Single-cell transcriptomics identifies adipose tissue CD271+ progenitors for enhanced angiogenesis in limb ischemia.

Therapeutic angiogenesis using mesenchymal stem/stromal cell grafts have shown modest and controversial effects in preventing amputation for patients with critical limb ischemia. Through single-cell transcriptomic analysis of human tissues, we identify CD271+ progenitors specifically from subcutaneous adipose tissue (AT) as having the most prominent pro-angiogenic gene profile distinct from other stem cell populations. AT-CD271+ progenitors demonstrate robust in vivo angiogenic capacity over conventional adipose stromal cell grafts, characterized by long-term engraftment, augmented tissue regeneration, and significant recovery of blood flow in a xenograft model of limb ischemia. Mechanistically, the angiogenic capacity of CD271+ progenitors is dependent on functional CD271 and mTOR signaling. Notably, the number and angiogenic capacity of CD271+ progenitors are strikingly reduced in insulin-resistant donors. Our study highlights the identification of AT-CD271+ progenitors with in vivo superior efficacy for limb ischemia. Furthermore, we showcase comprehensive single-cell transcriptomics strategies for identification of suitable grafts for cell therapy.

The widely used Ucp1-CreEvdr transgene elicits complex developmental and metabolic phenotypes.

Bacterial artificial chromosome transgenic models, including most Cre-recombinases, enable potent interrogation of gene function in vivo but require rigorous validation as limitations emerge. Due to its high relevance to metabolic studies, we performed comprehensive analysis of the Ucp1-CreEvdr line which is widely used for brown fat research. Hemizygotes exhibited major brown and white fat transcriptomic dysregulation, indicating potential altered tissue function. Ucp1-CreEvdr homozygotes also show high mortality, growth defects, and craniofacial abnormalities. Mapping the transgene insertion site revealed insertion in chromosome 1 accompanied by large genomic alterations disrupting several genes expressed in a range of tissues. Notably, Ucp1-CreEvdr transgene retains an extra Ucp1 gene copy that may be highly expressed under high thermogenic burden. Our multi-faceted analysis highlights a complex phenotype arising from the presence of the Ucp1-CreEvdr transgene independently of the intended genetic manipulations. Overall, comprehensive validation of transgenic mice is imperative to maximize discovery while mitigating unexpected, off-target effects.

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide.

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic homeostasis and other physiological and pathological processes. Contrary to other key metabolic pathways, such as glucose disposal, fatty acid synthesis is not routinely functionally assessed, leading to incomplete interpretations of metabolic status. In addition, there is a lack of publicly available detailed protocols suitable for newcomers in the field. Here, we describe an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo synthesis in brown adipose tissue in vivo. This method measures the synthesis of the products of fatty acid synthase independently of a carbon source, and it is potentially useful for virtually any tissue, in any mouse model, and under any external perturbation. Details on the sample preparation for GCMS and downstream calculations are provided. We focus on the analysis of brown fat due to its high levels of de novo fatty acid synthesis and critical roles in maintaining metabolic homeostasis.

Single cell transcriptomics identifies adipose tissue CD271+ progenitors for enhanced angiogenesis in limb ischemia.

Therapeutic angiogenesis using mesenchymal stem/stromal cell grafts have shown modest and controversial effects in preventing amputation for patients with critical limb ischemia. Through single-cell transcriptomic analysis of human tissues, we identified CD271 + progenitors specifically from subcutaneous adipose tissue (AT) as having the most prominent pro-angiogenic gene profile distinct from other stem cell populations. AT-CD271 + progenitors demonstrated robust in vivo angiogenic capacity, over conventional adipose stromal cell grafts, characterized by long-term engraftment, augmented tissue regeneration, and significant recovery of blood flow in a xenograft model of limb ischemia. Mechanistically, the angiogenic capacity of CD271 + progenitors is dependent on functional CD271 and mTOR signaling. Notably, the number and angiogenic capacity of CD271 + progenitors was strikingly reduced in insulin resistant donors. Our study highlights the identification of AT-CD271 + progenitors with in vivo superior efficacy for limb ischemia. Furthermore, we showcase comprehensive single-cell transcriptomics strategies for identification of suitable grafts for cell therapy. HIGHLIGHTS: Adipose tissue stromal cells have a distinct angiogenic gene profile among human cell sources. CD271 + progenitors in adipose tissue have a prominent angiogenic gene profile. CD271 + progenitors show superior therapeutic capacities for limb ischemia. CD271 + progenitors are reduced and functionally impaired in insulin resistant donors.

SEX-DEPENDENT EFFECTS OF ADIPOCYTE STAT3 INHIBITION ON THE INFLAMMATORY RESPONSE DURING SEVERE SEPSIS.

ABSTRACT: Introduction: Sepsis is a dysregulated host response to infection that can lead to life-threatening organ dysfunction. Clinical and animal studies consistently demonstrate that female subjects are less susceptible to the adverse effects of sepsis, demonstrating the importance of understanding how sex influences sepsis outcomes. The signal transducer and activator of transcription 3 (STAT3) pathway are a major signaling pathway that facilitates inflammation during sepsis. STAT3 is abundantly expressed in white adipose tissue; however, little is known about the contribution of white adipose tissue STAT3 activation during sepsis. We hypothesize that adipocyte STAT3 inhibition during severe sepsis will exaggerate the inflammatory response and impact organ injury, in a sex-dependent manner. Methods: We generated STAT3 flox/flox (wild-type [WT]) and adipocyte STAT3 knock out (A-STAT3 KO) mice using Cre-lox technology. Studies were done in 12- to 16-week-old male and female mice. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Control nonseptic mice did not undergo CLP (0 h CLP). Tissues were harvested 18 h after CLP. Body composition was determined by echo magnetic resonance imaging. Energy metabolism was determined by indirect calorimetry. White adipose tissue morphology was determined by hematoxylin and eosin staining, while STAT3 activation in the white adipose tissue was determined by western blot analysis and immunohistochemistry staining of STAT3 activation/phosphorylation at tyrosine 705. Plasma cytokines (TNF-α, IL-6, and leptin) were determined by luminex assay. Neutrophil infiltration of the lung and liver was assessed by myeloperoxidase activity assay. Histological signs of organ injury on lung and liver tissue were assessed by hematoxylin and eosin staining. Liver injury was further assessed by measuring plasma alanine and aspartate aminotransferase. In a separate cohort of mice, sepsis was induced by CLP and mice were monitored every 6-12 h over a 7-day period to assess survival rate. Results: We demonstrate that neither body composition nor energy metabolism is altered with adipocyte STAT3 inhibition in male or female mice, under nonseptic conditions. Sepsis was associated with reduced adipocyte size in female WT and A-STAT3 KO mice, suggesting that this event is STAT3 independent. Sepsis did not alter adipocyte size in male WT and A-STAT3 KO mice, suggesting that this event is also sex dependent. Although STAT3 phosphorylation at tyrosine 705 expression is negligible in male and female A-STAT3 KO mice, septic female WT and A-STAT3 KO mice have higher white adipose tissue STAT3 activation than male WT and A-STAT3 KO mice. Adipocyte STAT3 inhibition did not alter the proinflammatory cytokine response during sepsis in male or female mice, as measured by plasma TNF-α, IL-6, and leptin levels. Adipocyte STAT3 inhibition reduced lung neutrophil infiltration and histological signs of lung injury during sepsis in male mice. On the contrary, adipocyte STAT3 inhibition had no effect on lung neutrophil infiltration or lung injury in female mice. We further demonstrate that neither liver neutrophil infiltration nor histological signs of liver injury are altered by adipocyte STAT3 inhibition during sepsis, in male or female mice. Lastly, adipocyte STAT3 inhibition did not affect survival rate of male or female mice during sepsis. Conclusions: Our study demonstrates that sex influences white adipose tissue STAT3 activation and morphology during sepsis, which is not dependent on the presence of functional STAT3 in mature adipocytes. Furthermore, genetic inhibition of adipocyte STAT3 activation in male, but not female mice, results in reduced lung neutrophil infiltration and lung injury during sepsis. The results from our study demonstrate the importance of considering biological sex and the white adipose tissue as potential sources and targets of inflammation during sepsis.

Fluorescent Genetic Tools for Studying Brown Fat Development and Function in Mice.

Techniques to trace and isolate brown adipocyte precursor and adipocytes during development and disease are essential to fully understand brown adipose tissue development and function. Here we report several protocols using the R26R-mTmG reporter mice in thermogenic tissues based on confocal microscopy and fluorescence based flow cytometry. These techniques may be useful to understand the influence of genetic or environmental alterations in brown adipocyte precursors and adipocyte biology.

A BAFF/APRIL axis regulates obesogenic diet-driven weight gain.

The impact of immune mediators on weight homeostasis remains underdefined. Interrogation of resistance to diet-induced obesity in mice lacking a negative regulator of Toll-like receptor signaling serendipitously uncovered a role for B cell activating factor (BAFF). Here we show that overexpression of BAFF in multiple mouse models associates with protection from weight gain, approximating a log-linear dose response relation to BAFF concentrations. Gene expression analysis of BAFF-stimulated subcutaneous white adipocytes unveils upregulation of lipid metabolism pathways, with BAFF inducing white adipose tissue (WAT) lipolysis. Brown adipose tissue (BAT) from BAFF-overexpressing mice exhibits increased Ucp1 expression and BAFF promotes brown adipocyte respiration and in vivo energy expenditure. A proliferation-inducing ligand (APRIL), a BAFF homolog, similarly modulates WAT and BAT lipid handling. Genetic deletion of both BAFF and APRIL augments diet-induced obesity. Lastly, BAFF/APRIL effects are conserved in human adipocytes and higher BAFF/APRIL levels correlate with greater BMI decrease after bariatric surgery. Together, the BAFF/APRIL axis is a multifaceted immune regulator of weight gain and adipose tissue function.

The Lipid Handling Capacity of Subcutaneous Fat Is Programmed by mTORC2 during Development.

Overweight and obesity are associated with type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal, as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictor upregulate mature adipocyte markers but develop a striking lipid storage defect resulting in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARγ and ChREBP, including genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, which encodes a major mTORC2 substrate and insulin effector. Further exploring this pathway may uncover new strategies to improve insulin sensitivity.

Violet-light suppression of thermogenesis by opsin 5 hypothalamic neurons.

The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light1,2. In mice, OPN5 is a known photoreceptor in the retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.

Adaptive Thermogenesis in Mice Is Enhanced by Opsin 3-Dependent Adipocyte Light Sensing.

Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.

Non-canonical mTORC2 Signaling Regulates Brown Adipocyte Lipid Catabolism through SIRT6-FoxO1.

mTORC2 controls glucose and lipid metabolism, but the mechanisms are unclear. Here, we show that conditionally deleting the essential mTORC2 subunit Rictor in murine brown adipocytes inhibits de novo lipid synthesis, promotes lipid catabolism and thermogenesis, and protects against diet-induced obesity and hepatic steatosis. AKT kinases are the canonical mTORC2 substrates; however, deleting Rictor in brown adipocytes appears to drive lipid catabolism by promoting FoxO1 deacetylation independently of AKT, and in a pathway distinct from its positive role in anabolic lipid synthesis. This facilitates FoxO1 nuclear retention, enhances lipid uptake and lipolysis, and potentiates UCP1 expression. We provide evidence that SIRT6 is the FoxO1 deacetylase suppressed by mTORC2 and show an endogenous interaction between SIRT6 and mTORC2 in both mouse and human cells. Our findings suggest a new paradigm of mTORC2 function filling an important gap in our understanding of this more mysterious mTOR complex.

A Brain-Melanocortin-Vagus Axis Mediates Adipose Tissue Expansion Independently of Energy Intake.

The melanocortin system is a brain circuit that influences energy balance by regulating energy intake and expenditure. In addition, the brain-melanocortin system controls adipose tissue metabolism to optimize fuel mobilization and storage. Specifically, increased brain-melanocortin signaling or negative energy balance promotes lipid mobilization by increasing sympathetic nervous system input to adipose tissue. In contrast, calorie-independent mechanisms favoring energy storage are less understood. Here, we demonstrate that reduction of brain-melanocortin signaling actively promotes fat mass gain by activating the lipogenic program and adipocyte and endothelial cell proliferation in white fat depots independently of caloric intake via efferent nerve fibers conveyed by the common hepatic branch of the vagus nerve. Those vagally regulated obesogenic signals also contribute to the fat mass gain following chronic high-fat diet feeding. These data reveal a physiological mechanism whereby the brain controls energy stores that may contribute to increased susceptibility to obesity.

Brown fat organogenesis and maintenance requires AKT1 and AKT2.

OBJECTIVE: Understanding the signaling mechanisms that control brown adipose tissue (BAT) development is relevant to understanding energy homeostasis and obesity. The AKT kinases are insulin effectors with critical in vivo functions in adipocytes; however, their role in adipocyte development remains poorly understood. The goal of this study was to investigate AKT function in BAT development. METHODS: We conditionally deleted Akt1 and Akt2 either individually or together with Myf5-Cre, which targets early mesenchymal precursors that give rise to brown adipocytes. Because Myf5-Cre also targets skeletal muscle and some white adipocyte lineages, comparisons were made between AKT function in BAT versus white adipose tissue (WAT) and muscle development. We also deleted both Akt1 and Akt2 in mature brown adipocytes with Ucp1-Cre or Ucp1-CreER to investigate AKT1/2 signaling in BAT maintenance. RESULTS: AKT1 and AKT2 are individually dispensable in Myf5-Cre lineages in vivo for establishing brown and white adipocyte precursor cell pools and for their ability to differentiate (i.e. induce PPARγ). AKT1 and AKT2 are also dispensable for skeletal muscle development, and AKT3 does not compensate in either the adipocyte or muscle lineages. In contrast, AKT2 is required for adipocyte lipid filling and efficient downstream AKT substrate phosphorylation. Mice in which both Akt1 and Akt2 are deleted with Myf5-Cre lack BAT but have normal muscle mass, and doubly deleting Akt1 and Akt2 in mature brown adipocytes, either congenitally (with Ucp1-Cre), or inducibly in older mice (with Ucp1-CreER), also ablates BAT. Mechanistically, AKT signaling promotes adipogenesis in part by stimulating ChREBP activity. CONCLUSIONS: AKT signaling is required in vivo for BAT development but dispensable for skeletal muscle development. AKT1 and AKT2 have both overlapping and distinct functions in BAT development with AKT2 being the most critical individual isoform. AKT1 and AKT2 also have distinct and complementary functions in BAT maintenance.

Inflammation and Immunity: From an Adipocyte's Perspective.

Comprehension of adipocyte function has evolved beyond a long-held belief of their inert nature, as simple energy storing and releasing cells. Adipocytes, including white, brown, and beige, are capable mediators of global metabolic health, but their intersection with inflammation is a budding field of exploration. Evidence hints at a reciprocal relationship adipocytes share with immune cells. Adipocyte's capacity to behave in an "immune-like" manner and ability to sense inflammatory cues that subsequently alter core adipocyte function might play an important role in shaping immune responses. Clarifying this intricate relationship could uncover previously underappreciated contribution of adipocytes to inflammation-driven human health and disease. In this review, we highlight the potential of largely underappreciated adipocyte "immune-like" function and how it may contribute to inflammation, immunity, and pathology of various diseases.

Brown Adipose Tissue Development and Metabolism.

Brown adipose tissue is well known to be a thermoregulatory organ particularly important in small rodents and human infants, but it was only recently that its existence and significance to metabolic fitness in adult humans have been widely realized. The ability of active brown fat to expend high amounts of energy has raised interest in stimulating thermogenesis therapeutically to treat metabolic diseases related to obesity and type 2 diabetes. In parallel, there has been a surge of research aimed at understanding the biology of rodent and human brown fat development, its remarkable metabolic properties, and the phenomenon of white fat browning, in which white adipocytes can be converted into brown like adipocytes with similar thermogenic properties. Here, we review the current understanding of the developmental and metabolic pathways involved in forming thermogenic adipocytes, and highlight some of the many unknown functions of brown fat that make its study a rich and exciting area for future research.

Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues.

Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but are thought to be primarily diet derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity influencing acyl-chain diversity in the lipidome.

Brown Fat AKT2 Is a Cold-Induced Kinase that Stimulates ChREBP-Mediated De Novo Lipogenesis to Optimize Fuel Storage and Thermogenesis.

Brown adipose tissue (BAT) is a therapeutic target for metabolic diseases; thus, understanding its metabolic circuitry is clinically important. Many studies of BAT compare rodents mildly cold to those severely cold. Here, we compared BAT remodeling between thermoneutral and mild-cold-adapted mice, conditions more relevant to humans. Although BAT is renowned for catabolic ß-oxidative capacity, we find paradoxically that the anabolic de novo lipogenesis (DNL) genes encoding ACLY, ACSS2, ACC, and FASN were among the most upregulated by mild cold and that, in humans, DNL correlates with Ucp1 expression. The regulation and function of adipocyte DNL and its association with thermogenesis are not understood. We provide evidence suggesting that AKT2 drives DNL in adipocytes by stimulating ChREBPß transcriptional activity and that cold induces the AKT2-ChREBP pathway in BAT to optimize fuel storage and thermogenesis. These data provide insight into adipocyte DNL regulation and function and illustrate the metabolic flexibility of thermogenesis.

Adipose tissue mTORC2 regulates ChREBP-driven de novo lipogenesis and hepatic glucose metabolism.

Adipose tissue de novo lipogenesis (DNL) positively influences insulin sensitivity, is reduced in obesity, and predicts insulin resistance. Therefore, elucidating mechanisms controlling adipose tissue DNL could lead to therapies for type 2 diabetes. Here, we report that mechanistic target of rapamycin complex 2 (mTORC2) functions in white adipose tissue (WAT) to control expression of the lipogenic transcription factor ChREBPß. Conditionally deleting the essential mTORC2 subunit Rictor in mature adipocytes decreases ChREBPß expression, which reduces DNL in WAT, and impairs hepatic insulin sensitivity. Mechanistically, Rictor/mTORC2 promotes ChREBPß expression in part by controlling glucose uptake, but without impairing pan-AKT signalling. High-fat diet also rapidly decreases adipose tissue ChREBPß expression and insulin sensitivity in wild-type mice, and does not further exacerbate insulin resistance in adipose tissue Rictor knockout mice, implicating adipose tissue DNL as an early target in diet-induced insulin resistance. These data suggest mTORC2 functions in WAT as part of an extra-hepatic nutrient-sensing mechanism to control glucose homeostasis.

Emerging Complexities in Adipocyte Origins and Identity.

The global incidence of obesity and its comorbidities continues to rise along with a demand for novel therapeutic interventions. Brown adipose tissue (BAT) is attracting attention as a therapeutic target because of its presence in adult humans and high capacity to dissipate energy as heat, and thus burn excess calories, when stimulated. Another potential avenue for therapeutic intervention is to induce, within white adipose tissue (WAT), the formation of brown-like adipocytes called brite (brown-like-in-white) or beige adipocytes. However, understanding how to harness the potential of these thermogenic cells requires a deep understanding of their developmental origins and regulation. Recent cell-labeling and lineage-tracing experiments are beginning to shed light on this emerging area of adipocyte biology. We review here adipocyte development, giving particular attention to thermogenic adipocytes.

Highly selective in vivo labeling of subcutaneous white adipocyte precursors with Prx1-Cre.

The origins of individual fat depots are not well understood, and thus, the availability of tools useful for studying depot-specific adipose tissue development and function is limited. Cre drivers that selectively target only brown adipocyte, subcutaneous white adipocyte, or visceral white adipocyte precursors would have significant value because they could be used to selectively study individual depots without impacting the adipocyte precursors or intrinsic metabolic properties of the other depots. Here, we show that the majority of the precursor and mature subcutaneous white adipocytes in adult C57Bl/6 mice are labeled by Prx1-Cre. In sharp contrast, few to no brown adipocytes or visceral white adipocytes are marked by Prx1-Cre. This suggests that Prx1-Cre-mediated recombination may be useful for making depot-restricted genetic manipulations in subcutaneous white adipocyte precursor cells, particularly when targeting genes with fat-specific functions.