Skin is a regulatory hub for energy expenditure and metabolism, and alteration of lipid metabolism enzymes in skin impacts thermogenesis and obesogenesis in mice. Here we show that thermal properties of skin are highly reactive to diet: within three days, a high fat diet reduces heat transfer through skin. In contrast, a dietary manipulation that prevents obesity accelerates energy loss through skins. We find that skin is the largest target for dietary fat delivery, and that dietary triglyceride is assimilated by epidermis and dermal white adipose tissue, persisting for weeks after feeding. With caloric-restriction, mouse skins thin and assimilation of circulating lipids decreases. Using multi-modal lipid profiling, keratinocytes and sebocytes are implicated in lipid changes, which correlate with thermal function. We propose that skin should be routinely included in physiological studies of lipid metabolism, given the size of the skin lipid reservoir and its adaptable functionality.

Skin has been shown to be a regulatory hub for energy expenditure and metabolism: mutations of skin lipid metabolism enzymes can change the rate of thermogenesis and susceptibility to diet-induced obesity. However, little is known about the physiological basis for this function. Here we show that the thermal properties of skin are highly reactive to diet: within three days, a high fat diet reduces heat transfer through skin. In contrast, a dietary manipulation that prevents obesity accelerates energy loss through skins. We found that skin was the largest target in a mouse body for dietary fat delivery, and that dietary triglyceride was assimilated both by epidermis and by dermal white adipose tissue. Skin from mice calorie-restricted for 3 weeks did not take up circulating lipids and showed a highly depleted stratum corneum. Dietary triglyceride acyl groups persist in skin for weeks after feeding. Using multi-modal lipid profiling, we have implicated both keratinocytes and sebocytes in the altered lipids which correlate with thermal function. In response to high fat feeding, wax diesters and ceramides accumulate, and triglycerides become more saturated. In contrast, in response to the dramatic loss of adipose tissue that accompanies restriction of the branched chain amino acid isoleucine, skin becomes more heat-permeable, resisting changes induced by Western diet feeding, with a signature of depleted signaling lipids. We propose that skin should be routinely included in physiological studies of lipid metabolism, given the size of the skin lipid reservoir and its adaptable functionality.

Cytosolic citrate is imported from the mitochondria by SLC25A1, and from the extracellular milieu by SLC13A5. In the cytosol, citrate is used by ACLY to generate acetyl-CoA, which can then be exported to the endoplasmic reticulum (ER) by SLC33A1. Here, we report the generation of mice with systemic overexpression (sTg) of SLC25A1 or SLC13A5. Both animals displayed increased cytosolic levels of citrate and acetyl-CoA; however, SLC13A5 sTg mice developed a progeria-like phenotype with premature death, while SLC25A1 sTg mice did not. Analysis of the metabolic profile revealed widespread differences. Furthermore, SLC13A5 sTg mice displayed increased engagement of the ER acetylation machinery through SLC33A1, while SLC25A1 sTg mice did not. In conclusion, our findings point to different biological responses to SLC13A5- or SLC25A1-mediated import of citrate and suggest that the directionality of the citrate/acetyl-CoA pathway can transduce different signals.

Supplementary Methods, Figures 1-4, Table 1 from Wnt1 Expression Induces Short-Range and Long-Range Cell Recruitments That Modify Mammary Tumor Development and Are Not Induced by a Cell-Autonomous β-Catenin Effector

PDF file - 463K, Analysis of dataset GSE2990 using the RESTless Gene Signature and clustered using Euclidean distance.

<p>This file contains additional technical detail (antibodies used, gating strategy for FACS) and additional experimental data to complement that shown in the main figures.</p>

<p>PDF file - 29K, Tumors in GSE2990 and GSE2034 listed as RESTless or RESTfl.</p>

Low protein (LP) diets are associated with a decreased risk of diabetes in humans, and a low protein diet promotes leanness and glycemic control in both rodents and humans. While the effects of a LP diet on glycemic control are mediated by reduced dietary levels of the branched- chain amino acids (BCAAs), we have observed that reducing dietary levels of the other six essential amino acids leads to changes in body composition. Here, we find that dietary histidine plays a key role in the response to a LP diet in male C57BL/6J mice. Specifically reducing dietary levels of histidine by 67% reduces weight gain of young, lean male mice, reducing both adipose and lean mass gain, without altering glucose metabolism. Specifically reducing dietary histidine rapidly reverses diet-induced obesity and hepatic steatosis in diet-induced obese male mice, increasing insulin sensitivity; this normalization of metabolic health was associated not with caloric restriction or increased activity, but with increased energy expenditure. We find that the effects of histidine restriction surprisingly does not require the energy balance hormone Fgf21 . Histidine restriction started in mid-life promoted leanness and glucose tolerance in aged males but not females, but did not affect frailty or lifespan in either sex. Finally, we demonstrate that variation in dietary histidine levels helps to explain body mass index differences in humans. Overall, our findings demonstrate that dietary histidine is a key regulator of weight and body composition in male mice and in humans, and suggest that reducing dietary levels of histidine may be a highly translatable option for the treatment of obesity. Key Points Protein restriction (PR) promotes metabolic health in rodents and humans and extends rodent lifespan. Restriction of specific individual essential amino acids can recapitulate the benefits of PR. Reduced histidine promotes leanness and increased energy expenditure in mice. Reduced histidine does not extend the lifespan of mice when begun in mid-life. Dietary levels of histidine are positively associated with BMI in humans.

ABSTRACTMammalian skin impacts metabolic efficiency system-wide, controlling the rate of heat loss and consequent heat production. Here we compare the unique fat depots associated with mouse and human skin, to determine whether they have corresponding function and regulation. For human, we assay a skin-associated fat (SAF) body-wide depot to distinguish it from the subcutaneous fat pads characteristic of abdomen and upper limbs. We show that the thickness of SAF is not related to general adiposity; it is much thicker (1.6-fold) in women than men, and highly subject-specific. We used molecular and cellular assays of β-adrenergic induced lipolysis and found that dermal white adipose tissue (dWAT) in mice is resistant to lipolysis; in contrast, the body-wide human SAF depot becomes lipolytic, generating heat in response to β-adrenergic stimulation. In mice challenged to make more heat to maintain body temperature (either environmentally or genetically), there is a compensatory increase in thickness of dWAT: A corresponding β-adrenergic stimulation of human skin adipose (in vivoor in explant) depletes adipocyte lipid content. We summarize the regulation of skin-associated adipocytes by age, sex, and adiposity, for both species. We conclude that the body-wide dWAT depot of mice shows unique regulation that enables it to be deployed for heat preservation; combined with the actively lipolytic subcutaneous mammary fat pads they enable thermal defense. The adipose tissue that covers human subjects produces heat directly, providing an alternative to the brown adipose tissues.KEY POINTS SUMMARYSeveral distinct strategies produce and conserve heat to maintain body temperature of mammals, each associated with unique physiologies, with consequence for wellness and disease susceptibilityHighly regulated properties of skin offset the total requirement for heat productionWe hypothesize that the adipose component of skin is primarily responsible for modulating heat flux; here we evaluate the relative regulation of adipose depots in mouse and human, to test their recruitment to heat production and conservationWe found that insulating mouse dermal white adipose tissue accumulates in response to environmentally- and genetically-induced cool stress; this layer is one of two adipose depots closely apposed to mouse skin, where the subcutaneous mammary gland fat pads are actively recruited to heat productionIn contrast, the body-wide adipose depot associated with human skin produces heat directly, potentially creating an alternative to the centrally regulated brown adipose tissue

RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested "exclusion-based " purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP-RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro.

Thermoneutral housing has been shown to promote more accurate and robust development of several pathologies in mice. Raising animal housing temperatures a few degrees may create a relatively straightforward opportunity to improve translatability of mouse models. In this commentary, we discuss the changes of physiology induced in mice housed at thermoneutrality, and review techniques for measuring systemic thermogenesis, specifically those affecting storage and mobilization of lipids in adipose depots. Environmental cues are a component of the information integrated by the brain to calculate food consumption and calorie deposition. We show that relative humidity is one of those cues, inducing a rapid sensory response that is converted to a more chronic susceptibility to obesity. Given high inter-institutional variability in the regulation of relative humidity, study reproducibility may be improved by consideration of this factor. We evaluate a "humanized" environmental cycling protocol, where mice sleep in warm temperature housing, and are cool during the wake cycle. We show that this protocol suppresses adaptation to cool exposure, with consequence for adipose-associated lipid storage. To evaluate systemic cues in mice housed at thermoneutral temperatures, we characterized the circulating lipidome, and show that sera are highly depleted in some HDL-associated phospholipids, specifically phospholipids containing the essential fatty acid, 18:2 linoleic acid, and its derivative, arachidonic acid (20:4) and related ether-phospholipids. Given the role of these fatty acids in inflammatory responses, we propose they may underlie the differences in disease progression observed at thermoneutrality.

RNA binding proteins (RBPs) are critical regulators of cellular phenotypes, and dysregulated RBP expression is implicated in various diseases including cancer. A single RBP can bind to and regulate the expression of many RNA molecules via a variety of mechanisms, including translational suppression, prevention of RNA degradation, and alteration in subcellular localization. To elucidate the role of a specific RBP within a given cellular context, it is essential to first identify the group of RNA molecules to which it binds. This has traditionally been achieved using cross-linking-based assays in which cells are first exposed to agents that cross-link RBPs to nucleic acids and then lysed to extract and purify the RBP-nucleic acid complexes. The nucleic acids within the mixture are then released and analyzed via conventional means (e.g., microarray analysis, qRT-PCR, RNA sequencing, or Northern blot). While cross-linking-based ribonucleoprotein immunoprecipitation (RIP) has proven its utility within some contexts, it is technically challenging, inefficient, and suboptimal given the amount of time and resources (e.g., cells and antibodies) required. Additionally, these types of studies often require the use of over-expressed versions of proteins, which can introduce artifacts. Here, we describe a streamlined version of RIP that utilizes exclusion-based purification technologies. This approach requires significantly less starting material and resources compared to traditional RIP approaches, takes less time, which is tantamount given the labile nature of RNA, and can be used with endogenously expressed proteins. The method described here can be used to study RNA-protein interactions in a variety of cellular contexts. Graphical abstract.

Low-protein diets promote metabolic health in rodents and humans, and the benefits of low-protein diets are recapitulated by specifically reducing dietary levels of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we demonstrate that each BCAA has distinct metabolic effects. A low isoleucine diet reprograms liver and adipose metabolism, increasing hepatic insulin sensitivity and ketogenesis and increasing energy expenditure, activating the FGF21-UCP1 axis. Reducing valine induces similar but more modest metabolic effects, whereas these effects are absent with low leucine. Reducing isoleucine or valine rapidly restores metabolic health to diet-induced obese mice. Finally, we demonstrate that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal isoleucine as a key regulator of metabolic health and the adverse metabolic response to dietary BCAAs and suggest reducing dietary isoleucine as a new approach to treating and preventing obesity and diabetes.

Wnt signaling is an important morphogenetic signaling pathway best known for its essential role in determining embryonic cell fates; it is often activated to re-specify fetal cells or to maintain the lineage flexibility of somatic stem cells. In this review, we consider the role of this pathway in the remarkable process of differentiation, growth and morphogenesis of the mammary gland during embryogenesis, ductal outgrowth and pregnancy. Specifically, mammary stem cells are compared with stem cells from other tissues, to identify commonalities and differences. Wnt signaling is known to be required to maintain the bipotent basal stem cell present in adult mammary ductal trees, however, the absence of this stem cell has little effect on growth or morphogenesis, and Wnt signaling is not induced during the ductal/alveolar expansion during pregnancy. The evidence for pre-determined hierarchies of mammary epithelial cells is reviewed, together with the role of signaling between mixtures of specified mammary epithelial cells in the maintenance of Wnt-dependent clonagenic stem cells. The dazzling variety of Wnt signaling components expressed by mammary epithelial cells is presented, along with some potential stromal sources of Wnt proteins that may be important starting points for the induction of plasticity in the epithelium.

Zena Werb was a legendary scientist and remarkable human being who made defining contributions to the study of nearly every mammalian organ and stage of development and myriad human diseases. She had an encyclopedic knowledge of past literature and an open, creative orientation toward the future. She was a direct and unfiltered critic, a dear friend, a supportive mentor, and a pioneering advocate for women. She was born during World War II and raised by refugee parents in the midst of bigotry, racism, and xenophobia, yet she lived her life as an optimist. We remember her with a smile on her face—a knowing one—informed by hard experience. We start, as she would want, with the science. Zena made paradigm-shifting discoveries and reinvented herself every 5 years, displaying an exceptional ability to connect molecular function to cell, tissue, and organ consequences. Zena was a key leader in the discovery of the matrix metalloproteinases (MMPs) and the elucidation of their molecular function and physiological roles. She also contributed to realizing that extracellular matrix (ECM)-integrin connections transduce signals inside the cell. She led in the demonstration that fibroblasts and immune cells secrete diverse proteases and that proteolysis is a key driver of immune function and cancer progression. When Zena started her independent career in the 1970s, the intellectual focus in the broader field was on transcription factors, while proteases seemed limited, capable only of destroying proteins. It took real intellectual courage to insist, and real persistence to publish hundreds of papers that showed, proteolytic cleavage is a central regulator of cell, tissue, and organ structure and function. Next, Zena turned to understanding the in vivo role of matrix metalloproteinases. Together with her close friend and long-term collaborator Mina Bissell, she tested the effect of proteases on epithelial morphogenesis, initially focusing on their role in involution of the mammary epithelium after lactation. They revealed intricate paracrine conversations between the ECM, integrins, and transcriptional responses. The concept that emerged from her research was that proteolysis is a mechanism of regulating and accessing protein information; it could release bound growth factors, generate novel signaling molecules, and alter the physical properties of the microenvironment. Zena made important contributions to developmental biology, including early development (implantation, gastrulation), organogenesis (lung, limb, bone, and mammary gland), and angiogenesis. Her early work revealed the critical role of MMPs in embryonic implantation, trophoblast differentiation, and vascularization. She worked on the role of ECM synthesis and remodeling in bone and cartilage development. These studies highlighted the requirements for MMPs in endochondrial ossification and showed that ossification is tightly linked to angiogenesis. In the central nervous system, her contributions spanned developmental neurobiology, neural repair, and cancer. In mammary gland biology, she was the first to show that MMP-3 regulates branching morphogenesis, apoptosis, and stem cell homeostasis. Her contributions also extended beyond MMPs to transcription factors like GATA-3 that maintain normal mammary gland differentiation and also play an important role in metastatic progression. Zena’s understanding of disease was always grounded in a deep understanding of the normal biology of the tissue, with cancer being an example of development gone awry. Her seminal work on MMPs and the tissue microenvironment laid the critical foundation for advances in our understanding of tumor progression and metastasis. Together with Bissell, Zena showed that MMP-3 is sufficient to induce epithelial-to-mesenchymal transition and neoplastic progression. With Douglas Hanahan, she showed major roles of MMP-9 in tumor angiogenesis and cancer metastasis. These discoveries contributed to a frenzied search for MMP inhibitors as anti-cancer drugs. However, her own work across organs suggested a complex role for MMPs on tissue biology and showed that MMPs can inhibit, as well as promote, cancer in different contexts and can contribute via non-proteolytic functions. Over the past several years, Zena focused on metastasis, the primary cause of cancer death. “If you’re going to spend 80 hours a week thinking about a problem, you might as well spend it thinking about a problem with big significance and impact.” Zena Werb Zena gave her trainees intellectual freedom to pursue their own ideas and projects, even with long detours, because she knew that both the choice and the struggle were essential to finding the confidence and voice to think independently. She viewed her role as providing the counterpoint to our ideas, the chess partner in the game of science. We went to Zena not with questions we needed answered but with questions we needed help thinking about. Zena always tried to see data literally as soon as it was generated; she did “rounds,” visiting each trainee at the bench at least daily, asking for the very latest results and then relating our findings to data from a decades-old paper or a previous trainee. We were convinced that she had a photographic memory for data (although not for people’s names, which she readily admitted). She pushed her mentees to defend their ideas, assumptions, and conclusions and was direct in her criticism of underdeveloped ideas or mediocre projects. Once convinced, she was a powerful advocate, adding ideas, funding, and collaborators to push projects from idea to publication. She inspired us to want to know more, to investigate further, and always to read more. Her exuberance for science was contagious. Like her understanding of the importance of cellular microenvironment, Zena understood that her mentees’ environment was critical. Her office door was always open, and she celebrated and fought hard to provide a diverse and nurturing environment. Each lab meeting covered a breadth of topics that could include MMP biochemistry, bone development, breast cancer, immunology, or epithelial development and highlighted Zena’s deep knowledge base in normal physiology and pathophysiology. Her scientific and career advice were always on point. She encouraged us not to dwell on our problems but instead to focus on finding a way to get beyond them. She saw each of us as individuals, adapted her mentoring style to our strengths and weaknesses, and took great care in selecting speaking opportunities, journal invitations, and collaborations to advance our careers. Zena’s dedication to us was absolute and followed with us years and decades after leaving her lab, as we continued to seek her personal, scientific, and professional advice as we advanced in our careers. Each generation had a different name for the resulting broad extended family (Werbians, Werbies, Werbites, Zenagrafts), but we remain united in continuing to collaborate and to support one another. We all remember Zena with a vibrant smile and an optimistic attitude. This optimism was intentional and persisted despite her having to overcome many barriers. Zena started her career in a time of frank and unapologetic discrimination against women in science and medicine. She was not hired to tenure-track faculty positions initially, though she had many high-profile publications. She knew she had to work harder and publish more papers than her competitors for any promotion, award, or honor. She had a keen sense of fairness, and while she did not dwell on her personal obstacles, she did not forget how she was treated or how others with fewer talents were given easier paths. She chose, sometimes with great effort, to work around obstacles and to chart her course forward. Once she got her seat at the table in university and international leadership roles, she used her voice to speak up for underrepresented voices. She was a tireless advocate for equality around hiring, pay, and representation in seminars and conferences and for transparency and advice in the complex, normally opaque paths of the scientific career. She sought to make the process more transparent and fairer for the next generation. Her advice and perspective were especially transformational in the lives and careers of her female trainees. Those who knew Zena only professionally might not know that she was a marvelous cook and a gracious hostess. She always made sure the lab had a constant supply of free coffee, and while the focus in lab meetings was data-data-data, it was often combined with homemade treats, baked personally, despite her busy schedule. We all remember her Bubbie’s potato latkes, honey cake, biscotti, stuffed grape leaves, and holiday truffles. Her hospitality extended to our annual dim sum celebrations and to the wonderful parties she threw at her house as trainees were launched to their next career stage. Her passion for food was matched by her passion for travel and her love of visual and performing arts. A season ticketholder to the San Francisco Symphony, Zena would invite different lab members to join her for concerts and sometimes for a wonderful dinner beforehand. We each remember sitting and talking in her office, looking out at the view of the Golden Gate Bridge, and listening to classical music, cup of coffee or tea in hand. As we reflect on Zena’s exceptional life story, we note how much the world would have missed if even one small detail had gone slightly differently during World War II. Her parents were Polish refugees who met during the war. Her father was a mathematician whose university studies were cut short by the war, and her mother was a lithographer. Months before Zena was born, her parents were separated, and Zena was born March 24, 1945, in Bergen-Belsen, just weeks before the concentration camp was liberated and the war ended. After the war, her mother and Zena slowly made their way back to Poland, where they discovered that her father had avoided imprisonment by making his way via the underground to Italy and was looking for them. Her mother, carrying Zena, successfully walked across the Austrian Alps into Italy after multiple unsuccessful attempts at entering. Zena was 2 1/2 years old and afflicted with rickets when the family finally reunited in Italy at the displaced persons camp in Brindisi. They emigrated to Saskatchewan, Canada, raising pigs and chickens on their farm for a few years before moving to Toronto and then to a working farm and slaughterhouse on the Niagara Escarpment in Ontario. Living in rural Ontario, Zena attended a one-room schoolhouse through 8th grade and skipped two grades before entering the University of Toronto at 16 years of age. At her high school graduation, Zena gave an extremely prescient valedictory address that highlighted her lifelong quest for knowledge, commitment to hard work, and optimism for the future: “It takes a lifetime to become educated. Education is the fulfillment of the task of becoming a human being. We must learn to appreciate beauty, to contemplate truths, and to communicate with other people… Our futures depend on our initiative… Select a goal and work toward it with earnestness.” Zena lived her life oriented to the future, with a burning sense that her life’s work was contributing to something good that was greater than herself. As Zena’s former trainees, we hope to continue her legacy by learning from her fearless and bold life. She taught by example how to overcome obstacles on the way to visionary achievements, to foster and promote innovative science, to celebrate (especially unexpected) discoveries, and to take seriously our roles in mentoring the next generation of scientists. We hope that as Zena’s mentees and “lab children” we also will contribute to her life’s work of understanding the cellular microenvironment. We loved Zena and are forever grateful for having known her and for the positive impact she had on our lives and the lives of so many others. We were all her family.

The GLUT (SLC2) family of membrane-associated transporters are described as glucose transporters. However, this family is divided into three classes and, though the regulated transporter activity of class I proteins is becoming better understood, class III protein functions continue to be obscure. We have cataloged the relative expression and splicing of SLC2 mRNA isomers in tumors and normal tissues, with a focus on breast tumors and cell lines. mRNA for the class III protein GLUT8 is the predominant SLC2 species expressed alongside GLUT1 in many tissues, but GLUT8 mRNA exists mostly as an untranslated splice form in tumors. We confirm that GLUT8 is not presented at the cell surface and does not transport glucose directly. However, we reveal a lysosome-dependent reaction that cleaves the GLUT8 protein and releases the carboxy-terminal peptide to a separate vesicle population. Given the localization of GLUT8 at a major metabolic hub (the late endosomal/lysosomal interface) and its regulated cleavage reaction, we evaluated TXNIP-mediated hexosamine homeostasis and speculate that GLUT8 may function as a sensory component of this reaction.

OBJECTIVE:Elimination of food calories as heat could help redress the excess accumulation of metabolic energy exhibited as obesity. Prior studies have focused on the induction of thermogenesis in beige and brown adipose tissues as the application of this principle, particularly because the β-adrenergic environment associated with thermogenic activation has been shown to have positive health implications. The counterpoint to this strategy is the regulation of heat loss; we propose that mammals with inefficient heat conservation will require more thermogenesis to maintain body temperature. METHODS:Surface temperature thermography and rates of trans-epidermal water loss were integrated to profile the total heat transfer of genetically-engineered and genetically variable mice. RESULTS:These data were incorporated with energy expenditure data to generate a biophysical profile to test the significance of increased rates of evaporative cooling. CONCLUSIONS:We show that mouse skins vary considerably in their heat retention properties, whether because of naturally occurring variation (SKH-1 mice), or genetic modification of the heat-retaining lipid lamellae (SCD1, DGAT1 or Agouti Ay obese mice). In particular, we turn attention to widely different rates of evaporative cooling as the result of trans-epidermal water loss; higher rates of heat loss by evaporative cooling leads to increased demand for thermogenesis. We speculate that this physiology could be harnessed to create an energy sink to assist with strategies aimed at treating metabolic diseases.

BACKGROUND The induction of beige adipocytes in s.c. white adipose tissue (WAT) depots of humans is postulated to improve glucose and lipid metabolism in obesity. The ability of obese, insulin-resistant humans to induce beige adipose tissue is unknown. METHODS We exposed lean and obese research participants to cold (30-minute ice pack application each day for 10 days of the upper thigh) or treated them with the β3 agonist mirabegron. We determined beige adipose marker expression by IHC and quantitative PCR, and we analyzed mitochondrial bioenergetics and UCP activity with an Oxytherm system. RESULTS Cold significantly induced UCP1 and TMEM26 protein in both lean and obese subjects, and this response was not associated with age. Interestingly, these proteins increased to the same extent in s.c. WAT of the noniced contralateral leg, indicating a crossover effect. We further analyzed the bioenergetics of purified mitochondria from the abdominal s.c. WAT of cold-treated subjects and determined that repeat ice application significantly increased uncoupled respiration, consistent with the UCP1 protein induction and subsequent activation. Cold also increased State 3 and maximal respiration, and this effect on mitochondrial bioenergetics was stronger in summer than winter. Chronic treatment (10 weeks; 50 mg/day) with the β3 receptor agonist mirabegron induces UCP1, TMEM26, CIDEA, and phosphorylation of HSL on serine660 in obese subjects. CONCLUSION Cold or β3 agonists cause the induction of beige adipose tissue in human s.c. WAT; this phenomenon may be exploited to increase beige adipose in older, insulin-resistant, obese individuals. TRIAL REGISTRATION Clinicaltrials.gov NCT02596776, NCT02919176. FUNDING NIH (DK107646, DK112282, P20GM103527, and by CTSA grant UL1TR001998).

Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan normally expressed primarily by epithelia and plasma cells, is aberrantly induced in stromal fibroblasts of breast carcinomas. Stromal fibroblast-derived Sdc1 participates in paracrine growth stimulation of breast carcinoma cells and orchestrates stromal extracellular matrix fiber alignment, thereby creating a migration and invasion-permissive microenvironment. Here, we specifically tested the role of stromal Sdc1 in metastasis.