Global Energy Metabolism Research Articles

The Role of Proton-Coupled Amino Acid Transporter 2 (SLC36A2) in Cold-Induced Thermogenesis of Mice.

Brown adipocytes mainly utilize glucose and fatty acids to produce energy, which play key roles in thermogenesis. Furthermore, brown adipocytes also utilize other substrates, such as amino acids, for energy expenditure in various conditions. Here, we report the new physiological roles of proton-coupled amino acid transporters, SLC36A2 and SLC36A3, on global energy metabolism. The relative mRNA expression levels of both Slc36a2 and Slc36a3 were all highest in brown adipose tissue. We then generated global Slc36a2 and Slc36a3 knockout mice to investigate their functions in metabolism. Neither loss of Slc36a2 nor Slc36a3 affected the body weight and body composition of the mice. Slc36a2 knockout mice exhibited increased oxygen consumption during the daytime. After cold treatment, inhibition of Slc36a2 significantly decreased the mass of brown adipose tissue compared to wildtype mice, while it lowered the expression level of Cpt1a. Moreover, the serum lipid levels and liver mass were also decreased in Slc36a2 knockout mice after cold treatment. On the contrary, Slc36a3 knockout impaired glucose tolerance and up-regulated serum LDL-cholesterol concentration. Thus, SLC36A2 and SLC36A3 play central and different roles in the energy metabolism of the mice.

Fructose-enabled killing of antibiotic-resistant Salmonella enteritidis by gentamicin: Insight from reprogramming metabolomics

Salmonella enterica is a food-borne pathogen that poses a severe threat to both poultry production and human health. Antibiotics are critical for the initial treatment of bacterial infections. However, the overuse and misuse of antibiotics results in the rapid evolution of antibiotic-resistant bacteria, and the discovery and development of new antibiotics are declining. Therefore, understanding antibiotic resistance mechanisms and developing novel control measures are essential. In the present study, GC-MS-based metabolomics analysis was performed to determine the metabolic profile of gentamicin sensitive (SE-S) and resistant (SE-R) S. enterica. Fructose was identified as a crucial biomarker. Further analysis demonstrated a global depressed central carbon metabolism and energy metabolism in SE-R. The decrease in the pyruvate cycle reduces the production of NADH and ATP, causing a decrease in membrane potential, which contributes to gentamicin resistance. Exogenous fructose potentiated the effectiveness of gentamicin in killing SE-R by promoting the pyruvate cycle, NADH, ATP and membrane potential, thereby increasing gentamicin intake. Further, fructose plus gentamicin improved the survival rate of chicken infected with gentamicin-resistant Salmonella in vivo. Given that metabolite structures are conserved across species, fructose identified from bacteria could be used as a biomarker for breeding disease-resistant phenotypes in chicken. Therefore, a novel strategy is proposed for fighting against antibiotic-resistant S. enterica, including exploring molecules suppressed by antibiotics and providing a new approach to find pathogen targets for disease resistance in chicken breeding.

Osteocytes contribute via nuclear receptor PPAR-alpha to maintenance of bone and systemic energy metabolism.

The view that bone and energy metabolism are integrated by common regulatory mechanisms is broadly accepted and supported by multiple strands of evidence. This includes the well-characterized role of the PPARγ nuclear receptor, which is a common denominator in energy metabolism and bone metabolism. Little is known, however, about the role of PPARα nuclear receptor, a major regulator of lipid metabolism in other organs, in bone. A side-by-side comparative study of 5-15 mo old mice with global PPARα deficiency (αKO) and mice with osteocyte-specific PPARα deficiency (αOTKO) in order to parse out the various activities of PPARα in the skeleton that are of local and systemic significance. This study included transcriptome analysis of PPARα-deficient osteocytes, and analyses of bone mass and bone microarchitecture, systemic energy metabolism with indirect calorimetry, and differentiation potential of hematopoietic and mesenchymal bone cell progenitors. These analyses were paired with in vitro studies of either intact or silenced for PPARα MLO-A5 cells to determine PPARα role in osteocyte bioenergetics. In osteocytes, PPARα controls large number of transcripts coding for signaling and secreted proteins which may regulate bone microenvironment and peripheral fat metabolism. In addition, PPARα in osteocytes controls their bioenergetics and mitochondrial response to stress, which constitutes up to 40% of total PPARα contribution to the global energy metabolism. Similarly to αKO mice, the metabolic phenotype of αOTKO mice (both males and females) is age-dependent. In younger mice, osteocyte metabolism contributes positively to global energetics, however, with aging the high-energy phenotype reverts to a low-energy phenotype and obesity develops, suggesting a longitudinal negative effect of impaired lipid metabolism and mitochondrial dysfunction in osteocytes deficient in PPARα. However, bone phenotype was not affected in αOTKO mice except in the form of an increased volume of marrow adipose tissue in males. In contrast, global PPARα deficiency in αKO mice led to enlarged bone diameter with a proportional increase in number of trabeculae and enlarged marrow cavities; it also altered differentiation of hematopoietic and mesenchymal marrow cells toward osteoclast, osteoblast and adipocyte lineages, respectively. PPARα role in bone is multileveled and complex. In osteocytes, PPARα controls the bioenergetics of these cells, which significantly contributes to systemic energy metabolism and their endocrine/paracrine function in controlling marrow adiposity and peripheral fat metabolism.

Magnetic resonance imaging to assess the brain response to fasting in glioblastoma-bearing rats as a model of cancer anorexia

BackgroundGlobal energy balance is a vital process tightly regulated by the brain that frequently becomes dysregulated during the development of cancer. Glioblastoma (GBM) is one of the most investigated malignancies, but its appetite-related disorders, like anorexia/cachexia symptoms, remain poorly understood.MethodsWe performed manganese enhanced magnetic resonance imaging (MEMRI) and subsequent diffusion tensor imaging (DTI), in adult male GBM-bearing (n = 13) or control Wistar rats (n = 12). A generalized linear model approach was used to assess the effects of fasting in different brain regions involved in the regulation of the global energy metabolism: cortex, hippocampus, hypothalamus and thalamus. The regions were selected on the contralateral side in tumor-bearing animals, and on the left hemisphere in control rats. An additional DTI-only experiment was completed in two additional GBM (n = 5) or healthy cohorts (n = 6) to assess the effects of manganese infusion on diffusion measurements.ResultsMEMRI results showed lower T1 values in the cortex (p-value < 0.001) and thalamus (p-value < 0.05) of the fed ad libitum GBM animals, as compared to the control cohort, consistent with increased Mn2+ accumulation. No MEMRI-detectable differences were reported between fed or fasting rats, either in control or in the GBM group. In the MnCl2-infused cohorts, DTI studies showed no mean diffusivity (MD) variations from the fed to the fasted state in any animal cohort. However, the DTI-only set of acquisitions yielded remarkably decreased MD values after fasting only in the healthy control rats (p-value < 0.001), and in all regions, but thalamus, of GBM compared to control animals in the fed state (p-value < 0.01). Fractional anisotropy (FA) decreased in tumor-bearing rats due to the infiltrate nature of the tumor, which was detected in both diffusion sets, with (p-value < 0.01) and without Mn2+ administration (p-value < 0.001).ConclusionsOur results revealed that an altered physiological brain response to fasting occurred in hunger related regions in GBM animals, detectable with DTI, but not with MEMRI acquisitions. Furthermore, the present results showed that Mn2+ induces neurotoxic inflammation, which interferes with diffusion MRI to detect appetite-induced responses through MD changes.

Bedside interpretation of cerebral energy metabolism utilizing microdialysis in neurosurgical and general intensive care.

The microdialysis technique was initially developed for monitoring neurotransmitters in animals. In 1995 the technique was adopted to clinical use and bedside enzymatic analysis of glucose, pyruvate, lactate, glutamate and glycerol. Under clinical conditions microdialysis has also been used for studying cytokines, protein biomarkers, multiplex proteomic and metabolomic analyses as well as for pharmacokinetic studies and evaluation of blood-brain barrier function. This review focuses on the variables directly related to cerebral energy metabolism and the possibilities and limitations of microdialysis during routine neurosurgical and general intensive care. Our knowledge of cerebral energy metabolism is to a large extent based on animal experiments performed more than 40 years ago. However, the different biochemical information obtained from various techniques should be recognized. The basic animal studies analyzed brain tissue homogenates while the microdialysis technique reflects the variables in a narrow zone of interstitial fluid surrounding the probe. Besides the difference of the volume investigated, the levels of the biochemical variables differ in different compartments. During bedside microdialysis cerebral energy metabolism is primarily reflected in measured levels of glucose, lactate and pyruvate and the lactate to pyruvate (LP) ratio. The LP ratio reflects cytoplasmatic redox-state which increases instantaneously during insufficient aerobic energy metabolism. Cerebral ischemia is characterized by a marked increase in intracerebral LP ratio at simultaneous decreases in intracerebral levels of pyruvate and glucose. Mitochondrial dysfunction is characterized by a moderate increase in LP ratio at a very marked increase in cerebral lactate and normal or elevated levels of pyruvate and glucose. The patterns are of importance in particular for interpretations in transient cerebral ischemia. A new technique for evaluating global cerebral energy metabolism by microdialysis of the draining cerebral venous blood is discussed. In experimental studies it has been shown that pronounced global cerebral ischemia is reflected in venous cerebral blood. Jugular bulb microdialysis has been investigated in patients suffering from subarachnoid hemorrhage, during cardiopulmonary bypass and resuscitation after out of hospital cardiac arrest. Preliminary results indicate that the new technique may give valuable information of cerebral energy metabolism in clinical conditions when insertion of an intracerebral catheter is contraindicated.

Postnatal deletion of β-catenin in preosteoblasts regulates global energy metabolism through increasing bone resorption and adipose tissue fibrosis.

Many studies revealed bone can regulate global energy metabolism and our previous study also showed that Wnt/β-catenin pathway is involved in this process. To better understand the participation of canonical Wnt pathway in energy metabolism, we examined the β-catenin knock-out (β-cat KO) mice by crossing the osterix-cre transgenic mice with β-cateninflox/flox mice. First, we identified that postnatal deletion of β-catenin in preosteoblasts led to decreased fat mass and increased energy expenditure in mice. Osteoprotegerin administration largely rescued the decreased fat mass and partly normalized the energy expenditure accompanied by the inhibition of bone resorption. Anti-resorption with alendronate or RANKL-antibody could also partly rescued the decreased bone mass, decreased fat mass and increased energy expenditure in β-cat KO mice. We further found that the adipose cells in the inguinal fat tissue were smaller and the extracellular matrix components around adipocytes accumulated more in β-cat KO mice than their controls by histomorphology. Gene analysis by RT-PCR showed that the expression of collagen VI is 4.8 folds in adipose tissue of the β-cat KO mice compared with the control mice. We further detected the expression of cytokines which were related to fibrosis and the data showed that the level of TGF-beta1 was elevated in both of bone marrow serum and adipose tissue derived from the β-cat KO mice. After administration of TGF-beta1 neutralizing antibody, the impaired energy metabolism was partly rescued in β-cat KO mice. Besides, anti-resorption treatment and TGF-beta1 antibody could partly suppress the increased expression of genes related to fat tissue fibrosis. These results indicate that the abnormal global energy metabolism in β-cat KO mice may be attributed to increasing bone resorption and adipose tissue fibrosis.

The PhoPR two-component system responds to oxygen deficiency and regulates the pathways for energy supply in Corynebacterium glutamicum.

The PhoPR two-component system, a highly conserved system in corynebacteria and mycobacteria, is involved in the cellular response to environmental stress. When analysing the transcriptomic data of Corynebacterium glutamicum strains under different dissolved oxygen (DO) levels, PhoPR was found to be the most responsive two-component system to DO changes. Here, we systematically investigated the expression of PhoPR in response to different DO levels and its impact on genes related to global regulation and energy metabolism. Using Green fluorescent protein as a reporter, we confirmed that PhoPR was significantly upregulated upon decrease of DO. Through real-time quantitative PCR and electrophoretic mobility shift assay, we found that the effector protein PhoP directly activated glxR (encoding a global regulator), pfk and gapA (encoding the glycolytic enzymes) and ctaD (encoding cytochrome c in the electron transport chain), while downregulated aceE and gltA (encoding the TCA cycle enzymes). Overexpression of phoP or phoR resulted in a decreased intracellular NAD+/NADH ratio and increased intracellular ATP level, consistent with the gene expression changes regulated by PhoP. These results reveal the PhoPR system respond to oxygen deficiency and is responsible for the regulation of pathways involved in the sustainability of the energy levels required under low oxygen conditions. Our findings in this study not only provide new insights into the adaptation pathways of C. glutamicum in response to low oxygen conditions but also identify new possible genetic targets for the construction of the new cell factories aimed toward industrial applications.

The Importance of Metabolism for Immune Homeostasis in Allergic Diseases.

There is increasing evidence that the metabolic status of T cells and macrophages is associated with severe phenotypes of chronic inflammation, including allergic inflammation. Metabolic changes in immune cells have a crucial role in their inflammatory or regulatory responses. This notion is reinforced by metabolic diseases influencing global energy metabolism, such as diabetes or obesity, which are known risk factors of severity in inflammatory conditions, due to the metabolic-associated inflammation present in these patients. Since several metabolic pathways are closely tied to T cell and macrophage differentiation, a better understanding of metabolic alterations in immune disorders could help to restore and modulate immune cell functions. This link between energy metabolism and inflammation can be studied employing animal, human or cellular models. Analytical approaches rank from classic immunological studies to integrated analysis of metabolomics, transcriptomics, and proteomics. This review summarizes the main metabolic pathways of the cells involved in the allergic reaction with a focus on T cells and macrophages and describes different models and platforms of analysis used to study the immune system and its relationship with metabolism.

Pharmacologic Modulation of Mitochondrial Pathogenic Changes in Alzheimer’s Aβ‐Challenged Neurons

Mitochondria play essential roles in maintaining the high levels of brain energy demands required to maintain physiological ion gradients across membranes critical for the generation of action potentials and to sustain transport systems at endothelial barriers. About 90% of the energy requirement is produced through glucose oxidation, a tightly controlled mitochondrial process leading to ATP production. As the major consumers of oxygen, mitochondria are also the most important generators of reactive oxygen species (ROS) as by‐products of the electron transport chain. Thus, it is not surprising that mitochondrial dysfunction is emerging as a major contributor to the pathobiology of age‐associated neurodegenerative disorders and is being considered a crucial player in the pathophysiology of Alzheimer’s disease (AD). In spite of numerous studies, the mechanistic links among ROS homeostasis, cellular metabolic alterations, and changes in cell bioenergetics, particularly at the level of synapsis and in relation to oligomeric forms of amyloid‐β (oligAβ), still remain elusive. Through a combination of classical biochemical and immunocytochemical approaches together with the real‐time assessment of global energy metabolism our studies provide insights into the detrimental role of oligAβ in neuronal cell lines and murine primary cortical neurons. Our findings demonstrate that oligAβ induces detrimental changes in mitochondrial function with loss of mitochondrial membrane potential, release of cytochrome C, and enhanced ROS generation. Assessment of global energy metabolism in the Seahorse metabolic analyzer after sequential addition of different modulators of the mitochondrial metabolic pathways, demonstrated an oligAβ‐mediated reduction in oxygen consumption affecting basal and maximal respiration capacity and causing decreased ATP production. Pharmacologic targeting of Aβ‐challenged neurons with compounds of known antioxidant activity restored mitochondrial integrity/function, rescuing metabolic and bioenergetic changes induced by oligAβ. Overall, our studies provide insights into the complex molecular mechanisms triggered by oligAβ which profoundly affect mitochondrial performance and highlight the potential of antioxidant pharmacologic targeting to ameliorate the metabolic/bioenergetics alterations encompassing the complex and multifactorial pathways affected in AD.Support or Funding InformationNIH grants AG059595 and AG051266, the BrightFocus Foundation grant A2015275S, and the Alzheimer’s Association ZEN‐14‐283969

Nrf2 activation through the PI3K/GSK-3 axis protects neuronal cells from A\u03b2-mediated oxidative and metabolic damage

BackgroundMounting evidence points to a crucial role of amyloid-β (Aβ) in the pathophysiology of Alzheimer’s disease (AD), a disorder in which brain glucose hypometabolism, downregulation of central elements of phosphorylation pathways, reduced ATP levels, and enhanced oxidative damage coexist, and sometimes precede, synaptic alterations and clinical manifestations. Since the brain has limited energy storage capacity, mitochondria play essential roles in maintaining the high levels of energy demand, but, as major consumers of oxygen, these organelles are also the most important generators of reactive oxygen species (ROS). Thus, it is not surprising that mitochondrial dysfunction is tightly linked to synaptic loss and AD pathophysiology. In spite of their relevance, the mechanistic links among ROS homeostasis, metabolic alterations, and cell bioenergetics, particularly in relation to Aβ, still remain elusive.MethodsWe have used classic biochemical and immunocytochemical approaches together with the evaluation of real-time changes in global energy metabolism in a Seahorse Metabolic Analyzer to provide insights into the detrimental role of oligAβ in SH-SY5Y and primary neurons testing their pharmacologic protection by small molecules.ResultsOur findings indicate that oligomeric Aβ induces a dramatic increase in ROS production and severely affects neuronal metabolism and bioenergetics. Assessment of global energy metabolism in real time demonstrated Aβ-mediated reduction in oxygen consumption affecting basal and maximal respiration and causing decreased ATP production. Pharmacologic targeting of Aβ-challenged neurons with a set of small molecules of known antioxidant and cytoprotective activity prevented the metabolic/bioenergetic changes induced by the peptide, fully restoring mitochondrial function while inducing an antioxidant response that counterbalanced the ROS production. Search for a mechanistic link among the protective small molecules tested identified the transcription factor Nrf2—compromised by age and downregulated in AD and transgenic models—as their main target and the PI3K/GSK-3 axis as the central pathway through which the compounds elicit their Aβ protective action.ConclusionsOur study provides insights into the complex molecular mechanisms triggered by oligAβ which profoundly affect mitochondrial performance and argues for the inclusion of small molecules targeting the PI3K/GSK-3 axis and Nrf2-mediated pathways as part of the current or future combinatorial therapies.

Bone Research Society Abstracts

Bone Research Society Abstracts

Vhl deletion in osteoblasts boosts cellular glycolysis and improves global glucose metabolism.

The skeleton has emerged as an important regulator of systemic glucose homeostasis, with osteocalcin and insulin representing prime mediators of the interplay between bone and energy metabolism. However, genetic evidence indicates that osteoblasts can influence global energy metabolism through additional, as yet unknown, mechanisms. Here, we report that constitutive or postnatally induced deletion of the hypoxia signaling pathway component von Hippel-Lindau (VHL) in skeletal osteolineage cells of mice led to high bone mass as well as hypoglycemia and increased glucose tolerance, not accounted for by osteocalcin or insulin. In vitro and in vivo data indicated that Vhl-deficient osteoblasts displayed massively increased glucose uptake and glycolysis associated with upregulated HIF-target gene expression, resembling the Warburg effect that typifies cancer cells. Overall, the glucose consumption by the skeleton was increased in the mutant mice, as revealed by 18F-FDG radioactive tracer experiments. Moreover, the glycemia levels correlated inversely with the level of skeletal glucose uptake, and pharmacological treatment with the glycolysis inhibitor dichloroacetate (DCA), which restored glucose metabolism in Vhl-deficient osteogenic cells in vitro, prevented the development of the systemic metabolic phenotype in the mutant mice. Altogether, these findings reveal a novel link between cellular glucose metabolism in osteoblasts and whole-body glucose homeostasis, controlled by local hypoxia signaling in the skeleton.

Arsenic exposure induces glucose intolerance and alters global energy metabolism.

Environmental pollutants acting as endocrine-disrupting chemicals (EDCs) are recognized as potential contributors to metabolic disease pathogenesis. One such pollutant, arsenic, contaminates the drinking water of ~100 million people globally and has been associated with insulin resistance and diabetes in epidemiological studies. Despite these observations, the precise metabolic derangements induced by arsenic remain incompletely characterized. In the present study, the impact of arsenic on in vivo metabolic physiology was examined in 8-wk-old male C57BL/6J mice exposed to 50 mg/l inorganic arsenite in their drinking water for 8 wk. Glucose metabolism was assessed via in vivo metabolic testing, and feeding behavior was analyzed using indirect calorimetry in metabolic cages. Pancreatic islet composition was assessed via immunofluorescence microscopy. Arsenic-exposed mice exhibited impaired glucose tolerance compared with controls; however, no difference in peripheral insulin resistance was noted between groups. Instead, early insulin release during glucose challenge was attenuated relative to the rise in glycemia. Despite decreased insulin secretion, pancreatic β-cell mass was not altered, suggesting that arsenic primarily disrupts β-cell function. Finally, metabolic cage analyses revealed that arsenic exposure induced novel alterations in the diurnal rhythm of food intake and energy metabolism. Taken together, these data suggest that arsenic exposure impairs glucose tolerance through functional impairments in insulin secretion from β-cells rather than by augmenting peripheral insulin resistance. Further elucidation of the mechanisms underlying arsenic-induced behavioral and β-cell-specific metabolic disruptions will inform future intervention strategies to address this ubiquitous environmental contaminant and novel diabetes risk factor.

Osteoblasts regulate global energy metabolism

Skeleton has been proven to be an endocrine organ. Osteoblasts, the bone-forming cells in the skeleton, can secret osteocalcin which regulates global energy metabolism and male fertility in its undercarboxylated form and lipocalin 2 which influences appetite through binding to MC4R in the hypothalamus. Being a highly metabolic cell, osteoblast needs lots of energy through utilizing glucose and fatty acid to support its proliferation, differentiation, and mineralization. Recent data have shown the consumption change of fuels by osteoblasts, which contributes to global energy metabolism. Wnt/β-catenin signaling in osteoblasts is very important for bone formation, and disruption or constitutive activation of this pathway through gene editing may influence global energy metabolism. Through interacting with Wnt/β-catenin signaling, parathyroid hormone and sclerostin monoclonal antibody perform their anti-osteoporosis effect, and affect glucose homeostasis, insulin sensitivity, lipid metabolism and browning of peripheral adipose tissue. Though great progress has been made to understand the regulation of energy metabolism by bone during recent years, a lot of questions still remain to be answered. (Chin J Endocrinol Metab, 2017, 33: 811-815) Key words: Osteoblast; Energy metabolism; Osteocalcin; Wnt/β-catenin pathway

Skeletal energy homeostasis: a paradigm of endocrine discovery.

Throughout the last decade, significant developments in cellular, molecular and mouse models have revealed major endocrine functions of the skeleton. More recent studies have evolved the interplay between bone-specific hormones, the skeleton, marrow adipose tissue, muscle and the brain. This review focuses on literature from the last decade, addressing the endocrine regulation of global energy metabolism via the skeleton. In addition, we will highlight several recent studies that further our knowledge of new endocrine functions of some organs; explore remaining unanswered questions; and, finally, we will discuss future directions for this more complex era of bone biology research.

Bone: Osteoblasts and global energy metabolism - beyond osteocalcin.

There is mounting evidence that osteoblasts are involved in the regulation of global energy metabolism. Although osteocalcin signalling remains important, the complexity of systemic energy storage and expenditure makes it likely that hitherto unknown factors are also involved in osteoblast-mediated regulation of metabolism.

Wnt/β-catenin signaling in osteoblasts regulates global energy metabolism

Obesity, diabetes and osteoporosis have become a major public heath burden, and understanding the underlying mechanisms of these pathophysiological process will benefit their treatment. Osteoblast lineage cells in charge of the bone formation have been showed to participate in the whole-body energy metabolism. In this study, we identify that wnt/β-catenin signaling in osteoblasts could regulate global energy metabolism, including glucose homeostasis, fat accumulation and energy expenditure. Mice lacking β-catenin specifically in osteoblasts postnatally exhibit decreased bone mass, increased glucose level, decreased insulin production, decreased fat accumulation and increased energy expenditure. Osteocalcin supplement can rescue the impaired glucose balance by improving insulin production but cannot influence the abnormal fat accumulation and energy expenditure. Osteoprotegerin (OPG) overexpression exclusively in osteoblasts in β-catenin deletion mice can normalize not only the decreased bone mass but also the decreased fat accumulation and increased energy expenditure. The effect of β-catenin deletion and OPG overexpression in osteoblasts on global energy metabolism had no relation with inguinal fat browning. These results suggest that the regulation of bone on energy metabolism and fat accumulation is not mediated exclusively by osteocalcin. Our findings may provide a new insight into the regulation of bone on fat accumulation and energy metabolism.

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum.

SummaryMetabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor β signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.

Mitochondrial Fission 1 Regulates GSK3 and AMPK Signaling to Sustain Leukemia Stem Cell Function in Acute Myelogenous Leukemia

Abstract Mitochondrial dynamics describes the ability of mitochondria to switch between fission and fusion-active states to change morphology. Although it has long been thought that mitochondrial dynamics is downstream of various growth/differentiation signals and metabolic cues, recent studies have shown that mitochondrial morphology can also actively regulate cellular metabolism and the cell fate of normal stem cells including hematopoietic stem cells. In the present studies, we examined the role of mitochondrial dynamics in the biology of human leukemia stem cells (LSCs). We observe that LSCs are highly sensitive to the perturbation of mitochondrial morphology and that defined signal transduction pathways are involved in this process. We therefore propose that maintenance of the LSC state is critically linked to mitochondrial dynamics. Our data show that LSC enriched populations derived from primary human acute myeloid leukemia (AML) specimens have hyper-active mitochondrial fission regulators as evidenced by higher levels of mitochondrial fission 1 (FIS1) and activating phosphorylation of dynamin-related protein 1 (DRP1). LSCs also contain an increased number of smaller and globular-shaped mitochondria, indicating that they reside in a fission-active state relative to non-LSCs. Inhibition of mitochondrial fission through genetic knock-down of FIS1 (FIS1-KD) induces mitochondrial fusion and dramatically diminishes both colony-forming ability and serial engraftment potential of primary AML cells, suggesting a fission-active state of mitochondrial morphology is critical for LSC function. To dissect the mechanism by which inhibition of mitochondrial fission impairs the stem and progenitor potential of LSCs, we performed detailed analyses of molecular events following FIS1-KD. We determined that FIS1-KD in AML cells simultaneously induces activating phosphorylation of AMPK and inhibitory phosphorylation of GSK3. Consistent with AMPK activation, FIS1-depleted AML cells had elevated oxidative phosphorylation (OXPHOS) activity, increased cellular ATP, and dramatic changes of various metabolomic intermediates, indicating a clear shift to active global energy metabolism. With regard to GSK3 inhibition, we found that the FIS1-KD-induced gene expression signature strongly phenocopied the gene signature obtained using a pan-GSK3 inhibitor or shRNA-mediated knock down of GSK3. Further, FIS1-KD in AML cells also strongly induced hematopoietic differentiation as evidenced by increased surface expression of hematopoietic lineage markers, global upregulation of hematopoietic differentiation genes, and collapse of the HOXA9 transcriptional program. Importantly, both shRNA-mediated AMPK inhibition and overexpression of a constitutively active GSK3 allele can rescue the FIS1-KD-induced hematopoietic differentiation phenotype, suggesting FIS1-KD-induced differentiation is dependent on both AMPK activation and GSK3 inhibition. To investigate if FIS1-KD-induced hematopoietic differentiation depends on an altered state of mitochondrial morphology, we also studied other regulators of mitochondrial dynamics including fission player DRP1 and fusion players mitofusin 2 (MFN2) and Optic Atrophy 1 (OPA1). We showed that shRNA-mediated KD of fusion players MFN2 and OPA1 can also rescue FIS1-KD induced hematopoietic differentiation, suggesting the balance between the activity of fission and fusion regulators is critical in determining the fate of LSCs. In addition, we showed that shRNA-mediated KD of the fission player DRP1 could also lead to AMPK activation and GSK3 inhibition, in strong agreement with FIS1-KD. Finally, we showed that combined treatment of AMPK activator and GSK3 inhibitors can severely kill AML cells, suggesting a potential therapeutic strategy. Taken together, we propose that mitochondrial dynamics plays a previously unrealized yet important role in sustaining LSCs of AML. Further, inhibition of mitochondrial fission players can activate AMPK signaling, force active energy metabolism, collapse GSK3-mediated transcription programs, and induce hematopoietic differentiation, all of which lead to loss of stem and progenitor potential of AML cells. Thus the current study reveals a novel dependence of LSCs on mitochondrial dynamics, and provides novel insights towards improved therapeutic regimens. Disclosures Pollyea: Celgene: Other: advisory board, Research Funding; Ariad: Other: advisory board; Glycomimetics: Other: DSMB member; Pfizer: Other: advisory board, Research Funding; Alexion: Other: advisory board.

A technique for continuous bedside monitoring of global cerebral energy state.

BackgroundCerebral cytoplasmatic redox state is a sensitive indicator of cerebral oxidative metabolism and is conventionally evaluated from the extracellular lactate/pyruvate (LP) ratio. In the present experimental study of global cerebral ischemia induced by hemorrhagic shock, we investigate whether the LP ratio obtained from microdialysis of cerebral venous blood may be used as a surrogate marker of global cerebral energy state.MethodsSix female pigs were anesthetized and vital parameters were recorded. Microdialysis catheters were placed in the left parietal lobe, the superior sagittal sinus, and the femoral artery. Hemorrhagic shock was achieved by bleeding the animals to a mean arterial pressure (MAP) of approximately 40 mmHg and kept at a MAP of about 30–40 mmHg for 90 min. The animals were resuscitated with autologous whole blood followed by 3 h of observation.ResultsThe LP ratio obtained from the intracerebral and intravenous catheters immediately increased during the period of hemorrhagic shock while the LP ratio in the arterial blood remained close to normal levels. At the end of the experiment, median LP ratio (interquartile range) obtained from the intracerebral, intravenous, and intra-arterial microdialysis catheters were 846 (243–1990), 309 (103–488), and 27 (21–31), respectively. There was a significant difference in the LP ratio obtained from the intravenous location and the intra-arterial location (P < 0.001).ConclusionsDuring cerebral ischemia induced by severe hemorrhagic shock, intravascular microdialysis of the draining venous blood will exhibit changes of the LP ratio revealing the deterioration of global cerebral oxidative energy metabolism. In neurocritical care, this technique might be used to give information regarding global cerebral energy metabolism in addition to the regional information obtained from intracerebral microdialysis catheters. The technique might also be used to evaluate cerebral energy state in various critical care conditions when insertion of an intracerebral microdialysis catheter may be contraindicated, e.g., resuscitation after cardiac standstill, open-heart surgery, and multi-trauma.