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A common approach for understanding how drugs induce their therapeutic effects is to identify the genetic determinants of drug sensitivity. Because 'chemo-genetic profiles' are performed in a pooled format, inference of gene function is subject to several confounding influences related to variation in growth rates between clones. In this study, we developed Method for Evaluating Death Using a Simulation-assisted Approach (MEDUSA), which uses time-resolved measurements, along with model-driven constraints, to reveal the combination of growth and death rates that generated the observed drug response. MEDUSA is uniquely effective at identifying death regulatory genes. We apply MEDUSA to characterize DNA damage-induced lethality in the presence and absence of p53. Loss of p53 switches the mechanism of DNA damage-induced death from apoptosis to a non-apoptotic death that requires high respiration. These findings demonstrate the utility of MEDUSA both for determining the genetic dependencies of lethality and for revealing opportunities to potentiate chemo-efficacy in a cancer-specific manner., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Brown adipose tissue (BAT) engages futile fatty acid synthesis-oxidation cycling, the purpose of which has remained elusive. Here, we show that ATP-citrate lyase (ACLY), which generates acetyl-CoA for fatty acid synthesis, promotes thermogenesis by mitigating metabolic stress. Without ACLY, BAT overloads the tricarboxylic acid cycle, activates the integrated stress response (ISR) and suppresses thermogenesis. ACLY's role in preventing BAT stress becomes critical when mice are weaned onto a carbohydrate-plentiful diet, while removing dietary carbohydrates prevents stress induction in ACLY-deficient BAT. ACLY loss also upregulates fatty acid synthase (Fasn); yet while ISR activation is not caused by impaired fatty acid synthesis per se, deleting Fasn and Acly unlocks an alternative metabolic programme that overcomes tricarboxylic acid cycle overload, prevents ISR activation and rescues thermogenesis. Overall, we uncover a previously unappreciated role for ACLY in mitigating mitochondrial stress that links dietary carbohydrates to uncoupling protein 1-dependent thermogenesis and provides fundamental insight into the fatty acid synthesis-oxidation paradox in BAT., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Nonshivering thermogenesis by brown adipose tissue (BAT) is an adaptive mechanism for maintaining body temperature in cold environments. BAT is critical in rodents and human infants and has substantial influence on adult human metabolism. Stimulating BAT therapeutically is also being investigated as a strategy against metabolic diseases because of its ability to function as a catabolic sink. Thus, understanding how brown adipocytes and the related brite/beige adipocytes use nutrients to fuel their demanding metabolism has both basic and translational implications. Recent advances in mass spectrometry and isotope tracing are improving the ability to study metabolic flux in vivo. Here, we review how such strategies are advancing our understanding of adipocyte thermogenesis and conclude with key future questions., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Adaptive thermogenesis by brown adipose tissue (BAT) dissipates calories as heat, making it an attractive anti-obesity target. Yet how BAT contributes to circulating metabolite exchange remains unclear. Here, we quantified metabolite exchange in BAT and skeletal muscle by arteriovenous metabolomics during cold exposure in fed male mice. This identified unexpected metabolites consumed, released and shared between organs. Quantitative analysis of tissue fluxes showed that glucose and lactate provide ~85% of carbon for adaptive thermogenesis and that cold and CL316,243 trigger markedly divergent fuel utilization profiles. In cold adaptation, BAT also dramatically increases nitrogen uptake by net consuming amino acids, except glutamine. Isotope tracing and functional studies suggest glutamine catabolism concurrent with synthesis via glutamine synthetase, which avoids ammonia buildup and boosts fuel oxidation. These data underscore the ability of BAT to function as a glucose and amino acid sink and provide a quantitative and comprehensive landscape of BAT fuel utilization to guide translational studies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Few metabolites can claim a more central and versatile role in cell metabolism than acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is produced during nutrient catabolism to fuel the tricarboxylic acid cycle and is the essential building block for fatty acid and isoprenoid biosynthesis. It also functions as a signalling metabolite as the substrate for lysine acetylation reactions, enabling the modulation of protein functions in response to acetyl-CoA availability. Recent years have seen exciting advances in our understanding of acetyl-CoA metabolism in normal physiology and in cancer, buoyed by new mouse models, in vivo stable-isotope tracing approaches and improved methods for measuring acetyl-CoA, including in specific subcellular compartments. Efforts to target acetyl-CoA metabolic enzymes are also advancing, with one therapeutic agent targeting acetyl-CoA synthesis receiving approval from the US Food and Drug Administration. In this Review, we give an overview of the regulation and cancer relevance of major metabolic pathways in which acetyl-CoA participates. We further discuss recent advances in understanding acetyl-CoA metabolism in normal tissues and tumours and the potential for targeting these pathways therapeutically. We conclude with a commentary on emerging nodes of acetyl-CoA metabolism that may impact cancer biology., (© 2023. Springer Nature Limited.)

The signaling mechanisms underlying adipose thermogenesis have not been fully elucidated. Particularly, the involvement of adipokines that are selectively expressed in brown adipose tissue (BAT) and beige adipocytes remains to be investigated. Here we show that a previously uncharacterized adipokine (UPF0687 protein / human C20orf27 homolog) we named as Adissp (Adipose-secreted signaling protein) is a key regulator for white adipose tissue (WAT) thermogenesis and glucose homeostasis. Adissp expression is adipose-specific and highly BAT-enriched, and its secretion is stimulated by β3-adrenergic activation. Gain-of-functional studies collectively showed that secreted Adissp promotes WAT thermogenesis, improves glucose homeostasis, and protects against obesity. Adipose-specific Adissp knockout mice are defective in WAT browning, and are susceptible to high fat diet-induced obesity and hyperglycemia. Mechanistically, Adissp binds to a putative receptor on adipocyte surface and activates protein kinase A independently of β-adrenergic signaling. These results establish BAT-enriched Adissp as a major upstream signaling component in thermogenesis and offer a potential avenue for the treatment of obesity and diabetes., (© 2022. The Author(s).)

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Insulin stimulates glucose uptake into adipocytes via mTORC2/AKT signaling and GLUT4 translocation and directs glucose carbons into glycolysis, glycerol for TAG synthesis, and de novo lipogenesis. Adipocyte insulin resistance is an early indicator of type 2 diabetes in obesity, a worldwide health crisis. Thus, understanding the interplay between insulin signaling and central carbon metabolism pathways that maintains adipocyte function, blood glucose levels, and metabolic homeostasis is critical. While classically viewed through the lens of individual enzyme-substrate interactions, advances in mass spectrometry are beginning to illuminate adipocyte signaling and metabolic networks on an unprecedented scale, yet this is just the tip of the iceberg. Here, we review how 'omics approaches help to elucidate adipocyte insulin action in cellular time and space., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Brown adipose tissue (BAT) demonstrates extraordinary metabolic capacity. Previous research using conventional radio tracers reveals that BAT can act as a sink for a diverse menu of nutrients; still, the question of how BAT utilizes these nutrients remains unclear. Recent advances in mass spectrometry (MS) coupled to stable isotope tracing methods have greatly improved our understanding of metabolism in biology. Here, we have developed a BAT-tailored metabolomics and stable isotope tracing protocol using, as an example, the universally labeled 13 C-glucose, a key nutrient heavily utilized by BAT. This method enables metabolic roadmaps to be drawn and pathway fluxes to be inferred for each nutrient tracer within BAT and its application could uncover new metabolic pathways not previously appreciated for BAT physiology., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Active brown adipose tissue (BAT) consumes copious amounts of glucose, yet how glucose metabolism supports thermogenesis is unclear. By combining transcriptomics, metabolomics, and stable isotope tracing in vivo, we systematically analyze BAT glucose utilization in mice during acute and chronic cold exposure. Metabolite profiling reveals extensive temperature-dependent changes in the BAT metabolome and transcriptome upon cold adaptation, discovering unexpected metabolite markers of thermogenesis, including increased N-acetyl-amino acid production. Time-course stable isotope tracing further reveals rapid incorporation of glucose carbons into glycolysis and TCA cycle, as well as several auxiliary pathways, including NADPH, nucleotide, and phospholipid synthesis pathways. Gene expression differences inconsistently predict glucose fluxes, indicating that posttranscriptional mechanisms also govern glucose utilization. Surprisingly, BAT swiftly generates fatty acids and acyl-carnitines from glucose, suggesting that lipids are rapidly synthesized and immediately oxidized. These data reveal versatility in BAT glucose utilization, highlighting the value of an integrative-omics approach to understanding organ metabolism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3 H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Blaustein, Piegari, Martínez Calejman, Vila, Amante, Manese, Zeida, Abrami, Veggetti, Guertin, van der Goot, Corvi and Colman-Lerner.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR ( Rictor-iKO ) by stimulating each with insulin for a 30-min time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation because of chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR., (© 2020 Entwisle et al.)

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.

Purpose of Review: Obesity is a major risk factor for type 2 diabetes. Although adipose tissue allows storage of excess calories in periods of overnutrition, in obesity, adipose tissue metabolism becomes dysregulated and can promote metabolic diseases. This review discusses recent advances in understandings how adipocyte metabolism impacts metabolic homeostasis., Recent Findings: The ability of adipocytes to synthesize lipids from glucose is a marker of metabolic fitness, e.g., low de novo lipogenesis (DNL) in adipocytes correlates with insulin resistance in obesity. Adipocyte DNL may promote synthesis of special "insulin sensitizing" signaling lipids that act hormonally. However, each metabolic intermediate in the DNL pathway (i.e., citrate, acetyl-CoA, malonyl-CoA, and palmitate) also has second messenger functions. Mounting evidence suggests these signaling functions may also be important for maintaining healthy adipocytes. While adipocyte DNL contributes to lipid storage, lipid precursors may have additional second messenger functions critical for maintaining adipocyte health, and thus systemic metabolic homeostasis.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Mycobacterium tuberculosis has co-evolved with the human host, adapting to exploit the immune system for persistence and transmission. While immunity to tuberculosis (TB) has been intensively studied in the lung and lymphoid system, little is known about the participation of adipose tissues and non-immune cells in the host-pathogen interaction during this systemic disease., Methods: C57BL/6J mice were aerosol infected with M. tuberculosis Erdman and presence of the bacteria and the fitness of the white and brown adipose tissues, liver and skeletal muscle were studied compared to uninfected mice., Findings: M. tuberculosis infection in mice stimulated immune cell infiltration in visceral, and brown adipose tissue. Despite the absence of detectable bacterial dissemination to fat tissues, adipocytes produced localized pro-inflammatory signals that disrupted adipocyte lipid metabolism, resulting in adipocyte hypertrophy. Paradoxically, this resulted in increased insulin sensitivity and systemic glucose tolerance. Adipose tissue inflammation and enhanced glucose tolerance also developed in obese mice after aerosol M. tuberculosis infection. We found that infection induced adipose tissue Akt signaling, while inhibition of the Akt activator mTORC2 in adipocytes reversed TB-associated adipose tissue inflammation and cell hypertrophy., Interpretation: Our study reveals a systemic response to aerosol M. tuberculosis infection that regulates adipose tissue lipid homeostasis through mTORC2/Akt signaling in adipocytes. Adipose tissue inflammation in TB is not simply a passive infiltration with leukocytes but requires the mechanistic participation of adipocyte signals., (Copyright © 2019. Published by Elsevier B.V.)

Neural crest cells are embryonic progenitors that generate numerous cell types in vertebrates. With single-cell analysis, we show that mouse trunk neural crest cells become biased toward neuronal lineages when they delaminate from the neural tube, whereas cranial neural crest cells acquire ectomesenchyme potential dependent on activation of the transcription factor Twist1. The choices that neural crest cells make to become sensory, glial, autonomic, or mesenchymal cells can be formalized as a series of sequential binary decisions. Each branch of the decision tree involves initial coactivation of bipotential properties followed by gradual shifts toward commitment. Competing fate programs are coactivated before cells acquire fate-specific phenotypic traits. Determination of a specific fate is achieved by increased synchronization of relevant programs and concurrent repression of competing fate programs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Sugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, Acly FAT-/- females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas Acly FAT-/- males have only mild metabolic phenotypes. We find that ACLY is crucial for nutrient-dependent carbohydrate response element-binding protein (ChREBP) activation in adipocytes and plays a key role, particularly in females, in the storage of newly synthesized fatty acids in adipose tissue. The data indicate that adipocyte ACLY is important in females for the systemic handling of dietary carbohydrates and for the preservation of metabolic homeostasis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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.

In this issue of Molecular Cell, Krzysiak et al. (2018) describe a mechanism by which insulin signaling represses the NAD + -dependent SIRT1 deacetylase by promoting PACS-2 binding and provide structural clues to understanding how SIRT1 activating compounds (STACs) work., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

Productive coastal and estuarine habitats can be degraded by contaminants including persistent organic pollutants (POPs) such as PCBs, dioxins, and organochlorine insecticides to the extent of official designation as contaminated sites. Top-predatory wildlife may continue to use such sites as the habitat often appears suitable, and thus bioaccumulate POPs and other contaminants with potential consequences on their health and fitness. Victoria and Esquimalt harbours are located on southern Vancouver Island, British Columbia (BC) and are federally designated contaminated sites due mainly to past heavy industrial activities, such as from shipyards and sawmills. We collected scat samples from river otters (Lontra canadensis) throughout an annual cycle, and combined chemical analysis with DNA genotyping to examine whether the harbour areas constituted a contaminant-induced ecological trap for otters. We confirmed spatial habitat use by radio telemetry of a subsample of otters. Fifteen percent of otter scat contained PCB concentrations exceeding levels considered to have adverse effects on the reproduction of mink (Neovison vison), and there were significant positive correlations between concentrations of PCBs and of thyroid (T3) and sex (progesterone) hormones in fecal samples. Radio telemetry data revealed that otters did not show directional movement away from the harbours, indicating their inability to recognize the contaminated site as a degraded habitat. However, analysis and modeling of the DNA genotyping data provided no evidence that the harbour otters formed a sink population and therefore were in an ecological trap. Despite the highly POP-contaminated habitat, river otters did not appear to be adversely impacted at the population level. Our study demonstrates the value of combining chemical and biological technologies with ecological theory to investigate practical conservation problems., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

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 β-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 ChREBPβ 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Having healthy adipose tissue is essential for metabolic fitness. This is clear from the obesity epidemic, which is unveiling a myriad of comorbidities associated with excess adipose tissue including type 2 diabetes, cardiovascular disease, and cancer. Lipodystrophy also causes insulin resistance, emphasizing the importance of having a balanced amount of fat. In cells, the mechanistic target of rapamycin (mTOR) complexes 1 and 2 (mTORC1 and mTORC2, respectively) link nutrient and hormonal signaling with metabolism, and recent studies are shedding new light on their in vivo roles in adipocytes. In this review, we discuss how recent advances in adipose tissue and mTOR biology are converging to reveal new mechanisms that maintain healthy adipose tissue, and discuss ongoing mysteries of mTOR signaling, particularly for the less understood complex mTORC2., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Despite progress in our comprehension of the mechanisms regulating adipose tissue development, the nature of the factors that functionally characterize adipose precursors is still elusive. Defining the early steps regulating adipocyte development is needed for the generation of tools to control adipose tissue size and function. Here, we report the discovery of V-set and transmembrane domain containing 2A (VSTM2A) as a protein expressed and secreted by committed preadipocytes. VSTM2A expression is elevated in the early phases of adipogenesis in vitro and adipose tissue development in vivo. We show that VSTM2A-producing cells associate with the vasculature and express the common surface markers of adipocyte progenitors. Overexpression of VSTM2A induces adipogenesis, whereas its depletion impairs this process. VSTM2A controls preadipocyte determination at least in part by modulating BMP signaling and PPARγ2 activation. We propose a model in which VSTM2A is produced to preserve and amplify the adipogenic capability of adipose precursors., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 ChREBPβ. Conditionally deleting the essential mTORC2 subunit Rictor in mature adipocytes decreases ChREBPβ expression, which reduces DNL in WAT, and impairs hepatic insulin sensitivity. Mechanistically, Rictor/mTORC2 promotes ChREBPβ expression in part by controlling glucose uptake, but without impairing pan-AKT signalling. High-fat diet also rapidly decreases adipose tissue ChREBPβ 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.

Objective: Normal adipose tissue growth and function is critical to maintaining metabolic homeostasis and its excess (e.g. obesity) or absence (e.g. lipodystrophy) is associated with severe metabolic disease. The goal of this study was to understand the mechanisms maintaining healthy adipose tissue growth and function., Methods: Adipose tissue senses and responds to systemic changes in growth factor and nutrient availability; in cells mTORC1 regulates metabolism in response to growth factors and nutrients. Thus, mTORC1 is poised to be a critical intracellular regulator of adipocyte metabolism. Here, we investigate the role of mTORC1 in mature adipocytes by generating and characterizing mice in which the Adiponectin-Cre driver is used to delete floxed alleles of Raptor, which encodes an essential regulatory subunit of mTORC1., Results: Raptor (Adipoq-cre) mice have normal white adipose tissue (WAT) mass for the first few weeks of life, but soon thereafter develop lipodystrophy associated with hepatomegaly, hepatic steatosis, and insulin intolerance. Raptor (Adipoq-cre) mice are also resistant to becoming obese when consuming a high fat diet (HFD). Resistance to obesity does not appear to be due to increased energy expenditure, but rather from failed adipose tissue expansion resulting in severe hepatomegaly associated with hyperphagia and defective dietary lipid absorption. Deleting Raptor in WAT also decreases C/EBPα expression and the expression of its downstream target adiponectin, providing one possible mechanism of mTORC1 function in WAT., Conclusions: mTORC1 activity in mature adipocytes is essential for maintaining normal adipose tissue growth and its selective loss in mature adipocytes leads to a progressive lipodystrophy disorder and systemic metabolic disease that shares many of the hallmarks of human congenital generalized lipodystrophy.

Jumonji domain-containing protein 6 (JMJD6) is a nuclear protein involved in histone modification, transcription and RNA processing. Although JMJD6 is crucial for tissue development, the link between its molecular functions and its roles in any given differentiation process is unknown. We report that JMJD6 is required for adipogenic gene expression and differentiation in a manner independent of Jumonji C domain catalytic activity. JMJD6 knockdown led to a reduction of C/EBPβ and C/EBPδ protein expression without affecting mRNA levels in the early phase of differentiation. However, ectopic expression of C/EBPβ and C/EBPδ did not rescue differentiation. Further analysis demonstrated that JMJD6 was associated with the Pparγ2 and Cebpα loci and putative enhancers. JMJD6 was previously found associated with bromodomain and extra-terminal domain (BET) proteins, which can be targeted by the bromodomain inhibitor JQ1. JQ1 treatment prevented chromatin binding of JMJD6, Pparγ2 and Cebpα expression, and adipogenic differentiation, yet had no effect on C/EBPβ and C/EBPδ expression or chromatin binding. These results indicate dual roles for JMJD6 in promoting adipogenic gene expression program by post-transcriptional regulation of C/EBPβ and C/EBPδ and direct transcriptional activation of Pparγ2 and Cebpα during adipocyte differentiation., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

A lysosome-based mechanism of amino acid sensing by mTORC1 regulated by Rag GTPases has emerged. In this issue of Cancer Cell, Thomas and colleagues propose a Golgi-based and Rag-independent mechanism mediated by the Rab1A GTPase. Furthermore, Rab1A overexpression in colorectal cancers correlates with mTORC1 activity and sensitivity to mTOR inhibitors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The in vivo functions of mechanistic target of rapamycin complex 2 (mTORC2) and the signaling mechanisms that control brown adipose tissue (BAT) fuel utilization and activity are not well understood. Here, by conditionally deleting Rictor in the Myf5 lineage, we provide in vivo evidence that mTORC2 is dispensable for skeletal muscle development and regeneration but essential for BAT growth. Furthermore, deleting Rictor in Myf5 precursors shifts BAT metabolism to a more oxidative and less lipogenic state and protects mice from obesity and metabolic disease at thermoneutrality. We additionally find that Rictor is required for brown adipocyte differentiation in vitro and that the mechanism specifically requires AKT1 hydrophobic motif phosphorylation but is independent of pan-AKT signaling and is rescued with BMP7. Our findings provide insights into the signaling circuitry that regulates brown adipocytes and could have important implications for developing therapies aimed at increasing energy expenditure as a means to combat human obesity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Adipose tissue development is poorly understood. Here we use a lineage-tracing strategy optimal for adipocytes to provide evidence that Myf5 precursors are not the exclusive source of brown adipocytes and contribute more to the mature white and brite adipocyte populations than previously thought. Moreover, Myf5-lineage distribution in adipose tissue changes in response to modifiable and non-modifiable factors. We also find that the Pax3 lineage largely overlaps with the Myf5 lineage in brown fat and subcutaneous white fat, but exhibits gender-linked divergence in visceral white fat while the MyoD1 lineage does not give rise to any adipocytes. Finally, by deleting insulin receptor beta in the Myf5 lineage, we provide in vivo evidence that the insulin receptor is essential for adipogenesis and that adipocyte lineages have plasticity. These data establish a conceptual framework for adipose tissue development and could explain body fat patterning variations in healthy and lipodystrophic or obese humans.

The obesity epidemic has intensified efforts to understand the mechanisms controlling adipose tissue development. Adipose tissue is generally classified as white adipose tissue (WAT), the major energy storing tissue, or brown adipose tissue (BAT), which mediates non-shivering thermogenesis. It is hypothesized that brite adipocytes (brown in white) may represent a third adipocyte class. The recent realization that brown fat exist in adult humans suggests increasing brown fat energy expenditure could be a therapeutic strategy to combat obesity. To understand adipose tissue development, several groups are tracing the origins of mature adipocytes back to their adult precursor and embryonic ancestors. From these studies emerged a model that brown adipocytes originate from a precursor shared with skeletal muscle that expresses Myf5-Cre, while all white adipocytes originate from a Myf5-negative precursors. While this provided a rational explanation to why BAT is more metabolically favorable than WAT, recent work indicates the situation is more complex because subsets of white adipocytes also arise from Myf5-Cre expressing precursors. Lineage tracing studies further suggest that the vasculature may provide a niche supporting both brown and white adipocyte progenitors; however, the identity of the adipocyte progenitor cell is under debate. Differences in origin between adipocytes could explain metabolic heterogeneity between depots and/or influence body fat patterning particularly in lipodystrophy disorders. Here, we discuss recent insights into adipose tissue origins highlighting lineage-tracing studies in mice, how variations in metabolism or signaling between lineages could affect body fat distribution, and the questions that remain unresolved. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Naive T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic reprogramming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and T helper 2 (Th2) cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis, and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 costimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The mechanistic target of rapamycin (mTOR) kinase is a conserved regulator of cell growth, proliferation, and survival. In cells, mTOR is the catalytic subunit of two complexes called mTORC1 and mTORC2, which have distinct upstream regulatory signals and downstream substrates. mTORC1 directly senses cellular nutrient availability while indirectly sensing circulating nutrients through growth factor signaling pathways. Cellular stresses that restrict growth also impinge on mTORC1 activity. mTORC2 is less well understood and appears only to sense growth factors. As an integrator of diverse growth regulatory signals, mTOR evolved to be a central signaling hub for controlling cellular metabolism and energy homoeostasis, and defects in mTOR signaling are important in the pathologies of cancer, diabetes, and aging. Here we discuss mechanisms by which each mTOR complex might regulate cell survival in response to metabolic and other stresses.

The mechanistic target of rapamycin (mTOR) pathway serves as a key sensor of cellular-energetic state and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoiesis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, regulatory-associated protein of mTOR (Raptor), in mouse HSCs and its loss causes a nonlethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely nonredundant roles with rapamycin-insensitive companion of mTOR (Rictor) in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis and inform clinical strategies based on chronic mTORC1 inhibition., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The developmental origin of adipose tissue and what controls its distribution is poorly understood. By lineage tracing and gene expression analysis in mice, we provide evidence that mesenchymal precursors expressing Myf5--which are thought to give rise only to brown adipocytes and skeletal muscle--also give rise to a subset of white adipocytes. Furthermore, individual brown and white fats contain a mixture of adipocyte progenitor cells derived from Myf5(+) and Myf5(neg) lineages, the number of which varies with depot location. Subsets of white adipocytes originating from both Myf5(+) and Myf5(neg) precursors respond to β(3)-adrenoreceptor stimulation, suggesting "brite" adipocytes may also have multiple origins. We additionally find that deleting PTEN with myf5-cre causes lipomatosis and partial lipodystrophy by selectively expanding the Myf5(+) adipocyte lineages. Thus, the spectrum of adipocytes arising from Myf5(+) precursors is broader than previously thought, and differences in PI3K activity between adipocyte lineages alter body fat distribution.

Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo.

Extensive efforts are underway to develop small-molecule inhibitors of the mammalian target of rapamycin (mTOR) kinase. It is hoped that these inhibitors will have widespread clinical impact in oncology because mTOR is a major downstream effector of PI3K signaling, one of the most frequently activated pathways in cancer. In cells, mTOR is the catalytic core subunit of two distinct complexes, mTORC1 and mTORC2, which are defined by unique mTOR-interacting proteins and have unique functions downstream of PI3K. Two classes of mTOR inhibitors are currently being evaluated as cancer therapeutics: rapamycin and its analogs, which partially inhibit mTORC1 and in some cell types mTORC2, and the recently described ATP-competitive inhibitors, which inhibit the kinase activity of both complexes. Although small molecules that selectively target mTORC2 do not yet exist, experiments using mouse genetics suggest that a theoretical mTORC2 inhibitor may have significant therapeutic value. Here, we discuss an approach to model mTOR complex specific inhibitors using mouse genetics and how it can be applied to other gene products involved in oncogenic signaling to which inhibitors do not exist.

Soft-tissue sarcomas are heterogeneous cancers that can present with tissue-specific differentiation markers. To examine the cellular basis for this histopathological variation and to identify sarcoma-relevant molecular pathways, we generated a chimeric mouse model in which sarcoma-associated genetic lesions can be introduced into discrete, muscle-resident myogenic and mesenchymal cell lineages. Expression of Kirsten rat sarcoma viral oncogene [Kras(G12V)] and disruption of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16p19) in prospectively isolated satellite cells gave rise to pleomorphic rhabdomyosarcomas (MyoD-, Myogenin- and Desmin-positive), whereas introduction of the same oncogenetic hits in nonmyogenic progenitors induced pleomorphic sarcomas lacking myogenic features. Transcriptional profiling demonstrated that myogenic and nonmyogenic Kras; p16p19(null) sarcomas recapitulate gene-expression signatures of human rhabdomyosarcomas and identified a cluster of genes that is concordantly up-regulated in both mouse and human sarcomas. This cluster includes genes associated with Ras and mechanistic target of rapamycin (mTOR) signaling, a finding consistent with activation of the Ras and mTOR pathways both in Kras; p16p19(null) sarcomas and in 26-50% of human rhabdomyosarcomas surveyed. Moreover, chemical inhibition of Ras or mTOR signaling arrested the growth of mouse Kras; p16p19(null) sarcomas and of human rhabdomyosarcoma cells in vitro and in vivo. Taken together, these data demonstrate the critical importance of lineage commitment within the tumor cell-of-origin in determining sarcoma histotype and introduce an experimental platform for rapid dissection of sarcoma-relevant cellular and molecular events.

Under conditions of obesity and insulin resistance, the serine/threonine protein kinase Akt/PKB is required for lipid accumulation in liver. Two forkhead transcription factors, FoxA2 and FoxO1, have been suggested to function downstream of and to be negatively regulated by Akt and are proposed as key determinants of hepatic triglyceride content. In this study, we utilize genetic loss of function experiments to show that constitutive activation of neither FoxA2 nor FoxO1 can account for the protection from steatosis afforded by deletion of Akt2 in liver. Rather, another downstream target positively regulated by Akt, the mTORC1 complex, is required in vivo for de novo lipogenesis and Srebp1c expression. Nonetheless, activation of mTORC1 and SREBP1c is not sufficient to drive postprandial lipogenesis in the absence of Akt2. These data show that insulin signaling through Akt2 promotes anabolic lipid metabolism independent of Foxa2 or FoxO1 and through pathways additional to the mTORC1-dependent activation of SREBP1c., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The nutrient- and growth factor-responsive kinase mTOR complex 1 (mTORC1) regulates many processes that control growth, including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high-fat and -cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway., (Copyright © 2011 Elsevier Inc. All rights reserved.)