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.

Ketones and the Heart: Metabolic Principles and Therapeutic Implications.

The ketone bodies beta-hydroxybutyrate and acetoacetate are hepatically produced metabolites catabolized in extrahepatic organs. Ketone bodies are a critical cardiac fuel and have diverse roles in the regulation of cellular processes such as metabolism, inflammation, and cellular crosstalk in multiple organs that mediate disease. This review focuses on the role of cardiac ketone metabolism in health and disease with an emphasis on the therapeutic potential of ketosis as a treatment for heart failure (HF). Cardiac metabolic reprogramming, characterized by diminished mitochondrial oxidative metabolism, contributes to cardiac dysfunction and pathologic remodeling during the development of HF. Growing evidence supports an adaptive role for ketone metabolism in HF to promote normal cardiac function and attenuate disease progression. Enhanced cardiac ketone utilization during HF is mediated by increased availability due to systemic ketosis and a cardiac autonomous upregulation of ketolytic enzymes. Therapeutic strategies designed to restore high-capacity fuel metabolism in the heart show promise to address fuel metabolic deficits that underpin the progression of HF. However, the mechanisms involved in the beneficial effects of ketone bodies in HF have yet to be defined and represent important future lines of inquiry. In addition to use as an energy substrate for cardiac mitochondrial oxidation, ketone bodies modulate myocardial utilization of glucose and fatty acids, two vital energy substrates that regulate cardiac function and hypertrophy. The salutary effects of ketone bodies during HF may also include extra-cardiac roles in modulating immune responses, reducing fibrosis, and promoting angiogenesis and vasodilation. Additional pleotropic signaling properties of beta-hydroxybutyrate and AcAc are discussed including epigenetic regulation and protection against oxidative stress. Evidence for the benefit and feasibility of therapeutic ketosis is examined in preclinical and clinical studies. Finally, ongoing clinical trials are reviewed for perspective on translation of ketone therapeutics for the treatment of HF.

Loss of hepatic phosphoenolpyruvate carboxykinase 1 dysregulates metabolic responses to acute exercise but enhances adaptations to exercise training in mice.

Acute exercise increases liver gluconeogenesis to supply glucose to working muscles. Concurrently, elevated liver lipid breakdown fuels the high energetic cost of gluconeogenesis. This functional coupling between liver gluconeogenesis and lipid oxidation has been proposed to underlie the ability of regular exercise to enhance liver mitochondrial oxidative metabolism and decrease liver steatosis in individuals with nonalcoholic fatty liver disease. Herein we tested whether repeated bouts of increased hepatic gluconeogenesis are necessary for exercise training to lower liver lipids. Experiments used diet-induced obese mice lacking hepatic phosphoenolpyruvate carboxykinase 1 (KO) to inhibit gluconeogenesis and wild-type (WT) littermates. 2H/13C metabolic flux analysis quantified glucose and mitochondrial oxidative fluxes in untrained mice at rest and during acute exercise. Circulating and tissue metabolite levels were determined during sedentary conditions, acute exercise, and refeeding postexercise. Mice also underwent 6 wk of treadmill running protocols to define hepatic and extrahepatic adaptations to exercise training. Untrained KO mice were unable to maintain euglycemia during acute exercise resulting from an inability to increase gluconeogenesis. Liver triacylglycerides were elevated after acute exercise and circulating ß-hydroxybutyrate was higher during postexercise refeeding in untrained KO mice. In contrast, exercise training prevented liver triacylglyceride accumulation in KO mice. This was accompanied by pronounced increases in indices of skeletal muscle mitochondrial oxidative metabolism in KO mice. Together, these results show that hepatic gluconeogenesis is dispensable for exercise training to reduce liver lipids. This may be due to responses in ketone body metabolism and/or metabolic adaptations in skeletal muscle to exercise.NEW & NOTEWORTHY Exercise training reduces hepatic steatosis partly through enhanced hepatic terminal oxidation. During acute exercise, hepatic gluconeogenesis is elevated to match the heightened rate of muscle glucose uptake and maintain glucose homeostasis. It has been postulated that the hepatic energetic stress induced by elevating gluconeogenesis during acute exercise is a key stimulus underlying the beneficial metabolic responses to exercise training. This study shows that hepatic gluconeogenesis is not necessary for exercise training to lower liver lipids.

Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines.

FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact FA dimers formed in the LC-MS pipeline. These isobaric FA dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at subphysiological concentrations showed concentration-dependent formation of a diverse repertoire of FA dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species.  However, nonbiological FA dimers forming in the setting of high concentrations of FFAs can be misidentified as FAHFAs. Based on these results, we assembled a FA dimer database to identify nonbiological FA dimers in untargeted metabolomics datasets.

Metabolic and Signaling Roles of Ketone Bodies in Health and Disease.

Ketone bodies play significant roles in organismal energy homeostasis, serving as oxidative fuels, modulators of redox potential, lipogenic precursors, and signals, primarily during states of low carbohydrate availability. Efforts to enhance wellness and ameliorate disease via nutritional, chronobiological, and pharmacological interventions have markedly intensified interest in ketone body metabolism. The two ketone body redox partners, acetoacetate and D-ß-hydroxybutyrate, serve distinct metabolic and signaling roles in biological systems. We discuss the pleiotropic roles played by both of these ketones in health and disease. While enthusiasm is warranted, prudent procession through therapeutic applications of ketogenic and ketone therapies is also advised, as a range of metabolic and signaling consequences continue to emerge. Organ-specific and cell-type-specific effects of ketone bodies are important to consider as prospective therapeutic and wellness applications increase.

Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading.

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.

Isolation and characterization of peptides with antihypertensive activity in foodstuffs.

Hypertension is one of the main causes of cardiovascular diseases. Synthetic drugs inhibiting ACE activity present high effectiveness in the treatment of hypertension but cause undesirable side effects. Unlike these synthetic drugs, antihypertensive peptides do not show any adverse effect. These peptides are naturally present in some foods and since hypertension is closely related to modern diet habits, the interest for this kind of foods is increasing. Different methods for the purification, isolation, and characterization of antihypertensive peptides in foods have been developed. Nevertheless, there is no revision work summarizing and comparing these strategies. In this review, in vivo and in vitro pathways to obtain antihypertensive peptides have been summarized. The ACE mechanism and the methodologies developed to assay the ACE inhibitory activity have also been described. Moreover, a comprehensive overview on the isolation, purification, and identification techniques focusing on the discovery of new antihypertensive peptides with high activity has been included. Finally, it is worthy to highlight that the quantitation of antihypertensive peptides in foods is a new trend since genotype and processing conditions could affect their presence. Analytical methodologies using mass spectrometry constitute an interesting option for this purpose.

Gender-based heterogeneity of FAHFAs in trained runners.

Fatty acid esters of hydroxy fatty acid (FAHFA) are anti-diabetic and anti-inflammatory lipokines. Recently FAHFAs were also found to predict cardiorespiratory fitness in a cross-sectional study of recreationally trained runners. Here we report the influences of body composition and gender on static FAHFA abundances in circulation. We compared the association between circulating FAHFA concentrations and body composition, determined by dual x-ray absorptiometry, in female recreational runners who were lean (BMI < 25 kg/m2, n = 6), to those who were overweight (BMI ≥ 25 kg/m2, n = 7). To characterize the effect of gender we also compared circulating FAHFAs in lean male recreational runners (n = 8) to recreationally trained lean female (n = 6) runner group. Circulating FAHFAs were increased in females in a manner that was modulated by specific adipose depot sizes, blood glucose, and lean body mass. As expected, circulating FAHFAs were diminished in the overweight group, but strikingly, within the lean cohort, increases in circulating FAHFAs were promoted by increased fat mass, relative to lean mass, while the overweight group showed a significantly attenuated relationship. These studies suggest multimodal regulation of circulating FAHFAs and raise hypotheses to test endogenous FAHFA dynamic sources and sinks in health and disease, which will be essential for therapeutic target development. Baseline circulating FAHFA concentrations could signal sub-clinical metabolic dysfunction in metabolically healthy obesity.

Ketone Bodies in Right Ventricular Failure: A Unique Therapeutic Opportunity.

Ketone bodies are pleotropic metabolites that play important roles in multiple biological processes ranging from bioenergetics to inflammation regulation via suppression of the NLRP3 inflammasome, and epigenetic modifications. Ketone bodies are elevated in left ventricular failure (LVF) and multiple approaches that increase ketone concentrations exert advantageous cardiac effects in rodents and humans. However, the relationships between ketone bodies and right ventricular failure (RVF) are relatively unexplored. Moreover, the cardioprotective properties of ketones in preclinical RVF are unknown. Here, we show a compensatory ketosis is absent in pulmonary arterial hypertension (PAH) patients with RVF. In the monocrotaline (MCT) rat model of PAH-mediated RVF, a dietary-induced ketosis improves RV function, suppresses NLRP3 inflammasome activation, and combats RV fibrosis. The summation of these data suggest ketogenic therapies may be particularly efficacious in RVF, and therefore future studies evaluating ketogenic interventions in human RVF are warranted.

Metabolic Messengers: ketone bodies.

Prospective molecular targets and therapeutic applications for ketone body metabolism have increased exponentially in the past decade. Initially considered to be restricted in scope as liver-derived alternative fuel sources during periods of carbohydrate restriction or as toxic mediators during diabetic ketotic states, ketogenesis and ketone bodies modulate cellular homeostasis in multiple physiological states through a diversity of mechanisms. Selective signalling functions also complement the metabolic fates of the ketone bodies acetoacetate and D-ß-hydroxybutyrate. Here we discuss recent discoveries revealing the pleiotropic roles of ketone bodies, their endogenous sourcing, signalling mechanisms and impact on target organs, and considerations for when they are either stimulated for endogenous production by diets or pharmacological agents or administered as exogenous wellness-promoting agents.

Gender-based heterogeneity of FAHFAs in trained runners.

Fatty acid esters of hydroxy fatty acid (FAHFA) are anti-diabetic and anti-inflammatory lipokines. Recently FAHFAs were also found to predict cardiorespiratory fitness in trained runners. Here we compared the association between circulating FAHFA baseline concentrations and body composition, determined by dual x-ray absorptiometry, in female runners who were lean (BMI < 25 kg/m2, n = 6), to those who were overweight (BMI ≥ 25 kg/m2, n = 7). We also compared circulating FAHFAs in lean male runners (n = 8) to the same trained lean female (n = 6) runner group. Circulating FAHFAs were increased in females in a manner that was modulated by specific adipose depot sizes, blood glucose, and lean body mass. As expected, circulating FAHFAs were diminished in the overweight group, but, strikingly, in both lean and overweight cohorts, increases in circulating FAHFAs were promoted by increased fat mass, relative to lean mass. These studies suggest multimodal regulation of circulating FAHFAs and raise hypotheses to test endogenous FAHFA dynamic sources and sinks in health and disease, which will be essential for therapeutic target development. Baseline circulating FAHFA concentrations could signal sub-clinical metabolic dysfunction in metabolically healthy obesity.

ATP-Gated Potassium Channels Contribute to Ketogenic Diet-Mediated Analgesia in Mice.

Chronic pain is a substantial health burden and options for treating chronic pain remain minimally effective. Ketogenic diets are emerging as well-tolerated, effective therapeutic strategies in preclinical models of chronic pain, especially diabetic neuropathy. We tested whether a ketogenic diet is antinociceptive through ketone oxidation and related activation of ATP-gated potassium (KATP) channels in mice. We demonstrate that consumption of a ketogenic diet for one week reduced evoked nocifensive behaviors (licking, biting, lifting) following intraplantar injection of different noxious stimuli (methylglyoxal, cinnamaldehyde, capsaicin, or Yoda1) in mice. A ketogenic diet also decreased the expression of p-ERK, an indicator of neuronal activation in the spinal cord, following peripheral administration of these stimuli. Using a genetic mouse model with deficient ketone oxidation in peripheral sensory neurons, we demonstrate that protection against methylglyoxal-induced nociception by a ketogenic diet partially depends on ketone oxidation by peripheral neurons. Injection of tolbutamide, a KATP channel antagonist, prevented ketogenic diet-mediated antinociception following intraplantar capsaicin injection. Tolbutamide also restored the expression of spinal activation markers in ketogenic diet-fed, capsaicin-injected mice. Moreover, activation of KATP channels with the KATP channel agonist diazoxide reduced pain-like behaviors in capsaicin-injected, chow-fed mice, similar to the effects observed with a ketogenic diet. Diazoxide also reduced the number of p-ERK+ cells in capsaicin-injected mice. These data support a mechanism that includes neuronal ketone oxidation and activation of KATP channels to provide ketogenic diet-related analgesia. This study also identifies KATP channels as a new target to mimic the antinociceptive effects of a ketogenic diet.

Ketone bodies in right ventricular failure: A unique therapeutic opportunity.

Background: Ketone bodies are pleotropic metabolites that play important roles in multiple biological processes ranging from bioenergetics to inflammation regulation via suppression of the NLRP3 inflammasome, and epigenetic modifications. Ketone bodies are elevated in left ventricular failure (LVF) and multiple approaches that increase ketone concentrations exert advantageous cardiac effects in rodents and humans. However, the relationships between ketone bodies and right ventricular failure (RVF) are relatively unexplored. Methods: 51 PAH patients were dichotomized into preserved or impaired RV function based on a cardiac index of 2.2 L/min/m2. Impaired RV function patients were further segmented into intermediate or severe RV dysfunction based on a right atrial pressure of 8 mm Hg. Serum ketone bodies acetoacetate (AcAc) and beta-hydroxybutyrate (ßOHB) were quantified using ultra performance liquid chromatography and mass spectrometry. In rodent studies, male Sprague Dawley rats were assigned to three groups: control (saline injection), monocrotaline (MCT) standard chow diet (MCT-Standard), and MCT ketogenic diet (MCT-Keto). Immunoblots and confocal microscopy probed macrophage NLRP3 activation in RV extracts and sections. RV fibrosis was determined by Picrosirus Red. Echocardiography evaluated RV function. Pulmonary arteriole remodeling was assessed from histological specimens. Results: Human RVF patients lacked a compensatory ketosis as serum AcAc and ßOHB levels were not associated with hemodynamic, echocardiographic, or biochemical measures of RV dysfunction. In rodent studies, AcAc and ßOHB levels were also not elevated in MCT-mediated RVF, but the ketogenic diet significantly increased AcAc and ßOHB levels. MCT-Keto exhibited suppressed NLRP3 activation with a reduction in NLRP3, ASC (apoptosis-associated speck-like protein), pro-caspase-1, and interleukin-1 beta on immunoblots. Moreover, the number of ASC-positive macrophage in RV sections was reduced, RV fibrosis was blunted, and RV function was augmented in MCT-Keto rats. Conclusion: The ketogenic response is blunted in pulmonary arterial hypertension (PAH) patients with RVF. In the MCT rat model of PAH-mediated RVF, a dietary-induced ketosis improves RV function, suppresses NLRP3 inflammasome activation, and combats RV fibrosis. The summation of these data suggest ketogenic therapies may be particularly efficacious in RVF, and therefore future studies evaluating ketogenic interventions in human RVF are warranted.

ATP-gated potassium channels contribute to ketogenic diet-mediated analgesia in mice.

Chronic pain is a substantial health burden and options for treating chronic pain remain minimally effective. Ketogenic diets are emerging as well-tolerated, effective therapeutic strategies in preclinical models of chronic pain, especially diabetic neuropathy. We tested whether a ketogenic diet is antinociceptive through ketone oxidation and related activation of ATP-gated potassium (KATP) channels in mice. We demonstrate that consumption of a ketogenic diet for one week reduced evoked nocifensive behaviors (licking, biting, lifting) following intraplantar injection of different noxious stimuli (methylglyoxal, cinnamaldehyde, capsaicin, or Yoda1) in mice. A ketogenic diet also decreased the expression of p-ERK, an indicator of neuronal activation in the spinal cord, following peripheral administration of these stimuli. Using a genetic mouse model with deficient ketone oxidation in peripheral sensory neurons, we demonstrate that protection against methylglyoxal-induced nociception by a ketogenic diet partially depends on ketone oxidation by peripheral neurons. Injection of tolbutamide, a KATP channel antagonist, prevented ketogenic diet-mediated antinociception following intraplantar capsaicin injection. Tolbutamide also restored the expression of spinal activation markers in ketogenic diet-fed, capsaicin-injected mice. Moreover, activation of KATP channels with the KATP channel agonist diazoxide reduced pain-like behaviors in capsaicin-injected, chow-fed mice, similar to the effects observed with a ketogenic diet. Diazoxide also reduced the number of p-ERK+ cells in capsaicin-injected mice. These data support a mechanism that includes neuronal ketone oxidation and activation of KATP channels to provide ketogenic diet-related analgesia. This study also identifies KATP channels as a new target to mimic the antinociceptive effects of a ketogenic diet.

Ketolysis is Required for the Proper Development and Function of the Somatosensory Nervous System.

Ketogenic diets are emerging as protective interventions in preclinical and clinical models of somatosensory nervous system disorders. Additionally, dysregulation of succinyl-CoA 3-oxoacid CoA-transferase 1 (SCOT, encoded by Oxct1 ), the fate-committing enzyme in mitochondrial ketolysis, has recently been described in Friedreich's ataxia and amyotrophic lateral sclerosis. However, the contribution of ketone metabolism in the normal development and function of the somatosensory nervous system remains poorly characterized. We generated sensory neuron-specific, Advillin-Cre knockout of SCOT (Adv-KO-SCOT) mice and characterized the structure and function of their somatosensory system. We used histological techniques to assess sensory neuronal populations, myelination, and skin and spinal dorsal horn innervation. We also examined cutaneous and proprioceptive sensory behaviors with the von Frey test, radiant heat assay, rotarod, and grid-walk tests. Adv-KO-SCOT mice exhibited myelination deficits, altered morphology of putative Aδ soma from the dorsal root ganglion, reduced cutaneous innervation, and abnormal innervation of the spinal dorsal horn compared to wildtype mice. Synapsin 1-Cre-driven knockout of Oxct1 confirmed deficits in epidermal innervation following a loss of ketone oxidation. Loss of peripheral axonal ketolysis was further associated with proprioceptive deficits, yet Adv-KO-SCOT mice did not exhibit drastically altered cutaneous mechanical and thermal thresholds. Knockout of Oxct1 in peripheral sensory neurons resulted in histological abnormalities and severe proprioceptive deficits in mice. We conclude that ketone metabolism is essential for the development of the somatosensory nervous system. These findings also suggest that decreased ketone oxidation in the somatosensory nervous system may explain the neurological symptoms of Friedreich's ataxia.

Mitochondrial citrate metabolism and efflux regulates trophoblast differentiation.

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

Metformin impairs trophoblast metabolism and differentiation in dose dependent manner.

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing risks of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin's impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 µM (therapeutic range) and 2000 µM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 µM metformin treated cells, 2000 µM metformin impaired oxidative metabolism and increased abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 µM, but not 200 µM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impairs trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

Metformin impairs trophoblast metabolism and differentiation in a dose-dependent manner.

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing the risk of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin's impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 µM (therapeutic range) and 2000 µM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 µM metformin-treated cells, 2000 µM metformin impaired oxidative metabolism and increased the abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 µM, but not 200 µM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impair trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

Ketolysis is required for the proper development and function of the somatosensory nervous system.

Ketogenic diets are emerging as protective interventions in preclinical and clinical models of somatosensory nervous system disorders. Additionally, dysregulation of succinyl-CoA 3-oxoacid CoA-transferase 1 (SCOT, encoded by Oxct1), the fate-committing enzyme in mitochondrial ketolysis, has recently been described in Friedreich's ataxia and amyotrophic lateral sclerosis. However, the contribution of ketone metabolism in the normal development and function of the somatosensory nervous system remains poorly characterized. We generated sensory neuron-specific, Advillin-Cre knockout of SCOT (Adv-KO-SCOT) mice and characterized the structure and function of their somatosensory system. We used histological techniques to assess sensory neuronal populations, myelination, and skin and spinal dorsal horn innervation. We also examined cutaneous and proprioceptive sensory behaviors with the von Frey test, radiant heat assay, rotarod, and grid-walk tests. Adv-KO-SCOT mice exhibited myelination deficits, altered morphology of putative Aδ soma from the dorsal root ganglion, reduced cutaneous innervation, and abnormal innervation of the spinal dorsal horn compared to wildtype mice. Synapsin 1-Cre-driven knockout of Oxct1 confirmed deficits in epidermal innervation following a loss of ketone oxidation. Loss of peripheral axonal ketolysis was further associated with proprioceptive deficits, yet Adv-KO-SCOT mice did not exhibit drastically altered cutaneous mechanical and thermal thresholds. Knockout of Oxct1 in peripheral sensory neurons resulted in histological abnormalities and severe proprioceptive deficits in mice. We conclude that ketone metabolism is essential for the development of the somatosensory nervous system. These findings also suggest that decreased ketone oxidation in the somatosensory nervous system may explain the neurological symptoms of Friedreich's ataxia.

Mitochondrial citrate metabolism and efflux regulate BeWo differentiation.

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established BeWo cell culture model of trophoblast differentiation. Differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.