Anabolic Requirements Research Articles

Crosstalk between degradation and bioenergetics: how autophagy and endolysosomal processes regulate energy production.

Cells undergo metabolic reprogramming to adapt to changes in nutrient availability, cellular activity, and transitions in cell states. The balance between glycolysis and mitochondrial respiration is crucial for energy production, and metabolic reprogramming stipulates a shift in such balance to optimize both bioenergetic efficiency and anabolic requirements. Failure in switching bioenergetic dependence can lead to maladaptation and pathogenesis. While cellular degradation is known to recycle precursor molecules for anabolism, its potential role in regulating energy production remains less explored. The bioenergetic switch between glycolysis and mitochondrial respiration involves transcription factors and organelle homeostasis, which are both regulated by the cellular degradation pathways. A growing body of studies has demonstrated that both stem cells and differentiated cells exhibit bioenergetic switch upon perturbations of autophagic activity or endolysosomal processes. Here, we highlighted the current understanding of the interplay between degradation processes, specifically autophagy and endolysosomes, transcription factors, endolysosomal signaling, and mitochondrial homeostasis in shaping cellular bioenergetics. This review aims to summarize the relationship between degradation processes and bioenergetics, providing a foundation for future research to unveil deeper mechanistic insights into bioenergetic regulation.

Reprogrammed mitochondria: a central hub of cancer cell metabolism.

Mitochondria represent the metabolic hub of normal cells and play this role also in cancer but with different functional purposes. While cells in differentiated tissues have the prerogative of maintaining basal metabolism and support the biosynthesis of specialized products, cancer cells have to rewire the metabolic constraints imposed by the differentiation process. They need to balance the bioenergetic supply with the anabolic requirements that entail the intense proliferation rate, including nucleotide and membrane lipid biosynthesis. For this aim, mitochondrial metabolism is reprogrammed following the activation of specific oncogenic pathways or due to specific mutations of mitochondrial proteins. The main process leading to mitochondrial metabolic rewiring is the alteration of the tricarboxylic acid cycle favoring the appropriate orchestration of anaplerotic and cataplerotic reactions. According to the tumor type or the microenvironmental conditions, mitochondria may decouple glucose catabolism from mitochondrial oxidation in favor of glutaminolysis or disable oxidative phosphorylation for avoiding harmful production of free radicals. These and other metabolic settings can be also determined by the neo-production of oncometabolites that are not specific for the tissue of origin or the accumulation of metabolic intermediates able to boost pro-proliferative metabolism also impacting epigenetic/transcriptional programs. The full characterization of tumor-specific mitochondrial signatures may provide the identification of new biomarkers and therapeutic opportunities based on metabolic approaches.

Hindering NAT8L expression in hepatocellular carcinoma increases cytosolic aspartate delivery that fosters pentose phosphate pathway and purine biosynthesis promoting cell proliferation

N-acetylaspartate (NAA) is synthesized by the mitochondrial enzyme NAT8L, which uses acetyl-CoA and aspartate as substrates. These metabolites are fundamental for bioenergetics and anabolic requirements of highly proliferating cells, thus, NAT8L modulation may impinge on the metabolic reprogramming of cancer cells. Specifically, aspartate represents a limiting amino acid for nucleotide synthesis in cancer.Here, the expression of the NAT8L enzyme was modulated to verify how it impacts the metabolic adaptations and proliferative capacity of hepatocellular carcinoma. We demonstrated that NAT8L downregulation is associated with increased proliferation of hepatocellular carcinoma cells and immortalized hepatocytes. The overexpression of NAT8L instead decreased cell growth. The pro-tumoral effect of NAT8L silencing depended on glutamine oxidation and the rewiring of glucose metabolism. Mechanistically, NAT8L downregulation triggers aspartate outflow from mitochondria via the exporter SLC25A13 to promote glucose flux into the pentose phosphate pathway, boosting purine biosynthesis. These results were corroborated by the analyses of human and mouse hepatocellular carcinoma samples revealing a decrease in NAT8L expression compared to adjacent non-tumoral tissues. Overall, this work demonstrates that NAT8L expression in liver cells limits the cytosolic availability of aspartate necessary for enhancing the pentose phosphate pathway and purine biosynthesis, counteracting cell proliferation.

HIV-1 replication and latency are balanced by mTOR-driven cell metabolism.

Human Immunodeficiency virus type 1 (HIV-1) relies on host cell metabolism for all aspects of viral replication. Efficient HIV-1 entry, reverse transcription, and integration occurs in activated T cells because HIV-1 proteins co-opt host metabolic pathways to fuel the anabolic requirements of virion production. The HIV-1 viral life cycle is especially dependent on mTOR, which drives signaling and metabolic pathways required for viral entry, replication, and latency. As a central regulator of host cell metabolism, mTOR and its downstream effectors help to regulate the expression of enzymes within the glycolytic and pentose phosphate pathways along with other metabolic pathways regulating amino acid uptake, lipid metabolism, and autophagy. In HIV-1 pathogenesis, mTOR, in addition to HIF-1α and Myc signaling pathways, alter host cell metabolism to create an optimal environment for viral replication. Increased glycolysis and pentose phosphate pathway activity are required in the early stages of the viral life cycle, such as providing sufficient dNTPs for reverse transcription. In later stages, fatty acid synthesis is required for creating cholesterol and membrane lipids required for viral budding. Epigenetics of the provirus fueled by metabolism and mTOR signaling likewise controls active and latent infection. Acetyl-CoA and methyl group abundance, supplied by the TCA cycle and amino acid uptake respectively, may regulate latent infection and reactivation. Thus, understanding and exploring new connections between cellular metabolism and HIV-1 pathogenesis may yield new insights into the latent viral reservoirs and fuel novel treatments and cure strategies.

Ral GTPases are critical regulators of spinal cord myelination and homeostasis.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.

Blockade of Cellular Energy Metabolism through 6-Aminonicotinamide Reduces Proliferation of Non-Small Lung Cancer Cells by Inducing Endoplasmic Reticulum Stress.

Simple SummaryMetabolism targeting for cancer treatment is currently under research in an effort to classify molecules that may block major metabolic steps accompanying cancer development and malignant growth. The approach is to compromise or entirely inhibit the increased metabolic pathways in cancer cells by suppressing the enzymatic activity of the involved proteins. Targeting cancer metabolism unlocks the prospect of improving broadly appropriate drugs that can treat various cancer cell types and may facilitate an innovative class of anticancer molecules. Several analogs of metabolites are currently being tested as possible drug candidates for cancer metabolism. Determining the effect of these metabolites on lung cancer offers the potential for a new class of therapeutic agents for cancer treatment. Thus, the efficient use of metabolic inhibitors could be a clinically promising therapeutic scheme.The pentose phosphate pathway (PPP) is the most common pathway in most cancer cells and stimulates antioxidant defense mechanisms and synthesis of biomolecule precursors. It is believed that cancer cells persistently ameliorate glucose flux into the PPP to maintain their anabolic requirements and adjust oxidative stress. TCGA analyses have indicated the upregulation of enzymes involved in PPP in lung cancer. Hence, the present study aimed to determine whether the pharmacological blockade of glucose 6-phosphate dehydrogenase (G6PD), the primary and rate-limiting enzyme involved in PPP, using 6-aminonicotinamide (6-AN), could induce antiproliferative activity in two lung cancer cell lines. Exposure to 6-AN suppressed lactate production and glucose consumption, modified the mitochondrial potential and redox balance, and thereby induced the endoplasmic reticulum (ER) stress to reduce lung cancer cell proliferation and govern cellular apoptosis. Collectively, this is the first study in which PPP blockade by 6-AN causes reactive oxygen species (ROS)-mediated apoptosis by ER stress in lung cancer cells. Further preclinical studies will be conducted to validate the biological applicability of these findings.

The predominant role of glucose as a building block and precursor of reducing equivalents.

Stores of glucose (Glc) in our body are small compared with protein and lipid. Therefore, at times of famines or trauma/disease-related starvation, glucose utilization must be limited only to pathways that can only run with glucose carbon as substrate. We will try to outline how insulin resistance drives these pathways and inhibits glucose oxidation in the stressed organism. Glc is a basic substrate for a variety of other biomolecules like nucleic acids, amino acids, proteoglycans, mucopolysaccharides and lipids. It is essential for the formation of reducing equivalents, indispensable for anabolic, antioxidative, regulatory and immune processes. As a result, a continuous Glc turnover/cycle is essential to secure at all times the Glc requirements for nonoxidative pathways mentioned above but then requires introduction of extra glucose or other intermediates into the cycle. The production of ATP through complete Glc oxidation occurs only when Glc intake is higher than required for its nonoxidative metabolism. Insulin resistance and decreased Glc oxidation indicate that requirements of Glc for anabolic pathways are high. Glc is an important building block for anabolic reactions and substrate for reducing equivalents formation. Insulin resistance prevents irreversible Glc oxidation and stimulates Glc production during stress or growth. Glc is only oxidized when intake is in excess of its anabolic requirements.

Glycolytic Profiling of Mouse Embryonic Stem Cells (mESCs).

Mouse embryonic stem cells (mESCs) can be captured in vitro in different pluripotency states through media modulation, mimicking their natural environment during early embryo development. As highly proliferative cells, mESCs prefer to use glycolysis to support the energetic and biosynthetic demands, even in the presence of oxygen. Indeed, glycolysis can not only supply ATP at a much faster rate, when compared to other catabolic pathways, but also provides biosynthetic substrates to meet anabolic requirements. Considering that ESCs cultured in different media conditions display distinct metabolic requirements, it is of utmost importance to have a robust metabolic characterization methodology to understand how subtle metabolic variations may be coupled to ESC identity. Here we describe how to profile the glycolytic activity of naive mouse ESC, using the established Seahorse XFe24 Live-cell Metabolic Assay. This may be a useful protocol for understanding how the glycolytic function of mESCs changes in certain circumstances and how is it coupled to diverse pluripotency/differentiation phenotypes.

Remarkable metabolic reorganization and altered metabolic requirements in frog metamorphic climax

BackgroundMetamorphic climax is the crucial stage of amphibian metamorphosis responsible for the morphological and functional changes necessary for transition to a terrestrial habitat. This developmental period is sensitive to environmental changes and pollution. Understanding its metabolic basis and requirements is significant for ecological and toxicological research. Rana omeimontis tadpoles are a useful model for investigating this stage as their liver is involved in both metabolic regulation and fat storage.ResultsWe used a combined approach of transcriptomics and metabolomics to study the metabolic reorganization during natural and T3-driven metamorphic climax in the liver and tail of Rana omeimontis tadpoles. The metabolic flux from the apoptotic tail replaced hepatic fat storage as metabolic fuel, resulting in increased hepatic amino acid and fat levels. In the liver, amino acid catabolism (transamination and urea cycle) was upregulated along with energy metabolism (TCA cycle and oxidative phosphorylation), while the carbohydrate and lipid catabolism (glycolysis, pentose phosphate pathway (PPP), and β-oxidation) decreased. The hepatic glycogen phosphorylation and gluconeogenesis were upregulated, and the carbohydrate flux was used for synthesis of glycan units (e.g., UDP-glucuronate). In the tail, glycolysis, β-oxidation, and transamination were all downregulated, accompanied by synchronous downregulation of energy production and consumption. Glycogenolysis was maintained in the tail, and the carbohydrate flux likely flowed into both PPP and the synthesis of glycan units (e.g., UDP-glucuronate and UDP-glucosamine). Fatty acid elongation and desaturation, as well as the synthesis of bioactive lipid (e.g., prostaglandins) were encouraged in the tail during metamorphic climax. Protein synthesis was downregulated in both the liver and tail. The significance of these metabolic adjustments and their potential regulation mechanism are discussed.ConclusionThe energic strategy and anabolic requirements during metamorphic climax were revealed at the molecular level. Amino acid made an increased contribution to energy metabolism during metamorphic climax. Carbohydrate anabolism was essential for the body construction of the froglets. The tail was critical in anabolism including synthesizing bioactive metabolites. These findings increase our understanding of amphibian metamorphosis and provide background information for ecological, evolutionary, conservation, and developmental studies of amphibians.

Flavin dependency undermines proteome stability, lipid metabolism and cellular proliferation during vitamin B2 deficiency

Tumor cells adapt their metabolism to meet the energetic and anabolic requirements of high proliferation and invasiveness. The metabolic addiction has motivated the development of therapies directed at individual biochemical nodes. However, currently there are few possibilities to target multiple enzymes in tumors simultaneously. Flavin-containing enzymes, ca. 100 proteins in humans, execute key biotransformations in mammalian cells. To expose metabolic addiction, we inactivated a substantial fraction of the flavoproteome in melanoma cells by restricting the supply of the FMN and FAD precursor riboflavin, the vitamin B2. Vitamin B2 deficiency affected stability of many polypeptides and thus resembled the chaperone HSP90 inhibition, the paradigmatic multiple-target approach. In support of this analogy, flavin-depleted proteins increasingly associated with a number of proteostasis network components, as identified by the mass spectrometry analysis of the FAD-free NQO1 aggregates. Proteome-wide analysis of the riboflavin-starved cells revealed a profound inactivation of the mevalonate pathway of cholesterol synthesis, which underlines the manifold cellular vulnerability created by the flavoproteome inactivation. Cell cycle-arrested tumor cells became highly sensitive to alkylating chemotherapy. Our data suggest that the flavoproteome is well suited to design synthetic lethality protocols combining proteostasis manipulation and metabolic reprogramming.

Abstract 1687: Tumor methionine metabolism drives T-cell exhaustion in hepatocellular carcinoma

Abstract Introduction Exhausted T cells (ETC) denotes a hypofunctional state of T lymphocyte that is commonly seen in cancer. Chronic T-cell receptor stimulation and recruitment of immune suppressive cells from the tumor microenvironment have been implicated as the mechanisms driving ETC. However, the roles of tumor cells in promoting ETC has remained elusive. Tumor metabolism is a core cancer hallmark. While metabolic alterations of cancer cells are required to support the anabolic requirement of cellular growth and proliferation, they may also affect the composition and function of the non-cancer cells residing in the same microenvironment. Bioinformatically searching the interaction between ETC and tumor metabolism may facilitate the identification of tumor drivers and novel mechanisms attributing ETC in hepatocellular carcinoma (HCC). Method HCC-specific ETC molecular signature was identified from single cell RNA sequencing of HCC-infiltrating lymphocytes and global transcriptome of HCC tumors. Correlation analysis of ETC and tumor-associated genomics, transcriptome and metabolomics was carried out. The clinical impact of ETC and ETC-associated metabolic alterations were investigated. Biological functions of the ETC-associated tumor metabolites were also assessed in vitro on human CD8+ T cells isolated form health donors. Results By exploring the single-cell transcriptome profiles of 5,063 HCC-derived lymphocytes, we identified 82 ETC-specific genes and found that ETC status robustly predicts patient survival in three independent HCC cohort, comparing 675 patients. While the degree of ETC is negatively correlated with the activity of effector T cell in HCC tumor tissues and liver tissues with chronic inflammation, such relationship was not observed in non-cancerous patients with acute inflammatory conditions. An integrated analysis of tumor-associated genomics, transcriptomics and metabolomics identifies an oncogenic reprograming of HCC methionine recycling towards salvage pathway leads to the elevation of 5-methylthioadenosine (MTA) and S-adenosylmethionine (SAM), which are associated with ETC status and clinical prognosis of HCC patients. Consistent with these clinical findings, SAM and MTA inhibit T cell activation in vitro. Furthermore, using assay for transposase-accessible chromatin with high-throughput sequencing, we found that SAM/MTA treatment reduces global chromatin accessibilities in CD8+ T cells, leading to T cell dysfunction. Lastly, we showed that MTA level in the serum may serve as a biomarker of ETC and HCC patient survival. Conclusion Our results identify reprogramming of tumor methionine metabolism as a key metabolic mechanism by which HCC tumor drive ETC and evade an immune attack. Citation Format: Man Hsin Hung, Joo Sang Lee, Sophia Heinrich, Ching Wen Chan, Lichun Ma, Marshonna Forgues, Anuradha Budhu, Jittiporn Chaisaingmongko, Mathuros Ruchirawat, Eytan Ruppin, Xin Wei Wang. Tumor methionine metabolism drives T-cell exhaustion in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1687.

Serine Biosynthesis Pathway Supports MYC-miR-494-EZH2 Feed-Forward Circuit Necessary to Maintain Metabolic and Epigenetic Reprogramming of Burkitt Lymphoma Cells.

Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4, KLF4, CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo.

Abstract B080: PKM2 activation modulates metabolism and enhances immune response in solid tumor models

Abstract Pyruvate kinase functions as the key enzyme in the final step of glycolysis. Cancer cells largely utilize the M2 isoform of pyruvate kinase (PKM2) due to the ability of PKM2 to be allosterically regulated between fully active (tetramer) and less active (dimer) forms of the enzyme. This dynamic regulation is associated with metabolic reprogramming of cancer cells creating a balance between energy needs and anabolic cellular requirements to support cell growth and division. Furthermore, the allosteric regulation creates an opportunity to design a small molecule activator to reverse the metabolic reprogramming favoring cancer growth and immune evasion. TP-1454 is a novel PKM2 activator with low nanomolar PKM2 activation in biochemical assays (AC50 = 10 nM) and in A549 epithelial lung carcinoma cells (AC50 < 20 nM). TP-1454 potently suppresses A549 cell viability (IC50 = 19 nM) and inhibits the tumor growth (60%) under serine auxotrophy conditions both in vitro and in vivo. PKM2 also plays a critical role in the regulation of the adaptive metabolism required to mount an innate immune response. We hypothesized that PKM2 activation may reverse the immune-suppressive microenvironment often observed in many cancers in part by decreasing tumor lactate levels and favoring glucose utilization by immune cells over cancer cells. To test this hypothesis, we explored the combination of TP-1454 with immunotherapy in both immune-suppressive and immune-permissive mouse syngeneic tumors. TP-1454 combination with α-PD1 and α-CTLA4 resulted in tumor regression in the MC38 syngeneic mouse colorectal cancer model with no adverse toxicity or effects on body weights. TP-1454 combination with α-PD1, α-CTLA4 or triple combination with α-PD1 and α-CTLA4 resulted in tumor growth inhibition (TGI) of 76%, 96% and 99% respectively, in the MC38 model. We observed increased levels of glucose and decreased levels of glucose 6-phosphate, phosphoglycerate, phosphoenolpyruvate and lactate in TP-1454 treated compared to vehicle treated MC38 tumors. Kaplan Meier survival analysis revealed 90% and 100% survival for TP-1454 combination with α-CTLA-4 or triple combination with α-PD1 and α-CTLA4 respectively, a vast improvement over the 10% survival of the vehicle group. TP-1454 or α-PD1 alone demonstrated <10% TGI in a CT26 colorectal syngeneic model but synergized in combination resulting in a 68% TGI. Potential downstream biomarkers, including metabolism, immune gene alterations and immune phenotyping are currently under evaluation using LC-MS/MS, NanoString and flow cytometry. These preclinical studies strongly suggest the potential novel therapeutic activity of TP-1454 in cancer models through metabolism and tumor microenvironment modulation. The immune-modulatory and metabolic alterations by TP-1454 offer a unique mechanism to potentially activate the immune response in cancer patients when combined with immunotherapy. Citation Format: Satya Pathi, Peter Peterson, Ryan Mangelson, Ethika Tyagi, Jason M. Foulks, Clifford J. Whatcott, David J. Bearss, Steven L. Warner. PKM2 activation modulates metabolism and enhances immune response in solid tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B080. doi:10.1158/1535-7163.TARG-19-B080

Glycolytic metabolism is essential for CCR7 oligomerization and dendritic cell migration

Dendritic cells (DCs) are first responders of the innate immune system that integrate signals from external stimuli to direct context-specific immune responses. Current models suggest that an active switch from mitochondrial metabolism to glycolysis accompanies DC activation to support the anabolic requirements of DC function. We show that early glycolytic activation is a common program for both strong and weak stimuli, but that weakly activated DCs lack long-term HIF-1α-dependent glycolytic reprogramming and retain mitochondrial oxidative metabolism. Early induction of glycolysis is associated with activation of AKT, TBK, and mTOR, and sustained activation of these pathways is associated with long-term glycolytic reprogramming. We show that inhibition of glycolysis impaired maintenance of elongated cell shape, DC motility, CCR7 oligomerization, and DC migration to draining lymph nodes. Together, our results indicate that early induction of glycolysis occurs independent of pro-inflammatory phenotype, and that glycolysis supports DC migratory ability regardless of mitochondrial bioenergetics.

Proteomics Analysis to Assess the Role of Mitochondria in BRCA1-Mediated Breast Tumorigenesis.

Mitochondria are the organelles deputed to energy production, but they are also involved in carcinogenesis, cancer progression, and metastasis, playing a role in altered energy metabolism in cancer cells. Mitochondrial metabolism is connected with several mitochondrial pathways such as ROS signaling, Ca2+ homeostasis, mitophagy, and mitochondrial biogenesis. These pathways are merged in an interactive super-network that seems to play a crucial role in cancer. Germline mutations of the BRCA1 gene account for 5–10% of breast cancers and confer a risk of developing the disease 10- to 20-fold much higher than in non-carriers. By considering metabolic networks that could reconcile both genetic and non-genetic causal mechanisms in BRCA1 driven tumorigenesis, we herein based our study on the hypothesis that BRCA1 haploinsufficiency might drive metabolic rewiring in breast epithelial cells, acting as a push toward malignant transformation. Using 2D-DIGE we analyzed and compared the mitochondrial proteomic profile of sporadic breast cancer cell line (MCF7) and BRCA1 mutated breast cancer cell line (HCC1937). Image analysis was carried out with Decider Software, and proteins differentially expressed were identified by LC-MS/MS on a quadrupole-orbitrap mass spectrometer Q-Exactive. Ingenuity pathways analysis software was used to analyze the fifty-three mitochondrial proteins whose expression resulted significantly altered in response to BRCA1 mutation status. Mitochondrial Dysfunction and oxidative phosphorylation, and energy production and nucleic acid metabolism were, respectively, the canonical pathway and the molecular function mainly affected. Western blotting analysis was done to validate the expression and the peculiar mitochondrial compartmentalization of specific proteins such us HSP60 and HIF-1α. Particularly intriguing is the correlation between BRCA1 mutation status and HIF-1α localization into the mitochondria in a BRCA1 dependent manner. Data obtained led us to hypothesize an interesting connection between BRCA1 and mitochondria pathways, capable to trigger metabolic changes, which, in turn, sustain the high energetic and anabolic requirements of the malignant phenotype.

Meat-based enteral nutrition

Enteral nutrition is widely used in hospitals as a means of nutritional support and therapy for different diseases. Enteral nutrition must fulfil the energy needs of the body, be balanced by the nutrient composition and meet patient’s nutritional needs. Meat is a source of full-value animal protein, vitamins and minerals. On the basis of this research, recipes and technology for a meat-based enteral nutrition product were developed. The product is a ready-to-eat sterilised mixture in the form of a liquid homogeneous mass, which is of full value in terms of composition and enriched with vitamins and minerals, consists of particles with a size of not more than 0.3 mm and has the modified fat composition and rheological characteristics that are necessary for passage through enteral feeding tubes. The study presents experimental data on the content of the main macro- and micro-nutrients in the developed product. The new product is characterised by a balanced fatty acid composition, which plays an important role in correction of lipid metabolism disorders and protein-energy deficiency, and it is capable of satisfying patients’ daily requirements for vitamins and the main macro- and microelements when consuming 1500-2000 ml. Meat-based enteral nutrition can be used in diets as a standard mixture for effective correction of the energy and anabolic requirements of the body and support of the nutritional status of patients, including those with operated stomach syndrome.

Management of Multiple Nitrogen Sources during Wine Fermentation by Saccharomyces cerevisiae.

During fermentative growth in natural and industrial environments, Saccharomyces cerevisiae must redistribute the available nitrogen from multiple exogenous sources to amino acids in order to suitably fulfill anabolic requirements. To exhaustively explore the management of this complex resource, we developed an advanced strategy based on the reconciliation of data from a set of stable isotope tracer experiments with labeled nitrogen sources. Thus, quantifying the partitioning of the N compounds through the metabolism network during fermentation, we demonstrated that, contrary to the generally accepted view, only a limited fraction of most of the consumed amino acids is directly incorporated into proteins. Moreover, substantial catabolism of these molecules allows for efficient redistribution of nitrogen, supporting the operative de novo synthesis of proteinogenic amino acids. In contrast, catabolism of consumed amino acids plays a minor role in the formation of volatile compounds. Another important feature is that the α-keto acid precursors required for the de novo syntheses originate mainly from the catabolism of sugars, with a limited contribution from the anabolism of consumed amino acids. This work provides a comprehensive view of the intracellular fate of consumed nitrogen sources and the metabolic origin of proteinogenic amino acids, highlighting a strategy of distribution of metabolic fluxes implemented by yeast as a means of adapting to environments with changing and scarce nitrogen resources.IMPORTANCE A current challenge for the wine industry, in view of the extensive competition in the worldwide market, is to meet consumer expectations regarding the sensory profile of the product while ensuring an efficient fermentation process. Understanding the intracellular fate of the nitrogen sources available in grape juice is essential to the achievement of these objectives, since nitrogen utilization affects both the fermentative activity of yeasts and the formation of flavor compounds. However, little is known about how the metabolism operates when nitrogen is provided as a composite mixture, as in grape must. Here we quantitatively describe the distribution through the yeast metabolic network of the N moieties and C backbones of these nitrogen sources. Knowledge about the management of a complex resource, which is devoted to improvement of the use of the scarce N nutrient for growth, will be useful for better control of the fermentation process and the sensory quality of wines.

Ketones and brain development: Implications for correcting deteriorating brain glucose metabolism during aging

Brain energy metabolism in Alzheimer’s disease (AD) is characterized mainly by temporo-parietal glucose hypometabolism. This pattern has been widely viewed as a consequence of the disease, i . e . deteriorating neuronal function leading to lower demand for glucose. This review will address deteriorating glucose metabolism as a problem specific to glucose and one that precedes AD. Hence, ketones and medium chain fatty acids (MCFA) could be an alternative source of energy for the aging brain that could compensate for low brain glucose uptake. MCFA in the form of dietary medium chain triglycerides (MCT) have a long history in clinical nutrition and are widely regarded as safe by government regulatory agencies. The importance of ketones in meeting the high energy and anabolic requirements of the infant brain suggest they may be able to contribute in the same way in the aging brain. Clinical studies suggest that ketogenesis from MCT may be able to bypass the increasing risk of insufficient glucose uptake or metabolism in the aging brain sufficiently to have positive effects on cognition.

Flux balance analysis predicts essential genes in clear cell renal cell carcinoma metabolism.

Flux balance analysis is the only modelling approach that is capable of producing genome-wide predictions of gene essentiality that may aid to unveil metabolic liabilities in cancer. Nevertheless, a systemic validation of gene essentiality predictions by flux balance analysis is currently missing. Here, we critically evaluated the accuracy of flux balance analysis in two cancer types, clear cell renal cell carcinoma (ccRCC) and prostate adenocarcinoma, by comparison with large-scale experiments of gene essentiality in vitro. We found that in ccRCC, but not in prostate adenocarcinoma, flux balance analysis could predict essential metabolic genes beyond random expectation. Five of the identified metabolic genes, AGPAT6, GALT, GCLC, GSS, and RRM2B, were predicted to be dispensable in normal cell metabolism. Hence, targeting these genes may selectively prevent ccRCC growth. Based on our analysis, we discuss the benefits and limitations of flux balance analysis for gene essentiality predictions in cancer metabolism, and its use for exposing metabolic liabilities in ccRCC, whose emergent metabolic network enforces outstanding anabolic requirements for cellular proliferation.

HIF1α Modulates Cell Fate Reprogramming Through Early Glycolytic Shift and Upregulation of PDK1–3 and PKM2

Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor one alpha (HIF1α), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1α function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1α significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to upregulate the HIF1α target pyruvate dehydrogenase kinase (PDK) one and set in motion the glycolytic shift. However, additional HIF1α activation appears critical in the early upregulation of other HIF1α-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1α targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1α pathway as an enabling regulator of cellular reprogramming.