NAD+ centric mechanisms and molecular determinants of skeletal muscle disease and aging

Abstract

The nicotinamide adenine dinucleotide (NAD⁺) is an essential redox cofactor, involved in various physiological and molecular processes, including energy metabolism, epigenetics, aging, and metabolic diseases. NAD⁺ repletion ameliorates muscular dystrophy and improves the mitochondrial and muscle stem cell function and thereby increase lifespan in mice. Accordingly, NAD⁺ is considered as an anti-oxidant and anti-aging molecule. NAD⁺ plays a central role in energy metabolism and the energy produced is used for movements, thermoregulation, and defense against foreign bodies. The dietary precursors of NAD⁺ synthesis is targeted to improve NAD⁺ biosynthesis; however, studies have revealed conflicting results regarding skeletal muscle-specific effects. Recent advances in the activation of nicotinamide phosphoribosyltransferase in the NAD⁺ salvage pathway and supplementation of NAD⁺ precursors have led to beneficial effects in skeletal muscle pathophysiology and function during aging and associated metabolic diseases. NAD⁺ is also involved in the epigenetic regulation and post-translational modifications of proteins that are involved in various cellular processes to maintain tissue homeostasis. This review provides detailed insights into the roles of NAD⁺ along with molecular mechanisms during aging and disease conditions, such as the impacts of age-related NAD⁺ deficiencies on NAD⁺-dependent enzymes, including poly (ADP-ribose) polymerase (PARPs), CD38, and sirtuins within skeletal muscle, and the most recent studies on the potential of nutritional supplementation and distinct modes of exercise to replenish the NAD⁺ pool.

Keywords: Aging; Diabetes; Epigenetics; Muscle diseases; NAD+; Redox.

Figure 1.. The NAD⁺ de novo and salvage pathways.

(A) NAD⁺ is utilized by CD38 by hydrolyzing NAD⁺ to NAM. SIRTs and PARPs use NAD⁺ as a co-substrate for deacetylation and PARYlation, respectively. This process generates NAM as a byproduct and inhibits the activity of NAD-dependent enzymes; (B) NAM is salvaged to regenerate NAD⁺. NAMPT catalyzes NAM into NMN, which allows to regenerate NAD⁺ by NMNAT1-3. NR is salvaged into NMN by NKR1/2 and regenerate NAD⁺ [23, 26, 27]. (C) Chemical structures of the NAD⁺ precursors; tryptophan, nicotinamide, nicotinic acid, nicotinamide riboside, nicotinamide mononucleotide, and the Nampt enzymatic activator P7C3 compound.

Figure 2.. NAD⁺/NADH in skeletal muscle glycolysis.

(A) Glucose enters cells using GLUT 4 transporters. In the cytosol, glucose is further converted and processed by enzymes: hexokinase, phosphoglucose isomerase, phosphofructokinase, and aldolase in step-wise manner and converted into G3P. At this juncture, NAD⁺ is utilized by GAPDH to convert it into 1,3-BP (1,3-Bisphosphoglyceric acid). 1,3-BP is converted into pyruvate via four additional steps, each step involving the action of a unique enzyme. Pyruvate can then be reduced to form lactate by LDH (lactate dehydrogenase). LDH uses NADH and regenerates NAD⁺. Lower NAD⁺ levels that are accompanied with aging affect the production of 1,3-BP (1,3-bisphosphoglycerate), and consequently the production of pyruvate as an energy source to be used in the TCA cycle [64, 65]; (B) Cytosolic pyruvate enters the mitochondria where it is oxidized into acetyl-CoA by PDH (pyruvate dehydrogenase). PDH reduces NAD⁺ into NADH. Acetyl-CoA enters the TCA cycle and is converted into citrate. Citrate is converted into isocitrate by the enzyme aconitase. Isocitrate is oxidized by isocitrate dehydrogenase (IDH), reducing NAD⁺ into NADH and forming α-ketoglutorate. During this process NAD⁺ is reduced into its NADH form, by α-ketoglutorate dehydrogenase (α-KGDH), which converts α-ketoglutorate into succinyl-CoA. As shown in Figure 2, Succinyl-CoA then forms malate by enzymes succinyl-CoA synthetase, succinic dehydrogenase and fumarate. NAD⁺ is reduced to NADH to convert malate into oxaloacetate by malate dehydrogenase 2 (MDH2). Cytosolic oxaloacetate is then converted into malate by the enzyme malate dehydrogenase 1 (MDH1), which oxidizes NADH to NAD⁺. Cytosolic malate enters the mitochondria through the malate α-ketoglutorate antiporter and is used in the TCA cycle. Cytosolic DHAP (Dihydroxyacetone phosphate) is then converted into G3P by cytosolic GPDH, which oxidizes NADH to NAD⁺. G3P then enters the mitochondria and is converted into DHAP. This shuttling of G3P into the mitochondria is referred to as the G3P shuttle. Lower levels of NAD⁺ inhibit acetyl-CoA formation from pyruvate, α-ketoglutorate from isocitrate, succinyl-CoA from α-ketoglutorate, and oxaloacetate from malate. The inhibition of these steps prevents the formation of NADH and consequently effects redox reactions in the electron transport chain. Redox reactions are necessary to produce the proton gradient that is used to phosphorylate ADP (Adenosine diphosphate) into ATP and generate the energy that is required for the proper functioning of skeletal muscle [66, 68-70].

Figure 3.. Skeletal muscle inflammation results in decreased Nampt and NAD⁺ biosynthesis with increased DNA damage and altered mitochondrial biogenesis.

DMD is linked with a reduction in NAMPT activity and subsequent NAD⁺ biosynthesis. The DNA damage present in patients with DMD leads to heightened PARP activity. PARPs and SIRT1 both compete for NAD⁺, leading to a decrease in SIRT1 activity due to increased PARP activation. The reduction in SIRT1 activity results in decreased mitochondrial biogenesis, due to inhibition of the SIRT1/PGC-1α pathway. Inflammation of muscle and the consequent production of inflammatory cytokines in muscle diseases leads to reductions in NAMPT. Similarly, the inflammation associated with aging, also known as “inflamm-aging” leads to further reductions in NAMPT as more inflammatory cytokines are seen to circulate in the skeletal muscle. NAMPT regulates NAD⁺ levels and downstream activity of PARPs and sirtuins [166].

Figure 4.. The cyclic effects of inflammation and insulin resistance in skeletal muscle involving NAD⁺ biosynthesis.

Skeletal muscle inflammation results in the production of inflammatory cytokines. Inflammatory cytokines can lead to a reduction in NAD⁺ synthesis by downregulating NAD⁺ salvage enzymes. A reduction of NAD⁺ biosynthesis results in impaired sirtuin signaling and function, which is involved in insulin resistance. Insulin resistance has inflammatory effects that creates a cyclic effect, promoting further insulin resistance [90].

Figure 5.. Epigenetic regulation and metabolic links to NAD.

(A) The NAD-dependent epigenetic modifications of the DNA. The heterochromatin is associated with histones deacetylation and lower levels of gene transcription or gene silencing. (B) RNA modifications involved in mRNA splicing, translation, stability, and transport. The RNA methylases (Mettle3, and Mettle14 interact with WTAP), and demethylases (FTO and ALKBH5) are involved in the most commonly observed m⁶A mRNA modifications.