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The necroptosis-inducing pseudokinase mixed lineage kinase domain-like regulates the adipogenic differentiation of pre-adipocytes

Summary Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/β-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.


INTRODUCTION
Obesity is the first non-infectious epidemic outbreak in history. The World Health Organization (WHO) estimates that more than one billion people worldwide are overweight, including 300 million who are obese. Obesity increases the risk of several serious chronic diseases, such as type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD), cardiovascular diseases (CVD), and certain types of cancer (Field et al., 2001). Adipose tissue has been historically considered an inert tissue, which was only devoted to the storage and mobilization of lipids in response to various hormonal signals. However, over the last two decades, a considerable number of studies have demonstrated that it is an organ in its own right, with secretory functions regulating its own activity but also the whole-body energy metabolism (Kershaw and Flier, 2004).
Obesity causes major cellular alterations in the white adipose tissue (WAT), mainly adipocyte hypertrophy (i.e., an increase in the size of pre-existing adipocytes), leading to lipid-laden, dysfunctional adipocytes, which undergo cell death and contribute to adipose tissue inflammation (Sun et al., 2011). Adipocyte death is recognized as an important early event in the development of obesity complications, especially T2D (Cinti et al., 2005). Distant effects of adipose tissue inflammation such as the trigger of liver inflammation and hepatocyte injury have been documented and can contribute to the progression of diseases such as NAFLD . Therefore, a better understanding of the molecular mechanisms controlling and linking the death to inflammatory processes is necessary to identify new therapeutic targets in obesity and associated co-morbidities.
Until recently, two main forms of cell death were recognized: apoptosis, which occurs in a highly controlled manner, and necrosis which is accidentally triggered. However, during the past few years, it became clear that programmed cell death was not restricted to apoptosis, and comprised other forms of regulated cell death ( Vanden Berghe et al., 2014). Necroptosis is one of them, combining the molecular machinery of the extrinsic apoptotic pathways with an execution similar to necrosis (Vanden Berghe et al., 2014). Unlike apoptosis that requires the activation of aspartate-specific proteases known as caspases (McIlwain et al., 2013), necroptosis is primarily driven by the activation of the receptor-interacting protein kinase (RIPK) 1 and 3, followed by the activation of the pseudo kinase mixed lineage kinase domain-like (MLKL) (Vanden Berghe et al., 2014). Since its discovery in 2009, necroptosis has been involved in many human diseases by mediating cell death and inflammation including ischemic reperfusion injuries (Baidya et al., 2020(Baidya et al., , 2021Linkermann et al., 2013), neurodegeneration (Zhang et al., 2017), NAFLD (Afonso et al., 2015;Gautheron et al., 2014Gautheron et al., , 2015, progressive atherosclerotic lesions (Zhe-Wei et al., 2018), and so forth. However, reports of necroptotic-independent functions of necroptosis mediators which can regulate the interplay between cellular metabolism and death have recently emerged. Notably, we identified a paramount role of RIPK3 in maintaining WAT homeostasis in mice fed a high-fat diet (HFD) (Gautheron et al., 2016). Thus, Ripk3-ablation led to a caspase-8 mediated inflammation and apoptosis, driving insulin resistance and glucose intolerance in mice (Gautheron et al., 2016). The protective function of RIPK3 against insulin resistance seemed to be restricted to the adipose tissue, stressing the need to study its function specifically in adipocytes. Opposite results were reported in Mlkl-deficient mice, who were protected against dyslipidemia following obesogenic diets in comparison to Ripk3 À/À mice (Rasheed et al., 2020;Saeed et al., 2019). Moreover, Mlkl deficiency prevented obesity-induced insulin resistance and glucose intolerance in obese mice with significantly less body weight gain and lower visceral adipose tissue compartments than in wild-type littermates (Xu et al., 2019). In line with these latter findings, mice treated with RIPA-56 (i.e., an inhibitor of RIPK1) showed downregulation of MLKL expression in liver and reduced body weight gain when fed an HFD (Majdi et al., 2020). Necroptosis inhibition by targeting MLKL has also shown beneficial effects on hepatic steatosis both in vivo and in vitro Majdi et al., 2020;Wu et al., 2020b). Both MLKL and RIPK3 interact with enzymes participating in mitochondrial lipid metabolism (Yang et al., 2018;Zhang et al., 2009), and an upregulation of peroxisome-proliferator-activated receptor g (PPARg) results from their deletion (Afonso et al., 2021;Wu et al., 2020a). Therefore, there is evidence to suggest that RIPK3 and MLKL may have cell-death-independent effects in influencing the whole body and adipose-specific metabolism.
In the present study, to address RIPK3 and MLKL differential roles in adipose tissue homeostasis and adipogenesis, we applied functional genomic approaches in vitro using the 3T3-L1 murine cell line and primary human adipose stem cells (ASC). The impact of the loss of Ripk3 or Mlkl on adipocyte differentiation and function was evaluated by developing CRISPR/Cas9-mediated genome-editing approaches. Using the 3T3-L1 cell culture model, we demonstrated that Mlkl deficiency inhibits early white adipocyte differentiation by up-regulating the Wnt/b-catenin pathway and suppressing the expression of master gene regulators involved in adipogenesis. The absence of differentiation upon Mlkl deficiency could be recapitulated in human cells using ASC. In contrast, Ripk3 deficiency does not alter adipocyte differentiation but impacts fatty acid metabolism by upregulating the metabolic pathways that are found to be downregulated by Mlkl ablation. Finally, we show that neither Mlkl nor Ripk3 deficiency prevents beige adipocyte differentiation in 3T3-L1 cells. Altogether, our results expand our understanding of the regulation of adipogenesis and stress by necroptosis mediators and highlight MLKL inhibition as a potential therapeutic approach in obesity-related diseases.

Differential expression of mixed lineage kinase domain-like and RIPK3 in visceral adipose tissue of obese mice
We previously reported a strong induction of RIPK3 protein expression in the visceral adipose tissue (vis-WAT) but not in the liver nor in the skeletal muscle of mice fed an HFD (Gautheron et al., 2016). Additionally, increased Mlkl mRNA levels were found in the liver, muscle, and visceral adipose tissue of obese mice (Xu et al., 2019). To further understand the differential regulation of RIPK3 and MLKL in adipose tissue, we evaluated the expression pattern of MLKL and RIPK3 in the visWAT of 20-week-old mice with genetic-induced obesity. We used the leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, in which obesity is triggered by defects in the production or transduction of leptin, respectively (MacReady, 2014). As expected, RIPK3 was highly expressed in the visWAT of both genetic models compared to wild-type (WT) mice ( Figures 1A and 1B). Noteworthy, RIPK3 was not expressed in the vis-WAT of WT C57/BL6 mice ( Figures 1A and 1B). Conversely, MLKL was expressed in the visWAT of WT C57/BL6 mice and also, although to a lesser extent, increased in the visWAT of ob/ob and db/db mice ( Figures 1A and 1B). Higher RIPK3 levels in visWAT were associated with an increase of the monocyte chemoattractant protein-1 (MCP-1) in the visWAT of both genetic obese murine models compared to control mice ( Figure 1C), suggesting that the immunological fraction of the adipose tissue may also contribute to the increased expression of RIPK3 in these pathological conditions ( Figures 1A-1C). Finally, we investigated the expression pattern of MLKL and RIPK3 in the subcutaneous adipose tissue (subWAT) of these mice. We did not observe any significant increases, but instead a slight reduction, in the sub-WAT of both genetic models compared to WT mice, suggesting that RIPK3 and MLKL upregulation in iScience Article genetic-induced obesity occur specifically in the visWAT ( Figures 1D and 1E). Taken together, these results highlight the time-dependent expression of the necroptosis mediators in adipose tissue, which may impact either its development or remodeling. To first characterize the necroptotic function of RIPK3 and MLKL in pre-adipocytes, a CRISPR/Cas9-mediated knockout (KO) approach was developed. We used the murine 3T3-L1 pre-adipocytes as a cellular model owing to their ability to differentiate into mature adipocytes after stimulation (Green and Meuth, 1974). A custom-designed single-guide RNA (sgRNA)/Cas9 expression vector targeting the first exon of Mlkl or that of Ripk3 was used. 3T3-L1 cells transfected with a Cas9/scramble gRNA plasmid were used as a control (CTL). The efficiency of RIPK3 and MLKL knockout was confirmed by Western blot analysis, which showed an almost complete loss of their expression (Figures 2A and 2B). In addition, Sanger sequencing of Mlkl or Ripk3 exon 1 in genomic DNA from KO cells revealed a high level of on-target indels with 79.3% of insertions and 13.4% of deletions for Mlkl, and 90.5% of deletions for Ripk3 (Figures S1 and S2). Treatment of CTL and both KO cells with 20 ng/mL of Tumor Necrosis Factor (TNF)-a alone for 24 h did not result in a significant loss of cell viability as measured by microscopic analysis ( Figure 2C) and MTT assays ( Figure 2D). However, when added after pretreatment with zVAD, a pan-caspase inhibitor, TNF-a stimulation induced approximately 40% of necroptotic death in CTL cells. CTL cells became all necrotic when a cIAP1/2 inhibitor (BV6) was added to zVAD ( Figures 2C and 2D). In contrast, Ripk3 or Mlkl ablation efficiently rescued 3T3-L1 cells from necroptosis induced by zVAD + TNF-a or by zVAD + BV6+TNF-a ( Figures 2C and  2D). We enhanced cell death even more by blocking overall mRNA translation, including pro-survival factors, e.g., via NF-kB, using Cycloheximide (CHX). The majority of CTL cells died when pre-treated with CHX + BV6+ZVAD combined with TNF-a stimulation, whereas a significant part (20-35%) of both KO cell lines survived ( Figures 2C and 2D). Of note, a slight increase in the proliferation rate was detected in RIPK3-deficient cells ( Figure 2E). Altogether, these data validate our experimental setup and provide further evidence that the use of CRISPR/Cas9 did not globally deregulate the physiological functions of 3T3-L1 cells. They also confirmed that the ablation of either Mlkl or Ripk3 can prevent necroptotic death in 3T3-L1 pre-adipocytes, and validate the use of these KO cells to study the necroptotic independent functions of MLKL and RIPK3 in adipogenesis.
Mlkl deficiency, but not Ripk3 ablation, prevents white adipocyte differentiation To explore the roles of MLKL and RIPK3 in white adipocyte differentiation, we investigated the kinetics of expression of adipogenesis and fatty acid storage markers during a 10-day white adipocyte differentiation protocol ( Figure S3A). As shown in Figure 3, CTL cells, which expressed scrambled sgRNA or were Ripk3-KO, differentiated into adipocytes within 10 days (D10) ( Figure 3A) and displayed a strong accumulation of lipid droplets in the cytoplasm as well as an increase in the triglyceride content ( Figures 3B and 3C). Ripk3-KO adipocytes even showed a 20% higher triglyceride content than CTL adipocytes at D10 ( Figure 3C). In contrast, Mlkl deficiency led to a strong and significant decrease in lipid droplet formation associated with a 60% reduction of triglyceride content ( Figures 3A-3C). Next, we assessed the expression of adipocyte markers to evaluate adipocyte differentiation. Mlkl ablation suppressed the expression of CCAAT/ enhancer-binding protein alpha (C/EBPa), fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD1), whereas their expression was globally unaffected by Ripk3 ablation ( Figure 3D). The mRNA levels of these factors were significantly decreased in Mlkl-KO cells starting 3 days post-induction of differentiation and remained low at a later stage of differentiation (i.e., 10 days post-induction) ( Figure 3D). Of note, neither Ripk3 nor Mlkl ablation affected the ability of cells to enter cell-cycle arrest, which is needed prior to adipocyte differentiation ( Figure S4). The percentage of CTL and KO cells in G0/G1 phases increased as the cells reached confluence, while that in S and G2/M phases disappeared ( Figure S4).
Importantly, the effects of Mlkl ablation evidenced in 3T3-L1 cells were recapitulated in human adipose stem cells (ASC), which can be differentiated into adipocytes using an appropriate cocktail ( Figure S5). The efficiency of MLKL knockout was confirmed by Western blot analysis, which led to a complete loss of its expression compared to control (CTL) ASC ( Figure 3E). In addition, Sanger sequencing of MLKL exon 4 in genomic DNA from KO cells revealed a high level of on-target indels with 96% of insertions and deletions ( Figure S6). We found that MLKL-KO ASC displayed a lower accumulation of lipid droplet in their cytoplasm than CTL ASC ( Figures 3F and 3G). The effect of MLKL ablation on adipocyte was further evaluated by an expression study of adipogenic and mature adipocyte markers during the differentiation process. As compared to CTL ASC, MLKL-KO ASC displayed a sharp decrease in the expression of adipogenic markers, including the transcription factors PPARg and C/EBPa at D20 ( Figure 3H). The expression of mature adipocyte markers, such as fatty acid synthase (FAS), perilipin, and adiponectin was also markedly decreased in MLKL-KO cells ( Figure 3H). Altogether, these findings suggest divergent roles of MLKL and RIPK3 in controlling adipocyte differentiation and fatty acid metabolism. Because MLKL loss-of-function improves the metabolic profile and reduces body weight gain when mice are fed a high-fat diet (Majdi et al., 2020;Rasheed et al., 2020;Saeed et al., 2019;Xu et al., 2019), we next studied the impact of Mlkl ablation on beige adipocyte differentiation. The 3T3-L1 cells were differentiated into beige adipocytes by adding to the white adipocyte differentiation medium rosiglitazone and triiodothyronine (T3) (Asano et al., 2014;Wang et al., 2014) ( Figure S3B). As shown in Figure 4, CTL cells expressing scrambled sgRNA differentiated into beige adipocytes under these conditions within 10 days. They displayed a massive iScience Article accumulation of lipid droplets in the cytoplasm as well as increased triglyceride content (Figures 4A and 4B). As shown by immunofluorescence, the presence of lipid accumulation correlated with an increase of perilipin-1, a protein-coating lipid storage droplet in adipocytes ( Figures 4A and 4C). Moreover, immunofluorescence studies confirmed the increased abundance of UCP-1 protein and mRNA levels, further indicating the presence of beige adipocytes (Figures 4D-4F). Mlkl deficiency did not alter neither lipid droplet formation iScience Article nor accumulation of triglycerides when cells were grown in the beige adipocyte culture medium ( Figures 4A  and 4B). In agreement, Ucp-1 and Pparg mRNA levels were also increased in Mlkl-KO beige adipocytes compared with white adipocytes, together with increased expression of Pcg-1 ( Figures 4F and 4G), which plays a critical role to establish the thermogenic pathway in beige adipocytes (Uldry et al., 2006). Of note, Ripk3 deficiency did not alter beige adipocyte differentiation ( Figures S7A and S7B). Moreover, the addition of rosiglitazone to the beige adipocyte differentiation medium was not sufficient to explain the discrepancies in-between white and beige differentiation capacities of Mlkl-KO cells, as a short-term rosiglitazone boost (i.e., less than 3 days) did not overcome MLKL requirement for white adipocyte differentiation ( Figure S7C). Altogether, these data suggest that MLKL is a limiting factor for white but not beige adipocyte differentiation, highlighting a specific metabolic role of MLKL in white adipose tissue expansion.

Mixed lineage kinase domain-like and RIPK3 modulate regulatory networks involved in adipogenesis
To further investigate the differential impact of Mlkl and Ripk3 ablations in white adipocyte differentiation, gene expression profiles were determined by RNA sequencing of the 3T3-L1 pre-adipocytes deleted for either Mlkl or Ripk3 and induced to adipocyte differentiation for 7 days ( Figure S4A). Three biological replicates with 25-45 million reads per replicate were generated for undifferentiated (D0) or differentiated (D7) adipocytes per genotype. The good sequencing quality score ( Figure S8A) and a high consistency in gene expression distribution among samples confirmed the low technical variability across samples ( Figure S8B).
Additionally, principal component analysis demonstrated expected grouping among replicates in-between samples ( Figure S8C). A total of 5,167 and 3,055 differentially expressed genes (DEG), with a false discovery rate (FDR) below 0.05, were identified between undifferentiated (D0) Ripk3-KO or Mlkl-KO vs. CTL cells, respectively. Differentiation from pre-adipocytes to adipocytes resulted in drastic changes in DEG, with more than 12,000 DEG observed in CTL and Ripk3-KO cells D7 vs. D0 ( Figure 5A). However, the differentiation protocol seemed to have a minimal effect on Mlkl-KO cells with only 1,891 DEG at D7 compared to D0 ( Figure 5A). This was further confirmed in the Fragments Per Kilobase Million (FPKM) hierarchical clustering heatmap, indicating that Mlkl-KO cells at D7 post-differentiation are clustering with undifferentiated rather than differentiated Ripk3-KO or CTL cells ( Figure 5B). In addition, by comparing transcriptomes of differentiated cells (at D7) a total of 5,696 and 7,288 DEG were identified in Ripk3-KO or Mlkl-KO vs. CTL, respectively ( Figure 5C). To identify signatures specific to each genotype, we then performed gene ontology and functional enrichment analysis using STRING, Reactome, KEGG, and Wikipathways databases. At D0 and compared to CTL cells, the transcriptome of Mlkl-KO cells is specifically enriched for genes involved in regulatory networks controlling extracellular matrix organization and ossification, suggesting that Mlkl-KO preadipocytes might be oriented more toward trans-differentiation to bone cells formation rather than adipogenesis ( Figures S9A and S9B) (Zhang et al., 2012). Interestingly, Ripk3-KO pre-adipocytes present a completely different signature for which pathways are enriched for ''DNA replication'' and ''G1 to S cell cycle control'' ( Figures S9C and S9D). We further analyzed differences in gene regulatory network at D7 of differentiation. Wikipathways enrichment analysis revealed that most of the significantly enriched pathways contained down-regulated genes in Mlkl-KO cells vs. CTL cells ( Figure 5D). The signature of Mlkl-KO cells indicates a clear reduction in the expression of genes involved in the early or late stages of adipogenesis (C/ebpa, Pparg, Adipoq, Plin1, Lpl, Fabp4, Lipe) ( Figure 5E), PPAR signaling pathways, fatty acid and cholesterol biosynthesis, fatty acid-beta oxidation and TCA cycle ( Figure S10). Interestingly, C/ebpa and Pparg down-regulated expressions in our dataset correlated with an increase in expression of Wnt10b a known inhibitor of adipogenesis ( Figure 5E) (Cawthorn et al., 2012). We could further confirm by RT-qPCR that the expression of genes involved in fatty acid metabolism and PPAR signaling pathway was, indeed, reduced in Mlkl-KO cells at 7 days post-differentiation ( Figure 5F). On the contrary, Ripk3-KO differentiated cells present an enrichment in pathways involved in PPAR signaling, fatty acid beta-oxidation, electron transport chain, and TCA cycle compared to CTL cells, suggesting an increase in the expression of genes implicated in mitochondria bioenergetics and function ( Figures 5D and 5G, and S11). In agreement, we observed a significant increase in the mitochondrial metabolic rate of Ripk3-KO cells using the MTT assay ( Figure 2E). Taken together, these data suggest that Mlkl but not Ripk3 deficiency impaired adipogenesis of 3T3-L1 cells by reducing the expression of pro-adipogenic factors, genes involved in fatty acid metabolism, and possibly through the inhibitory effect of Wnt10b expression.

DISCUSSION
Despite a progressive awareness of the population toward the global obesity epidemic, obesity is the main risk factor for several diseases, including diabetes mellitus, NAFLD, cardiovascular diseases, and certain iScience Article forms of cancer. The present study aimed to demonstrate that the necroptosis regulators MLKL and RIPK3 could have necroptotic-independent functions in modulating adipogenesis, a key event to control adipose tissue expansion and remodeling. Our main findings demonstrate that MLKL, but not RIPK3, plays a determinant role in adipogenesis in pre-adipocytes. Notably, we show that Mlkl ablation markedly inhibits white adipocyte differentiation while surprisingly preserving the orientation of pre-adipocytes toward the beige phenotype. Therefore, this study highlights a novel function of MLKL in regulating adipogenesis. iScience Article To uncover the role of RIPK3 and MLKL during adipogenesis we used the murine 3T3-L1 pre-adipocytes in vitro model. 3T3-L1 cells are often used to study adipocyte differentiation and are known to recapitulate the pro-adipogenic transcriptional programs observed in vivo during WAT development (Birsoy et al., 2011;Sun et al., 2020). To study the function of MLKL and RIPK3 during adipogenesis, we deleted both genes using the CRISPR/Cas9 technology. To decrease potential off-target mutations resulting from Cas9 integration into the genome, we favored a transient transfection with a plasmid containing both the Cas9 and gRNA targeting Ripk3 or Mlkl genes. Moreover, to avoid the selection of clones with offtarget mutations, we analyzed a heterogeneous population of cells issued from FACS sorting. As expected, we observed that the gene expression pattern of Glu1, Glud1, Pgam5, or Camk2, all known targets of RIPK3 (Wang et al., 2012;Yang et al., 2018;Zhang et al., 2016), were upregulated in the Ripk3-KO cells while being downregulated in Mlkl-KO cells compared to CTL cells ( Figure S12).
To decipher how MLKL exerts its cell death-independent function in adipocyte differentiation, we attempted to study its phosphorylation profile in 3T3-L1 cells. We tested several commercial antibodies against known phosphorylated forms of MLKL, but failed to detect any phosphorylation using these antibodies. How can the lack of phosphorylation be explained? Most MLKL phosphorylation sites have been identified in the context of necroptosis. For instance, MLKL is phosphorylated on Ser-345 to translocate, accumulate in the plasma membrane, and cause necroptosis (Rodriguez et al., 2016). The apparent lack of cell death during adipocyte differentiation can likely explain the absence of phosphorylation on the known phospho-sites. Thus, we can hypothesize that the phospho-sites of MLKL, if needed for adipocyte differentiation, might be different from those typically targeted during necroptosis. Overall, this highlights the need for a large-scale proteomic analysis to identify MLKL phospho-sites during adipocyte differentiation.
Our study suggests a necroptotic-independent function of MLKL in regulating gene expression/mRNA stabilization, among them Wnt10b. Indeed, preceding necroptosis induction, MLKL is also located in the nucleus without being associated with the nuclear membrane (Yoon et al., 2016). Noticeably, preventing MLKL translocation at the plasma membrane using Necrosulfonamide (NSA) has no impact on its nuclear translocation, suggesting that the transit of MLKL to either the plasma membrane or the nucleus is independent (Yoon et al., 2016). Recently, MLKL was found to act as part of an RNA binding protein complex in association with RNA-binding motif protein 6 (RBM6) to stabilize a variety of transcripts including several adhesion molecules and demonstrating that MLKL is, indeed, involved in post-transcriptional regulation (Dai et al., 2020). We sought to establish the localization of MLKL in adipocytes ( Figure S13). Immunofluorescence analysis revealed that MLKL resides in both cytoplasm and nucleus in adipocytes ( Figure S13), suggesting that MLKL may regulate the stability/degradation of Wnt10b mRNA in order to drive adipocyte differentiation. However, further studies are needed to confirm this assumption and to decipher potential mechanisms.
Mlkl À/À mice were previously reported to resist HFD-induced obesity compared to WT littermates (Xu et al., 2019). These data were also in line with our previous report showing that the inhibition of RIPK1 in mice reduced MLKL expression, which correlated with a lower weight gain compared with wild-type littermates when fed an HFD (Majdi et al., 2020). Similarly, it has been reported in 2020 in a PISA (Pathobiology for investigators, students, & academicians) meeting (https://pisa20.asip.org; abstract A073) that an 8-week exposure to either RIPK1 or MLKL antisense oligonucleotides (ASO) therapy in obese mice markedly reduced weight and fat mass compared to controls. Therefore, these in vivo data corroborate a potential role of MLKL in promoting fat mass accumulation by regulating adipogenesis; however, conditional murine models, which are still lacking, are mandatory to confirm these observations and to analyze adipose tissuespecific functions of MLKL. In this line of work, our study provides further mechanistical explanation over the role of MLKL in controlling white adipocyte differentiation. Our data suggest that MLKL might differentially regulate white and beige adipose tissue development, stressing the need to study white vs. beige adipose tissue repartition, energy expenditure, and thermoregulation in adipose-specific Mlkl-deficient mice.
Beige adipocytes rely heavily on mitochondrial respiration to maintain their normal physiological function of thermogenesis (Cedikova et al., 2016). Previous work demonstrated that PPARg is a key regulator of both white and beige adipocytes. However, ectopic expression of PPARg alone gives rise to white adipocytes (Uldry et al., 2006). PGC-1, a co-activator of PPARg, is also involved in mitochondrial biogenesis, and known to induce UCP-1 expression, a key regulator of thermogenesis and beige adipocyte function (Uldry et al., 2006). We demonstrated previously that Mlkl-deficient hepatocytes displayed higher PGC-1 levels, which were associated with increased mitochondrial respiration and biogenesis (Majdi et al., 2020). In the present study, iScience Article we showed that Mlkl ablation did not impair Pgc-1 mRNA level induction in the beige adipocyte differentiation medium. Together with PPARg, PGC-1 can drive beige adipocyte differentiation in Mlkl-deficient cells.
We herein report that Ripk3 ablation slightly increased triglyceride content in white adipocytes. Moreover, Ripk3-KO adipocytes had an enrichment in pathways involved in PPAR signaling and fatty acid beta-oxidation compared to CTL adipocytes. These data are in line with our recent study showing that the deletion of Ripk3 impacted on hepatic lipidome by upregulating PPARg in a murine model of NAFLD (Afonso et al., 2021). In this study, PPARg was also negatively correlated with hepatic RIPK3 in patients with NAFLD (Afonso et al., 2021). Moreover, the inhibition of RIPK3 using a Glaxo Smith Kline inhibitor increased the expression of PPARg as well as triglyceride accumulation in ASC (Yang et al., 2022). Thus, RIPK3 appears as a lipid metabolism regulator, which activity might be targeted (e.g., by knocking down its expression or by the pharmacological inhibition of its kinase activity) to enhance lipid metabolism. However, deletion or inhibition of RIPK3 often triggers apoptosis, which may be deleterious in a pathological context (Orozco and Oberst, 2017). This phenomenon has been shown both in the liver and adipose tissue of obese mice (Gautheron et al., 2016;Roychowdhury et al., 2016). In that specific context, MLKL seems to be a more privileged target to counteract obesity. However, specific RIPK3 inhibitors deprived of pro-apoptotic effects could represent a relevant strategy.
The transcription factors that control the cascade of events leading to mature adipocytes are well characterized. For instance, it is established that the master regulators PPARg and C/EPBa coordinate the adipocyte program. Nevertheless, secreted or circulating extra-cellular factors may regulate preadipocyte differentiation as well (Rosen and MacDougald, 2006). One of the signaling pathways that involve secreted factors is the Wnt/b-catenin pathway (Nusse and Clevers, 2017). Wnt ligands are an evolutionarily conserved family of secreted, cysteine-rich glycoproteins, whose role in cell proliferation, differentiation, and polarity during embryogenesis is well established (Nusse and Clevers, 2017). More recently, Wnt signaling has been shown to modulate other physiological and developmental processes, including adipocyte differentiation (Prestwich and Macdougald, 2007). In pre-adipocytes, Wnt/b-catenin signaling pathway maintains the cells in an undifferentiated state primarily by suppressing key adipogenic transcription factors C/EBPa and PPARg (Prestwich and Macdougald, 2007). Among Wnt ligands, Wnt10b is expressed mostly in pre-adipocytes and its levels decline rapidly after the induction of differentiation (Ross et al., 2000). On the contrary, its overexpression in 3T3-L1 cells stabilizes b-catenin and blocks adipogenesis (Christodoulides et al., 2009). The association between Wnt10b and obesity has emerged with the identification of mutations resulting in the loss of Wnt10b function (Christodoulides et al., 2006). These mutations abrogated the ability of Wnt10b to activate canonical WNT signaling in vitro and blocked the differentiation of 3T3-L1 cells (Christodoulides et al., 2006). In the present study, we found that Mlkl deficiency in 3T3-L1 cells triggers a high expression of Wnt10b in 3T3-L1 pre-adipocytes and 7 days after the addition of the white adipocyte differentiation medium. However, Wnt10b overexpression could be a mere consequence of the downregulation of the pro-adipogenic factors seen in these cells. Indeed, PPARg agonists induce b-catenin inhibition, while the inhibition of the canonical Wnt/b-catenin pathway activates PPARg (Lecarpentier et al., 2017). A similar cross-talk can be found in-between C/EBPa and the Wnt/b-catenin pathway (Chung et al., 2012). Interestingly, in our dataset Wnt10b is already up-regulated in Mlkl-KO pre-adipocytes, while Pparg is unchanged compared to CTL cells. This strongly suggests that the impact of Mlkl deficiency on the Wnt/b-catenin pathway may occur before the engagement of cells toward adipogenesis.
Overall, we propose that MLKL can act as a molecular switch between these pathways in order to control adipogenesis. However, additional investigations are needed to decipher if the inhibition of MLKL regulates directly or indirectly the Wnt/b-catenin pathway.
In summary, we have shown that the ablation of Mlkl, but not Ripk3, specifically blocks white adipocyte differentiation. This study uncovers a novel metabolic function of MLKL independent of its major involvement in the necroptosis pathway that could be targeted in obesity and related diseases for therapeutic purposes.

LIMITATIONS OF THE STUDY
This study essentially relies on human and mouse pre-adipocytes cellular models. Therefore, in vivo mouse models, such as adipose-cell-specific Mlkl deficiency, are needed to corroborate these data and to study white vs. beige adipose tissue distribution, energy expenditure, and thermoregulation. Specifically, compensatory mechanisms owing to the absence of MLKL in mice could happen and contradict our results.
To overcome such discrepancies, the development of an inducible knockout mouse should be preferred. Further mechanistic studies are needed to corroborate and elucidate MLKL and Wnt10b interactions.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jé ré mie Gautheron at jeremie.gautheron@inserm.fr.

Materials availability
Unique materials generated in this study is available upon complete materials transfer agreement.

Data and code availability
All data reported in this paper will be shared by the lead contact upon request. Gene expression data are available through the Gene Expression Omnibus (GEO) repository under the accession number GSE201450. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animal model 12-week-old-male homozygous ob/ob mice (B6.V-Lepob/ob/JRj; leptin-deficient obese model), db/db mice (BKS-Lepr/db/db/JOrlRj; long-form leptin receptor-deficient obese model), and C57BL/6JRj wildtype mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France). All experiments were conducted in the CRSA animal facility (Agreement No. C-75-12-01), according to the European Communities Council Directive (2010/63/UE) for the care and use of animals for experimental procedures. All were approved (No. B751201) by Ethics Committee of Animal Experiments Charles Darwin.