Changes in Carbohydrate Metabolism of Cardiac and Striated Muscle in Hypothermic Rats

Transcription

1 Changes in Carbohydrate Metabolism of Cardiac and Striated Muscle in Rats By S. ITZHAKI, PH.D. AND E. WERTHEIMER, M.D. The following represents a study of the influence of hypothermia on the carbohydrate metabolism of rat heart slices in comparison to rat diaphragm. The effect of thyroid on the induced metabolic changes in both types of muscle is investigated. AFTER a short hypothermia the glycogen r\ stores of the heart muscle and the diaphragm are depleted. Smaller decreases are observed in gastrocnemius muscle. After thyroidectomy, no change in the glycogen content of the cardiac muscle is seen in hypothermia, while the depletion is maintained in diaphragm. An increase in glucose utilization and a lowering of the rate of endogenous oxygen consumption by heart slices occurs in hypothermia, whereas no such effect can be demonstrated in diaphragm. The lowering of the rate of endogenous oxygen consumption is caused only by the loss of oxidizable substrates from the heart muscle. A positive correlation exists between the initial glycogen content and the rate of endogenous respiration of heart muscle slices. All metabolic changes observed in the myocardium of the hypothermic rat disappear after thyroidectomy. METHODS Male albino of local strain (attaining a mature weight of 250 Gm. in 6-7 months), weighing between 90 and 120 Gm., were starved for 20 hours prior to the experiment. was induced by immersing the up to the neck in a large glass cylinder containing water at a temperature of 2-25 C. After half an hour the were removed from the water, dried, and used for the experiment. Their rectal temperatures averaged 29 C. such will subsequently be referred to as "hypothermic." At room temperature the hypothermia was reversed in less than 2 hours. The animals were killed under sodium pentobarbital anesthesia, the heart was then removed and From the Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel. Received for publication April 12, In partial fulfilment of Ph.D. Thesis by S. Itzhaki at Hebrew University, Jerusalem. placed into an ice-cold Ringer-Warburg solution containing 0.68S per cent NaCl, 0.1S per cent KC1, and 0.27 per cent CaCU, saturated with oxygen. Slices were cut from the heart chambers and transferred into ice cold, constantly aerated Krebs- Ringer-phosphate (KRP) solution made up as follows: 100 parts 0.9 per cent NaCl, parts 1.15 per cent KC1, 3 parts 1.22 per cent CaCU, 1 part 3.8 per cent MgSO -7H 2 O, 21 parts phosphate buffer ph (0 ml. 0.25M Na 2 HPO plus about 2 ml. IN HC1 plus distilled water up to a final volume of 100 ml. plus 0.2 per cent glucose). The incubation of the heart slices and the diaphragm, prepared according to Gemmill, 1 was carried out in 2 ml. and 1 ml. of medium, in Erlenmeyer flasks of 25 ml. and 10 ml. capacity respectively at 37 C, and shaken at a rate of 120 excursions/min. for 2\^ hours. utilization by the diaphragm was determined according to Krahl and Cori 2 ; the results being expressed in mg. glucose/100 mg. wet tissue/ 2}<; hours incubation. Glycogen synthesis in diaphragm was measured by the method used by Tuerkischer and Wertheimer, 3 values being given as mg. glycogen/100 mg. wet tissue/2^ hours incubation. Net glucose utilization was calculated by subtracting the value of lactic acid formation determined according to Barker and Summerson from the sum of glucose utilization plus glycogenolysis. Oxygen uptake of heart slices and diaphragm was measured and expressed in Qo 2, i.e., /zl. O 2 /100 mg. tissue/hr. 5 Heart slices or half diaphragm were placed into a Warburg vessel containing 2 ml. KRP solution enriched with 11111M substrate. Heart extract was prepared by heating a homogenate of 1 rat heart muscle in 1.2 ml. of physiologic saline at 60 C. for % hour. 0.3 ml. of the supernatant solution were placed into the side arm of the Warburg vessel and added to the incubation medium 20 min. after the beginning of the experiment. The chemical estimations were carried out according to Wertheimer, Ben-Tor and Wurzel. 6 The standard error of the mean, the statistical significance according to the t test, and the value of p taken from the t table 7 were found. The regression line (method of least squares) and the correlation coefficient were calculated according to Moore and co-workers Circulation Research, Volume V, July 1957

2 52 METABOLIC CHANGES IN CARDIAC MUSCLE IN HYPOTHERMIA TABLE 1. Effect of on Glycogen Content of Various Muscles in and Thyroidectomized Rals Glycogen (mg./loo mg. tissue) Heart Diaphragm Gastrocnemius Stomach muscle V Thyroidectomized V ± (73) ±0.012 (9) -72% 0.61 ± (22) 0. ± (15) -% ± (19) ± (19) -72% ±0.021 (7^ ± 0.01 (7) -65% 0.39 ± (7) ±0.01 (6) -33% 0.05 Results given as mean ± standard error of mean; figures in parentheses, number of ± (5) ± () nil TABLE 2. Effect of on Utilization by Heart Slices.. Thyroidectomized.. utilization (mg./loo mg tissue/2.5 hours) 0.5S ± ± (38) (3S).0.59 ± (15) 0.6 ± (1) RESULTS <%) P 0.30 in the rat caused a glycogen loss of 72 per cent in cardiac muscle, a much smaller loss in gastrocnemius, and none whatever in the smooth stomach muscle (table 1). Thyroidectomy prevented glycogen depletion in cardiac muscle, a fortnight after the operation the full effect could be observed. A shorter time interval after thryoidectomy showed only a partial glycogen loss. Thyroidectomy in hypothermia did not influence the glycogen loss in diaphragm (table 1). 2-2 hours after injection of 0.03 mg. thyroxin/100 Gm., the effect of thyroidectomy on heart muscle glycogen decreased in the hypothermic (9 experiments), the difference being 26 per cent (p = 0.03). utilization in cardiac muscle was found to be increased in hypothermia, whereas no such effect was obtained after thyroidectomy (table 2). Table 3 illustrates the carbohydrate balance for heart muscle slices in both hypothermic and normal. To determine the net glucose utilization, lactic acid formation was measured. This was found to be 20 per cent lower than in normal. Net glucose utilization in hypothermic was somewhat raised, whereas the glucose utilization by diaphragm in the same remained normal. Glycogen synthesis from glucose by diaphragm was increased in hypothermic. After thyroidectomy this increase was found to be much smaller (table ). utilization and glycogen values in heart muscle of recovering at room temperature were compared to values obtained with gastrocnemius. Glycogen recovery occurred more quickly in heart muscle than in striated muscle and so did the glucose utilization. Endogenous oxygen consumption by heart muscle slices was significantly lowered in hypothermic (table 5). Thyroidectomy canceled this effect. Upon addition of different substrates, oxygen consumption was raised to a greater extent in hypothermic than in normal ones (table 6). The effect of heart extract of normal and hypothermic on the oxygen consumption by heart slices of hypothermic is shown in figure 1. The increase in oxygen consumption was larger after addition of extract of normal. The relation between endogenous respiration and glycogen content of heart muscle of hypothermic

3 ITZHAKI AND WERTHEIMER 53 TABLE 3. Carbohydrate Balance in Heart Slices No. of Initial Glycogen Final utilization utilization -fglycogenolysis Lactic acid formation Net glucose utilization ± ± ± ± ± ± nil -20%, p + 11%, p 0.05 Results are expressed as mg./loo nig. tissue. TABLE. Effect of on Utilization and Glycogen Synthesis by Diaphragms No. of utilization (mg./loo mg. tissue/2.5 hour) Glycogen synthesis (mg./loo mg. tissue/2.5 hour) (Krebs-Ringer phosphate) ± ± ± ± (homologous serum) 5 1 ns ± 0?IO ± Thyroidectomized (homologous serum). TABLE V Thyroidectomized ± ± ± ± Effect of on the Endogenous Oxygen Consumption by Heart Slices No. of 1st hr 2nd hr. 3rd hr ± 0.36.S6 ± % 10.2 ± ± ± ± ± ± 0.5S No statistically significant difference between the two groups is demonstrated in figure 2, it is also seen in normal. When the glycogen range was mg. Qo 2 for the first hour was 9.52 (10 experiments). When the glycogen range was mg. Q 02 was (12 experiments). The effect of prolonged hypothermia on the relationship between glycogen content and oxygen consumption in heart muscle slices is illustrated in table 7. No significant change was observed in the oxygen consumption by diaphragm of normal and hypothermic.

4 5 METABOLIC CHANGES IN CARDIAC MUSCLE IN HYPOTHERMIA TABLE 6. Effect of Srtbstrales on Oxygen Consumption by Heart Slices Substrate* Type of No. of Mean Q^ for first hour Without substrate With substrate Effect of substrate Increase inqo, Per cent increase Lactate Lactate Pyruvate Pyruvate Acetate Acetate Acetoacetate.. Acetoacetate.. Glutamate Glutamate... Hypothermio S IS ' The concentration of substrate in the medium was 11 mm/l. in each case. DISCUSSION Endogenous respiration was appreciably lowered in heart slices, but not in diaphragm of hypothermic. In normal heart muscle the addition of different substrates caused increases of 3-27 per cent in the rate of respiration as already shown by other authors. 9 ' 12 Pyruvate caused a higher increase and seemed to maintain respiration rate better than the other substrates tested. A similar effect in heart slices of ducks was described by Miller and Olson. 13 The stimulating effect of substrates on respiration of heart slices was much higher in hypothermic., lactate, acetate and acetoacetate caused increases of about 50 per cent while the effect of pyruvate amounted to 10 per cent (see also, Wollenberger 11 ). The addition of substrate restored the lowered rate of oxygen uptake observed in hypothermia to its normal level. The lowering in endogenous respiration is therefore not due to the impairment of the oxidative enzyme system but seems to be caused by the loss of oxidizable substrates from the tissue, as confirmed by the stimulating effect of boiled heart extract on the respiration of hypothermic heart slices. Extract prepard from normal rat heart proved to be much more effective than from hypothermic heart in this respect caused an increase of 50 per cent in the rate of glucose utilization by heart muscle. On calculating the carbohydrate balance however, TABLE 7. Effect of Prolonged on Glycogen Content and Oxygen Consumption by Heart Muscle., 2 hours.., 3 hours.. No. of 2 2 Glycogen (mg./loo mg. tissue) Mean Qo 2 for first hour Endogenous S With the sum of "glucose utilization plus glycogenolysis" was found to be the same in both cases. "Net glucose utilization" was 10 per cent higher in the hypothermic series, probably due to the 20 per cent decrease in lactic acid formation observed in this case. When comparing the difference in glucose utilization by heart slices of hypothermic and normal to the increment of oxygen uptake caused by substrate addition, i.e., 0.28 mg. glucose/100 mg. wet tissue/2^ hours and 0.19 ml. O 2 /100 mg. wet tissue/23^ hours, respectively, the latter corresponds to 0.26 mg. glucose oxidized to CO 2 and water; the extra glucose utilized by hypothermic heart muscle is therefore oxidized to completion. It is interesting to note that there exists a positive high correlation between the initial glycogen content and the rate of endogenous respiration of hypothermic heart muscle while this relationship is also present but less pronounced in normal heart. It may be assumed

5 ITZHAKI AND WERTHEIMER 55 FIG. 2. Relationship between endogenous respiration and glycogen content of cardiac muscle of hypothermic. Ordinale, Qo, values for the first hour of respiration, abscissa, glycogen content (mg./loo mg. tissue), r = , p < 0.01, N = 32. Each point represents data from 1 rat. -So Fia. 1. Effect of heart extract on oxygen consumption by heart slices of hypothermic. Abscissa, minutes; ordinate, rate of oxygen consumption after tipping of extract (or saline) from the side-arm as per cent of rate during the preceding control period (25 min.). Three-tenths milliliter extract was added per flask: upper curve, heart extract from normothermic rat; middle curve, heart extract from hypothermic rat; lower curve control (0.3 ml. saline). Each curve, average of 3 experiments. that the glycogen level reflects the order of concentration of substrates present in the heart muscle. Endogenous respiration and glucose utilization in diaphragm was unchanged by hypothermia while glycogenolysis occurred to the same extent in both tissues. This may perhaps be explained as due to an accelerated hexokinase reaction in heart muscle only. The striking change in carbohydrate metabolism of diaphragm produced by hypothermia is the increased glycogen synthesis. According to Hansen, Rutter, and Samuels 1 glycogen synthesis by normal diaphragm could be related to the initial glycogen levels. But the variations in the initial glycogen level found by these investigators were much larger than the difference in glycogen content between diaphragms of normal and hypothermic observed by us. Thus the lowered glycogen level in diaphragms of hypothermic is probably neither the only nor perhaps the principal factor responsible for the increased glycogen ISo synthesis. Incubation in homologous serum for instance, caused a much greater difference in the glycogen synthesis of diaphragms of normal and hypothermic than with KRP as the incubation medium. The great decline in the glycogen of heart muscle cannot be caused by liberation of epinephrine. The data available in the literature indicates that the heart muscle, but not the striated muscle is relatively insensitive to the glycogenolytic action of epinephrine. Some authors have even reported a glycogen accumulation in the heart after epinephrine action. 15 After thyroidectomy the metabolic changes in heart muscle induced by hypothermia were found to be absent when tested 2 weeks after the operation. It is well known that the thyroid gland is actively associated with the mechanism of chemical heat regulation, particularly when the cold stimulus is continued over a long period. Wertheimer and Bentor 16 ' 17 found that exposure of to low temperatures, for a few hours only, caused an increased glycogen synthesis and glucose utilization in diaphragm which was later shown to be analogous to the effect of thyroid hormone, appearing long before the rise in basal metabolism. It might therefore be argued that hypothermia stimulates an enhanced secretion of thyroxine by the thyroid which in turn causes the aforementioned metabolic changes in the myocardium. That this, however, is not the case is evident from the following findings: Intravenous injection of 0.03 mg. of thyroxine had no effect on the glucose utilization of heart slices and, similarly, injected thyroidectomized

6 56 METABOLIC CHANGES IN CARDIAC MUSCLE IN HYPOTHERMIA, specially sensitive to thyroxine, 18 showed no change in heart glycogen content. It may be concluded that for the effects to be produced by hypothermia of short duration, an increased activity of the thyroid is not indispensable, but the presence of thyroid hormone is essential. Sellers and You 19 similarly postulated that the increased metabolic rate obtained in after exposure to cold is not due to a hyperthyroid state, but is caused in full strength only in the presence of thyroid hormone. There may be other factors which are activated by the hypothermic state, but are unnoticed in the absence of thyroxine. It is interesting to note that thyroidectomy blocks the decrease of glycogen in the heart, but not in diaphragm. All these observations point to a special role of the thyroid in the metabolism of the heart muscle, as may also be seen from studies on the influence of thyroid on the rigor of the heart, 20 but not on the rigor of striated muscle. SUMMARY The carbohydrate metabolism of cardiac muscle and diaphragm of hypothermic and normal was compared. was found to induce the following metabolic changes: (I) Depletion of glycogen in heart muscle and diaphragm, less so in grastrocnemius; (2) a 50 per cent increase in glucose utilization of heart slices but no change in glucose utilization of diaphragm; (3) a small decrease in lactic acid formation in heart slices; and () a lowering of the rate of endogenous respiration of heart slices but not of diaphragm. In the presence of various substrates, the rate of oxygen consumption of the hypothermic heart muscle did not differ from that of the normal. Pyruvate acted as a preferential substrate of the heart muscle. had no effect on the respiration of the diaphragm of normal and hypothermic. A positive correlation exists between the initial glycogen content and the rate of endogenous respiration of the hypothermic heart muscle. A similar relationship is seen in the normal heart but not in diaphragm. The metabolic changes observed in the myocardium of the hypothermic rat, are not observed in the heart slices of thyroidectomized. in thyroidectomized causes the same decrease in the diaphragm glycogen as in intact. causes a significant increase in glycogen synthesis by diaphragms of normal but not of thyroidectomized. SUMMARIO IN INTERLINGUA Esseva comparate le metabolismo de hydratos de carbon in le musculo cardiac e in le diaphragma de rattos hypothermic e de rattos normal. II esseva constatate que hypothermia induce le sequente cambiamentos metabolic: (1) Depletion de glycogeno in le musculo cardiac e in le diaphragma, minus in le gastrocnemio; (2) un augmento de 50 pro cento in le utilization de glucosa in sectiones cardiac, sed nulle cambiamento in le utilisation de glucosa in le diaphragma; (3) un parve reduction in le formation de acido lactic in sectiones cardiac; e () un reduction in le respiration endogene in sectiones cardiac sed non in le diaphragma. In le presentia de varie substratos, le grado de consumption de oxygeno in le myocardio hypothermic non differeva ab illo del myocardio normal. Pyruvato ageva como un substrato preferential del myocardio. Glucosa habeva nulle effecto super le respiration del diaphragma de rattos normal o de rattos hypothermic. Un correlation positive existe inter le initial contento de glycogeno e le respiration endogene del myocardio hypothermic. Un simile correlation es evidente in le myocardio normal, sed non in le diaphragma. Le cambiamentos metabolic observate in le myocardio del ratto hypothermic non es observabile in sectiones cardiac ab rattos thyroidectomisate. In rattos thyroidectomisate, hypothermia causa le mesme reduction in le glycogeno del diaphragma que es observate in rattos intacte. causa un augmento significative in le synthese de glycogeno in le diaphragmas de rattos normal sed non in le diaphragmas de rattos thyroidectomisate. REFERENCES 1 GBMMILL, C. L. AND HAMMAN, L., JR.: The effect of insulin on glycogen deposition and on glucose utilization by isolated muscle. Bull. Johns Hopkins Hosp. 68: 50, KRAHL, M. E. AND CORI, C. F.: The uptake of

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