Parallel control of cold-triggered adipocyte thermogenesis by UCP1 and CKB.

That uncoupling protein 1 (UCP1) is the sole mediator of adipocyte thermogenesis is a conventional viewpoint that has primarily been inferred from the attenuation of the thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue. To mitigate these potentially confounding ancillary changes, we constructed mice with inducible adipocyte-selective Ucp1 disruption. We find that, although germline Ucp1-/- mice succumb to cold-induced hypothermia with complete penetrance, most mice with the inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co-deletion of Ucp1 and creatine kinase b (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold intolerance. Following UCP1 deletion or UCP1/CKB co-deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature brown adipocytes that coordinately restore UCP1 and CKB expression. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy-through UCP1 and CKB-to promote cold-induced energy dissipation.

ADRA1A-Gαq signalling potentiates adipocyte thermogenesis through CKB and TNAP.

Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis1. Aside from cAMP signalling downstream of ß-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α1-adrenergic receptor (AR) and ß3-AR signalling induces the expression of thermogenic genes of the futile creatine cycle2,3, and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α1-AR subtype (ADRA1A) and Gαq to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gαq and Gαs signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A-Gαq-futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis.

A brown fat-selective mechanism of mitochondrial calcium import?

In this issue of Cell Metabolism, Xue et al. propose that the mitochondrial calcium uniporter (MCU) binds uncoupling protein 1 (UCP1) via the MCU regulator (EMRE) to form a protein complex that the authors term the "thermoporter." Through gain- and loss-of-function experiments, the authors infer that the thermoporter promotes calcium influx into the mitochondrial matrix to enhance NADH production, which supports thermogenesis in brown adipose tissue (BAT).

Creatine transport and creatine kinase activity is required for CD8+ T cell immunity.

The factors that promote T cell expansion are not fully known. Creatine is an abundant circulating metabolite that has recently been implicated in T cell function; however, its cell-autonomous role in immune-cell function is unknown. Here, we show that creatine supports cell-intrinsic CD8+ T cell homeostasis. We further identify creatine kinase B (CKB) as the creatine kinase isoenzyme that supports these T cell properties. Loss of the creatine transporter (Slc6a8) or Ckb results in compromised CD8+ T cell expansion in response to infection without influencing adenylate energy charge. Rather, loss of Slc6a8 or Ckb disrupts naive T cell homeostasis and weakens TCR-mediated activation of mechanistic target of rapamycin complex 1 (mTORC1) signaling required for CD8+ T cell expansion. These data demonstrate a cell-intrinsic role for creatine transport and creatine transphosphorylation, independent of their effects on global cellular energy charge, in supporting CD8+ T cell homeostasis and effector function.

Measurement of Futile Creatine Cycling Using Respirometry.

Thermogenic adipose tissue plays a vital function in regulating whole-body energy expenditure and nutrient homeostasis due to its capacity to dissipate chemical energy as heat, in a process called non-shivering thermogenesis. A reduction of creatine levels in adipocytes impairs thermogenic capacity and promotes diet-induced obesityKazak et al, Cell 163, 643-55, 2015; Kazak et al, Cell Metab 26, 660-671.e3, 2017; Kazak et al, Nat Metab 1, 360-370, 2019). Mechanistically, thermogenic respiration can be promoted by the liberation of an excess quantity of ADP that is dependent on addition of creatine. A model of a two-enzyme system, which we term the Futile Creatine Cycle, has been posited to support this thermogenic action of creatine. Futile creatine cycling can be monitored in purified mitochondrial preparations wherein creatine-dependent liberation of ADP is monitored through the measurement of oxygen consumption under ADP-limiting conditions. The current model proposes that, in thermogenic fat cells, mitochondria-targeted creatine kinase B (CKB) uses mitochondrial-derived ATP to phosphorylate creatine (Rahbani JF, Nature 590, 480-485, 2021). The creatine kinase reaction generates phosphocreatine and ADP, and ADP stimulates respiration. Next, a pool of mitochondrial phosphocreatine is directly hydrolyzed by a phosphatase, to regenerate creatine. The liberated creatine can then engage mitochondrial CKB to trigger another round of this cycle to support ADP-dependent respiration. In this model, the coordinated action of creatine phosphorylation and phosphocreatine hydrolysis triggers a futile cycle that produces a molar excess of mitochondrial ADP to promote thermogenic respiration (Rahbani JF, Nature 590, 480-485, 2021; Kazak and Cohen, Nat Rev Endocrinol 16, 421-436, 2020). Here, we provide a detailed method to perform respiratory measurements on isolated mitochondria and calculate the stoichiometry of creatine-dependent ADP liberation. This method provides a direct measure of the futile creatine cycle.

Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine.

Adaptive thermogenesis has attracted much attention because of its ability to increase systemic energy expenditure and to counter obesity and diabetes1-3. Recent data have indicated that thermogenic fat cells use creatine to stimulate futile substrate cycling, dissipating chemical energy as heat4,5. This model was based on the super-stoichiometric relationship between the amount of creatine added to mitochondria and the quantity of oxygen consumed. Here we provide direct evidence for the molecular basis of this futile creatine cycling activity in mice. Thermogenic fat cells have robust phosphocreatine phosphatase activity, which is attributed to tissue-nonspecific alkaline phosphatase (TNAP). TNAP hydrolyses phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation. Unlike in other cells, TNAP in thermogenic fat cells is localized to the mitochondria, where futile creatine cycling occurs. TNAP expression is powerfully induced when mice are exposed to cold conditions, and its inhibition in isolated mitochondria leads to a loss of futile creatine cycling. In addition, genetic ablation of TNAP in adipocytes reduces whole-body energy expenditure and leads to rapid-onset obesity in mice, with no change in movement or feeding behaviour. These data illustrate the critical role of TNAP as a phosphocreatine phosphatase in the futile creatine cycle.

Creatine kinase B controls futile creatine cycling in thermogenic fat.

Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease1. Thermogenesis by adipocytes can counteract obesity and metabolic diseases2,3. In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP-purportedly via a phosphorylation cycle4-to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.

Creatine-mediated crosstalk between adipocytes and cancer cells regulates obesity-driven breast cancer.

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.

Kinetics of Strand Displacement and Hybridization on Wireframe DNA Nanostructures: Dissecting the Roles of Size, Morphology, and Rigidity.

Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study.

Single-stranded templates as railroad tracks for hierarchical assembly of DNA origami.

DNA origami is one of the most effective tools for bottom-up construction of novel objects and devices at the nanometer-scale. However, many applications require larger structures than can be obtained with the conventional single-stranded scaffold, typically 7249 nucleotides. Here, we address this limitation by developing custom-made single-stranded scaffolds that bind pre-assembled origami tiles and induce their one-dimensional organization in high yields. Our synthetic method allows the conversion of multiple repetitive and unique sequences into correctly assembled, large backbones, and to finely tune the position and frequency of each building block. Granted with these regions, three and five origami tiles were successfully arranged in 1-D with the aid of one or two scaffolds, forming a nano-"railroad track". This new method increases length scale in DNA origami without increasing cost and complexity, and is anticipated to increase the yield of other approaches aiming to assemble large origami structures.

DNA Nanotubes with Hydrophobic Environments: Toward New Platforms for Guest Encapsulation and Cellular Delivery.

Natural systems combine different supramolecular interactions in a hierarchical manner to build structures. In contrast, DNA nanotechnology relies almost exclusively on DNA base pairing for structure generation. Introducing other supramolecular interactions can expand the structural and functional range of DNA assemblies, but this requires an understanding of the interplay between these interactions. Here, an economic strategy to build DNA nanotubes functionalized with lipid-like polymers is reported. When these polymers are linked to the nanotube using a spacer, they fold inside to create a hydrophobic environment within the nanotube; the nanotube can encapsulate small molecules and conditionally release them when specific DNA strands are added, as monitored by single-molecule fluorescence microscopy. When the polymers are directly linked to the nanostructure without spacers, they interact intermolecularly to form a network of DNA bundles. This morphological switch can be directly observed using a strand displacement strategy. The two association modes result in different cellular uptake behavior. Nanotubes with internal hydrophobic association show dye-mediated mitochondrial colocalization inside cells; while the bundles disassemble into smaller polymer-coated structures that reduce the extent of nonspecific cellular uptake. This approach uncovers parameters to direct the hierarchical assembly of DNA nanostructures, and produces promising materials for targeted drug delivery.

Encapsulation of Gold Nanoparticles into DNA Minimal Cages for 3D-Anisotropic Functionalization and Assembly.

Gold nanoparticles (AuNPs) endowed with anisotropic DNA valency are an important class of materials, as they can assemble into complex structures with a minimal number of DNA strands. However, methods to encode 3D DNA strand patterns on AuNPs with a controlled number of unique DNA strands in a predesigned spatial arrangement remain elusive. In this work, a simple one-step method to yield such DNA-decorated AuNPs is demonstrated, through encapsulating AuNPs into DNA minimal nanocages. The AuNP@DNA cage encapsulation complex inherits the 3D anisotropic molecular information from the DNA nanocage with enhanced structural stability. The DNA nanocage can be further functionalized and used as a building block for the self-assembly of complex architectures, such as dimers and trimers, programmed assemblies with sequential growth DNA backbones and DNA origami.

Dynamic DNA Nanotubes: Reversible Switching between Single and Double-Stranded Forms, and Effect of Base Deletions.

DNA nanotubes hold great potential as drug delivery vehicles and as programmable templates for the organization of materials and biomolecules. Existing methods for their construction produce assemblies that are entirely double-stranded and rigid, and thus have limited intrinsic dynamic character, or they rely on chemically modified and ligated DNA structures. Here, we report a simple and efficient synthesis of DNA nanotubes from 11 short unmodified strands, and the study of their dynamic behavior by atomic force microscopy and in situ single molecule fluorescence microscopy. This method allows the programmable introduction of DNA structural changes within the repeat units of the tubes. We generate and study fully double-stranded nanotubes, and convert them to nanotubes with one, two and three single-stranded sides, using strand displacement strategies. The nanotubes can be reversibly switched between these forms without compromising their stability and micron-scale lengths. We then site-specifically introduce DNA strands that shorten two sides of the nanotubes, while keeping the length of the third side. The nanotubes undergo bending with increased length mismatch between their sides, until the distortion is significant enough to shorten them, as measured by AFM and single-molecule fluorescence photobleaching experiments. The method presented here produces dynamic and robust nanotubes that can potentially behave as actuators, and allows their site-specific addressability while using a minimal number of component strands.

Sequential growth of long DNA strands with user-defined patterns for nanostructures and scaffolds.

DNA strands of well-defined sequence are valuable in synthetic biology and nanostructure assembly. Drawing inspiration from solid-phase synthesis, here we describe a DNA assembly method that uses time, or order of addition, as a parameter to define structural complexity. DNA building blocks are sequentially added with in-situ ligation, then enzymatic enrichment and isolation. This yields a monodisperse, single-stranded long product (for example, 1,000 bases) with user-defined length and sequence pattern. The building blocks can be repeated with different order of addition, giving different DNA patterns. We organize DNA nanostructures and quantum dots using these backbones. Generally, only a small portion of a DNA structure needs to be addressable, while the rest is purely structural. Scaffolds with specifically placed unique sites in a repeating motif greatly minimize the number of components used, while maintaining addressability. This combination of symmetry and site-specific asymmetry within a DNA strand is easily accomplished with our method.