centroacinar cells into the islet tissue were occassionally noticed (figure 8). Discussion The most striking feature of the present study is that the experimental superimposition of myocardial infarction in diabetes elicited distinctive exacerbation of the histophysiologic lesions which mimic clinical conditions such as atherosclerosis and acute and massive myocardial infarction. Further to this, the data give ample evidence to indicate that alterations in lipid components may play an important role in pathogenesis of the cardiac abnormalities seen in diabetic patients with ischaemic heart disease. The importance of lipid abnormalities in producing most of the clinical manifesttations of ischaemic damage to the myocardium has recently been reviewed (Katz and Messineo, 1981; Bajaj, 1984). The D plus MI combination in rats was associated with an evident reduction in TG and an unusual elevation of glucose and FFA. Animals with myocardial infarct alone showed changes similar to diabetes but for the relative efficacy of the two drugs, i.e., the SZ-induced metabolic alterations were more marked than

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those observed after ISO administration. These findings give substantial evidence to suggest that the combination of diabetes with MI leads to special conditions of lipid metabolism whereby circulating levels of FFA are elevated at the expense of TG. This could possibly happen through a number of mechanisms most of which are initiated by the exogeneously administered catecholamine (Wexler, 1975). In common, with the suggestions of Katz and Messineo (1981), selective depletion of cardiac glycogen by FFA might further prove to be of some significance to the pathogenesis of the cardiac damage, in view of the protective role of glycogen in the ischaemic heart. Results obtained in the present study are in apparent similarity with those reported by Miller and Miller (1975) and Gordon et al. (1977) in that considerable shifts occur in the lipoprotein levels of the diabetic animals. Ban-On and Eisenberg (1978) reported an interesting reciprocal relationship between plasma TG levels and HDL removal rates in humans. The HDL levels in the present study were not much altered while the percentage value of the circulating VLDL-TG increased significantly in diabetes. The cause of such discrepant results is difficult to interpret. These findings probably point to the property of HDL being held back with VLDL or with the most rapidly catabolisible fat-containing ‘remnant’ apoprotein particles—the chylomicrons (Wolinsky, 1980). These considerations favour the argument that the metabolic events leading to higher HDL in diabetes may be different and related more to increased synthesis rather than decreased catabolism (Tall and Small, 1978). The mechanism by which the relationships between the various forms of lipoproteins influence the development of vascular complications in experimental diabetes remain unidentified. The rats showed an avid response to LDL concentrations when subjected to the combined treatment. An inverse relationship between HDL and LDL levels suggests that the two lipoprotein classes are tied to a common enzyme, possibly a lipoprotein lipase (Tall and Small, 1978; Wolinsky, 1980). The data on HDL/LDL ratio support our contention that, rather than the absolute levels of HDL cholesterol, the ratios are better estimates of the risk factor and, therefore, are more closely related to the established myocardial injury in diabetes. Recent studies in infrahuman mammals and man have documented that Β cell destruction or dysfunction may result from environmental influences, such as exposure to toxic chemicals introduced into the environment or used as drugs or food additives (Longnecker et al., 1982; Srivastava et al., 1982; Tancrede et al., 1983). In mice, low-dose SZ treatment was associated with a major loss of Β cell function and islet mass (Bonnevie-Nielson and Lernmark, 1981). In our studies, maximum damage to Β cell seen in the animals exposed to combined treatment, is discernible through ‘glucose potentiation’ effect of the drugs, as observed by Halter et al. (1979). Thus, a prominent hyperglycemia and its modulatory effects on the non-glucose secretagogues, such as the breakdown products of lipid metabolism in D plus MI combination might have demanded an extra release of insulin from the metabolic integrator, the Β cell, resulting in their degeneration. Abnormality in the exocrine pancreatic function has recently been demonstrated in diabetic dogs (Yasuda et al., 1982). Though lacking in substantial evidence, the gross exocrine damage in D-ΜΙ rat pancreas reflect alterations in the activity of amylase and lipase. Long-term studies to determine whether structural adaptations of the exocrine pancreata in rats are derived from metabolic derangement are in progress.

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Structural signs of regeneration in drug-induced progressive diabetes (Wilander, 1975) suggest that physiological restitution to normal precedes that of the histological return. In rats, neogenesis of the pancreatic tissue occurred through the transformation of the so-called centroacinar cells which resembled the ‘neuro-insular complexes’ of alloxan diabetes (Patent and Alfert, 1967). The occurrence of mitotic features and lymphocytic infiltration in the islets of the rats provided further evidence to indicate that augmented protein biosynthetic activity may occur and that the damaged cells may still retain the ability of compensatory proliferation. Reports of islet cell hyperplasia in SZ-diabetic rats (Fox et al., 1978) lend further credence to this interpretation. However, studies employing microdensitometry and immunocytochemistry are planned to further define the light microscopic changes. Acknowledgements The authors thank the Council of Scientific and Industrial Research, New Delhi for financial support. The first author is also thankful to the University Grants Commission, New Delhi for providing facilities under its National Associateship Programme. References Bajaj, J. S. (1984) Diabetes mellitus in developing countries (New Delhi: Interprint), p. 444. Ban-On, H. and Eisenberg, S. (1978) Diabetologia, 14, 65. Bonnevie-Nielsen, V. and Lernmark, A. (1981) Acta Biol. Med. Germ., 40, 77. Bora, P. S. (1984) Diabetes mellitus in rats: a histophysiologic approach to myocardial dysfunction and ischaemia, Ph. D. Thesis, Kumaun University, Nainital. Brown, M/S., Kovanon, P. T. and Goldstein, J. L. (1981) Science, 212, 628. Burstein, M., Scholnuck, H. R. and Morfin, R. (1970) J. Lipid Res., 11, 583. Christopher, D. S. and Young, N. L. (1981) Diabetes, 30 (suppl.), 76. Christopher, S. F., Lowell, B. F. and Patrica, S. C. (1971) Clin. Chem., 17, 111. Chiamori, N. and Henry, R. J. (1959) Am. J. Clin. Pathol., 13, 305. Feteris, W. A. (1965) Am. J. Med. Tech., 31, 17. Fox, C, Willey, K. P., Doyle, D., Polak, J. Μ. and Sonksen, P. H. (1978) Diabetologia, 14, 232. Gordon, T., Castelli, W. P., Hjortland, M. G, Kannel, W. B. and Bawber, T. R. (1977) Am. J. Med., 62, 707. Halter, J. Β., Graf, R. J. and Porte, D. Jr. (1979) J. Clin. Endocrinol. Metab., 48, 946. Hohorst, H, J. (1974) in Methods in Enzymatic Analysis, (ed. Η. U. Bergmeyer) (New York: Academic Press), Vol. II, p. 261. Katz, A. M. and Messineo, F. C. (1981) Circ. Res., 48, 1. Longnecker, D. S. (1982) Am. J. Pathol., 107, 103. Lopez-Virella, M. C, Stone, P., Ellis, S. and Colwell, J. A. (1977) Clin. Chem., 23, 882. Miller, G. J. and Miller, Ν. Ε. (1975) Lancet, 1, 16. Montgomery, R. (1957) Arch. Biochem. Biophys., 67, 378. Novak, M. (1965) J. Lipid Res., 6, 431. Opie, L. H., Tansey, M. J. and Kennelly, B. M. (1979) S. A. Med. J., 56, 207. Patent, G. J. and Alfert, M. A. (1967) Acta Anat., 66, 481. Schettler, G. and Nussel, E. (1975) Arbeitsmed. Sozialmed. Praventivmed., 10, 25. Schnatz, J. D., Formaniak, J. M. and Chlouverakis, C. (1972) Diabetologia, 8, 125. Srivastava, L. M., Bora, P. S. and Bhatt, S. D. (1982) Trends Pharmacol. Sci., 3, 376. Tall, A. R. and Small, D. N. (1978) N Engl. J. Med., 299, 1232.


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Tancrede, G., Rousseau-Migneron, S. and Nadeau, A. (1983) Br. J. Exp. Pathol., 64, 117. Wexler, Β. C. (1975) Metabolism, 24, 1321. Wilander, Κ. (1975) Horm. Metab. Res., 7, 15. Wolinsky, H. (1980) Circ. Res., 47, 301. Yasuda, Η., Kakudo, Κ., Morino, Η., Kitamura, Η., Harano, Υ. and Shigeta, Υ. (1982)Acta Pathol. Jpn., 32, 783.