Free fatty acids, such as saturated fatty acids are now recognized as significant contributors to lipotoxicity pathology including insulin resistance, type 2 diabetes, and cardiomyopathy. Palmitate as a kind of saturated fatty acids can induce apoptosis in diverse cell types, such as cardiomyocytes. Apoptosis or programmed cell death is basically cellular suicide which occurs after sufficient cellular damage. Caspase-3, a member of cysteine-aspartic proteases that play a central role in the execution of the apoptotic program, exists as an inactive 32-kDa proenzyme in normally. The cleavage within the 19-kDa fragment generates a p17-kDa subunit (termed p17) as an activity form. During apoptosis, caspase-3 cleaved the 116-kDa PARP protein to yield a 24-kDa DNA-binding fragment and an 89-kDa catalytic fragment. In this study, our results showed that palmitate-induced apoptosis through increasing the activity of caspase-3 and PARP in H9c2 cells.
Adiponectin, an abundant circulating adipokine, is almost exclusively secreted from adipose tissue and exists in the range of 3–30 μg/mL in plasma. Although its physiological and pathological significance remains to be determined, numerous epidemiological studies have shown that the correlation between reduced adiponectin levels and increased morbidity/mortality of cardiovascular ischemic diseases and diabetes mellitus[29, 30]. Conversely, a higher plasma adiponectin concentration is associated with a lower risk of ischemic heart disease. Cardiomyocytes apoptosis is an important contributor to myocardial dysfunction and heart failure, so preventing cardiomyoytes apoptosis is an effective way to protect myocardial function. Recently some published in vivo studies demonstrated that adiponectin functioned as a cardioprotective molecule in myocardial ischemia-reperfusion injury[12, 18, 32]. The results of these research exhibited that exogenous adiponectin supplementation can significantly decrease myocardial apoptosis, infarct size and impaired cardiac function. More detailed in vitro studies regarding anti-apoptotic mechanisms of adiponectin have been performed in different cell types[33–35]. Although adiponectin also inhibited hypoxia/reoxygenation (H/R)-induced apoptosis through reducing cytochrome c release and decreasing the activity of caspase-3 in H9c2 cells, it is not clear that there is the effect of adiponectin on palmitate-induced apoptosis in H9c2 cells. In this study, our results showed that globular adiponectin inhibited palmitate-induced apoptosis in H9c2 cells through decreasing the activity of caspase-3 and PARP. Above data indicated that adiponectin might be a novel therapeutic molecule for anti-apoptosis in cardiomyopathy and myocardial damage.
Recently many studies showed that several signaling transduction pathways were shown to mediate both of pro- and anti-apoptosis effects in numerous tissues and cell types by adiponectin, such as (PI3K)/Akt signaling pathway, MAPK/ERK and AMPK.
Notably, PI3K/Akt signaling pathway has been shown to play a major role in the prevention of apoptosis, and acute activation of this signal pathway can promote both cardiomyocyte survival and function in vitro and in vivo. Previous studies have shown that adiponectin can activate the Akt signaling pathway to promote pro-survival or anti-apoptosis in several cell types[35, 37, 38]. Here, our results showed that globular adiponectin can attenuate apoptosis induced by palmitate in H9c2 cells through decreasing the activity of caspase-3 and PARP. This effect was abolished by LY294002, a highly specific inhibitor of PI3K/Akt. This data suggested that activation of PI3K/Akt signaling pathway was necessary for adiponectin mediated inhibition of H9c2 cells apoptosis induced by palmitate.
ERK1/2/MAPK is a well-known taking part in a signal transduction cascade in response to extracellular stimuli, and plays an important role in cell proliferation, growth and cell death. Research indicated that ERK1/2 signaling pathway would be activate by doxorubicin-induced apoptosis in H9c2 cells. Adiponectin mediates activation of the ERK1/2 signaling pathway in several cell types[19, 40]. Nevertheless, suppression of the activity of ERK1/2 signaling pathway by adiponectin has also been demonstrated. Therefore, it seems that the effect of adiponectin on ERK1/2 signaling pathway is controversial. In this study, our results showed that the p-ERK1/2 was increased after palmitate-induced apoptosis in H9c2 cells, and globular adiponectin decreased the level of p-ERK1/2, and then inhibited palmitate-induced apoptosis in H9c2 cells through decreasing the activity of caspase-3 and PARP. In our results also showed that the level of p-ERK1/2 was increased after palmitate-induced apoptosis in H9c2 cells, and ERK1/2 inhibitor U0126 can decrease the level of p-ERK1/2, and then attenuate palmitate-induced apoptosis in H9c2 cells. These results suggest that activation of ERK1/2 signaling pathway may be one of the reasons for palmitate-induced apoptosis in H9c2 cells.
Our findings in this study showed that PI3K/Akt inhibitor LY294002, not only inhibited the activity of PI3K/Akt signaling pathway, blocked adiponectin’s inhibition of palmitate-induced apoptosis in H9c2 cells, but also increased the activity of ERK1/2 signaling pathway. Similarly, ERK1/2 inhibitor U0126 also reduced palmitate-induced apoptosis in H9c2 cells, increased the activity of PI3K/Akt signaling pathway and thus promoted cells survival. This crosstalk of ERK1/2 and PI3K/Akt were observed in other study. These results suggested that ERK1/2 and PI3K/Akt signaling pathways maybe crosstalk regulates survival and apoptosis in H9c2 cells after treated with palmitate, but it regulates mechanism crosstalk in H9c2 cells require further investigation.