Redox biology and the interface between bioenergetics, autophagy and circadian control of metabolism

Abstract

Understanding molecular mechanisms that underlie the recent emergence of metabolic diseases such as diabetes and heart failure has revealed the need for a multi-disciplinary research integrating the key metabolic pathways which change the susceptibility to environmental or pathologic stress. At the physiological level these include the circadian control of metabolism which aligns metabolism with temporal demand. The mitochondria play an important role in integrating the redox signals and metabolic flux in response to the changing activities associated with chronobiology, exercise and diet. At the molecular level this involves dynamic post-translational modifications regulating transcription, metabolism and autophagy. In this review we will discuss different examples of mechanisms which link these processes together. An important pathway capable of linking signaling to metabolism is the post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc). This is a nutrient regulated protein modification that plays an important role in impaired cellular stress responses. Circadian clocks have also emerged as critical regulators of numerous cardiometabolic processes, including glucose/lipid homeostasis, hormone secretion, redox status and cardiovascular function. Central to these pathways are the response of autophagy, bioenergetics to oxidative stress, regulated by Keap1/Nrf2 and mechanisms of metabolic control. The extension of these ideas to the emerging concept of bioenergetic health will be discussed.

Keywords: Chronobiology; Keap1; Metabolic shift; Nrf2; Oxidative stress; Reserve capacity.

Figure 1. Redox and metabolic control of epigenetic modifications and molecular function

(A) Redox control of gene expression as explained in the text. Central focus is on cellular nitric oxide (•NO) signaling. (B). Metabolic control of gene expression through mitochondrial superoxide (O₂•⁻) signaling via hexosamine biosynthesis pathway (HBP) flux increasing UDP-GlcNAc and protein O-GlcNAcylation (O-GlcNAc). Including both cytosolic endothelial cell, nitric oxide synthase 3 (eNOS) to reduce nitric oxide signaling and mitochondrial proteins of oxidative phosphorylation (I, II, III, IV, and V). Additional recent focus has been on the tet methylcytosine dioxygenases (TETs) to regulate DNA methylation (5-m) to hydroxymethylation (5-hm).

Figure 2. The mitochondrial stress test

A typical profile is shown in which basal oxygen consumption rate (OCR) is allowed to stabilize before the sequential addition of oligomycin which inhibits ATP synthase preventing protons returning to the mitochondria and so decreasing OCR (ATP linked). The OCR remaining after oligomycin is ascribed to movement of ions across the mitochondrial inner membrane and proton translocation not involving the ATP synthase-collectively termed proton leak. The addition of the uncoupler, FCCP allows protons to flow into the mitochondrion increasing OCR to the level which can be sustained by endogenous substrates. The final addition of antimycin A and/or rotenone results in a residual OCR is ascribed to oxygen consuming processes outside mitochondrial electron transport. The insert shows a simplified proton circuit for oxidative phosphorylation showing how the proton gradient controls OCR. This time course is annotated to show the relative contribution of non-respiratory chain oxygen consumption, ATP-linked oxygen consumption, the maximal OCR after the addition of FCCP, and the reserve capacity (Maximal – basal OCR) of the cells.

Figure 3. Integrating Bioenergetics and Cellular Responses to Stress

These data have been adapted from [96] and show the injection of hemin (25 μM) onto bovine aortic endothelial cells. Panel A: measurement of OCR (oxygen consumption rate) following hemin injection at 24 minutes followed by the mitochondrial stress test following the protocol shown in Figure 2. Panel B: time dependent decreased in basal OCR showing detectable changes 30 min after hemin addition. Panel C: in a parallel plate protein samples were prepared at the times shown and the LC3 conversion to LC3-II determined. The arrow shows the 2 hour point at which time a detectable change in the levels of LC3-II were found. Panel D: Samples were also taken for measurement of the loss of Procaspase 9 as one marker of apoptosis and the arrow shows that these changes were not detectable until the 4 hour time point. Panel E: Reserve Capacity decreases dramatically after 4 hours exposure at the concentration which is the threshold for toxicity.

Figure 4. Autophagy and the response to oxidative stress

Redox, mitochondrial and proteotoxic stress damage cellular proteins and organelles and if not cleared by autophagy, cell death occurs and contributes to metabolic and cardiovascular pathologies. Upregulation of autophagy has been shown to provide beneficial effects on cell survival.

Figure 5. Mitophagy mechanisms have been shown to involve PINK1-PARKIN, DRP1, and MFN1/2 mediated mechanisms

Mitophagy plays an important role in mitochondrial quality control and the involvement of PARKIN-PINK1, as well as fission/fusion proteins has been demonstrated. PINK1 stabilization in the mitochondria is facilitated by mitochondrial membrane depolarization. PINK1 phosphorylates ubiquitin and enables PARKIN translocation to the mitochondria, leading to ubiquitination of several mitochondrial proteins, resulting in p62 and LC3 recruitment and autophagosomal engulfment of the mitochondrion. Interaction between MFN2 and PARKIN may play a key role in integrating fusion machinery and PARKIN mediated mitophagy.

Figure 6. Autophagy is regulated by epigenetics, protein O-GlcNAcylation, circadian clock, and cellular redox status

Histone deacetylases have been shown to participate in autophagy regulation although whether their activities in the nucleus or the cytosol are important for autophagy regulation is still being investigated. HDAC6 recruits an actin-remodeling machinery, and stimulates autophagosome-lysosome fusion and substrate degradation. P62 interaction with HDAC6 regulates its activity. Inhibition or disruption of HDAC1 leads to the conversion of LC3-I to LC3-II. Methylation of ATG16L2, ULK2, LC3A, BNIP3, and GABARAPL1 is associated with their downregulation. In addition, transcription regulation of p62 by antioxidant transcription factor Nrf2, and regulation of ATG14, ULK1, BNIP3, GABARAPL1, and LC3 by clock and BMAL1 have been demonstrated. Post-translational regulation of SNAP29 by O-GlcNAcylation has been shown to attenuate autophagosome-lysosome fusion.

Figure 7. Interactions between Redox Signaling and Metabolic Networks

In this review we have described the interactions between redox dependent pathways encompassing the signaling node controlled by the GlcNAc pathway, regulation by biological clocks, mitochondrial metabolism and epigenetics.