Ascorbic acid supplementation prevents hyperlipidemia and improves myocardial performance in streptozotocin-diabetic rats

ELSEVIER

DiabetesResearchand Clinical Practice27 (1995)11- 18

Ascorbic acid supplementation prevents hyperlipidemia and improves myocardial performance in streptozotocin-diabetic rats Soter Dai, John H. McNeill* Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, B.C., V6T 123. Canada

Received8 July 1994;revision received2 September1994;accepted30 September1994

Abstract

The present study investigated the effects of ascorbic acid (AA) supplementation on the cardiac performance and the plasma levels of glucose, insulin, triglycerides, cholesterol and free fatty acid in diabetic and non-diabetic rats. Diabetes was induced by intravenous injection of streptozotocin (STZ) 55 mg/kg. AA was given in drinking water in concentrations of 1 g/l or 2 g/l for 8 weeksafter STZ injection. Myocardial performance was determined using the isolated perfused working heart preparations. Following AA supplementation, there were no significant changesin any of the parametersmeasuredin non-diabetic rats; however, the occurrence of polydipsia, hyperphagia, hyperlipidemia and myocardial dysfunction in STZ-diabetic rats was significantly alleviated in a dose-dependentmanner. Nevertheless,the decreasedbody weight gain, hypoinsulinemia and hyperglycemia in diabetic animals were not affected. The data show that AA supplementation in STZ-diabetic rats improves both hyperlipidemia and cardiac function. However, the mechanismsof these effects and the correlation between these improvements are not clear. Keywords: Ascorbic acid; Diabetes; Cardiac dysfunction; Hypexlipidemia; Rat

1. Introduction Cellular uptake of ascorbic acid (AA) is an energy-dependent carrier-mediated process [ 11. Glucose and other monosaccharides share a common transport systemwith AA and dehydroascorbit acid [2]. Therefore, chronic hyperglycemia may impose an intracellular deficit of AA through competitive inhibition l

of membrane transport of AA

Correspondingauthor, Tel.: +l 6048222343;Fax: +l 604

8223035.

by ,the elevated plasma glucose [3]. Levels of AA in plasma and various tissues are decreased in diabetic patients and in animals with experimentally induced diabetes [4-71. Cellular deficiency of AA has been implicated in some of the cellular pathology and complications of diabetes mellitus such as angiopathy [2,8]. It has been suggested that AA supplementation may help to prevent the development of some diabetic complications [9-121. Cardiomyopathy is known to occur in chronic diabetics [13] and in animals with experimentally

0168-8227/95/$09.50 0 1995ElsevierScienceIreland Ltd. All rights reserved SSDI 0168-8227(94)01013-P

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S. Dai, J. H. McNeil1 / Diaberes Research and Clinical Practice 27 (1995) I I- I8

induced diabetes [ 14,151. The pathogenic mechanisms of this myocardial abnormality is uncertain but has been suggestedto be due to the metabolic alterations associatedwith diabetes [16]. The influence of AA supplementation on the occurrence of diabetes-induced cardiac dysfunction, however, has not yet been investigated. Results of some studies have suggestedthat AA may play a role in lipid metabolism [ 17- 191and is involved in regulating the activity of lipoprotein lipase [20,21]. There is evidence that AA deficiency is associated with increased plasma and tissue lipid levels which could be reversed by AA supplementation [19,20,22-251. It is well documented that diabetes mellitus in man and in experimental animals is associated with elevated plasma lipid level, in particular triglycerides [26,27]. This may be attributed to either the defective removal [28] or overproduction [29], or their combination, of one or more lipoproteins. Since decreasedAA levels in plasma and tissues is common in diabetic patients and in animals with experimentally induced diabetes [4-71, it is thus hypothesized that AA deficiency may contribute to the production of hyperlipidemia in diabetics and that the occurrence of hyperlipidemia in diabetics may then be prevented by AA supplementation. The present study was carried out in STZdiabetic rats to investigate the effects of AA supplementation in drinking water on the performance of the heart and on the plasma levels of triglyceride, cholesterol, free fatty acid, glucose and insulin. Myocardial performance of the rats was examined using the isolated perfused working heart preparations. Parallel studies were also carried out in non-diabetic rats.

halothane (Fluothane; Ayerst Lab., Montreal, Que., Canada) anesthesia. The occurrence of diabetes was confirmed by the presenceof hyperglycemia in blood drops obtained following tail snipping at 48 and 72 h after STZ injection, using Glucostix (Miles Canada Inc., Etobicoke, Ont., Canada) reagent strips read by a Glucometer II (Ames, Miles Laboratories, Elkhart, IN, USA). The minimal blood glucose value accepted for a diabetic rat was 13 mM. STZ was freshly dissolved in 0.9% saline immediately before use at a concentration of 55 mg/ml. An equivalent volume (1 ml/kg) of saline was administered by the same route to non-diabetic control animals.

2. Materials and methods

2.3. Isolated working heart preparation

2.1. Animals and induction of diabetes

Male Wistar rats (Animal Care Unit, University of British Columbia, Vancouver, B.C., Canada), weighing 210-240 g, were used. They were housed on a 12-h light-dark cycle and were allowed free access to standard laboratory diet (Purina rat chow) and drinking fluid. Diabetes was induced by intravenous injection of streptozotocin (STZ; Sigma, St. Louis, MO, USA) (55 mg/kg) via the caudal vein under

2.2. Ascorbic acid treatment

Treatment of the rats with ascorbic acid (AA) started 3 days after administration of STZ or saline. AA (Sigma, St. Louis, MO, USA) was prepared daily by dissolving in drinking tap water at a concentration of 1 g/l or 2 g/l. The animals were randomly divided into live groups and were given ordinary water or AA solution to drink ad libitum. The saline-injected non-diabetic rats drinking ordinary water were designated CON (n = 8), where those drinking 2 fl AA solution were designated CON-AA (n = 8). The STZinjected diabetic rats drinking water were designated DIA (n = 8), those drinking 1 g/l AA solution as DIA-AA1 (n = 8) and those drinking 2 g/l AA solution as DIA-AA2 (n = 10). Fluid intake, food intake and body weight of these animals were measured before STZ injection (week 0) and at weeks 2, 4, 6 and 8 after STZ injection.

Myocardial performance of the rats were investigated 8 weeksafter injection of STZ or saline, using the previously described isolated perfused working heart preparation [30,31] with some modifications. The perfusion fluid used was Chenoweth-Koelle solution which has the following composition (mM): NaCl, 120; KCl, 5.6; CaCl*, 2.18; MgC12, 2.1; glucose, 10. It was aerated with 95% 02-5% CO2 and was maintained at 37°C throughout the experimental period. Perfusion was provided by a peristaltic pump

S. Dai. J. H. McNeil1 / Diabetes Research and Clinical Practice 27 (1995) 1I - 18

(Masterflex, Cole-Parmer Instrument Co., Chicago, IL, USA). The rats were decapitated under deep anesthesia with intraperitoneally administered sodium pentobarbital (MTC Pharmaceuticals, Cambridge, Ont., Canada; 100mg/kg), their chests were opened and the hearts were quickly excised and mounted in a perfused working heart apparatus. The heart was first perfused in a retrograde manner through the aorta at a rate of 17 ml/min. Following cannulation of the pulmonary vein, cardiac work was initiated by switching the perfusion systemfrom retrograde mode to the working heart mode. The aortic flow was subjected to an afterload of 6.0 R.U. (Resistance Unit). Through a %O-gauge needle, which was inserted through the apex of the heart into the left ventricle and the sidearms of the aortic flow system and of the left atria1 cannula, left ventricular pressure, aortic pressure and left atria1 pressure were measured using Statham P23AA pressure transducers (Statham-Gould Instruments, Cleveland, OH, USA). The heart was paced at a rate of 300 beats/min by means of an electrode on the left atrium at suprathreshold voltage with pulses of 5ms duration supplied by a Grass SD9 square-wave stimulator. Left ventricular pressure, the first derivative of left ventricular pressure, aortic pressure and left atria1 pressure were displayed on a Grass model 79D polygraph. Cardiac function data, including left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), rate of pressuredevelopment (+dP/dT), rate of ventricular relaxation (-dP/dT) and heart rate were collected and analyzed by a microcomputer. Following a lO-min period of equilibration at a left atria1 filling rate of 34 ml/min, left ventricular function curves against various left atria1 tilling rates were recorded. The tilling rate was first decreased stepwise from 34 to 30, 25 and 17 ml/min, then increased stepwise to 25, 30, 34, 38, 42,46, and 50 ml/min. The pressureselicited in the left atria1 cannula in response to these perfusion rates were 3.0, 4.7, 5.5, 6.3, 7.5, 9.0, 10 and 11 mmHg, respectively. 2.4. Plasma analyses

When the rats were sacrificed for isolated heart

13

preparation, blood samples were collected from the decapitation wound into heparinized tubes. After centrifugation (3246 x g) at 4°C for 15 min, plasma samples were collected and stored at -20°C until assayed.Plasmaglucose, triglycerides, cholesterol and free fatty acid were determined using appropriate enzymatic calorimetric assay kits (Boehringer Mannheim, Laval, Que., Canada) and plasma insulin was measured with a radioimmunoassay kit (ICN Biochemicals, Costa Mesa,

1

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600

2

4

6

8

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2

4

6

8

2

4

6

a

0

l

-2 -

0

co.\ CON-A4

500

2 M .r(

400

;

0

Time

(week)

Fig. 1. (a) Fluid intakes, (b) food intakes and (c) body weights of untreated non-diabetic (CON) (n = 8), ascorbic acid-treated non-diabetic (CON-AA) (n = 8), untreated ST2 diabetic (DIA) (n = 8) and STZ-diabetic rats treated with ascorbic acid 1 g/l (DIA AAI) (n = 8) or 2 g/l (DIA-AA2) (n = 10) in drinking water. Values are means + S.E.M. *P c 0.05 vs. CON; +P < 0.05 vs. CON-AA; “P < 0.05 vs. DIA.

S. Dai, J.H. McNeil\/

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Diabetes Research and Clinical Practice 27 (1995) 11-18

CA, USA). The intra- and inter-assay C.V. of the insulin assay were 3.2% and 12.2%, respectively. 2.5. Statistical analyses

The data are expressed as means f standard error of the mean (S.E.M.) and were analysed using one-way or two-way analysis of variance, as appropriate, followed by the Newman-Keuls test. The level of significance was set at P < 0.05. 3. Results 3.1. Fluid intakes, food intakes and body weights

Treatment of non-diabetic rats with AA 2 g/l in drinking water did not remarkably affect the in-

‘O” m-.C= -

175

150

takes of fluid or food, or body weights. There were no significant differences in these variables between CON and CON-AA at any time point (Fig. 1). When compared with CON, DIA had significantly greater intakes of fluid and food and significantly slower and lower body weight gain throughout the experimental period. AA treatment dose-dependently alleviated the increased fluid and food intakes in diabetic rats but did not influence their body weights. Although the fluid and food intakes of both DIA-AA1 and DIA-AA2 were significantly greater than those of CON-AA at all time points after STZ injection, DIA-AA2 had significantly lower fluid intakes at weeks 2, 4, 6 and 8 and lower food intakes at weeks 6 and 8,

---

0 V

CON-AA DIA

T 0

DIA-AAL DIA-AA2

60

-

Left

atria1

filling

(ml/min)

Fig. 2. (a) Left ventricular developed pressure (LVDP), (b) left ventricular end-diastolic pressure (LVEDP), (c) +dP/dT and (d) -dP/dT of isolated perfused working hearts at various left atria1 filling rates. Hearts were isolated from untreated non-diabetic (CON) (n = 8), ascorbic acid-treated non-diabetic (CON-AA) (n = 6), untreated ST2 diabetic (DIA) (n = S), and STZ-diabetic rats treated with ascorbic acid 1 fl (DIA AAl) (n = 8) or 2 gA (DIA-AA2) (n = 8) in drinking water, Values are means f S.E.M. ‘P < 0.05 vs. CON; +P < 0.05 vs. CON-AA; “P c 0.05 vs. DIA.

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Diabetes Research and Clinical Practice 27 (1995) 11-18

than did DIA and DIA-AA1 had significantly lower fluid intakes than did DIA at weeks 4 and 8. The body weight of DIA-AA1 or DIA-AA2 were not significantly different from those of DIA and were significantly lower than those of CON-AA. The average daily intakes of AA from drinking water in various AA-treated groups were calculated from the average daily fluid intakes and body weights. They were 324 mg/kg per day in CON-AA, 664 mglkg per day in DIA-AA1 and 1054 mg/kg per day in DIA-AA2. 3.2. Myocardial performance

Fig. 2 shows the performance of the isolated perfused working hearts. In CON, there were progressive increases in LVDP, +dP/dT and -dP/dT and a slight increase in LVEDP in responseto the increasesin left atria1 filling rate. Performance of the normal heart was not affected by AA treatment. There were no significant differences in any parameters measured between CON-AA and CON. In DIA the increasesin LVDP, +dP/dT and -dP/dT at lower atria1 filling rates were followed by progressive decreasesat tilling rates higher than 34 ml/mm. There was a sharp rise in LVEDP at the same time. The values of LVDP, +dP/dT and -dP/dT in DIA at high tilling rates were significantly lower than those in CON. AA treat-

15

ment in diabetic rats remarkably improved these abnormalities. Both DIA-AA1 and DIA-AA2 had significantly higher LVDP than did DIA at high tilling rates and the values of both +dP/dT and -dP/dT in DIA-AA1 or DIA-AA2 were not significantly different from those in CON-AA, CON, or DIA. The differences in LVEDP at any tilling rate among the five groups of rats were not statistically significant. 3.3. Plasma concentrations of glucose, insulin, triglycerides, cholesterol and free fatty acid

Treatment of non-diabetic rats with AA did not significantly alter plasma concentrations of glucose, insulin, triglycerides, cholesterol, or free fatty acid. There were no significant differences in these variables between CON and CON-AA (Table 1). When compared with CON, DIA had significantly higher plasma levels of glucose, triglycerides, cholesterol and free fatty acid and a significantly lower plasma insulin level. AA treatment in diabetic rats did not alter the abnormalities in plasma glucose or insulin but dosedependently improved the elevated plasma triglycerides,cholesterol and free fatty acid. Plasma levels of glucose and insulin in DIA-AA1 and DIA-AA2 were not significantly different from those in DIA and were significantly higher and lower, respectively, than those in CON-AA. While

Table 1 Effects of ascorbic acid supplementation on plasma concentrations of glucose, insulin, triglycerides, cholesterol, and free fatty acid (FAA) in non-diabetic and STZ-diabetic rats Plasma concentration Glucose (mW CON (8) CON-AA (8) DIA (8) DIA-AA1 (8) DIA-AA2 (10)

8.16 f 8.57 f 19.79 l 19.49 f 21.11 f

Insulin WJW 0.37 0.28 1.09* 1.14+ 0.84+

71.69 f 64.10 f 16.45 f 24.18 f 28.99 f

5.99 5.80 3.12. 2.22+ 1.15+

Triglycerides WW

Cholesterol (mM)

FAA MM)

1.05 f 1.49 l 4.13 * 3.10 f 1.80 f

1.04 f 0.97 f 1.75 f 1.54 f 1.20 f

0.14 f 0.02 0.11 f 0.01 0.26 ct 0.04* 0.20 f 0.03 0.20 f 0.01

0.09 0.10 0.64* 0.66+ 0.16”

0.09 0.07 0.15* 0.07+ 0.06”

CON, untreated non-diabetic rats; CON-AA, ascorbic acid-treated non-diabetic rats; DIA, untreated diabetic rats; DIA-AAl, diabetic rats treated with ascorbic acid 1 @I in drinking water; DIA-AA2, diabetic rats treated with ascorbic acid 2 g/l in drinking water. Number of animals in each group is given in parentheses. Values are means f S.E.M. *P < 0.05 vs. CON. +P < 0.05 vs. CON-AA. ‘P < 0.05 vs. DIA.

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Diabetes Research and Clinical Practice 27 (199s) II-18

the levels of plasma triglycerides and cholesterol in DIA-AA1 were not significantly different from those in DIA and were significantly higher than those in CON-AA, these parameters in DIA-AA2 were significantly lower than those in DIA and were not significantly different from those in CON-AA or CON. Both DIA-AA1 and DIA-AA2 had plasma levels of free fatty acid which were not significantly different from those in CON, CONAA, or DIA. 4. Discussion Similar to previous studies [14,30,31], the current investigation showed that STZ-diabetic rats developed remarkable myocardial dysfunction. Interestingly, the impairment of myocardial performance in diabetic rats was significantly alleviated following AA supplementation and the protection appeared to be dose-dependent. The mechanisms of cardiac dysfunction in STZdiabetic rats remain unclear. It was suggestedthat the elevated circulating lipid levels might contribute to the production of cardiac dysfunction in STZ-diabetic rats [27,32]. However, the findings of the recent study of Rodrigues et al. [33] did not totally support this hypothesis. The current study also revealed an improvement in hyperlipidemia in STZ-diabetic rats following AA supplementation. However, evidence is lacking that the improvements in hyperlipidemia and in cardiac function are causally correlated and other factors should also be considered. There is evidence that AA deliciency is associated with impaired biosynthesis of carnitine and decreasedblood and tissue concentrations of carnitine [23,34,35]. Studies in diabetic patients [9] and in STZ-diabetic rats [36] or guinea-pigs [37] also showed that AA supplementation resulted in a decreasein erythrocyte or tissue concentrations of sorbitol, suggesting an interaction of the polyol and AA pathways in diabetes. Both carnitine depletion and sorbitol accumulation have been implicated in the pathogenesis of myocardial dysfunction in diabetics [32,38-411. It is thus possible that AA supplementation may help to preserve the cardiac function of the diabetic animals through an increase in carnitine concentration and/or a decreasein sorbitol

accumulation in the myocardium. Further studies are required. Furthermore, increased oxidative stresshas been suggestedto contribute to the development of some diabetic complications [42]. AA is known to be an antioxidant and the study of Young et al. [43] showed that AA supplementation in STZ-diabetic rats effectively reduced oxidative stress. Therefore, the possibility that the protective effects of AA supplementation against the occurrence of cardiac dysfunction in STZdiabetic rats may be mediated by an antioxidant activity should also be considered. The present study also showed that AA supplementation effectively prevented the occurrence of hyperlipidemia in STZ-diabetic rats in a dosedependent manner. Since STZ-induced diabetes in rats is associated with depletion of AA [5,12,42], these findings support the hypothesis that AA deficiency is involved in the pathogenesis of hyperlipidemia in diabetics, at least in STZdiabetic rats. They also provide support to the theory that AA may play a role in the regulation of lipid metabolism [ 17- 191. The influence of increasedAA intake may not be marked in the absenceof AA deficiency. Hence, administration of AA in non-diabetic rats as observed in the current investigation did not cause significant changes in plasma lipid levels. These findings agree with data found in guinea pigs. It was shown that guinea pigs fed a low vitamin C diet exhibited hyperlipidemia whereasthose fed a high vitamin C diet did not show abnormal plasma lipid level [24,44]. The role of AA in lipid metabolism and how AA deficiency causeshyperlipidemia are not yet clear. It has been suggestedthat AA may be involved in the regulation of the activity of lipoprotein lipase [20,21], or in the hydroxylation of cholesterol in the conversion of cholesterol to bile acid [45]. Becausethe AA-deficient guinea pigs exhibited significantly lower tissue levels of carnitine, Ha et al. [23] suggestedthat the decreased tissue carnitine levels resulting from AA deficiency led to impairment of the transport of long-chain fatty acid into mitochondria and subsequently shunting toward more triglyceride synthesis. On the other hand, one must also consider the possibility that the improvement in hyperlipidemia in STZ-diabetic rats as observed in the present

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Diabetes Research and Clinical Practice 27 (199s) II-18

study may just be the result of the decreasein food intake during AA supplementation. The current study revealed that AA supplementation in diabetic or non-diabetic rats did not alter the plasma levels of glucose or insulin, or the body weight. These findings agree with those of Young et al. [43]. It shows that the improvement of cardiac function and plasma lipid levels in diabetic rats by AA supplementation is not due to a total correction of the diabetic state. However, the present investigation observed a significant decrease in both fluid and food intakes in the AA-treated diabetic animals. The mechanism of this phenomenon is not clear. It is possible that AA does correct the diabetes-induced lipid changes by affecting metabolic processes.It is also possible there is an improvement in food absorption in the AA treated animals. It is not clear as to whether the decreases in food and caloric intakes in diabetic rats contribute to the improvement of circulating lipid levels and the cardiac performance. Further studies using the pair-fed diabetic rats are required. Previous studies have repeatedly shown that STZ-diabetes in rats is associated with AA depletion [5,12,43]. A recent study in our laboratory using separate batches of rats also showed 30% and 25% decreasesin plasma and myocardial AA levels, respectively, in STZ-diabetic rats that were restored by AA supplementation (data not shown). However, further studies are still required to define the correlation between the plasma or cardiac concentration of AA in diabetic animals and the occurrence of hyperlipidemia and myocardial dysfunction. In summary, the present study showed that the STZ-injected rats developed hyperphagia, polydipsia, reduced body weight gain, hypoinsulinemia, hyperglycemia, elevated levels of plasma triglycerides, cholesterol and free fatty acid and myocardial dysfunction. AA supplementation did not cause significant changes in any parameters measured in non-diabetic rats but dose dependently decreasedthe fluid and food intakes and the plasma levels of triglycerides, cholesterol and free fatty acid and improved the myocardial performance in STZ-diabetic animals. However, the reduced body weight gain, hypoinsulinemia and hyperglycemia in these animals were not

17

affected. The data show that AA supplementation in STZ-diabetic rats improves both the cardiac performance and hyperlipidemia. However, the mechanismsby which AA supplementation exerts these effects and the correlation between these improvements are not clear. Acknowledgements

The authors thank Ms. Violet Yuen and Stephanie Lee for their excellent technical assistance and Ms. Sylvia Chan for careful preparation of the manuscript. This work was supported by a grant from the Heart and Stroke Foundation of B.C. and Yukon. References 111Finn,

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