Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, ...the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.
Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, ...proliferation is suppressed in electron transport chain (ETC)–dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial β-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.
Editor’s summary When liver damage occurs, a key condition for recovery is ensuring the proliferation of healthy tissue without propagating sick or damaged cells. Wang et al . have identified a mechanism by which this happens through selective pressure on the mitochondria. In some pathogenic conditions, such as cirrhosis, the diseased areas contain mutations in the mitochondrial genome that lead to dysfunction in the electron transport chain required for cellular respiration. Although such dysfunction is not directly lethal, the authors have found that it puts the affected cells at a competitive disadvantage and reduces their metabolic flexibility. In addition, biliary epithelial cells can pitch in and transdifferentiate into hepatocytes when healthy liver cells are in short supply. —Yevgeniya Nusinovich
INTRODUCTION Mitochondrial electron transport chain (ETC) dysfunction is commonly observed in acquired human diseases, including in the setting of metabolic-associated liver diseases. During liver regeneration, proliferating hepatocytes compete and allow cells with increased fitness to more readily contribute to the composition of the regenerated organ. Although recent studies indicate that mitochondrial ETC function can affect stem cell behavior, it is unknown whether the ETC influences hepatocyte proliferation after liver injury and thereby contributes to the organ’s recovered condition and function. RATIONALE We used metabolite profiling of mitochondria and isotope tracing techniques in mice to investigate the metabolic response in hepatocytes under homeostatic and regenerative conditions. We then used a set of genetic mouse models targeting the mitochondrial ETC to dissect the contribution of each individual ETC complex (I to V) to liver regeneration. With this approach we aimed to examine the relative fitness of wild-type (WT) and ETC-dysfunctional hepatocytes during regeneration and identify mechanisms by which mitochondrial health is regulated in proliferating hepatocytes. RESULTS We found that mouse hepatocytes required a functional ETC to proliferate and compete with WT hepatocytes during liver regeneration. In the absence of an ETC, murine livers rapidly accumulated fatty acid species, resulting in steatosis. We additionally observed that transdifferentiation of cholangiocytes into hepatocytes was stimulated during regeneration of ETC-mutant livers. Metabolic tracing studies revealed that WT livers rely on mobilization and oxidation of peripheral fat stores to maintain acetyl-CoA levels during proliferation. In ETC-mutant livers, fatty acid oxidation was inhibited resulting in fat accumulation and decreased production of acetyl-CoA. Notably, mitochondrial complex I was not required for hepatocyte proliferation, suggesting that complex I is not the major electron donor to the ETC in regenerating hepatocytes. As fat accumulates in the setting of ETC dysfunction, the generation of acetyl-CoA from nonfatty acid sources (such as pyruvate or acetate) was suppressed as a result of induced expression of PDK4 (a negative regulator of pyruvate oxidation) and decreased expression of ACSS2 (the enzyme responsible for conversion of acetate to acetyl-CoA). This metabolic inflexibility (the inability to switch to an alternative nutrient for generation of acetyl-CoA) forces a reliance on fatty acid oxidation and thereby selects for proliferating hepatocytes with a functional ETC. To test this model we inhibited or deleted PDK4 to re-enable pyruvate oxidation to acetyl-CoA. In the absence of PDK4 activity, ETC-dysfunctional hepatocytes were able to proliferate during liver regeneration. CONCLUSION Our results support a model whereby the network topology regulating nutrient utilization in the liver encodes a metabolic inflexibility that promotes mitochondrial health during tissue regeneration. Specifically, the accumulation of fatty acids in the setting of ETC dysfunction inhibits the generation of acetyl-CoA from alternative substrates. We identify PDK4 expression downstream of fat accumulation as a key regulatory event that governs metabolic inflexibility in proliferating hepatocytes. Although metabolic flexibility has been largely proposed as beneficial to an organism’s survival and function, our model indicates that metabolic inflexibility can be used by the murine liver to promote the overall health of a population of proliferating cells. Suppressed acetyl-CoA production promotes mitochondrial health during liver regeneration. During liver injury and regeneration, fatty acids from adipose tissues transit to the liver to fuel mitochondrial beta oxidation in WT hepatocytes, which outcompete ETC-mutant hepatocytes. In the absence of a functional ETC, fatty acid buildup reduces acetyl-CoA through induction of PDK4. PDK4 inhibition restores flexibility for acetyl-CoA generation, allowing ETC-mutant hepatocytes to proliferate. Figure created with BioRender.com
The orphan nuclear receptor SHP (small heterodimer partner) is a well-known transcriptional corepressor of bile acid and lipid metabolism in the liver; however, its function in other tissues is ...poorly understood. Here, we report an unexpected role for SHP in the exocrine pancreas as a modulator of the endoplasmic reticulum (ER) stress response. SHP expression is induced in acinar cells in response to ER stress and regulates the protein stability of the spliced form of X-box-binding protein 1 (XBP1s), a key mediator of ER stress response. Loss of SHP reduces XBP1s protein level and transcriptional activity, which in turn attenuates the ER stress response during the fasting-feeding cycle. Consequently, SHP-deficient mice also are more susceptible to cerulein-induced pancreatitis. Mechanistically, we show that SHP physically interacts with the transactivation domain of XBP1s, thereby inhibiting the polyubiquitination and degradation of XBP1s by the Cullin3-SPOP (speckle-type POZ protein) E3 ligase complex. Together, our data implicate SHP in governing ER homeostasis and identify a novel posttranslational regulatory mechanism for the key ER stress response effector XBP1.
Mammalian embryogenesis requires rapid growth and proper metabolic regulation
. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development
. ...Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle
. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.
Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but ...generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.
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•MS-based isotopomer method requires less than 1% of the sample required for NMR•Isotopomer distributions provide detail about TCA cycle labeling from 13C tracers•MS method is particularly useful in human and mouse isotope-tracing experiments
Cai et al. developed a sensitive mass spectrometry method to report all 16 aspartate and 32 glutamate positional 13C isotopomers. In small tissue samples labeled with 13C, the method reveals aspects of TCA cycle metabolism difficult or impossible to detect with non-positional mass spectrometry or NMR.
Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients ...with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.
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•ETC inhibition rewires purine metabolism•Cytosolic NAD(H) imbalance induces purine accumulation in ETC-deficient cells•HPRT1-mediated purine salvage supports NSCLC growth during ETC inhibition•Purine uptake is required to supply salvage upon ETC inhibition
Wu et al. report that mitochondrial electron transport chain impairment induces a metabolic shift from de novo purine biosynthesis to purine salvage. Cancer cells with low electron transport chain activity require purine uptake and salvage to grow in culture and in vivo.