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On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [γ-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBβ-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBα/β siRNA or PKBα siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBα.

The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy +/- a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFkappaB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.