Functional genomic screens with death rate analyses reveal mechanisms of drug action.

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.

Dynamic interplay between AfadinS1795 phosphorylation and diet regulates glucose homeostasis in obese mice.

KEY POINTS: Afadin is a ubiquitously expressed scaffold protein with a recently discovered role in insulin signalling and glucose metabolism. Insulin-stimulated phosphorylation of Afadin at S1795 occurs in insulin-responsive tissues such as adipose tissue, muscle, liver, pancreas and heart. Afadin abundance and AfadinS1795 phosphorylation are dynamically regulated in metabolic tissues during diet-induced obesity progression. Genetic silencing of AfadinS1795 phosphorylation improves glucose homeostasis in the early stages of diet-induced metabolic dysregulation. AfadinS1795 phosphorylation contributes to the early development of obesity-related complications in mice. ABSTRACT: Obesity is associated with systemic insulin resistance and numerous metabolic disorders. Yet, the mechanisms underlying impaired insulin action during obesity remain to be fully elucidated. Afadin is a multifunctional scaffold protein with the ability to modulate insulin action through its phosphorylation at S1795 in adipocytes. In the present study, we report that insulin-stimulated AfadinS1795 phosphorylation is not restricted to adipose tissues, but is a common signalling event in insulin-responsive tissues including muscle, liver, pancreas and heart. Furthermore, a dynamic regulation of Afadin abundance occurred during diet-induced obesity progression, while its phosphorylation was progressively attenuated. To investigate the role of AfadinS1795 phosphorylation in the regulation of whole-body metabolic homeostasis, we generated a phospho-defective mouse model (Afadin SA) in which the Afadin phosphorylation site was silenced (S1795A) at the whole-body level using CRISPR-Cas9-mediated gene editing. Metabolic characterization of these mice under basal physiological conditions or during a high-fat diet (HFD) challenge revealed that preventing AfadinS1795 phosphorylation improved insulin sensitivity and glucose tolerance and increased liver glycogen storage in the early stage of diet-induced metabolic dysregulation, without affecting body weight. Together, our findings reveal that AfadinS1795 phosphorylation in metabolic tissues is critical during obesity progression and contributes to promote systemic insulin resistance and glucose intolerance in the early phase of diet-induced obesity.

Insulin resistance rewires the metabolic gene program and glucose utilization in human white adipocytes.

BACKGROUND: In obesity, adipose tissue dysfunction resulting from excessive fat accumulation leads to systemic insulin resistance (IR), the underlying alteration of Type 2 Diabetes. The specific pathways dysregulated in dysfunctional adipocytes and the extent to which it affects adipose metabolic functions remain incompletely characterized. METHODS: We interrogated the transcriptional adaptation to increased adiposity in association with insulin resistance in visceral white adipose tissue from lean men, or men presenting overweight/obesity (BMI from 19 to 33) and discordant for insulin sensitivity. In human adipocytes in vitro, we investigated the direct contribution of IR in altering metabolic gene programming and glucose utilization using 13C-isotopic glucose tracing. RESULTS: We found that gene expression associated with impaired glucose and lipid metabolism and inflammation represented the strongest association with systemic insulin resistance, independently of BMI. In addition, we showed that inducing IR in mature human white adipocytes was sufficient to reprogram the transcriptional profile of genes involved in important metabolic functions such as glycolysis, the pentose phosphate pathway and de novo lipogenesis. Finally, we found that IR induced a rewiring of glucose metabolism, with higher incorporation of glucose into citrate, but not into downstream metabolites within the TCA cycle. CONCLUSIONS: Collectively, our data highlight the importance of obesity-derived insulin resistance in impacting the expression of key metabolic genes and impairing the metabolic processes of glucose utilization, and reveal a role for metabolic adaptation in adipose dysfunction in humans.

Afadin is a scaffold protein repressing insulin action via HDAC6 in adipose tissue.

Insulin orchestrates metabolic homeostasis through a complex signaling network for which the precise mechanisms controlling its fine-tuning are not completely understood. Here, we report that Afadin, a scaffold protein, is phosphorylated on S1795 (S1718 in humans) in response to insulin in adipocytes, and this phosphorylation is impaired with obesity and insulin resistance. In turn, loss of Afadin enhances the response to insulin in adipose tissues via upregulation of the insulin receptor protein levels. This happens in a cell-autonomous and phosphorylation-dependent manner. Insulin-stimulated Afadin-S1795 phosphorylation modulates Afadin binding with interaction partners in adipocytes, among which HDAC6 preferentially interacts with phosphorylated Afadin and acts as a key intermediate to suppress insulin receptor protein levels. Adipose tissue-specific Afadin depletion protects against insulin resistance and improves glucose homeostasis in diet-induced obese mice, independently of adiposity. Altogether, we uncover a novel insulin-induced cellular feedback mechanism governed by the interaction of Afadin with HDAC6 to negatively control insulin action in adipocytes, which may offer new strategies to alleviate insulin resistance.

Restricting glycolysis impairs brown adipocyte glucose and oxygen consumption.

During thermogenic activation, brown adipocytes take up large amounts of glucose. In addition, cold stimulation leads to an upregulation of glycolytic enzymes. Here we have investigated the importance of glycolysis for brown adipocyte glucose consumption and thermogenesis. Using siRNA-mediated knockdown in mature adipocytes, we explored the effect of glucose transporters and glycolytic enzymes on brown adipocyte functions such as consumption of glucose and oxygen. Basal oxygen consumption in brown adipocytes was equally dependent on glucose and fatty acid oxidation, whereas isoproterenol (ISO)-stimulated respiration was fueled mainly by fatty acids, with a significant contribution from glucose oxidation. Knockdown of glucose transporters in brown adipocytes not only impaired ISO-stimulated glycolytic flux but also oxygen consumption. Diminishing glycolytic flux by knockdown of the first and final enzyme of glycolysis, i.e., hexokinase 2 (HK2) and pyruvate kinase M (PKM), respectively, decreased glucose uptake and ISO-stimulated oxygen consumption. HK2 knockdown had a more severe effect, which, in contrast to PKM knockdown, could not be rescued by supplementation with pyruvate. Hence, brown adipocytes rely on glucose consumption and glycolytic flux to achieve maximum thermogenic output, with glycolysis likely supporting thermogenesis not only by pyruvate formation but also by supplying intermediates for efferent metabolic pathways.

Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes.

BACKGROUND: Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. RESULTS: The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α. CONCLUSIONS: UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.

p53 controls choice between apoptotic and non-apoptotic death following DNA damage.

DNA damage can activate apoptotic and non-apoptotic forms of cell death; however, it remains unclear what features dictate which type of cell death is activated. We report that p53 controls the choice between apoptotic and non-apoptotic death following exposure to DNA damage. In contrast to the conventional model, which suggests that p53-deficient cells should be resistant to DNA damage-induced cell death, we find that p53-deficient cells die at high rates following DNA damage, but exclusively using non-apoptotic mechanisms. Our experimental data and computational modeling reveal that non-apoptotic death in p53-deficient cells has not been observed due to use of assays that are either insensitive to cell death, or that specifically score apoptotic cells. Using functional genetic screening - with an analysis that enables computational inference of the drug-induced death rate - we find in p53-deficient cells that DNA damage activates a mitochondrial respiration-dependent form of cell death, called MPT-driven necrosis. Cells deficient for p53 have high basal respiration, which primes MPT-driven necrosis. Finally, using metabolite profiling, we identified mitochondrial activity-dependent metabolic vulnerabilities that can be targeted to potentiate the lethality of DNA damage specifically in p53-deficient cells. Our findings reveal how the dual functions of p53 in regulating mitochondrial activity and the DNA damage response combine to facilitate the choice between apoptotic and non-apoptotic death.

Full activation of thermogenesis in brown adipocytes requires Basigin action.

Exploring mechanisms responsible for brown adipose tissue's (BAT) high metabolic activity is crucial to exploit its energy-dissipating ability for therapeutic purposes. Basigin (Bsg), a multifunctional highly glycosylated transmembrane protein, was recently proposed as one of the 98 critical markers allowing to distinguish 'white' and 'brown' adipocytes, yet its function in thermogenic brown adipocytes is unknown. Here, we report that Bsg is negatively associated with obesity in mice. By contrast, Bsg expression increased in the mature adipocyte fraction of BAT upon cold acclimation. Additionally, Bsg levels were highly induced during brown adipocyte maturation in vitro and were further increased upon ß-adrenergic stimulation in a HIF-1α-dependent manner. siRNA-mediated Bsg gene silencing in cultured brown adipocytes did not impact adipogenesis nor mitochondrial function. However, a significant decrease in mitochondrial respiration, lipolysis and Ucp1 transcription was observed in adipocytes lacking Bsg, when activated by norepinephrine. Furthermore, using gas chromatography/mass spectrometry-time-of-flight analysis to assess the composition of cellular metabolites, we demonstrate that brown adipocytes lacking Bsg have lower levels of intracellular lactate and acetoacetate. Bsg was additionally required to regulate intracellular AcAc and tricarboxylic acid cycle intermediate levels in NE-stimulated adipocytes. Our study highlights the critical role of Bsg in active brown adipocytes, possibly by controlling cellular metabolism.

Pyruvate kinase M2 represses thermogenic gene expression in brown adipocytes.

Utilizing the thermogenic capacity of brown adipose tissue is a potential anti-obesity strategy; therefore, the mechanisms controlling expression of thermogenesis-related genes are of interest. Pyruvate kinase (PK) catalyzes the last step of glycolysis and exists as four isoenzymes: PK, liver, PK, red blood cell, PK, muscle (PKM1 and PKM2). PKM2 has both glycolytic and nuclear functions. Here, we report that PKM2 is enriched in brown adipose compared with white adipose tissue. Specific knockdown of PKM2 in mature brown adipocytes demonstrates that silencing of PKM2 does not lead to a decrease in PK activity, but causes a robust upregulation of thermogenic uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) gene expression. This increase is not mediated by any of the known mechanisms for PKM2-regulated gene expression, thus implying the existence of a novel mechanism for PKM2-dependent effects on gene expression.

Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA.

OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations. METHODS: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes. RESULTS: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations. CONCLUSIONS: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.

Characterization of immortalized human brown and white pre-adipocyte cell models from a single donor.

Brown adipose tissue with its constituent brown adipocytes is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. The molecular control of brown adipocyte differentiation and function has been extensively studied in mice, but relatively little is known about such regulatory mechanisms in humans, which in part is due to lack of human brown adipose tissue derived cell models. Here, we used retrovirus-mediated overexpression to stably integrate human telomerase reverse transcriptase (TERT) into stromal-vascular cell fractions from deep and superficial human neck adipose tissue biopsies from the same donor. The brown and white pre-adipocyte cell models (TERT-hBA and TERT-hWA, respectively) displayed a stable proliferation rate and differentiation until at least passage 20. Mature TERT-hBA adipocytes expressed higher levels of thermogenic marker genes and displayed a higher maximal respiratory capacity than mature TERT-hWA adipocytes. TERT-hBA adipocytes were UCP1-positive and responded to ß-adrenergic stimulation by activating the PKA-MKK3/6-p38 MAPK signaling module and increasing thermogenic gene expression and oxygen consumption. Mature TERT-hWA adipocytes underwent efficient rosiglitazone-induced 'browning', as demonstrated by strongly increased expression of UCP1 and other brown adipocyte-enriched genes. In summary, the TERT-hBA and TERT-hWA cell models represent useful tools to obtain a better understanding of the molecular control of human brown and white adipocyte differentiation and function as well as of browning of human white adipocytes.

Regulation of glycolysis in brown adipocytes by HIF-1α.

Brown adipose tissue takes up large amounts of glucose during cold exposure in mice and humans. Here we report an induction of glucose transporter 1 expression and increased expression of several glycolytic enzymes in brown adipose tissue from cold-exposed mice. Accordingly, these genes were also induced after ß-adrenergic activation of cultured brown adipocytes, concomitant with accumulation of hypoxia inducible factor-1α (HIF-1α) protein levels. HIF-1α accumulation was dependent on uncoupling protein 1 and generation of mitochondrial reactive oxygen species. Expression of key glycolytic enzymes was reduced after knockdown of HIF-1α in mature brown adipocytes. Glucose consumption, lactate export and glycolytic capacity were reduced in brown adipocytes depleted of Hif-1α. Finally, we observed a decreased ß-adrenergically induced oxygen consumption in Hif-1α knockdown adipocytes cultured in medium with glucose as the only exogenously added fuel. These data suggest that HIF-1α-dependent regulation of glycolysis is necessary for maximum glucose metabolism in brown adipocytes.

MCT1 and MCT4 Expression and Lactate Flux Activity Increase During White and Brown Adipogenesis and Impact Adipocyte Metabolism.

Adipose tissue takes up glucose and releases lactate, thereby contributing significantly to systemic glucose and lactate homeostasis. This implies the necessity of upregulation of net acid and lactate flux capacity during adipocyte differentiation and function. However, the regulation of lactate- and acid/base transporters in adipocytes is poorly understood. Here, we tested the hypothesis that adipocyte thermogenesis, browning and differentiation are associated with an upregulation of plasma membrane lactate and acid/base transport capacity that in turn is important for adipocyte metabolism. The mRNA and protein levels of the lactate-H+ transporter MCT1 and the Na+,HCO3- cotransporter NBCe1 were upregulated in mouse interscapular brown and inguinal white adipose tissue upon cold induction of thermogenesis and browning. MCT1, MCT4, and NBCe1 were furthermore strongly upregulated at the mRNA and protein level upon differentiation of cultured pre-adipocytes. Adipocyte differentiation was accompanied by increased plasma membrane lactate flux capacity, which was reduced by MCT inhibition and by MCT1 knockdown. Finally, in differentiated brown adipocytes, glycolysis (assessed as ECAR), and after noradrenergic stimulation also oxidative metabolism (OCR), was decreased by MCT inhibition. We suggest that upregulation of MCT1- and MCT4-mediated lactate flux capacity and NBCe1-mediated HCO3-/pH homeostasis are important for the physiological function of mature adipocytes.

An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to ß-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

Glucagon-like peptide-1 (GLP-1) reduces mortality and improves lung function in a model of experimental obstructive lung disease in female mice.

The incretin hormone glucagon-like peptide-1 (GLP-1) is an important insulin secretagogue and GLP-1 analogs are used for the treatment of type 2 diabetes. GLP-1 displays antiinflammatory and surfactant-releasing effects. Thus, we hypothesize that treatment with GLP-1 analogs will improve pulmonary function in a mouse model of obstructive lung disease. Female mice were sensitized with injected ovalbumin and treated with GLP-1 receptor (GLP-1R) agonists. Exacerbation was induced with inhalations of ovalbumin and lipopolysaccharide. Lung function was evaluated with a measurement of enhanced pause in a whole-body plethysmograph. mRNA levels of GLP-1R, surfactants (SFTPs), and a number of inflammatory markers were measured. GLP-1R was highly expressed in lung tissue. Mice treated with GLP-1R agonists had a noticeably better clinical appearance than the control group. Enhanced pause increased dramatically at day 17 in all control mice, but the increase was significantly less in the groups of GLP-1R agonist-treated mice (P < .001). Survival proportions were significantly increased in GLP-1R agonist-treated mice (P < .01). SFTPB and SFTPA were down-regulated and the expression of inflammatory cytokines were increased in mice with obstructive lung disease, but levels were largely unaffected by GLP-1R agonist treatment. These results show that GLP-1R agonists have potential therapeutic potential in the treatment of obstructive pulmonary diseases, such as chronic obstructive pulmonary disease, by decreasing the severity of acute exacerbations. The mechanism of action does not seem to be the modulation of inflammation and SFTP expression.

ERRγ enhances UCP1 expression and fatty acid oxidation in brown adipocytes.

OBJECTIVE: Estrogen-related receptors (ERRs) are important regulators of energy metabolism. Here we investigated the hypothesis that ERRγ impacts on differentiation and function of brown adipocytes. DESIGN AND METHODS: We characterize the expression of ERRγ in adipose tissues and cell models and investigate the effects of modulating ERRγ activity on UCP1 gene expression and metabolic features of brown and white adipocytes. RESULTS: ERRγ was preferentially expressed in brown compared to white fat depots, and ERRγ was induced during cold-induced browning of subcutaneous white adipose tissue and brown adipogenesis. Overexpression of ERRγ positively regulated uncoupling protein 1 (UCP1) expression levels during brown adipogenesis. This ERRγ-induced augmentation of UCP1 expression was independent of the presence of peroxisome proliferator-activated receptor coactivator-1 (PGC-1α) but was associated with increased rates of fatty acid oxidation in adrenergically stimulated cells. ERRγ did not influence mitochondrial biogenesis, and its reduced expression in white adipocytes could not explain their low expression level of UCP1. CONCLUSIONS: Through its augmenting effect on expression of UCP1, ERRγ may physiologically be involved in increasing the potential for energy expenditure in brown adipocytes, a function that is becoming of therapeutic interest.