Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

OBJECTIVE: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. METHODS: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active Ucp1 expression in adult mice. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. RESULTS: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. However, Ucp1-CreERT2 showed no or only partial activation in these tissues of adult mice, indicating the potential for low or transient expression of endogenous Ucp1. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. CONCLUSIONS: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing.

Adipocyte size and fragility and commercial kit costs impose significant limitations on single-cell RNA sequencing of adipose tissue. Accordingly, we developed a workflow to isolate and sample-barcode nuclei from individual adipose tissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 10× Chromium controller. This approach can eliminate batch confounding, and significantly reduces poor-quality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content and cost efficiency.

Aging impairs cold-induced beige adipogenesis and adipocyte metabolic reprogramming.

The energy-burning capability of beige adipose tissue is a potential therapeutic tool for reducing obesity and metabolic disease, but this capacity is decreased by aging. Here, we evaluate the impact of aging on the profile and activity of adipocyte stem and progenitor cells (ASPCs) and adipocytes during the beiging process. We found that aging increases the expression of Cd9 and other fibro-inflammatory genes in fibroblastic ASPCs and blocks their differentiation into beige adipocytes. Fibroblastic ASPC populations from young and aged mice were equally competent for beige differentiation in vitro, suggesting that environmental factors suppress adipogenesis in vivo. Examination of adipocytes by single nucleus RNA-sequencing identified compositional and transcriptional differences in adipocyte populations with age and cold exposure. Notably, cold exposure induced an adipocyte population expressing high levels of de novo lipogenesis (DNL) genes, and this response was severely blunted in aged animals. We further identified natriuretic peptide clearance receptor Npr3, a beige fat repressor, as a marker gene for a subset of white adipocytes and an aging-upregulated gene in adipocytes. In summary, this study indicates that aging blocks beige adipogenesis and dysregulates adipocyte responses to cold exposure and provides a unique resource for identifying cold and aging-regulated pathways in adipose tissue.

Control of lipolysis by a population of oxytocinergic sympathetic neurons.

Oxytocin (OXT), a nine-amino-acid peptide produced in the hypothalamus and released by the posterior pituitary, has well-known actions in parturition, lactation and social behaviour1, and has become an intriguing therapeutic target for conditions such as autism and schizophrenia2. Exogenous OXT has also been shown to have effects on body weight, lipid levels and glucose homeostasis1,3, suggesting that it may also have therapeutic potential for metabolic disease1,4. It is unclear, however, whether endogenous OXT participates in metabolic homeostasis. Here we show that OXT is a critical regulator of adipose tissue lipolysis in both mice and humans. In addition, OXT serves to facilitate the ability of ß-adrenergic agonists to fully promote lipolysis. Most surprisingly, the relevant source of OXT in these metabolic actions is a previously unidentified subpopulation of tyrosine hydroxylase-positive sympathetic neurons. Our data reveal that OXT from the peripheral nervous system is an endogenous regulator of adipose and systemic metabolism.

Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

Objective: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. Methods: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice, to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active UCP1 expression. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. Results: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. However, endogenous Ucp1 was not actively expressed as Ucp1-CreERT2 failed to induce the reporter expression in the mammary glands. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. Conclusions: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

Exploring the heterogeneity of white adipose tissue in mouse and man.

Adipose tissue is a heterogeneous organ, comprising cell types, including mature adipocytes, progenitor cells, immune cells, and vascular cells. Here, we discuss the heterogeneity of human and mouse white adipose tissue in general and white adipocytes specifically, focusing on how our understanding of adipocyte subpopulations has expanded with the advent of single nuclear RNA sequencing and spatial transcriptomics. Furthermore, we discuss critical remaining questions regarding how these distinct populations arise, how their functions differ from one another, and which potentially contribute to metabolic pathophysiology.

Making the Case for Virtual Surgical Planning: Bilateral Sequential Fibula Flaps with Immediate Dental Implants for Maxillectomy.

SUMMARY: Oncologic maxillectomy defects requiring bony reconstruction are among the most challenging head and neck cases because of the complex three-dimensional geometry of the midface. Virtual surgical planning technology is advantageous in these cases because it provides superior positional precision and accuracy compared with traditional techniques and facilitates prosthodontic rehabilitation. Maxillary cancer recurrence after an initial fibula flap reconstruction presents a unique challenge. The authors report the first two cases of sequential fibula flaps after second or recurrent cancer of the maxilla. Virtual surgical planning facilitated resection with adequate tumor margins, optimized anatomic positioning of the fibula construct with three-dimensional printed plates, and enabled immediate functional dental implant placement.

Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

Combination intraoral radiation mouthguard-positioning stent.

The sequelae of head-and-neck radiation may include hyposalivation, dysgeusia, trismus, mucositis, and osteoradionecrosis. A mouthguard used during radiation therapy can mitigate the effects of backscatter radiation from dental restorations. In addition, an intraoral positioning stent can assist in repositioning oral structures, such as the tongue, away from the field of radiation during treatment, thereby limiting dose delivery. The purpose of this article is to provide a technique to fabricate a combination prosthesis, which functions to reposition oral structures as well as mitigate the effects of backscatter from dental restorations during head-and-neck radiation therapy.

Palmitate Potentiates Lipopolysaccharide-Induced IL-6 Production via Coordinated Acetylation of H3K9/H3K18, p300, and RNA Polymerase II.

IL-6 is elevated in obese individuals and participates in the metabolic dysfunction associated with that condition. However, the mechanisms that promote IL-6 expression in obesity are incompletely understood. Because elevated levels of palmitate and LPS have been reported in obesity, we investigated whether these agents interact to potentiate IL-6 production. In this study, we report that LPS induces higher levels of IL-6 in human monocytes in the presence of palmitate. Notably, the priming effect of palmitate is associated with enhanced p300 binding and transcription factor recruitment to Il6 promoter regions. Gene silencing of p300 blocks this action of palmitate. RNA polymerase II recruitment was also enhanced at the Il6 promoter in palmitate/LPS-exposed cells. Acetylation levels of H3K9 and H3K18 were increased in monocytes treated with palmitate. Moreover, LPS stimulation of palmitate-treated cells led to increased levels of the transcriptionally permissive acetylation marks H3K9/H3K18 in the Il6 promoter compared with LPS alone. The effect of palmitate on LPS-induced IL-6 production was suppressed by the inhibition of histone acetyltransferases. Conversely, histone deacetylase inhibitors trichostatin A or sodium butyrate can substitute for palmitate in IL-6 production. Esterification of palmitate with CoA was involved, whereas ß-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and H3K9/H3K18 acetylation. Monocytes of obese individuals showed significantly higher H3K9/H3K18 acetylation and Il6 expression. Overall, our findings support a model in which increased levels of palmitate in obesity create a setting for LPS to potentiate IL-6 production via chromatin remodeling, enabling palmitate to contribute to metabolic inflammation.

An Anatomic Analysis of Fibula Flap Mandible Reconstructions: Implications for Endosseous Implant Placement.

BACKGROUND: Precise planning and evaluation of the fibula bone are necessary if immediate endosseous implant placement is considered. Limited information is available on the anatomical dimensions or density of fibula used in mandibular reconstructions. This study aimed to describe the morphology and dimensions of the fibula used to reconstruct segmental mandibular defects and contrast the findings with the native mandible. METHODS: A retrospective analysis was performed of patients who underwent segmental mandibulectomy reconstructed with osteocutaneous fibula flaps and had at least one postoperative computed tomography scan. Fibula cross sectional dimensions and densities were evaluated with three-dimensional software. Radiographic measurements were obtained from the contralateral mandible medial to the first molar for comparison. RESULTS: Four hundred seventy-seven fibula cross sections from 159 segments were evaluated. Cross-sectional oval, quadrilateral, triangular, and pentagonal shapes differed significantly in proportion (p < 0.001). Thirty-eight percent of segments (95 percent CI, 30 to 46 percent) had differences in cross-section height greater than 1 mm (p < 0.001). Between segments within the same patient, the median height difference was 1.58 mm (range, 0.14 to 6 mm). The superior cortex density was significantly higher for the fibula than the native mandible; however, the medullary space density was significantly lower (p < 0.001). CONCLUSIONS: The current study comprises the most comprehensive description of fibula morphology in mandibular reconstructions and highlights the significant variability that exists. The findings provide justification for the added time and cost of computer-aided design and computer-aided manufacturing in centers interested in performing immediate dental implant placement, as the technology provides the necessary precision and accuracy.

Hepatic IRF3 fuels dysglycemia in obesity through direct regulation of Ppp2r1b.

Inflammation has profound but poorly understood effects on metabolism, especially in the context of obesity and nonalcoholic fatty liver disease (NAFLD). Here, we report that hepatic interferon regulatory factor 3 (IRF3) is a direct transcriptional regulator of glucose homeostasis through induction of Ppp2r1b, a component of serine/threonine phosphatase PP2A, and subsequent suppression of glucose production. Global ablation of IRF3 in mice on a high-fat diet protected against both steatosis and dysglycemia, whereas hepatocyte-specific loss of IRF3 affects only dysglycemia. Integration of the IRF3-dependent transcriptome and cistrome in mouse hepatocytes identifies Ppp2r1b as a direct IRF3 target responsible for mediating its metabolic actions on glucose homeostasis. IRF3-mediated induction of Ppp2r1b amplified PP2A activity, with subsequent dephosphorylation of AMPKα and AKT. Furthermore, suppression of hepatic Irf3 expression with antisense oligonucleotides reversed obesity-induced insulin resistance and restored glucose homeostasis in obese mice. Obese humans with NAFLD displayed enhanced activation of liver IRF3, with reversion after bariatric surgery. Hepatic PPP2R1B expression correlated with HgbA1C and was elevated in obese humans with impaired fasting glucose. We therefore identify the hepatic IRF3-PPP2R1B axis as a causal link between obesity-induced inflammation and dysglycemia and suggest an approach for limiting the metabolic dysfunction accompanying obesity-associated NAFLD.

A single-cell atlas of human and mouse white adipose tissue.

White adipose tissue, once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic and heterogenous, and is involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control and host defence1. High-fat feeding and other metabolic stressors cause marked changes in adipose morphology, physiology and cellular composition1, and alterations in adiposity are associated with insulin resistance, dyslipidemia and type 2 diabetes2. Here we provide detailed cellular atlases of human and mouse subcutaneous and visceral white fat at single-cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells, vascular and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease and provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. These data comprise an extensive resource for the exploration of genes, traits and cell types in the function of white adipose tissue across species, depots and nutritional conditions.

Crosstalk Between Adipose and Lymphatics in Health and Disease.

Adipose tissue, once thought to be an inert receptacle for energy storage, is now recognized as a complex tissue with multiple resident cell populations that actively collaborate in response to diverse local and systemic metabolic, thermal, and inflammatory signals. A key participant in adipose tissue homeostasis that has only recently captured broad scientific attention is the lymphatic vasculature. The lymphatic system's role in lipid trafficking and mediating inflammation makes it a natural partner in regulating adipose tissue, and evidence supporting a bidirectional relationship between lymphatics and adipose tissue has accumulated in recent years. Obesity is now understood to impair lymphatic function, whereas altered lymphatic function results in aberrant adipose tissue deposition, though the molecular mechanisms governing these phenomena have yet to be fully elucidated. We will review our current understanding of the relationship between adipose tissue and the lymphatic system here, focusing on known mechanisms of lymphatic-adipose crosstalk.

Intraoral radiation stents-Primer for clinical use in head and neck cancer therapy.

Intraoral radiation stents (IRS) are prosthetic devices that assist in the effective delivery of radiation to tumor tissues and aim to avoid unnecessary radiation to adjacent healthy tissues, thus limiting postradiotherapy toxicities. They are used to protect or displace vital structures, assist in positioning of the treatment beam for effective administration of radiotherapy, carry a radioactive material, shield healthy tissues of the oral cavity, and/or maintain the desired mouth opening during radiotherapy. With close collaboration between radiation oncologist and oral health care provider, several IRS can be fabricated by the latter for appropriate targeting and delivery of planned radiation dose and optimized treatment results. Modification of these IRS based on individual patient need is recommended to maximize prosthesis utility. The purpose of this review is to discuss the various types of IRS and highlight their clinical utility and benefits in patients receiving radiation therapy in the head and neck cancers.

Immediate Dental Implant Placement in the Oncologic Setting: A Conceptual Framework.

Historically, immediate dental implants have been reserved for patients with benign disease, with full dental rehabilitation rarely being accomplished in the oncologic setting due to concerns related to implant survival, flap compromise, and delay in initiation of adjuvant therapy. Recent developments in technology have made immediate dental implants using virtual surgical planning safe and reliable. At Memorial Sloan Kettering Cancer Center, we have implemented a workflow for immediate dental implant placement in the oncologic patient population that has become a routine part of maxillary and mandibular reconstruction. This approach begins with a multidisciplinary virtual surgical planning session and custom dental splints to be used for cutting and inset guides. Dental implants are placed intraoperatively at the time of tumor resection and reconstruction with the fibula flap. A temporary prosthesis, which can be worn during radiation therapy, is placed following a vestibuloplasty, approximately 4-6 weeks after the initial reconstruction. After the completion of radiation therapy and the resolution of edema, a permanent prosthesis is placed. When critically evaluating our experience, we have found that patients undergoing immediate dental implant placement have higher rates of implant survival and no delay in adjuvant therapy. The protocol described here in detail has successfully expanded the indications for immediate dental rehabilitation in the oncologic patient population.

Virtual Surgical Planning for Oncologic Mandibular and Maxillary Reconstruction.

Virtual surgical planning (VSP) with computer-aided design and computer-aided modeling (CAD/CAM) enables the opportunity to provide personalized medicine in complex head and neck reconstruction. This innovative technology allows ablative and reconstructive surgeons to virtually create and manipulate three-dimensional anatomic models to plan both the resection and reconstruction of complicated maxillofacial defects. Studies demonstrate improvements in preoperative planning, operative efficiency and accuracy, and postoperative outcomes. VSP facilitates immediate dental implantation in selected patients, which can improve the likelihood of achieving dental restoration. This article outlines strategies for technique optimization as well as the applications, advantages and disadvantages of VSP in complex oncologic head and neck reconstruction.