Sirtuin Activation: A Role for Plasma Membrane in the Cell Growth Puzzle

Abstract

For more than 20 years, the observation that impermeable oxidants can stimulate cell growth has not been satisfactorily explained. The discovery of sirtuins provides a logical answer to the puzzle. The NADH-dependent transplasma membrane electron transport system, which is stimulated by growth factors and interventions such as calorie restriction, can transfer electrons to external acceptors and protect against stress-induced apoptosis. We hypothesize that the activation of plasma membrane electron transport contributes to the cytosolic NAD⁺ pool required for sirtuin to activate transcription factors necessary for cell growth and survival.

THE puzzle is “why do so many oxidants and growth factors that stimulate transplasma membrane electron transport also stimulate cell growth?” The answer may lie in the activation of sirtuins that in turn activates functions involved in proliferation and prevention of apoptosis (1). NAD⁺ is a co-substrate that activates sirtuins leading to control metabolic homeostasis (2), and it becomes relevant that the compartmentalization of NAD⁺ inside the cell affect the variety of coordinated functions of NAD⁺-consuming enzymes. NAD⁺ levels are maintained by the cooperation between biosynthesis and consuming enzymes plus redox homeostasis by the bioenergetics pathways such as glycolysis and respiration (3). Up to now, the contribution of the transplasma membrane electron transport system to the bulk of NAD⁺ bioavailability has not been considered but evidence has accumulated to include this system in the NAD⁺-dependent cytosolic sirtuin activation (4).

Growth of cells is generally supported by supplementation with serum, which in turn contains agents such as insulin and transferrin (5,6). It is classically known that impermeable oxidants stimulate serum-free growth of cells in culture including ferricyanide (7), hexamine ruthenium (8), ascorbate free radical (9), dichloroindophenol (10), and tetrazolium WST-1 (11). Ferric transferrin alone can replace serum for the growth of many cells and stimulates the transplasma membrane electron transport system (12,13). The stimulation of growth by transferrin is thought to depend on iron supply but ferric lactoferrin, which does not contribute to iron uptake, also stimulated cell growth (14). The opposite has also been demonstrated. For example, inhibitors of the transplasma membrane electron transport system such as rhein and adriamycin inhibit both this system and cell growth (15). Adriamycin (doxorubicin) increased cell death in mouse embryonic fibroblasts and cardiomyocytes by SIRT1 dysfunction mediated by AMP-activated protein kinase inhibition (16).

The role of plasma membrane on growth control is particularly relevant in ρ° cells in which the maintenance of NAD⁺/NADH ratio is not dependent on respiratory chain. The transplasma membrane electron transport system is highly activated in these cells (17,18). ρ° cells require the addition of pyruvate and other oxidants to growth media that are reduced by the plasma membrane electron transport contributing to maintain both growth and cytosolic NAD⁺ availability (10). It has been just demonstrated that pyruvate and other treatments that increase NAD⁺ stimulate sirtuins in different cellular models (19). NAD⁺ homeostasis in ρ° cells is required for sirtuin activation because these cells maintain sirtuin levels as wild type (20). Also, it has been classically demonstrated that the plasma membrane diaphorase, currently NQO1-encoded enzyme, is induced and activated during the G1 and G2/M-phases (21). Furthermore, the inhibition of either respiratory chain or malate–aspartate shuttle enhanced plasma membrane electron transport contributing to the metabolic pathway in pancreatic β cells (22).

The transplasma membrane electron transport system integrates inner cytochrome b ₅ reductase that uses NADH as electron donor and NQO1-encoded enzyme that uses NAD(P)H as electron donor, reduces coenzyme Q intercalated in the membrane lipids, and maintains reduced membrane-bound antioxidants such as α-tocopherol and other soluble antioxidants such as ascorbate (Figure 1). The activation of this system increases the oxidation of NADH and contributes to raise the NAD⁺ concentration in the cytosol, contributing to the activation of NAD⁺-consuming enzymes such as sirtuins that are relevant to healthy age (23,24).

Figure 1.

Diagram that shows the possible arrangement of the components of the transplasma membrane electron transport system and the transferrin receptor. Generation of NAD⁺ by this transmembrane system would contribute to the activation of sirtuin in the cytosol.

The regulation of sirtuin activity by the cofactor NAD⁺ has been clearly demonstrated in interventions that improve aging such as calorie restriction (25). This intervention increases transplasma membrane electron transport system components such as cytochrome b ₅ reductase and NQO1, and also the antioxidant protection of membrane components, contributing to the increase of cytosolic NAD⁺ (26,27). Yeast NQR1 encodes for a homolog protein of the mammalian cytochrome b ₅ reductase that is also located at the plasma membrane (28). This enzyme is induced in low glucose media and adds to NAD⁺ homeostasis with increased respiration. As a whole, the transplasma membrane electron transport is supported by Nqr1p to improve replicative life span but only in the presence of Sir2 (28). There is a clear correlation of sirtuins transcriptional and enzyme activations by nuclear factor E2–related factor-2 (Nrf2) transcription factor and the improvement of both cellular life span and survival (29,30). Furthermore, pharmacological activation by β-lapachone of NADH oxidation by NQO1 resolves obesity and induces respiration (31).

The overall hypothesis is that growth of cells is closely related to control of the transplasma membrane electron transport system, which can maintain a high level of NAD⁺ in the cytosol, where it can activate vital transcription factors by supplying sirtuins with NAD⁺. In this way, NAD⁺ becomes a second messenger for sirtuin activation. This system is then a piece of a puzzle that can be now put together with NAD⁺ producer and consumers enzymes and bioenergetics mechanisms contributing to NAD⁺ homeostasis.

Funding

We would like to acknowledge financial support from Spanish Ministerio de Sanidad (FIS; grant PI11/00078), National Institutes of Health (NIH; grant 1R01AG028125-01A1), and the Intramural Research Program of the NIA/NIH.