Metabolic Reprogramming Is an Initial Step in Pancreatic Carcinogenesis That Can Be Targeted to Inhibit Acinar-to-Ductal Metaplasia.

Metabolic reprogramming is a hallmark of cancer and is crucial for cancer progression, making it an attractive therapeutic target. Understanding the role of metabolic reprogramming in cancer initiation could help identify prevention strategies. To address this, we investigated metabolism during acinar-to-ductal metaplasia (ADM), the first step of pancreatic carcinogenesis. Glycolytic markers were elevated in ADM lesions compared with normal tissue from human samples. Comprehensive metabolic assessment in three mouse models with pancreas-specific activation of KRAS, PI3K, or MEK1 using Seahorse measurements, nuclear magnetic resonance metabolome analysis, mass spectrometry, isotope tracing, and RNA sequencing analysis revealed a switch from oxidative phosphorylation to glycolysis in ADM. Blocking the metabolic switch attenuated ADM formation. Furthermore, mitochondrial metabolism was required for de novo synthesis of serine and glutathione (GSH) but not for ATP production. MYC mediated the increase in GSH intermediates in ADM, and inhibition of GSH synthesis suppressed ADM development. This study thus identifies metabolic changes and vulnerabilities in the early stages of pancreatic carcinogenesis. Significance: Metabolic reprogramming from oxidative phosphorylation to glycolysis mediated by MYC plays a crucial role in the development of pancreatic cancer, revealing a mechanism driving tumorigenesis and potential therapeutic targets. See related commentary by Storz, p. 2225.

Exploration of dietary interventions to treat mitochondrial fatty acid disorders in a mouse model.

Mitochondrial fatty acids synthesis (mtFAS) is a conserved metabolic pathway essential for mitochondrial respiration. The best characterized mtFAS product is the medium-chain fatty acid octanoate (C8) used as a substrate in the synthesis of lipoic acid (LA), a cofactor required by several mitochondrial enzyme complexes. In humans, mutations in the mtFAS component enoyl reductase MECR cause childhood-onset neurodegenerative disorder MEPAN. A complete deletion of Mecr in mice is embryonically lethal, while selective deletion of Mecr in cerebellar Purkinje cells causes neurodegeneration in these cells. A fundamental question in the research of mtFAS deficiency is if the defect is amenable to treatment by supplementation with known mtFAS products. Here we used the Purkinje-cell specific mtFAS deficiency neurodegeneration model mice to study if feeding the mice with a medium-chain triacylglycerol-rich formula supplemented with LA could slow down or prevent the neurodegeneration in Purkinje cell-specific Mecr KO mice. Feeding started at the age of 4 weeks and continued until the age of 9 months. The neurological status on the mice was assessed at the age of 3, 6, and 9 months with behavioral tests and the state of the Purkinje cell deterioration in the cerebellum was studied histologically. We showed that feeding the mice with medium chain triacylglycerols and LA affected fatty acid profiles in the cerebellum and plasma but did not prevent the development of neurodegeneration in these mice. Our results indicate that dietary supplementation with medium chain fatty acids and LA alone is not an efficient way to treat mtFAS disorders.

Neuroforensomics: metabolites as valuable biomarkers in cerebrospinal fluid of lethal traumatic brain injuries.

Traumatic brain injury (TBI) is a ubiquitous, common sequela of accidents with an annual prevalence of several million cases worldwide. In forensic pathology, structural proteins of the cellular compartments of the CNS in serum and cerebrospinal fluid (CSF) have been predominantly used so far as markers of an acute trauma reaction for the biochemical assessment of neuropathological changes after TBI. The analysis of endogenous metabolites offers an innovative approach that has not yet been considered widely in the assessment of causes and circumstances of death, for example after TBI. The present study, therefore, addresses the question whether the detection of metabolites by liquid-chromatography-mass spectrometry (LC/MS) analysis in post mortem CSF is suitable to identify TBI and to distinguish it from acute cardiovascular control fatalities (CVF). Metabolite analysis of 60 CSF samples collected during autopsies was performed using high resolution (HR)-LC/MS. Subsequent statistical and graphical evaluation as well as the calculation of a TBI/CVF quotient yielded promising results: numerous metabolites were identified that showed significant concentration differences in the post mortem CSF for lethal acute TBI (survival times up to 90 min) compared to CVF. For the first time, this forensic study provides an evaluation of a new generation of biomarkers for diagnosing TBI in the differentiation to other causes of death, here CVF, as surrogate markers for the post mortem assessment of complex neuropathological processes in the CNS ("neuroforensomics").

Structural enzymology studies with the substrate 3S-hydroxybutanoyl-CoA: bifunctional MFE1 is a less efficient dehydrogenase than monofunctional HAD.

Multifunctional enzyme, type-1 (MFE1) catalyzes the second and third step of the ß-oxidation cycle, being, respectively, the 2E-enoyl-CoA hydratase (ECH) reaction (N-terminal part, crotonase fold) and the NAD+-dependent, 3S-hydroxyacyl-CoA dehydrogenase (HAD) reaction (C-terminal part, HAD fold). Structural enzymological properties of rat MFE1 (RnMFE1) as well as of two of its variants, namely the E123A variant (a glutamate of the ECH active site is mutated into alanine) and the BCDE variant (without domain A of the ECH part), were studied, using as substrate 3S-hydroxybutanoyl-CoA. Protein crystallographic binding studies show the hydrogen bond interactions of 3S-hydroxybutanoyl-CoA as well as of its 3-keto, oxidized form, acetoacetyl-CoA, with the catalytic glutamates in the ECH active site. Pre-steady state binding experiments with NAD+ and NADH show that the kon and koff rate constants of the HAD active site of monomeric RnMFE1 and the homologous human, dimeric 3S-hydroxyacyl-CoA dehydrogenase (HsHAD) for NAD+ and NADH are very similar, being the same as those observed for the E123A and BCDE variants. However, steady state and pre-steady state kinetic data concerning the HAD-catalyzed dehydrogenation reaction of the substrate 3S-hydroxybutanoyl-CoA show that, respectively, the kcat and kchem rate constants for conversion into acetoacetyl-CoA by RnMFE1 (and its two variants) are about 10 fold lower as when catalyzed by HsHAD. The dynamical properties of dehydrogenases are known to be important for their catalytic efficiency, and it is discussed that the greater complexity of the RnMFE1 fold correlates with the observation that RnMFE1 is a slower dehydrogenase than HsHAD.

Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner.

The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; BrafCA; Ptenlox/+ melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.

7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Activation of the integrated stress response rewires cardiac metabolism in Barth syndrome.

Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.

The Molecular Basis of Catalysis by SDR Family Members Ketoacyl-ACP Reductase FabG and Enoyl-ACP Reductase FabI in Type-II Fatty Acid Biosynthesis.

The short-chain dehydrogenase/reductase (SDR) superfamily members acyl-ACP reductases FabG and FabI are indispensable core enzymatic modules and catalytic orientation controllers in type-II fatty acid biosynthesis. Herein, we report their distinct substrate allosteric recognition and enantioselective reduction mechanisms. FabG achieves allosteric regulation of ACP and NADPH through ACP binding across two adjacent FabG monomers, while FabI follows an irreversible compulsory order of substrate binding in that NADH binding must precede that of ACP on a discrete FabI monomer. Moreover, FabG and FabI utilize a backdoor residue Phe187 or a "rheostat" α8 helix for acyl chain length selection, and their corresponding triad residues Ser142 or Tyr145 recognize the keto- or enoyl-acyl substrates, respectively, facilitating initiation of nucleophilic attack by NAD(P)H. The other two triad residues (Tyr and Lys) mediate subsequent proton transfer and (R)-3-hydroxyacyl- or saturated acyl-ACP production.

Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells through HIF-1α-mediated glycolytic reprogramming.

T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.

LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2.

The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.

Design of Glycoengineered IL-4 Antagonists Employing Chemical and Biosynthetic Glycosylation.

Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.

Platelet-derived lipids promote insulin secretion of pancreatic ß cells.

Hyperreactive platelets are commonly observed in diabetic patients indicating a potential link between glucose homeostasis and platelet reactivity. This raises the possibility that platelets may play a role in the regulation of metabolism. Pancreatic ß cells are the central regulators of systemic glucose homeostasis. Here, we show that factor(s) derived from ß cells stimulate platelet activity and platelets selectively localize to the vascular endothelium of pancreatic islets. Both depletion of platelets and ablation of major platelet adhesion or activation pathways consistently resulted in impaired glucose tolerance and decreased circulating insulin levels. Furthermore, we found platelet-derived lipid classes to promote insulin secretion and identified 20-Hydroxyeicosatetraenoic acid (20-HETE) as the main factor promoting ß cells function. Finally, we demonstrate that the levels of platelet-derived 20-HETE decline with age and that this parallels with reduced impact of platelets on ß cell function. Our findings identify an unexpected function of platelets in the regulation of insulin secretion and glucose metabolism, which promotes metabolic fitness in young individuals.

USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

An engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration.

Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory function. MtFAS generates the octanoic acid precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. The structurally well-characterized component of mtFAS, human 2E-enoyl-ACP reductase (MECR) rescues respiratory growth and lipoylation defects of a Saccharomyces cerevisiae Δetr1 strain lacking native mtFAS enoyl reductase. To address the role of longer products of mtFAS, we employed in silico molecular simulations to design a MECR variant with a shortened substrate binding cavity. Our in vitro and in vivo analyses indicate that the MECR G165Q variant allows synthesis of octanoyl groups but not long chain fatty acids, confirming the validity of our computational approach to engineer substrate length specificity. Furthermore, our data imply that restoring lipoylation in mtFAS deficient yeast strains is not sufficient to support respiration and that long chain acyl-ACPs generated by mtFAS are required for mitochondrial function.

Glycolytic flux control by drugging phosphoglycolate phosphatase.

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.

c-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis.

Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3ß axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism.

Mass Spectrometric Metabolic Fingerprinting of 2-Deoxy-D-Glucose (2-DG)-Induced Inhibition of Glycolysis and Comparative Analysis of Methionine Restriction versus Glucose Restriction under Perfusion Culture in the Murine L929 Model System.

All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level.

Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma.

Communication between tumors and the stroma of tumor-draining lymph nodes (TDLN) exists before metastasis arises, altering the structure and function of the TDLN niche. Transcriptional profiling of fibroblastic reticular cells (FRC), the dominant stromal population of lymph nodes, has revealed that FRCs in TDLNs are reprogrammed. However, the tumor-derived factors driving the changes in FRCs remain to be identified. Taking an unbiased approach, we have shown herein that lactic acid (LA), a metabolite released by cancer cells, was not only secreted by B16.F10 and 4T1 tumors in high amounts, but also that it was enriched in TDLNs. LA supported an upregulation of Podoplanin (Pdpn) and Thy1 and downregulation of IL7 in FRCs of TDLNs, making them akin to activated fibroblasts found at the primary tumor site. Furthermore, we found that tumor-derived LA altered mitochondrial function of FRCs in TDLNs. Thus, our results demonstrate a mechanism by which a tumor-derived metabolite connected with a low pH environment modulates the function of fibroblasts in TDLNs. How lymph node function is perturbed to support cancer metastases remains unclear. The authors show that tumor-derived LA drains to lymph nodes where it modulates the function of lymph node stromal cells, prior to metastatic colonization.

Low Energy Status under Methionine Restriction Is Essentially Independent of Proliferation or Cell Contact Inhibition.

Nonlimited proliferation is one of the most striking features of neoplastic cells. The basis of cell division is the sufficient presence of mass (amino acids) and energy (ATP and NADH). A sophisticated intracellular network permanently measures the mass and energy levels. Thus, in vivo restrictions in the form of amino acid, protein, or caloric restrictions strongly affect absolute lifespan and age-associated diseases such as cancer. The induction of permanent low energy metabolism (LEM) is essential in this process. The murine cell line L929 responds to methionine restriction (MetR) for a short time period with LEM at the metabolic level defined by a characteristic fingerprint consisting of the molecules acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP. Here, we used mass spectrometry (LC/MS) to investigate the influence of proliferation and contact inhibition on the energy status of cells. Interestingly, the energy status was essentially independent of proliferation or contact inhibition. LC/MS analyses showed that in full medium, the cells maintain active and energetic metabolism for optional proliferation. In contrast, MetR induced LEM independently of proliferation or contact inhibition. These results are important for cell behaviour under MetR and for the optional application of restrictions in cancer therapy.