Comparative functional analysis reveals differential nucleotide sensitivity between human and mouse UCP1.

AIM: Mitochondrial uncoupling protein 1 (UCP1) is a unique protein of brown adipose tissue. Upon activation by free fatty acids, UCP1 facilitates a thermogenic net proton flux across the mitochondrial inner membrane. Non-complexed purine nucleotides inhibit this fatty acid-induced activity of UCP1. The most available data have been generated from rodent model systems. In light of its role as a putative pharmacological target for treating metabolic disease, in-depth analyses of human UCP1 activity, regulation, and structural features are essential. METHODS: In the present study, we established a doxycycline-regulated cell model with inducible human or murine UCP1 expression and conducted functional studies using respirometry comparing wild-type and mutant variants of human UCP1. RESULTS: We demonstrate that human and mouse UCP1 exhibit similar specific fatty acid-induced activity but a different inhibitory potential of purine nucleotides. Mutagenesis of non-conserved residues in human UCP1 revealed structural components in α-helix 56 and α-helix 6 crucial for uncoupling function. CONCLUSION: Comparative studies of human UCP1 with other orthologs can provide new insights into the structure-function relationship for this mitochondrial carrier and will be instrumental in searching for new activators.

From Planning Stage Towards FAIR Data: A Practical Metadatasheet For Biomedical Scientists.

Datasets consist of measurement data and metadata. Metadata provides context, essential for understanding and (re-)using data. Various metadata standards exist for different methods, systems and contexts. However, relevant information resides at differing stages across the data-lifecycle. Often, this information is defined and standardized only at publication stage, which can lead to data loss and workload increase. In this study, we developed Metadatasheet, a metadata standard based on interviews with members of two biomedical consortia and systematic screening of data repositories. It aligns with the data-lifecycle allowing synchronous metadata recording within Microsoft Excel, a widespread data recording software. Additionally, we provide an implementation, the Metadata Workbook, that offers user-friendly features like automation, dynamic adaption, metadata integrity checks, and export options for various metadata standards. By design and due to its extensive documentation, the proposed metadata standard simplifies recording and structuring of metadata for biomedical scientists, promoting practicality and convenience in data management. This framework can accelerate scientific progress by enhancing collaboration and knowledge transfer throughout the intermediate steps of data creation.

Lithium isotopes differentially modify mitochondrial amorphous calcium phosphate cluster size distribution and calcium capacity.

Lithium is commonly prescribed as a mood stabilizer in a variety of mental health conditions, yet its molecular mode of action is incompletely understood. Many cellular events associated with lithium appear tied to mitochondrial function. Further, recent evidence suggests that lithium bioactivities are isotope specific. Here we focus on lithium effects related to mitochondrial calcium handling. Lithium protected against calcium-induced permeability transition and decreased the calcium capacity of liver mitochondria at a clinically relevant concentration. In contrast, brain mitochondrial calcium capacity was increased by lithium. Surprisingly, 7Li acted more potently than 6Li on calcium capacity, yet 6Li was more effective at delaying permeability transition. The size distribution of amorphous calcium phosphate colloids formed in vitro was differentially affected by lithium isotopes, providing a mechanistic basis for the observed isotope specific effects on mitochondrial calcium handling. This work highlights a need to better understand how mitochondrial calcium stores are structurally regulated and provides key considerations for future formulations of lithium-based therapeutics.

Cold-stimulated brown adipose tissue activation is related to changes in serum metabolites relevant to NAD+ metabolism in humans.

Cold-induced brown adipose tissue (BAT) activation is considered to improve metabolic health. In murine BAT, cold increases the fundamental molecule for mitochondrial function, nicotinamide adenine dinucleotide (NAD+), but limited knowledge of NAD+ metabolism during cold in human BAT metabolism exists. We show that cold increases the serum metabolites of the NAD+ salvage pathway (nicotinamide and 1-methylnicotinamide) in humans. Additionally, individuals with cold-stimulated BAT activation have decreased levels of metabolites from the de novo NAD+ biosynthesis pathway (tryptophan, kynurenine). Serum nicotinamide correlates positively with cold-stimulated BAT activation, whereas tryptophan and kynurenine correlate negatively. Furthermore, the expression of genes involved in NAD+ biosynthesis in BAT is related to markers of metabolic health. Our data indicate that cold increases serum tryptophan conversion to nicotinamide to be further utilized by BAT. We conclude that NAD+ metabolism is activated upon cold in humans and is probably regulated in a coordinated fashion by several tissues.

High-Throughput Screening and Proteomic Characterization of Compounds Targeting Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and are implicated in multiple chronic diseases, which make them an attractive cell population for drug discovery. Here, we characterized the baseline proteomes and phospho-proteomes of mouse MDSC differentiated from a progenitor cell line to a depth of 7000 proteins and phosphorylation sites. We also validated the cellular system for drug discovery by recapitulating and identifying known and novel molecular responses to the well-studied MDSC drugs entinostat and mocetinostat. We established a high-throughput drug screening platform using a MDSC/T cell coculture system and assessed the effects of ∼21,000 small molecule compounds on T cell proliferation and IFN-γ secretion to identify novel MDSC modulator. The most promising candidates were validated in a human MDSC system, and subsequent proteomic experiments showed significant upregulation of several proteins associated with the reduction of reactive oxygen species (ROS). Proteome-wide solvent-induced protein stability assays identified Acyp1 and Cd74 as potential targets, and the ROS-reducing drug phenotype was validated by measuring ROS levels in cells in response to compound, suggesting a potential mode of action. We anticipate that the data and chemical tools developed in this study will be valuable for further research on MDSC and related drug discovery.

Chemoproteomic target deconvolution reveals Histone Deacetylases as targets of (R)-lipoic acid.

Lipoic acid is an essential enzyme cofactor in central metabolic pathways. Due to its claimed antioxidant properties, racemic (R/S)-lipoic acid is used as a food supplement but is also investigated as a pharmaceutical in over 180 clinical trials covering a broad range of diseases. Moreover, (R/S)-lipoic acid is an approved drug for the treatment of diabetic neuropathy. However, its mechanism of action remains elusive. Here, we performed chemoproteomics-aided target deconvolution of lipoic acid and its active close analog lipoamide. We find that histone deacetylases HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, and HDAC10 are molecular targets of the reduced form of lipoic acid and lipoamide. Importantly, only the naturally occurring (R)-enantiomer inhibits HDACs at physiologically relevant concentrations and leads to hyperacetylation of HDAC substrates. The inhibition of HDACs by (R)-lipoic acid and lipoamide explain why both compounds prevent stress granule formation in cells and may also provide a molecular rationale for many other phenotypic effects elicited by lipoic acid.

Thermogenic Capacity of Human Supraclavicular Brown Fat and Cold-Stimulated Brain Glucose Metabolism.

Human brain metabolism is susceptible to temperature changes. It has been suggested that the supraclavicular brown adipose tissue (BAT) protects the brain from these fluctuations by regulating heat production through the presence of uncoupling protein 1 (UCP-1). It remains unsolved whether inter-individual variation in the expression of UCP-1, which represents the thermogenic capacity of the supraclavicular BAT, is linked with brain metabolism during cold stress. Ten healthy human participants underwent 18F-FDG PET scanning of the brain under cold stimulus to determine brain glucose uptake (BGU). On a separate day, an excision biopsy of the supraclavicular fat-the fat proximal to the carotid arteries supplying the brain with warm blood-was performed to determine the mRNA expression of the thermogenic protein UCP-1. Expression of UCP-1 in supraclavicular BAT was directly related to the whole brain glucose uptake rate determined under cold stimulation (rho = 0.71, p = 0.03). In sub-compartmental brain analysis, UCP-1 expression in supraclavicular BAT was directly related to cold-stimulated glucose uptake rates in the hypothalamus, medulla, midbrain, limbic system, frontal lobe, occipital lobe, and parietal lobe (all rho ≥ 0.67, p < 0.05). These relationships were independent of body mass index and age. When analysing gene expressions of BAT secretome, we found a positive correlation between cold-stimulated BGU and DIO2. These findings provide evidence of functional links between brain metabolism under cold stimulation and UCP-1 and DIO2 expressions in BAT in humans. More research is needed to evaluate the importance of these findings in clinical outcomes, for instance, in examining the supporting role of BAT in cognitive functions under cold stress.

Steroidal Saponins─New Sources to Develop Potato (Solanum tuberosum L.) Genotypes Resistant against Certain Phytophthora infestans Strains.

Plant pathogens such as Phytophthora infestans that caused the Irish Potato Famine continue to threaten local and global food security. Genetic and chemical plant protection measures are often overcome by adaptation of pathogen population structures. Therefore, there is a constant demand for new, consumer- and environment-friendly plant protection strategies. Metabolic alterations induced by P. infestans in the foliage and tubers of six different potato cultivars were investigated. Using a combination of untargeted metabolomics, isolation techniques, and structure elucidation by MS and 1D/2D-NMR experiments, five steroidal glycoalkaloids, five oxylipins, and four steroidal saponins were identified. As the steroidal saponins showed antioomycete but no hemolytic activity, they may thus be considered as probably safe target substances for enrichment in breeding programs for disease resistance and as chemical lead structures for the production of nature-derived synthetic antioomycetes.

Loss of UCP1 function augments recruitment of futile lipid cycling for thermogenesis in murine brown fat.

OBJECTIVE: Classical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing. METHODS: In this study, we explore different potential sources of UCP1-independent thermogenesis and identify a futile ATP-consuming triglyceride/fatty acid cycle as the main contributor to cellular heat production in brown adipocytes lacking UCP1. We uncover the mechanism on a molecular level and pinpoint the key enzymes involved using pharmacological and genetic interference. RESULTS: ATGL is the most important lipase in terms of releasing fatty acids from lipid droplets, while DGAT1 accounts for the majority of fatty acid re-esterification in UCP1-ablated brown adipocytes. Furthermore, we demonstrate that chronic cold exposure causes a pronounced remodeling of adipose tissues and leads to the recruitment of lipid cycling capacity specifically in BAT of UCP1-knockout mice, possibly fueled by fatty acids from white fat. Quantification of triglyceride/fatty acid cycling clearly shows that UCP1-ablated animals significantly increase turnover rates at room temperature and below. CONCLUSION: Our results suggest an important role for futile lipid cycling in adaptive thermogenesis and total energy expenditure.

Uncoupling Protein 1 Does Not Produce Heat without Activation.

Mitochondrial uncoupling protein 1 (UCP1) is the crucial mechanistic component of heat production in classical brown fat and the newly identified beige or brite fat. Thermogenesis inevitably comes at a high energetic cost and brown fat, ultimately, is an energy-wasting organ. A constrained strategy that minimizes brown fat activity unless obligate will have been favored during natural selection to safeguard metabolic thriftiness. Accordingly, UCP1 is constitutively inhibited and is inherently not leaky without activation. It follows that increasing brown adipocyte number or UCP1 abundance genetically or pharmacologically does not lead to an automatic increase in thermogenesis or subsequent metabolic consequences in the absence of a plausible route of concomitant activation. Despite its apparent obviousness, this tenet is frequently ignored. Consequently, incorrect conclusions are often drawn from increased BAT or brite/beige depot mass, e.g., predicting or causally linking beneficial metabolic effects. Here, we highlight the inherently inactive nature of UCP1, with a particular emphasis on the molecular brakes and releases of UCP1 activation under physiological conditions. These controls of UCP1 activity represent potential targets of therapeutic interventions to unlock constraints and efficiently harness the energy-expending potential of brown fat to prevent and treat obesity and associated metabolic disorders.

Global Adipose Tissue Remodeling During the First Month of Postnatal Life in Mice.

During the first month of postnatal life, adipose tissue depots of mice go through a drastic, but transient, remodeling process. Between postnatal days 10 and 20, several white fat depots display a strong and sudden surge in beige adipocyte emergence that reverts until day 30. At the same time, brown fat depots appear to undergo an opposite phenomenon. We comprehensively describe these events, their depot specificity and known environmental and genetic interactions, such as maternal diet, housing temperature and mouse strain. We further discuss potential mechanisms and plausible purposes, including the tempting hypothesis that postnatal transient remodeling creates a lasting adaptive capacity still detectable in adult animals. Finally, we propose postnatal adipose tissue remodeling as a model process to investigate mechanisms of beige adipocyte recruitment advantageous to cold exposure or adrenergic stimulation in its entirely endogenous sequence of events without external manipulation.

LncRNA Ctcflos orchestrates transcription and alternative splicing in thermogenic adipogenesis.

The recruitment of thermogenic brite adipocytes within white adipose tissue attenuates obesity and metabolic comorbidities, arousing interest in understanding the underlying regulatory mechanisms. The molecular network of brite adipogenesis, however, remains largely unresolved. In this light, long noncoding RNAs (lncRNAs) emerged as a versatile class of modulators that control many steps within the differentiation machinery. Leveraging the naturally varying propensities of different inbred mouse strains for white adipose tissue browning, we identify the nuclear lncRNA Ctcflos as a pivotal orchestrator of thermogenic gene expression during brite adipocyte differentiation. Mechanistically, Ctcflos acts as a pleiotropic regulator, being essential for the transcriptional recruitment of the early core thermogenic regulatory program and the modulation of alternative splicing to drive brite adipogenesis. This is showcased by Ctcflos-regulated gene transcription and splicing of the key browning factor Prdm16 toward the isoform that is specific for the thermogenic gene program. Conclusively, our findings emphasize the mechanistic versatility of lncRNAs acting at several independent levels of gene expression for effective regulation of key differentiation factors to direct cell fate and function.

Fibroblast growth factor induced Ucp1 expression in preadipocytes requires PGE2 biosynthesis and glycolytic flux.

High uncoupling protein 1 (Ucp1) expression is a characteristic of differentiated brown adipocytes and is linked to adipogenic differentiation. Paracrine fibroblast growth factor 8b (FGF8b) strongly induces Ucp1 transcription in white adipocytes independent of adipogenesis. Here, we report that FGF8b and other paracrine FGFs act on brown and white preadipocytes to upregulate Ucp1 expression via a FGFR1-MEK1/2-ERK1/2 axis, independent of adipogenesis. Transcriptomic analysis revealed an upregulation of prostaglandin biosynthesis and glycolysis upon Fgf8b treatment of preadipocytes. Oxylipin measurement by LC-MS/MS in FGF8b conditioned media identified prostaglandin E2 as a putative mediator of FGF8b induced Ucp1 transcription. RNA interference and pharmacological inhibition of the prostaglandin E2 biosynthetic pathway confirmed that PGE2 is causally involved in the control over Ucp1 transcription. Importantly, impairment of or failure to induce glycolytic flux blunted the induction of Ucp1, even in the presence of PGE2 . Lastly, a screening of transcription factors identified Nrf1 and Hes1 as required regulators of FGF8b induced Ucp1 expression. Thus, we conclude that paracrine FGFs co-regulate prostaglandin and glucose metabolism to induce Ucp1 expression in a Nrf1/Hes1-dependent manner in preadipocytes, revealing a novel regulatory network in control of Ucp1 expression in a formerly unrecognized cell type.

Adenylate kinase derived ATP shapes respiration and calcium storage of isolated mitochondria.

The ratio of ADP and ATP is a natural indicator of cellular bioenergetic state and thus a prominent analyte in metabolism research. Beyond adenylate interconversion via oxidative phosphorylation and ATPase activities, ADP and ATP act as steric regulators of enzymes, e.g. cytochrome C oxidase, and are major factors in mitochondrial calcium storage potential. Consideration of all routes of adenylate conversion is critical to successfully predict their abundance in an experimental system and to correctly interpret many aspects of mitochondrial function. We showcase here how adenylate kinases elicit considerable impact on the outcome of a variety of mitochondrial assays through their drastic manipulation of the adenylate profile. Parameters affected include cytochrome c oxidase activity, P/O ratio, and mitochondrial calcium dynamics. Study of the latter revealed that the presence of ATP is required for mitochondrial calcium to be shaped into a particularly dense form of mitochondrial amorphous calcium phosphate.

Uncoupling protein-1 expression does not protect mice from diet-induced obesity.

We studied the metabolic phenotype of a novel Ucp1-LUC-iRFP713 knock-in reporter gene mouse model originally generated to monitor endogenous Ucp1 gene expression. Both reporter mice and reporter cells reliably reflected Ucp1 gene expression in vivo and in vitro. We here report an unexpected reduction in UCP1 content in homozygous knock-in (KI) reporter mice. As a result, the thermogenic capacity of KI mice stimulated by norepinephrine was largely blunted, making them more sensitive to an acute cold exposure. In return, these reporter mice with reduced UCP1 expression enabled us to investigate the physiological role of UCP1 in the prevention of weight gain. We observed no substantial differences in body mass across the three genotypes, irrespective of the type of diet or the ambient temperature, possibly due to the insufficient UCP1 activation. Indeed, activation of UCP1 by daily injection of the selective ß3-adrenergic receptor agonist CL316,243 resulted in significantly greater reduction of body weight in wild-type mice than in KI mice. Taken together, we conclude that the intact expression of UCP1 is essential for cold-induced thermogenesis but the presence of UCP1 per se does not protect mice from diet-induced obesity.NEW & NOTEWORTHY To study the functional role of UCP1-dependent brown adipose tissue thermogenesis for energy balance, new animal models are needed. By metabolic phenotyping of a novel mouse model with low UCP1 levels in brown fat, we demonstrate that the susceptibility to diet-induced obesity is not increased despite impaired cold-induced thermogenic capacity. Brown fat requires pharmacological activation to promote negative energy balance in diet-induced obese mice.

No Effect of Dietary Fish Oil Supplementation on the Recruitment of Brown and Brite Adipocytes in Mice or Humans under Thermoneutral Conditions.

SCOPE: Brown and brite adipocytes within the mammalian adipose organ provide non-shivering thermogenesis and thus, have an exceptional capacity to dissipate chemical energy as heat. Polyunsaturated fatty acids (PUFA) of the n3-series, abundant in fish oil, have been repeatedly demonstrated to enhance the recruitment of thermogenic capacity in these cells, consequently affecting body adiposity and glucose tolerance. These effects are scrutinized in mice housed in a thermoneutral environment and in a human dietary intervention trial. METHODS AND RESULTS: Mice are housed in a thermoneutral environment eliminating the superimposing effect of mild cold-exposure on thermogenic adipocyte recruitment. Dietary fish oil supplementation in two different inbred mouse strains neither affects body mass trajectory nor enhances the recruitment of brown and brite adipocytes, both in the presence and absence of a ß3-adrenoreceptor agonist imitating the effect of cold-exposure on adipocytes. In line with these findings, dietary fish oil supplementation of persons with overweight or obesity fails to recruit thermogenic adipocytes in subcutaneous adipose tissue. CONCLUSION: Thus, the authors' data question the hypothesized potential of n3-PUFA as modulators of adipocyte-based thermogenesis and energy balance regulation.

Isolation, Culture, and Functional Analysis of Murine Thermogenic Adipocytes.

Studying brown and brite adipose tissue requires precise and reliable quantification of cellular thermogenesis. This protocol describes the isolation of primary murine pre-adipocytes, differentiation into thermogenic brown and brite adipocytes, and subsequent oxygen consumption analysis. Commonly applied procedures only measure basal and maximal proton leak-linked oxygen consumption but not explicitly uncoupling protein 1 (UCP1)-dependent respiration. Meaningful oxygen consumption analyses require (1) the activation of UCP1, (2) control over intracellular free-fatty-acid levels, and (3) inhibition of ATP-consuming futile cycles. For complete details on the use and execution of this protocol, please refer to Li et al. (2014, 2017, 2018) and Schweizer et al. (2018).

Comparative assessment of purified saponins as permeabilization agents during respirometry.

Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.

Fatty Acid Metabolite Profiling Reveals Oxylipins as Markers of Brown but Not Brite Adipose Tissue.

Metabolites of omega-6 and omega-3 polyunsaturated fatty acids are important signaling molecules implicated in the control of adipogenesis and energy balance regulation. Some of these metabolites belonging to the group of oxylipins have been associated with non-shivering thermogenesis in mice mediated by brown or brite adipose tissue. We aimed to identify novel molecules with thermogenic potential and to clarify the relevance of these findings in a translational context. Therefore, we characterized and compared the oxylipin profiles of murine and human adipose tissues with different abundance of brown or brite adipocytes. A broad panel of 36 fatty acid metabolites was quantified in brown and white adipose tissues of C57BL/6J mice acclimatized to different ambient temperatures and in biopsies of human supraclavicular brown and white adipose tissue. The oxylipin profile of murine brite adipose tissue was not distinguishable from white adipose tissue, suggesting that adipose tissue browning in vivo is not associated with major changes in the oxylipin metabolism. Human brown and white adipose tissue also exhibited similar metabolite profiles. This is in line with previous studies proposing human brown adipose tissue to resemble the nature of murine brite adipose tissue representing a heterogeneous mixture of brite and white adipocytes. Although the global oxylipin profile served as a marker for the abundance of thermogenic adipocytes in bona fide brown but not white adipose tissue, we identified 5-HETE and 5,6-EET as individual compounds consistently associated with the abundance of brown or brite adipocytes in human BAT and murine brite fat. Further studies need to establish whether these candidates are mere markers or functional effectors of thermogenic capacity.

Purine Nucleotides in the Regulation of Brown Adipose Tissue Activity.

Non-shivering thermogenesis in mammalian brown adipose tissue is a powerful mechanism to defend normothermia in cold climates. To minimize the loss of chemical energy, the central functional component, mitochondrial uncoupling protein 1, UCP1, must be tightly regulated. The canonical pathway of UCP1 activation includes lipolytic release of free fatty acids in response to an adrenergic signal. Activating fatty acids overcome constitutive inhibition of UCP1 by the di- and triphosphate forms of purine nucleotides, i.e., ATP, ADP, GTP, and GDP. Cellular concentrations of inhibitory, free nucleotides are subject to significant, adrenergically induced alterations. The regulatory components involved may constitute novel drug targets to manipulate brown fat thermogenesis and thereby organismic energy balance. We here review evidence for and against a dominant role of nucleotides in thermogenic control, describe conceptual routes to endogenously and pharmacologically alter free nucleotide pool size, speculate on a signaling role of degradation products released from active brown fat, and highlight gaps in our understanding of signaling and metabolic pathways involved.