In the presence of high abundance of exogenous fatty acids, cells either store fatty acids in lipid droplets or oxidize them in mitochondria. In this study, we aimed to explore a novel and direct role of mitochondrial fission in lipid homeostasis in HeLa cells. We observed the association between mitochondrial morphology and lipid droplet accumulation in response to high exogenous fatty acids. We inhibited mitochondrial fission by silencing dynamin-related protein 1(DRP1) and observed the shift in fatty acid storage-usage balance. Inhibition of mitochondrial fission resulted in an increase in fatty acid content of lipid droplets and a decrease in mitochondrial fatty acid oxidation. Next, we overexpressed carnitine palmitoyltransferase-1 (CPT1), a key mitochondrial protein in fatty acid oxidation, to further examine the relationship between mitochondrial fatty acid usage and mitochondrial morphology. Mitochondrial fission plays a role in distributing exogenous fatty acids. CPT1A controlled the respiratory rate of mitochondrial fatty acid oxidation but did not cause a shift in the distribution of fatty acids between mitochondria and lipid droplets. Our data reveals a novel function for mitochondrial fission in balancing exogenous fatty acids between usage and storage, assigning a role for mitochondrial dynamics in control of intracellular fuel utilization and partitioning.

The targeting of metabolic pathways is emerging as an exciting new approach for modulating immune cell function and polarization states. In this study, carbon tracing and systems biology approaches integrating metabolomic and transcriptomic profiling data were used to identify adaptations in human T cell metabolism important for fueling pro-inflammatory T cell function. Results of this study demonstrate that T cell receptor (TCR) stimulation leads to a significant increase in glucose and amino acid metabolism that trigger downstream biosynthetic processes. Specifically, increased expression of several enzymes such as CTPS1, IL4I1, and ASL results in the reprogramming of amino acid metabolism. Additionally, the strength of TCR signaling resulted in different metabolic enzymes utilized by T cells to facilitate similar biochemical endpoints. Furthermore, this study shows that cyclosporine represses the pathways involved in amino acid and glucose metabolism, providing novel insights on the immunosuppressive mechanisms of this drug. To explore the implications of the findings of this study in clinical settings, conventional immunosuppressants were tested in combination with drugs that target metabolic pathways. Results showed that such combinations increased efficacy of conventional immunosuppressants. Overall, the results of this study provide a comprehensive resource for identifying metabolic targets for novel combinatorial regimens in the treatment of intractable immune diseases.

SUMMARY: Pancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies β-cell sensing of both glycolytic and mitochondrial fuels. PK present at the plasma membrane is sufficient to close K_ATPchannels and initiate calcium influx. Small-molecule PK activators increase β-cell oscillation frequency and potently amplify insulin secretion. By cyclically depriving mitochondria of ADP, PK restricts oxidative phosphorylation in favor of the mitochondrial PEP cycle with no net impact on glucose oxidation. Our findings support a compartmentalized model of β-cell metabolism in which PK locally generates the ATP/ADP threshold required for insulin secretion, and identify a potential therapeutic route for diabetes based on PK activation that would not be predicted by the β-cell consensus model.GRAPHICAL The consensus model for β-cell glucose sensing supports a dominant role for OxPhos. This model doesn’t fully explain the observed metabolic and electrophysiologic oscillations associated with glucose-stimulated insulin secretion. Lewandowskiet al. challenge this model by mechanistically connecting the anaplerotic PEP cycle to the electrically silent triggering phase, and OxPhos to the electrically active secretory phase. Here, the allosteric recruitment of pyruvate kinase directs metabolic traffic between the two cycles and identifies potential therapeutic strategies for diabetes based on pharmacologic pyruvate kinase activation.HIGHLIGHTS: Compartmentalized pyruvate kinase (PK) activity underlies β-cell fuel sensing Membrane-associated PK closes K_ATPchannels and controls calcium influx By lowering ADP, PK toggles mitochondria between OxPhos and PEP biosynthesis Pharmacologic PK activation increases oscillatory frequency and amplifies secretion