Resveratrol!

Caloric restriction (CR) has been of interest for some time due to its apparent slowing of the aging process. In addition to conferring enhanced longevity, it is known to correlate with the alleviation of a number of biomarkers associated with aging, as well as lowering age-associated disease incidences. In spite of this, its mechanism remains poorly characterized. Resveratrol, a polyphenol with antioxidant and antitumorigenic activity, is intriguing because it seems to mimic many of the affects of CR, and has been shown to provide a variety of palliative and neuroprotective effects in several model organisms. In this work¹, the authors show that one function of resveratrol is to stimulate AMP kinase (AMPK) activity in neurons. This is a significant step towards characterizing resveratrol's mechanism, which, in turn, may help provide a better understanding of how CR works.

The authors show that in mouse Neuro2a neuroblastoma as well as mouse dorsal root ganglia (DRG) sensory and cortical neurons, resveratrol correlates with an increase in both phosphorylated AMP and a downstream target of activated AMPK, phosphorylated acetyl-CoA carboxylase (ACC). These increases are comparable to those generated by a well-characterized AMPK activator, AICAR. This AMPK activity is not stimulated indirectly by decreased cellular energy levels (measured by the relative AMP:ATP ratio). Next, they show that resveratrol-mediated AMPK activation halts Neuro2a cell proliferation and induces an increase in neurite outgrowth. This is again similar to the AICAR-mediated AMPK activation effects, and is consistent with AMPK's activity in other cell types. AMPK activation is required and sufficient for this resveratrol-induced neurite growth. AMPK activation is also correlated with mitochondrial biogenesis, as measured by increases in mRNA levels of three markers for mitochondrial biogenesis.

In Neuro2a cells, resveratrol’s activation of AMPK is unaffected by the presence of inhibitors for SIRT1 (associated with stress response and cell cycle regulation, and with polyphenol activities) or CaMKKβ (a kinase upstream of AMPK). This suggested that another activating kinase upstream of AMPK, LKB1, is likely to effect AMPK activation. To test this, DRG sensory and cortical neurons from mouse embryos containing loxP sites flanking the Lkb1 exon were infected with lentivirus expressing a Cre recombinase capable of conditionally knocking out Lkb1 from this engineered site. LKB1 removal reduced AMPK and ACC activation in both cell types. Interestingly, while introduction of a CaMKKβ inhibitor did not affect DRG sensory neurons, it inhibited AMPK and ACC activation in cortical neurons. Finally, the authors demonstrated that directly injecting mice with resveratrol within two hours led to increased levels of AMPK and ACC phosphorylation in the brain.

A major question raised by this work is the difference in resveratrol-mediated AMPK activation in Neuro2a and DRG sensory cells versus activation in cortical neurons. LKB1, which appears to regulate AMPK activation in both cell types, is part of the three-protein AMPK kinase complex, and is known to regulate AMPK response to decreased cellular energy levels (AMP buildup).² As shown in this work, resveratrol-induced AMPK activation is independent from LKB1’s response to cellular AMP levels. CaMKKβ appears to control AMPK response to Ca²⁺ levels in the cell, which raises the question of whether this is also the mechanism by which resveratrol induces AMPK activation in cortical neurons. This could be tested by examining CaMKKβ’s resveratrol-effected regulation of AMPK in varying concentrations of Ca²⁺.

Is there a physical interaction between resveratrol and either of upstream regulators of AMPK examined in this work? One way to test this would be through fluorescence resonance energy transfer (FRET). Attaching a fluorescent protein to LKB1 or CaMKKβ and a small fluorophore, such as Cy3 or Cy5, to resveratrol would result in emission at the small fluorophore peak if resveratrol is in close proximity to the regulatory kinase. Although the specificity of this technique, and the fact that it can be used in vivo, make it well-suited for this, a potential problem is that, due to resveratrol’s small size, attachment of a small fluorophore might alter its interactions significantly. Another possibility would be to label resveratrol, which has three hydroxyl groups, with a radioactive oxygen isotope, then use anti-LKB1 antibodies to immunoprecipitate the complex out of solution. If resveratrol is bound to the complex, Western blotting followed by film exposure of the blot would detect radioactive signal.

Another logical follow-up to this study would be to examine whether resveratrol and CR complement each other. Since resveratrol is predicted to function as a CR mimetic, the expectation is that introducing resveratrol into a system affected by CR would have no effect. There are several assays that could be done to gauge this by comparing CR mice with and without resveratrol to ad libitum fed mice with and without resveratrol. Since CR functions to increase life-span, the most straightforward measurement is mortality. Another, potentially more informative, assay is a comparison between AMPK and ACC activation levels between these four groups. In light of the differences in regulation of AMPK in different mouse neurons, it would make sense to do these assays for a variety of cell types, as it is possible that the CR and resveratrol pathways overlap for some cell types, but not others.

References:

1. Dasgupta B, Milbrandt J. Resveratrol stimulates AMP kinase activity in neurons. Proc. Natl. Acad. Sci. U.S.A. 104: 7217-22 (2007).
2. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, DePinho RA, Montminy M, Cantley LC. The Kinase LKB1 Mediates Glucose Homeostasis in Liver and Therapeutic Effects of Metformin. Science 310: 1642-46 (2005).