A diverse proteome is present and enzymatically active in metabolite extracts.

Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.

NRF2-dependent regulation of the prostacyclin receptor PTGIR drives CD8 T cell exhaustion.

The progressive decline of CD8 T cell effector function-also known as terminal exhaustion-is a major contributor to immune evasion in cancer. Yet, the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here, we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis, which mediates cellular adaptations to oxidative stress, directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically, we identify PTGIR, a receptor for the circulating eicosanoid prostacyclin, as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover, lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-γ and granzyme production) to chronic infection and cancer. Together, these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking.

Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic acid and adrenic acid. These PUFAs then accumulate in phospholipids, particularly ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools dictates their sensitivity to ferroptosis.

Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides.

Targeting programmed cell death protein 1 (PD-1) is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment. Here, we find that αPD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to αPD-1, as decreasing Acod1 levels in αPD-1-resistant cancer cells can sensitize tumors to αPD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naive CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on the secretion of ITA but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

NF1 deficiency drives metabolic reprogramming in ER+ breast cancer.

OBJECTIVE: NF1 is a tumor suppressor gene and its protein product, neurofibromin, is a negative regulator of the RAS pathway. NF1 is one of the top driver mutations in sporadic breast cancer such that 27 % of breast cancers exhibit damaging NF1 alterations. NF1 loss-of-function is a frequent event in the genomic evolution of estrogen receptor (ER)+ breast cancer metastasis and endocrine resistance. Individuals with Neurofibromatosis type 1 (NF) - a disorder caused by germline NF1 mutations - have an increased risk of dying from breast cancer [1-4]. NF-related breast cancers are associated with decreased overall survival compared to sporadic breast cancer. Despite numerous studies interrogating the role of RAS mutations in tumor metabolism, no study has comprehensively profiled the NF1-deficient breast cancer metabolome to define patterns of energetic and metabolic reprogramming. The goals of this investigation were (1) to define the role of NF1 deficiency in estrogen receptor-positive (ER+) breast cancer metabolic reprogramming and (2) to identify potential targeted pathway and metabolic inhibitor combination therapies for NF1-deficient ER + breast cancer. METHODS: We employed two ER+ NF1-deficient breast cancer models: (1) an NF1-deficient MCF7 breast cancer cell line to model sporadic breast cancer, and (2) three distinct, Nf1-deficient rat models to model NF-related breast cancer [1]. IncuCyte proliferation analysis was used to measure the effect of NF1 deficiency on cell proliferation and drug response. Protein quantity was assessed by Western Blot analysis. We then used RNAseq to investigate the transcriptional effect of NF1 deficiency on global and metabolism-related transcription. We measured cellular energetics using Agilent Seahorse XF-96 Glyco Stress Test and Mito Stress Test assays. We performed stable isotope labeling and measured [U-13C]-glucose and [U-13C]-glutamine metabolite incorporation and measured total metabolite pools using mass spectrometry. Lastly, we used a Bliss synergy model to investigate NF1-driven changes in targeted and metabolic inhibitor synergy. RESULTS: Our results revealed that NF1 deficiency enhanced cell proliferation, altered neurofibromin expression, and increased RAS and PI3K/AKT pathway signaling while constraining oxidative ATP production and restricting energetic flexibility. Neurofibromin deficiency also increased glutamine influx into TCA intermediates and dramatically increased lipid pools, especially triglycerides (TG). Lastly, NF1 deficiency alters the synergy between metabolic inhibitors and traditional targeted inhibitors. This includes increased synergy with inhibitors targeting glycolysis, glutamine metabolism, mitochondrial fatty acid transport, and TG synthesis. CONCLUSIONS: NF1 deficiency drives metabolic reprogramming in ER+ breast cancer. This reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion, and these metabolic changes introduce novel metabolic-to-targeted inhibitor synergies.

Neutrophils resist ferroptosis and promote breast cancer metastasis through aconitate decarboxylase 1.

Metastasis causes breast cancer-related mortality. Tumor-infiltrating neutrophils (TINs) inflict immunosuppression and promote metastasis. Therapeutic debilitation of TINs may enhance immunotherapy, yet it remains a challenge to identify therapeutic targets highly expressed and functionally essential in TINs but under-expressed in extra-tumoral neutrophils. Here, using single-cell RNA sequencing to compare TINs and circulating neutrophils in murine mammary tumor models, we identified aconitate decarboxylase 1 (Acod1) as the most upregulated metabolic enzyme in mouse TINs and validated high Acod1 expression in human TINs. Activated through the GM-CSF-JAK/STAT5-C/EBPß pathway, Acod1 produces itaconate, which mediates Nrf2-dependent defense against ferroptosis and upholds the persistence of TINs. Acod1 ablation abates TIN infiltration, constrains metastasis (but not primary tumors), bolsters antitumor T cell immunity, and boosts the efficacy of immune checkpoint blockade. Our findings reveal how TINs escape from ferroptosis through the Acod1-dependent immunometabolism switch and establish Acod1 as a target to offset immunosuppression and improve immunotherapy against metastasis.

Acod1 Expression in Cancer Cells Promotes Immune Evasion through the Generation of Inhibitory Peptides.

Targeting PD-1 is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment (TME). Here, we find that αPD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to αPD-1, as decreasing Acod1 levels in αPD-1 resistant cancer cells can sensitize tumors to αPD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naïve CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on secretion of ITA, but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

Ketolysis drives CD8+ T cell effector function through effects on histone acetylation.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. ßOHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, ßOHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.

New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape.

The eukaryotic translation initiation factor 5A1 (eIF5A1) and 5A2 (eIF5A2) are important proteins in a variety of physiological and pathophysiological processes and their function has been linked to neurodevelopmental disorders, cancer, and viral infections. Here, we report two new genome-edited mouse models, generated using a CRISPR-Cas9 approach, in which the amino acid residue lysine 50 is replaced with arginine 50 (K50R) in eIF5A1 or in the closely related eIF5A2 protein. This mutation prevents the spermidine-dependent post-translational formation of hypusine, a unique lysine derivative that is necessary for activation of eIF5A1 and eIF5A2. Mouse brain lysates from homozygous eif5a2-K50R mutant mice (eif5a2K50R/K50R) confirmed the absence of hypusine formation of eIF5A2, and metabolomic analysis of primary mouse dermal fibroblasts revealed significant alterations in the metabolite landscape compared to controls including increased levels of tryptophan, kyrunenine, pyridoxine, nicotinamide adenine dinucleotide, riboflavin, flavin adenine dinucleotide, pantothenate, and coenzyme A. Further supported by new publicly available bioinformatics data, these new mouse models represent excellent in vivo models to study hypusine-dependent biological processes, hypusination-related disorders caused by eIF5A1 and eIF5A2 gene aberrations or mRNA expression dysregulation, as well as several major human cancer types and potential therapies.

PGC-1ß maintains mitochondrial metabolism and restrains inflammatory gene expression.

Metabolic programming of the innate immune cells known as dendritic cells (DCs) changes in response to different stimuli, influencing their function. While the mechanisms behind increased glycolytic metabolism in response to inflammatory stimuli are well-studied, less is known about the programming of mitochondrial metabolism in DCs. We used lipopolysaccharide (LPS) and interferon-ß (IFN-ß), which differentially stimulate the use of glycolysis and oxidative phosphorylation (OXPHOS), respectively, to identify factors important for mitochondrial metabolism. We found that the expression of peroxisome proliferator-activated receptor gamma co-activator 1ß (PGC-1ß), a transcriptional co-activator and known regulator of mitochondrial metabolism, decreases when DCs are activated with LPS, when OXPHOS is diminished, but not with IFN-ß, when OXPHOS is maintained. We examined the role of PGC-1ß in bioenergetic metabolism of DCs and found that PGC-1ß deficiency indeed impairs their mitochondrial respiration. PGC-1ß-deficient DCs are more glycolytic compared to controls, likely to compensate for reduced OXPHOS. PGC-1ß deficiency also causes decreased capacity for ATP production at steady state and in response to IFN-ß treatment. Loss of PGC-1ß in DCs leads to increased expression of genes in inflammatory pathways, and reduced expression of genes encoding proteins important for mitochondrial metabolism and function. Collectively, these results demonstrate that PGC-1ß is a key regulator of mitochondrial metabolism and negative regulator of inflammatory gene expression in DCs.

Principles of reproducible metabolite profiling of enriched lymphocytes in tumors and ascites from human ovarian cancer.

Identifying metabolites and delineating their immune-regulatory contribution in the tumor microenvironment is an area of intense study. Interrogating metabolites and metabolic networks among immune cell subsets and host cells from resected tissues and fluids of human patients presents a major challenge, owing to the specialized handling of samples for downstream metabolomics. To address this, we first outline the importance of collaborating with a biobank for coordinating and streamlining workflow for point of care, sample collection, processing and cryopreservation. After specimen collection, we describe our 60-min rapid bead-based cellular enrichment method that supports metabolite analysis between T cells and tumor cells by mass spectrometry. We also describe how the metabolic data can be complemented with metabolic profiling by flow cytometry. This protocol can serve as a foundation for interrogating the metabolism of cell subsets from primary human ovarian cancer.

Structure of an AMPK complex in an inactive, ATP-bound state.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.

Itaconate confers tolerance to late NLRP3 inflammasome activation.

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer.

Immune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few exceptions, their identities remain largely unknown. Here, we profiled tumor and T cells from tumor and ascites of patients with high-grade serous carcinoma (HGSC) to uncover the metabolomes of these distinct TME compartments. Cells within the ascites and tumor had pervasive metabolite differences, with a notable enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared with ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Functionally, MNA induces T cells to secrete the tumor-promoting cytokine tumor necrosis factor alpha. Thus, TME-derived MNA contributes to the immune modulation of T cells and represents a potential immunotherapy target to treat human cancer.

Repression of LKB1 by miR-17∼92 Sensitizes MYC-Dependent Lymphoma to Biguanide Treatment.

Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies.

Methionine Metabolism Shapes T Helper Cell Responses through Regulation of Epigenetic Reprogramming.

Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Here we identify methionine as a key nutrient affecting epigenetic reprogramming in CD4+ T helper (Th) cells. Using metabolomics, we showed that methionine is rapidly taken up by activated T cells and serves as the major substrate for biosynthesis of the universal methyl donor S-adenosyl-L-methionine (SAM). Methionine was required to maintain intracellular SAM pools in T cells. Methionine restriction reduced histone H3K4 methylation (H3K4me3) at the promoter regions of key genes involved in Th17 cell proliferation and cytokine production. Applied to the mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), dietary methionine restriction reduced the expansion of pathogenic Th17 cells in vivo, leading to reduced T cell-mediated neuroinflammation and disease onset. Our data identify methionine as a key nutritional factor shaping Th cell proliferation and function in part through regulation of histone methylation.

Disrupting Mitochondrial Pyruvate Uptake Directs Glutamine into the TCA Cycle away from Glutathione Synthesis and Impairs Hepatocellular Tumorigenesis.

Hepatocellular carcinoma (HCC) is a devastating cancer increasingly caused by non-alcoholic fatty liver disease (NAFLD). Disrupting the liver Mitochondrial Pyruvate Carrier (MPC) in mice attenuates NAFLD. Thus, we considered whether liver MPC disruption also prevents HCC. Here, we use the N-nitrosodiethylamine plus carbon tetrachloride model of HCC development to test how liver-specific MPC knock out affects hepatocellular tumorigenesis. Our data show that liver MPC ablation markedly decreases tumorigenesis and that MPC-deficient tumors transcriptomically downregulate glutathione metabolism. We observe that MPC disruption and glutathione depletion in cultured hepatomas are synthetically lethal. Stable isotope tracing shows that hepatocyte MPC disruption reroutes glutamine from glutathione synthesis into the tricarboxylic acid (TCA) cycle. These results support a model where inducing metabolic competition for glutamine by MPC disruption impairs hepatocellular tumorigenesis by limiting glutathione synthesis. These findings raise the possibility that combining MPC disruption and glutathione stress may be therapeutically useful in HCC and additional cancers.

Oxaloacetic acid mediates ADP-dependent inhibition of mitochondrial complex II-driven respiration.

We recently reported a previously unrecognized mitochondrial respiratory phenomenon. When [ADP] was held constant ("clamped") at sequentially increasing concentrations in succinate-energized muscle mitochondria in the absence of rotenone (commonly used to block complex I), we observed a biphasic, increasing then decreasing, respiratory response. Here we investigated the mechanism. We confirmed decades-old reports that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH). We then used an NMR method to assess OAA concentrations (known as difficult to measure by MS) as well as those of malate, fumarate, and citrate in isolated succinate-respiring mitochondria. When these mitochondria were incubated at varying clamped ADP concentrations, respiration increased at low [ADP] as expected given the concurrent reduction in membrane potential. With further increments in [ADP], respiration decreased associated with accumulation of OAA. Moreover, a low pyruvate concentration, that alone was not enough to drive respiration, was sufficient to metabolize OAA to citrate and completely reverse the loss of succinate-supported respiration at high [ADP]. Further, chemical or genetic inhibition of pyruvate uptake prevented OAA clearance and preserved respiration. In addition, we measured the effects of incremental [ADP] on NADH, superoxide, and H2O2 (a marker of reverse electron transport from complex II to I). In summary, our findings, taken together, support a mechanism (detailed within) wherein succinate-energized respiration as a function of increasing [ADP] is initially increased by [ADP]-dependent effects on membrane potential but subsequently decreased at higher [ADP] by inhibition of succinate dehydrogenase by OAA. The physiologic relevance is discussed.

Fibroblast growth factor 21 increases hepatic oxidative capacity but not physical activity or energy expenditure in hepatic peroxisome proliferator-activated receptor γ coactivator-1α-deficient mice.

NEW FINDINGS: What is the central question of this study? Does a reduction in hepatic peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which has been observed in an insulin-resistant obese state, impair the ability of fibroblast growth factor 21 (FGF21) to modulate metabolism? What is the main finding and its importance? A deficit in hepatic PGC-1α does not compromise the ability of FGF21 to increase hepatic fatty acid oxidation; however, the effects of FGF21 to regulate whole-body metabolism (i.e. total and resting energy expenditure), as well as ambulatory activity, were altered when hepatic PGC-1α was reduced. ABSTRACT: Fibroblast growth factor 21 (FGF21) treatment drives metabolic improvements, including increased metabolic flux and reduced hepatic steatosis, but the mechanisms responsible for these effects remain to be elucidated fully. We tested whether a targeted reduction in hepatic peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which has been shown to occur with obesity, had a negative impact on the metabolic effects of FGF21. We infused FGF21 (1 mg kg-1  day-1 ) or saline in chow-fed wild-type (WT) and liver-specific PGC-1α heterozygous (LPGC-1α) mice for 4 weeks. Administration of FGF21 lowered serum insulin and cholesterol (P ≤ 0.05) and tended to lower free fatty acids (P = 0.057). The LPGC-1α mice exhibited reduced complete hepatic fatty acid oxidation (FAO; LPGC-1α, 1788 ± 165 nmol g-1  h-1 compared with WT, 2572 ± 437 nmol g-1  h-1 ; P < 0.001), which was normalized by FGF21 treatment (2788 ± 519 nmol g-1  h-1 ; P < 0.001). FGF21 also increased hepatic incomplete FAO by 12% in both groups and extramitochondrial FAO by 89 and 56% in WT and LPGC-1α mice, respectfully (P = 0.001), and lowered hepatic triacylglycerol by 30-40% (P < 0.001). Chronic treatment with FGF21 lowered body weight and fat mass (P < 0.05), while increasing food consumption (P < 0.05), total energy expenditure [7.3 ± 0.60 versus 6.6 ± 0.39 kcal (12 h)-1 in WT mice; P = 0.009] and resting energy expenditure [5.4 ± 0.89 versus 4.6 ± 0.21 kcal (12 h)-1 in WT mice; P = 0.005]. Interestingly, FGF21 only increased ambulatory activity in the WT mice (P = 0.03), without a concomitant increase in non-resting energy expenditure. In conclusion, although reduced hepatic PGC-1α expression was not necessary for FGF21 to increase FAO, it does appear to mediate FGF21-induced changes in total and resting energy expenditure and ambulatory activity in lean mice.