- Email: [email protected]
Glucose uptake during ischemia and reperfusion in working rat hearts
Heather Fraser &?Alexander S. Clanachan, Department of Pharmacology, U of Alberta,
Canada.
ENDOTOXAEMIA INHIBITS MUSCLE- CARNITINE PALMITOYL TRANSFERASE I IN SUCKLING HEART Koji Fukumoto, Agostino Pierro, Lewis Spitz & Simon Eaton. Institute of Child Health, London, U.K.
Following uptake into cardiac muscle, glucose is directed towards either glycogen synthesis or lycolysis. Ischemia-induced stress alters lycogen aurnover and glucose metabolism that a wects the recovery of post-ischemic LV work, but the extent of changes in glucose uptake (GU) remains unclear. Studbs were performed In isolated, paced 5Hz) workrng rat hearts perfused with Krebs S$J Iutton contarnrng 1.2 mM palmitate, 11 mM [ H/ “C] lucos and 100 mu/L insulin. Rates (~mol.min~‘.g % wt -T! ) of GU, calculated as the sum of glycolysis (c?;) and glycogen synthesis (GS) from exogenous lucose, were measured dunt)g aerobic baseline 960 min), low-flow (0.5 ml.min- ischemia (LFI, 60 min) as well as aerobic repe d usion (R, 30 min). During aerobic baseline, when LV work and corona flow were stable, GU, GS, and GF were 5.86+0.7 8, 1.24+0.30 and 4.34kO.19, res ectively. Duriti severe VI, all detectable LV wo r/z ceased and 8 U was slightly depressed (3.98+0.48), due to small decreases in both GF and GS. During re erfusion, when the LV work recovered to only 2 & /o of aerobic baseline, GU remained depressed (2.75kO.35). The availability of rglucose was not rate-limiting in an phase of pe usron as glucose extraction was 0. 24&0.08% during baseline, 21+2% durin LFI and 2.8+0.7% durin R. LFI increased the AIA PlATP ratio727-fold) an % AMPK activity (4fold). Thus, in paced (~Hz), working hearts perfused with palmitate, glucose and insulin, severe rschemic stress has no marked acute effects on GU.
Heart is affected in sepsis-related multiple organ failure. Impairment of b-oxidation at the level of camitine palmitoyl transferase I (CPTI) during sepsis could lead to bioenergetic failure. Aim: To determine the effects of sepsis on neonatal cardiac CPTI. Methods: Sepsis was modelled in suckling rats by i.p. 300uglkg lipopolysaccharide. Controls received normal saline. Cardiac mitochondria were isolated after 2h. CPTI activity was measured radiochemically. Protein levels of muscle (M-) and liver (L-) isofomrs of CPTI were determined by western blotting. Results (mean * SEM) were compared by unpaired t-test. Results: CPTI activity was significantly decreased by endotoxaemia (control, 14.4 ?: 0.8 vs. endotoxaemia 20.4 f 0.8 nmol/minlU citrate synthase; n=33, pcO.0007). Lower CPTI activity was not due to decreases in immunoreactive protein, as neither M- nor L- isoform was altered (n=14 per group), suggesting a direct inhibition of CPTI activity. To determine whether M- or L- CPTI was inhibited, we carried out titrations with DNP-etomoxir-CoA. which at low concentrations specifically inhibits L-CPTI. Slopes of the titration curves with DNP-etomoxir-CoA were no different between control and endotoxaemia (n=15; p=O.25), suggesting that M-CPTI is specifically inhibited. This was supported by the findings that endotoxaemia increased the /CSO of malonyl-CoA on CPTI from 2.3 to 9.2PM and that endotoxaemia did not affect CPTI in kidney or liver mitochondna whereas it inhibited muscle mitochondria. Conclusions: M-CPTI is CPTI in inhibited during neonatal endotoxaemia, possibly by a direct free-radical related mechanism.
THE LINK BETWEEN INSULIN RESISTANCE AND HYPERTENSION DEPENDS ON SEX Denise Galipeau, Linfu Yao, John H. McNeill, Pharmaceutical Sciences, University of British Columbia.
COMFARTMENEXLIZilTON OF PYRlMiDINE NLKLEOi’TDE!JINANOXlCC4RDL4CMpOC~ TimothyP.Geisbuhler. Depxhn& of Physiology, Kirksville College of osteopathic Medicine, Kirksville, MO 63501.
To assess the effect of sex on the association between hyperinsulinemia / insulin resistance and hypertension, we chronically treated male and female rats with subcutaneous sustained release insulin (2 U/day) and measured insulin sensitivity (via oral glucose tolerance test - OGTT) and systolic blood pressure (via tail cuff). Male and females treated with insulin had significantly lower fasting plasma glucose levels compared to their sex-matched controls, but the hypoglycemic effects of insulin were significantly greater in females. Systolic blood pressure increased significantly in the male treated group only (123f2, 137f6*, 120+3, 120+3 mmHg for male, male treated, female, and female treated, *p<0.05). Results from the OG’IT demonstrate that (1) female rats are 4.5 fold more sensitive to insulin compared to males and (2) chronic insulin treatment impairs insulin sensitivity in both male and female rats, but to a greater degree in males. Concentration - response curves to U46619, a thromboxane mimetic, were constructed in aortae at termination. The potency of this agonist was two fold higher in tissues from male rats compared to females. It is concluded from these results that there are sex differences in the association between hyperinsulinemia / insulin resistance and hypertension and this may be related to differences in vascular smooth muscle responses to thromboxane. (Supported by the HSFBC&Y, DG by the Rx&D HRFKIHR)
Loss of 5’-nucleotides from cardiac myocytes is a distinguishiag lkature of myocardkl khemia. Previous work by ourselves and others ha9 documented dis1ocations of metabulic processes mediatedbybothpurineandpyrimidine nucleotides. This study was designed to establish the extent of anoxia-induced depletion of non-a&nine mlc1eotides in the cytosolic compartment of heart muscle cells. Cardiac myocytes were incubated aerobically (03 or anoxically (NJ for 30 or 60 min; anoxic cells at both time points were reoxygenated for 10 min. Roughly 8540% of CTI’ and UTP were cytosolic under aerobic conditions, compared with 62% of GTP under similar conditions. Similarly, the total cytidine and uridine nucleotide pool of aerobic myocytes was 70-90% cytosolicvs. 61%oftotalguaninenuc1eotides. Aftertheonset of ano* both cytidine and uridine nucleotides (principally the triphosphate forms) were quickly degraded. Reoxygenation of anoxic myocytes for 10 mitt allowed some recovery of both UTP and CTP. The resynthesized nucleotide appeared ahnost exclusively in the cytosol. These results support the concept that non-adenine nucleotides could reach ctitic.ally low levels in anoxic or ischemic heart in advance of adenine nucleotides. [Supported by a grant from the American Heart Association-Missouri ABiliate].
Al67
Canada.
ENDOTOXAEMIA INHIBITS MUSCLE- CARNITINE PALMITOYL TRANSFERASE I IN SUCKLING HEART Koji Fukumoto, Agostino Pierro, Lewis Spitz & Simon Eaton. Institute of Child Health, London, U.K.
Following uptake into cardiac muscle, glucose is directed towards either glycogen synthesis or lycolysis. Ischemia-induced stress alters lycogen aurnover and glucose metabolism that a wects the recovery of post-ischemic LV work, but the extent of changes in glucose uptake (GU) remains unclear. Studbs were performed In isolated, paced 5Hz) workrng rat hearts perfused with Krebs S$J Iutton contarnrng 1.2 mM palmitate, 11 mM [ H/ “C] lucos and 100 mu/L insulin. Rates (~mol.min~‘.g % wt -T! ) of GU, calculated as the sum of glycolysis (c?;) and glycogen synthesis (GS) from exogenous lucose, were measured dunt)g aerobic baseline 960 min), low-flow (0.5 ml.min- ischemia (LFI, 60 min) as well as aerobic repe d usion (R, 30 min). During aerobic baseline, when LV work and corona flow were stable, GU, GS, and GF were 5.86+0.7 8, 1.24+0.30 and 4.34kO.19, res ectively. Duriti severe VI, all detectable LV wo r/z ceased and 8 U was slightly depressed (3.98+0.48), due to small decreases in both GF and GS. During re erfusion, when the LV work recovered to only 2 & /o of aerobic baseline, GU remained depressed (2.75kO.35). The availability of rglucose was not rate-limiting in an phase of pe usron as glucose extraction was 0. 24&0.08% during baseline, 21+2% durin LFI and 2.8+0.7% durin R. LFI increased the AIA PlATP ratio727-fold) an % AMPK activity (4fold). Thus, in paced (~Hz), working hearts perfused with palmitate, glucose and insulin, severe rschemic stress has no marked acute effects on GU.
Heart is affected in sepsis-related multiple organ failure. Impairment of b-oxidation at the level of camitine palmitoyl transferase I (CPTI) during sepsis could lead to bioenergetic failure. Aim: To determine the effects of sepsis on neonatal cardiac CPTI. Methods: Sepsis was modelled in suckling rats by i.p. 300uglkg lipopolysaccharide. Controls received normal saline. Cardiac mitochondria were isolated after 2h. CPTI activity was measured radiochemically. Protein levels of muscle (M-) and liver (L-) isofomrs of CPTI were determined by western blotting. Results (mean * SEM) were compared by unpaired t-test. Results: CPTI activity was significantly decreased by endotoxaemia (control, 14.4 ?: 0.8 vs. endotoxaemia 20.4 f 0.8 nmol/minlU citrate synthase; n=33, pcO.0007). Lower CPTI activity was not due to decreases in immunoreactive protein, as neither M- nor L- isoform was altered (n=14 per group), suggesting a direct inhibition of CPTI activity. To determine whether M- or L- CPTI was inhibited, we carried out titrations with DNP-etomoxir-CoA. which at low concentrations specifically inhibits L-CPTI. Slopes of the titration curves with DNP-etomoxir-CoA were no different between control and endotoxaemia (n=15; p=O.25), suggesting that M-CPTI is specifically inhibited. This was supported by the findings that endotoxaemia increased the /CSO of malonyl-CoA on CPTI from 2.3 to 9.2PM and that endotoxaemia did not affect CPTI in kidney or liver mitochondna whereas it inhibited muscle mitochondria. Conclusions: M-CPTI is CPTI in inhibited during neonatal endotoxaemia, possibly by a direct free-radical related mechanism.
THE LINK BETWEEN INSULIN RESISTANCE AND HYPERTENSION DEPENDS ON SEX Denise Galipeau, Linfu Yao, John H. McNeill, Pharmaceutical Sciences, University of British Columbia.
COMFARTMENEXLIZilTON OF PYRlMiDINE NLKLEOi’TDE!JINANOXlCC4RDL4CMpOC~ TimothyP.Geisbuhler. Depxhn& of Physiology, Kirksville College of osteopathic Medicine, Kirksville, MO 63501.
To assess the effect of sex on the association between hyperinsulinemia / insulin resistance and hypertension, we chronically treated male and female rats with subcutaneous sustained release insulin (2 U/day) and measured insulin sensitivity (via oral glucose tolerance test - OGTT) and systolic blood pressure (via tail cuff). Male and females treated with insulin had significantly lower fasting plasma glucose levels compared to their sex-matched controls, but the hypoglycemic effects of insulin were significantly greater in females. Systolic blood pressure increased significantly in the male treated group only (123f2, 137f6*, 120+3, 120+3 mmHg for male, male treated, female, and female treated, *p<0.05). Results from the OG’IT demonstrate that (1) female rats are 4.5 fold more sensitive to insulin compared to males and (2) chronic insulin treatment impairs insulin sensitivity in both male and female rats, but to a greater degree in males. Concentration - response curves to U46619, a thromboxane mimetic, were constructed in aortae at termination. The potency of this agonist was two fold higher in tissues from male rats compared to females. It is concluded from these results that there are sex differences in the association between hyperinsulinemia / insulin resistance and hypertension and this may be related to differences in vascular smooth muscle responses to thromboxane. (Supported by the HSFBC&Y, DG by the Rx&D HRFKIHR)
Loss of 5’-nucleotides from cardiac myocytes is a distinguishiag lkature of myocardkl khemia. Previous work by ourselves and others ha9 documented dis1ocations of metabulic processes mediatedbybothpurineandpyrimidine nucleotides. This study was designed to establish the extent of anoxia-induced depletion of non-a&nine mlc1eotides in the cytosolic compartment of heart muscle cells. Cardiac myocytes were incubated aerobically (03 or anoxically (NJ for 30 or 60 min; anoxic cells at both time points were reoxygenated for 10 min. Roughly 8540% of CTI’ and UTP were cytosolic under aerobic conditions, compared with 62% of GTP under similar conditions. Similarly, the total cytidine and uridine nucleotide pool of aerobic myocytes was 70-90% cytosolicvs. 61%oftotalguaninenuc1eotides. Aftertheonset of ano* both cytidine and uridine nucleotides (principally the triphosphate forms) were quickly degraded. Reoxygenation of anoxic myocytes for 10 mitt allowed some recovery of both UTP and CTP. The resynthesized nucleotide appeared ahnost exclusively in the cytosol. These results support the concept that non-adenine nucleotides could reach ctitic.ally low levels in anoxic or ischemic heart in advance of adenine nucleotides. [Supported by a grant from the American Heart Association-Missouri ABiliate].
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