InteractiveFly: GeneBrief

NAD kinase 1a: Biological Overview | References

Gene name - NAD kinase 1a

Synonyms - CG6145, NAD kinase

Cytological map position - 50B1-50B1

Function - enzyme

Keywords - sustains lipogenesis by maintaining the pool of NADPH - NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals -NADK and fatty acid synthase 1 regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids.

Symbol - Nadk1a

FlyBase ID: FBgn0033853

Genetic map position - chr2R:13,537,371-13,540,797

Classification - NAD kinase, DAGK_cat: Diacylglycerol kinase catalytic domain

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Nadk1a orthologs: Biolitmine

Lipid storage in fat tissue is important for energy homeostasis and cellular functions. Through RNAi screening in Drosophila fat body, this study found that knockdown of a Drosophila NAD kinase (NADK), which phosphorylates NAD to synthesize NADP de novo, causes lipid storage defects. NADK sustains lipogenesis by maintaining the pool of NADPH. Promoting NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals. Furthermore, NADK and fatty acid synthase 1 (FASN1) regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids. Reducing the level of acetyl-CoA or increasing the synthesis of cardiolipin (CL), a mitochondrion-specific phospholipid, partially rescues the mitochondrial defects of NADK RNAi. Therefore, NADK- and FASN1-mediated fatty acid synthesis coordinates lipid storage and mitochondrial function (Xu, 2021).

Lipid homeostasis is important for human health, and its dysregulation is tightly associated with many metabolic diseases, such as type 2 diabetes, hepatic steatosis, cardiovascular disease, and cancer. Cellular lipid homeostasis is regulated by the opposing actions of lipid accumulation, including lipid uptake, de novo lipogenesis and lipid storage, and lipid mobilization, such as lipolysis, lipid oxidation, and lipid efflux. Excess lipid storage or insufficient lipid storage causes obesity or lipodystrophy, respectively (Xu, 2021).

Acetyl-CoA carboxylase (ACC) and FASN mediate fatty acid synthesis from acetyl-CoA during de novo lipogenesis. The fatty acids are then esterified for storage as neutral lipids such as triglycerides (TAGs). The lipid droplet, an organelle with a neutral lipid core and a phospholipid monolayer, is the hub for lipid storage. Understanding of the regulation of lipid storage and lipid droplet dynamics has significantly advanced in recent years. Many processes, including neutral lipid synthesis and degradation, composition of phospholipids, lipid droplet biogenesis and fusion, calcium homeostasis, and lipophagy, together determine lipid storage. Nevertheless, the mechanisms regulating lipid storage and lipid droplet dynamics in vivo are not completely clear (Xu, 2021).

To reduce lipid storage, TAG is mobilized through cytosolic lipolysis to release fatty acids, which are subsequently broken down, mainly in mitochondria, into acetyl-CoA units by lipid oxidation. Therefore, defective mitochondria often lead to lipid accumulation. For example, inhibition of β-oxidation in mitochondria causes lipid accumulation in Drosophila brain. Interestingly, besides conducting fatty acid oxidation, mitochondria also provide substrates and energy for de novo fatty acid synthesis. Both the acetyl-CoA and ATP required by fatty acid synthesis are derived from mitochondria. Impairment of mitochondrial function affects lipogenesis and lipid droplet accumulation. Therefore, impairment of mitochondrial function probably has a context-dependent effect on lipid storage (Xu, 2021).

Conversely, dysregulation of lipid storage also affects mitochondrial function. In the heart, cytoplasmic adipose TAG lipase (ATGL), which hydrolyzes TAG from lipid droplets, affects lipid storage and mitochondrial biogenesis and oxidative metabolism. Similarly, in islet β cells, ATGL knockdown impairs mitochondrial respiration and ATP production, and a PPARδ agonist rescues these mitochondrial defects (Xu, 2021).

Mechanistically, ATGL-mediated lipid droplet lipolysis induces the expression of genes involved in mitochondrial oxidation and respiration by activating the master regulators PPARα/PPARγ and PGC-1α. These studies pinpoint a close relationship between the mitochondrion and the lipid droplet, despite the compartmentalized features of lipid storage and lipid breakdown. Several metabolites, including acetyl-CoA and fatty acids, appear to mediate the two-way communication between these two organelles. De novo lipogenesis is tightly associated with acetyl-CoA and fatty acids. However, despite a few reports showing that lipogenesis inhibitors cause various mitochondrial dysfunctions in cancer, the question of whether and how de novo lipogenesis affects mitochondrial function has not been properly addressed (Xu, 2021).

Through an RNAi screen in Drosophila, this study found that CG6145, a cytosolic NAD kinase (NADK), affects lipid storage in fat body by providing NADPH, an essential reductant in lipogenesis. NADK RNAi causes similar de novo lipogenesis defects as FASN1 RNAi. More importantly, both NADK RNAi and FASN1 RNAi larvae exhibit reduced mitochondrial content. Finally, it was revealed that de novo fatty acid synthesis regulates mitochondrial mass, at least partially, by controlling PGC-1α acetylation and cardiolipin (CL) synthesis (Xu, 2021).

This study shows that NADK affects lipid storage and mitochondrial metabolism in Drosophila. NADK is essential for generating NADP and NADPH, the latter of which is important for de novo fatty acid synthesis. Besides lipid storage, NADK-mediated fatty acid synthesis also contributes to mitochondrial function, possibly through two different mechanisms: one is through acetyl-CoA and PGC-1α acetylation, and the other is through synthesis of the mitochondrion-specific phospholipid CL (Xu, 2021).

Despite the obvious requirement for NADPH in de novo fatty acid synthesis and other metabolic reactions, knowledge about the physiological function and impact of NADK on metabolic homeostasis in different organisms and tissues is limited. This study demonstrated the importance of NADK in animal lipid storage in vivo. NADK determines the level of NADP(H). Increasing NADPH availability rescues the defects in NADK RNAi, which confirms that NADPH is a key determinant of lipid storage. In support of this idea, NADPH-producing enzymes, such as G6PD and ME, promote lipid production in oleaginous microbes. The expression and activities of these enzymes are also correlated with lipid storage in mammals. These observations suggest that NADK and the level of NADPH are previously unappreciated regulators of organismal lipid storage. Interestingly, insulin, which promotes the synthesis and storage of lipids, activates NADK by Akt-mediated phosphorylation, which suggests that NADK may respond to physiological conditions to regulate lipid storage (Xu, 2021).

Besides lipogenesis, this stufy found that NADK also influences mitochondrial metabolism. The amounts of mitochondria and lipid droplets are decreased in both NADK RNAi and FASN1 RNAi, raising the possibility that these two closely linked organelles are co-regulated. Mitochondria regulate lipid metabolism by providing energy and substrates for lipogenesis and a site for fatty acid degradation. Lipid droplets, acting as an important organelle of lipid metabolism, also regulate mitochondrial function. Interestingly, elevating lipolysis by ATGL overexpression reduces the amount of lipid droplets, but it increases mitochondrial content, which suggests that reduced lipid storage per se is not the cause of the reduced mitochondrial mass in both NADK RNAi and FASN1 RNAi. Previous studies have shown that ATGL-mediated lipolysis promotes mitochondrial metabolism and biogenesis through activation of PPARs or Sirt1/PGC-1α. NADK RNAi and FASN1 RNAi exert a stronger effect on mitochondrial function than on lipolysis, which might be attributed to the severe decline in the level of fatty acids. Interestingly, PGC-1α acetylation mediates the regulation of mitochondrial function by both lipolysis and lipogenesis. Therefore, de novo fatty acid synthesis regulates the dynamics of both lipid droplets and mitochondria (Xu, 2021).

Fatty-acid-dependent activation of PPARs and Sirt1 is rather specific. The ligands of PPARs are primarily unsaturated and long-chain fatty acids, while Sirt1 is activated by monounsaturated fatty acids within a restricted range of concentrations. This study found that RNAi of the fat-body-specific PGC-1α homolog srl only moderately reduced mitochondrial mass, in contrast to the strong effect of NADK and FASN1 RNAi. In addition, knockdown of PPAR homologs in fat body caused no obvious mitochondrial phenotype. Therefore, it is likely that fatty acids also regulate mitochondrial function through other mechanism(s). In addition, the rescue of NADK RNAi and FASN1 RNAi by different exogenously supplied fatty acids (including saturated, monounsaturated, and odd-chain fatty acids) and by BMM overexpression suggests a general mechanism with limited or low fatty acid selectivity (Xu, 2021).

Phospholipid synthesis, which affects mitochondrial function in many ways, also requires fatty acids. CL is a mitochondrion-specific phospholipid and is important for almost every aspect of mitochondrial integrity, including crista organization, mitochondrial protein import, and assembly. It is a rather unique phospholipid, harboring four fatty acyl chains, and it undergoes remodeling, which makes it sensitive to the availability and composition of fatty acids. Importantly, the rescue of mitochondrial defects in NADK RNAi and FASN1 RNAi by several genetic manipulations to increase CL production suggests that decreased CL synthesis contributes to the mitochondrial phenotype in NADK RNAi and FASN1 RNAi. The mitochondrial morphology in NADK RNAi and FASN1 RNAi is not completely identical with CLS RNAi. In addition, the rescue effect of CLS overexpression is not comparable with fatty acid supplementation. These observations suggest that fatty acids might also regulate mitochondria through other mechanisms (Xu, 2021).

De novo fatty acid synthesis is important for many biological processes. For example, the activity of fatty acid synthesis is stimulated in some cancer cells or proliferating stem cells. Its inhibition suppresses cell proliferation and survival. It is generally thought that fatty acid synthesis mainly affects these processes by providing structural and signaling lipids\, and limited attention has been paid to the causative role of mitochondrial dysfunction, which is also important for cancer progression and stem cell homeostasis. For example, inhibition of PGC-1α or OXPHOS suppresses cancer cell survival and metastasis under oxidative or bioenergetic stress conditions. In addition, mitochondrial mass is associated with prostate cancer progression. Inhibition of mitochondrial biogenesis was identified as a therapeutic strategy for acute myeloid leukemia. Although OXPHOS activity is restricted in many cancer cells, mitochondrial content, dynamics, and metabolic activity are important for tumorigenesis and stem cell homeostasis (Xu, 2021).

Considering the findings of this study, it is possible that fatty acid synthesis-regulated mitochondrial function may be critical for cancer cell growth and stem cell differentiation. For example, fatty acid and lipid synthesis promote hepatocellular carcinoma development, accompanied by increased CL levels and OXPHOS activity. Inhibition of FASN or ACC reduces mitochondrial oxygen consumption, changes mitochondrial morphology, and affects the levels of mitochondrial proteins and metabolites in cancer and stem cells (Xu, 2021).

Both NADK and FASN are considered as potential targets for cancer therapy because of their lipogenic and other functions. NADK and FASN act as important regulators of lipid storage by restricting the capacity of fatty acid synthesis. This study showed that NADK- and FASN1-mediated fatty acid synthesis regulates mitochondrial function, probably by altering the levels of acetyl-CoA and CL. More physiological functions and molecular mechanisms of NADK and fatty acid synthesis may be revealed through the fatty-acid-mitochondrion link (Xu, 2021).

This study has demonstrated that increased PGC-1 acetylation and reduced CL synthesis are responsible for mitochondrial phenotype in NADK RNAi and FASN1 RNAi. However, reduced acetyl-CoA level and CLS overexpression only partially rescued mitochondrial phenotype. Exogenous fatty acid supplement completely restored mitochondrial mass in NADK RNAi and FASN1 RNAi, suggesting that fatty acid synthesis might regulate mitochondrial mass via other mechanisms as well. In addition, these studies were conducted in fat cells, which are specialized for lipid storage. It remains to be determined whether these findings apply to other cell types (Xu, 2021).

Functions of NADk orthologs in other species

Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology

NADK2 encodes the mitochondrial form of NAD Kinase, which phosphorylates nicotinamide adenine dinucleotide (NAD). Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. This study describes two chemically-induced mouse mutations in Nadk2, S326L and S330P, which cause a severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on brain, muscle, liver, and spinal cord at the same ages, and plasma at 5 weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency, and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. Findings from the Nadk model were compared to equivalent RNAseq and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled identification of disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve understanding of the pathophysiology of neuromuscular diseases, and describe mouse models that will be useful for future preclinical studies (Murray, 2022).

The regulation of ferroptosis by MESH1 through the activation of the integrative stress response

All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress survival mechanism is the stringent response triggered by the accumulation "alarmone" (p)ppGpp, whose level is regulated by RelA and SpoT. While metazoan genomes encode MESH1 (Metazoan SpoT Homolog 1) with ppGpp hydrolase activity, neither ppGpp nor the stringent response is found in metazoa. The deletion of Mesh1 in Drosophila triggers a transcriptional response reminiscent of the bacterial stringent response. However, the function of MESH1 remains unknown until a recent discovery of MESH1 as the first cytosolic NADPH phosphatase that regulates ferroptosis, a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides. To further understand whether MESH1 knockdown triggers a similar transcriptional response in mammalian cells, this study employed RNA-Seq to analyze the transcriptome response to MESH1 knockdown in human cancer cells. MESH1 knockdown induced different genes involving endoplasmic reticulum (ER) stress, especially ATF3, one of the ATF4-regulated genes in the integrative stress responses (ISR). Furthermore, MESH1 knockdown increased ATF4 protein, eIF2a phosphorylation, and induction of ATF3, XBPs, and CHOP mRNA. ATF4 induction contributes to ~30% of the transcriptome induced by MESH1 knockdown. Concurrent ATF4 knockdown re-sensitizes MESH1-depleted RCC4 cells to ferroptosis, suggesting its role in the ferroptosis protection mediated by MESH1 knockdown. ATF3 induction is abolished by the concurrent knockdown of NADK, implicating a role of NADPH accumulation in the integrative stress response. Collectively, these results suggest that MESH1 depletion triggers ER stress and ISR as a part of its overall transcriptome changes to enable stress survival of cancer cells. Therefore, the phenotypic similarity of stress tolerance caused by MESH1 removal and NADPH accumulation is in part achieved by ISR to regulate ferroptosis (Lin, 2021).

Direct stimulation of NADP(+) synthesis through Akt-mediated phosphorylation of NAD kinase

Nicotinamide adenine dinucleotide phosphate (NADP(+)) is essential for producing NADPH, the primary cofactor for reductive metabolism. Growth factor signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces acute synthesis of NADP(+) and NADPH. Akt phosphorylates NAD kinase (NADK), the sole cytosolic enzyme that catalyzes the synthesis of NADP(+) from NAD(+) (the oxidized form of NADH), on three serine residues (Ser(44), Ser(46), and Ser(48)) within an amino-terminal domain. This phosphorylation stimulates NADK activity both in cells and directly in vitro, thereby increasing NADP(+) production. A rare isoform of NADK (isoform 3) lacking this regulatory region exhibits constitutively increased activity. These data indicate that Akt-mediated phosphorylation of NADK stimulates its activity to increase NADP(+) production through relief of an autoinhibitory function inherent to its amino terminus (Hoxhaj, 2019).


Search PubMed for articles about Drosophila NAD kinase

Hoxhaj, G., Ben-Sahra, I., Lockwood, S. E., Timson, R. C., Byles, V., Henning, G. T., Gao, P., Selfors, L. M., Asara, J. M. and Manning, B. D. (2019). Direct stimulation of NADP(+) synthesis through Akt-mediated phosphorylation of NAD kinase. Science 363(6431): 1088-1092. PubMed ID: 30846598

Lin, C. C., Ding, C. C., Sun, T., Wu, J., Chen, K. Y., Zhou, P. and Chi, J. T. (2021). The regulation of ferroptosis by MESH1 through the activation of the integrative stress response. Cell Death Dis 12(8): 727. PubMed ID: 34294679

Murray, G., Bais, P., Hatton, C., Tadenev, A. L. D., Hoffmann, B. R., Stodola, T. J., Morelli, K. H., Pratt, S. L., Schroeder, D., Doty, R., Fiehn, O., John, S. W. M., Bult, C. J., Cox, G. A. and Burgess, R. W. (2022). Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology. Hum Mol Genet. PubMed ID: 35796562

Xu, M., Ding, L., Liang, J., Yang, X., Liu, Y., Wang, Y., Ding, M. and Huang, X. (2021). NAD kinase sustains lipogenesis and mitochondrial metabolism through fatty acid synthesis. Cell Rep 37(13): 110157. PubMed ID: 34965438

Biological Overview

date revised: 15 July 2022

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