Fatty Acid Desaturation Gets a NAD⁺ Reputation

Abstract

delta-5 desaturase and delta-6 desaturase are enzymes known to be involved in the synthesis of highly unsaturated fatty acids. In this issue, Kim et al. (2019) show that production of NAD⁺ by this desaturase reaction is an adaptive response to NAD⁺ depletion that may regulate cellular REDOX status.

Nicotinamide adenine dinucleotide (NAD⁺) is a ubiquitous cofactor used to transfer electrons among cellular reactions. Myriad metabolic reactions require NAD⁺, most notably those involved in energy metabolism. The reduction of NAD⁺ to NADH is necessary for transferring energy in nutrients to the electron transport chain (ETC) to produce the electrochemical gradient that drives ATP synthesis. Recent interest in NAD⁺ biology has been piqued by data on the beneficial effects of increasing cellular NAD⁺ concentrations. NAD⁺ can be synthesized de novo from amino acids or produced by a salvage pathway from vitamin precursors, including nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). Supplementation of NR and NMN increased lifespan in experimental models and also has shown promise as a treatment for metabolic disease (Yoshino et al., 2018). In addition to serving as a cofactor for electron transfer, NAD⁺ is also a regulator of signaling cascades that affect metabolism. It is believed that many beneficial effects are mediated by activation of the sirtuin family of deacetylases (Yoshino et al., 2011) that control gene expression and metabolic flux. Whereas NAD⁺ biosynthetic pathways may be therapeutically important, constant interconversion of NAD⁺ and NADH in metabolic pathways by oxidoreductase enzymes constitutes the overwhelming flux of NAD metabolism.

In order to energetically favor the reduction of NAD⁺ to NADH in metabolism, the cell normally maintains a high ratio of NAD⁺ to NADH (the REDOX state). Sustaining an appropriate REDOX state is critical to preserving cellular homeostasis. Since REDOX reactions are often in equilibrium, the relative concentrations of NAD⁺ and NADH are tightly linked to the relative concentrations of the related reduced and oxidized metabolites (Williamson et al., 1967). For example, the lactate dehydrogenase reaction (pyruvate + NADH → lactate + NAD⁺) links the lactate:pyruvate ratio to cytosolic REDOX state (i.e., [lactate]/[pyruvate] α [NAD⁺]/[NADH]). This system buffers cytosolic REDOX state and is essential for maintaining glycolytic NAD⁺ requirements during anaerobic conditions. The ETC normally generates the majority of NAD⁺ as part of ATP synthesis, but in the absence of oxygen, or other factors that limit mitochondrial function, NAD⁺ is maintained by lactate production. As long as lactate can be exported from the cell to maintain a reasonable lactate: pyruvate ratio, a sufficiently oxidized cytosolic REDOX state can sustain glycolysis. It is important to note that an expansive network of similar dehydrogenase reactions are linked by REDOX state.

In this issue of Cell Metabolism, Kim and colleagues (Kim et al., 2019) suggest that the reaction catalyzed to desaturate fatty acids is a possible mechanism for generating oxidized NAD⁺ in the cytosol in the absence of an active ETC (Figure 1). When mitochondrial ETC activity was compromised by rotenone or other mitochondrial inhibitors, the abundance of NAD⁺ decreased and cells increased the synthesis of highly unsaturated fatty acids (HUFAs) including arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. HUFAs are bioactive lipids enriched in fish oils and are believed to have a number of effects on cardiometabolic health (Li et al., 2018). HUFAs are synthesized from linoleic acid by a series of reactions to elongate and introduce double bonds along the carbon backbone in reactions catalyzed by delta-5/6 desaturase (D5D/D6D) enzymes (Cho et al., 1999a, 1999b). Among desaturase enzymes, this family is unique in having an NADH cytochrome b5 reductase domain to introduce a double bond in the carbon backbone of the fatty acid by utilizing NADH and producing NAD⁺. This led the authors to hypothesize that the activation of these desaturases was a compensatory response to produce NAD⁺. Indeed, other approaches that reduced cytosolic NAD⁺ content also stimulated HUFA synthesis, whereas approaches that increased NAD⁺ production blocked the effects of mitochondrial inhibition on HUFA synthesis. Transient overexpression of the genes encoding D5D and D6D also modestly increased the cellular NAD⁺/NADH ratio, while transient knockdown or pharmacologic inhibition had a reciprocal effect. Though modest in magnitude, these effects are consistent with the idea that generation of HUFA by these enzymes can affect the cellular REDOX state as a feedback mechanism in response to diminished NAD⁺ abundance.

Figure 1. Desaturases in the Regulation of NAD Homeostasis and HUFA Biosynthesis.

A schematic depicting the activation of D5D/D6D enzymes by metabolic stress, the compensatory responses, and the remaining mechanistic questions is shown.

These in vitro studies were also validated in mice where inhibition of complex I led to increased HUFA abundance in kidney and liver. Interestingly, the effects of NAD⁺ depletion on HUFA synthesis were also connected to a haplotype near the gene encoding a plasma membrane monocarboxylate transporter (SLC16A11) that is associated with increased risk of diabetes (Williams et al., 2014). SLC16A11 is one of several monocarboxylate transporters that import pyruvate and lactate (and likely other solutes) into the cell in a proton-coupled mechanism, and the disease haplotype is associated with multiple coding variants that reduce transporter activity (Rusu et al., 2017). In the present study, transient expression of the human SLC16A11 variant with reduced transport activity lowered the NAD⁺/NADH ratio and increased intracellular HUFA TAGs in kidney cells. Furthermore, in people harboring the SLC16A11 susceptibility haplotype in the Mexico City Diabetes Study, the proportion of plasma TAG containing HUFA was increased. Whether this observation plays a role in the disease phenotype associated with this genotype, or whether it’s a compensatory adaptive response, is not clear.

The work by Kim et al. provides valuable insight into regulatory mechanisms that control the REDOX state and link them to the generation of HUFAs. However, several important questions remain regarding the mechanism by which D5D/D6D enzymes are activated by energetic stress (Figure 1) as well as the functional implications of these observations. As noted by the authors, the contribution of this reaction to the total cellular NAD⁺ pool is probably very minor. Compared to D5D/D6D, the production of NAD⁺ from lactate dehydrogenase activity is conservatively 20 times higher than from D5D/D6D on a molar basis in most tissues. It’s not clear why HUFA synthesis is activated, rather than further increasing lactate synthesis to augment NAD⁺ production. Indeed, there are more than 200 known enzymes that catalyze the conversion of NAD⁺ to NADH; why is the HUFA synthesis pathway important? Could the HUFAs produced by this reaction also be a component of the adaptive response? Is the location or compartmentalization of NAD⁺ production at the endoplasmic reticulum important to the mechanism? Although several experimental paradigms were employed to modulate NAD⁺ levels, many were extreme interventions, which demonstrate the connectivity of the system, but also make it unclear whether the effects of D5D/D6D on NAD⁺ concentrations are broadly relevant in vivo. Genetic variations in the chromosomal region encoding the D5D/D6D enzymes have been linked to risk of diabetes (Dupuis et al., 2010). Do the effects of NAD⁺ synthesis contribute to the risk of diabetes, and are there additive effects if the disease-associated SLC16A11 haplotype is also present? Lastly, could targeting this pathway for activation be a mechanism to increase NAD⁺ levels and produce beneficial effects seen with NR and NMN supplementation?

While the numerous questions posed above remain to be answered, the present study identified a provocative potential role for HUFA synthesis in regulating REDOX status that could open the door to new lines of research.