Knocking Down CDKN2A in 3D hiPSC-Derived Brown Adipose Progenitors Potentiates Differentiation, Oxidative Metabolism and Browning Process.

Human induced pluripotent stem cells (hiPSCs) have the potential to be differentiated into any cell type, making them a relevant tool for therapeutic purposes such as cell-based therapies. In particular, they show great promise for obesity treatment as they represent an unlimited source of brown/beige adipose progenitors (hiPSC-BAPs). However, the low brown/beige adipocyte differentiation potential in 2D cultures represents a strong limitation for clinical use. In adipose tissue, besides its cell cycle regulator functions, the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus modulates the commitment of stem cells to the brown-like type fate, mature adipocyte energy metabolism and the browning of adipose tissue. Here, using a new method of hiPSC-BAPs 3D culture, via the formation of an organoid-like structure, we silenced CDKN2A expression during hiPSC-BAP adipogenic differentiation and observed that knocking down CDKN2A potentiates adipogenesis, oxidative metabolism and the browning process, resulting in brown-like adipocytes by promoting UCP1 expression and beiging markers. Our results suggest that modulating CDKN2A levels could be relevant for hiPSC-BAPs cell-based therapies.

Istradefylline protects from cisplatin-induced nephrotoxicity and peripheral neuropathy while preserving cisplatin antitumor effects.

Cisplatin is a potent chemotherapeutic drug that is widely used in the treatment of various solid cancers. However, its clinical effectiveness is strongly limited by frequent severe adverse effects, in particular nephrotoxicity and chemotherapy-induced peripheral neuropathy. Thus, there is an urgent medical need to identify novel strategies that limit cisplatin-induced toxicity. In the present study, we show that the FDA-approved adenosine A2A receptor antagonist istradefylline (KW6002) protected from cisplatin-induced nephrotoxicity and neuropathic pain in mice with or without tumors. Moreover, we also demonstrate that the antitumoral properties of cisplatin were not altered by istradefylline in tumor-bearing mice and could even be potentiated. Altogether, our results support the use of istradefylline as a valuable preventive approach for the clinical management of patients undergoing cisplatin treatment.

The transcription factor E2F1 controls the GLP-1 receptor pathway in pancreatic ß cells.

The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving ß cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating ß cell mass and function. Here, we report that ß cell-specific genetic ablation of E2f1 (E2f1ß-/-) impairs glucose homeostasis associated with decreased expression of the Glp-1 receptor (Glp1r) in E2f1ß-/- pancreatic islets. Pharmacological inhibition of E2F1 transcriptional activity in nondiabetic human islets decreases GLP1R levels and blunts the incretin effect of GLP1R agonist exendin-4 (ex-4) on insulin secretion. Overexpressing E2f1 in pancreatic ß cells increases Glp1r expression associated with enhanced insulin secretion mediated by ex-4. Interestingly, ex-4 induces retinoblastoma protein (pRb) phosphorylation and E2f1 transcriptional activity. Our findings reveal critical roles for E2f1 in ß cell function and suggest molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways.

Glucose Regulates m6A Methylation of RNA in Pancreatic Islets.

Type 2 diabetes is characterized by chronic hyperglycemia associated with impaired insulin action and secretion. Although the heritability of type 2 diabetes is high, the environment, including blood components, could play a major role in the development of the disease. Amongst environmental effects, epitranscriptomic modifications have been recently shown to affect gene expression and glucose homeostasis. The epitranscriptome is characterized by reversible chemical changes in RNA, with one of the most prevalent being the m6A methylation of RNA. Since pancreatic ß cells fine tune glucose levels and play a major role in type 2 diabetes physiopathology, we hypothesized that the environment, through variations in blood glucose or blood free fatty acid concentrations, could induce changes in m6A methylation of RNAs in pancreatic ß cells. Here we observe a significant decrease in m6A methylation upon high glucose concentration, both in mice and human islets, associated with altered expression levels of m6A demethylases. In addition, the use of siRNA and/or specific inhibitors against selected m6A enzymes demonstrate that these enzymes modulate the expression of genes involved in pancreatic ß-cell identity and glucose-stimulated insulin secretion. Our data suggest that environmental variations, such as glucose, control m6A methylation in pancreatic ß cells, playing a key role in the control of gene expression and pancreatic ß-cell functions. Our results highlight novel causes and new mechanisms potentially involved in type 2 diabetes physiopathology and may contribute to a better understanding of the etiology of this disease.

The multifunctional protein E4F1 links P53 to lipid metabolism in adipocytes.

Growing evidence supports the importance of the p53 tumor suppressor in metabolism but the mechanisms underlying p53-mediated control of metabolism remain poorly understood. Here, we identify the multifunctional E4F1 protein as a key regulator of p53 metabolic functions in adipocytes. While E4F1 expression is upregulated during obesity, E4f1 inactivation in mouse adipose tissue results in a lean phenotype associated with insulin resistance and protection against induced obesity. Adipocytes lacking E4F1 activate a p53-dependent transcriptional program involved in lipid metabolism. The direct interaction between E4F1 and p53 and their co-recruitment to the Steaoryl-CoA Desaturase-1 locus play an important role to regulate monounsaturated fatty acids synthesis in adipocytes. Consistent with the role of this E4F1-p53-Steaoryl-CoA Desaturase-1 axis in adipocytes, p53 inactivation or diet complementation with oleate partly restore adiposity and improve insulin sensitivity in E4F1-deficient mice. Altogether, our findings identify a crosstalk between E4F1 and p53 in the control of lipid metabolism in adipocytes that is relevant to obesity and insulin resistance.

Leptin brain entry via a tanycytic LepR-EGFR shuttle controls lipid metabolism and pancreas function.

Metabolic health depends on the brain's ability to control food intake and nutrient use versus storage, processes that require peripheral signals such as the adipocyte-derived hormone, leptin, to cross brain barriers and mobilize regulatory circuits. We have previously shown that hypothalamic tanycytes shuttle leptin into the brain to reach target neurons. Here, using multiple complementary models, we show that tanycytes express functional leptin receptor (LepR), respond to leptin by triggering Ca2+ waves and target protein phosphorylation, and that their transcytotic transport of leptin requires the activation of a LepR-EGFR complex by leptin and EGF sequentially. Selective deletion of LepR in tanycytes blocks leptin entry into the brain, inducing not only increased food intake and lipogenesis but also glucose intolerance through attenuated insulin secretion by pancreatic ß-cells, possibly via altered sympathetic nervous tone. Tanycytic LepRb-EGFR-mediated transport of leptin could thus be crucial to the pathophysiology of diabetes in addition to obesity, with therapeutic implications.

CDKN2A/p16INK4a suppresses hepatic fatty acid oxidation through the AMPKα2-SIRT1-PPARα signaling pathway.

In addition to their well-known role in the control of cellular proliferation and cancer, cell cycle regulators are increasingly identified as important metabolic modulators. Several GWAS have identified SNPs near CDKN2A, the locus encoding for p16INK4a (p16), associated with elevated risk for cardiovascular diseases and type-2 diabetes development, two pathologies associated with impaired hepatic lipid metabolism. Although p16 was recently shown to control hepatic glucose homeostasis, it is unknown whether p16 also controls hepatic lipid metabolism. Using a combination of in vivo and in vitro approaches, we found that p16 modulates fasting-induced hepatic fatty acid oxidation (FAO) and lipid droplet accumulation. In primary hepatocytes, p16-deficiency was associated with elevated expression of genes involved in fatty acid catabolism. These transcriptional changes led to increased FAO and were associated with enhanced activation of PPARα through a mechanism requiring the catalytic AMPKα2 subunit and SIRT1, two known activators of PPARα. By contrast, p16 overexpression was associated with triglyceride accumulation and increased lipid droplet numbers in vitro, and decreased ketogenesis and hepatic mitochondrial activity in vivo Finally, gene expression analysis of liver samples from obese patients revealed a negative correlation between CDKN2A expression and PPARA and its target genes. Our findings demonstrate that p16 represses hepatic lipid catabolism during fasting and may thus participate in the preservation of metabolic flexibility.

Emerging Roles for the INK4a/ARF (CDKN2A) Locus in Adipose Tissue: Implications for Obesity and Type 2 Diabetes.

Besides its role as a cell cycle and proliferation regulator, the INK4a/ARF (CDKN2A) locus and its associated pathways are thought to play additional functions in the control of energy homeostasis. Genome-wide association studies in humans and rodents have revealed that single nucleotide polymorphisms in this locus are risk factors for obesity and related metabolic diseases including cardiovascular complications and type-2 diabetes (T2D). Recent studies showed that both p16INK4a-CDK4-E2F1/pRB and p19ARF-P53 (p14ARF in humans) related pathways regulate adipose tissue (AT) physiology and adipocyte functions such as lipid storage, inflammation, oxidative activity, and cellular plasticity (browning). Targeting these metabolic pathways in AT emerged as a new putative therapy to alleviate the effects of obesity and prevent T2D. This review aims to provide an overview of the literature linking the INK4a/ARF locus with AT functions, focusing on its mechanisms of action in the regulation of energy homeostasis.

The nuclear receptor FXR inhibits Glucagon-Like Peptide-1 secretion in response to microbiota-derived Short-Chain Fatty Acids.

The gut microbiota participates in the control of energy homeostasis partly through fermentation of dietary fibers hence producing short-chain fatty acids (SCFAs), which in turn promote the secretion of the incretin Glucagon-Like Peptide-1 (GLP-1) by binding to the SCFA receptors FFAR2 and FFAR3 on enteroendocrine L-cells. We have previously shown that activation of the nuclear Farnesoid X Receptor (FXR) decreases the L-cell response to glucose. Here, we investigated whether FXR also regulates the SCFA-induced GLP-1 secretion. GLP-1 secretion in response to SCFAs was evaluated ex vivo in murine colonic biopsies and in colonoids of wild-type (WT) and FXR knock-out (KO) mice, in vitro in GLUTag and NCI-H716 L-cells activated with the synthetic FXR agonist GW4064 and in vivo in WT and FXR KO mice after prebiotic supplementation. SCFA-induced GLP-1 secretion was blunted in colonic biopsies from GW4064-treated mice and enhanced in FXR KO colonoids. In vitro FXR activation inhibited GLP-1 secretion in response to SCFAs and FFAR2 synthetic ligands, mainly by decreasing FFAR2 expression and downstream Gαq-signaling. FXR KO mice displayed elevated colonic FFAR2 mRNA levels and increased plasma GLP-1 levels upon local supply of SCFAs with prebiotic supplementation. Our results demonstrate that FXR activation decreases L-cell GLP-1 secretion in response to inulin-derived SCFA by reducing FFAR2 expression and signaling. Inactivation of intestinal FXR using bile acid sequestrants or synthetic antagonists in combination with prebiotic supplementation may be a promising therapeutic approach to boost the incretin axis in type 2 diabetes.

Improved synthesis, resolution, absolute configuration determination and biological evaluation of HLM006474 enantiomers.

An improved green synthesis of the E2F inhibitor HLM0066474 is described, using solvent-free and microwave irradiation conditions. The two enantiomers are separated using semi-preparative separation on Chiralpak ID and their absolute configuration is determined by vibrational circular dichroism (VCD) analysis. Biological evaluation of both enantiomers on E2F1 transcriptional activity reveals that the (+)-R, but not the (-)-S enantiomer is biologically active in repressing E2F1 transcriptional activity.

E2F1 inhibition mediates cell death of metastatic melanoma.

Melanoma is one of the most lethal cancers when it reaches a metastatic stage. Despite advancements in targeted therapies (BRAF inhibitors) or immunotherapies (anti-CTLA-4 or anti-PD1), most patients with melanoma will need additional treatment. Thus, there is an urgent need to develop new therapeutical approaches to bypass resistance and achieve more prolonged responses. In this context, we were interested in E2F1, a transcription factor that plays a major role in the control of cell cycle under physiological and pathological conditions. Here we confirmed that E2F1 is highly expressed in melanoma cells. Inhibition of E2F1 activity further increased melanoma cell death and senescence, both in vitro and in vivo. Moreover, blocking E2F1 also induced death of melanoma cells resistant to BRAF inhibitors. In conclusion, our studies suggest that targeting the E2F1 signaling pathway may be therapeutically relevant for melanoma.

E2F1 promotes hepatic gluconeogenesis and contributes to hyperglycemia during diabetes.

OBJECTIVE: Aberrant hepatic glucose production contributes to the development of hyperglycemia and is a hallmark of type 2 diabetes. In a recent study, we showed that the transcription factor E2F1, a component of the cell cycle machinery, contributes to hepatic steatosis through the transcriptional regulation of key lipogenic enzymes. Here, we investigate if E2F1 contributes to hyperglycemia by regulating hepatic gluconeogenesis. METHODS: We use different genetic models to investigate if E2F1 regulates gluconeogenesis in primary hepatocytes and in vivo. We study the impact of depleting E2F1 or inhibiting E2F1 activity in diabetic mouse models to evaluate if this transcription factor contributes to hyperglycemia during insulin resistance. We analyze E2F1 mRNA levels in the livers of human diabetic patients to assess the relevance of E2F1 in human pathophysiology. RESULTS: Lack of E2F1 impaired gluconeogenesis in primary hepatocytes. Conversely, E2F1 overexpression increased glucose production in hepatocytes and in mice. Several genetic models showed that the canonical CDK4-RB1-E2F1 pathway is directly involved in this regulation. E2F1 mRNA levels were increased in the livers from human diabetic patients and correlated with the expression of the gluconeogenic enzyme Pck1. Genetic invalidation or pharmacological inhibition of E2F1 improved glucose homeostasis in diabetic mouse models. CONCLUSIONS: Our study unveils that the transcription factor E2F1 contributes to mammalian glucose homeostasis by directly controlling hepatic gluconeogenesis. Together with our previous finding that E2F1 promotes hepatic steatosis, the data presented here show that E2F1 contributes to both hyperlipidemia and hyperglycemia in diabetes, suggesting that specifically targeting E2F1 in the liver could be an interesting strategy for therapies against type 2 diabetes.

Cdkn2a deficiency promotes adipose tissue browning.

OBJECTIVES: Genome-wide association studies have reported that DNA polymorphisms at the CDKN2A locus modulate fasting glucose in human and contribute to type 2 diabetes (T2D) risk. Yet the causal relationship between this gene and defective energy homeostasis remains elusive. Here we sought to understand the contribution of Cdkn2a to metabolic homeostasis. METHODS: We first analyzed glucose and energy homeostasis from Cdkn2a-deficient mice subjected to normal or high fat diets. Subsequently Cdkn2a-deficient primary adipose cells and human-induced pluripotent stem differentiated into adipocytes were further characterized for their capacity to promote browning of adipose tissue. Finally CDKN2A levels were studied in adipocytes from lean and obese patients. RESULTS: We report that Cdkn2a deficiency protects mice against high fat diet-induced obesity, increases energy expenditure and modulates adaptive thermogenesis, in addition to improving insulin sensitivity. Disruption of Cdkn2a associates with increased expression of brown-like/beige fat markers in inguinal adipose tissue and enhances respiration in primary adipose cells. Kinase activity profiling and RNA-sequencing analysis of primary adipose cells further demonstrate that Cdkn2a modulates gene networks involved in energy production and lipid metabolism, through the activation of the Protein Kinase A (PKA), PKG, PPARGC1A and PRDM16 signaling pathways, key regulators of adipocyte beiging. Importantly, CDKN2A expression is increased in adipocytes from obese compared to lean subjects. Moreover silencing CDKN2A expression during human-induced pluripotent stem cells adipogenic differentiation promoted UCP1 expression. CONCLUSION: Our results offer novel insight into brown/beige adipocyte functions, which has recently emerged as an attractive therapeutic strategy for obesity and T2D. Modulating Cdkn2a-regulated signaling cascades may be of interest for the treatment of metabolic disorders.

Cofactors As Metabolic Sensors Driving Cell Adaptation in Physiology and Disease.

Chromatin architectures and epigenetic fingerprint regulation are fundamental for genetically determined biological processes. Chemical modifications of the chromatin template sensitize the genome to intracellular metabolism changes to set up diverse functional adaptive states. Accumulated evidence suggests that the action of epigenetic modifiers is sensitive to changes in dietary components and cellular metabolism intermediates, linking nutrition and energy metabolism to gene expression plasticity. Histone posttranslational modifications create a code that acts as a metabolic sensor, translating changes in metabolism into stable gene expression patterns. These observations support the notion that epigenetic reprograming-linked energy input is connected to the etiology of metabolic diseases and cancer. In the present review, we introduce the role of epigenetic cofactors and their relation with nutrient intake and we question the links between epigenetic regulation and the development of metabolic diseases.

Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance.

We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.

E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis.

E2F transcription factors are known regulators of the cell cycle, proliferation, apoptosis, and differentiation. Here, we reveal that E2F1 plays an essential role in liver physiopathology through the regulation of glycolysis and lipogenesis. We demonstrate that E2F1 deficiency leads to a decrease in glycolysis and de novo synthesis of fatty acids in hepatocytes. We further demonstrate that E2F1 directly binds to the promoters of key lipogenic genes, including Fasn, but does not bind directly to genes encoding glycolysis pathway components, suggesting an indirect effect. In murine models, E2F1 expression and activity increased in response to feeding and upon insulin stimulation through canonical activation of the CDK4/pRB pathway. Moreover, E2F1 expression was increased in liver biopsies from obese, glucose-intolerant humans compared with biopsies from lean subjects. Finally, E2f1 deletion completely abrogated hepatic steatosis in different murine models of nonalcoholic fatty liver disease (NAFLD). In conclusion, our data demonstrate that E2F1 regulates lipid synthesis and glycolysis and thus contributes to the development of liver pathology.

CDK4 is an essential insulin effector in adipocytes.

Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4(R24C)). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.

Extracellular-signal-regulated kinase 5 modulates the antioxidant response by transcriptionally controlling Sirtuin 1 expression in leukemic cells.

Cancer cell metabolism differs from that of non-transformed cells in the same tissue. This specific metabolism gives tumor cells growing advantages besides the effect in increasing anabolism. One of these advantages is immune evasion mediated by a lower expression of the mayor histocompatibility complex class I molecules. The extracellular-signal-regulated kinase-5 regulates both mayor histocompatibility complex class I expression and metabolic activity. However, the mechanisms underlying are largely unknown. We show here that extracellular-signal-regulated kinase-5 regulates the transcription of the NADH(+)-dependent histone deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin 1) in leukemic Jurkat T cells. This involves the activation of the transcription factor myocyte enhancer factor-2 and its binding to the sirt1 promoter. In addition, extracellular-signal-regulated kinase-5 is required for T cell receptor-induced and oxidative stress-induced full Sirtuin 1 expression. Extracellular-signal-regulated kinase-5 induces the expression of promoters containing the antioxidant response elements through a Sirtuin 1-dependent pathway. On the other hand, down modulation of extracellular-signal-regulated kinase-5 expression impairs the anti-oxidant response. Notably, the extracellular-signal-regulated kinase-5 inhibitor BIX02189 induces apoptosis in acute myeloid leukemia tumor cells without affecting T cells from healthy donors. Our results unveil a new pathway that modulates metabolism in tumor cells. This pathway represents a promising therapeutic target in cancers with deep metabolic layouts such as acute myeloid leukemia.

Role of Ink4a/Arf locus in beta cell mass expansion under physiological and pathological conditions.

The ARF/INK4A (Cdkn2a) locus includes the linked tumour suppressor genes p16INK4a and p14ARF (p19ARF in mice) that trigger the antiproliferative activities of both RB and p53. With beta cell self-replication being the primary source for new beta cell generation in adult animals, the network by which beta cell replication could be increased to enhance beta cell mass and function is one of the approaches in diabetes research. In this review, we show a general view of the regulation points at transcriptional and posttranslational levels of Cdkn2a locus. We describe the molecular pathways and functions of Cdkn2a in beta cell cycle regulation. Given that aging reveals increased p16Ink4a levels in the pancreas that inhibit the proliferation of beta cells and decrease their ability to respond to injury, we show the state of the art about the role of this locus in beta cell senescence and diabetes development. Additionally, we focus on two approaches in beta cell regeneration strategies that rely on Cdkn2a locus negative regulation: long noncoding RNAs and betatrophin.