The addition of free fatty acids, in the form of 1.2 mM palmi- tate, to the isolated working heart perfusate had no effect on either control or diabetic heart function, ...
Effects of free fatty acids and dichloroacetate on isolated working diabetic rat heart TESSA
A. NICHOLL,
GARY
D. LOPASCHUK,
AND
JOHN
H. McNEILL
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 123; and Department of Pediatrics, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
NICHOLL,
TESSA
A.,
GARY
D.
LOPASCHUK,
AND
JOHN
H.
MCNEILL. Effects of free fatty acids and dichloroacetate on isolated working diabetic rat heart. Am. J. Physiol. 261 (Heart
Circ. Physiol. 30): H1053-H1059, 1991.-It is well established that cardiac dysfunction independent of atherosclerosisdevelops in both humans and animals with diabetes mellitus. The etiology is complex, involving many different processes,one of which may be increasedfatty acid utilization and/or a concomitant decreasein glucoseutilization by the diabetic heart. We comparedcontrol and 6-wk streptozotocin (STZ)-induced diabetic isolatedworking rat hearts and were able to demonstrate cardiac dysfunction in the diabetic as assessedby depressed heart rate (HR), heart rate peak systolic pressureproduct (HR. PSP), left ventricular developedpressure(LVDP), and rate of pressurerise (+dP/dt). Paralleling depressedcardiac function in the diabetic were hyperglycemia, hyperlipidemia, and decreasedbody weight gain comparedwith age-matchedcontrols. The addition of free fatty acids, in the form of 1.2 mM palmitate, to the isolated working heart perfusate had no effect on either control or diabetic heart function, with the exception of a depressiveeffect on +dP/dt of diabetic hearts. But diabetic hearts perfused with palmitate-containing perfusate plus the glucoseoxidation stimulator dichloroacetate (DCA) showeda marked improvement in function. HR and HR*PSP in spontaneously beating hearts, aswell asLVDP and +dP/dt in paced hearts were all restored to control heart values in diabetic hearts perfused in the presenceof DCA. Creatine phosphate and ATP levels were similar under all perfusion conditions, thus eliminating energy stores as the limiting factor in heart function. Results indicate that DCA will acutely reverse diabetic cardiac function depression.Therefore glucoseoxidation depressionin the diabetic heart may be a significant factor contributing to cardiac dysfunction. glucoseoxidation; cardiac function; streptozotocin-induced diabetes
of cardiac performance during diabetes due to a lesion in the cardiac muscle itself, independent of atherosclerotic artery disease, has been demonstrated in both clinical and experimental studies (8, 21, 32). For instance, if diabetes is produced in rats, a well-characterized depression in cardiac contractile performance is seen (7, 19, 23, 28, 30, 32). The etiology of these myocardial abnormalities is uncertain but has been suggested to be due to the metabolic alterations associated with diabetes. It is known that a prominent metabolic alteration in diabetes is an increased concentration of circulating fatty
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acids and concomitant increased reliance of the heart on fatty acids as an energy substrate (4,14,33). Experimental evidence suggests that normalization of various aspects of fatty acid metabolism is associated with beneficial effects on the mechanical function of the diabetic heart (23, 24, 28, 34). The mechanisms by which fatty acids exert this detrimental effect have not been completely delineated. One effect of high circulating free fatty acids (FFA) is to inhibit myocardial glucose use. Under nonfasting conditions, the heart utilizes -70% fatty acids and 30% glucose for energy production (15). But in the diabetic state glucose utilization is essentially abolished and fatty acids can account for up to 99% of the oxidative fuel of cardiac muscle if circulating fatty acids are elevated (5, 33). Recently, it was shown that glucose oxidation rates in isolated working hearts from diabetic rats were markedly depressed in the presence of physiological levels of fatty acids (33). An insulin lack resulting in a depression of glucose uptake is a contributor to the marked decrease in glucose utilization, but according to the concept of the glucosefatty acid cycle, elevated serum and tissue fatty acid levels in diabetes are accompanied by decreased glycolytic rates (20). First, increased citrate concentrations will inhibit phosphofructokinase, with glycogenolysis and glucose uptake being subsequently decreased due to an accumulation of glucose 6-phosphate (16). As well, fatty acids inhibit the pyruvate dehydrogenase (PDH) complex by increasing the products-to-substrates ratio of the reaction (5, 20). Dichloroacetate (DCA), a PDH activator, is a pharmacological agent that is effective in increasing myocardial glucose oxidation in normal and diabetic rat hearts perfused with glucose and insulin (11, 27). Direct addition of this agent to isolated working hearts from diabetic rats also markedly stimulates glucose oxidation. In a study yet to be publishd, we have demonstrated that DCA will increase glucose oxidation by 500% in diabetic rat hearts perfused in the presence of high FFA (G. D. Lopaschuk and J. J. McVeigh, unpublished observations). It is thus apparent that DCA could be a useful tool in investigating whether a depression of glucose oxidation is a contributor to diabetic cardiac dysfunction by examining any acute effects this agent may have on isolated working diabetic heart function. In this study, we attempted to determine whether fatty acids have a detrimental effect on diabetic heart function.
0 1991 the American
Physiological
Society
H1053
H1054
FFA AND DCA EFFECTS
This was done by investigating cardiac performance in both control and diabetic isolated working hearts with either glucose present as the available metabolic substrate or with both glucose and FFA present as substrates. Cardiac performance was also examined in control and diabetic FFA-perfused hearts in the presence of DCA. After analysis of heart function, each heart was examined for its ability to meet its energy requirements; each heart was assayed for ATP and creatine phosphate (CrP). MATERIALS
AND
METHODS
Animals. Diabetes was induced in male Wistar rats by a single tail vein injection of 60 mg/kg streptozotocin (STZ) dissolved in 0.9% saline. Control animals were injected with saline alone. Animals were provided with food and water ad libitum for 6 wk. STZ animals having serum glucose ~20 mM were used in this study. Heart perfusions. Hearts from pentobarbital sodiumanesthetized control and diabetic rats were initially perfused as a Langendorff preparation for 10 min with Krebs-Henseleit buffer gassed with 95% O&j% CO*. During this time, the opening to the left atria was cannulated. A cannula connected to a passive transducer was also inserted through the apex of the heart for the measurement of left ventricular developed pressure (LVDP). Hearts were then switched to a working-heart model of perfusion with perfusate containing glucose (11 mM) or glucose (11 mM) and palmitate (1.2 mM). All perfusions were done in the presence of 3% bovine serum albumin, with palmitate, when used, being prebound to the albumin. Hearts were perfused during initial work period at a left atria1 filling pressure of 15 cmH*O and a hydrostatic afterload of 90 cmH20. Spontaneously beating hearts were analyzed for function, described in terms of heart rate (HR) and HR-peak systolic pressure product (HR. PSP) measured through an aortic transducer connected to a Grass polygraph. Hearts were then paced at 240 beats/min and controlled function, based on LVDP and rate of pressure rise (+dP/dt) measured at various hydrostatic afterload pressures. The maximal work that could be performed by the heart was then determined by clamping off the aortic outflow line. At this maximal work load, hearts were frozen using Wollenberger clamps cooled to the temperature of liquid nitrogen. The same procedure was carried out on control, and diabetic hearts perfused in the presence of palmitate with and without 0.5 mM DCA. Tissue analyses. ATP and CrP were extracted with perchloric acid and enzymatically measured as described previously (9). Tissue triglycerides were extracted and measured as described by Bowyer and King (1). Statistical analysis. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by comparison of group means with a Newman-Keuls test. For heart function curves, repeated measures of ANOVA were used, followed by a Newman-Keuls test for comparison of group means at individual points on the curve. Statistical significance was set at P < 0.05.
ON DIABETIC
HEART
RESULTS
STZ-diabetic rat model. Diabetes was induced by a single intravenous injection of 60 mg/kg STZ. After 6 wk, at the time that the animals were killed, blood was collected and serum glucose levels were measured. Serum glucose levels of diabetic animals were found to be significantly higher than those of age-matched controls (Table 1). Serum fatty acid levels were also significantly elevated in diabetic rats compared with controls (Table 1) . Rats treated with STZ exhibited symptoms characteristic of the diabetic state. Diabetic rats gained less weight than age-matched controls and therefore had a significantly lower body weight at the time that they were killed (Table 1). Effect of FFA on control and diabetic isolated working rat hearts. To investigate the effects of FFA on diabetic
heart function, isolated working rat hearts were perfused in the presence of glucose (11 mM) alone or glucose (11 mM) plus palmitate (1.2 mM). In spontaneously beating hearts, at a medium work load (15 cmHzO filling pressure, 90 cmH20 afterload pressure), HR was found to be significantly lower in diabetic hearts compared with controls (Table 2). Table 2 also shows that FFA had no significant effect on HR. HR. PSP was measured at a medium work load and was found to be depressed in diabetics, regardless of available substrates (Table 2). Table 2 also shows that, TABLE
1. General features of 6-wk control
and diabetic rats
Control
Diabetic
Body wt change, g 179k6 90.96t9.65* Serum glucose, mM 11.23kO.25 25.66kO.34’ Serum FFA, mM 0.493~0.004 0.687kO.O05* Values are means t SE; n = 16-23. Body weight change was determined by the difference in weight of the rat at the time of streptozotocin or saline injection and the weight 6 wk after injection. * Significantly different from control, P < 0.05.
2. Effect of palmitate and DCA on heart function in spontaneously beating isolated working hearts from control and diabetic rats TABLE
Group
Perfusion Condition
Effect
Control Diabetic
of palmitate
Glucose Glucose-palmitate Glucose Glucose-palmitate Effect
Control
HR,
of DCA
beats/ min
HR.PSP, beats
l
mmHg* min-’
(n = 7-8) 185-ell 193k12 117217’ 134*17*
23,959+2,003 26,367&1,861 15,623+2,003* 16,423*1,873*
.
(n = 6-8)
26,367+1,861 Glucose-palmitate 193*12 27,644k 1,306 +DCA 191k6 l6,423*1,873t Diabetic Glucose-palmitate 133.8*16.6? 30,438k 1,524 +DCA 182.9k9.9 Values are means t SE. Heart rate (HR) and HR-peak systolic pressure product (HR*PSP) were determined in hearts perfused at a l5-cmHa0 preload and 90-cmHnO afterload. Perfusate concentrations: glucose, 11 mM; palmitate, 1.2 mM; dichloroacetate (DCA), 0.5 mM. * Significantly different from both control groups. t Significantly different from control hearts perfused under same conditions.
FFA AND DCA EFFECTS
in both controls and diabetics, the presence of FFA had no significant effect on HR. PSP. LVDP was measured at five different afterload pressures (60,70,90, and 140 cmHzO and maximal) and was found to be significantly depressed, at each afterload pressure, in both diabetic groups with respect to controls (Fig. 1). However, the presence of FFA had no significant effect. Positive dP/dt was measured at five different afterload pressures and was found to be depressed, at each afterload pressure, in diabetics perfused in the presence of FFA compared with controls perfused in either condition (Fig. 2). Positive dP/dt was also found to be depressed in diabetics perfused in the presence of FFA compared with diabetics perfused in the presence of glucose alone. This depression was found at each afterload pressure with the exception of 140 cmHzO. Only at an infinite afterload pressure did diabetic hearts perfused in the presence of glucose alone have a +dP/dt significantly depressed from controls perfused under either condition. FFA had no detrimental effect on +dP/dt in control hearts. 185
Control Control Diabetic Diabetic
l -e
o---o A-A
A .I.... A
Polmitate Glucose Palmitate Glucose
! /” .*‘I
I
* 95 ! 50
, 80
I 110 Afterload
Pressure
cmH20
MAX
FIG. 1. Effect of metabolic substrates on left ventricular developed pressure (LVDP) in control and diabetic isolated working rat hearts. LVDP was determined in hearts perfused at a 15cmH20 preload and at various afterload pressures. Glucose was used at a concentration of 11 mM, and palmitate, when used, was added to 11 mM glucose perfusate at a concentration of 1.2 mM. * Significantly different from both control groups.
0 .. ... . 0 a-* A .-w- A *...... *
ON DIABETIC
H1055
HEART
Effect of DCA on FFA-perfused control and diabetic isolated working rat hearts. DCA is a PDH activator that results in increased glucose oxidation rates when added to isolated working heart perfusate (Lopaschuk and McVeigh, unpublished observations). Table 2 demonstrates that, at a medium work load, the addition of 0.5 mM DCA to the FFA perfusate resulted in a significantly higher HR in diabetic hearts than in diabetic hearts perfused in the presence of FFA alone. The diabetic hearts perfused in the presence of DCA showed similar HR to those of controls. HR. PSP, measured at a medium work load, was also found to be significantly depressed in diabetics compared with controls and with diabetics perfused in the presence of 0.5 mM DCA (Table 2). Measured at five different afterload pressures, LVDP was found to be significantly lower in diabetic hearts, at 70 and 90 cmHz0 and maximal afterload pressures with respect to controls and to diabetics perfused in the presence of 0.5 mM DCA (Fig. 3). At a 60.cmHzO afterload diabetic hearts perfused in the absence of DCA had a significantly lower LVDP than control hearts only. Positive dP/dt was measured at five different afterload pressures and was found to be significantly depressed at each afterload pressure compared with controls and with diabetics perfused in the presence of 0.5 mM DCA (Fig. 4 . Effect of FFA and DCA on ventricular tissue metabolites in isolated working control and diabetic hearts. After the above-mentioned isolated working-heart perfusion protocol and at an infinite afterload, hearts were immediately frozen with Wollenberger clamps cooled to the temperature of liquid nitrogen. Tissue metabolites were extracted and assayed from the ventricular tissue as previously described (12). Table 3 demonstrates that ATP and CrP (pmol/g dry wt) were not significantly different in diabetic hearts compared with control hearts. In all hearts, CrP levels were lower than normally seen in hearts perfused at lower work loads (33), probably as a result of hearts being frozen during maximal work. Table 3 also shows that the presence of FFA or DCA in the working-heart perfusate
Control Giucose Control Palmitate Diabetic Glucose Diabetic Palmitate
185
l -e
0 .. ... . 0 A--A
* .. ... . I,
Control Control Diabetic Diabetic
+ .5mM + .5mM
DCA DCA
g510 f-1;:; ___.__ 83f‘.v -.-A* 80
110 140 '1 After-load Pressure cmH20
!~
N=8~
1
.II MAX
FIG. 2. Effect of metabolic substrates on rate of pressure rise (+dP/ dt) in control and diabetic isolated working rat hearts. +dP/dt was determined in hearts perfused at a 15cmHz0 preload and at various afterload pressures. Glucose was used at a concentration of 11 mM, and palmitate, when used, was added to 11 mM glucose perfusate at a concentration of 1.2 mM. * Significantly different from all other groups; + significantly different from diabetic palmitate; Q significantly different from control glucose; t significantly different from control palmitate.
Afterload
Pressure
cmH20
MAX
3. Effect of dichloroacetate (DCA) on LVDP in fatty acidperfused control and diabetic isolated working rat hearts. LVDP was determined in hearts perfused at a 15cmHzO preload and at various afterload pressures. The perfusate contained 11 mM glucose and 1.2 mM palmitate. DCA, when used, was added to perfusate to reach a final concentration of 0.5 mM. * Significantly different from all other S-OUPS; + significantly different from both control groups. FIG.
H1056 8000
FFA AND DCA EFFECTS
-,-
O-0
ON DIABETIC
HEART
tion in both normal and ischemic isolated working pig and rat hearts (6,9). We found that the addition of FFA, 7000 A---A P * .. ..-- * in the form of palmitate, to isolated working rat heart $ .5mM DCA , perfusate had some detrimental effects on diabetic heart 6000 4 Al . function. HR and HR. PSP were not affected by the presence of FFA in the perfusate of spontaneously beating control and diabetic hearts (Table 2). Similarly, 4000 .A----- -A LVDP measured in paced control and diabetic hearts at I increasing work loads was unaffected by the presence of * 3000 FFA (Fig. 1). However, in diabetic hearts +dP/dt was N=6-8 found to be depressed by FFA perfusion at four of five 2000 ! I I 50 80 110 afterload pressures but was found to be unchanged in MAX Afterload Pressure cmH20 control hearts (Fig. 2). In summary, FFA perfusion had FIG. 4. Effect of DCA on +dP/dt in fatty acid-perfused control and no detrimental effects on control or diabetic isolated diabetic isolated working rat hearts. +dP/dt was determined in hearts working hearts, except for a depressive action on +dP/ perfused at a 15cmHzO preload and at various afterload pressures. dt of diabetic hearts. Perfusate contained 11 mM glucose and 1.2 mM palmitate. DCA, when It is well documented that in the myocardium of the used, was added to perfusate to reach a final concentration of 0.5 mM. * Significantly different from all other groups. diabetic there is an increased amount of triglycerides (4). This buildup of triglycerides is suggested to occur from TABLE 3. Effect of palmitate and DCA on left increased levels of circulating FFA resulting in an inventricular tissue ATP, CrP and TG levels creased synthesis of and inhibited hydrolysis of triacylin isoluted working hearts from control and diabetic ruts glycerols (4, 17). In addition, total CoA levels of diabetic myocardium are elevated (B), favoring storage of longpmol/g dry wt chain fatty acids as triacylglycerols rather than transport Perfusion Group Condition across the mitochondrial membrane and subsequent oxTG ATP CrP idation (10). Control Glucose 17.14S.87 10.78t1.68 19.60A6.51 We measured myocardial triglyceride levels in control Glucose-palmitate 15.23t,1.58 6.9421.14 14.98t3.62 and diabetic rats after perfusion with either glucose or +DCA 13.3820.95 9.46t0.98 13.84t2.11 Diabetic Glucose 15.84*1.25 9.8721.18 23.36k2.90 glucose and palmitate as available metabolic substrates Glucose-palmitate 14.1621.86 8.2O-tl.46 23.822502 and found no significant difference between any of the +DCA 14.54k1.50 11.3Ok1.86 34.1428.89 groups (Table 3). Knowing that diabetic preperfusion Values are means t SE obtained from hearts freeze clamped after triglyceride levels in the heart are high suggests that perfusion and at a maximal work load; n = 5-9. Hearts are the same diabetic hearts are relying on endogenous triglyceride as those described in Figs. l-4. See Table 2 legend for perfusate stores for energy production. This proposition is further concentrations. TG, triglyceride. supported by work done by Wall and Lopaschuk (33), who demonstrated a decrease in glucose oxidation rates had no effect on the levels of ATP or CrP found in either in diabetic hearts perfused in the absence of added FFA control or diabetic hearts. compared with control hearts perfused under the same As shown in Table 3, tissue triglyceride levels were not conditions. This suggests that an increased metabolism changed by either the presence of diabetes or by the use of fatty acids from endogenous triglyceride stores must of FFA or DCA in the working-heart perfusate. be occurring to support ATP production. This same group found that the addition of palmitate to the perfusate of diabetic hearts resulted in an almost complete DISCUSSION suppression of glucose oxidation. However, a concomiA major metabolic alteration of diabetes is an in- tant increase in oxidation of exogenous fatty acids was creased concentration of circulating fatty acids and con- not found. Together, this information suggests that diacomitant increased reliance of heart cells on fatty acids betic hearts are relying mainly on endogenous stores of for oxidative phosphorylation (4, 14, 33). This alteration triglycerides for energy production with only a small in lipid metabolism may be responsible for some of the contribution from glucose oxidation. biochemical changes that take place in the diabetic myoThe finding of a depressive effect on +dP/dt in diabetic cardium (29). hearts perfused in the presence of FFA compared with In diabetic rats treatment with lipid-altering drugs diabetic hearts perfused in the absence of FFA may be such as carnitine (24), methyl palmoxirate (28), myo- attributed to further depression of glucose oxidation from inositol (34), methionine, and choline resulted in an that reported to already exist in the diabetic (33). Howimprovement in cardiac function associated with a de- ever, the presence of FFA did not affect HR, HR. PSP, crease in various lipid levels. This evidence strongly or LVDP of diabetic hearts, suggesting that energy from suggests that lipids may be contributing to diabetic dys- endogenous lipid stores was sufficient to maintain most function. aspects of function at the level of a diabetic heart perfused in the absence of FFA. On an acute basis, it has been shown that intravenous The depression of glucose oxidation by fatty acid meinfusion of FFA into anesthetized intact dogs had no effect on the mechanical activity of the heart (13), tabolism has led to much research interest regarding the possibility of increasing glucose oxidation in the diabetic whereas excess FFA were shown to impair cardiac func0 . -.-._- - 0
Control Control Diabetic Diabetic
,’ T
+ SmM
DCA
+
l
.
FFA
AND
DCA
EFFECTS
heart. The conseq uence 0 f this interest has been the developmen t of a number of hypoglycemic drugs that block carnitine palmitoyltransferase I, the enzyme responsible for catalyzing the key regulatory step in fatty acid oxidation. Recently, drugs in this class, incl uding phenylalkyloxi .rane carboxylic acid, the sulfonylurea tolbutamide, and etomoxir, have been demonstrated to increase glucose oxidation rates in isolated working hearts from chronically STZ-diabetic rats (25, 31, 33). In addition to stimulating glucose oxidation in the presence and absence of added palmitate, etomoxir was shown to significantly improve heart function in palmitate-perfused diabetic rats (33). DCA is another pharmacological agent that has proved capable of increasing myocardial glucose oxidation. It is now well established that DCA is an inhibitor of the protein kinase involved in the regulation of the PDH complex, a process resulting in PDH activation. As a result of this activation of PDH by DCA, an increase in glucose oxidation occurs (6, 11). We have also demonstrated that, even in the presence of elevated levels of fatty acids, DCA dramatically stimulates glucose oxidation ( 12). A marked stimulation of glucose oxidation also occurs in chronically diabetic rat hearts. In isolated working hearts obtained from lo-wk STZ rats that were perfused under conditions identical to those used in this study (i.e., high fatty acids), we have found that DCA will increase glucose oxidation from 54.6 t 12.8 to 513.7 t 28.4 nmol [ 14C]glucose oxidized min-’ . g dry wt-’ (Lopaschuk and McVeigh, unpublished observations). This compares with the findings in control hearts, in which DCA stimulates glucose oxidation from 374 t 121 to 1,205 t 2 13 nmol [ 14C]glucose oxidized min-’ . g dry wt-‘. It is thus apparent that DCA would be useful in investigating whether a depression of glucose oxidation is contributing to depressed cardiac function in the diabetic. We investigated the effects of enhanced glucose oxidation on diabetic cardiac function by directly adding 0.5 mM DCA to palmitate-containing i.solated working- heart perfusate. We were able to demonstrate restoration to control heart values of HR and HR. PSP in spontaneously beating diabetic hearts perfused in the presence of DCA (Table 2). In paced hearts, DCA was able to restore LVDP in diabetic hearts to control heart levels at three of five afterload pressures (Fig. 3). Also diabetic hearts perfused in the presence of DCA had +dP/dt values similar to those of control hearts at all afterload pressures measured (Fig. 4). In summary, these results indicate that DCA could acutely reverse the diabetic cardiac function depression. Why DCA acutely reverses the functional changes that occur in the chronically diabetic rat heart is not clear. Previous studies have demonstrated that dysfunctional changes in diabetic rat heart probably occur as a result of alterations in enzymes involved in excitation-contraction coupling, including myosin and actomyosin-adenosinetriphosphatase (ATPase) activities, sarcoplasmic reticular Ca”-ATPase, and Ca*’ transport activity, sarcolemmal Ca”’ pump activity, and mitochondrial activity (see Ref. 30 for review). If this is the case, then these studies demonstrate that the effects of these changes in enzymatic activity on myocardial function can be masked l
l
ON
DIABETIC
HEART
H1057
by acute stimulation of glucose oxidation. These subcellular changes are probably still present but not being manifested as a depression in heart function. Why a stimulation of glucose oxidation would overcome the cardiomyopathic changes is not clear. The fact that heart function can be significantly increased in chronically diabeti c rat hearts by the acute admini .stration of etomoxir, however, suppo rts the hypothesis that acute metabolic changes in diabetes are involved in diabetes-induced cardiac dysfunction (33). This is further supported by studies in ischemic hearts from diabetic rats, in which acute administration of etomoxir decreased the severity of ischemic injury in chronically diabetic rat hearts (9). To date, very little is known about the relationship between acute changes in myocardial energy substrate utilization and the activities of the enzymes involved in excitation-contraction coupling. It is clear, however, that chronic changes in myocardial metabolism alter the activity of these enzymes. Evidence for this is provided by the observations that normalization of various aspects of fatty acid metabolism (and the probable normalization of glucose oxidation secondary to 1these changes) reverses and prevents the changes in heart function associated with diabetes as well as the activities of many of the enzymes involved in excitation-contraction coupling (23, 24, 28, 34).
Why glucose oxidation is important in the heart of the diabetic is not well understood, but several possibilities have been proposed. A debatable explanation is that glycolytically produced ATP in the cytosol is important for membrane proteins, such as the sarcolemmal ATPases (2). Bunger and co-workers (3) offer another theory that increased activity of the PDH complex will prevent the accumulation of glycolytic products such as lactate, which may accumulate under ischemic conditions. Another explanation is that less oxygen is required in the production of ATP when glucose is the substrate. Complete oxidation of glucose produces 3.17 mol ATP/ mol 02, whereas palmitate produces 2.80 mol ATP/mol 02. It is clear that more work needs to be done to elucidate the mechanism by which depressed glucose oxidation has a detrimental effect on diabetic heart function. Other nonspecific effects of DCA on failing hearts also cannot be excluded. An important question is whether a decrease in myocardial ATP synthesis in chronically diabetic rats is contributing to a depression of function. Previous studies have suggested that reduced myocardial function in diabetes is associated with a loss of ATP and adenine nucleotides (19, 26). However, in one of these studies, all measured levels of ATP were extremely low (19). In contrast, Lopaschuk and Spafford (9) found that ATP levels were not depressed in hearts from chronically diabetic rats perfused with palmitate. Likewise, we demonstrated that postperfusion levels of both ATP and CrP, the transport form of ATP, were similar in the myocardium of control and diabetic rats regardless of available substrates or the presence of DCA (Table 3). This evidence suggests that energy concentration is not the limiting factor in diabetic heart function. However, the question of the relationship between ATP levels and myocardial function in the chronic diabetic requires fur-
H1058
FFA AND DCA EFFECTS
ther investigation. In particular, it requires that ATP synthetic rates be determined directly. In summary, we demonstrated that FFA had no detrimental effects on control or 6-wk diabetic isolated working rat hearts, with the exception of a depressive action on +dP/dt of diabetic hearts. We also revealed that addition of the glucose oxidation stimulator, DCA, to palmitate-perfused diabetic isolated working hearts resulted in a marked enhancement of cardiac function. This acute reversal of chronic cardiac depression in the diabetic by DCA suggests that inhibition of glucose oxidation, possibly due to high levels of circulating and endogenous fatty acids, potentiates cardiac func tion depression in the chronic diabetic. It is apparent from this study that good metabolic control of the diabetic, resulting in normalization of myocardial fatty acid metabolism and therefore normalization of glucose metabolism, may be beneficial to diabetic heart function. Chronic control of fatty acid metabolism has proved to lessen the severity of cardiomyopathy in diabetic animals, and as we have shown, reversal of glucose oxidation depression results in enhancement of cardiac function in diabetic rats. Together, this information suggests that therapy . long-term pharmacological aimed at control hng and normalizing fatty acid metabolism is an important clinical goal. This study was supported by a grant from the British Columbia and Yukon Heart and Stroke Foundation. Address for reprint requests: J. H. McNeill, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC V6T 123, Canada. Received 26 September 1990; accepted in final form 14 May 1991. REFERENCES D. E., AND J. P. KING. Methods for the rapid separation and estimation of the major lipids of arteries and other tissues by thin layer chromatography on small plates followed by microchemical assays. J. Chromatogr. 143: 473-490, 1977. 2. BRICKNELL, 0. L., AND L. H. OPIE. Effect of substrates on tissue metabolic changes in the isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion. Circ. Res. 43: 102-115, 1978. 3. BUNGER, R., B. SWINDALL, D. BRODIE, D. ZDUNEK, H. STIEGLER, AND G. WALTER. Pyruvate attenuation of hypoxia damage in isolated working guinea-pig heart. J. Mol. Cell. Cardiol. 18: 4231. BOWYER,
438,1986. 4. DENTON,
R. M., AND P. J. RANDLE. Concentration of glycerides and phospholipids in rat heart and gastrocnemius muscle. Effects of alloxan. Biochem. J. 104: 416-422, 1967. 5. GARLAND, P. B., E. A. NEWSHOLME, AND P. J. RANDLE. Effect of fatty acids, ketone bodies, diabetes and starvation on pyruvate metabolism in rat heart and diaphragm muscle. Nature Land. 195: 6.
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381-383,1962. HENDERSON, A. H., R. J. CRAIG, R. GORLIN, AND E. H. SONNENBLICK. Free fatty acids and myocardial function in perfused rat hearts. Cardiovasc. Res. 4: 466-472, 1970. INGEBRETSON, C. G., P. MOREAU, C. HAWELU-JOHNSON, AND W. R. INGEBRETSON. Performance of diabetic rat hearts: effects of anoxia and increased work. Am. J. Physiol. 239 (Heart Circ. Physiol. 8): H614-H620,1980. KANNEL, W. B., AND D. L. MCGEE. Diabetes and cardiovascular risk factors. The Framingham study. Circulation 59: 8-13, 1979. LOPASCHUK, G. D., AND M. SPAFFORD. Response of isolated work-
ing hearts to fatty acids and carnitine palmitoyltransferase I inhibition during reduction of coronary flow in acutely and chronically diabetic rats. Circ. Res. 65: 378-387, 1989. 10. LOPASCHUK, G. D., AND H. TSANG. Metabolism of palmitate in isolated working hearts from spontaneously diabetic “BB” Wistar
ON DIABETIC
HEART
rats. Circ. Res. 61: 853-858, 1987. 11. MCALLISTER, A., S. P. ALLISON,
AND P. J. RANDLE. Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo. Biochem. J. 134: 1067-1081, 1973. 12. MCVEIGH, J. J., AND G. D. LOPASCHUK. Dichloroacetate stimulation of glucose oxidation improves recovery of ischemic rat hearts. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1079-H1085, 1990. 13. MJOS, 0. D. Effect of free fatty acids on myocardial function and oxygen consumption in intact dogs. J. Clin. Invest. 50: 1386-1389, 1971. 14. MORGAN, H. E., E. CADERAS, D. M. REGAN, AND C. R. PARK.
Regulation of glucose uptake in muscle. III. Rate-limiting steps and effects of insulin and anoxia in heart muscle from diabetic rats. J. Biol. Chem. 236: 262-268, 1961. 15. NEELY, J. R., AND H. E. MORGAN. Relationship between carbohydrate metabolism and energy balance of heart muscle. Annu. Rev. Physiol. 16. NEWSHOLME,
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E. A., P. J. RANDLE, AND K. L. MANCHESTER. Inhibition of the phosphofructokinase reaction in perfused rat heart by respiration of ketone bodies, fatty acids and pyruvate.
Nature Lond. 193: 270-271, 17. PAULSON, D. J., AND M.
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