ß-Cell-Specific E2f1 Deficiency Impairs Glucose Homeostasis, ß-Cell Identity, and Insulin Secretion.

The loss of pancreatic ß-cell identity has emerged 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 ß-cell identity, insulin secretion, and glucose homeostasis. We show that the ß-cell-specific loss of E2f1 function in mice triggers glucose intolerance associated with defective insulin secretion, altered endocrine cell mass, downregulation of many ß-cell genes, and concomitant increase of non-ß-cell markers. Mechanistically, epigenomic profiling of the promoters of these non-ß-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 ß-cell dysfunctions, with E2F1 directly regulating several ß-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 ß-cell identity genes. Our data suggest that E2F1 is critical for maintaining ß-cell identity and function through sustained control of ß-cell and non-ß-cell transcriptional programs. ARTICLE HIGHLIGHTS: ß-Cell-specific E2f1 deficiency in mice impairs glucose tolerance. Loss of E2f1 function alters the ratio of α- to ß-cells but does not trigger ß-cell conversion into α-cells. Pharmacological inhibition of E2F activity inhibits glucose-stimulated insulin secretion and alters ß- and α-cell gene expression in human islets. E2F1 maintains ß-cell function and identity through control of transcriptomic and epigenetic programs.

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.

Hepatic PPARα is critical in the metabolic adaptation to sepsis.

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)α, which controls both lipid metabolism and inflammation. We aimed to elucidate the previously unknown role of hepatic PPARα in the response to sepsis. METHODS: Sepsis was induced by intraperitoneal injection of Escherichia coli in different models of cell-specific Ppara-deficiency and their controls. The systemic and hepatic metabolic response was analyzed using biochemical, transcriptomic and functional assays. PPARα 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 Ppara-deficiency in mice decreased survival upon bacterial infection. Livers of septic Ppara-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 PPARα impaired the metabolic response to sepsis and was sufficient to decrease survival upon bacterial infection. Hepatic PPARA 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, Ppara-deficiency in hepatocytes is deleterious as it impairs the adaptive metabolic shift from glucose to FA utilization. Metabolic control by PPARα 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 PPARα 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.

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.

KAT2B Is Required for Pancreatic Beta Cell Adaptation to Metabolic Stress by Controlling the Unfolded Protein Response.

The endoplasmic reticulum (ER) unfolded protein response (UPR(er)) pathway plays an important role in helping pancreatic ß 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 ß 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 ß cell function. Importantly, Kat2b expression is decreased in mouse and human diabetic ß cells and correlates with UPR(er) gene expression in normal human islets. In conclusion, Kat2b is a crucial transcriptional regulator for adaptive ß cell function during metabolic stress by controlling UPR(er) and represents a promising target for T2D prevention and treatment.

Functional genomics of the CDKN2A/B locus in cardiovascular and metabolic disease: what have we learned from GWASs?

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.

Cdkn2a/p16Ink4a regulates fasting-induced hepatic gluconeogenesis through the PKA-CREB-PGC1α pathway.

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 p16(Ink4a), which have not yet been implicated in the control of hepatic glucose homeostasis. Here, we show that p16(Ink4a) deficiency enhances fasting-induced hepatic glucose production in vivo by increasing the expression of key gluconeogenic genes. p16(Ink4a) downregulation leads to an activation of PKA-CREB-PGC1α signaling through increased phosphorylation of PKA regulatory subunits. Taken together, these results provide evidence that p16(Ink4a) controls fasting glucose homeostasis and could as such be involved in T2D development.

Bone marrow p16INK4a-deficiency does not modulate obesity, glucose homeostasis or atherosclerosis development.

OBJECTIVE: A 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 RESULTS: Bone 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. CONCLUSION: Bone marrow p16(INK4a)-deficiency does not affect plasma lipids, obesity, glucose tolerance or atherosclerosis in mice.

p16INK4a deficiency promotes IL-4-induced polarization and inhibits proinflammatory signaling in macrophages.

The CDKN2A locus, which contains the tumor suppressor gene p16(INK4a), 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 p16(INK4a) 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,ß phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,ß. These findings identify p16(INK4a) as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases.