Akt2 knockout preserves cardiac function in high-fat diet-induced obesity by rescuing cardiac autophagosome maturation

Dear Editor, Accumulating studies have demonstrated that the autophagy–lysosome pathway, a major pathway governing protein and organelle degradation and recycling, is a house keeper in cardiomyocytes under physiological conditions (Mizushima and Klionsky, 2007). However, the role of autophagy in the heart under pathological conditions is still controversial (Nemchenko et al., 2011). In vivo studies depicted that inhibition of mammalian target of rapamycin (mTOR), a primary inhibitory regulator of autophagy, is capable of attenuating pressure overload-induced cardiac dysfunction (McMullen et al., 2004). To the contrary, recent studies have also indicated that suppressing autophagy is beneficial for cardiac hypertrophy (Cao et al., 2011). Along the same line, activated autophagy has been proved detrimental for pressure overload-induced heart failure (Zhu et al., 2007). However, the role of autophagy in the heart in high-fat diet (HFD)-induced obesity is poorly understood. To date, there is little evidence suggesting a role of autophagy in heart anomalies associated with diet-induced obesity, although a number of upstream regulators of autophagy have been identified to play a role in HFD-induced obesity. For example, the primary inhibitor of autophagy mTOR may be hyperactivated by an HFD and contribute to the development of cardiac dysfunction (Birse et al., 2010). As the major activator of mTOR, the Akt family of serine–threonine kinases is also activated by an HFD in the heart. However, the precise role of Akt2, one of the three Akt isoforms predominantly found in the heart, in autophagy regulation in HFD-induced obesity still remains elusive. To this end, the present study was designed to evaluate the role of autophagy and autophagy flux in HFD feeding-induced cardiac geometric and functional changes with a special focus on Akt2 signaling. HFD intake significantly increased body and organ (heart, liver, kidney and adipose tissue) weights compared with low-fat diet (LFD) feeding (Supplementary Table S2). Western blot analysis confirmed the absence of Akt2 in hearts from Akt2 mice (Supplementary Figure S1A and B). Interestingly, HFD feeding upregulated cardiac expression of Akt2 (Supplementary Figure S1A and B) but not that of Akt1 (Supplementary Figure S7A and B) and Akt3 (Supplementary Figure S7A and C). Akt2 knockout did not affect body or organ weight in LFD-fed mice (Supplementary Table S2). However, Akt2 knockout effectively nullified HFD-induced gain in body and organ/tissue weights, in particular the heart (Supplementary Table S2). Accumulating studies have demonstrated that Akt regulates cell growth and lipid biosynthesis through mTORC1. Accordingly, we found that an HFD-activated Akt (Supplementary Figure S6A and B) and mTORC1 (Supplementary Figure S7A and I) in the heart, both of which were mitigated by Akt2 knockout. These data depict a beneficial effect of Akt2 knockout against HFD-induced weight gain possibly through the inhibition of Akt-mTORC1 activation. In addition, HFD feeding significantly increased the level of triglyceride, the effect of which was ablated by Akt2 knockout (Supplementary Figure S1C). Further scrutiny of glucose metabolism using intraperitoneal glucose tolerance test revealed overt glucose intolerance following HFD intake in the wild type (WT) which was partially attenuated in the Akt2 mice (Supplementary Figure S1D and E). HFD feeding significantly compromised myocardial geometry and function as evidenced by overtly increased LV ESD, LV EDD and LV mass, as well as decreased fractional shortening associated with unchanged septum and posterior wall thickness. Interestingly, Akt2 knockout ameliorated HFD feeding-induced cardiac geometric and contractile anomalies (Figure 1A and B and Supplementary Figure S2A–E). Further assessment of cardiomyocyte contractile function revealed consistent findings. HFD feeding dampened cardiomyocyte contractile capacity (decreased peak shortening and maximal velocity of shortening/re-lengthening) associated with unchanged duration of shortening and re-lengthening, which was recovered by Akt2 knockout (Figure 1C and D and Supplementary Figures S2F–I and S3A–C). Besides, Akt2 knockout significantly ameliorated intracellular Ca2+ handling dysfunction induced by an HFD in the WT mice (Supplementary Figure S2J–O). Additionally, HFD feeding induced cardiac hypertrophy (Supplementary Figure S4A–H), interstitial fibrosis (Supplementary Figure S5A and B), and activated cardiac protein synthesis pathway (Supplementary Figures S6A–K and S7A, J, and K), which were obliterated by Akt2 knockout. Taken together, these results supported that Akt2 knockout protected murine hearts against HFD-induced cardiac pathological hypertrophy. Interestingly, our data revealed that the expression of LC3B I (microtubule-associated protein light chain 3 I, type B) was dramatically increased following HFD feeding in both the WT and Akt2 mice, indicating that HFD feeding may trigger the initial autophagy steps (Figure 1E and Supplementary Figure S8A, C–G, J, and L– O). LC3B II integrates onto the autophagosomal membrane, and is widely used as a marker of autophagosomes. Nonetheless, an increase in LC3B II may represent either an increase in autophagosome formation (initiation of autophagy) or a doi:10.1093/jmcb/mjs055 Journal of Molecular Cell Biology (2013), 5, 61–63 | 61 Published online December 19, 2012