Pretreatment with insulin before ischaemia reduces infarct size in Langendorff-perfused rat hearts B. N. Fuglesteg, 1 C. Tiron, 2 A. K. Jonassen, 2 O. D. Mjøs 1 and K. Ytrehus 1 1 Department of Medical Physiology, Faculty of Medicine, University of Tromsø, Tromsø, Norway 2 Section of Physiology, Faculty of Medicine, University of Bergen, Bergen, Norway Received 13 March 2008, revision requested 10 April 2008, final revision received 3 September 2008, accepted 12 September 2008 Correspondence: B. N. Fuglesteg, Department of Medical Physiology, Faculty of Medicine, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. E-mail: brittf@fagmed.uit.no Abstract Aim: To compare the possible role of Akt and mammalian target of rapa- mycin (mTOR) in mediating cardioprotection against ischaemia under three different conditions: (1) During ischaemic preconditioning (IPC), (2) when insulin was given as a pretreatment agent (InsPC) and (3) when insulin was given as a reperfusion cell survival agent (Ins R ). Methods: Isolated perfused rat hearts were subjected to IPC (3 · 5 min) or InsPC (50 mU mL )1 ;3 · 5 min), before 30 min of regional ischaemia fol- lowed by 120 min of reperfusion Æ 1L-6-hydroxymethyl-chiro-inositol-2- [(R)-2-O-methyl-3-O-octadecylcarbonate] (HIMO) (20 lm; Akt inhibitor) or rapamycin (1 nm; mTOR inhibitor). In addition, insulin (3 mU mL )1 ) was given at the onset of reperfusion, ÆHIMO or rapamycin. Risk zone (R) and infarct size (I) were determined with Evans blue and tetrazolium staining respectively. Western blot analysis was performed on tissue from Lange- ndorff-perfused rat hearts and cell lysates from cultured HL1 cells. Results: IPC, InsPC and Ins R treatment resulted in a significant reduction in infarct size compared to controls (all P < 0.05). This protective effect of IPC and insulin was abolished by the inhibitors. However, the putative Akt inhibitor, although capable of abolishing cardioprotection induced by insu- lin, was not able to inhibit insulin-induced phosphorylation of Akt in Langendorff-perfused rat hearts and cultured HL1 cells. The target for this compound therefore remains to be determined. Conclusion: IPC and insulin (either as InsPC or Ins R ) appear to activate mTOR, and this kinase seems to play an essential role in cardioprotection against ischaemia and reperfusion injury as rapamycin blocked the protection. Keywords Akt, insulin, ischaemia, mTOR. In recent years, pre-ischaemic as well as post-ischaemic treatments with short lasting ischaemia or selected receptor agonists have been shown to protect the heart against infarction and promote cell survival in stan- dardized experimental protocols. Insulin administered at ischaemic reperfusion is one example (Jonassen et al. 2001). The effect of insulin on substrate metabolism in the heart is well studied, but the interest in insulin as a possible therapeutic agent in connection with myocar- dial infarction stems back to 1962 where treatment with glucose-insulin-potassium (GIK) after acute myocardial infarction (AMI) was found to be beneficial (Sodi- Pallares et al. 1962). Almost 30 years later, a meta- analysis of nine trials revealed that in-hospital mortality after AMI was reduced by GIK therapy (Fath-Ordou- badi & Beatt 1997), and one study reported a 66% reduction in the relative in-hospital mortality risk when adding GIK upon reperfusion during AMI (Diaz et al. 1998). GIK therapy has also been found to expedite recovery and prevent myocardial infarction after coronary artery bypass grafting (Lazar et al. 1997), and enhance left ventricular function during AMI Acta Physiol 2009, 195, 273–282 Ó 2008 The Authors Journal compilation Ó 2008 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2008.01901.x 273