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Effects of myo-inositol and, or triiodothyronine (T₃) treatment on cardiac dysfunction and elevated myocardial… Xiang, Hong 1987

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E F F E C T S O F M Y O I N O S I T O L A N D , O R T R I I O D O T H Y R O N I N E (Tg) T R E A T M E N T O N C A R D I A C D Y S F U N C T I O N A N D E L E V A T E D M Y O C A R D I A L LIPID L E V E L S IN S T Z - D I A B E T I C R A T S by Hong Xiang B .Med . , Beijing Medica l Universi ty , P . R . C . , 1984 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E STUDIES Divis ion of Pharmacology and Toxicology of the Facul ty of Pharmaceut ical Sciences We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A September, 1987 © Xiang Hong, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i A B S T R A C T A number of experimental studies have implied a link between diabetes-induced l ipid accumulation in the myocardium and the development of cardiomyopathy. Since diabetics excrete large amounts of myo-inositol which is a l ipotropic agent, this study was undertaken to investigate the effects of myo-inositol on the elevated myocardial l ip id levels and the depressed cardiac performance of diabetic rats. Diabetes was induced in female Wistar rats (190-215 g) wi th streptozotocin (STZ) (55 mg/kg, i .v.) . Three days after diabetes induction, myo-inositol was administered in the drinking water (2.5 g/kg/day) for a 8 week period. Untreated diabetics exhibited a loss of body weight, hyperglycemia, hypoinsulinemia and hypothyroidism. These effects were not altered after myo-inositol treatment. STZ-diabetes also produced a significant elevation of plasma and myocardial t r iacylg lycerol , cholesterol and phospholipid. Myo-inos i to l treatment decreased these l ip id levels. In addition, hearts from diabetic animals had a decreased abi l i ty to develop left ventricular developed pressure (LVDP) and both the rate of pressure rise (+dP/dt) and the rate of pressure decline (-dP/dt) were also reduced. Hearts from myo-inositol-treated diabetic animals showed a part ial but definite improvement of cardiac function. A s diabetes-induced hypothyroidism was not altered after myo-inositol supplementation, a combination treatment of both myo-inositol (2.5 g/kg/day, p.o. daily) and T^ (30 ug/kg/day, s.c. daily) was then undertaken to determine whether heart function of diabetic rats could be further improved. STZ-diabet ic rats were character ized by a loss of body weight, hyperglycemia and hypoinsulinemia; none of which were altered by either T„ or myo-inositol plus i i i Tg treatment. treatment normalized the thyroid state of diabetic animals as shown by Tahi l iani and M c N e i l l (1984). However, plasma and myocardial t r iacylg lycero l , cholesterol and phospholipid levels of diabetic rats either remained elevated or were further increased wi th or myo-inositol plus treatment. In addition, treatment alone did not prevent cardiac dysfunction in diabetic rats. There was, however, some improvement in heart function in the groups treated wi th both myo-inositol and T^, but the improvement was not as pronounced as wi th myo-inositol treatment alone. John H . M c N e i l l , Ph .D . Thesis Supervisor iv T A B L E O F C O N T E N T S P A G E A B S T R A C T i i LIST O F T A B L E S v i LIST O F F I G U R E S v i i LIST O F A B B R E V I A T I O N S ix A C K N O W L E D G E M E N T S x i D E D I C A T I O N x i i I N T R O D U C T I O N 1 I. Overview of diabetes melli tus 1 II. Diabet ic cardiomyopathy 5 III. Diabetes and altered l ipid metabolism in the heart and adipose tissue 8 IV. Treatments employed to correct l ipid metabolism in diabetics 11 V . Myo-inosi to l and diabetes melli tus 13 VI . Thyroid hormones 17 SPECIFIC G O A L S O F T H E INVESTIGATION 20 M A T E R I A L S A N D M E T H O D S 22 I. Mater ia ls 22 1. Animals 22 2. Chemicals 22 3. Assay ki ts 22 P A G E II. Methods 23 1. Induction of experimental diabetes 23 2. Treatment protocols 23 (1) Myo-inosi to l preliminary study 23 (2) Myo-inosi tol treatment study 24 (3) Myo-inosi tol and triiodothyronine (Tg) treatment study 25 3. Isolated working heart perfusion 26 4. Measurement of plasma t r iacylglycerol , cholesterol and phospholipid levels 28 5. Determinat ion of myocardial t r iacylg lycerol , cholesterol and phospholipid levels 28 6. Pro te in assay 29 7. Blood analysis 29 8. S ta t i s t ica l analysis 30 R E S U L T S 31 DISCUSSION 85 C O N C L U S I O N S 102 R E F E R E N C E S 104 v i LIST O F T A B L E S P A G E I. Character is t ics of Type I and Type II diabetes melli tus 3 II. Effect of different concentrations of myo-inositol treatment on plasma glucose, plasma and myocardial lipids in 8 week diabetic rats 41 Na. Effect of different concentrations of myo-inositol treatment on fluid intake and estimated inositol intake of 8 week diabetic rats 42 III. General features of experimental rats used for myo-inositol treatment study 43 IV. F lu id and myo-inositol intake of experimental rats used for myo-inositol treatment study 44 V . Thyroid status of experimental rats used for myo-inositol treatment study 45 VI. General features of experimental rats used for myo-inositol and triiodothyronine treatment study 64 VII. F lu id and myo-inositol intake of experimental rats used for myo-inositol and triiodothyronine treatment study 65 VIII. Thyroid status of experimental rats used for myo-inositol and triiodothyronine treatment study 66 v i i LIST O F F I G U R E S P A G E 1. Possible metabolic changes in diabetes 9 2. Myo-inosi tol metabolism cycle 14 3. Effec t of myo-inositol treatment on plasma tr iacylglycerols of control and STZ-diabet ic rats 46 4. Effects of myo-inositol treatment on plasma cholesterol of control and STZ-diabet ic rats 48 5. Effec t of myo-inositol treatment on plasma phospholipid of control and STZ-diabet ic rats 50 6. Effec t of myo-inositol treatment on myocardial t r iacylglycerols of control and STZ-diabet ic rats 52 7. Effec t of myo-inositol treatment on myocardial cholesterol of control and STZ-diabet ic rats 54 8. Effect of myo-inositol treatment on myocardial phospholipid of control and STZ-diabet ic rats 56 9. Effect of diabetes and myo-inositol treatment on left ventricular developed pressure of isolated perfused working hearts at various f i l l ing pressures 58 10. Effec t of diabetes and myo-inositol treatment on positive dP/dt of isolated perfused working hearts at various f i l l ing pressures 60 11. Effec t of diabetes and myo-inositol treatment on negative dP/dt of isolated perfused working hearts at various f i l l ing pressures 62 v i i i P A G E 12. Levels of t r iacylglycerols in plasma obtained from control and diabetic rats treated wi th myo-inositol and triiodothyronine (Tg) 67 13. Levels of cholesterol in plasma obtained from control and diabetic rats treated wi th myo-inositol and T 3 69 14. Levels of phospholipid in plasma obtained from control and diabetic rats treated wi th myo-inositol and T 3 71 15. Measurement of myocardial t r iacylglycerol levels in control and diabetic rats administered myo-inositol and 73 16. Measurement of myocardial cholesterol levels in control and diabetic rats administered myo-inositol and 75 17. Measurement of myocardial phospholipid levels in control and diabetic rats administered myo-inositol and 77 18. Ef fec t of 8 week myo-inositol and treatment in diabetic rats on diabetes-induced depression of L V D P 79 19. Effec t of 8 week myo-inositol and treatment in diabetic rats on diabetes-induced depression of +dP/dt 81 20. Effec t of 8 week myo-inositol and treatment in diabetic rats on diabetes-induced depression of -dP/dt 83 ix LIST O F A B B R E V I A T I O N S MI myo-inositol STZ streptozotocin L V D P left ventricular developed pressure +dP/dt rate of development of left ventricular pressure -dP/dt rate of decline of left ventricular pressure T 3 3,5,3'triiodothyronine T 4 3,5,3',5' tetraiodothyronine C O N untreated controls COI myo-inositol-treated controls C O T Tg-treated controls CIT myo-inositol-plus-Tg-treated controls DIA untreated diabetics DII myo-inositol-treated diabetics DIT Tg-treated diabetics DIIT myo-inositol-plus-Tg-treated diabetics F F A free fatty acid L C A C long-chain acylcarnit ine c m H 2 0 centimeter of water g gram K ki lo kg kilogram L l i ter m m i l l i X mg mil l igram m L mi l l i l i t r e mmHg mi l l imeter of mercury mm Hg/s mi l l imeter of mercury per second ng nanogram nmole nanomole U unit u micro SR sarcoplasmic ret iculum I P 3 inositol-l ,4,5-triphosphate x i A C K N O W L E D G E M E N T I wish to express my deepest gratitude to my supervisor Dr . John H . M c N e i l l for moral support, knowledgeable direction and understanding which have enabled me to complete this work. I would also l ike to express my sincere thanks to Dr . Clayton Heyliger , M r . Brian Rodrigues and M r . A l l a n Prakash who have encouraged me and given me inspiration and invaluable support in this effort. The suggestions and scient i f ic input from the members of the supervisory committee (Dr. J . Diamond, Dr . K . MacLeod , Dr . F .S . Abbott) are gratefully acknowledged. A special thanks to Ms. Judy Wyne for typing this thesis. The financial support of the B . C . Heart Foundation is gratefully acknowledged. A l s o my thanks to a l l my colleagues and associates for their enthusiasm and help. Xiang Hong x i i D E D I C A T I O N To my parents and brothers, for their love and a l l they have done. 1 I N T R O D U C T I O N I. O V E R V I E W O F D I A B E T E S M E L L I T U S . Diabetes melli tus is a very old disease of mankind. A s far back as the Ebers papyrus (about 1500 B . C . ) , the phenomenon of polyuria was observed in Egypt and "honey urine" was noted by Sushrutha in India in 400 B . C . The first good c l in ica l description of the disease was provided by Celsus (30 B . C . to A . D . 50), and the term "diabetes", which is Ionic Greek and means "to run through a siphon", was first introduced by another Roman physician, Aretaeus ( A . D . 30 to A . D . 90). Descriptions of a disease (such as "malady of thirst") suggesting diabetes were made by Chinese and Japanese in these early centuries. A n Arab physician Avicenna in about 1000 A . D . gave a very good description of diabetes as wel l as some of its complications such as gangrene (for reviews, see Best, 1960 and Marble , 1971). During the 17th century, the observations of Wil l i s marked the beginning of the "diagnostic period" in the history of diabetes. He observed that the urine of diabetes was "wonderfully sweet" and Dobson (1775) stated that the sweetness was due to sugar. These led Cul len (1710-1790) to add "melli tus" to "diabetes" to separate diabetes mellitus from diabetes insipidus. Rol lo (1797) started the period of diabetes treatment by prescribing a dietary regimen (low carbohydrate, high protein and fat). In 1869 Langerhans discovered the islet In pancreatic tissue which was named In his honour by Laguesse. The experimental period was ini t iated by Mering and Minkowski (1889) who produced diabetes in dogs by total pancreatectomy and first connected the diabetes wi th the pancreas. The greatest step forward came in 1921 when Banting 2 and Best succeeded in extract ing from the islets of Langerhans a substance wi th hypoglycemic properties, which was later given the name "insulin". Subsequently oral hypoglycemic agents were introduced in 1955. It was not until 1965 that Gepts showed that major abnormalities occurred in beta cells in the islets of Langerhans in Type I diabetic patients. The avai labi l i ty of insulin radioimmunoassay (Yalow and Berson, 1960) and further research in the last two or three decades made it possible to differentiate Type I and Type II diabetes melli tus and to give the current definition of diabetes melli tus (for reviews, see Best, 1960 and Marble , 1971). Diabetes melli tus is characterized by an absolute or relat ive deficiency of insulin and subsequent disorders in the metabolism of carbohydrate, fat and protein, as wel l as structural and functional abnormalities in a variety of tissues. There are two common types of diabetes mellitus: Type I (insulin-dependent diabetes melli tus, IDDM), and type II (non-insulin-dependent diabetes melli tus, NIDDM). Three other types are also included in a new classif icat ion system adopted in 1979, which are: impaired glucose tolerance, gestational diabetes, and diabetes mellitus associated wi th other conditions or syndromes. Table 1 summarizes two common types of diabetes melli tus, their etiology, epidemiology and c l in ica l features. In type I, genetics, viruses and/or autoimmune factors have been suggested to be involved in the pathogenesis of the disease. Genet ical ly , type I has been shown to be frequently associated wi th cer tain human leukocyte antigen (HLA) genes (Cudworth and Woodrow, 3 T A B L E I. C H A R A C T E R I S T I C S O F T Y P E I A N D T Y P E II D I A B E T E S M E L L I T U S Type I (IDDM) Type II (NIDDM) Etiology genetic, v i ra l or auto-immune abnormalities - a decreased affinity of the receptors for insulin or a "down regulation" at insulin receptors, genetic or v i ra l factors may be involved. Epidemiology C l i n i c a l features 5%-10% of al l diabetics any age but usually <30 non-obese rapid onset ketosis common - 90%-95% of al l diabetics - Any age but usually >40 - 80% of type II obese - slow onset - ketosis rare except in stress or infection l i t t l e or no endogenous insulin varying amounts of endogenous insulin, often higher than normal levels. 4 1975; Nerup, 1978). A diabetogenic variant of Coxsackie B4 virus has been identified to be able to damage the pancreatic beta cells (Yoon et a l . , 1979). Ci rcu la t ing islet ce l l antibodies capable of destroying beta cells in the pancreatic islets are found in type I diabetics (Lernmark et a l . , 1978). In type II, either a "post receptor defect", a decreased affini ty of the receptors for insulin (Bar et a l . , 1978), or a "down regulation" of the insulin receptors as a consequence of increased levels of insulin (Kahn et a l . , 1977) are proposed to be possible factors in the pathogenesis of the disease. Regardless of the etiology of diabetes mellitus, absolute or relat ive deficiency of insulin can result in abnormal metabolism of carbohydrate, protein and fat, leading to a series of c l in ica l symptoms. Glucose uptake into muscle and adipose tissue, and glycogenesis in l iver and muscle are reduced. A t the same t ime, glycogenolysis and gluconeogenesis increase. A l l of these changes result in markedly elevated blood glucose levels and glucosuria occurs when the blood glucose levels exceed the renal threshold for glucose. Increased amounts of glucose in the urine causes osmotic diuresis giving rise to the symptom of polyuria, followed by polydipsia and polyphagia due to metabolic imbalance. Synthesis of fatty acids and t r iacylglycerol in l iver and adipose tissue decreases and lipolysis in adipose tissue increases, resulting in hyperlipidemia. Insulin deficiency also reduces protein synthesis and weight loss follows (Ganong, 1985). A large number of secondary complications are also associated with diabetes mell i tus. A c u t e complications include ketoacidosis (McGarry and Foster, 1977) and non-ketotic diabetic coma (Arief f and C a r o l l , 1972). Much 5 more common now are chronic complications including microangiopathy which is probably responsible for diabetic retinopathy (Davis, 1974) and nephropathy (Balodimos, 1971); macroangiopathy which is perhaps related to atherosclerosis, coronary artery disease, myocardial infarct ion, hypertension and stroke (Renold et a l . , 1978); diabetic neuropathy (Sibley, 1982) and an increased incidence of urinary tract infections (Kass, 1956; Ooi et a l . , 1974). Diabet ic cardiomyopathy is another complicat ion whose importance has become recognized in recent years (Fein et a l . , 1980). Most current evidence indicates that there is a common mechanism which induces these diverse complications of diabetes. It is suggested that insulin deficiency, hyperglycemia and other metabolic derangements play a central role in the pathogenesis of diabetic complications (Brownlee and Cerami , 1981; Ger ich , 1986; Winegrad, 1987). II. D I A B E T I C C A R D I O M Y O P A T H Y . One of the major causes of morbidity and mortal i ty in diabetics is cardiovascular disease, which accounts for about 80% of al l diabetic deaths (Kannel, 1978). Besides coronary artery disease as a common complicat ion, c l in ica l (Ahmed et a l . , 1975; Regan et a l . , 1977), pathological (Hamby et a l . , 1974; Regan et a l . , 1977), epidemiological (Kannel et a l . , 1974), and experimental data (Regan et a l . , 1974; Fe in et a l . , 1980) have strongly suggested that diabetes mellitus is associated wi th the development of a cardiomyopathy - diabetic cardiomyopathy. Diabet ic cardiomyopathy is character ized as myocardial failure independent of atherosclerotic coronary 6 artery disease, valvular disease or hypertension. C l i n i c a l l y , and pathologically, Hamby et a l . (1974) reported evidence of myocardial dysfunction in the absence of large coronary disease in 16 diabetic patients and suggested that diabetic cardiomyopathy might be due to pathological changes in small coronary vessels. A shorter left ventricular ejection t ime, a longer pre-ejection period and a higher rat io of pre-ejection period/left ventricular ejection t ime ( P E P / L V E T ) were shown in diabetic subjects without myocardial ischemia or other cardiovascular disease (Ahmed et a l . , 1975). Post-mortem examination by Regan et a l . (1977) showed nine out of eleven diabetics had no significant coronary disease, although six had bad heart failure at death. Shapiro et a l . (1981) also observed an increased P E P / L V E T rat io in diabetic patients. Furthermore, the Framingham study (Kannel et a l . , 1974) demonstrated that diabetic men had a two-fold increased risk and diabetic women a f ive-fold increased risk of congestive heart failure even without coronary or rheumatic heart disease present. In experimental animal studies, a primary myocardial abnormality in diabetes was first shown in dog, a species known to have a low incidence of spontaneous arterial disease, by Regan and co-workers (1974). The end-diastolic volume and the stroke volume response were significantly less in eleven-month alloxan-diabetic dogs associated with a two-fold greater end-diastolic pressure increment, suggesting a decreased ventricular compliance in the absence of coronary artery disease. Similar abnormalities in ventricular function were reported in 18-month diabetic rhesus monkeys (Haider, et a l . , 1978). Studies on the effects of the diabetic state on myocardial mechanics (Fein et a l . , 1980) showed a slowing of relaxation as well as 7 a depression of shortening ve loc i ty in left ventr icular papillary muscles from severely diabetic rats. Using the isolated working heart preparation, a decreased rate of contraction and relaxation at higher f i l l ing pressures (Penpargkul et a l . 1980; Vadlamudi et a l . , 1982) and afterload (Ingebretson et a l . , 1980) have been reported. More recently, studies on papillary muscles from alloxan-diabetic rabbits demonstrated a markedly prolonged duration of isometric and isotonic contraction and relaxation (Fein et a l . 1985). The mechanisms involved in diabetic cardiomyopathy may include alterations in subcellular membranes and in a number of enzyme systems. These include depressed C a ^ + st imulated ATPase ac t iv i ty in sarcoplasmic ret iculum (SR) (Lopaschuk et a l . , 1983*), lower oxidative phosphorylation capaci ty and M g 2 + - A T P a s e ac t iv i ty in mitochondria (Pierce and Dhal la , 1986), reduced N a + , K + - A T P a s e act ivi t ies in sarcolemma (SL) (Ku and Sellers, 1982; P ie rce and Dhal la , 1986). A l l of these changes result in a decreased capacity to transport C a ^ + and subsequent depressed membrane function. A n elevation in long chain acylcarnit ine ( L C A C ) levels is correlated with the depression in C a 2 + - A T P a s e in SR (Adam et a l . , 1978; Lopaschuk et a l . , 1983 1) and N a + , K + - A T P a s e in SL (Wood et a l . , 1977). A depression in myocardial myosin and actomyosin ATPase has also been shown (Dil lman, 1980; Malhotra et a l . , 1981), which may be due to changes in the myosin isozyme distribution 2+ 2+ (Dil lman, 1980). In addition, myofibr i l lar basal (Mg dependent) and C a st imulated ATPase act ivi t ies in diabetic rat hearts were reported to be lower as compared to controls (Pierce and Dhal la , 1981). Other studies have shown a correlat ion between elevated levels of c i rcula t ing and myocardial 8 fats and the development of diabetic cardiomyopathy (Rodrigues et a l . , 1985; Rodrigues et a l . , 1986; Heyliger et a l . , 1986). III. D I A B E T E S A N D A L T E R E D LIPID M E T A B O L I S M IN T H E H E A R T A N D A D I P O S E TISSUE. A number of experimental studies have implied a link between diabetes-induced metabolic changes and the development of cardiomyopathy (Regan et a l . , 1973; Shipp et a l . , 1973). One prominent metabolic change seen in diabetes is a decrease in glucose u t i l iza t ion by the heart due to lack of insulin. A s a result, the heart derives the energy required for its mechanical ac t iv i ty almost entirely from l ip id sources (Morgan et a l . , 1961; Randle et a l . , 1966). Numerous alterations in the energy substrate metabolism pathways of diabetic myocardium have been documented. Glucose u t i l iza t ion is inhibited secondary to a decreased glucose transport. The increase in c i t ra te levels due to increased fatty acid metabolism also decreases glycolysis by inhibiting phosphofructokinase (Randle et a l . , 1966). Increased fatty acid metabolism is associated with increased tissue levels of acyl C o A , acylcarnit ine and tr iacylglycerols (fig. 1) (Denton and Randle, 1967; Feuvray et a l . , 1979). Another biochemical abnormality in diabetes is a marked increase in adipose tissue lipolysis (McGarry and Foster, 1977). Tr iacylg lycero l lipase is the ra te- l imi t ing enzyme in adipose tissue lipolysis. It is an insulin-inhibited, cyc l i c AMP-dependent enzyme; while catecholamines, glucagon and a number of other hormones can stimulate this enzyme resulting in hydrolysis of t r iacylglycerol accompanied by enhanced release of free 9 F I G U R E 1 Possible metabolic changes in diabetes - arrow (T , i ) indicates diabetes-induced changes. MEMBRANE m E R MITOCHONDRIAL MEMBRANE 11 fatty acids (FFA) (Steinberg, 1972; Fa in , 1973). During insulin deficiency, t r iacylglycerol lipase is not inhibited and therefore increased F F A are produced. The fatty acids may diffuse out of the ce l l or they may be re-esterif ied into t r iacylglycerol within the adipocyte. The re-ester if icat ion process, however, requires alpha-glycerophosphate which is provided by insulin promoting the flux of glucose intracel lular ly . Thus, large quantities of F F A are released from the adipose tissue during insulin deficiency (Saudek and Eder, 1979). Diabetes is also associated with elevated plasma catecholamine levels (Paulson et a l . , 1980). Catecholamines bind to beta receptors of the adipocytes and increase c A M P which in turn leads to phosphorylation and act ivat ion of t r iacylglycerol lipase (William-Olsson et a l . , 1979). Hence, both lack of insulin and elevation of catecholamines contribute to the increased adipose tissue lipolysis in diabetes. IV. T R E A T M E N T S E M P L O Y E D TO A F F E C T LIPID M E T A B O L I S M IN D I A B E T I C S . Since altered l ipid metabolism in the diabetic myocardium may be involved in the diabetic cardiomyopathy, various attempts have been made to return l ip id metabolism to normal in diabetic animals. Insulin treatment (9 U/kg/day) has been shown to normalize cardiac SR levels of long-chain acylcarnit ines associated wi th a recovery of calcium transport ac t iv i ty . Heart function in diabetic rats treated with insulin was similar to control rat hearts (Lopaschuk et a l . , 1983 ). However, hypoglycemia remains a major c l in ica l problem wi th insulin treatment. A more direct approach is carnitine treatment. Carni t ine administration ( 3 g/kg/day) to diabetic rats effect ively 12 prevented the accumulation of L C A C within the myocardium and the depression of ca lc ium uptake in the SR. However, unlike insulin, carnit ine was total ly ineffective in improving cardiac function in diabetic rats (Lopaschuk et a l . , 1983 ). Another treatment that has been attempted in order to prevent l ip id alterations is that of methyl palmoxirate. Methy l palmoxirate, a fatty acid analog, has been shown to inhibit fatty acid metabolism due to irreversible inhibition of carnitine acyl transferase, thus forcing carbohydrate u t i l iza t ion (Pearce et a l . , 1979). However, methyl palmoxirate treatment (25 mg/kg/day) was unable to control diabetes-induced changes in plasma glucose, insulin, lipids as well as functional myocardial depression; even though elevations of L C A C and depression of ca lc ium uptake in SR of diabetic rat hearts could be prevented by the treatment (Tahiliani and M c N e i l l , 1985). Hydralazine, a vasodilator used for the treatment of hypertension and congestive heart failure, has been shown to have the unusual effect of lowering blood lipids (Perry and Schroeder, 1955; Deming et a l . , 1958). Hydralazine treatment in diabetic animals also successfuly prevented diabetes-induced hyperlipidemia. In addition, the functional depression seen in diabetic rat hearts was prevented by the treatment (Rodrigues et a l . , 1986). A s myocardial l ip id buildup has been suggested to be more closely related to the cardiac dysfunction associated with diabetes, further efforts have been made to control l ip id metabolism in the diabetic myocardium. Choline and methionine, both of which are l ipotropic agents (Young et a l . , 1956; Kuksis and Mookerjea, 1978), were reported to drast ical ly reduce myocardial t r iacylglycerol and cholesterol levels in diabetic rats (Heyliger 13 et a l . , 1986). In addition, a significant improvement in heart function in treated diabetics was seen relat ive to the untreated ones. V . M Y O - I N O S I T O L (INOSITOL) A N D D I A B E T E S M E L L I T U S . Myo-inosi tol is a dietary component as wel l as a precursor of cel lular phosphatidylinositol. During the normal process of metabolism, myo-inositol is absorbed across the intestinal mucosa by a specific, Na + -dependent transport system (Caspary and Crane, 1970), c i rculated freely in plasma, f i l tered by the renal glomerulus and reabsorbed by a high-affinity Na + -dependent carrier-mediated transport system in the renal brush border membrane (Greene and La t t imer , 1986). Myo-inosi tol is also synthesized in many cells from glucose 6-phosphate by a specific cyclase and phosphatase (Burton and Wells, 1974; Chen and Eisenberg, 1975; Mauck et a l . , 1980). In the kidney, inositol is degraded to glucuronic acid by myo-inositol oxygenase (Howard and Anderson, 1967; Clements and Diethelm, 1979), while the major pathway for inositol metabolism in other tissues is reversible incorporation into the phosphoinositides, a specific class of membrane phospholipids, as shown in figure 2 (Berridge, 1985). Research suggests that the interact ion of external agonists (e.g. alpha-1-adrenergic and muscarinic cholinergic agonists) with cell-surface receptors may stimulate phosphoinositide turnover in a variety of tissues and organ systems (Michel l , 1975; M i c h e l l , 1979) including the heart (Quist, 1982; Brown et a l . , 1985). Two important second messengers are then released, inositol-1,4,5- trisphosphate ( I P J which has been implicated in the release 14 F I G U R E 2. M Y O - I N O S I T O L M E T A B O L I S M C Y C L E EXTERNAL AGONISTS eg. ALPHA j-ADRENERGIC OR MUSCARINIC CHOLINERGIC AGONISTS PHOSPHOLIPASE C r INOSITOL TRIPHOSPHATE (IP3) t INOSITOL BISPHOSPHATE (IP2) 1 INOSITOL PHOSPHATE (IPj) MYO-INOSITOL T PHOSPHATIDYLINOSITOL 4,5 BISPHOSPHATE (PIP2) PHOSPHATIDYLINOSITOL 4-PHOSPHATE (PIP) 1 PHOSPHATIDYLINOSITOL (PI) 1 D1ACYLGLYCEROL (DG) PHOSPHATIDE ACID (PA) 15 of calcium from intracellular storage sites in many tissues (Streb et a l . , 1983; Berridge, 1984; Berridge and Irvine, 1984) and diacylglycerol (DG) which can act ivate protein kinase C and result in phosphorylating specific membrane proteins (Nishizuka, 1983; Nishizuka, 1984; Haslam and Davidson, 1984). There is some evidence that IP^ can mobil ize calcium in the heart (Hirata et a l . , 1984; Fabiato, 1986; Nosek et a l . , 1986), and it is proposed that IPg may be involved in exci tat ion-contract ion coupling in cardiac muscle (Poggioli et a l . , 1986). Myo-inosi to l was first shown to be a l ipotropic agent in various animal species in 1941 when Gavin and McHenry reported that inositol could prevent the development of the fatty l iver and the accumulation of cholesterol in l iver . Subsequently Do t t i et a l . (1951) reported that 0.5 g of inositol daily added to the high cholesterol diet el iminated the expected rise in blood cholesterol and phospholipid in rabbits. L ive r t r iacylglycerols were shown to accumulate in experimental animals under conditions of inositol deficiency (Hayashi et a l . , 1974). Research on the relationship between diabetes melli tus and myo-inositol metabolism goes back to 1858 when Vohl first discovered that human diabetics excreted large amounts of myo-inositol in their urine. This was confirmed by Freinkel et a l . (1960) and Pitkanen (1972). The inosituria in diabetics may result from the competi t ively inhibitory effect of hyperglycemia on renal tubular reabsorption (Clements and Diethelm, 1979; Hammerman et a l . , 1980) or the inhibitory effect of diabetes on the act iv i ty of inositol oxygenase, the enzyme responsible for metabolizing inositol in 16 the kidney (Whiting et a l . , 1979). Furthermore, Clements and Reynertson (1977) showed that a widespread intracel lular deficiency of myo-inositol was present in untreated diabetics. The deficiency was shown to select ively exist in some tissues in which glucose transport is not ra te- l imi t ing for its metabolism, such as peripheral nerve (Greene et a l . , 1975), retina (MacGregor et a l . , 1984), ar ter ial wal l (Morrison, 1984) and renal glomerulus (Beger-Mears et a l . , 1984). However, plasma inositol was significantly higher in uncontrolled diabetics which may be due to enhanced gastrointestinal absorption and impaired intracel lular transport, both of which may result from hyperglycemia as insulin treatment could correct these abnormalities (Clements and Reynertson, 1977). Myo-inosi tol has been used in the treatment of diabetics. Clements and Reynertson (1977) showed that a 3 —g oral load of inositol could significantly elevate plasma inositol concentrations in human subjects with diabetics showing a greater response. The administration of inositol , 0.5 g twice daily for two weeks, has been reported to be beneficial in the treatment of diabetic neuropathy (Greene et a l . , 1975; Salway et a l . , 1978) through increased N a + - K + - A T P a s e ac t iv i ty with resulting improvement of nerve conduction veloci ty (Green and La t t imer , 1983). The above observations suggest that myo-inositol may play a role in regulating l ipid metabolism and may be beneficial in the prevention and treatment of cardiomyopathy associated with diabetes melli tus. We therefore investigated the effect of myo-inositol on elevated plasma and myocardial lipids and depressed cardiac performance in diabetic rats. 17 VI . T H Y R O I D H O R M O N E S . Thyroid hormones, including thyroxine (T^) and 3,5,3'-triiodothyronine (Tg), are produced and secreted under st imulation of thyroid-stimulating hormone (TSH) from the anterior pituitary, which, in turn, is st imulated by thyrotropin-releasing hormone (TRH) from the hypothalamus. A negative feedback mechanism operates to decrease the release of T S H in the presence of high levels of T^ or T^ (Spaulding and Utiger , 1981). Thyroxine is degraded by monodeiodination to form either T^ (Pi t t -Rivers , 1955; Braverman, 1970) or 3,3' ,5'-triiodothyronine (reverse T^, rT^) (Flock, 1961; Gavin , 1977). T^ is biological ly more potent than T^, while rT^ has insignificant biological ac t iv i ty (Spaulding and Utiger , 1981). Diabetes mellitus is a metabolic disorder which is related to the alterations not only in insulin levels but also in levels of several other hormones (Johansen and Hansen, 1969; Lefebvre and Luyckx, 1979; Tzagournis, 1982) including thyroid hormones (Cooppan, 1982). Plasma thyroid hormones have been reported to be normal (Pittman et al , 1979 ,), sl ightly reduced (Naeije et a l . , 1978) or substantially reduced in uncontrolled diabetic patients (Saunders et a l . , 1978). Studies wi th animal models of chemically-induced diabetes have shown decreased plasma T^ and T^ levels in diabetic rats (Zaninovich et a l . , 1977; Boado et a l . , 1978). Extrathyroidal conversion of T^ to T^ in diabetic rats was reduced (Balsam et a l . , 1978; P i t tman et a l . , 1979 ), and was correlated wi th reduced act iv i ty of T ,-5'-deiodinase in the l iver of diabetic rats (Gavin et a l . , 1981). In addition, 18 deficient TSH secretion has been demonstrated in diabetic animals during a low iodine diet regime (Periscas and Jol in , 1977). Furthermore, Wilber et a l . (1981) found that diabetes mellitus is also associated with a reduction in c i rcula t ing T R H . Various suggestions regarding the mechanisms of these reductions have been proposed. Reduced production of from may be related to decreased glucose uptake and ut i l iza t ion (Saunders et a l . , 1978), as insulin therapy improved the conversion from to which correlated well wi th the normal level of blood glucose (Pit tman et a l . , 1979*). Changes in T S H secretion could result from the interference with the normal negative feedback mechanism, either by altering the relationship of free and protein bound circulat ing thyroid hormone due to the increased F F A displacing thyroid hormone from their protein binding (Liewendahl and Helenius, 1976), or by changing the u t i l iza t ion and disposal of the thyroid hormones by peripheral tissues. Moreover, diabetes may direct ly influence pituitary T S H secretion by some local ized metabolic effect on thyrotroph cel ls . This, is supported by the histological findings of Desclaux et a l . (1948), who observed degranulation of the acidophil cells and a decreased number of basophil cells in the pi tui tary of alloxan-diabetic rats. Furthermore, the increase in the pituitary-adrenal axial ac t iv i ty in diabetic rats and mice (L 'Age et a l . , 1940; Coleman & Burkart , 1977) may result in a decreased T S H secretion (Sakiz and Gui l lemin , 1965). F ina l ly , the reduced TSH secretion may be secondary to lowered hypothalamic secretion of T R H . A s discussed above, a significant decrease of c i rculat ing T R H was observed in diabetes (Wilber 19 et a l . , 1981). The mechanism(s) by which the diabetic state affects T R H secretion remains to be investigated. The amount of c i rculat ing thyroid hormones has profound effects on the responsiveness of the cardiovascular system. There is evidence that thyroid hormones have a direct positive inotropic effect on the heart (Markowitz and Ya te r , 1932), which is exerted by binding with myocardial nuclear receptors and subsequent promotion of the synthesis of R N A and protein (Tata and Windell , 1966; Surks et a l . , 1973; Tsai and Samuels, 1974; Car te r et a l . , 1985). Philipson and Edelman (1977) have documented that the ac t iv i ty of N a + - K + - A T P a s e in the heart is increased when T^ is administered to hypothyroid rats. The ac t iv i ty of the SR Ca^ + - s t imula ted ATPase has also been shown to be increased (Suko, 1973; Limas , 1978). Another important enzyme, whose ac t iv i ty is controlled by the level of thyroid hormone and is related to the positive inotropic effect of thyroid hormone, is ventricular myosin ATPase . The myosin ATPase has been shown to exist in three isozymic forms ( V j , Vg , V^) in order of decreasing electrophoretic mobil i ty and ATPase ac t iv i ty in rats, rabbits and a number of other species (Hoh et a l . , 1977; Mork in et a l . , 1983). In the adult euthyroid state, V j strongly predominates in rats (85%) while in rabbits Vg represents 85% of the total enzyme (Lompre, et a l . , 1981). The relat ive proportions of these isozymic forms in rats and rabbits have been shown to depend upon the function of the thyroid. In general, thyroid hormone enhances the synthesis of the V j form which results in increased myosin ATPase ac t iv i ty (Hoh et a l . , 1977; Mar t in , et a l . , 1982). In man the myosin 20 isozyme distribution patterns were suggested to be s imilar to rabbits but the effects of thyroid hormone on the patterns were re la t ively less (Morkin et a l . , 1983). Thyroid hormone exerts its effect on the heart not only direct ly but also indirect ly through adrenergic st imulation (Graves, 1835; Levey, 1971). A n increased number of beta-adrenergic binding sites have been shown in membranes from hyperthyroid hearts while the affinity -of the receptor for catecholamines remained unchanged (Williams et a l . , 1977; Banerjee and Kung, 1977; Cia ra ld i and Mar ine t t i , 1977). However, the number of alpha-adrenergic receptors was decreased (Sharma and Banerjee, 1978; Wil l iams and Lefkowi tz , 1979). Diabetes-induced hypothyroidism has been shown to result in altered myosin isoenzyme distribution (from V j to Vg) (Dillman, 1980). The Vg form is the slowest to hydrolyze A T P and to form cross bridges. The predominance of the V3 form (about 68% of the total in diabetic rats) would therefore result in a reduced myosin ATPase ac t iv i ty . Thus, it appears that thyroid hormones play some role in the regulation of myocardial contract iv i ty . In addition, diabetes is associated with a hypothyroid state. It was therefore of interest for us to investigate the effect of both Tg and myo-inositol on the cardiac dysfunction of diabetic rats. S P E C I F I C G O A L S O F T H E P R E S E N T INVESTIGATION. 1. To determine i f plasma and myocardial t r iacylglycerols , cholesterol and 21 phospholipid levels are elevated in streptozotocin (STZ)-diabetic rats. 2. To study the role of elevated myocardial l ipid levels in the development of diabetic cardiomyopathy. 3. To investigate the effect of myo-inositol on cardiac dysfunction and elevated myocardial l ipid levels in diabetic rats. 4. To determine i f myo-inositol plus T^ treatment could further improve heart function and myocardial l ipid levels in STZ diabetic rats. 22 M A T E R I A L S A N D M E T H O D S I. M A T E R I A L S 1. A N I M A L S . Female Wistar rats weighing between 190-215 g were used throughout the study. The rats were obtained from U . B . C . A n i m a l Care faci l i t ies . 2. C H E M I C A L S The following chemicals were purchased from Sigma Chemica l Co . : Bovine Serum Albumin , c i t r i c acid, copper sulfate, ether, Fol in-Cioca l teu ' s phenol reagent, myo-inositol , sodium bicarbonate, sodium potassium tartrate, streptozotocin (STZ), sucrose, L - 3,5,3'-triiodothyronine, Tris-base. Ca lc ium chloride, chloroform, D-glucose, hydrochloric acid, magnesium chloride, methanol, potassium chloride and sodium chloride were purchased from B D H . 3. A S S A Y KITS. Insulin radioimmunoassay (RIA) kits , Amer lex T^ R I A kits and Amer l ex T^ R I A kits were purchased from Amersham. Glucose assay kits, cholesterol assay kits , phospholipid assay kits and t r iacylglycerol assay kits were purchased from Boehringer-Mannheim. II. M E T H O D S 23 (1) I N D U C T I O N O F E X P E R I M E N T A L D I A B E T E S . Female Wistar rats between 190-215 g were used. A l l animals were anesthetized with ether prior to injection of either STZ or its vehicle into the ta i l vein. Diabetes was induced by a single intravenous injection of STZ (55 mg/kg) dissolved in 0.1 M ci t ra te buffer (pH 4.5). Control rats were injected with c i t ra te buffer alone. A l l rats injected wi th STZ survived and were housed three to four per cage. Food and water were provided ad l ibi tum throughout the study period. Diabetes was detected three days later by estimating the extent of glucosuria wi th the aid of L i l l y Tes-tape. 2. T R E A T M E N T P R O T O C O L S . 1. M Y O - I N O S I T O L P R E L I M I N A R Y S T U D Y . Three days after STZ injection, the diabetic rats were randomly divided into six groups: untreated diabetic, myo-inositol-treated diabetic (with myo-inositol concentrations of 2, 3, 4, 6 or 8 mg/ml respectively). Myo-inosi tol was added to the drinking water of the treated animals. Treatment was carr ied out for 8 weeks and at the end of this period the animals were sacrif iced. Blood samples were col lected in heparinized tubes, centrifuged at 1300 g for 20 min to separate cells from plasma. The plasma obtained was stored at -20°C unti l assayed. Plasma was analyzed 24 for glucose, cholesterol and tr iacylglycerols . Hearts were immediately frozen wi th a Wollenberger clamp previously cooled in l iquid nitrogen and stored at -25°C for the determination of myocardial cholesterol and t r iacylglycerols . (2) M Y O - I N O S I T O L T R E A T M E N T S T U D Y . Contro l and diabetic rats were subdivided into two groups, each at random, 3 days after diabetes induction. Each group drank one of the following fluids: 1) control group: water; 2) control with myo-inositol-treat-ment group: water containing myo-inositol , 3) diabetic group: water; 4) diabetic wi th myo-inositol treatment group: water containing myo-inosi tol . For diabetic rats, the concentration of myo-inositol given was 3.5 mg/ml , which was determined from the preliminary study as the most effective concentration to treat diabetics. Controls received concentrations three times higher because their fluid intake was approximately three times lower than that of diabetics. The concentration of myo-inositol in water was adjusted to give the same dose of myo-inositol (2.5 g/kg/day) to controls and diabetics. The intake of agents for each group (mg/kg~*/day~*) was calculated wi th the following formula: Average fluid intake/day (ml) x Intake ( m g / k g _ 1 / d a y _ I ) = concentrations of agent (mg/ml) x 1000 body weight (g) A l l animals were sacrif iced after eight weeks of treatment. Hearts were excised quickly and perfused in the working heart mode as described below and then frozen wi th a Wollenberger clamp previously cooled in liquid nitrogen and stored at -25°C for the determination of myocardial lipids. 25 In the myocardial l ipid study, t r iacylglycerols , cholesterol and phospholipid were measured. Blood was col lected in heparinized tubes, centrifuged at 1300 g for 20 min. The plasma obtained was stored at -20°C unti l assayed for glucose, insulin, Tg, T^, t r iacylglycerols , cholesterol and phospho-lipids. (3) M Y O - I N O S I T O L A N D T R I I O D O T H Y R O N I N E (T g ) T R E A T M E N T S T U D Y . Contro l and diabetic rats were randomly divided into eight groups: controls; myo-inositol-treated controls; Tg-treated controls; myo-inositol plus Tg-treated controls: diabetic; myo-inositol-treated diabetic; Tg-treated diabetic; and myo-inositol plus Tg-treated diabetic. Myo-inosi tol at a dose of 2.5 g/kg/day was administered in the drinking water to myo-inositol-treat-ed and myo-inositol plus Tg-treated control or diabetic rats. The intake of agents for each group (mg/kg Vday"*) was calculated wi th the same formula as shown in the "myo-inositol treatment study" part. This myo-inositol dose was chosen as our myo-inositol treatment study had shown that it could decrease myocardial t r iacylg lycero l , cholesterol and phospholipid levels and part ial ly improve cardiac function in diabetic rats. Tg at a dose of 30 ug/kg~*/day~* was injected subcutaneously to Tg-treated and myo-inositol plus Tg -treated control or diabetic rats. This dose was selected as previous studies have shown that i t can effect ively prevent the depression of myosin ATPase (Dillmann, 1982). Rats were treated for eight weeks and then decapitated. Blood was col lected in heparinized tubes, then centrifuged at 1300 x g for 20 min. The plasma obtained was stored at -20°C for the later determination of glucose, insulin, Tg , T^, t r i acy l -glycerols, cholesterol and phospholipid levels. Hearts were perfused in the 26 working heart mode and then stored at -24°C for myocardial l ipid measure-ment. 3. I S O L A T E D W O R K I N G H E A R T P E R F U S I O N Cardiac performance was assessed using a modificat ion of Neely 's isolated working heart preparation, as described by Vadlamudi et a l . (1982). The perfusion fluid used was Chenoweth-Koelle (CK) buffer of the following composition (mil l imolar concentrations of solutes): N a C l , 120 ; KC1 , 5.6; C a C l 2 , 2.18; M g C l 2 , 2.1; NaHCOg, 19; glucose 10. The C K buffer was oxygenated continuously wi th a 95% 0g and 5% COg mixture. The pH of the buffer was maintained at 7.4, and temperature kept constant at 37°±1°C. Rats were sacrif iced by decapitation wi th a guillotine. Hearts were quickly excised from the rats and placed in warm aerated C K buffer where extraneous tissue was dissected free. The aort ic stump was located and tied to a 15-gauge stainless steel aortic perfusion cannula. Perfusion was ini t ia ted in the retrograde manner through the aorta at 45 cm H^O (30 mm Hg) aortic f i l l ing pressure. The left atrium was then attached through the pulmonary vein to a 16-gauge stainless steel cannula which was connected to atr ia l f i l l ing reservoirs. Cardiac work was started by switching the perfusion system from the retrograde mode to the working heart mode. In the working heart mode, the perfusate entered the left ventr ic le through the left atrium and was pumped out into the aort ic stump. The aort ic outflow was connected to a compliance chamber containing 2-3 ml of air. Hearts were subjected to an afterload of 75 cm H 9 0 . L V D P was measured 27 by means of a Statham P 2 3 A A transducer (Statham-Gould Instruments) attached to a 20-gauge needle through a 3 cm piece of polyethylene (PE-90) tubing. The needle was inserted through the apex of the heart into the left ventr ic le . Intraaortic pressure was measured by means of another Statham P 2 3 A A transducer attached to a sidearm of the aortic outflow system. L V D P , intraaort ic pressure and the first derivative of L V D P (positive and negative dP/dt) were recorded on a Grass Model 79D polygraph. The hearts were stimulated by means of a platinum electrode placed on the left atrium at twice the threshold voltage with square-wave pulses of 5 msec duration from a Grass model SD9D stimulator to give a rate of 300 beats/min. Data were col lected and analyzed with a microcomputer according to the method of Harr is et a l . (1983). The pressure transducer signal from the polygraph was sampled at 667 Hz over 1.5 seconds at each function point. This resulted in data being col lected for six complete cardiac pulses and three of these were analyzed by use of curve-f i t t ing techniques to determine pulse height, area, start and finish. Values from these three pulses were averaged to produce data values at each function point. The hearts were equilibrated at 15 cm ^ 0 atr ia l f i l l ing pressure for about 10 min before function curves were performed. Studies were performed by estimating the left ventricular function against varying left a t r ia l f i l l ing pressures (through varying the height of the left a t r ia l f i l l ing reservoir) from 7.5 cm H^O to 20.0 cm ^ 0 in 2.5 cm steps. The f i l l ing pressure was first reduced stepwise from 15.0 to 7.5 cm HjO, then increased stepwise to 20.0 cm H 9 0 and finally decreased stepwise to 15.0 cm H 9 0 . 28 A t each point, pressure development was allowed to stabil ize before i t was recorded. In general, stable pressure development was achieved within 2 min after the left a t r ia l f i l l ing pressure was changed. A complete function curve took about 20-30 min and the total t ime of perfusion was approximately 40 min. 4. M E A S U R E M E N T O F P L A S M A T R I A C Y L G L Y C E R O L S , C H O L E S T E R O L A N D P H O S P H O L I P I D L E V E L S Plasma tr iacylglycerols , cholesterol and phospholipids were determined using the respective Boehringer-Mannheim diagnostic reagent ki ts , a l l of which rely on enzymatic reactions followed by color imetr ic determinations. 5. D E T E R M I N A T I O N O F M Y O C A R D I A L T R I A C Y L G L Y C E R O L S , C H O L E S T E R O L A N D P H O S P H O L I P I D L E V E L S . The frozen hearts were thawed and the a t r i a , fat and aorta were removed. The ventricles were then diced in 25 ml of ice-cold homogenization buffer (0.25 M sucrose, 5.0 m M Tris , 1.0 m M M g C l 2 , pH 7.4 with HC1), and homogenized with a Polytron PT-10 homogenizer (3x20 s; setting 5). A n aliquot (1.0 ml) of this homogenate was used for l ip id analysis. Lipids were extracted from heart homogenates wi th a 2:1 chloroform-methanol solution for 3 hours. A f t e r aspirating the methanol and removing the denatured protein that precipitated at the chloroform-methanol interface, a 400 ul aliquot of the remaining chloroform in which the lipids were dissolved was evaporated in a stream of nitrogen. The residue remaining was then assayed for cholesterol, phospholipids and tr iacylglycerols using 29 the respective kits from Boehringer Mannheim Canada Inc. 6. P R O T E I N A S S A Y The protein in the homogenate was quantified using the standard Lowry (1951) protein assay. Bovine serum albumin was used as a standard. A 25 ul aliquot of the ventricular tissue homogenate was added to 0.975 ml of dis t i l led water. To this was added 5.0 ml of copper reagent (1 ml 2% N a 2 C 0 3 in 0.1 N NaOH. 0.01 ml 2% N a + - K + tar trate and 0.01 ml 1% CuSO^ per ml of reagent), the tubes were vortexed and incubated at room temperature for 10 minutes. This was followed by the addition of 0.5 ml of a 50/50 Fol in-Cioca l teu ' s phenol reagent/water mixture. Thi r ty min after adding copper reagent, the absorbance was read at 623 nm. 7. B L O O D A N A L Y S I S (1) Plasma glucose was determined by the glucose oxidase method using a Boehringer-Mannheim glucose kit* (2) Plasma immunoreactive insulin was assayed by the radioimmunoassay method of Herbert et a l . (1965) using the Amersham insulin radioimmunoassay (RIA) k i t . The insulin assay reagents consisted of human insulin standards, 125 [ I] porcine insulin and an insulin antibody raised against porcine insulin, for which human insulin exhibits 100% and rat insulin 90% cross react iv i ty . Human insulin standards were used since the error made in the estimation of rat insulin levels was minimal due to the very high cross react iv i ty 30 wi th rat insulin (about 90%). (3) Plasma triiodothyronine (T^) and thyroxine (T^) levels were measured using the Amer lex T-3 and T-4 radioimmunoassay (RIA) kits of Amersham. 8. S T A T I S T I C A L A N A L Y S I S The data are expressed as mean+standard error of the mean (S.E.M.) . S ta t is t ica l significance was determined by a two way analysis of variance followed by a Newman-Keul ' s test. The level of s tat is t ical significance was set at a probability of less than 0.05 (p<0.05). 31 R E S U L T S A . P R E L I M I N A R Y S T U D Y In the preliminary study, the most effective concentration of MI in decreasing l ip id levels in the diabetic rats was determined. Diabetes was induced by injection wi th STZ (55 mg/kg, i.v.). Three days after diabetes induction, myo-inositol was administered at different concentrations (2,3,4,6,8 mg/ml) in the drinking water and treatment continued for 8 weeks. Results showed that plasma glucose, plasma and myocardial t r iacylglycerol levels tended to decrease in diabetics treated wi th myo-inositol at concentrations of 3 and 4 mg/ml (table 2). There was no significant difference in plasma and myocardial cholesterol among al l six groups. B . M Y O - I N O S I T O L T R E A T M E N T S T U D Y 1. E F F E C T O F S T Z - I N D U C E D D I A B E T E S O N C A R D I A C F U N C T I O N , P L A S M A A N D M Y O C A R D I A L LIPID L E V E L S IN F E M A L E WISTAR R A T S In al l of our studies, diabetes was induced by a single i .v. injection of 55 mg/kg STZ. The general features of STZ-diabet ic rats were quite character is t ic of the disease and are summarized in table 3. Body weight gain during the study period was significantly less in the diabetic groups than in the control rats so that the diabetic rats had significantly lower body weights upon sacrif ice. Plasma glucose levels measured at the time of sacrif ice were significantly higher in the STZ-injected rats accompanied by a marked depression in plasma insulin levels. A n elevated urine glucose 32 (>2%) was observed in diabetic rats throughout the study period. In addition, during the study period the diabetic rats exhibited polyuria, polydipsia (table 4) and polyphagia. When thyroid status of experimental rats were measured, i t was observed that plasma and levels were significantly lower in the untreated diabetic rats compared to controls (table 5). Plasma l ip id profiles in the experimental rats are shown in figures 3-5. Plasma tr iacylglycerols (figure 3), cholesterol (figure 4) and phospholipid (figure 5) were markedly elevated in the untreated diabetic group relative to controls. Myocardia l t r iacylglycerols (figure 6), cholesterol (figure 7) and phospho-l ipid (figure 8) were elevated in untreated diabetic rats relat ive to controls. When the hearts were isolated and perfused in the working heart mode, it was observed that cardiac performance assessed by measuring L V D P , rate of pressure development (+dP/dt) and rate of pressure decline (-dP/dt) was significantly depressed in 8-week diabetic rats at a l l f i l l ing pressures (figures 9-11) compared to controls. 2. E F F E C T O F M Y O - I N O S I T O L T R E A T M E N T O N C A R D I A C D Y S F U N C T I O N A N D E L E V A T E D P L A S M A A N D M Y O C A R D I A L LIPID L E V E L S IN S T Z - D I A B E T I C R A T S . Myo-inosi to l treatment in control and diabetic rats was ini t ia ted three days after injecting them with buffer or STZ (55 mg/kg i.v.) and was continued for 8 weeks. There was no significant difference between body weight gain, fluid intake, plasma glucose, plasma insulin, plasma T^ or T . of untreated and myo-inositol treated controls (table 3-5). Both untreated 33 and treated diabetic rats gained less weight than the controls. In addition, both untreated and treated diabetic rats had significantly higher fluid intake, markedly elevated blood glucose levels and severe hypoinsulinemia (table 3 and 4). Therefore i t seemed that myo-inositol treatment did not al ter the diabetic status of rats. T^ and T^ levels of the diabetic rats were significantly lower than controls, and myo-inositol treatment did not affect T^and T^ levels in diabetic rats (table 5). Plasma tr iacylglycerols (figure 3), cholesterol (figure 4) and phospho-l ip id (figure 5) were markedly elevated in the untreated diabetic group. In contrast, myo-inositol treatment of the diabetic rats restored plasma tr iacylglycerols , cholesterol and phospholipids back to normal, while the treated controls did not exhibit lowered plasma tr iacylglycerols , cholesterol and phospholipid levels compared to untreated controls. Tr iacylglycerols , cholesterol and phospholipid were increased in the myocardium of untreated diabetics (figure 6-8). Supplementation with myo-inositol also affected the diabetic myocardial l ip id levels. The t r iacylglycerols content of the treated STZ animals was significantly lower than that of the untreated diabetics and was even lower than untreated controls (figure 6). Myocardia l t r iacylglycerols of treated controls were also reduced relat ive to the untreated controls. Myocardia l cholesterol was also reduced in treated diabetics relat ive to their untreated counterparts (figure 7). Myocardia l cholesterol was s imilar in both untreated and treated controls. Myocardia l phospholipid of treated diabetics was significantly lower than that of untreated diabetics, but was s t i l l higher than both 34 controls (figure 7). Hence it seems that myo-inositol controlled the elevated myocardial t r iacylglyerols , cholesterol and phospholipid of the diabetic rats. Myocardia l t r iacylglycerols of the myo-inositol-treated controls also appeared to be affected. Figures 9-11 show the effect of myo-inositol treatment on diabetes-induced depression of L V D P , +dP/dt and -dP/dt . Hearts from untreated diabetic rats showed lower L V D P , +dP/dt and -dP/dt compared to both untreated and treated controls at a l l f i l l ing pressures examined (from 7.5 cm HgO to 20.5 cm HgO). There was no significant difference in L V D P , +dP/dt and -dP/dt between untreated and treated controls. Treatment of the diabetic rats with myo-inositol did not appear to have any significant effect on L V D P at 7.5 cm H 2 0 of f i l l ing pressure (figure 9). A t f i l l ing pressure more than 10 cm H 2 0 , the pressure developed in myo-inositol treated diabetics was significantly higher than untreated diabetics but was s t i l l significantly lower than both controls. Similar results were also seen for the rate of pressure development (+dP/dt) (figure 10). However, in the case of treated diabetics, the +dP/dt at 7.5 cm HgO of f i l l ing pressure was part ial ly improved and +dP/dt at 17.5 cm H^O of f i l l ing pressure was not different from untreated diabetics. For the rate of pressure decline (-dP/dt) (figure 11), myo-inositol treatment of the diabetic rats restored the depressed -dP/dt back to normal at f i l l ing pressures of 10 cm H2<D and 12.5 cm H 2 0 , par t ia l ly reversed this depressed parameter at f i l l ing pressures of 7.5 cm HgO and 15 cm HgO, and had no effect on -dP/dt at f i l l ing pressures greater than 17.5 cm H 9 0 . These results 35 confirm the observation that untreated diabetic rats are characterized by a depression of cardiac function (Penpargkul et a l . , 1980; Vadlamudi et a l . , 1982). Myo-inosi tol treatment part ial ly improved the cardiac function of STZ-diabet ic rats. C . M Y O - I N O S I T O L A N D / O R Tg T R E A T M E N T S T U D Y . 1. E F F E C T O F S T Z - I N D U C E D D I A B E T E S O N C A R D I A C F U N C T I O N , P L A S M A A N D M Y O C A R D I A L LIPID L E V E L S IN F E M A L E WISTAR R A T S . The general features of STZ (55 mg/kg, i .v.)-diabetic rats are summarized in table 6. The diabetic rats exhibited reduced body weights compared to a l l controls. Plasma glucose levels were markedly elevated in STZ-injected rats accompanied by a depression in plasma insulin levels. A n elevated urine glucose (>2%) was observed in diabetic rats throughout the study period. In addition, the diabetic rats exhibited polyuria, polydipsia (table 7) and polyphagia during the 8 week study period. When thyroid status was studied, untreated diabetics exhibited lower plasma Tg and T^ levels compared to controls (table 8). Figures 12-14 show the plasma l ipid profiles in the experimental rats. Plasma tr iacylgycerols (figure 12), cholesterol (figure 13) and phospholipids (figure 14) were significantly higher in the untreated diabetic rats compared to a l l controls. Myocardia l t r iacylglycerols (figure 15), cholesterol (figure 16) and phospholipids (figure 17) were also elevated in untreated diabetic rats relative 36 to al l controls. Hearts obtained from rats diabetic for eight weeks exhibited depressed cardiac function. The L V D P , +dP/dt and -dP/dt were a l l significantly depressed in the diabetic hearts at al l f i l l ing pressures studied (figure 18-20). 2. E F F E C T O F M Y O - I N O S I T O L A N D / O R T g T R E A T M E N T O N C A R D I A C D Y S F U N C T I O N A N D E L E V A T E D P L A S M A A N D M Y O C A R D I A L LIPID L E V E L S IN S T Z - D I A B E T I C R A T S . Myo-inosi to l (2.5 g/kg/day) and/or T^ (30 ug/kg/day) treatment in control and diabetic rats were ini t ia ted three days after injecting rats with buffer or STZ (55 mg/kg, i.v.) and was continued for 8 weeks. There was no significant difference in body weight gain, f luid intake, plasma glucose, plasma insulin, and plasma T^ among untreated control , myo-inositol-treated control , Tg-treated control and myo-inositol plus T^-treated control groups (tables 6-8). Untreated diabetic rats gained weight significantly less than al l controls while myo-inositol and/or T^ treatment had no effect on the body weight gain in diabetic rats (table 6). F lu id intake was markedly elevated in the untreated diabetic rats. Myo-inosi tol treated diabetic rats had lower fluid intake than untreated diabetics, but s t i l l higher than controls (table 7). T^-treated and myo-inositol plus T^-treated diabetic animals had even higher fluid intake than untreated diabetics. Untreated diabetic rats exhibited severe hyperglycemia and hypoinsulinemia. Myo-inosi tol and/or Tg treatment did not affect plasma glucose and plasma insulin levels in diabetic rats (table 6). It thus seemed that the diabetic status of rats was 37 not altered by either myo-inositol or Tg treatment, or both. Plasma Tg levels of the diabetic rats were significantly lower compared to al l controls. Tg alone or myo-inositol plus Tg-treatment reversed Tg levels back to normal in diabetic rats, whereas myo-inositol treated diabetic rats s t i l l had lower Tg values relat ive to controls. While diabetic rats had lower T^ values than controls, al l animals treated wi th Tg (controls and diabetics) had significantly lower values as compared with the respective untreated controls or diabetics. Plasma tr iacylglycerols , cholesterol and phospholipids were markedly elevated in the untreated diabetics compared to a l l controls (figures 12-14). Myo-inosi to l treatment alone restored the plasma tr iacylglycerols , cholesterol and phospholipids of diabetic rats back to normal. Tg treatment alone and myo-inositol plus Tg treatment could not lower plasma tr iacylglycerols and cholesterol levels of diabetic rats. Plasma phospholipids of diabetic rats were not affected by myo-inositol plus Tg treatment and was further increased wi th Tg treatment alone. Plasma tr iacylglycerols , cholesterol and phospholipids of control rats were not affected by either myo-inositol or Tg treatment or both. Hence it seems that myo-inositol treatment alone controlled elevated plasma lipids of the diabetic rats having no effect in the controls. Tg treatment or the combination treatment did not affect or further increased the plasma lipids of the diabetic rats. Myocardia l t r iacylglycerols , cholesterol and phospholipids of untreated diabetic rats were markedly elevated compared to a l l control rats (figures 15-17). Myocardia l t r iacylglycerols , cholesterol and phospholipid contents 38 were s imilar in controls and diabetics treated wi th myo-inositol alone. Myocardia l t r iacylglycerols and phospholipid concentrations of diabetic rats were not affected by Tg treatment alone, while myocardial cholesterol was further increased wi th Tg treatment alone relat ive to untreated diabetics. Myocardia l t r iacylglycerols of diabetic rats were lowered by myo-inositol plus Tg treatment, but s t i l l higher than controls. Myocardia l cholesterol and phospholipids were further increased wi th myo-inositol plus Tg treatment compared to untreated diabetics. Myocardia l lipids of control rats appeared not to be affected by myo-inositol and/or Tg treatment. This study shows that myo-inositol treatment alone controlled elevated myocardial lipids of STZ diabetic rats. Tg treatment or myo-inositol plus Tg treatment did not affect or further increased myocardial lipids of diabetic rats. Nei ther myo-inositol or Tg or both had any effect in the controls. Figures 18-20 show the effect of myo-inositol and/or Tg treatment on diabetes-induced depression of L V D P , +dP/dt and -dP/dt . Hearts from untreated diabetic rats showed lower L V D P , +dP/dt and -dP/dt compared to untreated controls at a l l f i l l ing pressures. Myo-inosi to l treatment alone in the diabetic rats restored the L V D P back to normal only at 7.5 cm HgO of f i l l ing pressure (figure 18). A t f i l l ing pressures greater than 10 cm H 2 O , the pressure developed in myo-inositol treated diabetics was significantly higher than untreated diabetics but was s t i l l s ignificantly lower than untreated and myo-inositol treated controls. L V D P was significantly higher in the myo-inositol treated controls relat ive to their untreated counterparts at f i l l ing pressures greater than 12.5 cm H 9 0 . In the T„ treated 39 controls or diabetics no significant elevation or depression of L V D P was seen as compared to untreated controls or diabetics, respectively. Myo- inos i to l plus Tg treatment of the diabetic rats restored L V D P to normal at 7.5 cm H 2 0 of f i l l ing pressure, and part ial ly improved L V D P at f i l l ing pressures greater than 10.0 cm HgO. However, at f i l l ing pressures greater than 12.5 cm H 2 0 , myo-inositol plus Tg treated diabetic rats had lower L V D P values as compared to myo-inositol treated diabetic rats. L V D P in hearts from myo-inositol plus Tg treated controls did not differ significantly from untreated controls. For the rate of pressure development (+dP/dt) (figure 19), there was no significant difference among untreated, myo-inositol treated, Tg-treated and myo-inositol plus Tg-treated controls. Myo-inosi tol pretreatment of the diabetic rats part ial ly reversed the depression in +dP/dt at a l l f i l l ing pressures. Tg treatment did not appear to have any significant effect on +dP/dt in diabetic rats. Myo-inosi tol plus Tg treatment of the diabetic rats part ial ly improved +dP/dt at al l f i l l ing pressures. However, at f i l l ing pressures greater than 17.5 cm H 2 0 , myo-inositol plus Tg treated diabetic rats had lower +dP/dt values relat ive to myo-inositol treated diabetic rats. S imi lar results were also seen for the rate of pressure decline (-dP/dt) (figure 19). However, in this case, myo-inositol treated controls had higher -dP/dt values relat ive to untreated controls at 10 cm H^O and 12.5 cm H 2 0 of f i l l ing pressures. In addition, myo-inositol plus Tg treated diabetic rats had lower -dP/dt values at f i l l ing pressures greater than 15 cm H 2 0 when compared to myo-inositol treated diabetic rats. Thus it is evident that STZ-treatment resulted in a depressed cardiac function, and 40 that cardiac functions of diabetic rats improved wi th myo-inositol treatment alone and also wi th myo-inositol plus Tg treatment. However, the former treatment was more efficacious than the lat ter . Tg treatment did not appear to have any effect on cardiac dysfunction of diabetic rats. T A B L E II E F F E C T OF D I F F E R E N T C O N C E N T R A T I O N S O F M Y O - I N O S I T O L T R E A T M E N T O N P L A S M A G L U C O S E , P L A S M A A N D M Y O C A R D I A L LIPIDS IN 8 W E E K D I A B E T I C R A T S . Untreated Inositol-treated diabetic diabetic Inositol concentration (n=4) (mg/ml) 2 (n=4) 3 (n=4) 4 (n=4) 6 (n=4) 8 (n=4) Plasma glucose 297 303 180 196 292 254 (mg/100 ml) ±47 ±35 ±22 ±51 ±53 ±62 Plasma triacylglycerols 320 251 75 82 215 244 (mg/100 ml) ±14 ±17* ±12*t ± 1 6 * t ±7* ± 2 1 * Plasma cholesterol 81 77 99 97 123 87 (mg/100 ml) ±15 ±7 ±10 ±12 ±19 ±14 Myocardial 4.70 4.55 1.99 2.35 2.06 2.55 tr iacylglycerols ±0.23 ±0.14 ±0.50* ±0.54* ±0 .48* ±0.36* (nmol/mg protein) Myocardial cholesterols 17.1 16.3 15.8 16.1 13.7 15.1 (nmol/mg protein) ±0.3 ±0.8 ±0.7 ±0.6 ±0.8 ±1.2 Oral myo-inositol treatment at different concentrations (2,3,4,6,8 mg/ml) was ini t iated 3 days after induction of diabetes and conducted for 8 weeks in inositol-treated diabetic rats. Results are the mean±S.E.M. at sacrif ice for number of samples indicated in parentheses; n = number of animals. •Signif icant ly different from untreated diabetics, p<0.05. tS igni f icant ly different from inositol-treated diabetics at inositol concentrations of 2,6 and 8 mg/ml , p<0.05. T A B L E Ha E F F E C T O F D I F F E R E N T C O N C E N T R A T I O N S O F M Y O - I N O S I T O L O N F L U I D I N T A K E A N D E S T I M A T E D INOSITOL I N T A K E O F 8-WEEK DIABETIC R A T S Inositol-treated diabetic inositol concentration (mg/dl) 2 3 4 6 8 (n=4) (n=4) (n=4) (n=4) (n=4) F lu id intake 150+3 107±2 128±3 171+4 106±2 (ml/day) Est imated inositol dose (g/kg/day) 1.27 1.40 2.27 4.64 3.58 1. The calculat ion of "estimated inositol dose" was based on the average fluid intake and final body weight. 2. Results shovv' that inositol dose increased to a certain extent when inositol concentrations increased. 43 T A B L E III G E N E R A L F E A T U R E S O F E X P E R I M E N T A L R A T S U S E D F O R M Y O - I N O S I T O L S T U D Y . C O N COI DIA DII (n=6) (n=6) (n=5) (n=6) B O D Y WEIGHT (g) 282 285 229 221 ±10 ±9 ± 6 * t ± 3 * t P L A S M A G L U C O S E 123 133 390 374 (mg/dl) ±8 ±5 ±13* t ±20* t P L A S M A INSULIN 49 43 17 16 (uU/ml) ±4 ±5 ±0 .3* t ± 2 * t Myo-inosi tol (2.5 g/kg/day) was administered orally for 8 weeks to rats 3 days after induction of diabetes with 55 mg/kg i .v. STZ; C O N , untreated controls; COI , myo-inositol-treated controls; DIA, untreated diabetics; DII, myo-inositol-treated diabetics; n = number of animals; results are expressed as the mean±S.E.M. •Signif icant ly different from untreated controls, p<0.05. tS ign i f ican t ly different from myo-inositol-treated controls, p<0.05. 44 T A B L E IV F L U I D A N D M Y O - I N O S I T O L I N T A K E O F E X P E R I M E N T A L R A T S U S E D F O R M Y O - I N O S I T O L T R E A T M E N T S T U D Y C O N COI DIA DII (n=6) (n=6) (n=5) (n=6) F L U I D I N T A K E 48 50 157 161 (ml/day) ±1 ±1 ± 2 * t ±4* t INOSITOL I N T A K E 0 2.50 0 2.49 (g/kg/day) ±0.06 ±0.05 The concentration of myo-inositol in water was adjusted to give the myo-inositol dose at 2.5 g/kg/day. Myo-inosi tol (2.5 g/kg/day) was given in the drinking water for 8 weeks to rats 3 days after induction of diabetes with 55 mg/kg i .v. STZ; C O N , untreated controls; COI , myo-inositol-treated controls; DIA, untreated diabetics; DII, myo-inositol-treated diabetics; n, number of animals; results are expressed as mean±S.E.M. •Signif icant ly different from untreated controls, p<0.05. tS ign i f i can t ly different from myo-inositol-treated controls, p<0.05. 45 T A B L E V T H Y R O I D S T A T U S O F E X P E R I M E N T A L R A T S U S E D F O R M Y O - I N O S I T O L S T U D Y C O N COI DIA DII (n=6) (n=6) (n=5) (n=6) P L A S M A T , (ng/ml) * P L A S M A T , (ug/dl) 4 0.457 0.477 0.330 0.311 +0.043 ±0.040 ±0.012* t ±0.040* t 3.20 3.73 2.19 2.28 ±0.13 ±0.38 ±0 .23* t ± 0 . 1 1 * t Myo-inosi tol (2.5 g/kg/day) was given in the drinking water for 8 weeks to rats 3 days after induction of diabetes with 55 mg/kg i .v . STZ; C O N , untreated controls; COI , myo-inositol-treated controls; DIA, untreated diabetics; DII, myo-inositol-treated diabetics; n = number of animals; results are expressed as the mean±S.E.M. •Signif icant ly different from untreated controls, p«=0.05. tS ign i f i can t ly different from myo-inositol-treated controls, p<0.05. 46 F I G U R E 3 Effect of myo-inositol treatment on plasma tr iacylglycerols of control and STZ-diabet ic rats. Myo-inosi to l was given at a dose of 2.5 g/kg/day for 8 weeks. Plasma samples were col lected at the t ime of sacr i f ice in untreated controls ( C O N , • , n=6), myo-inositol- treated controls (COI, f=§ n=6), untreated diabetics (DIA, 0 , n=5), and myo-inositol- treated diabetics (DII, ^ , n=6). Measurement of t r iacylglycerols is as described in Methods. Results shown are the mean±S.E.M. §Signif icant ly different from the other three groups. P L A S M A T R I A C Y L G L Y C E R O L S 1—( E 7 0 0 - 1 • O t—i \ 07 5 6 0 -E w V) i—) o 4 2 0 -L Q) U X »—t 2 8 0 -07 >—» X u D r-t 1 4 0 -L 1-0 E to 0 - -0 Q. C O N < N - 6 > • I I ( N - 6 ) • 0 C O I CN = 6 ) 0 M Y O - I N O S I T O L T R E A T M E N T 48 F I G U R E 4 Effect of myo-inositol-treatment on plasma cholesterol of control and STZ-diabet ic rats. Myo-inosi to l was given at a dose of 2.5 g/kg/day for 8 weeks. Plasma samples were col lected at the t ime of sacr i f ice in untreated controls ( C O N , D , n=6), myo-inositol- treated controls (COI, § , n=6), untreated diabetics (DIA, 0 , n=5), and myo-inositol-treated diabetics (DII, ^ , n=6). Measurement of cholesterol is as described in Methods. Results shown are the mean±S.E.M. §Signif icant ly different from the other three groups, p<0.05. ^ 120 t - l E O a T-i \ 00 07 E 0 L a a 60 0 J: u 30 0 E U) 0 o on <N-e> P L A S M A D 0 C H O L E S T E R O L CON <N-e> CO I cN-e> D IA <N-5> MYO-INOSITOL TREATMENT 50 F I G U R E 5 Effec t of myo-inositol treatment on plasma phospholipids of control and STZ-diabet ic rats. Myo-inos i to l was given at a dose of 2.5 g/kg/day for 8 weeks. Plasma samples were col lec ted at the t ime of sacr i f ice in untreated controls ( C O N , • , n=6), myo-inositol-treated controls (COI, @ , n=6), untreated diabetics (DIA, Q » n=5), and myo-inositol- treated diabetics (DII, 0 , n=6). Measurement of t r iacylglycerols is as described in methods. Results shown are the mean±S.E.M. §Signif icant ly different from the other three groups, p<0.05. 2 300 O Q n 07 E 2 4 0 0 180 TJ LL 0 r Q. <D 0 x: CL 0 E W 0 ft CL 120 BO P L A S M A P H O S P H O L I P I D S CON D CO! § DIA E l <N-6> CN-6> CN-5) o n 0 CN-0> § I 4 m MYO-INOSITOL TREATMENT 52 F I G U R E 6 Effec t of myo-inositol treatment on myocardial t r iacylglycerols of control and STZ-diabet ic rats. Myo-inosi to l was given at a dose of 2.5 g/kg/day for 8 weeks. Lipids were extracted from heart homogenates. Measurement of t r iacylglycerols is as described in Methods. Results shown are the mean±S.E .M. •Signi f icant ly different from untreated controls, p<0.05 tS ign i f i can t ly different from myo-inositol- treated controls, p<0.05. + Signif icantly different from untreated diabetics, p<0.05. C O N , untreated controls; COI , myo-inositol- treated controls; DIA , untreated diabetics; DII, myo-inositol- treated diabetics. M Y O C A R D I A L T R I A C Y L G L Y C E R O L S C O N D CN = 6) C O I CN = B) D I A CN = 5 ) • I I (N = B) X • ••• • ••• • < • ••• • ••« »••• • ••• .•»« • • »••• en CO V/// M Y O - I N O S I T O L T R E A T M E N T 54 F I G U R E 7 Effect of myo-inositol treatment on myocardial cholesterol of control and STZ-diabet ic rats. Myo-inosi to l was given a a dose of 2.5 g/kg/day for 8 weeks. Lipids were extracted from heart homogenates. Measurement of cholesterol is as described in Methods. Results shown are the mean±S.E.M. §Signif icant ly different from the other three groups, p<0.05. C O N , untreated controls; COI , myo-inositol-treated controls; DI, untreated diabetics; DII, myo-inositol-treated diabetics. 15 UJ a 1 2 DL 07 E N. _J Q UJ 01 UJ a i u g o E C M Y O C A R D I A L C H O L E S T E R O L CON <N~6> DII CN»B> • 0 COI i l ~ '/A/, y/A X '/A A/< DIA M Y O - I N O S I T O L T R E A T M E N T 56 F I G U R E 8 Effec t of myo-inositol treatment on myocardial phospholipids of control and STZ-diabet ic rats. Myo-inosi to l was given at a dose of 2.5 g/kg/day for 8 weeks. Lipids were extracted from heart homogenates. Measurement of phospholipids is as described in Methods. Results shown are the mean±S.E.M. •Signi f icant ly different from untreated controls, P<0.05. tS ign i f ican t ly different from myo-inositol-treated controls, p<0.05. t Significantly different from untreated diabetics, p<0.05. C O N , untreated controls, COI , myo-inositol-treated controls; DIA , untreated diabetics; DII, myo-inositol-treated diabetics. n m o l P H O S P H O L I P I D S / m g P R O T E I N a OJ cn 10 I\J tn I I L I I I < Q I »—t Z • if) • r m > —i m z H Z Z p • i n cn >— oi o i-i ^ z E 3 • • s : • t * : J i i : • : • : • : • i • : ' j J : • : • : z i n oi o Lin -< • n > • i—i > r TJ I O cn TJ I • r »—» TJ i—i • in z i a ui >-^ > E 3 AS 58 F I G U R E 9 Effect of diabetes and myo-inositol treatment on L V D P of isolated perfused working hearts at various f i l l ing pressures. Hearts were isolated from control ( C O N , 0 , n=5), myo-inositol- treated control (COI, • , n=5), diabetic (DIA, A , n=4) and myo-inositol-treated diabetic (DII, • , n=5) rats 60 days after the induction of diabetes wi th 55 mg/kg i .v . S T Z . Myo-inosi tol- t reated control and diabetic rats received 2.5 g/kg/day myo-inositol oral ly commencing 3 days after induction of diabetes. Results are the mean±S.E.M. for number of hearts shown in parentheses. •Signi f icant ly different from untreated controls, p<0.05. tS ign i f i can t ly different from myo-inositol-treated controls, p<0.05. tS ign i f ican t ly different from untreated diabetics, p<0.05. 59 L V D P 1 5 0 - i 12SH U) X E E <y 1 DO • > _1 7 5 5 0 C • ) C O N c •> c o i C A ) D I A C N - 5 5 CN=5> I 1 1 1 1 O 5 10 15 2 0 2 5 F I L L I N G P R E S S U R E ( c m H x O ) 60 F I G U R E 10 Effec t of diabetes and myo-inositol-treatment on +dP/dt of isolated perfused working hearts at various f i l l ing pressures. Hearts were isolated from control ( C O N , 0 , n=5); myo-inositol- treated control (COI, • , n=5); diabetic (DIA, A , n=4) and myo-inositol-treated diabetic (DII, • , n=5) rats 60 days after the induction of diabetes 55 mg/kg i .v . STZ. Myo-inosi tol- t reated control and diabetic rats received 2.5 g/kg/day myc-inosi tol oral ly commencing 3 days after induction of diabetes. Results are the mean+S.E.M. for number of hearts shown in parentheses. •Signi f icant ly different from untreated controls, p<0.05 tS ign i f ican t ly different from myo-inositol-treated control , p<0.05. tS ign i f i can t ly different from untreated diabetics, p<0.05. 61 P O S I T I V E d P / d T 4 5 0 0 - i 4 0 0 0 H w cn ^ 3 5 0 0 E TJ \ 3 0 0 0 D_ TJ UJ > h-in • Q_ 2 5 0 0 H 2 0 0 0 -i 1 5 0 0 C • ) C O N C • 5 C O I C A> D I A < • > P I I CN«=5!> CN=»5> CN=4> CN=5> C O N COI i DM * t * DIA I 1 1 1 1 0 5 10 15 2 D 2 5 F I L L I N G P R E S S U R E ( c m H . D ) 62 F I G U R E 11 Effec t of diabetes and myo-inositol-treatment on -dP/dt of isolated perfused working hearts at various f i l l ing pressures. Hearts were isolated from control ( C O N , 0 , n=5), myo-inositol-treated control (COI, • , n=5), diabetic (DIA, A., n=4) and myo-inositol-treated diabetic (DII, • , n=5) rats 60 days after the induction of diabetes with 55 mg/kg i .v. STZ. Myo-inosi tol- t reated control and diabetic rats received 2.5 g/kg/day myo-inositol oral ly commencing 3 days after induction of diabetes. Results are the mean±S.E.M. for number of hearts shown in parentheses. •Signi f icant ly different from untreated controls, p<0.05. tS ign i f i can t ly different from myo-inositol- treated controls, p<0.05 tS ign i f i can t ly different from untreated diabetics, p<0.05. §Signif icant ly different from the other three groups, p<0.05. 63 N E G A T I V E d P / d T <• • > CON T A B L E VI G E N E R A L F E A T U R E S OF E X P E R I M E N T A L R A T S USED F O R M Y O - I N O S I T O L (MI) A N D T R I I O D O T H Y R O N I N E ( T J T R E A T M E N T S T U D Y C O N COI C O T CIT DIA DII DIT DIIT (n=5) (n=4) (n=7) (n=7) (n=5) (n=4) (n=6) (n=7) B O D Y WEIGHT (g) 254 259 255 266 196 220 218 216 ±13 ±11 ±8 ±6 ±16 t ±14* * ± 8 * * ± 6 * t P L A S M A G L U C O S E 135 141 125 114 421 411 419 385 (mg/dl) ±11 ±13 ±7 ±2 ± 1 7 t ± 1 2 t ± 4 t ± 1 0 t P L A S M A INSULIN 41 40 37 47 15 16 17 15 (uU/ml) ±4 ±3 ±4 ±5 ± l t ± 2 t ± l t ± 2 t Myo-inosi tol (2.5 g/kday, orally) and/or T\, (30 ug/kg/day, s.c.) were given for 8 weeks to rats 3 days after induction of diabetes with 55 mg/kg i.v. STZ. The different groups are represented by the following letters: C O N , untreated controls; COI , Mi-treated controls; C O T , T „ - t r e a t e d controls; CIT, MI + T „ - t r e a t e d controls; DIA, untreated diabetics; DII, Mi-treated diabetics; DIT, T „ - t r e a t e d diabetics; DIIT, MI + T „ - t r e a t e d diabetics; n, number of animals. Results are expressed as the mean±S.E.M. •Signif icant ly different from untreated controls, (p<0.05). tS igni f icant ly different from all controls, (p<0.05). tS igni f icant ly different from controls of same treatment group (p<0.05). T A B L E VII F L U I D A N D M Y O - I N O S I T O L I N T A K E O F E X P E R I M E N T A L R A T S U S E D F O R M Y O - I N O S I T O L (MI) A N D T R I I O D O T H Y R O N I N E ( T J - T R E A T M E N T S T U D Y . C O N COI C O T CIT DIA DII DIT DIIT (n-5) (n=4) (n=7) (n=7) (n=5) (n-4) (n-6) (n=7) F L U I D I N T A K E 44 37 47 49 170 127 194 186 (ml/day) ±1 ±1 * ±1 * ±1 * ±2 t ± 2 * § * ± 5 * § * ±3*§ * INOSITOL I N T A K E 0 2.54 0 2.53 0 2.51 0 2.52 (g/kg/day) ±0.09 ±0.06 ±0.04 ±0.03 The concentration of myo-inositol in water was adjusted to give the myo-inositol dose at 2.5 g/kg/day. Myo-inosi tol (2.5 g/kg/day, orally) and/or T„ (30 ug/kg/day, s.c.) were given for 8 weeks to rats 3 days after induction of diabetes with 55 mg/kg i .v. STZ. The different groups are represented by the following letters: C O N , untreated controls; COI, Mi- t reated controls; C O T , T „ - t r e a t e d controls; CIT , M l + T „ - t r e a t e d controls; DIA , untreated diabetics; DII, Mi- t rea ted diabetics; DIT, Tg-treated diabetics; DIIT, MI + Tg-treated diabetics; n, number of animals. Results are expressed as the mean±S.E.M. •Signif icant ly different from untreated controls (p<0.05). tS igni f icant ly different from al l controls (p<0.05). tS igni f icant ly different from controls of the same treatment group (p<0.05). §Signif icant ly different from untreated diabetics (p<0.05). T A B L E VIII T H Y R O I D S T A T U S OF E X P E R I M E N T A L R A T S U S E D F O R M Y O - I N O S I T O L (MI) A N D T R I I O D O T H Y R O N I N E (T g ) - T R E A T M E N T S T U D Y . C O N COI C O T CIT DIA DII DIT DIIT (n=5) (n=4) (n=7) (n=7) (n=5) (n=4) (n=6) (n=7) P L A S M A T„ 0.483 0.588 0.734 0.676 0.340 0.355 0.506 0.510 (ng/ml) 6 ±0.023 ±0.046 ±0.042 ±0.035 ±0.015f ±0.016+ ±0.023 ±0.024 P L A S M A T4 53 50 16 22 39 37 20 16 (nmol/1) ±4 ±4 ± 1 * ± 1 * ±3+ ± l t ±3*§ ±1*§ Myo-inosi tol (2.5 g/kg/day, orally) and/or T„ (30 ug/kg/day, s.c.) were given for 8 weeks to rats 3 days after induction of diabetes with 55 mg/kg i.v. STZ. The different groups are represented by the following letters: C O N , untreated controls; COI, Mi- t reated controls; C O T , T „ - t r e a t e d controls; CIT, MI + T^-treated controls; DIA, untreated diabetics; DII, Mi- t reated diabetics; DIT, T^-treated diabetics; DIIT, MI + T^-treated diabetics; n, number of animals. Results are expressed as the mean±S.E.M. •Signif icant ly different from untreated controls (p<0.05). tS igni fcant ly different from al l controls (p<0.05). tS igni f icant ly different from controls of the same treatment group (p<0.05). §Signif icant ly different from untreated diabetics (p<0.05). 67 F I G U R E 12 Levels of t r iacylglycerols in plasma obtained from control and diabetic rats treated with myo-inositol and Tg . Plasma samples were col lected at t ime of sacr if ice in untreated controls ( C O N , • , n=5), myo-inositol-treated controls (COI, § , n=4), Tg-treated controls (COT, M , n=7), myo-inositol plus Tg-treated controls (CIT, H , n=7), untreated diabetics (DIA, E3 , n=5), myo-inositol treated diabetics (DII, E l , n=4), Tg-treated diabetics (DIT, S , n=6), and myo-inositol-plus-Tg-treated diabetics (DIIT, H , n=7). Measurement of t r iacylglycerols is as described in Methods. Results shown are the mean±S.E.M. •Signi f icant ly different from untreated controls, p<0.05. tS ign i f i can t ly different from al l controls, p<0.05. tS ign i f i can t ly different from controls of same treatment group, p<0.05. §Signif icant ly different from untreated diabetics, p<0.05. P L A S M A T R I A C Y L G L Y C E R O L S 7 8 0 - 1 • Q N 6 5 0 -0) E 5 2 0 -0 L 0 390 H (J X 0) r—I X 0 0 L h-0 E (0 0 Q. 260-1 3 0 -CON • COI s CN-5) CN-4) s CIT g DIA CN-7) s CN-5) DIT DIIT CN-6) CN-7) t ** » • • • • 1 • ••• > ••• COT CN-7) OI I CN-4) MYO-INOSITOL AND T 3 TREATMENT 69 F I G U R E 13 Levels of cholesterol in plasma obtained from control and diabetic rats treated wi th myo-inositol and T g . Plasma samples were col lec ted at the t ime of sacr if ice in untreated controls ( C O N , • , n=5), myo-inositol-treated controls (COI, @ , n=4), Tg-treated controls (COT, ID) , n=7), myo-inositol plus Tg-treated controls (CIT, ffl , n=7), untreated diabetics (DIA, 0 , n=5), myo-inositol- treated diabetics (DII, W , n=4), Tg-treated diabetics (DIT, S3, n=6) and myo-inositol plus Tg-treated diabetics (DIIT, B§ , n=7). Measurement of cholesterol is as described in Methods. Results shown are the mean+S.E.M. •Signi f icant ly different from untreated controls, p<0.05. tS ign i f i can t ly different from al l controls, p<0.05. tS ign i f i can t ly different from controls of same treatment group, p<0.05. §Signif icant ly different from untreated diabetics, p<0.05. P L A S M A C H O L E S T E R O L CON CN-5) COT <N-7> • II CN-4) MYO-INOSITOL AND T 3 TREATMENT 71 F I G U R E 14 Levels of phospholipids in plasma obtained from control and diabetic rats treated with myo-inositol and Tg . Plasma samples were co l lec ted at the t ime of sacr if ice in untreated controls ( C O N , • , n=5), myo-inositol-treated controls (COI, @ , n=4), Tg-treated controls (COT, OH , n=7), myo-inositol plus Tg treated controls (CIT, H , n=7), untreated diabetics (DIA, E3 , n=5), myo-inositol- treated diabetics (DII, 23 , n=4), Tg-treated diabetics (DIT, S3, n=6) and myo-inositol plus Tg-treated diabetics (DIIT, H3 , n=7). Measurement of phospholipids is as described in Methods. Results are shown as the mean±S.E.M. •Signi f icant ly different from untreated controls, p<0.05. tS igni f icant ly different from al l controls, p<0.05. tSignif icant ly different from controls of same treatment group, p<0.05. iS ign i f ican t ly different from untreated diabetics, p<0.05. P L A 5 M A P H O S P H O L I P I D S 73 F I G U R E 15 Measurement of myocardial t r iacylglycerol levels in control and diabetic rats administered myo-inositol and T g . Lipids were extracted from heart homogenates. Results are shown as the mean±S.E.M.; C O N , untreated controls; C O I , Mi- t rea ted controls; C O T , Tg-treated controls; CIT, MI plus Tg-treated controls; DIA , untreated diabetics; DII, Mi- t rea ted diabetics; DIT, Tg-treated diabetics; DIIT, MI plus Tg-treated diabetics. •Signi f icant ly different from untreated controls, p<0.05. tS ign i f ican t ly different from al l controls, p<0.05. tS ign i f ican t ly different from controls of the same treatment group, p<0.05. §Signif icant ly different from untreated diabetics, p<0.05. M Y O C A R D I A L T R I A C Y L G L Y C E R O L S z I D - . C O N C N - 5 ) C I T (N=7) D I T <N=6) • s C O I ( N " 4 ) DIA CN=-5> D I I T C N - 7 ) C O T C N - 7 ) D I I CN«*4) 8 -6-4 -2 -O-_X_ m I • ••• M Y O - I N O S I T O L A N D T 3 T R E A T M E N T 75 F I G U R E 16 Measurement of myocardial cholesterol levels in control and diabetic rats administered myo-inositol and Tg. Lipids were extracted from heart homogenates. Results shown are the mean±S.E.M.; C O N , untreated controls; COI , Ml- t rea ted controls; C O T , Tg-treated controls; CIT, MI plus Tg-treated controls; D I A , untreated diabetics; DII, MJ-treated diabetics; DIT, Tg-treated diabetics; DIIT, MI plus Tg-treated diabetes. •Signi f icant ly different from untreated controls, p<0.05. tS ign i f i can t ly different from al l controls, p<0.05. tS ign i f i can t ly different from controls of same treatment group, p<0.05. §Signif icant ly different from untreated diabetics, p<0.05. z UJ h-• 2 0 -o_ 07 E \ 1 5 -_) • cc UJ \-1 0 -Ul UJ _J a X u 5 -r - H 0 E n M Y O C A R D I A L C O N D S C H O L E S T E R O L CN=»5) C I T CN=7> D I T C N - 6 ) C O I 01 A CN-5> D I I T <N=»7> 0 t •••• i V/// v • V S H '/.'.> / f y * /''•>' '//,-"/// '''/' 22. C O T 0 <N-7> DII 0 <N~4> M Y D - I N O S I T O L A N D T ? T R E A T M E N T 77 F I G U R E 17 Measurement of myocardial phospholipid levels in control and diabetic rats administered myo-inositol and Tg . Lipids were extracted from heart homogenates. Results shown are the mean±S.E.M.; C O N , untreated controls; C O I , Mi- t rea ted controls; C O T , Tg-treated controls; CIT, MI plus Tg-treated controls; DIA, untreated diabetics; DII, Mi- t rea ted diabetics; DIT, Tg-treated diabetics; DIIT, M l plus Tg-treated diabetics. •Signi f icant ly different from untreated controls, p<0.05 tS ign i f i can t ly different from al l controls, p<0.05 tS ign i f ican t ly different from controls of same treatment group, p<0.05 §Signif icant ly different from untreated diabetics, p<0.05. M Y O C A R D I A L P H O S P H O L I P I D S Z 25-, uJ • or Q. 20-01 E \ U) IS-• _J a i 0_ V) • I 0_ 0 E C 10-5-CON 0 COI CN-5) rm CN-4) CIT 1 DIA CN-7) CN-5) OIT DI IT CN-5) CN-7) • • • ••< »••• • ••< > ••• • • ••• ft •'/••> //A /••>$ Y/A w Y/A ys? COT CN-7) DII CN-4) M Y O - I N O S I T O L A N D T 3 T R E A T M E N T 79 F I G U R E 18 Effect of 8 weeks of myo-inositol and Tg treatment in diabetic rats on diabetes-induced depression of L V D P . Results shown are the mean±S.E.M.; C O N , untreated controls; COI , Mi- t rea ted controls; C O T , Tg-treated controls; CIT, MI plus Tg-treated controls; DIA , untreated diabetics; DII, Mi- t rea ted diabetics; DIT, Tg-treated diabetics; DIIT, M l plus Tg treated diabetics. •Signi f icant ly different from untreated controls, p<0.05. tS ign i f ican t ly different from all controls, p<0.05. tS ign i f ican t ly different from controls of the same treatment group, p<0.05. §Signif icant ly different from untreated diabetics, p<0.05. TSignificantly different from myo-inositol-treated diabetics, p<0.05. 80 L V D P 200-c 0 5 CON c • > c o r CN-4> c COT CN=7> c V > CIT CN=7> c • > DIA CN=5> c • > DII CN-4) c DIT c T > DIIT <N-=7> 17CH ^ 1 4 0 -E E 0_ • 1 1 0 -> 80 H 50-( C O I ) ( C O N , C O T , C I T ) / * + § | * + § j # + § f w s § r r (diit) 4 / 'X-^ t ( D I A , D I T ) / t r t — i 1 1 1 • — — i 5 ID 15 20 25 F I L L I N G P R E S S U R E ( c m H 2 C 0 81 F I G U R E 19 Effect of 8 weeks of myo-inositol and Tg treatment in diabetic rats on diabetes-induced depression of +dP/dt. Results are shown as the mean±S.E.M.; C O N , untreated controls; C O I , Mi- t rea ted controls; C O T , Tg-treated controls; CIT, MI plus Tg-treated controls; DIA, untreated diabetics; DII, Mi- t rea ted diabetics; DIT, Tg-treated diabetics; DIIT, MI plus Tg-treated diabetics. •Signif icant ly different from untreated controls, p<0.05. tS igni f icant ly different from al l controls, p<0.05. tS igni f icant ly different from controls of same treatment group, p<0.05. §Signif icant ly different from untreated diabetics, p<0.05. tSignif icant ly , different from myo-inositol-treated diabetics. 82 P O S I T I V E d P / d T 4.500-1 u> \ r E E h TJ 3 B Q O -3 1 0 0 -2 4 0 0 -• 1 7 0 0 -c o > COM <N-=5> c • 5 C D I CN«=45 c A ) C O T CN=7> c V ) C I T CN-=7) c • > D I A CN-=5> c • 5 D I I < N » 4 5 c A > D I T CN=5) c T 5 D I I T C N ° 7 > ( C O K , C O I , C O T , C I T ) * * § ^ 1 ( D I I ) — T 1 ]L£ ~ J J ( D I I T ) ^ ^ ^ q (DIA, D I T ) * t + t I D O D - f 1 1 r 5 10 1 5 2 0 T I L L I N G P R E S S U R E ( c m H i O ) 83 F I G U R E 20 Effect of 8 weeks of myo-inositol and Tg treatment in diabetic rats on diabetes-induced depression of -dP/d t . Results are shown as the mean±S.E.M.; C O N , untreated controls; C O I , Mi- t rea ted controls; C O T , Tg-treated controls; CIT, MI plus Tg-treated controls; DIA, untreated diabetics; DII, Ml- t rea ted diabetics; DIT, Tg-treated diabetics; DIIT, MI plus Tg-treated diabetics. •Signif icant ly different from untreated controls, p<0.05. tS ign i f ican t ly different from controls, p<0.05. tS ign i f ican t ly different from controls of the same treatment group, p<0.05. §Signif icantIy different from untreated diabetics, p<0.05. tSignif icant ly different from myo-inositol-treated diabetics, p<0.05. 84 N E G A T I V E d P / d T 5 0 0 0 -(ft r E E •o 3 0 D D H EL U Hi > 1-4 <£ 2 0 D 0 -UJ z 1 0 D D -c 0 > CON CN=5> ( D> CD I CN=4> c COT <N=7> c V > CIT CN=7> c • > DIA CN=5> c • > DII CN»4.) c A > DIT CN=6> c T > DIIT (N=7> g ^ ^ 5 (coi) ( C O N , C O T , C I T ) * * § £ - r ^ I ( D I I T ) * * § ^ * * § ( D I A , D I T ) r t — i 1 1 1 1 5 ID 15 2 D F I L L I N G P R E S S U R E ( c m H i D ) 2 5 85 DISCUSSION The present study was designed to investigate the effect of myo-inositol and/or Tg treatment on cardiac dysfunction and elevated myocardial l ip id levels in STZ-diabet ic rats. Diabetes was induced by the intravenous injection of STZ (55 mg/kg) which direct ly destroys pancreatic beta cel ls . The diabetic state was character ized by hyperglycemia and loss of body weight as early as 3 days after STZ injection. Absolute or relat ive deficiency of insulin can lead to a marked decrease in the cel lular rate of glucose uptake, resulting in elevated levels of blood glucose. The decrease in body weight at the t ime of sacrif ice occurred in spite of increased fluid and food intake and was probably due to dehydration and protein and fat catabolism during diabetes (Oakley, 1968). In our study, the choice of an 8 week study period was based on previous work done in our laboratory as wel l as the work of others. Fe in et a l . (1980) reported that changes in papillary muscle mechanics of STZ-diabet ic rats were seen five weeks after the onset of diabetes. A study by Penpargkul et al . (1980) demonstrated that eight weeks after STZ treatment diabetic rats exhibited depressed cardiac function. Simi lar ly , Vadlamudi et a l . (1982) found that the onset of depression of cardiac performance of STZ diabetic rats was between 30 days and 3 months of diabetes. In a study by Tahi l iani et a l . (1983), STZ treated rats exhibited the depression 6 weeks after the induction of diabetes. Thus it appears 86 that myocardial changes occur 4 to 8 weeks after the induction of diabetes. Results from our study showed that hearts from 8 week diabetic rats exhibited a depression in cardiac function compared to controls which is in general agreement wi th the above observations. A n association between hyperlipidemia and diabetic ketoacidosis was first demonstrated by Bloor (1916). Since then, elevated blood lipids have been widely reported among patients wi th poorly controlled diabetes melli tus. It has been estimated that the frequency of elevated plasma l ip id levels in diabetic patients is between 20 and 90% (Bil lmoria et a l . , 1976; Simpson et a l . , 1979) depending on the degree of diabetic control and the type of diabetes. The most commonly and markedly elevated fasting plasma l ipid level is t r iacylglycerol (New et a l . 1963; N ikk i l a , 1974) associated with a less pronounced increase in plasma cholesterol and phospholipid in the diabetic population (Adlersberg and Eis ler , 1959; Albr ink et a l . 1963; Medal ie et a l . , 1974). L i p i d alterations have also been reported in chemically-induced animal models. In non-human primates during insulin deprivation, plasma tr iacylglycerols increase first and pr imari ly , followed by plasma cholesterol and phospholipids (Howard, 1975). S imi lar ly , an elevation of plasma free fat ty acid, t r iacylglycerols , cholesterol and phospholipids were shown in STZ diabetic rats (Rodrigues et a l . 1986). Elevat ion of plasma lipids indicates either the defective removal or overproduction or both of one or more lipoproteins. Because of the role of insulin in both production and removal of t r iacylg lycero l - r ich lipoproteins, insulin deficiency may be the major cause of l ip id disorders of diabetes. Evidence strongly suggests that there 87 is a defect in the removal of t r iacylg lycero l - r ich lipoproteins in insulin-deficient animals and man that is restored to normal by the administration of insulin (Nikki la et a l . , 1977). Furthermore, lipoprotein lipase (LPL) , which is the enzyme responsible for the removal of t r iacylg lycero l - r ich lipoprotein, has been demonstrated to fal l after insulin withdrawal in insulin-dependent diabetic animals (Elkeles and Hambley, 1977) and man (Bagdade et a l . 1968) and is low in poorly controlled diabetic patients (Nikki la et a l . 1977). Adminis t ra t ion of insulin to patients was found to increase L P L ac t iv i ty above normal (Nikki la et a l . 1977). Overproduction of lipoproteins is another biochemical abnormality involved in increased plasma lipids in diabetes. Evidence suggests that increased very low density lipoproteins (VLDL) production by the l iver is more commonly shown in patients with insulin resistance (Kissebah et a l . , 1974). In addition, it is suggested that during insulin resistance insulin is ineffect ive in suppressing lipolysis in adipose tissue, which may also account for the elevation in plasma cholesterol and tr iacylglycerols (Goldberg, 1981). C l i n i c a l evidence has shown that thyroid hormones also play a role in plasma lipoprotein and cholesterol metabolism. Hypercholesterolaemia is almost a c l in ica l prerequisite of severe hypothyroidism. Studies in cultured cells (Chait et a l . 1979) and the intact organism (Thompson et a l . 1981) indicate that T^ or thyroxine deficiency is associated with defective expression of low density lipoprotein (LDL) receptor ac t iv i ty , leading to abnormal catabolism of lipoprotein and cholesterol. Replacement therapy, on the other hand, increased receptor ac t iv i ty , promoted L D L catabolism 88 and therefore reduced cholesterol levels in the blood. Diabetes is known to result in a state of hypothyroidism (Pit tman et a l . 1979*). It is thus possible that elevated plasma cholesterol levels could be a result of diabetes-induced hypothyroidism. Our results regarding the effects of STZ diabetes on plasma l ip id levels suggest that elevated levels of plasma l ipid could result from either low plasma insulin levels and an associated decreased removal of lipoproteins, or diabetes-induced hypothyroidism. The involvement of elevated levels of c i rcula t ing lipids in the development of vascular and heart diseases has been suggested in both diabetic man and animals. Pomeranze and Kunkel (1950) showed a general correlat ion between arteriosclerosis and high total l ip id , high cholesterol or reduced phospholipid/cholesterol rat io. Adlersberg and Eis ler (1959) suggested that elevation of plasma fatty acid and t r iacylglycerol levels were related to the presence of vascular complications in diabetic patients. S imi lar ly , studies by New et a l . (1963) showed a relationship between complications (such as myocardial infarction and peripheral vascular disease) and elevated plasma t r iacylglycerol concentration in diabetics aged 31 to 50. A l so it is of interest that non-diabetics in this middle age group were found to have elevated t r iacylglycerol levels which have been correlated wi th the early development of ischemic heart disease (Antonis and Bersohn, 1960; Carlson, 1960). One line of evidence in the animal studies that implicates elevated levels of c i rculat ing fats in the production of diabetic cardiomyopathy is that insulin treatment in diabetic animals not only causes 89 a glucose lowering effect but also decreases the levels of c i rculat ing fats which is accompanied by a recovery of cardiac function (Mochizuki et a l . , 1984). In a study by Rodrigues and M c N e i l l (1986), Wistar and Wis tar -Kyoto (WKY) rats were injected wi th identical doses of STZ and both strains of rats exhibited hyperglycemia and hypoinsulinemia. However, only the Wistar rats exhibited hyperlipidemia and depressed myocardial function, while neither hyperlipidemia nor cardiac dysfunction was seen in rats of the W K Y strain. In another study, hydralazine treatment normalized c i rcula t ing l ip id levels as wel l as the function of hearts in diabetic rats (Rodrigues et a l . , 1986). However, a combination of thyroid hormone and methylpalmoxirate which was able to part ial ly improve cardiac function did not have any effect on elevated levels of c i rculat ing lipids in diabetic rats (Tahiliani and M c N e i l l , 1985). Furthermore, Heyl iger et a l . (1986) showed that choline and/or methionine improved the cardiac performance of diabetic rats which was accompanied by a reduction in both plasma and myocardial lipids. These results suggest that c i rculat ing lipids are not the only determinant of cardiac function in diabetes and myocardial lipids may be more direct ly related to the cardiac function in diabetes. Myocardia l l ipid levels in diabetic rats were also measured in our study. Our results demonstrated significant elevations of myocardial lipids in STZ-diabet ic rats. A s imilar l ipid accumulation has also been reported by other investigators. Regan et a l . (1973), Shipp et a l . (1973) and Murthy and Shipp (1977) showed that there was an abnormal t r iacylglyerol buildup in the diabetic myocardium. In 1986, Heyliger et a l . demonstrated an 90 accumulation of both t r iacylglycerol and cholesterol in the myocardium in STZ-diabet ic rats. Several factors could be involved in this myocardial l ip id buildup. Accumula t ion of t r iacylglycerol may reside in a defect in the lipase system, which may then lead to a decreased removal of t r iacylglycerol from the myocardium (Atkin and Meng, 1972). A diminished uptake of chylomicron t r iacylglycerol has been observed in diabetic rat hearts (Kreisberg, 1966). Evidence of reduced lipoprotein lipase ac t iv i ty supplied by A t k i n and Meng (1972) also supports the view that extracel lular t r iacylg lycerol does not contribute to the cardiac l ip id buildup. Increased plasma F F A levels could also be . involved in the myocardial l ip id accumulation. In fact both the uptake and ester if icat ion of F F A are increased when the perfusate F F A concentration is increased (Opie 1969; Neely and Morgan, 1974). Furthermore, myocardial t r iacylglycerol content is known to be increased in diabetes in which plasma F F A concentrations are increased (Frederickson and Gordon, 1958; Shipp et a l . , 1973). However, Regan et a l . (1973) failed to observe a close correlat ion between the increase in plasma F F A and the myocardial buildup of t r iacylg lycero l . On the other hand, studies suggest that increased uptake of plasma F F A could inhibit cardiac t r iacylglycerol moblization, which may also contribute to t r iacylglycerol accumulation in the myocardium of diabetics (Paulson and Crass, 1982; Murthy et a l . , 1983). In addition it is possible that increased synthesis of t r iacylglycerol could account for t r iacylglycerol accumulation in the myocardium of diabetics. However, increased biosynthesis of t r iacylg lycerol is shown to be a factor in its buildup in the myocardium 91 only in ketot ic diabetic rats (Murthy and Shipp, 1977). There is no evidence for the increased myocardial t r iacylglycerol or fatty acid synthesis in nonketotic diabetic animals (Whereat and Orishimo, 1969). Thus, i t seems that impaired t r i acy lg lycero l lipase ac t iv i ty and/or inhibited mobil izat ion of cardiac t r iacylglycerol may play a role in the myocardial t r iacylglyerol buildup in diabetes. Myocardia l cholesterol buildup could result from either increased synthesis or increased uptake, or both. Increased uptake and associated accumulation of cholesterol in the myocardial cells can downregulate de  novo endogenous cholesterol synthesis by inhibiting H M G C o A reductase, the ra te- l imi t ing enzyme in cholesterol synthesis. Suppression of this enzyme leaves the ce l l dependent on external cholesterol (Brown and Goldstein, 1984). Furthermore, the incoming cholesterol promotes the storage of this l ip id in the ce l l by act ivat ing the enzyme a c y l - C o A : cholesterol acyltransferase ( A C A T ) . In the diabetic state, plasma cholesterol is elevated which may result in increased uptake of this l ipid by the heart and accumulation of this l ip id in the myocardium. Hence, increased uptake of cholesterol by the heart, rather than increased de novo synthesis in the heart, could be mainly contributed to the increased myocardial cholesterol content. There is increasing evidence that cardiac function can be affected by al terat ion in the l ip id levels of the myocardium. Henderson et a l . (1970) demonstrated that F F A perfusion depressed myocardial cont rac t i l i ty in rat hearts subjected to hypoxia. Regan et a l . (1973) showed that cardiac dysfunction in chronical ly diabetic dogs was accompanied by t r iacylglycerol 92 increases in cardiac cells . In addition, Peterson et a l . (1979) presented evidence that myocardial cont rac t i l i ty in papillary muscles was lowered in atherosclerotic rabbits. From our studies, it also appears that changes in the l ip id levels of the myocardium are correlated wi th the depressed performance in the diabetic hearts. Several mechanisms have been postulated for the depressed cardiac function as a result of myocardial l ip id levels. The increase in t r iacylglycerol and cholesterol in the myocardium may be involved in the left ventricular stiffness of the diabetic heart (Regan et a l . , 1973). Accumulat ions of more cholesterol than phospholipid in the ce l l could increase cholesterol/phospholipid rat io, resulting in a decrease in membrane f luidi ty. The changes in membrane fluidity may further alter the microenvironment and affect the integri ty of both the cel lular and subcellular membranes (Pezzol i et a l . , 1983). The alterations in the integrity of the myocardial membranes could depress the ac t iv i ty of various myocardial enzymes and other transporting systems that play important roles in the normal contract ion of the heart on a beat to beat basis. Depressed N a + - K + A T P a s e (Kar l i et a l . 1979) and C a 2 + - M g 2 + - d e p e n d e n t ATPase (Panagia et a l . 1982) act ivi t ies in the rat heart SL have been reported. In addition it has been suggested that defective SR calcium transport is related to diabetes-induced metabolic derangements in lipids and concomitant membrane alterations (Ganguly et a l . 1983). It is also known that myocardial l ip id buildup can increase cardiac l ip id metabolism. The resulting elevated levels of L C A C in the SR preparations derived from chronically diabetic rats could inhibit membrane 93 transport proteins including SR calc ium ATPase (Lopaschuk et al . 1983 ). Increased cardiac l ip id metabolism could also increase cardiac oxygen demand which may lead to anoxia. In diabetes, glycolysis is inhibited by increased F F A and the only substrate available for energy is F F A . F F A cannot be u t i l i zed for energy in anoxia and may accumulate intracel lular ly as t r iacylglycerol or F F A which may lead to decreased energy supply and decreased oxygen supply in the diabetic myocardium (Opie, 1972). The present study aimed at examining the effect of myo-inositol alone or in combination wi th Tg in STZ diabetic rats. Da i ly oral treatment wi th myo-inositol improved cardial function in STZ diabetic rats. Furthermore, S T Z diabetes elevated plasma and myocardial l ipid levels. Our results showed that myo-inositol treatment is effective in decreasing these levels in STZ diabetic animals. Treatment wi th STZ resulted in a diabetic state character ized by hyperglycemia, hypoinsulinemia, loss of body weight and increased fluid intake. Similar alterations were also reported by other investigators (Vadlamudi et a l . 1982; Rodrigues et a l . , 1986). Myo-inosi tol treatment had no effect on these parameters indicating myo-inositol could not alter the diabetic state of rats. The pronounced effect of myo-inositol in lowering plasma and myocardial t r iacylg lycerol , cholesterol and phospholipids in the S T Z diabetic rats are, in general, consistent wi th previous reports. Gavin and McHenry in 1941 first indicated that myo-inositol could serve as a l ipotropic factor. They fed a beef l iver fraction to rats which caused the production of acutely 94 fatty livers containing large amounts of cholesterol. However, when myo-inositol was given at the same t ime, the development of fatty l iver and the accumulation of cholesterol in the l iver were prevented. In 1951 D o t t i showed that myo-inositol inhibited the expected rise in plasma cholesterol and phospholipid levels in rabbits on high cholesterol diets. Furthermore, Hayashi (1974) observed an accumulation of t r i a cy l -glycerol and cholesterol in the l iver and elevated levels of plasma F F A in rats fed a myo-inositol deficient diet, which was corrected by supplement-ing the diet wi th myo-inositol . The mechanisms by which myo-inositol decreased plasma l ipid levels of the diabetic rats is a matter of speculation. Myo-inosi tol treated diabetic rats, although they s t i l l had low plasma insulin, did not have the elevated plasma t r iacylglycerols , cholesterol and phospholipid seen in the untreated diabetic group. It thus seems that the action of myo-inositol on plasma lipids of diabetic rats may be independent of insulin. Myo-inosi tol could have induced its effect on adipose tissue lipolysis through the sympathetic nerve system. Hayashi et a l . (1974) observed an increased mobil izat ion of fatty acid from adipose tissue to the l iver in the inositol-deficient rats. Subsequent work by these investigators (1978) suggested that the increased lipolysis associated with an inositol-deficient state may be mediated by an exci ta t ion of the sympathetic nerve terminals innervating in the adipose tissues and the resulting act ivat ion of hormone-sensitive lipase. Hence, i t is possible that pretreatment wi th myo-inositol could inhibit lipolysis in adipose tissue by depressing sympathetic nerve terminals, which may then decrease the plasma l ipid levels of the diabetic rats. 95 The myocardial l ipid-lowering effect of myo-inositol in these diabetic animals is s t i l l not clear. However, myo-inositol is involved in the synthesis of phosphatidylinositol (Paulus and Kennedy, 1960), which further increases the synthesis and secretion of lipoproteins in the l iver (Hasan et a l . , 1970; Hasan et a l . , 1971). A depression in the levels of total plasma lipoprotein and plasma PI in inositol-deprived dams during lactat ion has been observed (Burton and Wells, 1971). Thus, myo-inositol supplementation could have enhanced the removal of cholesterol from the myocardium by increasing the levels of lipoproteins in these animals. Tr iacy lg lycero l also decreased in the myocardium in these myo-inositol treated rats. This could have been achieved by a decrease in plasma F F A as pointed out earlier. The effect of myo-inositol on myocardial l ip id levels could also be attr ibuted to decreased synthesis of lipids in the myocardium. In fact, increased d£ novo synthesis of lipogenetic enzymes in the l iver during myo-inositol deficiency has been observed (Beach and F l i c k , 1982). It could be speculated that myo-inositol treatment may decrease the synthesis of lipogenic enzymes in the myocardium. Synthesis of lipogenic enzymes may also be depressed in the l iver and adipose tissues. Thus, less lipids would be available for the uptake of the myocardium. Since elevated myocardial l ip id level has been suggested to be one of the important contributors in the development of cardiac dysfunction in diabetic animals, myo-inositol may improve cardiac performance of diabetic rats by lowering myocardial l ipid levels, wi th a concomitant decrease in left ventr icular stiffness, decrease in L C A C level and cardiac oxygen demand, and an improvement in myocardial membrane integrity and 96 subcellular act ivi t ies . These possible effects of myo-inositol on the cardiac performances of diabetic rats requires further investigation. In addition, improvements in other cel lular defects of the diabetic state by myo-inositol treatment could also be involved in the improved cardiac function of the treated diabetic animals. Myo-inosi tol is involved in the synthesis of phosphatidyl-inositol which is an integral part of the membrane structure and has important biochemical functions at the membrane level including the mediation of cel lular responses to external s t imul i , nerve transmission, and the regulation of enzyme ac t iv i ty through specific interactions with various proteins (Holub, 1982). Thus, myo-inositol supplementation may enhance the integri ty of the membrane structure of myocardial cells associated with an improvement of biochemical function of the membrane. Myo-inosi tol is also involved in the synthesis of inositol trisphosphate (IPg) under muscarinic and alpha-adrenergic receptor st imulation (Brown and Jones, 1986). IPg has been shown by some investigators (Hirata et a l . , 1984; Fabiato, 1986; Nosek et a l . , 1986) to be able to release calc ium from cardiac SR, although this has been a subject of controversy. Release of ca lc ium from the SR may then lead to exci tat ion-contract ion coupling in cardiac cel ls . Hence, myo-inositol may improve cardiac performance by releasing calc ium from the SR through IP^. Myo-inos i to l metabolism has been reported to be altered in diabetic patients (Clements and Reynertson, 1977) and STZ diabetic rats (Goldfarb et a l . , 1986). A n increased renal excret ion of myo-inositol (Vohl, 1958; Daughaday et a l . , 1953) and an intracellular deficiency of myo-inositol (Clements and Reynertson, 1977) have been well documented. Thus it is 97 possible that supplementation with myo-inositol could have simply improved the myo-inositol status of the diabetic animals, which subsequently restored heart function. Our study showed that myo-inositol treatment alone par t ia l ly improved cardiac function and decreased myocardial and plasma l ipid levels, while hypothyroidism associated wi th the diabetes state was s t i l l present. Hypothyroidism is known to slow relaxation and to depress the force-veloci ty relat ion in papillary muscles (Buccino et a l . , 1967). Ca lc ium transport by the SR has also been shown to be depressed in the hypothyroid state (Suko, 1973). On the other hand, the increase in calc ium accumulating abi l i ty of the SR has been observed in hyperthyroid animals (Suko, 1971; Limas , 1978). In addition, greater cont rac t i l i ty has been shown in papillary muscles from hearts of guinea pigs treated with thyroxine (Goodkind et a l . , 1974). Furthermore, Di l lman (1982) studied the effect of thyroid hormone replacement therapy on diabetes-induced depression of myosin ATPase . T^ treatment at a pharmacological dose (30 ug/kg~*/day~*) successfully prevented diabetes-induced hypothyroidism and normalized cardiac myosin A T P a s e ac t iv i ty . The decrease in heart rate occurring as a result of diabetes is also correctable by thyroid hormone treatment (Goyal and M c N e i l l , unpublished results). Thus i t is possible that cardiac dysfunction seen in diabetics could be due to hypothyroidism associated with the disease. Therefore, in the third study, both myo-inositol and T^ were administered to the diabetic rats. We wanted to see i f the combination treatment could further improve cardiac function in diabetic animals. 98 A s wi th myo-inositol treatment alone, Tg alone or in combination wi th myo-inositol could not alter STZ-induced diabetic state in rats, which was character ized by a loss of body weight and hyperglycemia wi th corresponding hypoinsulinemia. In addition, untreated diabetic rats had significantly higher fluid intake, which was further increased wi th Tg alone or in combination with myo-inositol . The further increase in fluid intake wi th Tg treatment may be attributed to the high metabolic state associated wi th Tg administration. It is well established that the oxygen consumption and metabolic rate are increased by T^ and Tg (Ganong, 1985). Therefore endogenous protein and fat stores are catabolized, which can result in diuresis. Thus increased fluid intake by Tg treatment may be due to diuresis and a high metabolic state. When plasma and myocardial l ipid levels of the diabetic rats were examined, the l ipid levels were found to be unchanged or even increased wi th Tg and MI plus Tg treatment. Cardiac function was depressed in untreated diabetic rats and Tg treatment alone did not appear to have any effect on cardiac dysfunction. There was, however, some improvement in heart function in the groups treated wi th both myo-inositol and Tg, but the improvement was not as pronounced as wi th myo-inositol treatment alone. The failure of Tg to improve heart function shown in our study was not unexpected and was consistent with previous work. Fe in et a l . (1981) observed that there was a dissociation between papillary muscle mechanics and thyroid hormone levels. Furthermore, Tahil iani and M c N e i l l (1984) were 99 unable to improve cardiac function of diabetic hearts by treating the diabetic rats wi th thyroid hormone. On the other hand, previous reports have indicated that treating normal (euthyroid) animals wi th thyroid hormone did result in enhanced cardiac function as compared wi th untreated euthyroid animals in at least some species (Taylor, 1970; Brooks et a l . , 1981). In our study, a dose of 30 ug/kg~*/day~* of T^ was administered to diabetic rats as T^ treatment at this dose has been previously shown to prevent diabetes-induced hypothyroidism and normalize cardiac myosin ATPase ac t iv i ty . However, cardiac function of diabetic rats remain depressed. These observations confirm the suggestions made by Tahi l iani and M c N e i l l (1984) that cardiac dysfunction of diabetic animals does not seem to result from hypothyroidism and decreased myosin ATPase ac t iv i ty . A less pronounced improvement of cardiac function of diabetic animals was observed wi th myo-inositol plus T^ treatment compared to myo-inositol alone. This could be related to the calorigenic and diabetogenic effects of thyroid hormones. A s discussed above, a major effect of thyroid hormones is to stimulate oxygen consumption and increase metabolic rate (calorigenic effect) Thus, i t is possible that when the metabolic rate is increased, the need for myo-inositol is increased. Myo-inosi tol supplementation at the same dose as when myo-inositol was given alone may not be sufficient for the diabetic animals who received T^ at the same t ime. Thyroid hormones have also been shown to have a diabetogenic effect. Thyrotoxicosis was reported to aggravate c l in ica l diabetes and thyroid diabetes can be 100 produced in animals with decreased pancreatic reserve (Ganong, 1985). Several mechanisms have been postulated for this diabetogenic effect of thyroid hormones. Thyroid hormones may increase absorption of glucose from the intestine (Ganong, 1985). The hormones may also cause some degree of hepatic glycogen depletion probably by potentiating the effects of catcholamines. Glycogen-depleted l iver cells are easily damaged. When the l iver is damaged, it takes up less of the absorbed glucose. In addition, thyroid hormones may enhance glycolysis and gluconeogenesis (Guyton, 1986). Furthermore, thyroid hormones may accelerate the degradation of insulin. A l l these actions have a hyperglycemic effect and, when the pancreatic reserve is already low, may make experimental diabetes worse. Therefore, even though myo-inositol was supplemented, the combination of both could not further improve heart function of diabetic animals. Tg alone or in combination wi th myo-inositol did not have any effect on or even further increase the l ip id levels of diabetic rats compared to untreated diabetic rats. Several mechanisms may be involved in the elevation of l ip id levels of plasma and myocardium of diabetic rats treated wi th Tg . Thyroid hormones may stimulate cholesterol synthesis in the l iver (Goodman and Middlesworth, 1980). Thyroid hormones may also increase the synthesis and mobil izat ion of F F A from adipose tissue. There is evidence that in hypothyroid rats the synthesis of long-chain fatty acids from glucose and other carbohydrate precursors is diminished. S imi lar ly , oxidation of long-chain fatty acids is reduced (Bray and Goodman, 1968). Thus, administration of Tg may increase myocardial lipids by increasing synthesis and release of F F A from adipose tissue and increasing the concentrations of F F A in plasma. 101 The present study shows that myo-inositol treatment influenced l ipid metabolism and improved cardiac performance in STZ diabetic rats. While it appears that control of l ipid levels is an important factor in the improvement of cardiac function of diabetic animals, it should be noted that the myo-inositol-plus-Tg treatment did not affect the elevated l ipid levels of diabetic rats even though the heart function of these diabetic animals was part ial ly improved. It w i l l nevertheless be of interest in future studies to investigate the mechanism of action of myo-inositol in l ipid metabolism. Since myo-inositol metabolism is altered in diabetes and myo-inositol and its derivatives have been suggested to act as second messengers in mediating a positive inotropic response in the heart, myo-inosi-tol supplementation could be of great importance in regulating myocardial cont rac t i l i ty at the cel lular and subcellular levels in diabetic animals and man. 102 C O N C L U S I O N S 1. STZ-induced 8-week diabetic rats exhibited cardiac dysfunction as assessed by a depression of the L V D P , rate of pressure rise and rate of pressure decline in the isolated working heart. 2. Paral le l ing the- depression of cardiac function, an elevation in plasma and myocardial t r iacylglycerols , cholesterol and phospholipids levels was seen in diabetic animals. 3. Myo-inosi tol treatment part ial ly prevented the depression of heart function of diabetic rats. The treatment was also effective in lowering plasma as well as myocardial tr iglycerols, cholesterol and phospholipid levels. The mechanism by which myo-inositol lowered lipids and improved heart function remains to be explored. 4. Myo-inosi tol failed to correct diabetes-induced hypothyroidism. However, improvement of cardiac function of diabetic rats was less pronounced with a combination treatment of both myo-inositol and Tg compared to myo-inositol treatment alone, which may be related to the calorigenic and diabetogenic effects of thyroid hormones. 5. Tg alone or in combination wi th myo-inositol had no effect on, or even further increased plasma and myocardial l ip id levels of diabetic rats, even though heart function of these diabetic animals was part ial ly improved. In summary, myo-inositol appeared to control l ipid metabolism as well as modify cardiac dysfunction of diabetic rats. However, myo-inositol-treatment in combination with Tg did not lower the l ipid levels of the diabetic rats, even though cardiac function of these diabetic animals 103 was part ial ly improved. These findings do not support a causal relationship between elevated l ip id levels and cardiac dysfunction in diabetic rats. 104 R E F E R E N C E S A d a m , R . J . , Cohen, D.W. , Gupte, J . , Johnson, D . , Wal l ick , E .T . , Wang, T. and Schwartz, A . : In vi t ro effects of palmitylcarni t ine op+ cardiac plasma ^membrane Na , K - A T P a s e , and sarcoplasmic ret iculum C a -ATPase and C a -transport. J . B i o l . Chem. 254: 12404-12410, 1978. Adlersberg, D . and Eisler , L . : Ci rcula t ing lipids in diabetes melli tus. J A M A 170: 1261-1265, 1959. Ahmed, S.S., Jafer i , G . A . , Narang, R . M . and Regan, T . J . : P rec l in ica l abnormality of left ventricular function in diabetes mell i tus. A m . Heart J . 89: 153-158, 1975. Albr iuk , M . J . , Lavietes, P . H . and Man, E . B . : Vascular disease and serum lipids in diabetes melli tus. Observations over 30 years (1931-1961). Ann . Intern. Med . 58: 305-323, 1963. Antonis, A . and Bersohn, I.: Serum-triglyceride levels in South Af r i can Europeans and Bantu and in ischemic heart disease. Lancet , I: 998-1002, 1960. Ar ie f f , A . I . and Ca ro l l , H . J . : Nonketot ic hyperosmolar coma wi th hyperglycemia: c l in ica l features, pathophysiology, renal function, acid base balance, plasma cerebrospinal f luid equilibria and the effects of therapy in 37 cases. Medicine 51: 73-94, 1972. A t k i n , E . and Meng, H . C . Release of clearing factor lipase (lipoprotein lipase) in vivo and from isolated perfused hearts of alloxan diabetic rats. Diabetes 21 (Suppl. 3): 149-156, 1972. Bagdade, J .D . , Porte , D . and Bierman, E . L . : Acu te insulin withdrawal and the regulation of plasma tr iglyceride removal in diabetic subjects. Diabetes 17: 127-132, 1968. Balodimos, M . C . : Diabet ic Nephropathy. In: "Joslin's Diabetes melli tus." Ed . Marble, A . , White, P . , Bradley, R . F . and Kra l l s , R . F . Lea & Febinger, Philadelphia. 1971. Balsam, A . , Inbar, S .H. and Sexton, F . : The influence of fasting, diabetes and several pharmacological agents on the pathways of thyroxine metabolism in rat l iver . J . C l i n . Invest. 62: 415-424, 1978. Banerjee, S.P. and Kung, L .S . : Beta-adrenergic receptors in rat heart: Effects of thyroidectomy. Eur. J . Pharmacol . 13: 207-208, 1977. Banting, F . G . and Best, C . H . : Pancreat ic extracts. J . Lab . C l i n . Med. 7: 464, 1922. 105 Bar, R .S . , Muggeo, M . , Roth , J . , Kahn, C . R . , Havrankova, J . and Imperato-McGinley, J . : Insulin resistance, acanthosis nigricans and normal insulin receptors in a young woman: evidence for a post-receptor defect. J . C l i n . Endocrinol. Metab. 47: 620-625, 1978. Beach, D . C . and F l i c k , P . K . Ear ly effect of myo-inositol deficiency on fatty acid synthetic enzymes of rat l iver . B ioch im. Biophys. A c t a 711: 452-459, 1982. Berridge, M . J . : Inositol triphosphate and diacylglycerol as second messengers. Biochem. J . 220: 345-360, 1984. Berridge, M . J . : The molecular basis of communication within the ce l l . Sc i . A m e r . 253 (4): 142-152, 1985. Berridge, M . J . and Irvine, R . F . : Inositol triphosphate, a novel second messenger in cel lular signal transduction. Nature 312: 315-321, 1984. Best, C . H . : Epochs in the history of diabetes. In: Diabetes. Ed . R . H . Wil l iams, pp. 1-13, Paul B . Hoeber, Inc., Medica l Divis ion of Harper & Brothers, N . Y . , 1960. Beyer-Mears, A . , K u , L . and Cohen, M . P . : Glomerular polyol accumulation in diabetes and its prevention by oral sorbinil . Diabetes 33: 604-607, 1984. B i l l imor i a , J . D . , Issacs, A . J . and M e l k i , K . : A l ipid and lipoprotein profile of treated and untreated diabetics. Ann . C l i n . Biochem. 13: 315-321, 1976. Bloor, W.R. : The lipoids of the blood in diabetes. J . B i o l . Chem. 26: 417-430, 1916. Boado, R . , Brown, T . J . , Bromage, N . R . , Mat ty , A . J . and Zaninovich, A . A . : Triiodothyronine metabolism in diabetic rats. A c t a Endocrinol . 89: 323-328, 1978. Braverman, L . E . , Ingbar, S .H. and Sterl ing, K . : Conversion of thyroxine ( T j to triiodothyronine (T„) in athyreotic human subjects. J . C l i n . Invest. 49: 855-864, 1970. J Bray, G . A . and Goodman, H . M . : Metabolism of adipose tissue from normal and hypothyroid rats. Endocrinology 82: 860-864, 1968. Brooks, I., Flynn, S.B. and Underwood, A . H . : Effects of L-3,3 ' ,5 ' - t r i iodothyronine on rat cardiac function. Br . J . Pharmacol . 73: 138P, 1981. 106 Brown, J . H . , Buxton, I .L . and Brunton, L . L . : Alpha-adrenergic and muscarinic cholinergic st imulation of phosphoinositide hydrolysis in adult rat cardiomyocytes. C i r c . Res. 57: 532-537, 1985. Brown, J . H . and Jones, L . G . Phosphoinositide metabolism in the heart. In: Phosphoinositides and receptor mechanisms, Ed . Putney, J .W., pp. 245-270, A l a n R . Liss , Inc., New York , 1986. Brown, M S . and Goldstein, J . L . : How L D L receptors influence cholesterol and atherosclerosis. S c i . A m . 251: 58-66, 1984. Brownlee, M . and Cerami , A . : The biochemistry of the complications of diabetes melli tus. Annu. Rev . Biochem. 50: 385-432, 1981. Buccino, R . A . , Spann, J .F . , Pool , P . E . , Sonnenblick, E . H . and Braunwald, E . : Influence of the thyroid state on the intrinsic contract i le properties and energy stores of the myocardium. J . C l i n . Invest. 46: 1669-1682, 1967. Burton, L . E . , Wells, W.W.: Character izat ion of the lactation-dependent fatty l iver in myo-inositol deficient rats. J . Nutr . 107: 1871-1883, 1977. Burton, L . E . and Wells, W.W.: Studies on the developmental pattern of the enzymes converting glucose 6-phosphate to myo-inositol in the rat. Dev. B i o l . 37: 35-42, 1974. Carlson, L . A . : Serum lipids in man wi th myocardial infarct ion. A c t a . Med. Scand. 167: 399-413, 1960. Car ter , W . J . , Van der Weijden Banjamin, W.S. and Faas, F . H . : Effect of thyroid hormone on protein turnover in cultured cardiac myocytes. J . M o l . C e l l . Card io l . 17: 897-905, 1985. Caspary, W.F . and Crane, R . K . : A c t i v e transport of myo-inositol and its relat ion to the sugar transport system in hamster small intestine. Bioch im. Biophys. A c t a 203: 308-316, 1970. Chai t , A . , Bierman, E . L . and Albers , J . J . : Regulating role of triiodothyronine in the degradation of low density lipoproteins by cultured human skin fibroblasts. J . C l i n . Endocrinol . Metab. 48: 887-889, 1979. Chen, C . H . J , and Eisenberg, F . : Myoinosose-2-1-phosphate: an intermediate in the myoinositol-l-phosphate synthase reaction. J . B i o l . Chem. 250: 2963-2967, 1975. C ia ra ld i , T. and Mar ine t t i , G . V . : Thyroxine and propylthiouracil effects in vivo on alpha- and beta-adrenergic receptors in rat heart. Biochem. Biophys. Res. Comm. 74: 984-991, 1977. 107 Clements, R .S . and Diethelm, A . G . : The metabolism of myo-inositol by the human kidney. J . Lab. C l i n . Med . 93: 210-219, 1979. Clements, R . S . and Reynertson, R . : Myoinosi tol metabolism in diabetes melli tus: Effect of insulin treatment. Diabetes 26: 215-221, 1977. Coleman, D . L . and Burkart , D . L . : Plasma corticosterone concentrations in diabetic (db) mice. Diabetologia 13: 25-26, 1977. Cooppan, R . : Recognit ion of hypothyroidism in diabetes. Prac t . Card io l . 8 (7): 113-115, (1982). Cudworth, A . G . and Woodrow, J . C . : Evidence for H L A - l i n k e d genes in "juvenile" diabetes melli tus. Br i t . Med . J . 3: 133, 1975. Cul len , W.: A treatise of the materia medica. 2 vols. Edinburgh, C . E l l io t , 1789. Daughaday, W . H . , Larner, J . and Houghton, E . : The renal excretion of inositol in normal and diabetic human beings. J . C l i n . Invest. 33: 326-332, 1953. Davis, M . D . : Defini t ion, classif icat ion and course of diabetic retinopathy. In: Diabet ic Retinopathy, Ed. J .R . L y n , W.B. Snyder and A . Vassier, pp. 145-170, Grune and Stratton, New York , 1974. Deming, Q .B . , Hodes, M . E . , Bal tzar , A . , Edreira , J . G . and Torosdag, S.: The changes in concentration of cholesterol in the serum of hypertensive patients during antihypertensive therapy. A m . J . Med. 24: 882-892, 1958. Denton, R . M . and Randle, R . J . : Concentrat ion of glycerides and phospholipids in rat heart and gastrocnemius muscles. Effects of alloxan-diabetes and perfusion. Biochem. J . 104: 416-422, 1967. Desclaux, P . , Soulairac, A . and Teyseyre, J . : Modifications hypophysaires au cours du diabete alloxanique du rat. C . R . Soc. B i o l . (Paris) 142: 491-493, 1948. (Comptes Rendus des Seances. Societe de Biologie et de Ses Fi l ia les) . D i l lman , W . H . : Diabetes mellitus induces changes in cardiac myosin of the rat. Diabetes 29: 579-582, 1980. Di l lman , W . H . : Influence of thyroid hormone administration on myosin A T P a s e ac t iv i ty and myosin isoenzyme distribution in the heart of diabetic rats. Metabolism 31: 199-204, 1982. 108 Dobson, M . : Experiments and observations on the urine in diabetics. Med. Obs. Ingu. 5: 298-316, 1776. D o t t i , L . B . , Fe lch , W . C . and Ilka, S.J . : Inhibiting effect of inositol on serum cholesterol and phospholipids following cholesterol feeding in rabbits. Proc . Soc. Exp. B i o l . Med. 78: 165-167, 1951. Elkeles, R .S . and Hambley, J . : The effects of fasting and streptozotocin diabetes on hepatic t r iglyceride lipase ac t iv i ty in the rat. Diabetes 26: 58-64, 1977. Fabiato, A . : Inositol (l,4,5)-trisphosphate-induced release of C a 2 + from the sarcoplasmic ret iculum of skinned cardiac cel ls . Biophys. J . 49: 190a, 1986. Fa in , J . N . : Biochemical aspects of drug and hormone action on adipose tissue. Pharmacol . Rev . 25: 67-118, 1973. Fe in , F .S . , Kornstein, L . B . , Strobeck, J . E . , Capasso, T . N . and Sonnenblick, E . H . : A l t e r e d myocardial mechanics in diabetic rats. C i r c . Res. 47: 922-933, 1980. Fe in , F .S . , Mi l l e r -Green , B . and Sonnenblick, E . H . : A l t e r e d myocardial mechanics in diabetic rabbits. A m . J . Physiol . 248: H729-H736, 1985. Fe in , F .S . , Strobeck, J . E . , Malhotra , A . , Scheuer, J . and Sonnenblick, E . H . : Revers ib i l i ty of diabetic cardiopathy with insulin in rats. C i r c . Res. 49: 1251-1261, 1981. Feuvray, D . , Idell-Wenger, J . A . and Neely, J .R . : Effects of ischemia on rat myocardial function and metabolism in diabetes. C i r c . Res. 44: 322-329, 1979. F lock , E . V . , Bol lman, J . L . , Grindlay, J . H . and Stoble, G . H . : Par t i a l deiodination of L-thyroxine. Endocrinology 69: 626-637, 1961. Frederickson, D.S. and Gordon, R.S . : Transport of fatty acids. Physiol . Rev . 38: 585-630, 1958. Fre inkel , N . , Goodner, C . J . and Dawson, R . M . C . : The relationship between renal glucose and inositol transport (abstract). C l i n . Res. 8: 240, 1960. Ganguly, P . K . , Pierce , G . N . , Dhal la , K . S . and Dhal la , N .S . : Defect ive sarcoplasmic ret icular calcium transport in diabetic cardiomyopathy. A m . J . Physiol . 244: E528-E535, 1983. Ganoug, W.F . : Review of medical physiology. 12th Ed . , pp. 258-292, Lange Medica l Publications, Los Al tos , C A , 1985. 109 Gavin , L . , Cas t le , J . , McMahon, F . , Mar t in , P . , Hammond, M . and Cava l i e r i , R . R . : Extrathyroidal conversion of thyroxine to 3,3' ,5'-triiodothyronine ( R e v e r s e - T j and to 3,5,3'-triiodothyronine (Tg) in humans. J . C l i n . Endocrinol . Metab. 44: 733-742, 1977. Gavin , G . and McHenry, E.W.: Inositol: A l ipotropic factor. J . B i o l . Chem. 139: 485, 1941. Gavin , L . , McMahon, F . and Moel ler , M . : The mechanism of impaired Tg production from in diabetes. Diabetes 30: 694-699, 1981. Gepts, W.: Pathologic anatomy of the pancreas in juvenile diabetes melli tus. Diabetes 14: 619-633, 1965. Ger ich , J .E . : Insulin-dependent diabetes mellitus: pathophysiology. Mayo C l i n . P roc . 61: 787-791, 1986. Goldberg, R . B . : L i p i d disorders in diabetes. Diabetes Care 4(5): 561-572, 1981. Goldfarb, S., Simmons, D . A . and Kern , E . : Amel iora t ion of glomerular hyperfi l t rat ion in acute experimental diabetes by dietary myo-inositol and by an aldose reductase inhibitor. C l i n . Res. 34(2): 725A, 1986. Goodkind, H . J . , Damback, G . E . , Thyrum, P .T . and L u c k i , R . : Effect of thyroxine on ventricular myocardial contract i l i ty and ATPase ac t iv i ty in guinea pigs. A m . J . Physiol . 226: 66-72, 1974. Goodman, H . M . and Middlesworth, L . V . : The thyroid gland. In: Medica l Physiology, Ed . Mountcastle, V . B . 14th ed., pp. 1510, the C . V . Mosby Company, St. Louis, Missouri , 1980. Graves, R . J . : Newly observed affection of the thyroid gland in females. London Medica l and Surgical Journal 7: 516-517, 1835. Greene, D . A . , Dejesus, P . V . and Winegrad, A . I . : Effects of insulin and dietary myoinositol on impaired peripheral motor nerve conduction veloci ty in acute streptozotocin diabetes. J . C l i n . Invest. 55: 1326-1336, 1975. Greene, D . A . and La t t imer , S .A. : Biochemical alterations and complications in diabetes. C l i n . Chem. 32 (10B): B42-B47, 1986. Greene, D . A . and La t t imer , S .A. : Impaired rat sc ia t ic nerve sodium-potassium adenosine triphosphatase in acute streptozotocin diabetes and its correct ion by dietary myo-inositol supplementation. J . C l i n . Invest. 72: 1058-1063, 1983. 110 Guyton, A . C . : The thyroid metabolic hormones. In: Textbook of Medica l Physiology, Ed . Dreibelbis, D . 6th Ed . , pp. 901, W.B. Saunders Company, Philadelphia, P A , 1986. Haider, B . , Y e h , C , Thomas, G . , et a l . : A l t e r ed myocardial function and collagen in diabetic rhesus monkeys on atherogenic diet. Trans. Assoc. A m . Physicians 91: 197-203, 1978. Hamby, R . I . , Zoneraich, S. and Sherman, S.: Diabet ic cardiomyopathy. J A M A 229: 1749-1754, 1974. Hammerman, M . R . , Sacktor, B . and Daughaday, W . H . : Myo-inosi tol transport in renal brush border vesicles and its inhibit ion by D-glucose. A m . J . Physiol . 239: F l 13-120, 1980. Harris , D . P . , Marr io t t , M . L . and M c N e i l l , J . H . : Microcomputer acquisition and analysis system for the isolated working heart preparation. J . Pharmacol . Methods 10: 65-73, 1983. Hasan, S .H. , Ko tak i , A . and Y a g i , K . : Studies on myo-inositol . VI. Effect of myoinositol on plasma lipoprotein metabolism of rats suffering from fatty l iver . J . Vi taminol . 16: 144-148, 1970. Hasan, S .H. , Nakagawa, Y . , Nishigaki , I. and Y^agi, K . : Studies on myoinositol . VIII. The incorporation of H-myoinosi tol into phosphatidylinositol of fatty l iver . J . Vi tamino l . 17: 159-162, 1971. Haslam, R . J . and Davidson, M . M . L . : Receptor-induced diacylglycerol formation in permeabilized platelets; possible role for a GTP-binding protein. J . Recept . Res. 4: 605-629, 1984. Hayashi, E . , Maeda, T. , Hasegawa, R . and Tomi ta , T.: The effect of myo-inositol deficiency on l ip id metabolism in rats. HI. The mechanism of an enhancement in lipolysis due to myo-inositol deficiency in rats. Biochem. Biophys. A c t a 531: 197-205, 1978. Hayashi, E . , Maeda, T. and Tomi ta , T.: The effect of myo-inositol deficiency on l ip id metabolism in rats. I. The al terat ion of l ip id metabolism in myo-inositol-deficient rats. B ioch im. Biophys. A c t a 360: 134-145, 1974. Henderson, A . H . , Most, A . S . , Parnully, W.W., Gor l in , R . and Sonnenblick, E . H . : Depression of myocardial cont rac t i l i ty in rats by free fatty acids during hypoxia. C i r c . Res. 26: 439-448, 1970. I l l Herbert , V . , Lau , K . S . and Parient, F .W. : Coated charcoal immunoassay of insulin. J . C l i n . Endocrinol . 25: 1375-1384, 1965. Heyliger , C . E . , Rodrigues, B . B . and M c N e i l l , J . H . : Effect of choline and methionine treatment on cardiac dysfunction of diabetic rats. Diabetes 35(10): 1152-1157, 1986. Hi ra ta , M . , Suejnatsu, E . , Hashimoto, T. , Hamachi , T. and Koga, T.: Release of C a from a non-mitochondrial store site in peritoneal macrophages treated with saponin by inositol 1,4,5-trisphosphate. Biochem. J . 223: 229-236, 1984. Hoh, F . Y . , M c G r a t h , P . A . and Hale, P .T . : Electrophoret ic analysis of mult iple forms of rat cardiac myosin: effects of hypophysectomy and thyroxine replacement. J . M o l . C e l l . Card io l . 10: 1053-1076, 1977. Holub, B . J . : The nutrit ional significance, metabolism, and function of myo-inositol and phosphatidylinositol in health and disease. Adv . Nutr . Res. 4: 107-141, 1982. Howard, C . F . : Diabetes and l ip id metabolism in nonhuman primates. Advances in L i p i d Research 13: 91-134, 1975. Howard, C . F . and Anderson, L . : Metabolism of myo-inositol in animals. II. Complete catabolism of myo-inosi tol- C by rat kidney slices. A r c h . Biochem. Biophys. 118: 332-339, 1967. Ingebretsen, C . G . , Moreau, P . , Hawelu-Johnson, C . and Ingebretsen, W.: Performance of diabetic rat hearts: effects of anoxia and increased work. A m . J . Physiol . 239: H614-H620, 1980. Jadhav, A . L . and Jandhyala, B.S. : Effects of chronic administration of vanadate on blood-pressure, heart rate and N a / K -ATPase ac t iv i ty in adult male Wistar rats. A r c h . Int. Pharmacodyn. Ther. 263: 74-84, 1983. Johansen, K . and Hansen, A a . P . : High 24-hour level of serum growth hormone in juvenile diabetics. Br . Med. J . 2: 356-357, 1969. Kahn, C . R . , Meghesi, K . and Barr , R .S . : Receptors for peptide hormones. A n n . Intern. Med. 86: 205-219, 1977. Kannel , W.B. : Role of diabetes in cardiac disease: conclusions from population studies. In: Diabetes and the heart, Ed . S. Zoneraich, pp. 97-112, Charles C . Thomas, Springfield, 1978. Kannel , W.B. , Hjortland, M . and Cas te l l i , W.P. : Role of diabetes in congestive heart failure: The Framingham Study. A m . J . C a r d i o l . 34: 29-35, 1974. 112 K a r l i , J . N . , Kar ikas , G . A . , Hatzipavlou, P . K . , Levis , G . M . and Moulopoulos, S .N. : The inhibition of Na and K stimulated ATPase ac t iv i ty of rabbit and dog heart sarcolemma by lysophosphatidyl choline. L i f e S c i . 24: 1869-1876, 1979. Kass, E . H . : Asymptomat ic infections of the urinary tract . Trans. Assoc. A m . Physicians. 119: 56-63, 1956. Kissebah, A . H . , Adams, P .W. and Wynn, V . : Inter-relationship between insulin secretion and plasma free fatty acid and tr iglyceride transport kinetics in maturi ty onset diabetes and the effect of phenethylbiguanide (phenformin). Diabetologia 10: 119-130, 1974. Kreisberg, R . A . : Effect of diabetes and starvation on myocardial tr iglyceride and free fatty acid u t i l iza t ion . A m . J . Physiol . 210: 379-384, 1966. K u , D . D . and Sellers, B . M . : Effect of streptozotocin diabetes and insulin treatment on myocardial sodium pump ac t iv i ty and cont rac t i l i ty of the rat heart. J . Pharmacol . Exp. Ther. 222: 395-400, 1982. Kuksis, A . and Mookerjea, S.: Choline. Nutr . Rev . 36: 201-207, 1978. L ' A g e , M . , Langholz, J . , Fechner, W. and Salzmann, H . : Disturbances of the hypothalamo-hypophysiol-adrenocortical system in the alloxan diabetic rat. Endocrinology 95: 760-765, 1974. Langerhans, P . : Contributions to the Microscopic Anatomy of the Pancreas. Ber l in , G . , Lange, 1869. Reprint of the German original with an English translation and an Introductory Essay by H . Morrison; Bal t imore , The Johns Hopkins Press, 1937. Lefebvre, P . J . and Luyckx, A . S . : Glucagon and diabetes: A reappraisal. Diabet-ologia 16: 347-354, 1979. Lernmark, A . , Freedman, Z . R . , Hofmann, C , Rubenstein, A . H . , Steiner, D . F . , Jackson, R . L . , Winter, R . J . and Fraisman, H.S. : Islet-cell surface antibodies in juvenile diabetes. N . Engl . J . Med. 299: 375-380, 1978. Levey, G.S.: Catecholamine sensitivity, thyroid hormone and the heart. Can. J . Med . 50: 413-420, 1971. Liewendahl, K . and Helenius, T.: Effect of fatty acids on thyroid function tests in vi t ro and in vivo. C l i n . C h i m . A c t a 72: 301-313, 1976. Limas , C . J . : Ca lc ium transport ATPase of cardiac sarcoplasmic reticulum in experimental hyperthyroidism. A m . J . Physiol . 235: H745-751, 1978. Lompre, A . M . , Mercadier , J . J . and Wisnensky, C : Species and age-depen-dent changes in the relat ive amounts of cardiac myosin isoenzymes in mam-mals. Dev. B i o l . 84: 286-290, 1981. Lopaschuk, G . D . , K a t z , S. and M c N e i l l , J . H . : The effect of alloxan and strep-tozotocin-induced diabetes on calcium transport in rat cardiac sarcoplasmic re t iculum. The possible involvement . of long chain acylcarnitines. A m . J . Physiol . Pharmacol . 61: 439-448, 1983. 113 Lopaschuk, G . D . , Tahl l iani , A . G . , Vadlamudi, R . V . S . V . , K a t z , S. and M c N e i l l , J . H . : Cardiac sarcoplasmic reticulum function in insulin- or carnit ine-treated diabetic rats. A m . J . Physiol . 245: H969-H976, 1983. Lowry , O . H . , Rosebrough, N . J . , Farr , A . L . and Randal l , R . J . : Prote in measurement wi th the Fo l in phenol reagent. J . B i o l . Chem. 193: 265-275, 1951. Malhotra , A . , Penpargkul, S., Fe in , F .S . , Sonnenblick, E . H . and Scheuer, J . : The effect of streptozotocin-induced diabetes in rats on cardiac contract i le proteins. C i r c . Res. 49: 1243-1250, 1981. Marble , A . : Current concepts of diabetes. In: Joslin's Diabetes Mel l i tus . 11th ed., Ed . A . Marble, P . White, R . F . Bradley, and L . P . K r a l l , pp. 1-2, Lea & Febiger, Philadelphia, 1971. Markowi tz , C . and Ya te r , W . M . : Response of explanted cardiac muscle to thyroxine. A m . J . Physiol . 100: 162-166, 1932. Mar t in , A . F . , Pagani, E . D . and Solaro, J .F . : Thyroxine-induced redistribution of isoenzymes of rabbit ventricular myosin. C i r c . Res. 50: 117-124, 1982. Mauck, L . A . , Wong, Y . H . and Sherman, W.R. : L-myo-inositol-1-phosphate synthase from bovine testis: purification to homogeneity and part ial character izat ion. Biochemistry 19: 3623-3629, 1980. Medal ie , J . H . , Papier, C , Herman, J .B . , Goldbourt, U . , Tamir , S., Neufeld, H . N . and Riss, E . : Diabetes melli tus among 10,000 adult men. 1. Five-year incidence of associated variables. Isr. J . Med . Sc i . 10: 681-697, 1974. Mering, J . von and Minkowski , O.: Diabetes mellitus nach Pankreasextirpation. A r c h . Exp. Path. Pharmak. 26: 371-387, 1890. M i c h e l l , R . H . : Inositol phospholipids and ce l l surface receptor function. Biochim. Biophys. A c t a 415: 87-147, 1975. M i c h e l l , R . H . : Inositol phospholipids in membrane function. Trends Biochem. Sc i . 4: 128-131, 1979. Mochizuk i , S., Ishikawa, Shin-Ichiro and Abe M . : Insulin treatment and myocardial function in isolated, perfused heart from diabetic rat. Jpn. C i r c . J . 48: 255-265, 1984. Morgan, H . E . , Cadenas, E . , Regen, D . M . and Park, C . R . : Regulat ion of glucose uptake in muscles. III. Ra te l imi t ing step and effects of insulin and anoxia in heart muscle from diabetic rats. J . B i o l . Chem. 236: 262-268, 1961. Mork in , E . , Fl ink, I .L. and Goldman, S.: Biochemical and physiologic effects of thyroid hormone on cardiac performance. Prog. Cardiovasc. Dis . 25: 435-464, 1983. 114 Morrison, A . D . : A o r t i c smooth muscle metabolism: effect of polyol pathway inhibit ion (abstract). C l i n . Res. 32: 851A, 1984. Murthy, V . K . , Bauman, M . D . and Shipp, J . C . : Regulation of t r iacylglycerol lipolysis in the perfused hearts of normal and diabetic rats. Diabetes 32: 718-722, 1983. Murthy, V . K . and Shipp, J . C . : Accumula t ion of myocardial tr iglycerides in ketot ic diabetes. Evidence for increased biosynthesis. Diabetes 26: 222-229, 1977. MacGregor , L . C . , Rosecan, L . R . , Lat ies , A . M . and Matchinsky, F . M . : Microanalysis of total l ip id , glucose, sorbitol and myo-inositol in individual ret inal layers of normal and alloxan diabetic rabbits (abstract). Diabetes 33: 89A, 1984. McGar ry , J . D . and Foster, D.W.: Hormonal control of ketogenesis. A r c h . Intern. Med. 137: 495-501, 1977. Naeije, R . , Golstein, J . , Clumeck, N . , Meinhold, H . , Wenzel, K . W . and Vanhaelst, L . : A low T~ syndrome in diabetic ketoacidosis. C l i n . Endocrinol . 8: 467-472, 1978: Neely, J .R . and Morgan, H . E . : Relationship between carbohydrate and l ip id metabolism and the energy balance of heart muscle. A n n . Rev . Physiol . 34: 413-459, 1974. Nerup, J . : H L A studies in diabetes mellitus: a review. A d v . Metab. Disord. 9: 263-277, 1978. New, M. I . , Roberts, T . N . , Bierman, E . L . and Reader, G . G . : The significance of blood l ip id alterations in diabetes mell i tus. Diabetes 12: 208-212, 1963. N i k k i l a , E . A . : Plasma triglycerides in human diabetes. Proc . Roy . Soc. Med. 67: 18-21, 1974. N i k k i l a , E . A . , Huttunen, J . K . and Ehnholm, C : Post-heparin plasma lipoprotein lipase and hepatic lipase in diabetes mell i tus. Diabetes 26(1): 11-21, 1977. Nishizuka, Y . : Phospholipid degradation and signal translation for protein phosphorylation. Trends Biochem. Sc i . 8: 13-16, 1983. Nishizuka, Y . : Prote in kinases in signal transduction. T I B A 9: 163-166, 1984. 115 Nosek, T . M . , Wil l iams, WjF. , Zeigler , S.T. and Godt, R . E . : Inositol trisphosphate enhances C a release from the sarcoplasmic ret iculum of skinned cardiac and skeletal muscle. Biophys. J . 49: 191a, 1986. Oakley, W . G . : C l i n i c a l manifestations and associations. In: C l i n i c a l diabetes and its biochemical basis, ed. W . G . Oakley, D . A . Ryke and K . W . Taylor , pp. 252-283, Blackwel l , Oxford, 1968. Ooi , B.S. , Chen, B . T . M . and Y u , M . : Prevalence and site of bacteriuria in diabetes melli tus. Postgrad. Med. 50: 497-499, 1974. Opie, L . H . : Metabolism of the heart in health and disease. A m . Heart J . 76: 685-698, 1969. Opie, L . H . : Metabol ic response during impending myocardial infarct ion. I. Relevance of studies of glucose and fatty acid metabolism in animals. Ci rcu la t ion 45: 483-490, 1972. Paija^gia, V . , Lamers, J . M . J . , Singal, P . K . and Dhal la , N .S . : C a 2 + - and M g -dependent ATPase act ivi t ies in the deoxycholate-treated rat heart sarcolemma. Int. J . Biochem. 14: 387-397, 1982. Paulson, D . J . and Crass, M . F . : Endogenous t r iacylglycerol metabolism in diabetic heart. A m . J . Physiol . 242: HI084-1094, 1982. Paulson, D . J . , Shetlar, D . and Light , K . E . : Catecholamine levels in the heart, serum and adrenals of experimental diabetic rats. Fed. Proc . 39: 637, 1980. Paulus, H . and Kennedy, E . P . : The enzymatic synthesis of inositol monophosphatide. J . B i o l . Chem. 235: 1303-1311, 1960. Pearce, F . J . , Forster, J . , Deleeuw, G . , Will iamson, J .R . and Tutwiler , G . F . : Inhibition of fatty acid oxidation in normal and hypoxic perfused rat hearts by 2-tetradecylglycidic acid. J . M o l . C e l l . Card io l . 11: 893-915, 1979. Penpargkul, S., Schaible, T. , Yip in t so i , T. and Scheuer, J . : The effect of diabetes on performance and metabolism of rat hearts. C i r c . Res. 47: 911-921, 1980. Periscas, I. and Jol in , T.: Effect of streptozotocin-induced diabetes on pi tui tary- thyroid axis in goitrogenic treated rats. A c t a Endocrinol . 86: 128-138, 1977. Perry, H . M . and Schroeder, H . A . : Depression of cholesterol levels in human plasma following ethylene diamine tetraacetate and hydralazine. J . Chronic Dis . 21: 520-533, 1955. 116 Peterson, D.W. , Gr i f f i t h , D.W. and Napolitano, C A . : Decreased myocardial cont rac t i l i ty in papillary muscle from atherosclerotic rabbits. C i r c . Res. 45: 338-346, 1979. P e z z o l i , C , F io r in i , R . M . , Curatola , G . and Stramentinoli , G . : S-adenosylmethionine protects against erythrocyte membrane alterations induced in rabbits by cholesterol-rich diet. Pharmacol . Res. Commun. 15: 785-795, 1983. Phil ipson, K . D . and Edelman, I.S.: Thyroid hormone control of N a - K - A T P a s e and K-dependent phosphatase in rat heart. A m . J . Physiol . 232: C196-C201, 1977. P ie rce , G . N . and Dhal la , N .S . : Membrane defects during the development of diabetic cardiomyopathy. J . M o l . C e l l . Card io l . 18 (Supp. 3): 56, 1986. Pierce , G . N . and Dhal la , N .S . : Cardiac myofibr i l lar A T P a s e ac t iv i ty in diabetic rats. J . M o l . C e l l . Card io l . 13: 1-7, 1981. Pi tkanen, E . : The serum polyol pattern and the urinary polyol excret ion in diabetic and in uremic patients. C l i n . C h i m . A c t a 38: 221-230, 1972. P i t tman, C .S . , Suda, A . K . , Chambers, J .B . , McDan ie l , H . G . , Ray, G . Y . and Preston, B . K . : Abnormali t ies of thyroid hormones, turnover in patients wi th diabetes mellitus before and after insulin therapy. J . C l i n . Endocrinol . Metab. 48: 854-860, 1979.1 Pi t tman, C .S . , Suda, A . K . , Chambers, J . B . and Ray, G . Y . : Impaired 3,5,3'-triiodothyrorune (T„) production in diabetic patients. Metabolism 28: 333-338, 1979. J Pi t t -R ive r s , R . , Stanbury, J . B . and Rapp, B . : Conversion of thyroxine to 3,5,3'-triiodothyronine in vivo. J . C l i n . Endocrinol . Metab. 15: 616-620, 1955. Poggiol i , J . , Sulpice, J . C . and Vassort, G . : Inositol phosphate production following alpha-1-adrenergic, muscarinic or e lec t r ica l s t imulation in isolated rat heart. F E B S Let ters , 206(2): 292-298, 1986. Pomeranze, J . and Kunkel , H . G . : Serum lipids in diabetes melli tus. Proc. A m e r . Diabetes Assoc. 10: 217-231, 1950. Quist, E . E . : Evidence for a carbachol st imulated phosphatidylinositol effect in heart. Biochem. Pharmacol . 31(19): 3130-3133, 1982. Randle, P . J . , Garland, P . B . , Hales, C . N . , Newsholme, E . A . , Denton, R . M . and Pogson, C.I . : Interaction of metabolism and the physiological role of insulin. Recent Prog. Horm. Res. 22: 1-44, 1966. 117 Regan, T . J . , Et t inger, P . O . , Khan, M. I . , Jesrani, M . V . , Lyons, M . M . , Oldewurtel , H . A . and Weber, M . : A l t e r e d myocardial function and metabolism in chronic diabetes melli tus without ischemia in dogs. C i r c . Res. 35: 222-237, 1974. Regan, T . J . , Khan, M.I . , Jesrani, M . U . , Oldewurtel , H . A . and Ett inger, P .O . : Al tera t ions of myocardial function and metabolism in chronic diabetes melli tus. In: Recent Advances in Studies on Cardiac Structure and Metabol ism. Dhal la , N . S . , E d . Bal t imore, M D , Univers i ty Park Press, pp. 169-178, 1973. Regan, T . J . , Lyons, M . M . , Ahmed, S.S., Levinson, G . E . , Oldewurtel , H . A . , Ahmed, M . R . and Haider, B . : Evidence for cardiomyopathy in famil ia l diabetes melli tus. J . C l i n . Invest. 60: 885-899, 1977. Renold, A . E . , M i n t z , D . H . , Mul ler , W . A . and C a h i l l , G . F . : Diabetes mell i tus. In: The metabolic basis of inherited disease. Eds. Stanburg, J .B . , Wyagaarden, J . B . and Fredrickson, D.S. M c G r a w - H i l l , New York , pp. 79-109, 1978. Rodrigues, B . B . , Agrawal , D . K . and M c N e i l l , J . H . : A r e elevated plasma lipids and diabetic cardiomyopathy related? Fed. Proc . 44: 1657 (abstract), 1985. Rodrigues, B . , Goyal , R . K . and M c N e i l l , J . H . : Effects of hydralazine on streptozotocin-induced diabetic rats: prevention of hyperlipidemia and improvement in cardiac function. J . Pharmacol . Exp. Ther. 237: 292-299, 1986. Rodrigues, B . and M c N e i l l , J . H . : Cardiac function in spontaneously hypertensive diabetic rats. A m . J . Physiol . 251: H571-H580, 1986. Ro l lo , J . : Uber die honigartiger Harnruhr. Translated by J . H . Jugler. Stendal, Franzen and Grosse, 1801. Sakiz , E . and Gui l lemin , R . : Inverse effects of purified hypothalamic T R F on the acute secretion of T S H and A C T H . Endocrinology 77: 797-801, 1965. Salway, J . G . , Finnegan, J . A . , Barnett , D . , Whitehead, L . , Karunanayaka, A . and Payne, R . B . : Effect of myo-inositol on peripheral-nerve function in diabetes. Lancet 2: 1282-1284, 1978. Saudek, C D . and Eder, H . A . : L i p i d metabolism in diabetes mell i tus. A m . J . M e d 66: 843-852, 1979. 118 Saunders, J . , H a l l , S. and Sonksen, P . : Thyroid hormones in insulin requiring diabetes before and after treatment. Diabetologia 15: 29-32, 1978. Shapiro, L . M . , Howat, A . P . and Cal te r , M . M . : Lef t ventr icular function in diabetes mell i tus. I: Methodology, and prevalence and spectrum of abnormalities. Br i t i sh Heart Journal 45: 122-128, 1981. Sharma, V . K . and Banerjee, S.P.: Alpha-adrenergic receptor in rat heart. Effects of thyroidectomy. J . B i o l . Chem. 253: 5277-5279, 1978. Shipp, J . C . , Menahan, L . A . , Crass, M . F . and Chandhuri, S .N. : Heart tr iglycerides in health and disease. In: Recent advances in studies on cardiac structure and metabolism. Dhal la , N .S . , Ed . Bal t imore , M D , Universi ty Park Press, pp. 179-204, 1973. Sibley, W . A . : The neuropathies of diabetes melli tus. In: Management of diabetes melli tus, Ed . R . Bressler and D . G . Johnson, pp. 249-269, John Wright, Boston, Br i s to l , London, 1982. Simpson, R . W . , Mann, J.I., Hockaday/ T . D . , Hockaday, J . M . , Turner, R . C . and Jelfs , R . : L i p i d abnormalities in untreated maturity-onset diabetics and the effect of treatment. Diabetologia 16: 101-106, 1979. Spaulding, S.W. and Utiger , R . D . : The thyroid: physiology, hyperthyroidism, hypothyroidism, and the painful thyroid. In: Endocrinology and Metabol ism. Fe l ig , P . , Baxter, J .D . , Broadus, A . E . and Frohman, L . A . (Ed.), pp 281-301, M c G r a w - H i l l Book Company, New Y o r k , 1981. Steinberg, D . : Hormonal control of lipolysis in adipose tissue. A d v . Exp. Med . B i o l . 26: 77-88, 1972. Streb, H . , Irvine, R . F . , Berridge, M . J . and Schulz, I.: Release of C a 2 + from a non-mitochondrial intracel lular store in pancreatic acinar cells by inositol-l ,4,5-triphosphate. Nature 306: 67-69, 1983. 2+ 2+ Suko, J . : Al tera t ions of C a uptake and C a act ivated A T P a s e of cardiac sarcoplasmic ret iculum in hyper- and hypothyroidism. Bioch im. Biophys. A c t a . 252: 324-327, 1971. Suko, J . : The calcium pump of cardiac sarcoplasmic ret iculum: functional alterations at different levels of thyroid state in rabbits. J . Physiol . (Lond) 228: 563-582, 1973. 119 Surks, M. I . , Koerner, D . , Di l lman , W. and Oppenheimer, J . H . : L i m i t e d capacity binding sites for L-tr i iodothyronine in rat l iver nuclei . Loca l i za t ion to the chromatin and part ial character izat ion of the L-tr iodothyronine-chromatin complex. J . B i o l . Chem. 248: 7066-7072, 1973. Tahi l iani , A . G . and M c N e i l l , J . H . : Lack of effect of thyroid hormone on diabetic rat heart function and biochemistry. Can . J . Physiol . Pharmacol . 62 (6): 617-621, 1984. Tahi l iani , A . G . and M c N e i l l , J . H . : Prevention of diabetes-induced myocardial dysfunction in rats by methyl palmoxirate and triiodothyronine treatment. Can . J . Physiol . Pharmacol . 63 (8): 925-931, 1985. Tahi l iani , A . G . , Vadlamudi, R . V . S . V . and M c N e i l l , J . H . : Prevention and reversal of altered myocardial function in diabetic rats by insulin treatment. Can . J . Physiol . Pharmacol . 61: 516-523, 1983. Ta ta , J .R . and Windell , C . C . : Ribonucleic acid synthesis during the early action of thyroid hormones. Biochem. J . 98: 604-620, 1966. Taylor , R . R . : Cont rac t i le properties of cardiac muscle in hyperthyroidism. C i r c . Res. 27: 539-549, 1970. Thompson, G . R . , Soutar, A . K . , Spengel, F . A . , Jadhav, A . , Garigan, S.J .P. and Myant , N . B . : Defects of receptor mediated low density lipoprotein catabolism in homozygous fami l ia l hypercholesterolemia and hypothyroidism in v ivo . Proc . N a t l . A c a d . Sc i . U . S . A . 78: 2591-2595, 1981. Tsai , J.S. and Samuels, H . H . : Thyroid hormone action: demonstration of putative nuclear receptors in human lymphocytes. J . C l i n . Endocrinol . Metab. 39: 919-922, 1974. Tzagournis, M . : Insulin, glucagon, growth hormone and somatostatin. Their relat ion to diabetes and heart disease. In: C l i n i c a l cardiology and diabetes, V o l . 1, part 1: Fundamental Considerations in Cardiology and Diabetes, Ed . Scott , R . C . Futura Publishing C o . , New York , pp. 171-190, 1982. Vadlamudi, R . V . S . V . , Rodgers, R . L . and M c N e i l l , J . H . : The effect of chronic alloxan and streptozotocin-induced diabetes on isolated rat heart performance. Can . J . Physiol . 60: 902-911, 1982. Vohl , H . : Ueber das Auft re ten des Inosits in H a m bei Nierenkrankheiten und die Verwandlung des Diabetes inositus. A r c h . F . Physiol . Hei lk . Stut tg. n .F. 2: 410-412, 1858. 120 Whereat, A . F . and Orishimo, M.W. : Effects of fasting and diabetes on fatty acid synthesis by heart mitochondria. A m e r . J . Physiol . 217: 998-1003, 1969. Whiting, P . H . , Palmano, K . Y . and Hawthorne, J . N . : Enzymes of myo-inositol l ip id metabolism in rats wi th streptozotocin-induced diabetes. Biochem. J . 179: 549-553, 1979. Wilber, J .F . , Banerji , A . , Prasad, C . and M o r i , M . : Al tera t ions in hypothalamic-pitui tary-thyroid regulation produced by diabetes mell i tus. L i f e S c i . 28: 1757-1763, 1981. Wil l iam-Olsson, T. , Fellenius, E . , Bjorntorp, P . and Smith, V . : Differences in metabolic responses to beta-adrenergic st imulation after propranolol or metoprolol administration. A c t a Med. Scand. 205: 201-206, 1979. Wil l iams, L . T . , Lefkowi tz , R . J . and Watanabe, A . M . , Hathaway, D . R . and Besch, H . R . : Thyroid hormone regulation of beta-adrenergic number. J . B i o l . Chem. 252: 2787-2789, 1977. Wil l iams, R .S . and Lefkowi tz , R . J . : Thyroid hormone regulation of < alpha-receptors. Studies in rat myocardium. Cardiovasc. Pharm. 1: 181-189, 1979. Wil l i s , T. : Opera omnia. Genevae, 1676-80. Winegrad, A . I.: Does a common mechanism induce the diverse complications of diabetes? Diabetes 36: 396-406, 1987. Wood, J . M . , Bush, B . , P i t t s , B . J . R . and Schwartz, A . : Inhibition of bovine heart N a , K - A T P a s e by palmityl carnit ine and palmityl C o A . Biochem. Biophys. Res. Commun. 74: 677-684, 1977. Ya low, R . S . and Berson, S .A. : Immunoassay of endogenous plasma insulin in man. J . C l i n . Invest. 39: 1157-1175, 1960. Yoon , J .W., Aus t in , M . , Onodera, T. and Notkins, A . L . : Virus-induced diabetes-mellitus: isolation of a virus from the pancreas of a chi ld with diabetic ketoacidosis. N . Engl . J . Med . 300: 1173-1179, 1979. Young, J .R . , Luca , C C , Patterson, J . M . and Best, C . H . : Lipotropic dose-response studies in rats: Comparisons of choline, betaine and methionine. Can . J . Biochem. Biophys. 34: 713-720, 1956. Zaninovich, A . A . , Brown, J . J . , Boado, R . , Bromage, N . R . and Mat ty , A . J . : Thyroxine metabolism in diabetic rats. A c t a Endocrinol . 86: 336-343, 1977. 

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