Futile Cycle Research Articles

The dynamic regulatory network of phosphatidic acid metabolism: a spotlight on substrate cycling between phosphatidic acid and diacylglycerol.

Mammalian cells utilize over 1000 different lipid species to maintain cell and organelle membrane properties, control cell signaling and processes, and store energy. Lipid synthesis and metabolism are mediated by highly interconnected and spatiotemporally regulated networks of lipid-metabolizing enzymes and supported by vesicle trafficking and lipid-transfer at membrane contact sites. However, the regulatory mechanisms that achieve lipid homeostasis are largely unknown. Phosphatidic acid (PA) serves as the central hub for phospholipid biosynthesis, acting as a key intermediate in both the Kennedy pathway and the CDP-DAG pathway. Additionally, PA is a potent signaling molecule involved in various cellular processes. This dual role of PA, both as a critical intermediate in lipid biosynthesis and as a significant signaling molecule, suggests that it is tightly regulated within cells. This minireview will summarize the functional diversity of PA molecules based on their acyl tail structures and subcellular localization, highlighting recent tools and findings that shed light on how the physical, chemical, and spatial properties of PA species contribute to their differential metabolic fates and functions. Dysfunctional effects of altered PA metabolism as well as the strategies cells employ to maintain PA regulation and homeostasis will also be discussed. Furthermore, this review will explore the differential regulation of PA metabolism across distinct subcellular membranes. Our recent proximity labeling studies highlight the possibility that substrate cycling between PA and DAG may be location-dependent and have functional significance in cell signaling and lipid homeostasis.

Brown fat ATP-citrate lyase links carbohydrate availability to thermogenesis and guards against metabolic stress.

Brown adipose tissue (BAT) engages futile fatty acid synthesis-oxidation cycling, the purpose of which has remained elusive. Here, we show that ATP-citrate lyase (ACLY), which generates acetyl-CoA for fatty acid synthesis, promotes thermogenesis by mitigating metabolic stress. Without ACLY, BAT overloads the tricarboxylic acid cycle, activates the integrated stress response (ISR) and suppresses thermogenesis. ACLY's role in preventing BAT stress becomes critical when mice are weaned onto a carbohydrate-plentiful diet, while removing dietary carbohydrates prevents stress induction in ACLY-deficient BAT. ACLY loss also upregulates fatty acid synthase (Fasn); yet while ISR activation is not caused by impaired fatty acid synthesis per se, deleting Fasn and Acly unlocks an alternative metabolic programme that overcomes tricarboxylic acid cycle overload, prevents ISR activation and rescues thermogenesis. Overall, we uncover a previously unappreciated role for ACLY in mitigating mitochondrial stress that links dietary carbohydrates to uncoupling protein 1-dependent thermogenesis and provides fundamental insight into the fatty acid synthesis-oxidation paradox in BAT.

Quantification of nutrient fluxes during acute exercise in mice.

Despite the known metabolic benefits of exercise, an integrated metabolic understanding of exercise is lacking. Here, we use invivo steady-state isotope-labeled infusions to quantify fuel flux and oxidation during exercise in fasted, fed, and exhausted female mice, revealing several novel findings. Exercise strongly promoted glucose fluxes from liver glycogen, lactate, and glycerol, distinct from humans. Several organs spared glucose, a process that broke down in exhausted mice despite concomitant hypoglycemia. Proteolysis increased markedly, also divergent from humans. Fatty acid oxidation dominated during fasted exercise. Ketone production and oxidation rose rapidly, seemingly driven by a hepatic bottleneck caused by gluconeogenesis-induced cataplerotic stress. Altered fuel consumption was observed in organs not directly involved in muscle contraction, including the pancreas and brown fat. Several futile cycles surprisingly persisted during exercise, despite their energy cost. In sum, we provide a comprehensive, integrated, holistic, and quantitative accounting of metabolism during exercise in an intact organism.

The activity of the ribonucleotide monophosphatase UmpH is controlled by interaction with the GlnK signaling protein in Escherichia coli

The PII signaling proteins are ubiquitous in prokaryotes serving as crucial metabolic hubs in different metabolic pathways due to their ability to sense and integrate signals of the cellular nitrogen, carbon, and energy levels. In this study we used ligand fishing assays to identify the ribonucleotide monophosphatase UmpH enzyme as a novel target of the PII signaling protein GlnK in Escherichia coli. In vitro analyses showed that UmpH interacts specifically with the PII protein GlnK but not with its paralogue protein GlnB. The UmpH – GlnK complex is modulated by the GlnK uridylylation status and by the levels of the GlnK allosteric effectors ATP, ADP and 2-oxoglutarate. Upon engaging interaction with GlnK, UmpH becomes less active towards its substrate uridine 5'-monophosphate (UMP). We suggest a model where GlnK will physically interact to reduce the UmpH activity during the transition from N-starvation to N-sufficient conditions. Such a mechanism may help the cells to reprogram the fate of UMP from catabolism to anabolism avoiding futile cycling of key nutrients.

HDAC5 controls a hypothalamic STAT5b-TH axis, the sympathetic activation of ATP-consuming futile cycles and adult-onset obesity in male mice

With age, metabolic perturbations accumulate to elevate our obesity burden. While age-onset obesity is mostly driven by a sedentary lifestyle and high calorie intake, genetic and epigenetic factors also play a role. Among these, members of the large histone deacetylase (HDAC) family are of particular importance as key metabolic determinants for healthy ageing, or metabolic dysfunction. Here, we aimed to interrogate the role of class 2 family member HDAC5 in controlling systemic metabolism and age-related obesity under non-obesogenic conditions. Starting at 6 months of age, we observed adult-onset obesity in chow-fed male global HDAC5-KO mice, that was accompanied by marked reductions in adrenergic-stimulated ATP-consuming futile cycles, including BAT activity and UCP1 levels, WAT-lipolysis, skeletal muscle, WAT and liver futile creatine and calcium cycles, and ultimately energy expenditure. Female mice did not differ between genotypes. The lower peripheral sympathetic nervous system (SNS) activity in mature male KO mice was linked to higher dopaminergic neuronal activity within the dorsomedial arcuate nucleus (dmARC) and elevated hypothalamic dopamine levels. Mechanistically, we reveal that hypothalamic HDAC5 acts as co-repressor of STAT5b over the control of Tyrosine hydroxylase (TH) gene transactivation, which ultimately orchestrates the activity of dmARH dopaminergic neurons and energy metabolism in male mice under non-obesogenic conditions.

Abstract A070: Time-Dependent Oxidation and Aggregation of Vimentin upon Binding by β-lap Induced Immunogenic Cell Death

Abstract β-lapachone (β-lap) has been demonstrated as an NAD(P)H:quinone oxidoreductase1 (NQO1) bioactivatable compound. It initiated a futile redox cycle catalyzed by NQO1, resulting in reactive oxygen species (ROS) generation, with consumption of the reductive NAD(P)H. After a 4-h pulse of sufficiently lethal dose of β-lap, high levels of ROS diffused into nuclei to induce tumor-selective DNA damage and programmed necrosis in cancers overexpressing NQO1. Recently, our experiments showed that prolonged low dose β-lap administration exerted significant and NQO1 independent antitumor efficacy in mouse models with no overt signs of toxicity. Thus, we synthesized a photoactive β-lap probe to identify the molecular target of β-lap. Vimentin, a major constituent of the intermediate filament family of proteins, was identified as the direct binding target of β-lap. Vimentin was highly upregulated in multiple cancers including PDAC, and was a binding partner of β-lap with a dissociation constant at micromolar levels. Ligand binding assays in Panc1 cells employing β-lap as a specific competitor showed that vimentin was bound by β-lap-probe in a β-lap-competitive manner, and further confirmed that the in vivobinding target of β-lap was vimentin. Moreover, it was manifested that endogenous vimentin mRNA levels directly correlated with sensitivity to prolonged exposure of β-lap in a panel of pancreatic cancer cell lines, and knockout of vimentin in Panc1 cells markedly decreased lethality after prolonged exposure of β-lap. Mechanistically, we revealed that β-lap bound to vimentin and induced disulfide bonds that participated in disulfide cross-linking between a pair of vimentin dimers in a concentration and time dependent manner, forming vimentin oligomers of approximately 150 kDa. Prolonged exposure of β-lap promoted the disruption of filament architecture and vimentin degradation, ultimately resulting in cell death. In addition, the cell death by targeting vimentin exhibited a more inflammatory condition and enhanced DC activation. Together, our findings unveiled an unknown molecular target for β-lap and provided valuable insights into its mechanism of action. Targeting vimentin also presented a new strategy that inhibited the tumor growth and synergized with immunotherapy to disrupt immune evasion and reduce immune suppression against pancreatic cancers. Citation Format: Feng Qian, Lei Dou, Kaixin Wang, Yi Xia, Zhidong Chen, Yumeng Zu, Shuang Xi. Time-Dependent Oxidation and Aggregation of Vimentin upon Binding by β-lap Induced Immunogenic Cell Death [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A070.

Simultaneous Targeting of NQO1 and SOD1 Eradicates Breast Cancer Stem Cells via Mitochondrial Futile Redox Cycling.

Triple negative breast cancer (TNBC) contains the highest proportion of cancer stem-like cells (CSCs), which display intrinsic resistance to currently available cancer therapies. This therapeutic resistance is partially mediated by an antioxidant defense coordinated by the transcription factor NRF2 and its downstream targets including NQO1. Here, we identified the antioxidant enzymes NQO1 and SOD1 as therapeutic vulnerabilities of ALDH+ epithelial-like CSCs and CD24-/loCD44+/hi mesenchymal-like CSCs in TNBC. Effective targeting of these CSC states was achieved by utilizing IB-DNQ, a potent and specific NQO1-bioactivatable futile redox cycling molecule, which generated large amounts of reactive oxygen species (ROS) including superoxide and hydrogen peroxide. Furthermore, the CSC killing effect was specifically enhanced by genetic or pharmacological inhibition of SOD1, a copper-containing superoxide dismutase highly expressed in TNBC. Mechanistically, a significant portion of NQO1 resided in the mitochondrial intermembrane space, catalyzing futile redox cycling from IB-DNQ to generate high levels of mitochondrial superoxide, and SOD1 inhibition markedly potentiated this effect resulting in mitochondrial oxidative injury, cytochrome c release, and activation of the caspase 3-mediated apoptotic pathway. Treatment with IB-DNQ alone or together with SOD1 inhibition effectively suppressed tumor growth, metastasis, and tumor-initiating potential in xenograft models of TNBC expressing different levels of NQO1. This futile oxidant-generating strategy, which targets CSCs across the epithelial-mesenchymal continuum, could be a promising therapeutic approach for treating TNBC patients.

Inverse blebs operate as hydraulic pumps during mouse blastocyst formation.

During preimplantation development, mouse embryos form a fluid-filled lumen. Pressurized fluid fractures cell-cell contacts and accumulates into pockets, which coarsen into a single lumen. How the embryo controls intercellular fluid movement during coarsening is unknown. Here we report inverse blebs growing into cells at adhesive contacts. Throughout the embryo we observed hundreds of inverse blebs, each filling with intercellular fluid and retracting within a minute. Inverse blebs grow due to pressure build-up resulting from fluid accumulation and cell-cell adhesion, which locally confines fluid. Inverse blebs retract due to actomyosin contraction, practically pushing fluid within the intercellular space. Importantly, inverse blebs occur infrequently at contacts formed by multiple cells, which effectively serve as fluid sinks. Manipulation of the embryo topology reveals that without sinks inverse blebs pump fluid into one another in futile cycles. We propose that inverse blebs operate as hydraulic pumps to promote luminal coarsening, thereby constituting an instrument used by cells to control fluid movement.

Chronic β3-AR stimulation activates distinct thermogenic mechanisms in brown and white adipose tissue and improves systemic metabolism in aged mice.

Adipose thermogenesis has been actively investigated as a therapeutic target for improving metabolic dysfunction in obesity. However, its applicability to middle-aged and older populations, which bear the highest obesity prevalence in the United States (approximately 40%), remains uncertain due to age-related decline in thermogenic responses. In this study, we investigated the effects of chronic thermogenic stimulation using the β3-adrenergic (AR) agonist CL316,243 (CL) on systemic metabolism and adipose function in aged (18-month-old) C57BL/6JN mice. Sustained β3-AR treatment resulted in reduced fat mass, increased energy expenditure, increased fatty acid oxidation and mitochondrial activity in adipose depots, improved glucose homeostasis, and a favorable adipokine profile. At the cellular level, CL treatment increased uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT). However, in white adipose tissue (WAT) depots, CL treatment increased glycerol and lipid de novo lipogenesis (DNL) and turnover suggesting the activation of the futile substrate cycle of lipolysis and reesterification in a UCP1-independent manner. Increased lipid turnover was also associated with the simultaneous upregulation of proteins involved in glycerol metabolism, fatty acid oxidation, and reesterification in WAT. Further, a dose-dependent impact of CL treatment on inflammation was observed, particularly in subcutaneous WAT, suggesting a potential mismatch between fatty acid supply and oxidation. These findings indicate that chronic β3-AR stimulation activates distinct cellular mechanisms that increase energy expenditure in BAT and WAT to improve systemic metabolism in aged mice. Considering that people lose BAT with aging, activation of futile lipid cycling in WAT presents a novel strategy for improving age-related metabolic dysfunction.

Allosteric Regulation of Pyruvate Kinase Enables Efficient and Robust Gluconeogenesis by Preventing Metabolic Conflicts and Carbon Overflow.

Glycolysis and gluconeogenesis are reciprocal metabolic pathways that utilize different carbon sources. Pyruvate kinase catalyzes the irreversible final step of glycolysis, yet the physiological function of its regulation is poorly understood. Through metabolomics and enzyme kinetics studies, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis in the soil bacterium Bacillus subtilis . This regulation involves an extra C-terminal domain (ECTD) of pyruvate kinase, which is essential for autoinhibition and regulation by metabolic effectors. Introducing a pyruvate kinase mutant lacking the ECTD into B. subtilis resulted in defects specifically under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects are not caused by the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Instead, we identified two significant metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: increased carbon overflow into the medium and failure to expand glycolytic intermediates such as phosphoenolpyruvate (PEP). In silico analysis revealed that in wild-type cells, an expanded PEP pool enabled by pyruvate kinase regulation is critical for the thermodynamic feasibility of gluconeogenesis. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient energy utilization, and antimicrobial resistance.

Elucidation of the mechanism underlying the sequential catalysis of inulin by fructotransferase

Glycoside hydrolase family 91 (GH91) inulin fructotransferase (IFTases) enables biotransformation of fructans into sugar substitutes for dietary intervention in metabolic syndrome. However, the catalytic mechanism underlying the sequential biodegradation of inulin remains unelusive during the biotranformation of fructans. Herein we present the crystal structures of IFTase from Arthrobacter aurescens SK 8.001 in apo form and in complexes with kestose, nystose, or fructosyl nystose, respectively. Two kinds of conserved noncatalytic binding regions are first identified for IFTase-inulin interactions. The conserved interactions of substrates were revealed in the catalytic center that only contained a catalytic residue E205. A switching scaffold was comprised of D194 and Q217 in the catalytic channel, which served as the catalytic transition stabilizer through side chain displacement in the cycling of substrate sliding in/out the catalytic pocket. Such features in GH91 contribute to the catalytic model for consecutive cutting of substrate chain as well as product release in IFTase, and thus might be extended to other exo-active enzymes with an enclosed bottom of catalytic pocket. The study expands the current general catalytic principle in enzyme-substrate complexes and shed light on the rational design of IFTase for fructan biotransformation.

Development of a functional beige fat cell line uncovers independent subclasses of cells expressing UCP1 and the futile creatine cycle

Although uncoupling protein 1 (UCP1) is established as a major contributor to adipose thermogenesis, recent data have illustrated an important role for alternative pathways, particularly the futile creatine cycle (FCC). How these pathways co-exist in cells and tissues has not been explored. Beige cell adipogenesis occurs invivo but has been difficult to model invitro; here, we describe the development of a murine beige cell line that executes a robust respiratory response, including uncoupled respiration and the FCC. The key FCC enzyme, tissue-nonspecific alkaline phosphatase (TNAP), is localized almost exclusively to mitochondria in these cells. Surprisingly, single-cell cloning from this cell line shows that cells with the highest levels of UCP1 express little TNAP, and cells with the highest expression of TNAP express little UCP1. Immunofluorescence analysis of subcutaneous fat from cold-exposed mice confirms that the highest levels of these critical thermogenic components are expressed in distinct fat cell populations.

Single-nucleus transcriptomics identifies separate classes of UCP1 and futile cycle adipocytes

Adipose tissue can recruit catabolic adipocytes that utilize chemical energy to dissipate heat. This process occurs either by uncoupled respiration through uncoupling protein 1 (UCP1) or by utilizing ATP-dependent futile cycles (FCs). However, it remains unclear how these pathways coexist since both processes rely on the mitochondrial membrane potential. Utilizing single-nucleus RNA sequencing to deconvolute the heterogeneity of subcutaneous adipose tissue in mice and humans, we identify at least 2 distinct subpopulations of beige adipocytes: FC-adipocytes and UCP1-beige adipocytes. Importantly, we demonstrate that the FC-adipocyte subpopulation is highly metabolically active and utilizes FCs to dissipate energy, thus contributing to thermogenesis independent of Ucp1. Furthermore, FC-adipocytes are important drivers of systemic energy homeostasis and linked to glucose metabolism and obesity resistance in humans. Taken together, our findings identify a noncanonical thermogenic adipocyte subpopulation, which could be an important regulator of energy homeostasis in mammals.

Human NQO1 as a Selective Target for Anticancer Therapeutics and Tumor Imaging.

Human NAD(P)H-quinone oxidoreductase1 (HNQO1) is a two-electron reductase antioxidant enzyme whose expression is driven by the NRF2 transcription factor highly active in the prooxidant milieu found in human malignancies. The resulting abundance of NQO1 expression (up to 200-fold) in cancers and a barely detectable expression in body tissues makes it a selective marker of neoplasms. NQO1 can catalyze the repeated futile redox cycling of certain natural and synthetic quinones to their hydroxyquinones, consuming NADPH and generating rapid bursts of cytotoxic reactive oxygen species (ROS) and H2O2. A greater level of this quinone bioactivation due to elevated NQO1 content has been recognized as a tumor-specific therapeutic strategy, which, however, has not been clinically exploited. We review here the natural and new quinones activated by NQO1, the catalytic inhibitors, and the ensuing cell death mechanisms. Further, the cancer-selective expression of NQO1 has opened excellent opportunities for distinguishing cancer cells/tissues from their normal counterparts. Given this diagnostic, prognostic, and therapeutic importance, we and others have engineered a large number of specific NQO1 turn-on small molecule probes that remain latent but release intense fluorescence groups at near-infrared and other wavelengths, following enzymatic cleavage in cancer cells and tumor masses. This sensitive visualization/quantitation and powerful imaging technology based on NQO1 expression offers promise for guided cancer surgery, and the reagents suggest a theranostic potential for NQO1-targeted chemotherapy.

Accidental Encounter of Repair Intermediates in Alkylated DNA May Lead to Double-Strand Breaks in Resting Cells.

In clinics, chemotherapy is often combined with surgery and radiation to increase the chances of curing cancers. In the case of glioblastoma (GBM), patients are treated with a combination of radiotherapy and TMZ over several weeks. Despite its common use, the mechanism of action of the alkylating agent TMZ has not been well understood when it comes to its cytotoxic effects in tumor cells that are mostly non-dividing. The cellular response to alkylating DNA damage is operated by an intricate protein network involving multiple DNA repair pathways and numerous checkpoint proteins that are dependent on the type of DNA lesion, the cell type, and the cellular proliferation state. Among the various alkylating damages, researchers have placed a special on O6-methylguanine (O6-mG). Indeed, this lesion is efficiently removed via direct reversal by O6-methylguanine-DNA methyltransferase (MGMT). As the level of MGMT expression was found to be directly correlated with TMZ efficiency, O6-mG was identified as the critical lesion for TMZ mode of action. Initially, the mode of action of TMZ was proposed as follows: when left on the genome, O6-mG lesions form O6-mG: T mispairs during replication as T is preferentially mis-inserted across O6-mG. These O6-mG: T mispairs are recognized and tentatively repaired by a post-replicative mismatched DNA correction system (i.e., the MMR system). There are two models (futile cycle and direct signaling models) to account for the cytotoxic effects of the O6-mG lesions, both depending upon the functional MMR system in replicating cells. Alternatively, to explain the cytotoxic effects of alkylating agents in non-replicating cells, we have proposed a "repair accident model" whose molecular mechanism is dependent upon crosstalk between the MMR and the base excision repair (BER) systems. The accidental encounter between these two repair systems will cause the formation of cytotoxic DNA double-strand breaks (DSBs). In this review, we summarize these non-exclusive models to explain the cytotoxic effects of alkylating agents and discuss potential strategies to improve the clinical use of alkylating agents.

Research on Gas Injection Limits and Development Methods of CH4/CO2 Synergistic Displacement in Offshore Fractured Condensate Gas Reservoirs

Gas injection for enhanced oil and gas reservoir recovery is a crucial method in offshore Carbon Capture, Utilization, and Storage (CCUS). The B6 buried hill condensate gas reservoir, characterized by high CO2 content, a deficit in natural energy, developed fractures and low-pressure differentials between formation and saturation pressures, requires supplementary formation energy to mitigate retrograde condensation near the wellbore area through gas injection. However, due to the connected fractures, the B6 gas reservoir exhibits strong horizontal and vertical heterogeneity, resulting in severe gas channeling and a futile cycle, which affects the gas injection efficiency at various levels of fracture development. Based on these findings, we conducted gas injection experiments and numerical simulations on fractured cores. A characterization method for oil and gas relative permeability considering dissolution was established. Additionally, the gas injection development boundary for this type of condensate gas reservoir was quantified according to the degree of fracture development, and the gas injection mode of the B6 reservoir was optimized. Research indicates that the presence of fractures leads to the formation of a dominant gas channel; the greater the permeability difference, the poorer the gas injection effect. The permeability gradation (fracture permeability divided by matrix permeability) in the gas injection area should be no higher than 15; gas injection in wells A1 and A2 is likely to achieve a better development effect under the existing well pattern. Moreover, early gas injection timing and pulse gas injection prove beneficial in enhancing the recovery rate of condensate oil. The study offers significant guidance for the development of similar gas reservoirs and for reservoirs with weakly connected fractures; advancing the timing of gas injection can mitigate the retrograde condensation phenomenon, whereas initiating gas injection after depletion may reduce the impact of gas channeling for reservoirs with strongly connected fractures.

The metabolic benefits of thermogenic stimulation are preserved in aging.

Adipose thermogenesis has been actively investigated as a therapeutic target for improving metabolic dysfunction in obesity. However, its applicability to middle-aged and older populations, which bear the highest obesity prevalence in the US (approximately 40%), remains uncertain due to age-related decline in thermogenic responses. In this study, we investigated the effects of chronic thermogenic stimulation using the β3-adrenergic (AR) agonist CL316,243 (CL) on systemic metabolism and adipose function in aged (18-month-old) C57BL/6JN mice. Sustained β3-AR treatment resulted in reduced fat mass, increased energy expenditure, increased fatty acid oxidation and mitochondrial activity in adipose depots, improved glucose homeostasis, and a favorable adipokine profile. At the cellular level, CL treatment increased uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT). However, in white adipose tissue (WAT) depots, CL treatment increased glycerol and lipid de novo lipogenesis (DNL) and turnover suggesting the activation of the futile substrate cycle of lipolysis and reesterification in a UCP1-independent manner. Increased lipid turnover was also associated with the simultaneous upregulation of proteins involved in glycerol metabolism, fatty acid oxidation, and reesterification in WAT. Further, a dose-dependent impact of CL treatment on inflammation was observed, particularly in subcutaneous WAT, suggesting a potential mismatch between fatty acid supply and oxidation. These findings indicate that chronic β3-AR stimulation activates distinct cellular mechanisms that increase energy expenditure in BAT and WAT to improve systemic metabolism in aged mice. Our study provides foundational evidence for targeting adipose thermogenesis to improve age-related metabolic dysfunction.

How to sensitize glioblastomas to temozolomide chemotherapy: a gap-centered view.

Temozolomide (TMZ) is a methylating agent used as the first-line drug in the chemotherapy of glioblastomas. However, cancer cells eventually acquire resistance, necessitating the development of TMZ-potentiating therapy agents. TMZ induces several DNA base adducts, including O 6 -meG, 3-meA, and 7-meG. TMZ cytotoxicity stems from the ability of these adducts to directly (3-meA) or indirectly (O 6 -meG) impair DNA replication. Although TMZ toxicity is generally attributed to O 6 -meG, other alkylated bases can be similarly important depending on the status of various DNA repair pathways of the treated cells. In this mini-review we emphasize the necessity to distinguish TMZ-sensitive glioblastomas, which do not express methylguanine-DNA methyltransferase (MGMT) and are killed by the futile cycle of mismatch repair (MMR) of the O 6 -meG/T pairs, vs. TMZ-resistant MGMT-positive or MMR-negative glioblastomas, which are selected in the course of the treatment and are killed only at higher TMZ doses by the replication-blocking 3-meA. These two types of cells can be TMZ-sensitized by inhibiting different DNA repair pathways. However, in both cases, the toxic intermediates appear to be ssDNA gaps, a vulnerability also seen in BRCA-deficient cancers. PARP inhibitors (PARPi), which were initially developed to treat BRCA1/2-deficient cancers by synthetic lethality, were re-purposed in clinical trials to potentiate the effects of TMZ. We discuss how the recent advances in our understanding of the genetic determinants of TMZ toxicity might lead to new approaches for the treatment of glioblastomas by inhibiting PARP1 and other enzymes involved in the repair of alkylation damage (e.g., APE1).

Molecular Regulation of Thermogenic Mechanisms in Beige Adipocytes.

Adipose tissue is conventionally recognized as a metabolic organ responsible for storing energy. However, a proportion of adipose tissue also functions as a thermogenic organ, contributing to the inhibition of weight gain and prevention of metabolic diseases. In recent years, there has been significant progress in the study of thermogenic fats, particularly brown adipose tissue (BAT). Despite this progress, the mechanism underlying thermogenesis in beige adipose tissue remains highly controversial. It is widely acknowledged that beige adipose tissue has three additional thermogenic mechanisms in addition to the conventional UCP1-dependent thermogenesis: Ca2+ cycling thermogenesis, creatine substrate cycling thermogenesis, and triacylglycerol/fatty acid cycling thermogenesis. This paper delves into these three mechanisms and reviews the latest advancements in the molecular regulation of thermogenesis from the molecular genetic perspective. The objective of this review is to provide readers with a foundation of knowledge regarding the beige fats and a foundation for future research into the mechanisms of this process, which may lead to the development of new strategies for maintaining human health.

Relative sarcolipin (SLN) and sarcoplasmic reticulum Ca2+ ATPase (SERCA1) transcripts levels in closely related endothermic and ectothermic scombrid fishes: Implications for molecular basis of futile calcium cycle non-shivering thermogenesis (NST)

Regional endothermy is the ability of an animal to elevate the temperature of specific regions of the body above that of the surrounding environment and has evolved independently among several fish lineages. Sarcolipin (SLN) is a small transmembrane protein that uncouples the sarcoplasmic reticulum calcium ATPase pump (SERCA1b) resulting in futile Ca2+ cycling and is thought to play a role in non-shivering thermogenesis (NST) in cold-challenged mammals and possibly some fishes. This study investigated the relative expression of sln and serca1 transcripts in three regionally-endothermic fishes (the skipjack, Katsuwonus pelamis, and yellowfin tuna, Thunnus albacares, both of which elevate the temperatures of their slow-twitch red skeletal muscle (RM) and extraocular muscles (EM), as well as the cranial endothermic swordfish, Xiphias gladius), and closely related ectothermic scombrids (the Eastern Pacific bonito, Sarda chiliensis, and Pacific chub mackerel, Scomber japonicus). Using Reverse Transcription quantitative PCR (RT-qPCR) and species-specific primers, relative sln expression trended higher in both the RM and EM for all four scombrid species compared to white muscle. In addition, relative serca1 expression was found to be higher in RM of skipjack and yellowfin tuna in comparison to white muscle. However, neither sln nor serca1 transcripts were higher in swordfish RM, EM or cranial heater tissue in comparison to white muscle. A key phosphorylation site in sarcolipin, threonine 5, is conserved in the swordfish, but is mutated to alanine or valine in tunas and the endothermic smalleye Pacific opah, Lampris incognitus, which should result in increased uncoupling of the SERCA pump. Our results support the role of potential SLN-NST in endothermic tunas and the lack thereof for swordfish.