Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney Disease Progression, investigated with targeted tracing metabolomics analysis in MEF cells using 15N2-glutamine.
STUDY_SUMMARY
Polycystic Kidney Disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD. Of interest, in these tracing studies we could confirm that the pyrimidine biosynthesis pathway is increased and rescued by silencing of Asns.
Disruption of Glucose Homeostasis by Bacterial Infection Orchestrates Host Innate Immunity Through NAD+/NADH Balance
STUDY_SUMMARY
Metabolic reprogramming is crucial for activating innate immunity in macrophages, and the accumulation of immunometabolites is thought essential for effective defense against infection. The NAD+/NADH redox couple serves as a critical node that integrates metabolic pathways and signaling events, but how this metabolite couple engages macrophage activation remains unclear. Here, we showed that the NAD+/NADH ratio serves as a molecular signal that regulates proinflammatory responses and type I interferon (IFN) responses divergently. Salmonella Typhimurium infection led to a decreased NAD+/NADH ratio by inducing the accumulation of NADH. Further investigation showed that an increased NAD+/NADH ratio correlates to attenuated proinflammatory responses and enhanced type I IFN responses. Conversely, a decreased NAD+/NADH ratio is linked to intensified proinflammatory responses and restrained type I IFN responses. These results showed the NAD+/NADH ratio is an essential cell-intrinsic factor that orchestrates innate immunity, which enhanced our understanding of how metabolites fine-tune innate immunity.
Measuring PEMT activity in HCMV infected fibroblasts using 13C-methionine.
STUDY_SUMMARY
HCMV promotes the levels of PC lipids in infection. The Kennedy pathway is the canonical host pathway for de novo PC lipid synthesis. Host cells encode alternative pathways for PC synthesis such as the Phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Here, we tested if HCMV promotes metabolic activity in the PEMT pathway. We used 13C-methionine to track the metabolism of labeled tracer molecule into the head group of PC lipids thus, measuring PEMT pathway activity. We find that HCMV and uninfected cells exhibit similarly low PEMT metabolic activity and percentage of labeled PCs. Moreover, when cells were engineered to overexpress the host PEMT enzyme, PC labeling via PEMT increased when expression was induced with doxycycline. Overexpressing GFP and feeding labeled molecule resulted in labeling similar to mock and infected labeling percentages, <5%. Thus, PEMT is not an active pathway in the primary human fibroblasts used in our studies and HCMV infection does not increase metabolic activity of PEMT to synthesize PCs.