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Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration ([O2]) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous [O2] within the intracellular environments. The mitochondrial [O2] at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria.

Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity.

The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK), an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE) in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.