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Glucose utilization in cultured adult heart cells
71 193 LIPOPROTEIN LIPASE (LPL) ACTIVITY IN HEARTS FROM DIABETIC AND INSULIN-TREATED DIABETIC RATS. S.Mochizuki, ~T.Murase, M.Taniguchi and M.Nagano. D e p a r t m e n t of Medicine, Aoto Medical Center, Jikei U n i v e r s i t y School of M e d i c i n e and ~Department of Medicine, University of Tokyo, Tokyo, Japan. Effects of diabetes mellitus and insulin treatment on two types of LPL, functional LPL and non-functional LPL were studied in isolated, pelf used hearts from streptozotoein-induced diabetic and insulin treated diabetic mats. The hearts were perfused by working heart mode and h e p a r i n (5u/ml) was added to the p e r f u s a t e followed by 20 min aerobic perfusion. Coronary effluent was collected with 5 min intervals and functional LPL was determined using glycerol tri (I-14C) oleate as a substrate. The hearts were homogenized in ammonium buffer and extracted to measure non-functional LPL. Functional LPL activities in diabetic mats were S07+55 and 116+15 nmoles FFA/min at 5 and i0 min following h e p a r i n a d m i n i s t r a t i o n respectively and they were significantly lower than in control (406• and 251• However, non-functional LFL in diabetic mats (77.2+10.9 nmoles FFA/min/whole tissue) was significantly higher than in control(29.S+8). Insulin treatment raised the functional LPL and lessened the nonfunctional LPL, however, they were not statistically significant when compared with diabetic rats. It was suggested that theme was a conversion defect from the precursor of LPL to the functional form of LPL in diabetic myoeardium. High level of plasma NEFA could be related to the inhibition of the conversion.
194 ENZYMATIC CONTROL OF S T I M U L A T I O N IN RATS.
CARDIAC
GLYCOGEN
RESTORATION
S.Grably and A.Rossi. L a b o r a t o i r e de p h y s i o l o g i e 38402 S T - M A R T I N d ' H E R E S C~dex, France.
FOLLOWING
BETA-RECEPTOR
animale, ERA CNRS 1046, BP 68,
Following subcutaneous i n j e c t i o n of i s o ~ r e n a l i n e into rats (5 mg.kg-l) the cardiac glycogen stores were reduced by 88% in less than 15 min. The complete restoration of myocardial glycogen is a slow process (more than 7-8 hours) despite a fast activation of glycogen synthetase and the maintenance for several hours of a high p e r c e n t a g e of the active form (max 5h : 55% vs 8.2% in controls). Since p h o s p h o r y l a s e was also a c t i v a t e d (lh : 34% active form vs 19% in controls) it may ~e assumed that g l y c o g e n synthesis operates at high rates but is counterbalanced by a continuous glycogenolysis. Indeed the reconstitution of g l y c o g e n stores was a c c e l e r a t e d as soon as the percentage of the active form of p h o s p h o r y l a s e regained to control values (5h : phosphorylase 15%, glycogen synthetase : 55%). W~en a cardioselective b e t a - b l o c k e r (atenolol 20 m g . k g - 1 ) was a d m i n i s t e r e d 30 win following the injection of isoprenaline cardiac glycogen stores were completely restored by 30 min, the enzymes activations being : phosphorylase 12%, G l y c o g e n synthetase 45%. In both conditions, the increase in g l y c o g e n levels was paralleled by a progressive deactivation of glycogen synt~etase. It can be concluded that the potential capabilities of myocardial tissue for g l y c o g e n o g e n e s i s are probably high a l t o u g h obscured by the activity of phosphorylase. Supported by a grant from MRI 821 07 55
195 GLUCOSE UTILIZATION IN CULTURED ADULT HEART CELLS. *) R. Spahr, H.M. Piper, I. Probst, P.G. Spieckermann. Zentren Physiologic, Biochemie, Univerait~t G~ttingen, FRG. Cultured adult eardiocytes (3MCC 14 (1982) 597) consisting of nearly 100 % intact cells are mechanically inactive and in a slate of basal metabolism. In aerobic Tyrode solution (5 mM glucose, I mM CaC1 , 57 ~ they produce lactate (t) and CO 2 at a ratio of 2.5. Under I00 % 0 they do no~ change this behaviour indicating that L-production 2 is not due to partial hypoxia. That it is due to low PDH activation is suggested by the finding that DCA may decrease the L/CO ratio to 0~ while only slightly 2 influencing glycolytic flux. A 4-fold stimulation of C02-production by I0 mH DCA shows that there is no critical limitation of the mitoehondrial pyruvate carrier activity. Insulin maximally increases L-production 4-fold, but C02Production only by 50 ~; (half max. at i0-- M). Thus L-formation seems to be the main route for reoxygenalien of cytosolic NAOH. In the presence of insutin DCA and e x o g e n o u s L the L/CO ratio can be varied between 0.5 and 16 indicating that there is no close coupling ~etween glycolytic flux and pyruvate oxidation in this system. Both exogeneous L and palmitate (P) decrease glucose (G) oxidation. However, P-induced inhibition of C02-production from G further linearly increases when P-oxidation is already saturated (100 pH). This shows that P does not simply compete for endoxidation but most probably inhibits PDH. P-oxidation at lO0 pM P was not influenced by insulin or DCA, although this resulted in increase of G-oxidation. This again indicates that increase in glucose utilization cannot counteract fatty acid oxidation. *)DFG, SFB 89
194 ENZYMATIC CONTROL OF S T I M U L A T I O N IN RATS.
CARDIAC
GLYCOGEN
RESTORATION
S.Grably and A.Rossi. L a b o r a t o i r e de p h y s i o l o g i e 38402 S T - M A R T I N d ' H E R E S C~dex, France.
FOLLOWING
BETA-RECEPTOR
animale, ERA CNRS 1046, BP 68,
Following subcutaneous i n j e c t i o n of i s o ~ r e n a l i n e into rats (5 mg.kg-l) the cardiac glycogen stores were reduced by 88% in less than 15 min. The complete restoration of myocardial glycogen is a slow process (more than 7-8 hours) despite a fast activation of glycogen synthetase and the maintenance for several hours of a high p e r c e n t a g e of the active form (max 5h : 55% vs 8.2% in controls). Since p h o s p h o r y l a s e was also a c t i v a t e d (lh : 34% active form vs 19% in controls) it may ~e assumed that g l y c o g e n synthesis operates at high rates but is counterbalanced by a continuous glycogenolysis. Indeed the reconstitution of g l y c o g e n stores was a c c e l e r a t e d as soon as the percentage of the active form of p h o s p h o r y l a s e regained to control values (5h : phosphorylase 15%, glycogen synthetase : 55%). W~en a cardioselective b e t a - b l o c k e r (atenolol 20 m g . k g - 1 ) was a d m i n i s t e r e d 30 win following the injection of isoprenaline cardiac glycogen stores were completely restored by 30 min, the enzymes activations being : phosphorylase 12%, G l y c o g e n synthetase 45%. In both conditions, the increase in g l y c o g e n levels was paralleled by a progressive deactivation of glycogen synt~etase. It can be concluded that the potential capabilities of myocardial tissue for g l y c o g e n o g e n e s i s are probably high a l t o u g h obscured by the activity of phosphorylase. Supported by a grant from MRI 821 07 55
195 GLUCOSE UTILIZATION IN CULTURED ADULT HEART CELLS. *) R. Spahr, H.M. Piper, I. Probst, P.G. Spieckermann. Zentren Physiologic, Biochemie, Univerait~t G~ttingen, FRG. Cultured adult eardiocytes (3MCC 14 (1982) 597) consisting of nearly 100 % intact cells are mechanically inactive and in a slate of basal metabolism. In aerobic Tyrode solution (5 mM glucose, I mM CaC1 , 57 ~ they produce lactate (t) and CO 2 at a ratio of 2.5. Under I00 % 0 they do no~ change this behaviour indicating that L-production 2 is not due to partial hypoxia. That it is due to low PDH activation is suggested by the finding that DCA may decrease the L/CO ratio to 0~ while only slightly 2 influencing glycolytic flux. A 4-fold stimulation of C02-production by I0 mH DCA shows that there is no critical limitation of the mitoehondrial pyruvate carrier activity. Insulin maximally increases L-production 4-fold, but C02Production only by 50 ~; (half max. at i0-- M). Thus L-formation seems to be the main route for reoxygenalien of cytosolic NAOH. In the presence of insutin DCA and e x o g e n o u s L the L/CO ratio can be varied between 0.5 and 16 indicating that there is no close coupling ~etween glycolytic flux and pyruvate oxidation in this system. Both exogeneous L and palmitate (P) decrease glucose (G) oxidation. However, P-induced inhibition of C02-production from G further linearly increases when P-oxidation is already saturated (100 pH). This shows that P does not simply compete for endoxidation but most probably inhibits PDH. P-oxidation at lO0 pM P was not influenced by insulin or DCA, although this resulted in increase of G-oxidation. This again indicates that increase in glucose utilization cannot counteract fatty acid oxidation. *)DFG, SFB 89