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Traditional views of cellular metabolism imply that it is passively adapted to meet the demands of the cell. It is becoming increasingly clear, however, that metabolites do more than simply supply the substrates for biological processes; they also provide critical signals, either through effects on metabolic pathways or via modulation of other regulatory proteins. Recent investigation has also uncovered novel roles for several metabolites that expand their signalling influence to processes outside metabolism, including nutrient sensing and storage, embryonic development, cell survival and differentiation, and immune activation and cytokine secretion. Together, these studies suggest that, in contrast to the prevailing notion, the biochemistry of a cell is frequently governed by its underlying metabolism rather than vice versa. This important shift in perspective places common metabolites as key regulators of cell phenotype and behaviour. Yet the signalling metabolites, and the cognate targets and transducers through which they signal, are only beginning to be uncovered. In this Review, we discuss the emerging links between metabolism and cellular behaviour. We hope this will inspire further dissection of the mechanisms through which metabolic pathways and intermediates modulate cell function and will suggest possible drug targets for diseases linked to metabolic deregulation.

Metabolism is intertwined with various cellular processes, including controlling cell fate, influencing tumorigenesis, participating in stress responses and more. Metabolism is a complex, interdependent network, and local perturbations can have indirect effects that are pervasive across the metabolic network. Current analytical and technical limitations have long created a bottleneck in metabolic data interpretation. To address these shortcomings, we developed Metaboverse, a user-friendly tool to facilitate data exploration and hypothesis generation. Here we introduce algorithms that leverage the metabolic network to extract complex reaction patterns from data. To minimize the impact of missing measurements within the network, we introduce methods that enable pattern recognition across multiple reactions. Using Metaboverse, we identify a previously undescribed metabolite signature that correlated with survival outcomes in early stage lung adenocarcinoma patients. Using a yeast model, we identify metabolic responses suggesting an adaptive role of citrate homeostasis during mitochondrial dysfunction facilitated by the citrate transporter, Ctp1. We demonstrate that Metaboverse augments the user’s ability to extract meaningful patterns from multi-omics datasets to develop actionable hypotheses.

Callyspongiolide is a marine-derived macrolide that kills cells in a caspase-independent manner. NCI COMPARE analysis of human tumor cell line toxicity data for synthetic callyspongiolide indicated that its pattern of cytotoxicity correlated with that seen for concanamycin A, an inhibitor of the vacuolar-type H+-ATPase (V-ATPase). Using yeast as a model system, we report that treatment with synthetic callyspongiolide phenocopied a loss of V-ATPase activity including (1) inability to grow on a nonfermentable carbon source, (2) rescue of cell growth via supplementation with Fe2+, (3) pH-sensitive growth, and (4) a vacuolar acidification defect visualized using the fluorescent dye quinacrine. Crucially, in an in vitroassay, callyspongiolide was found to dose-dependently inhibit yeast V-ATPase (IC50= 10 nM). Together, these data identify callyspongiolide as a new and highly potent V-ATPase inhibitor. Notably, callyspongiolide is the first V-ATPase inhibitor known to be expelled by Pdr5p.

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The field of bioenergetics is rapidly expanding with new discoveries of mechanisms and potential therapeutic targets. The 2023 Keystone symposium on ‘Bioenergetics in Health and Disease’, which was jointly held with the symposium ‘Adipose Tissue: Energizing Good Fat’, consisted of a powerhouse line-up of researchers who shared their insights.

Insulin and insulin-like peptides play a pivotal role in a wide variety of cellular and physiological events, including energy storage, proliferation, aging, and differentiation. Variants of insulin and insulin-like peptides may therefore be probes for studying the insulin signaling pathway and therapeutic candidates for treating metabolic diseases. Here, we report a method for genetically displaying single-chain insulin-like peptides on the surface of Saccharomyces cerevisiaestrain DY1632. Using a previously reported single-chain insulin analogue, SCI-57, as a model, we demonstrate that nearly 70% of yeast binds to insulin receptor (IR), suggesting that SCI-57 is folded correctly and maintains its IR binding property. Furthermore, the interaction between displayed SCI-57 and IR can be weakened using increasing concentrations of native insulin as a soluble competitor, suggesting that the interaction is insulin-dependent. We further applied this methodology to three other single-chain insulin analogues with various lengths and confirmed their interactions with IR. In summary, we successfully displayed a number of insulin-like peptides on a yeast surface and demonstrated insulin-dependent interactions with IR. This method may, therefore, be used for construction of libraries of insulin-like peptides to select for chemical probes or therapeutic molecules.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.

Upon antigen-specific T Cell Receptor (TCR) engagement, human CD4+T cells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that the generation of extra-mitochondrial pyruvate is an important step for acetyl-CoA production and subsequent H3K27ac-mediated remodeling of histone acetylation. Histone modification, transcriptomic, and carbon tracing analyses of pyruvate dehydrogenase (PDH)-deficient T cells show PDH-dependent acetyl-CoA generation as a rate-limiting step during T activation. Furthermore, T cell activation results in the nuclear translocation of PDH and its association with both the p300 acetyltransferase and histone H3K27ac. These data support the tight integration of metabolic and histone-modifying enzymes, allowing metabolic reprogramming to fuel CD4+ T cell activation. Targeting this pathway may provide a therapeutic approach to specifically regulate antigen-driven T cell activation.

Due to their varied metabolic and signalling roles, mitochondria are important in mediating cell behaviour. By altering mitochondrial function, two studies now identify metabolite-induced epigenetic changes that have profound effects on haematopoietic stem cell fate and function.

Elevated hepatic synthesis of fatty acids and triglycerides, driven by hyperactivation of the SREBP-1c transcription factor, has been implicated as a causal feature of metabolic syndrome. SREBP-1c activation requires the proteolytic maturation of the endoplasmic-reticulum-bound precursor to the active, nuclear transcription factor, which is stimulated by feeding and insulin signaling. Here, we show that feeding and insulin stimulate the hepatic expression of PASK.We also demonstrate, using genetic and pharmacological approaches, that PASK is required for the proteolytic maturation of SREBP-1c in cultured cells and in the mouse and rat liver. Inhibition of PASK improves lipid and glucose metabolism in dietary animal models of obesity and dyslipidemia. Administration of a PASK inhibitor decreases hepatic expression of lipogenic SREBP-1c target genes, decreases serum triglycerides, and partially reverses insulin resistance. While the signaling network that controls SREBP-1c activation is complex, we propose that PASK is an important component with therapeutic potential.

A highly conserved middle region of Vms1 is necessary and sufficient for mitochondrial targeting (MTD). MTD-mediated mitochondrial targeting of Vms1 is negatively regulated by a direct interaction with the Vms1 N-terminus. Under conditions in which selected mitochondria are damaged, Vms1 is selectively recruited to damaged mitochondria.

The inability to coordinate cellular metabolic processes with the cellular and organismal nutrient environment leads to a variety of disorders, including diabetes and obesity. Nutrient-sensing protein kinases, such as AMPK and mTOR, play a pivotal role in metabolic regulation and are promising therapeutic targets for the treatment of disease. In this Extra View, we describe another member of the nutrient-sensing protein kinase group, PAS kinase, which plays a role in the regulation of glucose utilization in both mammals and yeast. PAS kinase deficient mice are resistant to high fat diet-induced weight gain, insulin resistance, and hepatic triglyceride hyperaccumulation, suggesting a role for PAS kinase in the regulation of glucose and lipid metabolism in mammals. Likewise, PAS kinase deficient yeast display altered glucose partitioning, favoring glycogen biosynthesis at the expense of cell wall biosynthesis. As a result, PAS kinase deficient yeast are sensitive to cell wall perturbing agents. This partitioning of glucose in response to PAS kinase activation is due to phosphorylation of Ugp1, the enzyme primarily responsible for UDP-glucose production. The two yeast PAS kinase homologs, Psk1 and Psk2, are activated by two stimuli, cell integrity stress and nonfermentative carbon sources. We review what is known about yeast PAS kinase and describe a genetic screen that may help elucidate pathways involved in PAS kinase activation and function.

Acute myocardial infarction (AMI) may progress to heart failure (HF). Ischemic-reperfusion injury (I/R) accounts for ~50% of the damage post-AMI. Currently, there are minimal interventions to mitigate I/R and prevent HF. Mitochondrial Pyruvate Carrier (MPC) is considered cardio-protective, and deletion causes HF. The purpose of this investigation was to determine if cardiac MPC deletion (MPC-/-) in mice limits myocardial salvage following I/R. We hypothesized MPC-/-mice would have more necrosis and less myocardial salvage compared to wild type (WT) littermates.MPC1 was targeted in C57Bl/6 mice by placing loxP sites in the introns flanking the exon 3-5 of MPC1. A cardiac-specific deletion was used by intraperitoneal injections of tamoxifen at 8 weeks old. Four weeks post injection, prior to development of HF, mice were intubated, ventilated, and the left anterior descending artery was occluded for 30 minutes then reperfused for 2 hours. Next, Evans-Blue was injected into the heart to define the ischemic area. Hearts of paired littermates between WT (n=6) and MPC-/-(n=6) were excised and cut into 1-mm transverse slices. Slices were stained with triphenyltetrazolium-chloride (TTC) to define myocardial salvage. Next, slices were imaged and quantified by independent researchers using planimetry. Paired t-tests with an α-level of 0.05 were used to compare groups.Litters not subjected to I/R showed significant (p<0.001) MPC1 deletion (WT: 0.28±0.11 au vs. MPC-/-: 0.06±0.02 au). The area at risk (AAR) was comparable (p=0.12) between groups (WT: 46.7±5.9% vs. MPC-/-: 54.3±13.3), indicating equivalent coronary ligation-induced ischemia. Within the AAR, MPC-/-mice showed significantly (p=0.04) more necrosis (MPC-/-: 52.8±10.4% vs. WT: 42.7±17.4%) with less myocardial salvage (MPC-/-: 47.2±10.4% vs. WT: 57.3±10.6%). MPC-/-mice also had significantly (p=0.002) more absolute necrosis (28.4±7.8%) compared to WT mice (19.8±7.8%). There were no significant differences (p>0.05) in percent viable or salvageable tissue in relation to total area.In conclusion, following I/R, MPC-/-mice showed more absolute and standardized necrosis with less myocardial salvage, suggesting when cardiac MPC is reduced it causes more myocardial damage leading to HF.

Introduction:Diabetes is a major cause of heart failure with preserved ejection fraction (HFpEF), which has limited diagnostic tools and therapeutic options. Abnormal remodeling in the t-tubule calcium handling cardiac bridging integrator 1 (cBIN1)-microdomains has emerged as key to the pathophysiology of failing cardiomyocytes, which can be targeted for cardiac functional improvement. It remains unclear whether cBIN1 replacement therapy is effective in restoring cardiac function in diabetic HFpEF.Hypothesis:We hypothesized that cBIN1 gene therapy rescues diastolic failure in mice with diabetic cardiomyopathy.Methods:9-wks old male and female db/db mice that received retro-orbital injection of 1x1011vg of AAV9 transducing GFP or cBIN1 were compared to non-diabetic control db/m littermates injected with AAV9-GFP (N = 16 mice per group). Mice were terminated 8-wks later for tissue acquisition, imaging, as well as biochemical analysis. Echocardiography, exercise tolerance, and plasma cBIN1 score (CS, an inverse index of plasma cBIN1 concentration) were obtained before and after gene therapy.Results:db/db mice developed diastolic failure with reduced E/A, increased E/e’, exercise intolerance, and cardiac hypertrophy with reduced tissue cBIN1 levels and corresponding elevations in plasma CS. Supporting our hypothesis, AAV9-cBIN1 normalized E/A and E/e’ and improved exercise tolerance in db/db mice, which can be distinguished by plasma CS as an inverse marker of cBIN1-microfolds and cardiomyocyte remodeling (Figure 1). Preservation of cBIN1-microdomains in db/db cardiomyocytes reorganized functional SERCA2a and rescued mitochondrial respiration with diminished oxidative stress.Conclusions:Exogenous cBIN1 normalizes lusitropic function of diabetic hearts through microdomain-organized effective calcium handling and mitochondrial respiration. cBIN1 gene therapy is a new and effective method for guided treatment of diabetic HFpEF.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αβ T cell numbers. MPC1-deficient T cells have defective thymic development at the β-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αβ T cell development and that its deficiency results in reduced but activated peripheral T cell populations.

Background:Significant improvements in myocardial structure and function have been reported in some advanced heart failure (HF) patients (Responders-R) upon left ventricular assist devices (LVAD)- induced mechanical unloading. This is reported to affect the energy balance, suggesting its potential to reverse the deleterious metabolic adaptations of the failing heart. Specifically, our prior work demonstrated a post-LVAD glycolysis upregulation with no subsequent increase in pyruvate mitochondrial oxidation through the TCA cycle. The underlying mechanisms responsible for this mismatch are not known.Aim:We hypothesized that the upregulated glycolytic intermediates are channeled instead of the TCA cycle into metabolic cardioprotective and repair pathways driving the observed myocardial recovery in Responders.Methods:We prospectively obtained LV apical myocardial tissue obtained from normal donors and HF patients at LVAD implant (Pre-LVAD) and transplantation (Post-LVAD) and segregated them as R and Non-R based on post-LVAD clinical recovery.Results:Western blot analysis of the key PPP and OCM enzymes, glucose-6-phosphate dehydrogenase and transketolase, phosphoglycerate dehydrogenase and mitochondrial serine hydroxymethyl transferase showed significant increase in Post-R as compared to post-Non-responders. In agreement with the elevated enzyme levels, the metabolite levels of the enzyme substrates such as sedoheptulose-6-phosphate (PPP) and serine and glycine (OCM) were decreased in post-R. Furthermore, post-R had significantly reduced ROS levels, high NADPH levels, improved mitochondrial dynamics and enhanced ECM protein, ?-dystroglycan, further indicating increased flux of glucose into PPP and OCM and its correlation with recovery.Conclusions:The recovering human heart appears to direct the increased glycolytic metabolites into PPP and OCM, both of which play a role in cardioprotection by generating NADPH thereby reducing ROS and metabolites for repair. Further confirmation of these findings with metabolic flux studies are warranted.

Mitochondria are crucial for many cellular functions. In this issue of Cell Reports, studies from Lanning et al. and Wolf and Mootha describe RNAi approaches to screening the mitochondrial proteome. Unexpectedly, they uncover key roles for two poorly characterized mitochondrial proteins: AK4 and FASTKD4. These studies provide examples of the power of forward genetic screens, even when screening a subset of genes, in deciphering functions of previously mysterious mitochondrial proteins.

Comment on: Hao HX, et al. Science 2009; 325:1139-42.

The Rho1-activating complex formed by yPASK-phosphorylated Ugp1 enables yeast to overcome loss of Tor2 function.