Myocardial substrate metabolism: Implications for diabetic Cardiomyopathy

J Mol Cell Cardiol 27, 1169-179 (1995)

Review

Myocardial Substrate Metabolism: Implications for Diabetic Cardiomyopathy Brian Rodrigues, Margaret C. Cam and John H. McNeill Division of Pharmacolog!t and ~lbxicologfl, Facultfl of Pharmaceutical Sciences, The Universitfl of Britislz Columbia, ¼u~couvel; British Columbia, Canada V6T 1Z3

(Received 1 April I994, accepted in n'~vised form 13 lul!! 1994)

Kt.:Y Worms: Diabetes; Cardiomyopathy; Glucose oxidation: Vanadium.

Introduction

Numerous clinical and epidemiological reports have confirmed that despite insulin therapy, the Type l (I1)DM) diabetic population appears particularly sus- ceptible to heart failure, which is a leading cause of death in these patients (Galderisi et al., 1991 ; Dubrey et al., 1994). Several factors appear to largely account for this increased incidence of cardiovascular disease during diabetes, including atherosclerosis of the coronary arteries, macroangiopathy and autonomic neuropathy (]aneczko et a/., 1991 ). However, it has also become apparent that these thctors, although important, are not exclusive determinants of the cardiac problems associated with diabetes. In- deed, a significant number of diabetic patients who do not develop the aforementioned vascular or neural deflects continue to suffer from cardiomegaly, left ventricular dysfunction and clinically overt con- gestive heart failure (Zoneraich and Mollura, ] 993). These observations suggest that a specific cardiac muscle disease (diabetic cardiomyopathy) may also occur (Regan and Weisse, 1.992) and that this path- ology is probably a direct consequence of the insulin- deficient state on myocardial cell function. Using animal models of Type I diabetes, the chemically induced [via streptozotocin (STZ) or alloxan] and spontaneously diabetic (BB) rats were found to develop myocardial abnormalities comparable to that seen in some human diabetic patients (Miller, ] 979; Rodrigues and McNeill, 1990). Similarly, the cardiac dysfunction in most of these animal studies was not

accompanied by a significant reduction in myocardial oxygenation and coronary flow or the presence of major vessel disease, ttence, given the parallel observations in both humans and diabetic animal models, it can be proposed that the chronic metabolic state of diabetes mellitus can negatively alter myocardial function independent of vascular det'ects.

Pathogenesis of diabetic cardiomyopathy

Chronic changes

The etiology of diabetic cardiornyopathy is complex and a number of factors have been suggested to be involved in the development of this disease stale. The myocardial events which occur over a chronic period are often irreversible changes including mi- croan giopathy (Shapiro, 1985), abnormal vascular sensitivity and reactivity to various ligands (Fried- man, 1989), and altered cardiac autonomic function (ttulper and Wilms, 1980). tn addition, there is increased stiffness of the ventricular wall associated with perivascular thickening of basement membranes and interstitial accumulat ion of gly- coprotein and insoluble collagen in the myocardium (Regan et al., 19 74). Lastly, there are abnormalities of various proteins which control ion movements , specifically intracellular calcium. The

Please address all correspondence to: Dr I. H. McNeill, Division of Pl~armacology and Toxicology. Faculty of Pharmaceutical Sciences. The University of British Columbia, Vancouver, B.C.. Canada V6T 17,3.

0022=2828/95/010169 + 11 $08.00/0 169 631995 Academic Press Limited

170 B. Rodrigues et al.

cardiac contraction and relaxation cycle is gen- erally viewed as the consequence of raising and lowering the intracellular concentration of t?ee calcium. The significance of the sarcolemma in this beat-by-beat phenomenon is dependent on its association with several enzymatic and non- enzymatic systems. Hence, any alterations in the composition and structure of this subcellular or- ganelle could change it s ability to transport calcium effectively and, as a result, the cardiac contraction and relaxation process may be significantly modi- fied. Several sarcolemmal changes have been iden- tified during diabetes and they include alterations in sarcolemmal calcium binding (Pierce et al., 1 9 8 3 ) , Na+-K+-ATPase (Pierce and Dhalla, 1983) and Ca 2 + pump activity (Heyliger e t al., 1987). Calcium transport by the sarcoplasmic reticulum (SR) is another major mechanism by which myocardial levels of Ca 2 +, and thereby tension development, are mod- ulated. The SR participates in the relaxation of the heart by actively accmnulating calcium from the cytoplasmic space, and any alteration in the capacity of this membrane to sequester Ca 2 + efficiently would, therefore, be expected to have an important impact on the relaxaiion of the myocardial muscles. [n diabetic rat hearts, SR Ca 2~ binding and Ca" '-- Mg :z~ ATPase activity are depressed, leading to a defect in SR Ca ~ transport (Bouchard and Bose, 1991), which then correlates with a slower relaxation (Penpargkul et at., 1981). Two cardiac enzyme systems extensively studied during diabetes arc myosin and actomyosin ATPases, whose activities are known lo correlate closely with contractility. A number of studies have shown that the Ca e ~-ATPase activities of myosin and actomyosin are depressed, thus accounting for the decreased shortening velocity of cardiac muscle, which is associated with a myosin isoenzyme shift from more active V, to the less active V ~ form (Dillmann, ] 9 8 0). Lastly, mitochondria are the chief source of myocardial ATP and, in addil!ion, have been suggested to have calcium accumulating properties and may hence act as a reserw)ir or "sink" to modulate intracellular calcium stores. Mitochondrial oxidative capacity, Mg~' ~--A'FPase activity and Ca 2+ uptake activity are all depressed in the diabetic myocardium (Pierce and Dhalla, 19 8 5).

Acute changes

During the early, acute phase of diabetes, alterations in contractile fl.mction and cardiac ul- trastructure are probably minimal, ttowever, metabolic abnormalities may already exist in the

myocardium. These metabolic derangements in both fuel supply and utilization by heart tissue precede chronic changes and could potentially be the primary biochemical lesion in the pathogenesis of diabetic cardiomyopathy. A plethora of metabolic changes result from insulin deficiency, and so far in animal studies, the most successful treatment regimen in preventing or retarding cardiac dysfunction associated with diabetes con- tinues to be insulin replacement therapy (Fein et al., 1981). However, in a chronic canine model of diabetes, both collagen accumulation and a diminished myocardial compliance were either unaffected or only partly reversed by insulin control of postprandial hyperglycemia (Regan et al., 1981; Pogasta et al., 1979). Furthermore, clinical studies show that myocardial abnormalities remained despite insulin treatment and rigid glucose control (University Group Diabetes Program, 1975). These studies raise the question as to whether the altered cardiovascular function in diabetes is secondary to hyperglycemia and/or to the presence of other factors induced by a deficiency in insulin. In this regard, aberrant plasma lipoprotein levels persist in spite of insulin therapy. [t is proposed that such lipid and lipoprotein abnormalities, with a subsequent alteration in myocardial energy production, could be important factors for the development of diabetic cardiomyopathy. In lhct, numerous human and animal studies have attempted to correlate changes in lipid and lipoprotein metabolism seen during diabetes to the observed excess in cardiovascular risk (Tan, 1993). In the Framingham Study, diabetic subjects who had a higher incidence of cardiovascular disease also presented with higher VH)L-triglyceride levels (Kannel and McGee, 1979). The WHO study found that of the variables measured in diabetics, serum triglyceride was the strongest indicator for cardiovascular disease (West et tfl., 1983). Rosenstock et al. (1987) have reported that meticulous control ol' Type 1 diabetic patients resulted in consistently reduced cardiovascular risk factors (i.e. altered plasma lipid and lipoprotein levels) for as long as four years. With animal studies, some treatments that lower blood lipids in STZ-diabetic rats were ef[~ctive in im- proving cardiac function (Rodrigues et al., 1986, 1988). However, several agents such as clofibrate, prazosin and enalapril, which produced triglyceride-lowering effects in the STZ-diabetic rats were ineffective in preserving normal myocardial function (Rodrigues et al., 1994) and suggest that factors in addition to elevated triglyceride levels

Myocardial Substrate Metabolism 1 71

70

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.~ 40

.~ a0

20

10

I I

insulin FFA

0.5

0.4

0.3

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0.1

0.0

E

Figure I l?tasma insulin and free fatty acids (FFA) in control (D) and diabetic (11) rats. 1)iabetes was induced by a streplozotocin injection (100 mg/kg, i.v., 0.9% sa- line). After 4 days, blood samples were obtained I}:om the tail vein prior to death. Plasma glucose was elevated in the diabetic rats (control---7.3 +- / -0 . ] , diabetic= 17.3 + / - -0 .4 mmol/l). Data is mean ± S.I.:.M.

may be responsible for the diabetic cardiac disease.

Cardia c lipid metabolism

In the heart, the source of cellular energy in the lbrm of ATP is obtained via the oxidation of various substrates including free fatty acids (FFA), glucose, lactate and ketone bodies, with FFA being the prin- cipal substrate utilized by the heart, Indeed, in rats, the heart muscle accounts for a disproportionately large consumption of FFA with respect to body weight (van der Vusse el al, 1992). Transport of FFA into the myocyte occurs by saturable, carrier- mediated transporters (fatty acid binding proteins, FABPs) or by nonsaturable diffusion across the plasma membrane (Schulz, 1991), FABPs, in addition to increasing the influx of fatty acids into the cell, have also been proposed to facilitate the diffusion of FFA in the aqueous phase of the cytosol and to act as an intracellular "bufl~r" to attenuate the inhibitory effect: of fatty acids on enzymes (Stew- art et al., 1991). Fatty acids are supplied to the heart from several sources: either through lipolysis of endogenous triglyceride stores or from the blood where they are carried as free acid bound to albumin or as triglyceride in chylomicrons and very low density lipoproteins. During insulin deficiency, there is a marked increase in adipose tissue lipolysis with a subsequent outflow of free fatty acids, which become greatly elevated in diabetic p lasma (Fig. 1), When the rate of FFA uptake exceeds the rate of disposal, myocardial triglyceride content is increased, as has been observed in perfused hearts (Denton and Randle, 1967; Paulson and Crass,

1982) and in cardiac myocytes (Kenno and Sev- erson, 1985) obtained from diabetic rats. There has been some evidence to suggest that high circulating fa|ty acids inhibit endogenous myocardial triglyceride hydrolysis and, in fact, results in greater triglyceride synthesis (Saddik and Lopaschuk, 199t) . It has been proposed that the observed reduction in triglyceride hydrolysis results l?om an inhibition of myocardial triglycerideqipase by long chain acyl CoA (Mcl)onough and Neely, 1988). An increase in myocardial enzymes catalysing the synthesis of triglyceride (Murthy and Shipp, 1980), ill addition to an increase in CoA levels (Reibel et al., 1981), serve to shunt the fatty acids away I>om mitochondrial oxidation and toward the production of triglycerides (Lopaschuk et ~fl., 1986), Subsequent hydrolysis of this expanded intracellular store of triglyceride could eventually lead to high tissue FFA levels (Chattopadhyay et ~fl°, 1990) and, indeed, the rate of lipolysis is increased in the diabetic heart (Kenno and Severson, 1985). This has a direct consequence on the heart, and FFA at high concentrations have been associated with a reduced cardiac contractile lbrce and greater cardiac susceptibility to arrhythmias in both control and diabetic hearts (()pie, 1970). On the other hand, as an expected consequence of elevated myocardial FFA and a marked increase in the FABP content of diabetic rat hearts (Glatz et (ft., ] 994), oxidation of Ntty acids of both endogenous and exogenous origin by the diabetic heart is prolbundly enhanced. This has two potentially deleterious consequences: an abnormally high requirement for oxygen tbr cata- bolism and an intracellular accumulation of potentially toxic intermediates of l~ltty acid metabolism (e.g. long chain acyl-CoA and acylcarnitine). Whether accumulation of long chain acyl-CoA or acyl carnitine can contribute to diabetic heart Nil- are is controversial. Lopaschuk and Spaftbrd (1989) have reported that in palmitate-perfi~sed control hearts, long chain acylearnitine levels were mark-- edly elevated after 1 5 rain of ischemia, while mechanical function was only slightly decreased. In addition, they showed that Etomoxir (a CPT 1 inhibitor) markedly decreased the rate of mechanical failure in both acutely and chronically diabetic rat hearts, even though long chain acyl-CoA and acylcarnitine levels were not decreased. The authors concluded that it was the impaired glucose use in diabetic rat hearts, rather than an accumulation of metabolic intermediates, which con- tributes to the increased susceptibility to ischemic damage. However, other reports ihave suggested that these intermediates, if sufficiently large, may have adverse electrophysiological, biochemical and

.172 B. Rodrigues et al.

mechanical effects on the heart, in particular, it is postulated that they can: (a) cause conduction disturbances and ventricular arrhythmia (Liedtke et al., 1978); (b) reduce both the basal and insulin- stimulated glucose transport and metabolism in the heart (Randle et al., 1963); (c) modify the structure of sarcolemmal and other subceflular membranes and by their "detergentqike" effects alter membrane fluidity and molecular dynamics (Katz and Mes- sineo, 1981); (d) interfere with various cellular functions by specifically inhibiting critical enzyme systems such as Ca-'+-ATPase of sarcoplasmic reticulum (Adams et aI., 1979), Na +, K ~-ATPase, Na+/Ca2+-exchange and Ca ~ ~ pump in myocardial sarcolemma (Kramer and Weglicki, 1985; Ashavaid et al., 1985; Dhalla et al., 1 9 9 1 ) , cardiac cyclic AMP phosphodiesterase and myosin light chain kinase (Katoh et al., 1982) and protein kinase C (Katoh et al., 1981); (e) inhibit the adenine nucleotide translocator in isolated mitochondria resulting in a reduction in the myocardial levels of ATP (Vaartjes et al., 1972): (f) mediate an increase in alpha-adrenoceptor sensitivity to norepinephrine (tteathers et al., 1987) leading to a mobilization of Ca ~'+ from intracellular stores; and (g) interact

Ca ~ -channels directly with voltage-dependent ' ' (Inoue and Pappano, 198~; Spedding and Mir, .1987) or indirectly via the Na~-Ca ~ exchanger (Wu and Corr, 19941). A consequence of these etlbcts is an aberrant intracellular handling of Ca" ~ leading to Ca~' ~ overload with subsequent alterations in membrane permeability, activation of Ca" %stimulated proteases, phospholipases and lysosomal enzyme activities, mitochondrial calcitication with depletion of cellular ATP stores~ cell death and eventual cardiac dysfunction (Orrenius et al., l 989). The rise in cytoplasmic calcium is also a major contributor to muscle stiflness (Brady and Farnsworth, 1986). Overall. there appears to be a "lipid paradox" in that low c()ncentrations of FFA are essential tbr the proper functioning of the heart whereas excessive amounts arc potentially deleterious.

Cardiac carbohydrate metabolism

Intracellular glucose disposal occurs through several major pathways. Nonoxidative glucose disposal primarily reflects the conversion to glycogen (1)eFronzo et al., 1 9 8 1 ), whereas the oxidative pathway involves either the complete oxidation of glucose-derived carbon atoms to carbon dioxide or the conversion to fatty acids in lipogenic tissues. Whereas glycolysis, or the breakdown of glucose or glycogen to pyruvate provides a limited amount

3.5

3.0

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.~ .~ 2.0

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0.5

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Figure 2 Effect of diabeles on basal ([2]) and insulin- stimulated (~N) glucose oxidation in isolated cardiomyocytes. Ca2*-tolerant myocytes were isolated from control and diabetic rat hearts as previously described (Rodrigues et al., 1992). Glucose oxidation was initiated by incubating myocytes with labeled D-[U-I4C]glucose at 37°C for 60 rain in the presence and absence of insulin (100 ng/ml). The amount of ~COe produced was measured. Data is mean ± S.e.M.

of ATP, it is the subsequent entry of pyruvate into the mitochondria and its oxidation that provides the majority of energy obtained from glucose. Insulin affects all areas of carbohydrate metabolism chiefly by controlling the transport of glucose. In insulin- responsive tissues (i.e. muscle, tilt and heart), it has beell shown that insulin can induce el rapid reversible translocation of glucose transporter proteins from a latent intracellular pool to the plasma membrane and a possible enhancement in the in- trinsic activity of the transporters (Suzuki and Kono, 1980; Cushman and Wardzala, 1980: Karnieli et al., 1981). Activation of glucose transport by insulin is Ibllowed by intracellular processes which are also further enhanced by insulin: glycogen syn[hesis, glycolysis and glucose oxidation, llence, in the hypoinsulinemic condition, there is a sig- nilicant reduction in the basal myocardial glucose utilizaliom as observed in isolated diabetic car- diomyocyles (Chen et al., 11984) (Fig. 2). The maior restrict:tim to glucose utilization in the diabetic heart is the sk)w rate of glucose transport across the sarcolemmal membrane into the myocardimn, which probably results from the cellular depletion of glucose transporters. Recently, Eckel and Reinauer (1990) and Garvey et al. (1993) have reported that in insulin-deficient rats, it was the insulin- responsive glucose transporter (GLUT-4) protein and mRNA that was specilically reduced in cardiomyocytes and sarcolemmal vesicles. The diabetes-associated reduction in glucose transporter protein/activity is poorly understood. [t has been


demonstrated that the activity of the glucose transporter in vitro is influenced by the composition (specifically membrane lipid) and the struclure of ceil membranes (Melchoir and Czech, 1979; Pilch et al., 1980). Since the diabetic state is associated with hyperlipidemia, a considerably altered fatty acid profile of membranes could accomH, tbr the decrease in glucose transporter translocation/activity and result in an observed reduction in insulin- stimulated myocardial glucose utilization ( then et

al., 1984) (Fig. 2). The impaired glucose oxidation in tlhe diabetic

heart can also result t~om a decreased rate of phosphorylation of glucose which can subsequently limit the entry of glucose into the cell. The reduced phosphorylation has been proposed to result from the increased metabolism of FFA (Das, 1973). An excessive FFA oxidation is at least partly responsible for the insulin resistance and depression of cardiac glucose oxidation, a notion introduced by the classic studies of Randle et aL (11963). They suggest that an increased availability of FFA can stimulate the TCA cycle and increase citrate levels. The citrate tbrmed inhibits phosphofructokinase, thereby re- ducing the rate of glycolysis which leads to a decrease in glucose uptake and oxidation. Fur- thermore, the reduction in substrate flow through the glycolytic pathway results in an eventual buildup in the tissue levels of glucose-6-phosphate which activates glycogen synthase and inhibits phosphorylase. These changes in enzyme activity appear to account for glycogen accumulation as the small amount of glucose that is transported is diverted toward glycogen production (Chen and Ianuzzo, 1982). Another explanation for the reduced oxidation of glucose by the diabetic heart is that elevated FleA oxidation increases the acetyl CoA to CoA ratio which activates the pyruvate dehydrogenase kinase to phosphorylate and in- activate the pyruvate dehydrogenase complex (PDtl) (Kerbey et al., 1985). The end result is a diminished oxidation of pyruvate. Inhibition of glucose oxidation in the diabetic heart could also be due to a direct alteration in PDH activity. Weiland et aL (1971) found that total PI)H activity was reduced in hearts from diabetic animals. Several studies have recently confirmed the earlier findings of Randle and his colleagues. For example, it was shown in several tissue types that: insulin suppressed plasma FFA availability and stimulated the activation of the PDH complex (Cooney et al., 1993). Wall and Lopaschuk (198% have demonstrated that in the presence of relevant concentrations of fatty acids, as observed in chronically diabetic rats, myocardial glucose oxidation is essentially ab-

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Figure 3 Effect of oleic acid on glucose oxidation in cardiac myocyies from c(mtrol and diabetic rals. Myocyt;es were incubated for 60 rain in the absence ([-]) arid presence (Ill) of 0.9 mM oleate (6:1 molar ratio to BSA) and glucose oxidation measured as described in Fig. 2. Data is mean ¢-S.V.M.

olished. Furthermore, with high concentrations of fatty acids, the glycolytic rate is more than 13 times the rate of glucose oxidation, thus supporting the idea that fatty acids inhibition of glucose utilization occurs to a greater extent at the level of pyruvate dehydrogenase than at the level of phospho- l?cuctokinase (Saddik and Lopaschuk, 1991 ). Indeed, normal cardiomyocYtes cultured in the presence of pahnitate exhibit a markedly depressed (67%) insulin-stimulated glucose oxidation which is par- tially restored by the inhibition of fatty acid oxidation (Eckel et al., 19911). We (and others, Chert et al., 1984-) have also observed a reduction in basal glucose oxidation in cardiac myocytes isolated from acutely (.3 day) diabetic rats (Fig. 3). In addition, the inhibitory elli;ct of fatty acids is demonstrated when incubation with exogenous oleate fl:lrther reduced basal and insulin-stimulated glucose oxidation in both control and diabetic myocytes (Fig. 4). Interestingly. in control myocytes, the inhibitory effect of oleate on basal glucose oxidation was reversed by washing, whereas the inhibition of insulin-stimulated glucose oxidation remained. This is in contrast to the results of Muter et al. (1992) wherein the oleate-induced inhibition of insulin- stimulated glucose oxidation in isolated rat adipocytes was lost with washing. It: is possible that, in our study, the high concentrations of FFA that we used has a prolonged effect to decrease insulin binding and action in the cardiomyocytes, in this regard, Svedbcrg et al. (19921) have demonstrated that elevated FFA levels are associated with impaired insulin cell surface binding to isolated hepatocytes, possibly through an effect of lipid oxidation

174 B. Rodrigues el al.

100

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.6

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Figure 4 Effects of oleic acid and insulin on glucose oxidation. Myocytes were incubated tbr I h with oleic acid, washed, and reincubated tbr another hour with or without [(b) basal acl:ivityl insulin. Results are I)om a single representative experiment.

on the internalization/recycling of the insulin receptor complex without any perturbation of the receptor tyrosine kinase activity.

Additional evidence thai. changes in FFA metabolism can affect carbohydrate metabolism have come from the demonstration that pharmacologic agents which strongly inhibit the activity of CPT1. and hence the hepatic oxidation of long-chain fatty

acids (e.g. 2-tetradecylglycidic acid; 2[5(4-chloro- phenyl) pentyl oxirane-2-carboxylic acid; etomoxir; B 827- 33), can also oppose the fatty acid-induced inhibition of glucose oxidation, and are effective hypoglycemic agents in STZ-diabetic animals (Rosen and Reinauer, 1984). The hypoglycemic potency of these agents appears to be independent of insulin and is attributed to their ability to increase glucose oxidation by insulin sensitive tissues via the reactivation of the PDH complex, or to a sup- pression of hepatic gluconeogenesis (Martin et d., 1991 ). In inhibiting fatty acid and ketone breakdown, these compounds reduce intracellular ace@ CoA/CoA and NADH/NAD ratios, leading to a reduced end product inhibition of PDH (Foley, 1992). Recently, Saddik et al. (1993) have proposed that activating the PDH complex will increase the supply of acetyl-CoA for carnitine acetyltransferase. The resulting rise in cytosolic acetyl CoA levels in turn stimulates acetyl-CoA carboxylase activity. In- creased malonyl-CoA production will subsequently inhibit CPT1 activity, thus decreasing the rate of fatty acid oxidation. Since NfP and acetyl CoA are necessary tbr hepatic glucose production, an inhibition of fatty acid oxidation would result in a reduction in fasting blood glucose levels. Thus, a switch from predominantly fatty acid to carbohydrate oxidation could be thought to produce the salutary effects of these drugs on cardiac performance (Dillmann, 1985). Other agents that directly alter lipid metabolism have also been shown to have similar ell'eels. For example, acute administration of nicotinic acid (a potent inhibitor of lipolysis) has been shown to lower elevated plasma FFA, fatty acid oxidation and plasma glucose levels in both diabetic rats (Reaven et d., 1988) and man (Neuwahl, 1943),

Given the supportive data so far, the complex relationship between glucose and triglyceride breakdown and utilization requires more detailed ex- amination. However, there is little dispute that a more rigorous attempt should be made to decrease the abnormally high rates of FFA oxidation. This directed therapy would serve to overcome the accumulation of toxic fatty acid intermediates and hence prevent the inhibition of myocardial glucose utilization. Glucose oxidation in the diabetic heart: is important lbr the following reasons: (1) ATP produced from glycolysis could be preferentially used by membrane ion pumps such as sarcolemmal ATPases (Bunger et d., 1986) and hence a deficiency in glycolytic ATP could damage the in- tegrity of cellular membranes, (2) increasing flux through the PDH complex will prevent the accumulation of potentially toxic glycolytic end-

Myocardial Substrate Metabolism 175

products such as lactate, and (3) thtty acids are known to have an "oxygen-wasting" effect when compared to carbohydrates, which results in a higher ratio between myocardial oxygen consumption and cardiac work, i.e. an increase in ()2 consumed per ATP produced. Indeed, Rosen et aL (1986) have reported that, in chronically diabetic hearts, the diminished glucose conversion and depressed energy production from glucose are associated with an impaired in vivo cardiac pertbrmance. Similarly, perfusion of the chronically diabetic rat heart with a stimulator of glucose oxidation [dicholoroacetate, a P1)tt complex ac- tivator (Stacpoole and Greene, 1992)11 was shown to acutely reverse the diabetic cardiac dysfunction (Nicholl et al., 1991) whereas hypoglycemic drugs like etomoxir, which block CPT1, have also been demonstrated to significantly improve heart tunc- tion in diabetic rats (Wall and Lopaschuk, 1989). Treatmenl of diabetic rats with agents like hydralazine (Rodrigues et aI., 1986) and carnitine (Ro- drigues et M., 1988) lowered plasma lipid levels and improved cardiac/unction. In addition, these agents have recently been reported to have stimulatory effects on glucose oxidation in Ntty acid perfllsed hearts (Burns et al., 1991; Broderick et d., 1992).

Although several factors could potentially contribute to the ultimate failure of the heart, it was of interest to us to study compomlds which have been shown to prevent cardiomyopathy in vivo in diabetic rats, particularly with regards to their abib ity to enhance glucose oxidation in isolated myocyte preparations. A potential disadvantage of using isolated cardiac myocytes is that the metabolic fate of substrates like glucose and FFA could be different in the non-beating myocytes as compared to beating hearts. On the other hand, the advantage of using isolated cardiomyocytes for metabolic studies is that these cells are tully differentiated and mor- phologically similar to cells in the intact: heart, but lack interstitial tissue and contaminating cell types which can complicate measurements in whole tissues. In addition, because they are not spontaneously active, cellular behavior can be investigated under well-defined environmental con- ditions. Thus, the effect of an insulin-mimetic agent vanadium on glucose oxidation in isolated myocytes was investigated. Vanadium has been demonstrated to have potent insulin-mimetic effects both in vitro and in vivo. Chronic treatment with oral vanadium in the form of vanadate (+ 5) and vanadyl (+4 ) produces a remarkable improvement in glucose homeostasis in several animal models of diabetes: streptozotocin-diabetic (Iteyliger et d., 1985), par- tially pancreatectomized (Rossetti and Laughlin,

1989), genetically obese and hyperinsulinemic fi# j~ rats (Brichard et al., i1989) and ob/ob mice (Brichard et al., 199[)). More importantly, chronic treatment with wmadium results in improved car~ diac function in STZ-diabetic rats (lteyliger et aL, 1985: Ramandham et aL, 1989). Vanadate has been shown to activate glucose oxidation in vitro in rat adipocytes, in the same manner as insulin (Shechter and Karlish, 1980). Our preliminary result:s now also indicate that vanadate potentiates insulin-stimulated glucose oxidation in isolated diabetic cardiomyocytes (see Fig. 5). Since the rate of glucose oxidation is limited by its rate of transport into the cell, a potential mechanism lbr increased oxidation of glucose could be a stimulatory eflbct on glucose transport. Vanadate has been shown to increase glucose transport in vitro in a variety of rat tissue types: adipocytes (Dubyak and KleinzelIer, 1980), skeletal muscle (Okumura and Shimazu, 1992), and in vivo in brain (Meyerovilch et aL, 1989), and in liver and muscle (Meyerovitch et al., 1987). This effect might be attributed to an insulin- like effect on stimulating the translocation of GLUT- 4 to the plasma membrane which has been shown in vitro (Paquet et M., 1992). However, recent studies have failed to demonstrate any effect of either vanadate alone (Lonnroth et al., 1993) or with insulin on glucose transport activity (Shisheva and Shechter, 1993) in isolated adipocytes, and more studies are yet. needed to contkm these ef[i~cts.

Summary

The incidence of mortality from cardiovascular dis- eases in higher in diabetic patients. The cause of this accelerated cardiovascular disease is multitactorial and, although atherosclerotic cardiovascular disease in association with well-defined risk factors has an influence on morbidity and mortality in diabetics, myocardial ceil dysflmction independent of vascular defects have also been defined. We postulate that these adverse cardiac effects could presumably result as a consequence of the following sequence of events. Major abnormalities in myocardial carbohydrate and lipid metabolism occur as a result of insulin deficiency. These changes are closely linked to the accumulation of various acylcarnitine and coenzyme derivatives. Abnormally high amounts of metabolic intermediates could cause disturbances in calcium homeostasis ei ther directly or indirectly through structural and func- tional subcellular membrane alterations. Over time, chronic abnormalities such as reduced myosin

1 76 B. Rodrigues et al.

i.2

~c7 8 o

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Figure 5 l:~ffecl of wmadate and insulin on glucose oxidation. Myocytes were incubated with ( ~ ) or without ([2) insulin (100 ng/mt) for 60 rain, either alone or after preincabation for I h with ] aiM vanadate. Glucose oxidation was measured as described in Fig, 2, Data is mean _+S.f,:.M.

ATPase activity, decreased abil i ly of tlhe sarcoplasmic ret;iculum to take up ca lc ium as well as depresskm of o ther m e m b r a n e enzymes such as Na~-K + ATPase and Ca 2 +-ATPase leads to changes in ca lc ium homeostas is and eventua l ly to cardiac dyslhnct ion, More impor tan t ly from the point of view of pharmaco log ica l in tervent ion, dur ing the initial stages, acute d is turbances in both the glucose and FFA oxidat ive p a t h w a y s m a y provide the initial b iochemical lesion from which fur ther events ensue, Thus therapies which ta rge t these metabol ic ab- er ra t ions in the hea r t dur ing the ear ly stages of diabetes, in ettiecl, can potent ia l ly delay or impede the progression of more p e r m a n e n t sequelae which could ensue from otherwise uncontrol led de- r angemen t s in card iac metabol ism. There is little dispute tha t an attempt: should be made to lower raised p lasma tr iglyceride and FFA levels. This would decrease the hear t ' s re l iance on fatty acids and, hence, overcome the fatty acid inhibi t ion of myocard ia l glucose uti l ization. In this regard, the likely appl ica t ion of fat ty acid oxidat ion inhibi tors (CPT inhibitors, be ta -oxida t ion inhibitors, sequest ra t ion of mi tochondr ia l CoA) is also apparent .

Acknowledgements

The studies described in this paper were suppor ted by gran ts from the B.C. and Yukon Hear t Found- ation, the Medical Research Council (MRC) of Ca-

nada. the Canadian Diabetes Associat ion and the B,C. t t ea l th Care Research Founda t ion , MCC is an MRC s tudentsh ip holder. BR is a Canadian Diabetes Associat ion Scholar,

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Myocardial substrate metabolism: Implications for diabetic Cardiomyopathy