We have previously shown IGF-I to be effective at attenuating ceramide-induced apoptotic cell death in cultured neonatal rat cardiomyocytes and in the H9c2 rat heart-derived cell line (Singh et al. 2000). However, in these models insulin proved to be ineffective at suppressing apoptosis, even at higher concentrations.

In this study we investigated whether IGF-I or insulin could limit apoptotic damage induced by ischaemia/ reperfusion in isolated perfused hearts. Immuno-histochemistry was used to detect the presence of the apoptotic markers p85 PARP (poly ADP-ribose polymerase) and the cleaved form of caspase 3.

Male Wistar rats (250-275 g) were humanely killed and their hearts removed and perfused in the Langendorff mode at 11 ml min^-1 with Tyrode buffer (pH 7.4) bubbled with O₂ at 37 °C. Hearts were treated during equilibrium with Tyrode buffer containing IGF-I at 50 ng ml^-1 (n = 4) or insulin at 100 ng ml^-1 (n = 4) before 30 min of ischaemia and 60 min reperfusion. Immediately following reperfusion hearts were fixed using 10 % formalin in phosphate-buffered saline after which time four lateral sections were taken from each heart. The tissue sections were immunostained using anti-p85 PARP and anti-cleaved caspase 3. The positive staining and total tissue area of each section was measured using Image-Pro Plus, and positive staining was quantified as percentage of the total tissue area.Ischaemia/reperfusion induced a significant amount (P < 0.05) of apoptosis based on p85 PARP and cleaved caspase 3 staining (Fig. 1). This apoptosis was significantly attenuated (P < 0.05) by IGF-I (50 ng ml^-1). Insulin (100 ng ml^-1) also reduced the amount of apoptosis, although this reduction was only significant in the case of the p85 PARP staining (P < 0.05).

IGF-I reduces apoptosis that results from ischaemia/ reperfusion injury of perfused rat hearts, as does insulin, but at a higher concentration than IGF-I. At this concentration it is possible that the anti-apoptotic effect of insulin may be mediated by the IGF-I receptor.

Figure 1. A, percentage of area that stained positive for p85 PARP; B, percentage of area that stained positive for p85 PARP. Mean ± S.E.M. *Significantly different from hearts subjected to ischaemia/ reperfusion in the absence of treatment with insulin or IGF-I (P < 0.05). Unpaired t test.

Singh, R.J.R., Stewart, C.E.H., Holly, J.M.P. & Suleiman, M.-S. (2000). J. Physiol. 526.P, 94P.