The major new findings in this study are that: 1) OB displayed a beneficial effect against the alterations of contractility and β-adrenergic response in the heart of STZ-treated diabetic rats; 2) the protective effect is related to the ability of OB to restore oxidative balance and to promote phosphorylation/modulation of AMPK and pro-survival kinases such as Akt, ERK1/2 and GSK3β. To our knowledge, this is the first report showing that OB is able to exert a protective action in myocardial tissue affected by experimentally induced diabetes in adult animals.
OB is a newly discovered peptide, which derives from the ghrelin peptide precursor pre-pro-ghrelin [1, 2, 4]. Although it has been suggested that both OB and ghrelin participate in a complex regulatory system , the intracellular pathways activated by OB and the role it plays in physiological and in pathophysiological conditions are largely unknown. Several studies have already indicated that, in common with ghrelin and other growth hormone secretagogues, OB protects cardiac muscle against I/R injury . Moreover, it has been shown that ghrelin reduces oxidative stress and inhibits the production of reactive oxygen species [34, 35], biological events that have been implicated in various pathologies, including hypertension , cardiac ischemia  and myocardial fibrosis [19, 20, 23, 29]. A similar protective effect has been also reported in primary cardiomyocytes, as well as in H9c2 cardiomyoblastic cell line and endothelial cells, in which both ghrelin  and OB  inhibit apoptosis induced by doxorubicin or I/R, by activating ERK1/2, PKC and PI3K/Akt dependent mechanisms. Several evidences indicate that ghrelin gene-derived peptides prevent the development of diabetes in STZ-treated newborn rats. Indeed, both ghrelin and OB promote regeneration of β-cells and improve glucose metabolism in STZ-treated newborn rats, granting a therapeutic potential in medical conditions associated with impaired β-cell function [10–13]. Moreover, a recent paper showed that ghrelin ameliorated the reductions in motor and sensory nerve conduction velocities and reduced oxidative stress in STZ-treated mice , suggesting that ghrelin gene-derived peptides could also be able to protect against dysfunction in experimentally-induced diabetes in adult animals, in which it is likely these peptides have lost their ability to induce β-cell regeneration. In addition, ghrelin improves both the metabolic functions and the disturbed energy metabolism in the cardiac muscle of obese diabetic rats .
Among all diabetic complications, cardiovascular disease continues to be the primary cause of morbidity and mortality. In agreement with other studies [16, 40], we previously reported that diabetic cardiomyopathy induced by chronic hyperglycemia is characterized by myocyte loss and myocardial fibrosis, which lead to decreased elasticity and impaired contractile function . Moreover, the enhanced oxidative stress reduced peak contractile amplitude and maximal velocity of contraction and relaxation under basal condition, as well as the β-adrenergic response in diabetic myocardial tissue [29, 30].
This study demonstrates that OB exerts a protective effect against the derangement of contractility observed in papillary muscles from diabetic rats (−65% of mechanical tension vs control). The beneficial effect of OB could be due to its ability to counteract the switch of cardiac myosin heavy chain gene expression from the α- to the β-MHC isoform, and the increase of pro-fibrogenic growth factors, such as TGFβ1 and CTGF, observed in diabetic myocardial tissue. In addition, OB was also able to restore the β-adrenergic response by promoting recovery of β1-adrenoreceptors protein expression. The molecular mechanisms leading to myocardial dysfunction observed in diabetic myocardial tissue include an unbalance between the pro-oxidant and antioxidant compounds and increased inflammatory process, in terms of TNF-α plasma levels and NFkB activation. We observed that OB corrects oxidative unbalance and reduces inflammatory processes in diabetic myocardium, although, on the basis of our data, we do not provide a clear evidence that the initial mechanism upon which OB acts is related to a direct antioxidative action. We thus investigated the possible molecular mechanisms and intracellular pathway involved in the protective effect of OB. OB enhanced AMPK phosphorylation and up-regulated pro-survival kinases Akt, ERK1/2 and GSK3β in diabetic myocardial tissue. The ability of OB to enhance pAMPK levels in myocardial cells was confirmed by in both in vivo and in vitro models after acute administration of this peptide. Interestingly, the effects of OB were comparable to those induced by metformin, an anti-diabetic drug known to stimulate AMPK. AMPK has recently emerged as an important intracellular signaling pathway in the heart . In the heart, AMPK is modulated by hormones such as adiponectin, leptin and ghrelin, or cytokines like TNFα. A recent paper by Paiva , showing that the administration of metformin during the early reperfusion significantly enhances AMPK phosphorylation, concomitantly with the reduction of infarct size, highlighted the important role of AMPK activation in cardioprotective mechanisms. It has been proposed that AMPK may protect against reperfusion injury by increasing glucose uptake, if the potential negative consequences of increased fatty acid oxidation are not present . Since ATP from glycolysis may be preferentially used to support membrane activities such as ion pumping, a shift in glucose metabolism may play an important role in cardiac contractile function, metabolic activity and calcium homeostasis under conditions of calcium overload, such as post-ischemic recovery  or during β-adrenergic stimulation .
In addition, AMPK activation has been involved in the reduction of inflammatory markers such as TNFα and NFkB transcription factor . Together with Akt, AMPK is considered the main signaling molecule controlling cardiac functions . The observed decrease of cardiac AMPK phosphorylation may therefore contribute, together with reduced Akt signaling, to the depressed myocardial contractility observed in diabetic rats. Indeed, it has been recently reported that AMPK activation may induce an enhancement of L-type calcium current I(Ca) and a prolongation of the action potential duration in cardiomyocytes .
Besides AMPK, OB induced phosphorylation of Akt, ERK1/2 and GSK3β, pro-survival kinases known to exert a protective role against oxidative stress [20, 23, 48], and proposed as integral components of a protective cascade involved in myocardial preconditioning against I/R [49, 50]. The involvement of these kinases in the action of OB has been already shown in the proliferation of human retinal pigment epithelial and gastric cancer cells [51, 52], and in rodent β-cells and human pancreatic islets cell survival [10, 11].
Oxidative stress can trigger the opening of the mitochondrial membrane permeability transition pore (mPTP) and lead to a significant loss of mitochondrial NAD+ stores and subsequent derangement of cellular energy reserves and intact cellular functions. Mitochondrial dysfunction plays an important role in the development of diabetes and insulin resistance, and proper cellular function during diabetes requires the maintenance of mitochondrial membrane potential . The present study shows that OB also promoted phosphorylation/inactivation of GSK3β. GSK3β is a substrate of multiple pro-survival protein kinases, including Akt and ERK1/2, and is therefore a step to which multiple protective signaling pathways converge . Since phosphorylated GSK3β limits the opening of mPTP, it is reasonable to think that GSK3β inactivation plays a crucial role in the protective effect afforded by OB in diabetic myocardial tissue.
We cannot exclude, however, that the protective effect of OB may result from additional mechanisms not investigated in the present study, such as the nitric oxide (NO) pathway activation. Recent results, indeed, indicate that increased NO availability attenuates some alterations in metabolism and gene expression associated with insulin resistance induced by a high fat diet .
A still open question regards the earliest receptor-initiated mechanisms involved in the action of OB. Although OB was initially claimed to activate the G protein-coupled receptor-39 (GPR39) , subsequent studies were unable to demonstrate binding of OB to GPR39 or a stimulatory function of the OB peptide on GPR39-transfected cells [8, 9], and the original proposal that OB acts as a ligand of GPR39 has been retracted . We have recently provided evidence that specific OB receptors are present in ventricular myocardial cells , with a receptor density and binding affinity quite close to those previously found in rodent and human pancreatic β-cells, where OB promotes cell survival and induces expression of genes involved in the regulation of β-cell mass and function [10, 11]. In conclusion, although to date the precise role of OB in cardiovascular pathophysiology remains still partially unknown, the observed beneficial effect in diabetic myocardial tissue confirms the relevance of this peptide as a physiological agent exerting protective effects against cardiac dysfunction and oxidative stress, already shown in the case of ischemic/reperfused heart.