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The loss of pancreatic β-cell identity emerges as an important feature of type 2 diabetes development, but the molecular mechanisms are still elusive. Here, we explore the cell-autonomous role of the cell cycle regulator and transcription factor E2F1 in the maintenance of β-cell identity, insulin secretion and glucose homeostasis. We show that the β-cell-specific loss of E2f1 function in mice triggers glucose intolerance associated with defective insulin secretion, an altered endocrine cell mass, a downregulation of many β-cell genes and a concomitant increase of non-β-cell markers. Mechanistically, the epigenomic profiling of promoters of these non-β-cell upregulated genes identified an enrichment of bivalent H3K4me3/H3K27me3 or H3K27me3 marks. Conversely, promoters of downregulated genes were enriched in active chromatin H3K4me3 and H3K27ac histone marks. We find that specific E2f1 transcriptional, cistromic and epigenomic signatures are associated with these β-cell dysfunctions, with E2F1 directly regulating several β-cell genes at the chromatin level. Finally, the pharmacological inhibition of E2F transcriptional activity in human islets also impairs insulin secretion and the expression of β-cell identity genes. Our data suggest that E2F1 is critical for maintaining β-cell identity and function through a sustained control of β-cell and non β-cell transcriptional programs.

The loss of pancreatic β-cell identity emerges as an important feature of type 2 diabetes development, but the molecular mechanisms are still elusive. Here, we explore the cellautonomous role of the cell cycle regulator and transcription factor E2F1 in the maintenance of β-cell identity, insulin secretion and glucose homeostasis. We show that the β-cell-specific loss of E2f1 function in mice triggers glucose intolerance associated with defective insulin secretion, an altered endocrine cell mass, a downregulation of many β-cell genes and a concomitant increase of non-β-cell markers. Mechanistically, the epigenomic profiling of promoters of these non-β-cell upregulated genes identified an enrichment of bivalent H3K4me3/H3K27me3 or H3K27me3 marks. Conversely, promoters of downregulated genes were enriched in active chromatin H3K4me3 and H3K27ac histone marks. We find that specific E2f1 transcriptional, cistromic and epigenomic signatures are associated with these β-cell dysfunctions, with E2F1 directly regulating several β-cell genes at the chromatin level. Finally, the pharmacological inhibition of E2F transcriptional activity in human islets also impairs insulin secretion and the expression of β-cell identity genes. Our data suggest that E2F1 is critical for maintaining β-cell identity and function through a sustained control of β-cell and non β-cell transcriptional programs.

The loss of pancreatic β-cell identity emerges as an important feature of type 2 diabetes development, but the molecular mechanisms are still elusive. Here, we explore the cell-autonomous role of the cell cycle regulator and transcription factor E2F1 in the maintenance of β-cell identity and insulin secretion. We show that the β-cell-specific loss ofE2f1function in mice triggers glucose intolerance associated with defective insulin secretion, an altered α-to-β-cell ratio, a downregulation of many β-cell genes and a concomitant increase of non-β-cell markers. Mechanistically, the epigenomic profiling of non-beta cell upregulated gene promoters identified an enrichment of bivalent H3K4me3/H3K27me3 or H3K27me3 marks. Conversely, downregulated genes were enriched in active chromatin H3K4me3 and H3K27ac histone marks. We find that histone deacetylase inhibitors modulate E2F1 transcriptional and epigenomic signatures associated with these β-cell dysfunctions. Finally, the pharmacological inhibition of E2F transcriptional activity in human islets also impairs insulin secretion and the expression of β-cell identity genes. Our data suggest that E2F1 is critical for maintaining β-cell identity through a sustained repression of non β-cell transcriptional programs.

Background & Aims: Although the role of inflammation to combat infection is known, the contribution of metabolic changes in response to sepsis is poorly understood. Sepsis induces the release of lipid mediators, many of which activate nuclear receptors such as the peroxisome proliferator-activated receptor (PPAR)alpha, which controls both lipid metabolism and inflammation. We aimed to elucidate the previously unknown role of hepatic PPAR alpha in the response to sepsis.Methods: Sepsis was induced by intraperitoneal injection of Escherichia coli in different models of cell-specific PPAR alpha-deficiency and their controls. The systemic and hepatic metabolic response was analyzed using biochemical, transcriptomic and functional assays. PPAR alpha expression was analyzed in livers from elective surgery and critically ill patients and correlated with hepatic gene expression and blood parameters.Results: Both whole body and non-hematopoietic PPAR alpha-deficiency in mice decreased survival upon bacterial infection. Livers of septic PPAR alpha-deficient mice displayed an impaired metabolic shift from glucose to lipid utilization resulting in more severe hypoglycemia, impaired induction of hyperketonemia and increased steatosis due to lower expression of genes involved in fatty acid catabolism and ketogenesis. Hepatocyte-specific deletion of PPAR alpha impaired the metabolic response to sepsis and was sufficient to decrease survival upon bacterial infection. Hepatic PPAR alpha expression was lower in critically ill patients and correlated positively with expression of lipid metabolism genes, but not with systemic inflammatory markers.Conclusion: During sepsis, PPAR alpha-deficiency in hepatocytes is deleterious as it impairs the adaptive metabolic shift from glucose to FA utilization. Metabolic control by PPAR alpha in hepatocytes plays a key role in the host defense against infection.Lay summary: As the main cause of death in critically ill patients, sepsis remains a major health issue lacking efficacious therapies. While current clinical literature suggests an important role for inflammation, metabolic aspects of sepsis have mostly been overlooked. Here, we show that mice with an impaired metabolic response, due to deficiency of the nuclear receptor PPAR alpha in the liver, exhibit enhanced mortality upon bacterial infection despite a similar inflammatory response, suggesting that metabolic interventions may be a viable strategy for improving sepsis outcomes. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Genome-wide association studies (GWASs) provide an unprecedented opportunity to examine, on a large scale, the association of common genetic variants with complex diseases like type 2 diabetes (T2D) and cardiovascular disease (CVD), thus allowing the identification of new potential disease loci. Using this approach, numerous studies have associated SNPs on chromosome 9p21.3 situated near the cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) locus with the risk for coronary artery disease (CAD) and T2D. However, identifying the function of the nearby gene products (CDKN2A/B and ANRIL) in the pathophysiology of these conditions requires functional genomic studies. We review the current knowledge, from studies using human and mouse models, describing the function of CDKN2A/B gene products, which may mechanistically link the 9p21.3 risk locus with CVD and diabetes.

The genomic CDKN2A/B locus, encoding p16(INK4a) among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16(INK4a) is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16(INK4a) knock-down in 3T3/L1 preadipocytes or p16(INK4a) deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16(INK4a) in adipose tissue formation. p16(INK4a)-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16(INK4a)-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16(INK4a)-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16(INK4a) gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16(INK4a) modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

Type 2 diabetes (T2D) is hallmarked by insulin resistance, impaired insulin secretion, and increased hepatic glucose production. The worldwide increasing prevalence of T2D calls for efforts to understand its pathogenesis in order to improve disease prevention and management. Recent genome-wide association studies have revealed strong associations between the CDKN2A/B locus and T2D risk. The CDKN2A/B locus contains genes encoding cell cycle inhibitors, including p16Ink4a, which have not yet been implicated in the control of hepatic glucose homeostasis. Here, we show that p16Ink4a deficiency enhances fasting-induced hepatic glucose production in vivo by increasing the expression of key gluconeogenic genes. p16Ink4a downregulation leads to an activation of PKA-CREB-PGC1α signaling through increased phosphorylation of PKA regulatory subunits. Taken together, these results provide evidence that p16Ink4a controls fasting glucose homeostasis and could as such be involved in T2D development.

The CDKN2A locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize toward classically (CAMφ) or alternatively (AAMφ) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. Here, we show that p16INK4a deficiency (p16−/−) modulates the macrophage phenotype. Transcriptome analysis revealed that p16−/− BM-derived macrophages (BMDMs) exhibit a phenotype resembling IL-4–induced macrophage polarization. In line with this observation, p16−/− BMDMs displayed a decreased response to classically polarizing IFNγ and LPS and an increased sensitivity to alternative polarization by IL-4. Furthermore, mice transplanted with p16−/− BM displayed higher hepatic AAMφ marker expression levels on Schistosoma mansoni infection, an in vivo model of AAMφ phenotype skewing. Surprisingly, p16−/− BMDMs did not display increased IL-4–induced STAT6 signaling, but decreased IFNγ-induced STAT1 and lipopolysaccharide (LPS)–induced IKKα,β phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,β. These findings identify p16INK4a as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases.

The endoplasmic reticulum (ER) unfolded protein response (UPR(er)) pathway plays an important role in helping pancreatic β cells to adapt their cellular responses to environmental cues and metabolic stress. Although altered UPR(er) gene expression appears in rodent and human type 2 diabetic (T2D) islets, the underlying molecular mechanisms remain unknown. We show here that germline and β cell-specific disruption of the lysine acetyltransferase 2B (Kat2b) gene in mice leads to impaired insulin secretion and glucose intolerance. Genome-wide analysis of Kat2b-regulated genes and functional assays reveal a critical role for Kat2b in maintaining UPR(er) gene expression and subsequent β cell function. Importantly, Kat2b expression is decreased in mouse and human diabetic β cells and correlates with UPR(er) gene expression in normal human islets. In conclusion, Kat2b is a crucial transcriptional regulator for adaptive β cell function during metabolic stress by controlling UPR(er) and represents a promising target for T2D prevention and treatment.

ObjectiveA genomic region near the CDKN2A locus, encoding p16(INK4a), has been associated to type 2 diabetes and atherosclerotic vascular disease, conditions in which inflammation plays an important role. Recently, we found that deficiency of p16(INK4a) results in decreased inflammatory signaling in murine macrophages and that p16(INK4a) influences the phenotype of human adipose tissue macrophages. Therefore, we investigated the influence of immune cell p16(INK4a) on glucose tolerance and atherosclerosis in mice.Methods and resultsBone marrow p16(INK4a)-deficiency in C57Bl6 mice did not influence high fat diet-induced obesity nor plasma glucose and lipid levels. Glucose tolerance tests showed no alterations in high fat diet-induced glucose intolerance. While bone marrow p16(INK4a)-deficiency did not affect the gene expression profile of adipose tissue, hepatic expression of the alternative markers Chi3l3, Mgl2 and IL10 was increased and the induction of pro-inflammatory Nos2 was restrained on the high fat diet. Bone marrow p16(INK4a)-deficiency in low density lipoprotein receptor-deficient mice did not affect western diet-induced atherosclerotic plaque size or morphology. In line, plasma lipid levels remained unaffected and p16(INK4a)-deficient macrophages displayed equal cholesterol uptake and efflux compared to wild type macrophages.ConclusionBone marrow p16(INK4a)-deficiency does not affect plasma lipids, obesity, glucose tolerance or atherosclerosis in mice.

Human adipose tissue macrophages (ATMs) display an alternatively activated (M2) phenotype, but are still able to produce excessive inflammatory mediators. However, the processes driving this particular ATM phenotype are not understood. Genome-wide association studies associated the CDKN2A locus, encoding the tumour suppressor p16(INK4A), with the development of type 2 diabetes. In the present study, p16(INK4A) levels in human ATMs and the role of p16(INK4A) in acquiring the ATM phenotype were assessed.Gene expression of p16 ( INK4A ) in ATMs was analysed and compared with that in monocyte-derived macrophages (MDMs) from obese patients or with macrophages from human atherosclerotic plaques (AMs). Additionally, p16(INK4A) levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16(INK4A) in MDMs from healthy donors was investigated by small interfering (si)RNA-mediated silencing or adenovirus-mediated overproduction of p16(INK4A).Compared with MDMs and AMs, ATMs from obese patients expressed lower levels of p16 ( INK4A ). In vitro, IL-4-induced M2 polarisation resulted in lower p16(INK4A) protein levels after differentiation of monocytes from healthy donors in macrophages. Silencing of p16(INK4A) in MDMs mediated by siRNA increased the expression of M2 marker genes and enhanced the response to lipopolysaccharide (LPS), to give a phenotype resembling that of ATM. By contrast, adenovirus-mediated overproduction of p16(INK4A) in MDMs diminished M2 marker gene expression and the response to LPS. Western blot analysis revealed that p16(INK4A) overproduction inhibits LPS- and palmitate-induced Toll-like receptor 4 (TLR4)-nuclear factor of κ light polypeptide gene enhancer in B cells (NF-κB) signalling.These results show that p16(INK4A) inhibits the acquisition of the ATM phenotype. The age-related increase in p16(INK4A) level may thus influence normal ATM function and contribute to type 2 diabetes risk.

Genome wide association studies have been linked the CDKN2A/B locus to an increased risk for atherosclerosis, type 2 diabetes and aneurysm development. These conditions all are linked to perivascular adipose tissue (pAT) accumulation, which has an inflammatory phenotype.

Introduction Le diabete de type 2 (T2D) est un trouble metabolique de l'homeostasie du glucose. Il est caracterise par une hyperglycemie chronique qui resulte en partie d'une production excessive de glucose par le foie consequence au developpement d'une resistance a l'insuline. Le T2D est une pathologie multifactorielle a la fois genetique et environnementale. Recemment des etudes d'associations de genes (GWAS) dans differentes cohortes ont mis en evidence une forte correlation entre le locus CDKN2A et le risque de developpement du T2D en se basant sur certains parametres metaboliques tel que la glycemie a jeun. Le locus CDKN2A code pour des proteines regulatrices du cycle cellulaire dont la proteine p16INK4a. p16INK4a est largement decrite dans la litterature pour son role suppresseur de tumeurs et comme marqueur de senescence, cependant son role dans le controle de l'homeostasie hepatique du glucose n'a jamais ete rapporte Materiels et methodes Afin de determiner le role de p16INK4a dans le metabolisme hepatique du glucose, nous avons utilise in vivo des souris sauvages (p16+/+) et deficientes pour p16INK4a (p16-/-) et in vitro des hepatocytes primaires ainsi que la lignee AML12. Resultats Nous avons montre qu'apres un jeune, les souris p16-/- presentent une hypoglycemie moins prononcee qui se traduit par une expression hepatique plus elevee de genes de la neoglucogenese tels que PEPCK, G6Pase et PGC1a. De plus, les hepatocytes primaires de souris p16-/- presentent une meilleur reponse au glucagon que ceux des p16+/+. Enfin, nous avons montre que la diminution d'expression de p16INK4a par siRNA dans les AML12 suffit a induire l'expression des genes de la neoglucogenese et potentialise la reponse de ces cellules a differents stimuli gluconeogenique. L'effet observe depend de l'activation de la voie PKA-CREB-PGC1A. Conclusion L'ensemble de ces donnees montrent pour la premiere fois que p16INK4a pourrait jouer un role un cours du developpement du T2D.

Introduction Des analyses d'association de genes montrent que le locus CDKN2A/B qui code notamment la proteine p16INK4a, pourrait etre associe au developpement des maladies coronariennes, de l'atherosclerose et du diabete de type 2. Ces pathologies sont associees a l'expansion et a l'inflammation de differents depots de tissu adipeux (TA), notamment le tissu adipeux perivasculaire (PVAT). p16INK4a est un regulateur du cycle cellulaire cependant sa fonction dans l'adipogenese reste encore inconnue. Materiels et methodes Dans cette etude, nous avons etudie le role de p16INK4a dans l'adipogenese in vitro en utilisant des preadipocytes 3T3L1 transfectes par un siRNA-CDKN2A ou des fibroblastes embryonnaires (MEF) isoles des souris p16+/+ et p16−/−, et in vivo, chez l'homme, en etudiant l'expression de p16INK4a dans le PVAT, et chez la souris, en etudiant le developpement des depots de TA induit par un traitement Rosiglitazone. Le role de p16INK4a dans le developpement du PVAT a partir de la moelle osseuse a ete etudie chez des souris chimeres p16−/−LDLRKO et p16+/+LDLRKO soumises a un regime western. Resultats La diminution de l'expression de p16INK4a dans les pre-adipocytes 3T3L1 augmente l'adipogenese, mesuree par une augmentation de l'expression de PPARgamma, adiponectine, perilipine et CEBPalpha et une accumulation de lipides, sans affecter l'expansion clonale. Des resultats similaires ont ete obtenus dans les MEF p16−/−. Chez l'homme, p16INK4a est fortement exprime dans le PVAT cardiaque compare aux autres depots de TA. Chez la souris, bien que la deficience de p16INK4a n'influence pas le developpement des differents depots de TA, le traitement des souris p16−/− par la Rosiglitazone augmente specifiquement le developpement du PVAT, associe a une augmentation de l'expression de marqueurs de cellules precurseurs des adipocytes de la moelle. La deficience de p16INK4a dans la moelle osseuse de souris chimeres p16−/− LDLRKO augmente le developpement du PVAT induit par un regime western. Conclusion L'ensemble de ces donnees demontre un nouveau role de p16INK4a dans l'adipogenese et le developpement du PVAT.