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.

Metabolic Profiling Using Stable Isotope Tracing Reveals Distinct Patterns of Glucose Utilization by Physiologically Activated CD8+ T Cells.

Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.

Prior metabolite extraction fully preserves RNAseq quality and enables integrative multi-'omics analysis of the liver metabolic response to viral infection.

Here, we provide an in-depth analysis of the usefulness of single-sample metabolite/RNA extraction for multi-'omics readout. Using pulverized frozen livers of mice injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh), we isolated RNA prior (RNA) or following metabolite extraction (MetRNA). RNA sequencing (RNAseq) data were evaluated for differential expression analysis and dispersion, and differential metabolite abundance was determined. Both RNA and MetRNA clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Over 85% of LCMV versus Veh differentially expressed genes were shared between extraction methods, with the remaining 15% evenly and randomly divided between groups. Differentially expressed genes unique to the extraction method were attributed to randomness around the 0.05 FDR cut-off and stochastic changes in variance and mean expression. In addition, analysis using the mean absolute difference showed no difference in the dispersion of transcripts between extraction methods. Altogether, our data show that prior metabolite extraction preserves RNAseq data quality, which enables us to confidently perform integrated pathway enrichment analysis on metabolomics and RNAseq data from a single sample. This analysis revealed pyrimidine metabolism as the most LCMV-impacted pathway. Combined analysis of genes and metabolites in the pathway exposed a pattern in the degradation of pyrimidine nucleotides leading to uracil generation. In support of this, uracil was among the most differentially abundant metabolites in serum upon LCMV infection. Our data suggest that hepatic uracil export is a novel phenotypic feature of acute infection and highlight the usefulness of our integrated single-sample multi-'omics approach.

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.

eNOS deletion impairs mitochondrial quality control and exacerbates Western diet-induced NASH.

Dysregulated mitochondrial quality control leads to mitochondrial functional impairments that are central to the development and progression of hepatic steatosis to nonalcoholic steatohepatitis (NASH). Here, we identify hepatocellular localized endothelial nitric oxide synthase (eNOS) as a novel master regulator of mitochondrial quality control. Mice lacking eNOS were more susceptible to Western diet-induced hepatic inflammation and fibrosis in conjunction with decreased markers of mitochondrial biogenesis and turnover. The hepatocyte-specific influence was verified via magnetic activated cell sorting purified primary hepatocytes and in vitro siRNA-induced knockdown of eNOS. Hepatic mitochondria from eNOS knockout mice revealed decreased markers of mitochondrial biogenesis (PPARγ coactivator-1α, mitochondrial transcription factor A) and autophagy/mitophagy [BCL-2-interacting protein-3 (BNIP3), 1A/1B light chain 3B (LC3)], suggesting decreased mitochondrial turnover rate. eNOS knockout in primary hepatocytes exhibited reduced fatty acid oxidation capacity and were unable to mount a normal BNIP3 response to a mitophagic challenge compared with wild-type mice. Finally, we demonstrate that eNOS is required in primary hepatocytes to induce activation of the stress-responsive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). Thus, our data demonstrate that eNOS is an important regulator of hepatic mitochondrial content and function and NASH susceptibility.

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.

Transcriptomic differences in intra-abdominal adipose tissue in extremely obese adolescents with different stages of NAFLD.

Mechanisms responsible for progression of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH) remain poorly defined. To examine the potential contribution of adipose tissue to NAFLD progression, we performed a complete transcriptomic analysis using RNA sequencing (RNA-Seq) on intra-abdominal adipose tissue (IAT) from severely obese adolescents [Mage 16.9 ± 0.4 yr, body mass index (BMI) z-score 2.7 ± 0.1] undergoing bariatric surgery and liver biopsy categorized into three groups: no steatosis (normal, n = 8), steatosis only (n = 13), or NASH (n = 10) by liver histology. Age, body weight, and BMI did not differ among groups, but subjects with NASH were more insulin resistant (increased homeostatic model assessment/insulin resistance, P < 0.05 vs. other groups). RNA-Seq revealed 175 up- and 492 downregulated mRNA transcripts (≥±1.5-fold, false discovery rate <0.10) in IAT between NASH vs. Normal, with "mitochondrial dysfunction, P = 4.19E-7" being the top regulated canonical pathway identified by Ingenuity Pathway Analysis; only 19 mRNA transcripts were up- and 148 downregulated when comparing Steatosis vs. Normal, with suppression of "EIF2 signaling, P = 1.79E-27" being the top regulated pathway indicating increased cellular stress. A comparison of IAT between NASH vs. Steatosis found 515 up- and 175 downregulated genes, with "antigen presentation, P = 6.03E-18" being the top regulated canonical pathway and "inflammatory response" the top diseases and disorders function. Unique transcriptomic differences exist in IAT from severely obese adolescents with distinct stages of NAFLD, providing an important resource for identifying potential novel therapeutic targets for childhood NASH.

Aerobic exercise training in the treatment of non-alcoholic fatty liver disease related fibrosis.

KEY POINTS: Physiologically relevant rodent models of non-alcoholic steatohepatitis (NASH) that resemble the human condition are limited. Exercise training and energy restriction are first-line recommendations for the treatment of NASH. Hyperphagic Otsuka Long-Evans Tokushima fatty rats fed a western diet high in fat, sucrose and cholesterol for 24 weeks developed a severe NASH with fibrosis phenotype. Moderate intensity exercise training and modest energy restriction provided some improvement in the histological features of NASH that coincided with alterations in markers of hepatic stellate cell activation and extracellular matrix remodelling. The present study highlights the importance of lifestyle modification, including exercise training and energy restriction, in the regulation of advanced liver disease. ABSTRACT: The incidence of non-alcoholic steatohepatitis (NASH) is rising but the efficacy of lifestyle modifications to improve NASH-related outcomes remain unclear. We hypothesized that a western diet (WD) would induce NASH in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat and that lifestyle modification would improve this condition. Eight-week-old Long-Evans Tokushima Otsuka (L) and OLETF (O) rats consumed a control diet (10% kcal fat, 3.5% sucrose) or a WD (45% kcal fat, 17% sucrose, 1% cholesterol) for 24 weeks. At 20 weeks of age, additional WD-fed OLETFs were randomized to sedentary (O-SED), food restriction (O-FR; ∼25% kcal reduction vs. O-SED) or exercise training (O-EX; treadmill running 20 m min(-1) with a 15% incline, 60 min day(-1) , 5 days week(-1) ) conditions for 12 weeks. WD induced a NASH phenotype in OLETFs characterized by hepatic fibrosis (collagen 1α1 mRNA and hydroxyproline content), as well as elevated inflammation and non-alcoholic fatty liver disease activity scores, and hepatic stellate cell activation (α-smooth muscle actin) compared to Long-Evans Tokushima Otsuka rats. FR and EX modestly improved NASH-related fibrosis markers (FR: hydroxyproline content, P < 0.01; EX: collagen 1α1 mRNA, P < 0.05; both: fibrosis score, P < 0.01) and inflammation (both: inflammation score; FR: interleukin-1ß and tumor necrosis factor α) vs. O-SED. FR reduced hepatic stellate cell activation markers (transforming growth factor-ß protein and α-smooth muscle actin mRNA), whereas EX increased the hepatic stellate cell senescence marker CCN1 (P < 0.01 vs. O-SED). Additionally, both FR and EX normalized extracellular matrix remodelling markers to levels similar to L-WD (P > 0.05). Although neither EX nor FR led to complete resolution of the WD-induced NASH phenotype, both independently benefitted liver fibrosis via altered hepatic stellate cell activation and extracellular matrix remodelling.

Gestational exercise protects adult male offspring from high-fat diet-induced hepatic steatosis.

BACKGROUND & AIMS: Mounting evidence indicates that maternal exercise confers protection to adult offspring against various diseases. Here we hypothesized that maternal exercise during gestation would reduce high-fat diet (HFD)-induced hepatic steatosis in adult rat offspring. METHODS: Following conception, pregnant dams were divided into either voluntary wheel running exercise (GE) or wheel-locked sedentary (GS) groups throughout gestation (days 4-21). Post-weaning, offspring received either normal chow diet (CD; 10% fat, 70% carbohydrate, 20% protein) or HFD (45% fat, 35% carbohydrate, and 20% protein) until sacrificed at 4- or 8-months of age. RESULTS: GE did not affect offspring birth weight or litter size. HFD feeding in offspring increased weight gain, body fat percentage, and glucose tolerance test area under the curve (GTT-AUC). Male offspring from GE dams had reduced body fat percentage across all ages (p<0.05). In addition, 8-month male offspring from GE dams were protected against HFD-induced hepatic steatosis, which was associated with increased markers of hepatic mitochondrial biogenesis (PGC-1α and TFAM), autophagic potential (ATG12:ATG5 conjugation) and hepatic triacylglycerol secretion (MTTP). CONCLUSIONS: The current study provides the first evidence that gestational exercise can reduce susceptibility to HFD-induced hepatic steatosis in adult male offspring.

Fibroblast growth factor 21 and exercise-induced hepatic mitochondrial adaptations.

Exercise stimulates hepatic mitochondrial adaptations; however, the mechanisms remain largely unknown. Here we tested whether FGF21 plays an obligatory role in exercise induced hepatic mitochondrial adaptations by testing exercise responses in FGF21 knockout mice. FGF21 knockout (FGF21-KO) and wild-type (WT) mice (11-12 wk of age) had access to voluntary running wheels for exercise (EX) or remained sedentary for 8 wk. FGF21 deficiency resulted in greater body weight, adiposity, serum cholesterol, insulin, and glucose concentrations compared with WT mice (P < 0.05). In addition, hepatic mitochondrial complete palmitate oxidation, ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activity, and nuclear content of PGC-1α were 30-50% lower in FGF21-KO mice compared with WT mice (P < 0.01). EX effectively lowered body weight, adiposity, serum triglycerides, free fatty acids, and insulin and normalized mitochondrial complete palmitate oxidation in the FGF21-KO mice, whereas the reduced hepatic ß-HAD activity and lowered nuclear content of PGC-1α in FGF21-KO mice were not restored by EX. In addition, EX increased hepatic CPT-1α mRNA expression and ACC phosphorylation (a marker of increased AMPK activity) and reduced hepatic triacylglycerol content in both genotypes. However, FGF21-KO mice displayed a lower EX-induced increase in the mRNA expression of the hepatic gluconeogenic gene, PEPCK, compared with WT. In conclusion, FGF21 does not appear necessary for exercise-induced systemic and hepatic mitochondrial adaptations, but the increased adiposity, hyperinsulinemia, and impairments in hepatic mitochondrial function induced by FGF21 deficiency can be partially rescued by daily wheel running exercise.

Ablation of eNOS does not promote adipose tissue inflammation.

Adipose tissue (AT) inflammation is a hallmark characteristic of obesity and an important determinant of insulin resistance and cardiovascular disease; therefore, a better understanding of factors regulating AT inflammation is critical. It is well established that reduced vascular endothelial nitric oxide (NO) bioavailability promotes arterial inflammation; however, the role of NO in modulating inflammation in AT remains disputed. In the present study, 10-wk-old C57BL6 wild-type and endothelial nitric oxide synthase (eNOS) knockout male mice were randomized to either a control diet (10% kcal from fat) or a Western diet (44.9% kcal from fat, 17% sucrose, and 1% cholesterol) for 18 wk (n= 7 or 8/group). In wild-type mice, Western diet-induced obesity led to increased visceral white AT expression of inflammatory genes (e.g., MCP1, TNF-α, and CCL5 mRNAs) and markers of macrophage infiltration (e.g., CD68, ITGAM, EMR1, CD11C mRNAs, and Mac-2 protein), as well as reduced markers of mitochondrial content (e.g., OXPHOS complex I and IV protein). Unexpectedly, these effects of Western diet on visceral white AT were not accompanied by decreases in eNOS phosphorylation at Ser-1177 or increases in eNOS phosphorylation at Thr-495. Also counter to expectations, eNOS knockout mice, independent of the diet, were leaner and did not exhibit greater white or brown AT inflammation compared with wild-type mice. Collectively, these findings do not support the hypothesis that reduced NO production from eNOS contributes to obesity-related AT inflammation.

Chronic NOS inhibition accelerates NAFLD progression in an obese rat model.

The progression in nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis is a serious health concern, but the underlying mechanisms remain unclear. We hypothesized that chronic inhibition of nitric oxide (NO) synthase (NOS) via N(ω)-nitro-L-arginine methyl ester (L-NAME) would intensify liver injury in a rat model of obesity, insulin resistance, and NAFLD. Obese Otsuka Long-Evans Tokushima fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats received control or L-NAME (65-70 mg·kg(-1)·day(-1))-containing drinking water for 4 wk. L-NAME treatment significantly (P < 0.05) reduced serum NO metabolites and food intake in both groups. Remarkably, despite no increase in body weight, L-NAME treatment increased hepatic triacylglycerol content (+40%, P < 0.05) vs. control OLETF rats. This increase was associated with impaired (P < 0.05) hepatic mitochondrial state 3 respiration. Interestingly, the opposite effect was found in LETO rats, where L-NAME increased (P < 0.05) hepatic mitochondrial state 3 respiration. In addition, L-NAME induced a shift toward proinflammatory M1 macrophage polarity, as indicated by elevated hepatic CD11c (P < 0.05) and IL-1ß (P = 0.07) mRNA in OLETF rats and reduced expression of the anti-inflammatory M2 markers CD163 and CD206 (P < 0.05) in LETO rats. Markers of total macrophage content (CD68 and F4/80) mRNA were unaffected by L-NAME in either group. In conclusion, systemic NOS inhibition in the obese OLETF rats reduced hepatic mitochondrial respiration, increased hepatic triacylglycerol accumulation, and increased hepatic inflammation. These findings suggest an important role for proper NO metabolism in the hepatic adaptation to obesity.

Mother's exercise during pregnancy programmes vasomotor function in adult offspring.

The intrauterine environment is influenced by maternal behaviour and programmes atherosclerotic disease susceptibility in offspring. The aim of this investigation was to test the hypothesis that mothers' exercise during pregnancy improves endothelial function in 3-, 5- and 9-month-old porcine offspring. The pregnant sows in the exercise group ran for an average of 39.35 ± 0.75 min at 4.81 ± 0.35 km h(-1) each day for 5 days per week for all but the last week of gestation. This induced a significant reduction in resting heart rate (exercised group, 89.3 ± 3.5 beats min(-1); sedentary group, 102.1 ± 3.1 beats min(-1); P < 0.05) but no significant differences in gestational weight gain (65.8 ± 2.1 versus 63.3 ± 1.9%). No significant effect on bradykinin-induced vasorelaxation with and without l-NAME was observed. A significant main effect was identified on sodium nitroprusside-induced vasorelaxation (P = 0.01), manifested by a reduced response in femoral arteries of all age groups from exercised-trained swine. Nitric oxide signalling was not affected by maternal exercise. Protein expression of MYPT1 was reduced in femoral arteries from 3-month-old offspring of exercised animals. A significant interaction was observed for PPP1R14A (P < 0.05) transcript abundance and its protein product CPI-17. In conclusion, pregnant swine are able to complete an exercise-training protocol that matches the current recommendations for pregnant women. Gestational exercise is a potent stimulus for programming vascular smooth muscle relaxation in adult offspring. Specifically, exercise training for the finite duration of pregnancy decreases vascular smooth muscle responsiveness in adult offspring to an exogenous nitric oxide donor.

A high-throughput screening platform for discovering bacterial species and small molecules that modify animal physiology.

The gut microbiome has been proposed to influence many aspects of animal development and physiology. However, both the specific bacterial species and the molecular mechanisms by which bacteria exert these effects are unknown in most cases. Here, we established a high throughput screening platform using the model animal Caenorhabditis elegans for identifying bacterial species and mechanisms that influence animal development and physiology. From our initial screens we found that many Bacillus species can restore normal animal development to insulin signaling mutant animals that otherwise do not develop to adulthood. To determine how Bacilli influence animal development we screened a complete non-essential gene knockout library of Bacillus subtilis for mutants that no longer restored development to adulthood. We found the Bacillus gene speB is required for animal development. In the absence of speB, B. subtilis produces excess N1-aminopropylagmatine. This polyamine is taken up by animal intestinal cells via the polyamine transporter CATP-5. When this molecule is taken up in sufficient quantities it inhibits animal mitochondrial function and causes diverse species of animals to arrest their development. To our knowledge, these are the first observations that B. subtilis can produce N1-aminopropylagmatine and that polyamines produced by intestinal microbiome species can antagonize animal development and mitochondrial function. Given that Bacilli species are regularly isolated from animal intestinal microbiomes, including from humans, we propose that altered polyamine production from intestinal Bacilli is likely to also influence animal development and metabolism in other species and potentially even contribute developmental and metabolic pathologies in humans. In addition, our findings demonstrate that C. elegans can be used as a model animal to conduct high throughput screens for bacterial species and bioactive molecules that alter animal physiology.

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.

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 and adrenic acid. These PUFAs then accumulate in phospholipids, including 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 contributes to their sensitivity to ferroptosis.

ASCT2 is a major contributor to serine uptake in cancer cells.

The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo, others are auxotrophic and therefore reliant on serine uptake. Importantly, despite several transporters being known to be capable of transporting serine, the transporters that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (SLC1A5) as a major contributor to serine uptake in cancer cells. ASCT2 is well known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that estrogen receptor α (ERα) promotes serine uptake by directly activating SLC1A5 transcription. Collectively, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target.

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.

A hierarchical hepatic de novo lipogenesis substrate supply network utilizing pyruvate, acetate, and ketones.

Hepatic de novo lipogenesis (DNL) is a fundamental physiologic process that is often pathogenically elevated in metabolic disease. Treatment is limited by incomplete understanding of the metabolic pathways supplying cytosolic acetyl-CoA, the obligate precursor to DNL, including their interactions and proportional contributions. Here, we combined extensive 13C tracing with liver-specific knockout of key mitochondrial and cytosolic proteins mediating cytosolic acetyl-CoA production. We show that the mitochondrial pyruvate carrier (MPC) and ATP-citrate lyase (ACLY) gate the major hepatic lipogenic acetyl-CoA production pathway, operating in parallel with acetyl-CoA synthetase 2 (ACSS2). Given persistent DNL after mitochondrial citrate carrier (CiC) and ACSS2 double knockout, we tested the contribution of exogenous and leucine-derived acetoacetate to acetoacetyl-CoA synthetase (AACS)-dependent DNL. CiC knockout increased acetoacetate-supplied hepatic acetyl-CoA production and DNL, indicating that ketones function as mitochondrial-citrate reciprocal DNL precursors. By delineating a mitochondrial-cytosolic DNL substrate supply network, these findings may inform strategies to therapeutically modulate DNL.

Functional overlap of inborn errors of immunity and metabolism genes defines T cell metabolic vulnerabilities.

Inborn errors of metabolism (IEMs) and immunity (IEIs) are Mendelian diseases in which complex phenotypes and patient rarity have limited clinical understanding. Whereas few genes have been annotated as contributing to both IEMs and IEIs, immunometabolic demands suggested greater functional overlap. Here, CRISPR screens tested IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable previously unappreciated crossover. Analysis of IEMs showed that N-linked glycosylation and the hexosamine pathway enzyme Gfpt1 are critical for T cell expansion and function. Further, T helper (TH1) cells synthesized uridine diphosphate N-acetylglucosamine more rapidly and were more impaired by Gfpt1 deficiency than TH17 cells. Screening IEI genes found that Bcl11b promotes the CD4 T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. Thus, a high degree of functional overlap exists between IEM and IEI genes, and immunometabolic mechanisms may underlie a previously underappreciated intersection of these disorders.