How quickly a year flys by. I was hoping to have this post done before the first month of a year has passed! 2013 was a noteworth year with the publishing of my first big post on the topic of Redox and GSH ( and part 2) which was also accepted onto here.

My fellow authors there recently posted up a recap of their new insights over the last year at:

Having gained a decent understanding of the GSH piece of the puzzle, I’ve started to look further into redox and the interactions with other the couples (NAD+/NADH, NADP/NADPH) and how they interact with other key areas of interest, ATP, Caloric Restriction(CR), AMPK and SIRTs.

One thing which was confusing originally is that NAD/NADH ratio is predominately NAD+ as an energy acceptor/oxidiser and maintained ‘opposite’ to the GSH/GSSG and NADP/NADPH couples which provide reducing power. This required brushing up on some basic biochem to make sense of it. The following exceprt from Molecular Biology of the Cell. 4th edition. was enlightening.

Like ATP, NADPH is an activated carrier that participates in many important biosynthetic reactions that would otherwise be energetically unfavorable.

The difference of a single phosphate group has no effect on the electron-transfer properties of NADPH compared with NADH, but it is crucial for their distinctive roles. The extra phosphate group on NADPH is far from the region involved in electron transfer and is of no importance to the transfer reaction. It does, however, give a molecule of NADPH a slightly different shape from that of NADH, and so NADPH and NADH bind as substrates to different sets of enzymes. Thus the two types of carriers are used to transfer electrons (or hydride ions) between different sets of molecules.

Why should there be this division of labor? The answer lies in the need to regulate two sets of electron-transfer reactions independently. NADPH operates chiefly with enzymes that catalyze anabolic reactions, supplying the high-energy electrons needed to synthesize energy-rich biological molecules. NADH, by contrast, has a special role as an intermediate in the catabolic system of reactions that generate ATP through the oxidation of food molecules, as we will discuss shortly. The genesis of NADH from NAD+ and that of NADPH from NADP+ occur by different pathways and are independently regulated, so that the cell can independently adjust the supply of electrons for these two contrasting purposes. Inside the cell the ratio of NAD+ to NADH is kept high, whereas the ratio of NADP+ to NADPH is kept low. This provides plenty of NAD+ to act as an oxidizing agent and plenty of NADPH to act as a reducing agent—as required for their special roles in catabolism and anabolism, respectively.

A big story in late 2013 was David Sinclair and colleagues from both Harvard and Australia publication showing that supplementation with an NAD+ precursor could reverse this mitochondrial dysfunction and “Warburg-like” metabolic state in mice in just one week.

Keeping that in mind, one of the key references (The Redox Stress Hypothesis of Aging Free Radic Biol Med. 2012) in my first post stated:

Furthermore, transgenic studies have shown that augmentation of reducing power, provided by NADPH and GSH, is the most effective currently known experimental manipulation for the prolongation of lifespan.
Given Sinclair’s results I would be be interested to see the results of supplementing both with a GSH precursor and NAD+ precursor(s), ensuring sufficient levels of both oxidising and reducing activated carrier molecules. 2013 also marked the introduction of a new commercial Nicotinamide riboside product called Niagen which also acts as a NAD precursor. This could be an interesting one to stack with the newer GSH/Cysteine pro-drugs available such as Ribose-Cysteine.

Looking back at an article I had previously referenced indicates this to be true. Dual-energy precursor and nuclear erythroid-related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species. Neurobiol Aging. 2014 Jan

By combining the Nrf2 activator together with the NADH[/NAD+] precursor, nicotinamide, we increased neuron survival against amyloid beta stress in an additive manner.
The alcohol dehydrogenase and aldehyde dehydrogenase enzymes both require NAD+ for the two catabolic steps to break down ethanol. Perhaps some Nicotinamide Riboside wouldn’t go astray after enjoying a few alcoholic beverages. I’ll be personally testing this hypothesis this year.


While NAD+ has been in the limelight again recently, I’ve been piecing together some articles looking at NAPDH, which is critical for maintaining redox homoeostasis as it is required to recycle GSSG back to 2GSH.

This following article was particularly interesting. While it is in regards to tumor cell survial, the energy stress (caloric restriction?) situation seems applicable for normal cells too.

AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress (Nature. 2012 May)

The underlying mechanisms of cell death and survival under metabolic stress are not well understood. A key signalling pathway involved in metabolic adaptation is the liver kinase B1 (LKB1)–AMP-activated protein kinase (AMPK) pathway2,3. Energy stress conditions that decrease intracellular ATP levels below a certain level promote AMPK activation by LKB1. Previous studies showed that LKB1-deficient or AMPK-deficient cells are resistant to oncogenic transformation and tumorigenesis4–6, possibly because of the function of AMPK in metabolic adaptation. However, the mechanisms by which AMPK promotes metabolic adaptation in tumour cells are not fully understood. Here we show that AMPK activation, during energy stress, prolongs cell survival by redox regulation. Under these conditions, NADPH generation by the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. The inhibition of the acetyl-CoA carboxylases ACC1 and ACC2 by AMPK maintains NADPH levels by decreasing NADPH consumption in fatty-acid synthesis and increasing NADPH generation by means of fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas the activation of ACC1 or ACC2 attenuates these processes. Thus AMPK, in addition to its function in ATP homeostasis, has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo.
Briefly back to NAD+, AMPK also increase the NAD+/NADH ratio, a nice double whammy, by increasing mitochondrial ß-oxidation. From AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity Nature 2009.
To determine how AMPK acutely increases the NAD+/NADH ratio, we pharmacologically targeted different possible sources of cellular NAD+ production. Inhibition of the glycolytic enzyme lactate dehydrogenase with oxamic acid did not affect the ability of AICAR to increase NAD+ levels and the NAD+/NADH ratio. In contrast, inhibition of mitochondrial fatty acid oxidation with etomoxir was enough to hamper the increase in NAD+/NADH induced by AMPK, indicating that an increase in mitochondrial ß-oxidation is required for AMPK to increase the NAD+/NADH ratio.

From Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-related Hearing Loss under Caloric Restriction Cell. 2010 November

Collectively, these results provide evidence that during CR, Sirt3 induces the deacetylation and activation of Idh2 [isocitrate dehydrogenase], leading to increased levels of NADPH in mitochondria of multiple tissues.

This recent thinking took me back to an article I referenced which stood out in my mind when doing my previous redox/GSH research.

Time line of redox events in aging postmitotic cells eLife. 2013;

We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death.

Thioredoxin reductase: an early oxidation target in yeast

Oxidation of at least 28 proteins significantly preceded the general oxidation of proteins under standard or caloric restriction conditions (Figure 3, clusters D and E and Table 1). Of these early-oxidized proteins, 20 had oxidation states of more than 45% at day 2 of cultivation, which was 1.5- to 3.8-fold higher than their oxidation status during exponential growth. One of these early oxidation targets is the highly conserved enzyme thioredoxin reductase, the key component of the thioredoxin system. Although we cannot exclude that oxidation of any one of the other early oxidation targets directly or indirectly affects or even controls S. cerevisiae lifespan, we decided to focus our subsequent studies on thioredoxin reductase, as this enzyme is the central player in maintaining cellular redox homeostasis. Loss of thioredoxin reductase activity has been shown to cause widespread protein oxidation.

This is one example where redox stress disables the enzymes required to maintain redox homoeostasis, further increasing the stress. Is there hope?
It was intriguing to observe that early protein oxidation is, at least in its initial stage, a fully reversible event in yeast. Moreover, we found that more than 80% of viable cells were recovered from day 3- and day 4-old cultures despite an almost fully oxidized thiol proteome.

More on Redox Stress

One article published at the start of this year was on SirT1 mutants which replaced oxidation-sensitive cysteine with serine to stop inactivation by redox stress.

This appears to be another one of many pathways in the viscous cycle of aging, as increased redox stress inactives SIRT1 (possibly by S-glutathiolation of a SirT1 cysteine residue decreasing the binding affinity of NAD+). In the second two quoted sections also note that Glutaredoxin-1 (Glrx) uses glutathione as a co-factor.

A redox-resistant sirtuin-1 mutant protects against hepatic metabolic and oxidant stress J Biol Chem. 2014 Jan.

We show in SirT1 overexpressing HepG2 cells that oxidants (nitrosocysteine or hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivate SirT1 by reversible oxidative post-translational modifications (OPTM) on cysteines.

To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 (Glrx) was overexpressed to remove this modification. Glrx overexpression maintains endogenous SirT1 activity and prevents proapoptotic signaling in metabolically stressed HepG2 cells.

By demonstrating that SIRT1 can be regulated by S-glutathiolation of specific Cys residues, our results have uncovered the potential for SIRT1 to be directly regulated by oxidative modifications that may link its activity to GSH redox and production of cellular reactive oxygen and nitrogen species. These novel findings also indicate that the GSH redox state may affect the response of SIRT1 to polyphenols. These results imply that antioxidants might significantly potentiate the response to small molecule activators of SIRT1.


Further reading AMPK and SIRT1: a long-standing partnership? Am J Physiol Endocrinol Metab. 2010 April;