Metabolic effects of treadmill on the diabetic heart DENNIS Department
J. PAULSON,
ROBERT
MATHEWS,
JEFFERY
BOWMAN,
AND
JIANSHENG
ZHAO
of Physiology, Chicago College of Osteopathic Medicine, Downers Grove, Illinois 60515
PAULSON, DENNIS J., ROBERT MATHEWS, JEFFERY BowMm, mm JIAN~HENG 2~~0. Metabolic effects of treadmill exercise training on the diabetic heart. J. Appl. Physiol. 73(l): 265
271,1992.-This study determinedwhether exercisetraining in rats would prevent the accumulation of lipids and depressed glucoseutilization found in hearts from diabetic rats. Diabetes was induced by intravenous streptozotocin (60 mg/kg). Trained diabetic rats were run on a treadmill for 60 min, 27 m/min, 10%grade, 6 days/wk for 10 wk. Training of diabetic rats had no effect on glycemic control but decreasedplasma lipids. In vivo myocardial long-chain acylcarnitine, acyl-CoA, and high-energy phosphate levels were similar in sedentary control, sedentary diabetic, and trained diabetic groups. The levels of myocardial triacylglycerol were similar in sedentary control and diabetic rats but decreasedin trained diabetic rats. Hearts were perfused with buffer containing diabetic concentrations of glucose(22 mM) and palmitate (1.2 mM). D-[U-‘*Cl glucoseoxidation rates (‘*CO, production) were depressedin hearts from sedentary diabetic rats relative to sedentary control rats. Hearts from trained diabetic rats exhibited increased glucoseoxidation relative to those of sedentary diabetic rats, but this improvement wasbelow that of the sedentary control rats. [9,10-3H]palmitate oxidation rates (3H,0 production) wereidentical in all three groups. These findings suggestthat exercisetraining resulted in a partial normalization of myocardial glucoseutilization in diabetic rats. glucoseand fatty acid oxidation; hyperlipidemia; diabetic cardiomyopathy
PATIENTS suffer from an increased incidence of myocardial infarction (1) and are less likely to survive an ischemic insult (10). In addition to coronary artery disease, diabetic patients are more likely to develop heart failure than nondiabetic patients (8). In many cases, the depression in heart contractile function has been shown to occur in the absence of any discernable alteration in coronary blood flow. Numerous studies (6,27) have demonstrated that experimental animal models of diabetes characteristically exhibit significant alterations in myocardial contractile performance in response to changing preloads, afterloads, extracellular calcium ion concentration, or @-adrenergic receptor stimulation. These contractile abnormalities occur without evidence of coronary artery disease. The term diabetic cardiomyopathy has been used to describe this condition. The cause of these diabetes-inducedcardiac abnormalities is uncertain but may also be related to alterations in glucose or lipid metabolism by the diabetic heart (20,27, 32). The diabetic heart is characterized by impaired glyco-
DIABETIC
exercise training
lytic flux and oxidation, whereas fatty acid oxidation is enhanced (21). This shift in the substrate utilization pattern may affect overall myocardial oxygen consumption as well as alter the energetics of cytoplasmic systems, which preferentially utilize glycolytic ATP (12, 34). Within the diabetic myocardium, there is reportedly an accumulation of glycogen, triacylglycerol, free fatty acids, long-chain acyl-CoA and long-chain acylcarnitine esters (3, 15, 18). Because the accumulation of lipid intermediates has been suggested to interfere with a number of key metabolic processes (9,19,25,35), it has been suggested that the accumulation of these compounds is an important contributing factor to the diabetes-induced cardiac depression. Exercise training of streptozotocin-induced diabetic rats has been shown to be effective in normalizing cardiac contractile pump performance (16) and in improving the recovery of cardiac contractile performance after a period of ischemia and then reperfusion (17). These beneficial effects of exercise training were associated with an increase in myocardial total carnitine content and with a lowering of plasma lipids. Plasma glucose levels were not affected by exercise training, suggesting that the exercise training protocol used in these studies did not affect glycemic control. However, because a single time point in glucose measurement is not a sensitive index of overall glycemic control, it is still uncertain whether the exercise training protocol used in these studies was effective in improving glucose homeostasis. The purpose of this study was to further investigate possible metabolic mechanisms that may account for the beneficial effects of exercise training on the diabetic heart. The hypothesis tested was that exercise training of diabetic rats would have a hypolipidemic action but would not alter overall glycemic control. A more comprehensive assessment of plasma lipids was performed and blood glycosylated hemoglobin levels were measured to accomplish this goal. In addition, we investigated whether exercise training of diabetic rats 1) prevents the accumulation of lipids within the diabetic myocardium, 2) alters myocardial high-energy phosphate levels, and 3) modifies the ability of the diabetic heart to utilize glucose and/or palmitate as metabolic substrates. METHODS
Experimental protocols. Male Sprague-Dawley rats weighing between 125 and 175 g were obtained from Sasco/King Animal Laboratories (Oregon, WI) and housed
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266
EFFECTS
OF
TRAINING
under quarantined conditions for a period of 9 days. All rats were exercised each morning for a 2-wk exercise stress adaptation period. Treadmill speed and exercise duration were gradually increased during this period until all rats were adapted to running at a speed of 18 m/ min for 20 min. Rats were divided into three groups: sedentary control, sedentary diabetic, and exercise-trained diabetic. Induction of diabetes. Rats from the exercise-adapted group and sedentary group were selected at random, anesthetized with ether, and made diabetic by intravenous injection of streptozotocin (60 mg/kg) via the tail vein. The streptozotocin was dissolved in a 0.1 M citrate buffer (pH 4.5) and injected within 5 min after preparation. The control group received sham injection of an equal volume of vehicle. Seventy-two hours after the injection was performed, the induction of diabetes was confirmed by glucose testing of the urine using AmesKeto-Diastix. At this time, the treadmill exercise regimen was reinstated for the exercise-trained diabetic group. Exercise training regimen. The rats were run on the treadmill 6 days a week at a 10% grade. The speed and duration were gradually increased during the next 2 wk until each rat was running continuously for a period of 1 h/day at 27 m/min. The duration of diabetes and exercise training was -10 wk. Excision of hearts. In rats from each of the three groups (sedentary control, sedentary diabetic, and trained diabetic), hearts were freeze-clamped in situ to measure the effects of diabetes with and without exercise training on in vivo levels of cellular metabolites: long-chain acylcarnitine, long-chain acyl-CoA, triacylglycerol, and high-energy phosphate compounds. Before removal of the heart, each rat was anesthetized with ketamine (80 mg/kg) and xylazine (8 mg/kg). A midline incision was made at the throat, and the trachea was isolated and connected to a Phipps and Bird small-animal respirator. The chest was opened, and a suture was placed through the apex of the heart. Cold 30 mM KC1 was injected into the left ventricle, and the heart was freeze-clamped in situ. Tissue analysis. The frozen heart was assayed for ATP, ADP, AMP, long-chain acyl carnitine, and long-chain acyl-CoA. The heart was weighed and fragmented into a powdered tissue. A 50.mg aliquot was used for determination of the dry-to-wet weight ratio. Another 200 mg of powdered frozen tissue were mixed with 1 ml of cold 6% perchloric acid and immediately sonicated for 2 s. This homogenate was centrifuged at 16,000 g for 2 min, and the supernatant saved. To the pellet another 1 ml of cold 6% perchloric acid was added and then sonicated for 1 s. After centrifugation the supernatants were pooled, pH was adjusted to 5.5-6.5 with 2 M tris(hydroxymethyl)aminomethane and assayed for ATP, ADP, and AMP using the high-performance liquid chromatography (HPLC) procedure of Guranowski et al. (7). For this procedure, a VYDAC column, 303NT405 ion-exchange column and Gilson HPLC system were used. The mobile phase consisted of a gradient of two solvents: solvent A, 0.045 M NH,COOH, pH 4.6; and sobent B, 0.50 M NaH,PO,, pH 2.7. Over a 15-min period the percentage of solvent B increased from 0 to 100%. Flow rate was 2.0 ml/min, and
ON
DIABETIC
HEART
the detector was set at 254 nm. Peak area was used to quantify content. To the acid insoluble pellet, 1 ml of 0.5 M KOH was added, and the pellet was sonicated and incubated at 50°C for 1 h. The sample was then cooled, pH was adjusted to 7.0-7.4 with 3-(N-morpholino)propanesulfonic acid HCl, and the sample was centrifuged. Long-chain acylcarnitine was determined from this solution by assaying for free carnitine by the method described by Parvin and Pande (14). Long-chain acyl-CoA esters (100 mg) were extracted using the procedure described by Paulson and Shug (19), and assayed according to the method of Veloso and Veech (31). Myocardial total lipids were extracted from a 200-mg portion of powdered hearts using 20 volumes of chloroform and methanol (2:l). The tubes were gassed with 100% nitrogen and set in the dark for 24 h. The extract was quantitatively poured through Whatman filter paper and then evaporated to dryness under nitrogen. The extract was redissolved in 1 ml of chloroform. A loo-p1 aliquot was evaporated to dryness under nitrogen. Triacylglycerol content was determined using a calorimetric assay kit (Sigma Chemical). Blood analyses. Immediately after excision of the heart, the pooled blood in the chest cavity was transferred into a heparinized test tube. After centrifugation, the plasma was removed and analyzed for the concentrations of glucose, triacylglycerol, cholesterol, and insulin by using kits purchased from Sigma Chemical. Plasma free fatty acids were measured using a Waco NEFA kit. Glycosylated hemoglobin levels were measured in the packed red blood cells using the Helena Glyco-Tek affinity column method (normal values: 7-S%). Perfusion of hearts. A different set of rats was used to measure myocardial substrate utilization. Each rat was anesthetized as described above and injected with 500 U of heparin. The chest was opened, and the heart was excised, placed in cold perfusion buffer, and mounted on the perfusion apparatus (13) within 60 s. Each heart was perfused initially in a nonrecirculating Langendorff manner for 10 min. The initial perfusion medium consisted of a Krebs-Henseleit bicarbonate buffer [(in mM): 118 NaCl, 4.7 KCl, 2.5 CaC&, 1.2 MgSO,, 1.2 KH,PO,, 25 NaHCO, (pH 7.4)] containing 11 mM glucose, gassed with 95% O,-5% CO,, and maintained at 37°C. The heart was then switched to perfusion medium containing the above concentrations of electrolytes with 3% albumin bound to 1.2 mM [9,10-3H]palmitate (850,000 disintegrations per min (dpm) /ml) and containing 22 mM [U14C] glucose (400,000 dpm/ml). During perfusion, aortic pressure and heart rate were continuously monitored. The hearts were paced throughout at 300 beats/min using a Grass S88 stimulator. The perfusion apparatus was a closed system that permitted quantitative collection of 14C0, produced from the oxidation of exogenous glucose. Liquid-phase 14C0, was also determined at 15min intervals by acidifying an aliquot of the perfusion medium. Tritiated water production was used to estimate exogenous fatty acid oxidation. An additional l-ml aliquot of the perfusion medium was also taken at 15-min intervals and added to 5 ml of Dole-Meinertz extraction solution. The solution was vortexed for 15 s and allowed to stand l
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EFFECTS
OF TRAINING
ON DIABETIC
267
HEART
1. Effects of diabetes and exercise training on blood glycosylated hemoglobin, plasma glucose, triglycerides, total cholesterol, high-density-lipoprotein cholesterol, and free fatty acids
TABLE
HDL
Group
n
Control Diabetic Exdiabetic
14 15 13
gHb, %
7.4kO.4 18.4t0.7* 20.6t0.7*
Glu, mM
10.6t0.8 26.Ot_O.9* 26.5-+0.7*
TG, mM
0.73t0.10 2.46*0.38* 1.31+0.24*t
Cholesterol, mM
Cholesterol, mM
FFA, mM
1.71t0.13 2.82*0.26* 1.86+_0.18t
0.93kO.08 1.50+0.16* 1.03t_o.10
0.49t0.06 1.03t0.15* 0.74kO.08
Insulin, jJJ/ml
40+3 27t2* 24+_2*
Values are means t SE; n, no. of rats. gHb, glycosylated hemoglobin; Glu, glucose; TG, triglycerides; HDL, high-density lipoprotein; fatty acids. * Significantly different from control, P < 0.05. t Significantly different from diabetic, P < 0.05.
for 15 min. To this solution 2 ml of water and 3 ml of heptane were added, vortexed for 15 s, and allowed to stand for 15 min. The upper organic phase was removed. The lower aqueous phase contained [14C]glucose and 3H 0 To separate these isotopes, a l-ml aliquot of the loier phase was re-extracted with 5 ml of Dole-Meinertz solution and treated as above. The organic phase was discarded while 0.5 ml of the lower aqueous phase was added to columns containing Dowex LX4 anion-exchange resin (200-400 mesh) and eluted with 1 ml of water. These columns were prepared by incubating the Dowex in 1 M boric acid overnight. The Dowex was rinsed several times with water and then added to Pasteur pipettes containing a small glass wool plug. The height of the column was -5 cm and width was 0.5 cm. Radioactivity determinations. All samples were counted on a Packard 1900CA Tricarb liquid scintillation counter using double-isotope counting techniques. Statistical analyses. Tissue metabolite values on nonperfused hearts were expressed on a per gram wet weight basis. Substrate oxidation rates on perfused hearts were expressed on a per gram dry weight basis. Data obtained for each group were compared to determine statistically significant differences using a one-way analysis of variance test and a least significant difference test for post hoc comparisons among treatment groups. A P value < 0.05 was accepted as significant. RESULTS
The effects of diabetes and exercise training on blood metabolic profile are reported in Table 1. Diabetic rats exhibited a marked elevation in blood glycosylated hemoglobin and plasma glucose relative to control rats. Exercise training of diabetic rats had no effect on these indexes of glycemic control. Diabetic rats were characterized by significantly increased levels of plasma lipids: triacylglycerol, total cholesterol, high-density lipoprotein cholesterol, and nonesterified fatty acids. With the exception of nonesterified fatty acids, exercise training produced a significant lowering of these lipids. Plasma insulin levels were significantly decreased in both sedentary and trained diabetic rats. Exercise training had no effect on plasma insulin. The effects of diabetes with and without exercise training on heart and body weights are shown in Table 2. Both sedentary and exercised-trained diabetic rats had significantly lowered body and heart weights compared with control rats. The heart-to-body weight ratios of all three groups were similar.
FFA, free
Despite the elevation of plasma lipids, the sedentary diabetic rats did not demonstrate an increased in vivo myocardial content of long-chain acylcarnitine, longchain acyl-CoA, or triacylglycerol levels (Table 3). Exercise training of diabetic rats had no effect on long-chain acylcarnitine or CoA esters but produced a significant lowering of myocardial triacylglycerol levels. The effects of diabetes and exercise training on in vivo myocardial ATP, ADP, and AMP content are shown in Table 4. There were no significant differences in myocardial high-energy phosphate content among the three groups. Another set of hearts was perfused under Langendorff conditions with buffer containing the concentrations of glucose (22 mM) and fatty acids (1.2 mM) characteristic of poorly controlled diabetes. To ensure that the cardiac work and myocardial oxygen consumption would be identical between all three groups, the hearts were perfused under these low-work-load conditions and paced at 300 beats/min. Aortic systolic pressure among the three groups was virtually identical during the 60-min perfusion protocol (data are not shown). D- [u-‘“C] glucose oxidation was estimated by the amount of gas-phase and liquid-phase 14C0, produced. The oxidation of [9,10-3H] palmitate was determined by the amount of 3H,0 produced. Glucose oxidation, as measured by the production of gas-phase 14C02, was depressed in hearts from sedentary diabetic rats relative to those from sedentary control rats (Fig. 1, top). Total glucose oxidation (gas-phase plus liquid-phase 14C02; Fig. 1, bottom) in hearts from control rats was 19 t 1 pmol. g dry wt-’ 60 min. The rate of total glucose oxidation was significantly depressed in hearts from sedentary diabetic rats (10 t 1 pmol . g dry wt-l. 60 min-l). Hearts from trained diabetic rats exhibited improved glucose oxidation rates (14 t 1 pmol . g dry wt-l. 60 min-‘) relative to sedentary diabetic hearts; however, the rate of l
2. Effects of diabetes and exercise training on body weight, heart weight, and heart-to-body weight ratio TABLE
Group
n
Control Diabetic Exdiabetic
14 15 13
Body Weight,
442k57 276t40* 277k29”
g
Heart Weight,
279k16 192tl1* 179+8*
mg
Heart-to-Body Weight Ratio, wk
0.615kO.037 0.695kO.045 0.648kO.024
Values are means t SE; n, no. of rats. * Significantly different from control, P < 0.05.
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268
EFFECTS
OF TRAINING
ON DIABETIC 12-
3. Effects of diabetes and exercise training on in vivo myocardial long-chain acyl carnitine, long-chain acyl-CoA esters, and triacylglycerol levels
TABLE
Long-Chain Acyl Carnitine, pmol/g wet wt
Group
n
Control Diabetic Exdiabetic
12
172t23
9 13
176~24 167t25
HEART O-A -0
0 Control Diabetic Ex-Diabetic
Long Chain AcylCoA, pmol/g wet wt
20.7k5.8 32.3k5.9 23.9k3.6
Values are means t SE; n, no. of rats. * Significantly different from control, P < 0.05. t Significantly different from diabetic, P < 0.05.
glucose oxidation remained below that of control levels. During the 60-min period of perfusion, the amount of fatty acid taken up and oxidized in the heart was considerably greater than the molar amount of glucose metabolized in all three groups. Palmitate oxidation rates were also identical among the three groups (-24 pmol g dry wt-l .60 min-‘; Fig. 2). These rates for glucose and fatty acid oxidation are consistent with previously published reports (26, 32) and indicate that hearts from both control and diabetic rats perfused under diabetic substrate conditions utilize exogenous free fatty acids as the predominant energy substrate. Even though all hearts were perfused under identical substrate conditions, glucose oxidation rates were depressed to a greater extent in hearts from sedentary diabetic rats. Exercise training of diabetic rats partially improved the ability of diabetic hearts to utilize glucose. The relative contribution of exogenous glucose and palmitate oxidation to ATP generation is shown in Table 5. ATP generation was calculated using the theoretical values of 38 molecules of ATP produced per molecule of glucose oxidized and 129 molecules ATP produced per molecule of palmitate oxidized. In sedentary diabetic hearts the yield of ATP from glucose oxidation was markedly depressed relative to sedentary control hearts. Exercise-trained diabetic hearts had greater rates of ATP production via glucose oxidation; however, this rate was still below that of the sedentary control hearts. In all three groups, the overwhelming majority of the ATP produced from the oxidation of exogenous substrates came from palmitate. In the sedentary controls, the ratio of ATP produced from the oxidation of exogenous glucose to palmitate was 26%. In sedentary and exercisedtrained rats the values for these ratios were 15 and 19%, respectively.
30 Time
(min)
20-m
k gl 4 l8 u
s
16-m
g
$
14--
l
g$
12--
8;
lo--
$ k ‘is .I z )-
*+ +
8-&. 4-2 OControl
Diabetic
Ex-Diabetic
1. [U-14C]glucose oxidation as estimated by production of 14C0,. Top: gas-phase 14C0, production; bottom: both gas- and liquidphase 14C0,. All values are means k SE for 13-14 rats. * Significantly different from control, P < 0.05. + Significantly different from diabetic rats, P < 0.05. FIG.
function associated with the streptozotocin-induced diabetic rat. Exercise training of diabetic rats was found to I) prevent the depression in cardiac pump function of isolated perfused working hearts subjected to varying left atria1 filling pressures (16) and 2) improve the ability of hearts from diabetic rats to recover pump function after a 75min period of low-flow ischemia followed by 30 min of reperfusion (17). In these experiments, all hearts were perfused with buffer containing the concentrations of glucose (22 mM) and palmitate (1.2 mM) characteristic 30 c .-0 % 73% l
Previously, we have shown that exercise training is beneficial in limiting some of the alterations in cardiac
60
22-
l
DISCUSSION
45
0 -0 A -A
Control Diabetic
25
-
6
F20
I
4. Effects of diabetes and exercise training on in vivo myocardial ATP, ADP, and AMP content TABLE
Group
n
ATP, pmol/g wet wt
Control Diabetic Exdiabetic
12 12 13
3.35kO.37 4.31kO.34 4.09t0.45
ADP, pmol/g wet wt
AMP, pmol/g wet wt
1.03t0.15
0.39t0.07 0.34t0.06 0.25kO.03
Values are means t SE; n, no. of rats.
1.13t0.10 1.45t0.11
i0 Time
(min)
2. [9,10-3H]palmitate oxidation as estimated by production 3H,0. All values are means t SE for 13-14 rats. FIG.
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of
EFFECTS
OF TRAINING
ON DIABETIC
HEART
269
sible mechanism for the harmful effect of elevated exogenous lipids is that they may cause an excessive accumulation of lipid compounds such as nonesterified free fatty acids, long-chain acylcarnitine, long-chain acyl-CoA esters, and triacylglycerol. The excessive accumulation of ATP Production these compounds has been suggested to be harmful to the myocardium because of either nonspecific detergent acFrom glucose, From palmitate, pmol g dry prnol g dry tions or specific inhibition of a variety of metabolic prowt-’ 60 min-’ Group n wt-’ 60 min-’ Glucose/palmitate cesses (9). For example, long-chain acyl-CoA esters have been shown to inhibit the adenine nucleotide transloca724t52 Control 14 3,162+375 0.26kO.03 384*23* 3,119&362 Diabetic 14 0.15*0.02* tor of isolated mitochondria (19). Nonesterified free fatty 520+49*t Exdiabetic 13 3,079t340 0.19to.O2* acids reportedly have a decoupling effect on isolated mitochondria (25). Long-chain acylcarnitine has been Values are means t SE; n, no. of rats. * Significantly different from control, P < 0.05. 7 Significantly different from diabetic, P < 0.05. shown to depress sarcolemmal Na+,K+-adenosinetriphosphatase (ATPase) activity (35). These inhibitions of of poorly controlled di abetes mellitus. This buffer comkey metabolic processes may result in altered energy meposition was also used in the present study. The benefitabolism and contractile function. Our results indicated cial cardiac effects of exercise training were associated that even though plasma lipids were elevated, the hearts with a lowering of plasma triacylglycerol and total cholesfrom sedentary diabetic rats did not demonstrate an in terol concentration while having no apparent effect on vivo accumulation of triacylglycerol, long-chain acylplasma glucose. The decrease in myocardial total carniCoA, and carnitine esters, nor were there any differences tine content found in the sedentary diabetic rat was pre- in myocardial ATP levels among control, sedentary diavented by exercise training. betic, and trained diabetic rats. These findings appear to The results of the present study confirmed our pre- be in conflict with previous studies (3, 15, 18) that have vious finding th.at exercise training of streptozotocin-inshown that diabetic animals produce an increase in these duced diabetic rats (60 mg/kg) has no effect on overall lipid compounds; however, it should be pointed out that glycemic control as assessed by a single time point these previous studies were performed in relatively plasma glucose or by the level of blood dYCOSY lated hemodiabetic animals (12 wk). It appears that globin. The decrease in plasma insulin levels found in short-term these lipids may accumulate initially after the induction diabetic animals was also unaffected by exercise training. of diabetes but that they eventually return to normal Similar results have been obtained by others (2). Allevels. Exercise training of diabetic rats had no effect on though it is true that other studies have shown a glucosemyocardial long-chain acyl-CoA and carnitine esters but lowering effect of exercise training in human diabetics produced a lowering of triacylglycerol content. These and experimental diabetic animals, the severity of diafindings suggest that there may not be a correlation bebetes in these studies was relatively mild (5, 28, 29). In tween the accumulation of lipids and the depressed carsevere studies utilizing a more form of diabetes (16, 17, diac contractile performance associated with diabetes 30,33), exercise training has no effect on overall glycemic mellitus. levels. Another possible mechanism that may account for the As shown in previous studies (16, 17, 28), the exercise correlation between diabetic hyperlipidemia and detraining protocol used in this study did have a significant hypolipidemic effect. Exercise-trained diabetic rats have pressed cardiac performance may be the effect of exogenous lipids on myocardial glucose utilization. According significantly decreased plasma levels of triacylglycerol, to this postulated mechanism an increase in circulating total cholesterol and high-density-lipoprotein choleslipids results in the inhibition of glucose utilization with terol, Plasma nonesterified fatty acids tended to be lower fatty acid oxidation becoming the predominant source of in this group, but the decrease was not statistically signifenergy (21). Because the ratio of mitochondrial produced icant. This lowering of plasma lipids may have a number ATP to oxygen molecule consumed for glucose oxidation of metabolic consequences on the diabetic heart that is higher than the ratio for fatty acid oxidation, the decould account for the beneficial effects of exercise crease in glucose utilization may cause an imbalance betraining. A number of studies have suggested that elevated exog- tween the supply and demand for oxygen. This shift in the substrate utilization pattern of the heart may also enous lipid levels may contribute to the cardiac dysfunccontribute to the insulin resistance and hyperglycemia tion found in experimental diabetic animals (20, 23, 27). found in diabetic rats (22,24); thus the inherent ability of Long-term treatment with lipid-lowering drugs or agents that -inhibit fatty acid oxidation have been shown to pre- the diabetic heart to utilize glucose may be impaired by As shown in the present study, vent the cardiac dysfu n&ion of hearts from diabetic rats. chronic hyperlipidemia. In addition, acute lowering of exogenous free fatty acid this imbalance is not enough to affect total myocardial ATP levels, but it may be sufficient to affect metabolic concentration has also been shown to improve cardiac performance of the hearts of diabetic rabbits (4) and processes that require glycolytic ATP. For example, it has been proposed that glycolytic ATP is the principle rats (20). (12). The A number of mechanisms have been proposed to ex- substrate for the sarcolemmal Na+,K+-ATPase level of glycolytic ATP may also regulate ATP-sensitive plain why the hyperlipidemia associated with diabetes mellitus is harmful to the diabetic myocardium. One pos- potassium channels (34). Alterations in the function of
5. Calculated ATP produced from oxidation of exogenousglucose and palmitate in isolated Langendorff perfused hearts from sedentary control, sedentary diabetic, and exercise-trained diabetic rats TABLE
l
l
l
l
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270
EFFECTS
OF TRAINING
these processes may adversely affect cardiac contractile function. By lowering plasma lipids, exercise training should enhance glucose oxidation by the diabetic heart. Exercise training also reportedly enhances insulin sensitivity as well as the ability of various tissues to utilize glucose (2). The results of the present study showed that myocardial glucose oxidation rate was decreased by 50% in sedentary diabetic rats relative to sedentary control rats. For all groups, hearts were perfused under substrate concentrations that are characteristically found in poorly controlled diabetes mellitus. Exercise training of diabetic rats significantly improved the ability of diabetic hearts to utilize glucose as a substrate; however, the rates of glucose oxidation in this group were not returned completely to levels found in hearts of the sedentary control rats. There were no differences in the ability of these hearts to utilize exogenous free fatty acids as a substrate for energy metabolism. From a total energy output standpoint, exogenous glucose oxidation theoretically should yield ~724 pmol g-l. h-l of ATP in control hearts, while fatty acid oxidation should produce considerably more ATP, ~3,162 pm01 g-l h--l. Thus, under the perfusion conditions of this study, exogenous glucose oxidation contributes only a modest amount of ATP to the total pool. The yield of ATP from glucose oxidation in the sedentary diabetic rat was calculated to be - 384 vs. 3,119 pmol g-l h-l for palmitate oxidation. In exercise-trained diabetic rats, exogenous glucose oxidation yielded slightly more ATP, 520 pmol g-l 6h-l, but was still considerably lower than the ATP obtained from the oxidation of exogenous palmitate, 3,079 pm01 g-l h-l. This small increase in glucose-derived ATP does not affect myocardial total ATP content, but it may have important implications of those metabolic processes that require glycolytic ATP. It should be pointed out that our experiment only measured glucose oxidation through the pyruvate dehydrogenase pathway. It is possible that glycolytic flux to lactate may have been increased even further in hearts of exercise-trained diabetic rats. Evidence in support of this hypothesis to explain the beneficial effects of exercise training on the diabetic heart comes from a number of other studies. Rosen and Reinauer (24) showed that addition of sodium 2-[5-(4chlorophenyl)-pentyl-oxirane-2-carboxylate, a selective inhibitor of carnitine palmitoyltransferase I (CPT-I), to the perfusion medium of diabetic hearts increased glucose uptake, glycolysis, pyruvate oxidation and insulin sensitivity. Wall and Lopaschuk (32) showed that Etomoxir, another CPT-I inhibitor, treatment of diabetic rats partially reversed the decrease in glucose oxidation and improved cardiac function. Another study by this laboratory (11) showed that Etomoxir improved the recovery of hearts subjected to ischemia and reperfusion. This beneficial effect of Etomoxir was not associated with affects of myocardial long-chain acyl carnitine but rather with an increase in glucose utilization by the diabetic heart. Treatment of diabetic rats with antilipolytic agents such as nicotinic acid and phenylisopropyladenosine were shown by Reaven et al. (22) to lower both plasma free fatty acids and glucose. Thus the inhibition l
l
l
l
l
l
l
l
ON DIABETIC
HEART
of glycolysis by elevated exogenous lipid may contribute to the impaired contractile function associated with diabetes mellitus. In summary, the results of the present study extend and support previous findings indicating that exercise training of diabetic rats lowers plasma lipids but does not affect glycemic control. Neither diabetes nor exercise training had any effect on the myocardial accumulation of lipids or ATP content. The lowering of plasma lipids in exercise-trained diabetic rats was associated with an improved ability of hearts from these animals to utilize glucose as a metabolic substrate. This effect of exercise training on plasma lipids and myocardial glucose utilization may be a mechanism to account for the beneficial effects of this treatment in preventing some of the cardiac abnormalities associated with diabetes mellitus. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39200. Address for reprint requests: D. J. Paulson, Dept. of Physiology, Chicago College of Osteopathic Medicine, 555 31st St., Downers Grove, IL 50515. Received 19 August 1991; accepted in final form 17 January 1992. REFERENCES G., AND H. SCHULTE. Diabetes mellitus and hypertension in the elderly: concomitant hyperlipidemia and coronary heart disease risk. Am. J. Curdiol. 63: 33H-37H, 1989. 2. DALLAGLIO, E., F. CHANG, H. CHANG, D. WRIGHT, AND G. M. REAVEN. Effect of exercise training and sucrose feeding on insulin-stimulated glucose uptake in rats with streptozotocin-induced insulindeficient diabetes. Diabetes 32: 165-168, 1983. 3. FEUVRAY, D., J. A. IDELL-WENGER, AND J. R. NEELY. Effects of ischemia on rat myocardial function and metabolism in diabetes. Circ. Res. 44: 322-329, 1979. 4. FIELDS, L. E., A. DAUGHERTY, AND S. R. BERGMANN. Effects of fatty acid on performance and lipid content of hearts from diabetic rabbits. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H10781. ASSMANN,
H1085,1986. 5. GOODYEAR, L. J., M. F. HIRSHMAN, E. SON, L. J. WARDZALA, AND E. S, HORTON.
D. HORTON, S. M. KNUTExercise training normalizes glucose metabolism in a rat model of impaired glucose tolerance. Metabolism 40: 455-464, 1991. 6. GOTZSCHE, 0. Myocardial cell dysfunction in diabetes mellitus: a review of clinical and experimental studies. Diabetes 35: 1158-1162, 1986. 7. GURANOWSKI, A., J. CHIANG. Adenosine
A. MONTGOMERY, G. L. CANTONI, AND P. K. analogues as substrates and inhibitors of Sadensylhomocysteine hydrolase. Biochemistry 20: llO-115,198l. 8. KANNEL, W. B. Role of diabetes in cardiac disease: conclusions from population studies. In: Diabetes and the Heart, edited by S. Zonaraich. Springfield, IL: Thomas, 1978, p. 97-112. 9. KATZ, A. M., AND F. C. MESSINEO. Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ. Res. 48: 1-16, T. 10. KOTLER, 11.
12. 13.
14. 15.
1981.
S., AND G. A. DIAMOND. Myocardial ischemia in diabetic patients. Ann. Intern. Med. 109: 678-678, 1988. LOPASCHUK, G. D., S. R. WALL, P. M. OLLEY, AND N. J. DAVIES. Etomoxir, a carnitine palmitoyltransferase I inhibitor, protects hearts from fatty acid-induced ischemic injury independent of changes in long chain acylcarnitine. Circ. Res. 63: 1036-1043,1988. MERCER, R. W., AND P. B. DUNHAM. Membrane-bound ATP fuels the Na/K pump. J. Gen. Physiol. 78: 547-568,198l. NEELY, J. R., H. LIEBERMEISTER, E. J. BATTERSBY, AND H. E. MORGAN. Effect of pressure development on oxygen consumption by isolated rat heart. Am. J. Physiol. 212: 804-814, 1967. PARVIN, R., AND S. V. PANDE. Microdetermination of (-) carnitine and carnitine acetyltransferase. Anal. Biochem. 79: 190-201, 1977. PAULSON, D. J., AND M. F. CRASS III. Endogenous triacylglycerol metabolism in diabetic heart. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H1084-H1094,
1982.
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EFFECTS
OF TRAINING
D. J., S. J. KOPP, D. G. PEACE, AND J. P. Tow. Myocardial adaptation to endurance exercise training in diabetic rats. Am.
16. PAULSON,
J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): Rl073Rl081, 1987. 1% PAULSON, D. J., S. J. KOPP, D. G. PEACE, AND J. P. Tow. Improved
postischemic recovery of cardiac pump function in exercisedtrained diabetic rats. J. Appl. Physiol. 65: 187-193, 1988. 18. PAULSON, D. J., M. J. SCHMIDT, J. S. TRAXLER, M. T. RAMACCI, AND A. L. SHUG. Improvement of myocardial function in diabetic rats after treatment with L-carnitine. Metabolism 33: 358-363, 1984. 19. PAULSON,
D. J., AND A. L. SHUG. Inhibition of the adenine nucleotide translocator by matrix-localized palmityl-CoA in rat heart mitochondria. Biochim. Biophys. Acta 766: 70-76, 1984. 20. PAULSON, D. J., A. L. SHUG, R. JURAK, AND M. SCHMIDT. The role of altered lipid metabolism in the cardiac dysfunction associated with diabetes. In: The Diabetic Heart, edited by M. Nagano and N. S. Dhalla. New York: Raven, 1991, p. 395-407. 21. RANDLE, P. J., C. N. HALES, P. B. GARLAND, AND E. A. NEWSHOLME. The glucose fatty acid cycle. Its role in insulin sensitivity and the metabolic disturbance of diabetes mellitus. Lancet 1: 785789,1963. 22. REAVEN, G. M., H. CHANG, H. Ho, C. Y. JENG, AND B. B. HOFFMAN. Lowering of plasma glucose in diabetic rats by antilipolytic agents. Am. J. Physiol. 254 (Endocrinol. Metab. 17): E23-E30,1988. 23. RODRIGIJES, B., R. K. GOYAL, AND J. H. MCNEILL. Effects of hy-
dralazine on streptozotocin-induced diabetic rats: prevention of hyperlipidemia and improvement of cardiac function. J. Pharmacol. Exp. Ther. 237: 292-299, 1986. 24. ROSEN, P., AND H. REINAIJER.
Influence of the carnitine palmitoyltransferase inhibitor POCA on myocardial performance and metabolism of insulin resistant rats. Acta Physiol. Hung. 71: 271-280,
1988. 25. ROTTENBERG,
H., AND S. STEINER-M•
RDOCH.
Free fatty acids de-
ON DIABETIC
HEART
271
couple oxidative phosphorylation by dissipating intramembranal protons without inhibiting ATP synthesis driven by the proton electrochemical gradient. FEBS Lett. 202: 314-318, 1986. 26. SADDIK, M., AND G. D. LOPASCHUK. Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts. J. Biol. Chem. 266: 8162-8170, 1991. 27. TAHILIANI, A. G., AND J. H. MCNEILL. Minireview, diabetes-induced abnormalities in the myocardium. Life Sci. 38: 959-974, 1986. 28. TAN,
M. H., A. BONEN, J. B. GARNER, AND A. N. BELCASTRO. Physical training in diabetic rats: effects on glucose tolerance and serum lipids. J. Appl. Physiol. 52: 1514-1518, 1982. 29. TANCREDE, G., S. ROUSSEAU-MIGNERON, AND A. NADEAU. Beneficial effects of physical training in rats with a mild streptozotocininduced diabetes mellitus. Diabetes 31: 406-409, 1982. 30. VALLERAND, A. L., J. LUPIEN, Y. DESHAIES, AND L. J. BUKOWIECKI. Intense exercise training does not improve intravenous glucose tolerance in severely diabetic rats. Horm. Metab. Res. 18: 79-81,1986. 31. VELOSO, D., AND R. L. VEECH.
Stoichiometric hydrolysis of long chain acyl CoA and measurement of the CoA formed with an enzymatic cycling method. Anal. Biochem. 62: 449-450, 1974. 32. WALL, S. R., AND G. D. LOPASCHUK. Glucose oxidation rates in fatty acid-perfused isolated working hearts from diabetic rats. Biochim. Biophys. Acta 1006: 97-103, 1989. 33. WALLBERG-HENRIKSSON, H., R. GIJNNARSSON, S. ROSSNER, AND J. WAHREN. Long-term physical training in female type 1 (insulin-
dependent) diabetic patients: absence of significant effects on glycaemic control and lipoprotien levels. Diabetologia 29: 53-57, 1986. 34. WEISS, J. N., AND S. T. LAMP. Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis. J. Gen. Physiol. 94: 911-935,1989. 35. WOOD, J. M., B. BUSH,
B. J. R. PITTS, AND A. SCHWARTZ. Inhibition of bovine heart Na+,K+-ATPase by palmitylcarnitine and palmityl-CoA. Biochem. Biophys. Res. Commun. 74: 677-684, 1977.
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J. PAULSON,
ROBERT
MATHEWS,
JEFFERY
BOWMAN,
AND
JIANSHENG
ZHAO
of Physiology, Chicago College of Osteopathic Medicine, Downers Grove, Illinois 60515
PAULSON, DENNIS J., ROBERT MATHEWS, JEFFERY BowMm, mm JIAN~HENG 2~~0. Metabolic effects of treadmill exercise training on the diabetic heart. J. Appl. Physiol. 73(l): 265
271,1992.-This study determinedwhether exercisetraining in rats would prevent the accumulation of lipids and depressed glucoseutilization found in hearts from diabetic rats. Diabetes was induced by intravenous streptozotocin (60 mg/kg). Trained diabetic rats were run on a treadmill for 60 min, 27 m/min, 10%grade, 6 days/wk for 10 wk. Training of diabetic rats had no effect on glycemic control but decreasedplasma lipids. In vivo myocardial long-chain acylcarnitine, acyl-CoA, and high-energy phosphate levels were similar in sedentary control, sedentary diabetic, and trained diabetic groups. The levels of myocardial triacylglycerol were similar in sedentary control and diabetic rats but decreasedin trained diabetic rats. Hearts were perfused with buffer containing diabetic concentrations of glucose(22 mM) and palmitate (1.2 mM). D-[U-‘*Cl glucoseoxidation rates (‘*CO, production) were depressedin hearts from sedentary diabetic rats relative to sedentary control rats. Hearts from trained diabetic rats exhibited increased glucoseoxidation relative to those of sedentary diabetic rats, but this improvement wasbelow that of the sedentary control rats. [9,10-3H]palmitate oxidation rates (3H,0 production) wereidentical in all three groups. These findings suggestthat exercisetraining resulted in a partial normalization of myocardial glucoseutilization in diabetic rats. glucoseand fatty acid oxidation; hyperlipidemia; diabetic cardiomyopathy
PATIENTS suffer from an increased incidence of myocardial infarction (1) and are less likely to survive an ischemic insult (10). In addition to coronary artery disease, diabetic patients are more likely to develop heart failure than nondiabetic patients (8). In many cases, the depression in heart contractile function has been shown to occur in the absence of any discernable alteration in coronary blood flow. Numerous studies (6,27) have demonstrated that experimental animal models of diabetes characteristically exhibit significant alterations in myocardial contractile performance in response to changing preloads, afterloads, extracellular calcium ion concentration, or @-adrenergic receptor stimulation. These contractile abnormalities occur without evidence of coronary artery disease. The term diabetic cardiomyopathy has been used to describe this condition. The cause of these diabetes-inducedcardiac abnormalities is uncertain but may also be related to alterations in glucose or lipid metabolism by the diabetic heart (20,27, 32). The diabetic heart is characterized by impaired glyco-
DIABETIC
exercise training
lytic flux and oxidation, whereas fatty acid oxidation is enhanced (21). This shift in the substrate utilization pattern may affect overall myocardial oxygen consumption as well as alter the energetics of cytoplasmic systems, which preferentially utilize glycolytic ATP (12, 34). Within the diabetic myocardium, there is reportedly an accumulation of glycogen, triacylglycerol, free fatty acids, long-chain acyl-CoA and long-chain acylcarnitine esters (3, 15, 18). Because the accumulation of lipid intermediates has been suggested to interfere with a number of key metabolic processes (9,19,25,35), it has been suggested that the accumulation of these compounds is an important contributing factor to the diabetes-induced cardiac depression. Exercise training of streptozotocin-induced diabetic rats has been shown to be effective in normalizing cardiac contractile pump performance (16) and in improving the recovery of cardiac contractile performance after a period of ischemia and then reperfusion (17). These beneficial effects of exercise training were associated with an increase in myocardial total carnitine content and with a lowering of plasma lipids. Plasma glucose levels were not affected by exercise training, suggesting that the exercise training protocol used in these studies did not affect glycemic control. However, because a single time point in glucose measurement is not a sensitive index of overall glycemic control, it is still uncertain whether the exercise training protocol used in these studies was effective in improving glucose homeostasis. The purpose of this study was to further investigate possible metabolic mechanisms that may account for the beneficial effects of exercise training on the diabetic heart. The hypothesis tested was that exercise training of diabetic rats would have a hypolipidemic action but would not alter overall glycemic control. A more comprehensive assessment of plasma lipids was performed and blood glycosylated hemoglobin levels were measured to accomplish this goal. In addition, we investigated whether exercise training of diabetic rats 1) prevents the accumulation of lipids within the diabetic myocardium, 2) alters myocardial high-energy phosphate levels, and 3) modifies the ability of the diabetic heart to utilize glucose and/or palmitate as metabolic substrates. METHODS
Experimental protocols. Male Sprague-Dawley rats weighing between 125 and 175 g were obtained from Sasco/King Animal Laboratories (Oregon, WI) and housed
0161-7567192 $2.00 Copyright0 1992 the AmericanPhysiological Society
265
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266
EFFECTS
OF
TRAINING
under quarantined conditions for a period of 9 days. All rats were exercised each morning for a 2-wk exercise stress adaptation period. Treadmill speed and exercise duration were gradually increased during this period until all rats were adapted to running at a speed of 18 m/ min for 20 min. Rats were divided into three groups: sedentary control, sedentary diabetic, and exercise-trained diabetic. Induction of diabetes. Rats from the exercise-adapted group and sedentary group were selected at random, anesthetized with ether, and made diabetic by intravenous injection of streptozotocin (60 mg/kg) via the tail vein. The streptozotocin was dissolved in a 0.1 M citrate buffer (pH 4.5) and injected within 5 min after preparation. The control group received sham injection of an equal volume of vehicle. Seventy-two hours after the injection was performed, the induction of diabetes was confirmed by glucose testing of the urine using AmesKeto-Diastix. At this time, the treadmill exercise regimen was reinstated for the exercise-trained diabetic group. Exercise training regimen. The rats were run on the treadmill 6 days a week at a 10% grade. The speed and duration were gradually increased during the next 2 wk until each rat was running continuously for a period of 1 h/day at 27 m/min. The duration of diabetes and exercise training was -10 wk. Excision of hearts. In rats from each of the three groups (sedentary control, sedentary diabetic, and trained diabetic), hearts were freeze-clamped in situ to measure the effects of diabetes with and without exercise training on in vivo levels of cellular metabolites: long-chain acylcarnitine, long-chain acyl-CoA, triacylglycerol, and high-energy phosphate compounds. Before removal of the heart, each rat was anesthetized with ketamine (80 mg/kg) and xylazine (8 mg/kg). A midline incision was made at the throat, and the trachea was isolated and connected to a Phipps and Bird small-animal respirator. The chest was opened, and a suture was placed through the apex of the heart. Cold 30 mM KC1 was injected into the left ventricle, and the heart was freeze-clamped in situ. Tissue analysis. The frozen heart was assayed for ATP, ADP, AMP, long-chain acyl carnitine, and long-chain acyl-CoA. The heart was weighed and fragmented into a powdered tissue. A 50.mg aliquot was used for determination of the dry-to-wet weight ratio. Another 200 mg of powdered frozen tissue were mixed with 1 ml of cold 6% perchloric acid and immediately sonicated for 2 s. This homogenate was centrifuged at 16,000 g for 2 min, and the supernatant saved. To the pellet another 1 ml of cold 6% perchloric acid was added and then sonicated for 1 s. After centrifugation the supernatants were pooled, pH was adjusted to 5.5-6.5 with 2 M tris(hydroxymethyl)aminomethane and assayed for ATP, ADP, and AMP using the high-performance liquid chromatography (HPLC) procedure of Guranowski et al. (7). For this procedure, a VYDAC column, 303NT405 ion-exchange column and Gilson HPLC system were used. The mobile phase consisted of a gradient of two solvents: solvent A, 0.045 M NH,COOH, pH 4.6; and sobent B, 0.50 M NaH,PO,, pH 2.7. Over a 15-min period the percentage of solvent B increased from 0 to 100%. Flow rate was 2.0 ml/min, and
ON
DIABETIC
HEART
the detector was set at 254 nm. Peak area was used to quantify content. To the acid insoluble pellet, 1 ml of 0.5 M KOH was added, and the pellet was sonicated and incubated at 50°C for 1 h. The sample was then cooled, pH was adjusted to 7.0-7.4 with 3-(N-morpholino)propanesulfonic acid HCl, and the sample was centrifuged. Long-chain acylcarnitine was determined from this solution by assaying for free carnitine by the method described by Parvin and Pande (14). Long-chain acyl-CoA esters (100 mg) were extracted using the procedure described by Paulson and Shug (19), and assayed according to the method of Veloso and Veech (31). Myocardial total lipids were extracted from a 200-mg portion of powdered hearts using 20 volumes of chloroform and methanol (2:l). The tubes were gassed with 100% nitrogen and set in the dark for 24 h. The extract was quantitatively poured through Whatman filter paper and then evaporated to dryness under nitrogen. The extract was redissolved in 1 ml of chloroform. A loo-p1 aliquot was evaporated to dryness under nitrogen. Triacylglycerol content was determined using a calorimetric assay kit (Sigma Chemical). Blood analyses. Immediately after excision of the heart, the pooled blood in the chest cavity was transferred into a heparinized test tube. After centrifugation, the plasma was removed and analyzed for the concentrations of glucose, triacylglycerol, cholesterol, and insulin by using kits purchased from Sigma Chemical. Plasma free fatty acids were measured using a Waco NEFA kit. Glycosylated hemoglobin levels were measured in the packed red blood cells using the Helena Glyco-Tek affinity column method (normal values: 7-S%). Perfusion of hearts. A different set of rats was used to measure myocardial substrate utilization. Each rat was anesthetized as described above and injected with 500 U of heparin. The chest was opened, and the heart was excised, placed in cold perfusion buffer, and mounted on the perfusion apparatus (13) within 60 s. Each heart was perfused initially in a nonrecirculating Langendorff manner for 10 min. The initial perfusion medium consisted of a Krebs-Henseleit bicarbonate buffer [(in mM): 118 NaCl, 4.7 KCl, 2.5 CaC&, 1.2 MgSO,, 1.2 KH,PO,, 25 NaHCO, (pH 7.4)] containing 11 mM glucose, gassed with 95% O,-5% CO,, and maintained at 37°C. The heart was then switched to perfusion medium containing the above concentrations of electrolytes with 3% albumin bound to 1.2 mM [9,10-3H]palmitate (850,000 disintegrations per min (dpm) /ml) and containing 22 mM [U14C] glucose (400,000 dpm/ml). During perfusion, aortic pressure and heart rate were continuously monitored. The hearts were paced throughout at 300 beats/min using a Grass S88 stimulator. The perfusion apparatus was a closed system that permitted quantitative collection of 14C0, produced from the oxidation of exogenous glucose. Liquid-phase 14C0, was also determined at 15min intervals by acidifying an aliquot of the perfusion medium. Tritiated water production was used to estimate exogenous fatty acid oxidation. An additional l-ml aliquot of the perfusion medium was also taken at 15-min intervals and added to 5 ml of Dole-Meinertz extraction solution. The solution was vortexed for 15 s and allowed to stand l
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EFFECTS
OF TRAINING
ON DIABETIC
267
HEART
1. Effects of diabetes and exercise training on blood glycosylated hemoglobin, plasma glucose, triglycerides, total cholesterol, high-density-lipoprotein cholesterol, and free fatty acids
TABLE
HDL
Group
n
Control Diabetic Exdiabetic
14 15 13
gHb, %
7.4kO.4 18.4t0.7* 20.6t0.7*
Glu, mM
10.6t0.8 26.Ot_O.9* 26.5-+0.7*
TG, mM
0.73t0.10 2.46*0.38* 1.31+0.24*t
Cholesterol, mM
Cholesterol, mM
FFA, mM
1.71t0.13 2.82*0.26* 1.86+_0.18t
0.93kO.08 1.50+0.16* 1.03t_o.10
0.49t0.06 1.03t0.15* 0.74kO.08
Insulin, jJJ/ml
40+3 27t2* 24+_2*
Values are means t SE; n, no. of rats. gHb, glycosylated hemoglobin; Glu, glucose; TG, triglycerides; HDL, high-density lipoprotein; fatty acids. * Significantly different from control, P < 0.05. t Significantly different from diabetic, P < 0.05.
for 15 min. To this solution 2 ml of water and 3 ml of heptane were added, vortexed for 15 s, and allowed to stand for 15 min. The upper organic phase was removed. The lower aqueous phase contained [14C]glucose and 3H 0 To separate these isotopes, a l-ml aliquot of the loier phase was re-extracted with 5 ml of Dole-Meinertz solution and treated as above. The organic phase was discarded while 0.5 ml of the lower aqueous phase was added to columns containing Dowex LX4 anion-exchange resin (200-400 mesh) and eluted with 1 ml of water. These columns were prepared by incubating the Dowex in 1 M boric acid overnight. The Dowex was rinsed several times with water and then added to Pasteur pipettes containing a small glass wool plug. The height of the column was -5 cm and width was 0.5 cm. Radioactivity determinations. All samples were counted on a Packard 1900CA Tricarb liquid scintillation counter using double-isotope counting techniques. Statistical analyses. Tissue metabolite values on nonperfused hearts were expressed on a per gram wet weight basis. Substrate oxidation rates on perfused hearts were expressed on a per gram dry weight basis. Data obtained for each group were compared to determine statistically significant differences using a one-way analysis of variance test and a least significant difference test for post hoc comparisons among treatment groups. A P value < 0.05 was accepted as significant. RESULTS
The effects of diabetes and exercise training on blood metabolic profile are reported in Table 1. Diabetic rats exhibited a marked elevation in blood glycosylated hemoglobin and plasma glucose relative to control rats. Exercise training of diabetic rats had no effect on these indexes of glycemic control. Diabetic rats were characterized by significantly increased levels of plasma lipids: triacylglycerol, total cholesterol, high-density lipoprotein cholesterol, and nonesterified fatty acids. With the exception of nonesterified fatty acids, exercise training produced a significant lowering of these lipids. Plasma insulin levels were significantly decreased in both sedentary and trained diabetic rats. Exercise training had no effect on plasma insulin. The effects of diabetes with and without exercise training on heart and body weights are shown in Table 2. Both sedentary and exercised-trained diabetic rats had significantly lowered body and heart weights compared with control rats. The heart-to-body weight ratios of all three groups were similar.
FFA, free
Despite the elevation of plasma lipids, the sedentary diabetic rats did not demonstrate an increased in vivo myocardial content of long-chain acylcarnitine, longchain acyl-CoA, or triacylglycerol levels (Table 3). Exercise training of diabetic rats had no effect on long-chain acylcarnitine or CoA esters but produced a significant lowering of myocardial triacylglycerol levels. The effects of diabetes and exercise training on in vivo myocardial ATP, ADP, and AMP content are shown in Table 4. There were no significant differences in myocardial high-energy phosphate content among the three groups. Another set of hearts was perfused under Langendorff conditions with buffer containing the concentrations of glucose (22 mM) and fatty acids (1.2 mM) characteristic of poorly controlled diabetes. To ensure that the cardiac work and myocardial oxygen consumption would be identical between all three groups, the hearts were perfused under these low-work-load conditions and paced at 300 beats/min. Aortic systolic pressure among the three groups was virtually identical during the 60-min perfusion protocol (data are not shown). D- [u-‘“C] glucose oxidation was estimated by the amount of gas-phase and liquid-phase 14C0, produced. The oxidation of [9,10-3H] palmitate was determined by the amount of 3H,0 produced. Glucose oxidation, as measured by the production of gas-phase 14C02, was depressed in hearts from sedentary diabetic rats relative to those from sedentary control rats (Fig. 1, top). Total glucose oxidation (gas-phase plus liquid-phase 14C02; Fig. 1, bottom) in hearts from control rats was 19 t 1 pmol. g dry wt-’ 60 min. The rate of total glucose oxidation was significantly depressed in hearts from sedentary diabetic rats (10 t 1 pmol . g dry wt-l. 60 min-l). Hearts from trained diabetic rats exhibited improved glucose oxidation rates (14 t 1 pmol . g dry wt-l. 60 min-‘) relative to sedentary diabetic hearts; however, the rate of l
2. Effects of diabetes and exercise training on body weight, heart weight, and heart-to-body weight ratio TABLE
Group
n
Control Diabetic Exdiabetic
14 15 13
Body Weight,
442k57 276t40* 277k29”
g
Heart Weight,
279k16 192tl1* 179+8*
mg
Heart-to-Body Weight Ratio, wk
0.615kO.037 0.695kO.045 0.648kO.024
Values are means t SE; n, no. of rats. * Significantly different from control, P < 0.05.
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268
EFFECTS
OF TRAINING
ON DIABETIC 12-
3. Effects of diabetes and exercise training on in vivo myocardial long-chain acyl carnitine, long-chain acyl-CoA esters, and triacylglycerol levels
TABLE
Long-Chain Acyl Carnitine, pmol/g wet wt
Group
n
Control Diabetic Exdiabetic
12
172t23
9 13
176~24 167t25
HEART O-A -0
0 Control Diabetic Ex-Diabetic
Long Chain AcylCoA, pmol/g wet wt
20.7k5.8 32.3k5.9 23.9k3.6
Values are means t SE; n, no. of rats. * Significantly different from control, P < 0.05. t Significantly different from diabetic, P < 0.05.
glucose oxidation remained below that of control levels. During the 60-min period of perfusion, the amount of fatty acid taken up and oxidized in the heart was considerably greater than the molar amount of glucose metabolized in all three groups. Palmitate oxidation rates were also identical among the three groups (-24 pmol g dry wt-l .60 min-‘; Fig. 2). These rates for glucose and fatty acid oxidation are consistent with previously published reports (26, 32) and indicate that hearts from both control and diabetic rats perfused under diabetic substrate conditions utilize exogenous free fatty acids as the predominant energy substrate. Even though all hearts were perfused under identical substrate conditions, glucose oxidation rates were depressed to a greater extent in hearts from sedentary diabetic rats. Exercise training of diabetic rats partially improved the ability of diabetic hearts to utilize glucose. The relative contribution of exogenous glucose and palmitate oxidation to ATP generation is shown in Table 5. ATP generation was calculated using the theoretical values of 38 molecules of ATP produced per molecule of glucose oxidized and 129 molecules ATP produced per molecule of palmitate oxidized. In sedentary diabetic hearts the yield of ATP from glucose oxidation was markedly depressed relative to sedentary control hearts. Exercise-trained diabetic hearts had greater rates of ATP production via glucose oxidation; however, this rate was still below that of the sedentary control hearts. In all three groups, the overwhelming majority of the ATP produced from the oxidation of exogenous substrates came from palmitate. In the sedentary controls, the ratio of ATP produced from the oxidation of exogenous glucose to palmitate was 26%. In sedentary and exercisedtrained rats the values for these ratios were 15 and 19%, respectively.
30 Time
(min)
20-m
k gl 4 l8 u
s
16-m
g
$
14--
l
g$
12--
8;
lo--
$ k ‘is .I z )-
*+ +
8-&. 4-2 OControl
Diabetic
Ex-Diabetic
1. [U-14C]glucose oxidation as estimated by production of 14C0,. Top: gas-phase 14C0, production; bottom: both gas- and liquidphase 14C0,. All values are means k SE for 13-14 rats. * Significantly different from control, P < 0.05. + Significantly different from diabetic rats, P < 0.05. FIG.
function associated with the streptozotocin-induced diabetic rat. Exercise training of diabetic rats was found to I) prevent the depression in cardiac pump function of isolated perfused working hearts subjected to varying left atria1 filling pressures (16) and 2) improve the ability of hearts from diabetic rats to recover pump function after a 75min period of low-flow ischemia followed by 30 min of reperfusion (17). In these experiments, all hearts were perfused with buffer containing the concentrations of glucose (22 mM) and palmitate (1.2 mM) characteristic 30 c .-0 % 73% l
Previously, we have shown that exercise training is beneficial in limiting some of the alterations in cardiac
60
22-
l
DISCUSSION
45
0 -0 A -A
Control Diabetic
25
-
6
F20
I
4. Effects of diabetes and exercise training on in vivo myocardial ATP, ADP, and AMP content TABLE
Group
n
ATP, pmol/g wet wt
Control Diabetic Exdiabetic
12 12 13
3.35kO.37 4.31kO.34 4.09t0.45
ADP, pmol/g wet wt
AMP, pmol/g wet wt
1.03t0.15
0.39t0.07 0.34t0.06 0.25kO.03
Values are means t SE; n, no. of rats.
1.13t0.10 1.45t0.11
i0 Time
(min)
2. [9,10-3H]palmitate oxidation as estimated by production 3H,0. All values are means t SE for 13-14 rats. FIG.
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of
EFFECTS
OF TRAINING
ON DIABETIC
HEART
269
sible mechanism for the harmful effect of elevated exogenous lipids is that they may cause an excessive accumulation of lipid compounds such as nonesterified free fatty acids, long-chain acylcarnitine, long-chain acyl-CoA esters, and triacylglycerol. The excessive accumulation of ATP Production these compounds has been suggested to be harmful to the myocardium because of either nonspecific detergent acFrom glucose, From palmitate, pmol g dry prnol g dry tions or specific inhibition of a variety of metabolic prowt-’ 60 min-’ Group n wt-’ 60 min-’ Glucose/palmitate cesses (9). For example, long-chain acyl-CoA esters have been shown to inhibit the adenine nucleotide transloca724t52 Control 14 3,162+375 0.26kO.03 384*23* 3,119&362 Diabetic 14 0.15*0.02* tor of isolated mitochondria (19). Nonesterified free fatty 520+49*t Exdiabetic 13 3,079t340 0.19to.O2* acids reportedly have a decoupling effect on isolated mitochondria (25). Long-chain acylcarnitine has been Values are means t SE; n, no. of rats. * Significantly different from control, P < 0.05. 7 Significantly different from diabetic, P < 0.05. shown to depress sarcolemmal Na+,K+-adenosinetriphosphatase (ATPase) activity (35). These inhibitions of of poorly controlled di abetes mellitus. This buffer comkey metabolic processes may result in altered energy meposition was also used in the present study. The benefitabolism and contractile function. Our results indicated cial cardiac effects of exercise training were associated that even though plasma lipids were elevated, the hearts with a lowering of plasma triacylglycerol and total cholesfrom sedentary diabetic rats did not demonstrate an in terol concentration while having no apparent effect on vivo accumulation of triacylglycerol, long-chain acylplasma glucose. The decrease in myocardial total carniCoA, and carnitine esters, nor were there any differences tine content found in the sedentary diabetic rat was pre- in myocardial ATP levels among control, sedentary diavented by exercise training. betic, and trained diabetic rats. These findings appear to The results of the present study confirmed our pre- be in conflict with previous studies (3, 15, 18) that have vious finding th.at exercise training of streptozotocin-inshown that diabetic animals produce an increase in these duced diabetic rats (60 mg/kg) has no effect on overall lipid compounds; however, it should be pointed out that glycemic control as assessed by a single time point these previous studies were performed in relatively plasma glucose or by the level of blood dYCOSY lated hemodiabetic animals (12 wk). It appears that globin. The decrease in plasma insulin levels found in short-term these lipids may accumulate initially after the induction diabetic animals was also unaffected by exercise training. of diabetes but that they eventually return to normal Similar results have been obtained by others (2). Allevels. Exercise training of diabetic rats had no effect on though it is true that other studies have shown a glucosemyocardial long-chain acyl-CoA and carnitine esters but lowering effect of exercise training in human diabetics produced a lowering of triacylglycerol content. These and experimental diabetic animals, the severity of diafindings suggest that there may not be a correlation bebetes in these studies was relatively mild (5, 28, 29). In tween the accumulation of lipids and the depressed carsevere studies utilizing a more form of diabetes (16, 17, diac contractile performance associated with diabetes 30,33), exercise training has no effect on overall glycemic mellitus. levels. Another possible mechanism that may account for the As shown in previous studies (16, 17, 28), the exercise correlation between diabetic hyperlipidemia and detraining protocol used in this study did have a significant hypolipidemic effect. Exercise-trained diabetic rats have pressed cardiac performance may be the effect of exogenous lipids on myocardial glucose utilization. According significantly decreased plasma levels of triacylglycerol, to this postulated mechanism an increase in circulating total cholesterol and high-density-lipoprotein choleslipids results in the inhibition of glucose utilization with terol, Plasma nonesterified fatty acids tended to be lower fatty acid oxidation becoming the predominant source of in this group, but the decrease was not statistically signifenergy (21). Because the ratio of mitochondrial produced icant. This lowering of plasma lipids may have a number ATP to oxygen molecule consumed for glucose oxidation of metabolic consequences on the diabetic heart that is higher than the ratio for fatty acid oxidation, the decould account for the beneficial effects of exercise crease in glucose utilization may cause an imbalance betraining. A number of studies have suggested that elevated exog- tween the supply and demand for oxygen. This shift in the substrate utilization pattern of the heart may also enous lipid levels may contribute to the cardiac dysfunccontribute to the insulin resistance and hyperglycemia tion found in experimental diabetic animals (20, 23, 27). found in diabetic rats (22,24); thus the inherent ability of Long-term treatment with lipid-lowering drugs or agents that -inhibit fatty acid oxidation have been shown to pre- the diabetic heart to utilize glucose may be impaired by As shown in the present study, vent the cardiac dysfu n&ion of hearts from diabetic rats. chronic hyperlipidemia. In addition, acute lowering of exogenous free fatty acid this imbalance is not enough to affect total myocardial ATP levels, but it may be sufficient to affect metabolic concentration has also been shown to improve cardiac performance of the hearts of diabetic rabbits (4) and processes that require glycolytic ATP. For example, it has been proposed that glycolytic ATP is the principle rats (20). (12). The A number of mechanisms have been proposed to ex- substrate for the sarcolemmal Na+,K+-ATPase level of glycolytic ATP may also regulate ATP-sensitive plain why the hyperlipidemia associated with diabetes mellitus is harmful to the diabetic myocardium. One pos- potassium channels (34). Alterations in the function of
5. Calculated ATP produced from oxidation of exogenousglucose and palmitate in isolated Langendorff perfused hearts from sedentary control, sedentary diabetic, and exercise-trained diabetic rats TABLE
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these processes may adversely affect cardiac contractile function. By lowering plasma lipids, exercise training should enhance glucose oxidation by the diabetic heart. Exercise training also reportedly enhances insulin sensitivity as well as the ability of various tissues to utilize glucose (2). The results of the present study showed that myocardial glucose oxidation rate was decreased by 50% in sedentary diabetic rats relative to sedentary control rats. For all groups, hearts were perfused under substrate concentrations that are characteristically found in poorly controlled diabetes mellitus. Exercise training of diabetic rats significantly improved the ability of diabetic hearts to utilize glucose as a substrate; however, the rates of glucose oxidation in this group were not returned completely to levels found in hearts of the sedentary control rats. There were no differences in the ability of these hearts to utilize exogenous free fatty acids as a substrate for energy metabolism. From a total energy output standpoint, exogenous glucose oxidation theoretically should yield ~724 pmol g-l. h-l of ATP in control hearts, while fatty acid oxidation should produce considerably more ATP, ~3,162 pm01 g-l h--l. Thus, under the perfusion conditions of this study, exogenous glucose oxidation contributes only a modest amount of ATP to the total pool. The yield of ATP from glucose oxidation in the sedentary diabetic rat was calculated to be - 384 vs. 3,119 pmol g-l h-l for palmitate oxidation. In exercise-trained diabetic rats, exogenous glucose oxidation yielded slightly more ATP, 520 pmol g-l 6h-l, but was still considerably lower than the ATP obtained from the oxidation of exogenous palmitate, 3,079 pm01 g-l h-l. This small increase in glucose-derived ATP does not affect myocardial total ATP content, but it may have important implications of those metabolic processes that require glycolytic ATP. It should be pointed out that our experiment only measured glucose oxidation through the pyruvate dehydrogenase pathway. It is possible that glycolytic flux to lactate may have been increased even further in hearts of exercise-trained diabetic rats. Evidence in support of this hypothesis to explain the beneficial effects of exercise training on the diabetic heart comes from a number of other studies. Rosen and Reinauer (24) showed that addition of sodium 2-[5-(4chlorophenyl)-pentyl-oxirane-2-carboxylate, a selective inhibitor of carnitine palmitoyltransferase I (CPT-I), to the perfusion medium of diabetic hearts increased glucose uptake, glycolysis, pyruvate oxidation and insulin sensitivity. Wall and Lopaschuk (32) showed that Etomoxir, another CPT-I inhibitor, treatment of diabetic rats partially reversed the decrease in glucose oxidation and improved cardiac function. Another study by this laboratory (11) showed that Etomoxir improved the recovery of hearts subjected to ischemia and reperfusion. This beneficial effect of Etomoxir was not associated with affects of myocardial long-chain acyl carnitine but rather with an increase in glucose utilization by the diabetic heart. Treatment of diabetic rats with antilipolytic agents such as nicotinic acid and phenylisopropyladenosine were shown by Reaven et al. (22) to lower both plasma free fatty acids and glucose. Thus the inhibition l
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of glycolysis by elevated exogenous lipid may contribute to the impaired contractile function associated with diabetes mellitus. In summary, the results of the present study extend and support previous findings indicating that exercise training of diabetic rats lowers plasma lipids but does not affect glycemic control. Neither diabetes nor exercise training had any effect on the myocardial accumulation of lipids or ATP content. The lowering of plasma lipids in exercise-trained diabetic rats was associated with an improved ability of hearts from these animals to utilize glucose as a metabolic substrate. This effect of exercise training on plasma lipids and myocardial glucose utilization may be a mechanism to account for the beneficial effects of this treatment in preventing some of the cardiac abnormalities associated with diabetes mellitus. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39200. Address for reprint requests: D. J. Paulson, Dept. of Physiology, Chicago College of Osteopathic Medicine, 555 31st St., Downers Grove, IL 50515. Received 19 August 1991; accepted in final form 17 January 1992. REFERENCES G., AND H. SCHULTE. Diabetes mellitus and hypertension in the elderly: concomitant hyperlipidemia and coronary heart disease risk. Am. J. Curdiol. 63: 33H-37H, 1989. 2. DALLAGLIO, E., F. CHANG, H. CHANG, D. WRIGHT, AND G. M. REAVEN. Effect of exercise training and sucrose feeding on insulin-stimulated glucose uptake in rats with streptozotocin-induced insulindeficient diabetes. Diabetes 32: 165-168, 1983. 3. FEUVRAY, D., J. A. IDELL-WENGER, AND J. R. NEELY. Effects of ischemia on rat myocardial function and metabolism in diabetes. Circ. Res. 44: 322-329, 1979. 4. FIELDS, L. E., A. DAUGHERTY, AND S. R. BERGMANN. Effects of fatty acid on performance and lipid content of hearts from diabetic rabbits. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H10781. ASSMANN,
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D. J., AND A. L. SHUG. Inhibition of the adenine nucleotide translocator by matrix-localized palmityl-CoA in rat heart mitochondria. Biochim. Biophys. Acta 766: 70-76, 1984. 20. PAULSON, D. J., A. L. SHUG, R. JURAK, AND M. SCHMIDT. The role of altered lipid metabolism in the cardiac dysfunction associated with diabetes. In: The Diabetic Heart, edited by M. Nagano and N. S. Dhalla. New York: Raven, 1991, p. 395-407. 21. RANDLE, P. J., C. N. HALES, P. B. GARLAND, AND E. A. NEWSHOLME. The glucose fatty acid cycle. Its role in insulin sensitivity and the metabolic disturbance of diabetes mellitus. Lancet 1: 785789,1963. 22. REAVEN, G. M., H. CHANG, H. Ho, C. Y. JENG, AND B. B. HOFFMAN. Lowering of plasma glucose in diabetic rats by antilipolytic agents. Am. J. Physiol. 254 (Endocrinol. Metab. 17): E23-E30,1988. 23. RODRIGIJES, B., R. K. GOYAL, AND J. H. MCNEILL. Effects of hy-
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