Molecular glues of the regulatory ChREBP/14-3-3 complex protect beta cells from glucolipotoxicity.

The Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor (TF) that is characterized by two major splice isoforms (α and ß). In acute hyperglycemia, both ChREBP isoforms regulate adaptive ß-expansion; however, during chronic hyperglycemia and glucolipotoxicity, ChREBPß expression surges, leading to ß-cell dedifferentiation and death. 14-3-3 binding to ChREBPα results in its cytoplasmic retention and concomitant suppression of transcriptional activity, suggesting that small molecule-mediated stabilization of this protein-protein interaction (PPI) via molecular glues may represent an attractive entry for the treatment of metabolic disease. Here, we show that structure-based optimizations of a molecular glue tool compound led not only to more potent ChREBPα/14-3-3 PPI stabilizers but also for the first time cellular active compounds. In primary human ß-cells, the most active compound stabilized the ChREBPα/14-3-3 interaction and thus induced cytoplasmic retention of ChREBPα, resulting in highly efficient ß-cell protection from glucolipotoxicity while maintaining ß-cell identity. This study may thus not only provide the basis for the development of a unique class of compounds for the treatment of Type 2 Diabetes but also showcases an alternative 'molecular glue' approach for achieving small molecule control of notoriously difficult targetable TFs.

NRF2 is required for neonatal mouse beta cell growth by maintaining redox balance and promoting mitochondrial biogenesis and function.

AIMS/HYPOTHESIS: All forms of diabetes result from insufficient functional beta cell mass. Due to the relatively limited expression of several antioxidant enzymes, beta cells are highly vulnerable to pathological levels of reactive oxygen species (ROS), which can lead to the reduction of functional beta cell mass. During early postnatal ages, both human and rodent beta cells go through a burst of proliferation that quickly declines with age. The exact mechanisms that account for neonatal beta cell proliferation are understudied but mitochondrial release of moderated ROS levels has been suggested as one of the main drivers. We previously showed that, apart from its conventional role in protecting beta cells from oxidative stress, the nuclear factor erythroid 2-related factor 2 (NRF2) is also essential for beta cell proliferation. We therefore hypothesised that NRF2, which is activated by ROS, plays an essential role in beta cell proliferation at early postnatal ages. METHODS: Beta cell NRF2 levels and beta cell proliferation were measured in pancreatic sections from non-diabetic human cadaveric donors at different postnatal ages, childhood and adulthood. Pancreatic sections from 1-, 7-, 14- and 28-day-old beta cell-specific Nrf2 (also known as Nfe2l2)-knockout mice (ßNrf2KO) or control (Nrf2lox/lox) mice were assessed for beta cell NRF2 levels, beta cell proliferation, beta cell oxidative stress, beta cell death, nuclear beta cell pancreatic duodenal homeobox protein 1 (PDX1) levels and beta cell mass. Seven-day-old ßNrf2KO and Nrf2lox/lox mice were injected daily with N-acetylcysteine (NAC) or saline (154 mmol/l NaCl) to explore the potential contribution of oxidative stress to the phenotypes seen in ßNrf2KO mice at early postnatal ages. RNA-seq was performed on 7-day-old ßNrf2KO and Nrf2lox/lox mice to investigate the mechanisms by which NRF2 stimulates beta cell proliferation at early postnatal ages. Mitochondrial biogenesis and function were determined using dispersed islets from 7-day-old ßNrf2KO and Nrf2lox/lox mice by measuring MitoTracker intensity, mtDNA/gDNA ratio and ATP/ADP ratio. To study the effect of neonatal beta cell-specific Nrf2 deletion on glucose homeostasis in adulthood, blood glucose, plasma insulin and insulin secretion were determined and a GTT was performed on 3-month-old ßNrf2KO and Nrf2lox/lox mice fed on regular diet (RD) or high-fat diet (HFD). RESULTS: The expression of the master antioxidant regulator NRF2 was increased at early postnatal ages in both human (1 day to 19 months old, 31%) and mouse (7 days old, 57%) beta cells, and gradually declined with age (8% in adult humans, 3.77% in adult mice). A significant correlation (R2=0.568; p=0.001) was found between beta cell proliferation and NRF2 levels in human beta cells. Seven-day-old ßNrf2KO mice showed reduced beta cell proliferation (by 65%), beta cell nuclear PDX1 levels (by 23%) and beta cell mass (by 67%), and increased beta cell oxidative stress (threefold) and beta cell death compared with Nrf2lox/lox control mice. NAC injections increased beta cell proliferation in 7-day-old ßNrf2KO mice (3.4-fold) compared with saline-injected ßNrf2KO mice. Interestingly, RNA-seq of islets isolated from 7-day-old ßNrf2KO mice revealed reduced expression of mitochondrial RNA genes and genes involved in the electron transport chain. Islets isolated from 7-day old ßNrf2KO mice presented reduced MitoTracker intensity (by 47%), mtDNA/gDNA ratio (by 75%) and ATP/ADP ratio (by 68%) compared with islets from Nrf2lox/lox littermates. Lastly, HFD-fed 3-month-old ßNrf2KO male mice displayed a significant reduction in beta cell mass (by 35%), a mild increase in non-fasting blood glucose (1.2-fold), decreased plasma insulin (by 14%), and reduced glucose tolerance (1.3-fold) compared with HFD-fed Nrf2lox/lox mice. CONCLUSIONS/INTERPRETATION: Our study highlights NRF2 as an essential transcription factor for maintaining neonatal redox balance, mitochondrial biogenesis and function and beta cell growth, and for preserving functional beta cell mass in adulthood under metabolic stress. DATA AVAILABILITY: Sequencing data are available in the NCBI Gene Expression Omnibus, accession number GSE242718 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242718 ).

Transcriptional activation of the Myc gene by glucose in ß-cells requires a ChREBP-dependent 3-D chromatin interaction between the Myc and Pvt1 genes.

OBJECTIVE: All forms of diabetes result from insufficient functional ß-cell mass. Thus, achieving the therapeutic goal of expanding ß-cell mass requires a better mechanistic understanding of how ß-cells proliferate. Glucose is a natural ß-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. METHODS: Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. RESULTS: We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated ß-cell proliferation. CONCLUSIONS: The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.

Does Iodine Intake Modify the Effect of Maternal Dysglycemia on Birth Weight in Mild-to-Moderate Iodine-Deficient Populations? A Mother-Newborn Prospective Cohort Study.

It is unclear how maternal glycemic status and maternal iodine status influence birth weight among individuals with mild-to-moderate iodine deficiency (ID). We studied the association between birth weight and both maternal glucose levels and iodine intake among pregnant women with mild-to-moderate ID. Glucose values were assessed using a glucose challenge test (GCT) and non-fasting glucose levels that were determined before delivery; individuals' iodine statuses were assessed using an iodine food frequency questionnaire; and serum thyroglobulin (Tg) and urinary iodine concentrations (UIC) were used to assess each group's iodine status. Thyroid antibodies and free thyroxine (FT4) levels were measured. Obstetric and anthropometric data were also collected. Large-for-gestational age (LGA) status was predicted using a Cox proportional hazards model with multiple confounders. Tg > 13 g/L was independently associated with LGA (adjusted hazard ratio = 3.4, 95% CI: 1.4-10.2, p = 0.001). Estimated iodine intake correlated with FT4 among participants who reported consuming iodine-containing supplements (ICS) after adjusting for confounders (ß = 0.4, 95% CI: 0.0002-0.0008, p = 0.001). Newborn weight percentiles were inversely correlated with maternal FT4 values (ß = -0.2 95% CI:-0.08--56.49, p = 0.049). We conclude that in mild-to-moderate ID regions, insufficient maternal iodine status may increase LGA risk. Iodine status and ICS intake may modify the effect that maternal dysglycemia has on offspring weight.

T3 and glucose increase expression of phosphoenolpyruvate carboxykinase (PCK1) leading to increased ß-cell proliferation.

OBJECTIVES: Thyroid hormone (T3) and high glucose concentrations are critical components of ß-cell maturation and function. In the present study, we asked whether T3 and glucose signaling pathways coordinately regulate transcription of genes important for ß-cell function and proliferation. METHODS: RNA-seq analysis was performed on cadaveric human islets from five different donors in response to low and high glucose concentrations and in the presence or absence of T3. Gene expression was also studies in sorted human ß-cells, mouse islets and Ins-1 cells by RT-qPCR. Silencing of the thyroid hormone receptors (THR) was conducted using lentiviruses. Proliferation was assessed by ki67 immunostaining in primary human/mouse islets. Chromatin immunoprecipitation and proximity ligation assay were preformed to validate interactions of ChREBP and THR. RESULTS: We found glucose-mediated expression of carbohydrate response element binding protein alpha and beta (ChREBPα and ChREBPß) mRNAs and their target genes are highly dependent on T3 concentrations in rodent and human ß-cells. In ß-cells, T3 and glucose coordinately regulate the expression of ChREBPß and PCK1 (phosphoenolpyruvate carboxykinase-1) among other important genes for ß-cell maturation. Additionally, we show the thyroid hormone receptor (THR) and ChREBP interact, and their relative response elements are located near to each other on mutually responsive genes. In FACS-sorted adult human ß-cells, we found that high concentrations of glucose and T3 induced the expression of PCK1. Next, we show that overexpression of Pck1 together with dimethyl malate (DMM), a substrate precursor, significantly increased ß-cell proliferation in human islets. Finally, using a Cre-Lox approach, we demonstrated that ChREBPß contributes to Pck1-dependent ß-cell proliferation in mouse ß-cells. CONCLUSIONS: We conclude that T3 and glucose act together to regulate ChREBPß, leading to increased expression and activity of Pck1, and ultimately increased ß-cell proliferation.

Maladaptive positive feedback production of ChREBPß underlies glucotoxic ß-cell failure.

Preservation and expansion of ß-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPß) is a nuclear effector of hyperglycemic stress occurring in ß-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPß is necessary for adaptive ß-cell expansion in response to metabolic challenges. Conversely, chronic excessive ß-cell-specific overexpression of ChREBPß results in loss of ß-cell identity, apoptosis, loss of ß-cell mass, and diabetes. Furthermore, ß-cell "glucolipotoxicity" can be prevented by deletion of ChREBPß. Moreover, ChREBPß-mediated cell death is mitigated by overexpression of the alternate CHREBP gene product, ChREBPα, or by activation of the antioxidant Nrf2 pathway in rodent and human ß-cells. We conclude that ChREBPß, whether adaptive or maladaptive, is an important determinant of ß-cell fate and a potential target for the preservation of ß-cell mass in diabetes.

Nrf2 Regulates ß-Cell Mass by Suppressing ß-Cell Death and Promoting ß-Cell Proliferation.

Finding therapies that can protect and expand functional ß-cell mass is a major goal of diabetes research. Here, we generated ß-cell-specific conditional knockout and gain-of-function mouse models and used human islet transplant experiments to examine how manipulating Nrf2 levels affects ß-cell survival, proliferation, and mass. Depletion of Nrf2 in ß-cells results in decreased glucose-stimulated ß-cell proliferation ex vivo and decreased adaptive ß-cell proliferation and ß-cell mass expansion after a high-fat diet in vivo. Nrf2 protects ß-cells from apoptosis after a high-fat diet. Nrf2 loss of function decreases Pdx1 abundance and insulin content. Activating Nrf2 in a ß-cell-specific manner increases ß-cell proliferation and mass and improves glucose tolerance. Human islets transplanted under the kidney capsule of immunocompromised mice and treated systemically with bardoxolone methyl, an Nrf2 activator, display increased ß-cell proliferation. Thus, by managing reactive oxygen species levels, Nrf2 regulates ß-cell mass and is an exciting therapeutic target for expanding and protecting ß-cell mass in diabetes.

Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances ß-cell proliferation and identity and relieves oxidative damage.

OBJECTIVE: Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes ß-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance ß-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). METHODS: Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and ß-cell proliferation and ß-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. RESULTS: EP3 blockade increased ß-cell mass in db/db mice through enhanced ß-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. CONCLUSIONS: The current study suggests that EP3 blockade promotes ß-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage.

Adaptive and maladaptive roles for ChREBP in the liver and pancreatic islets.

Excessive sugar consumption is a contributor to the worldwide epidemic of cardiometabolic disease. Understanding mechanisms by which sugar is sensed and regulates metabolic processes may provide new opportunities to prevent and treat these epidemics. Carbohydrate Responsive-Element Binding Protein (ChREBP) is a sugar-sensing transcription factor that mediates genomic responses to changes in carbohydrate abundance in key metabolic tissues. Carbohydrate metabolites activate the canonical form of ChREBP, ChREBP-alpha, which stimulates production of a potent, constitutively active ChREBP isoform called ChREBP-beta. Carbohydrate metabolites and other metabolic signals may also regulate ChREBP activity via posttranslational modifications including phosphorylation, acetylation, and O-GlcNAcylation that can affect ChREBP's cellular localization, stability, binding to cofactors, and transcriptional activity. In this review, we discuss mechanisms regulating ChREBP activity and highlight phenotypes and controversies in ChREBP gain- and loss-of-function genetic rodent models focused on the liver and pancreatic islets.

Nrf2: The Master and Captain of Beta Cell Fate.

Prolonged hyperglycemia is toxic to pancreatic ß cells, generating excessive reactive oxygen species, defective glucose-stimulated insulin secretion, decreased insulin production, and eventually ß cell death and diabetes. Nrf2 is a master regulator of cellular responses to counteract dangerous levels of oxidative stress. Maintenance of ß cell mass depends on Nrf2 to promote the survival, function, and proliferation of ß cells. Indeed, Nrf2 activation decreases inflammation, increases insulin sensitivity, reduces body weight, and preserves ß cell mass. Therefore, numerous pharmacological activators of Nrf2 are being tested in clinical trials for the treatment of diabetes and diabetic complications. Modulating Nrf2 activity in ß cells is a promising therapeutic approach for the treatment of diabetes.

The many lives of Myc in the pancreatic ß-cell.

Diabetes results from insufficient numbers of functional pancreatic ß-cells. Thus, increasing the number of available functional ß-cells ex vivo for transplantation, or regenerating them in situ in diabetic patients, is a major focus of diabetes research. The transcription factor, Myc, discovered decades ago lies at the nexus of most, if not all, known proliferative pathways. Based on this, many studies in the 1990s and early 2000s explored the potential of harnessing Myc expression to expand ß-cells for diabetes treatment. Nearly all these studies in ß-cells used pathophysiological or supraphysiological levels of Myc and reported enhanced ß-cell death, dedifferentiation, or the formation of insulinomas if cooverexpressed with Bcl-xL, an inhibitor of apoptosis. This obviously reduced the enthusiasm for Myc as a therapeutic target for ß-cell regeneration. However, recent studies indicate that "gentle" induction of Myc expression enhances ß-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for ß-cell regeneration. Furthermore, although it has been known for decades that Myc is induced by glucose in ß-cells, very little is known about how this essential anabolic transcription factor perceives and responds to nutrients and increased insulin demand in vivo. Here we summarize the previous and recent knowledge of Myc in the ß-cell, its potential for ß-cell regeneration, and its physiological importance for neonatal and adaptive ß-cell expansion.

HB-EGF Signaling Is Required for Glucose-Induced Pancreatic ß-Cell Proliferation in Rats.

The molecular mechanisms of ß-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced ß-cell proliferation in rats is dependent on epidermal growth factor receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the ß-cell proliferative response to glucose, a ß-cell mitogen and key regulator of ß-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates ß-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks ß-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in ß-cells in response to glucose in a carbohydrate-response element-binding protein (ChREBP)-dependent manner. In addition, chromatin immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced ß-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in ß-cell compensation to increased metabolic demand.

Myc Is Required for Adaptive ß-Cell Replication in Young Mice but Is Not Sufficient in One-Year-Old Mice Fed With a High-Fat Diet.

Failure to expand pancreatic ß-cells in response to metabolic stress leads to excessive workload resulting in ß-cell dysfunction, dedifferentiation, death, and development of type 2 diabetes. In this study, we demonstrate that induction of Myc is required for increased pancreatic ß-cell replication and expansion during metabolic stress-induced insulin resistance with short-term high-fat diet (HFD) in young mice. ß-Cell-specific Myc knockout mice fail to expand adaptively and show impaired glucose tolerance and ß-cell dysfunction. Mechanistically, PKCζ, ERK1/2, mTOR, and PP2A are key regulators of the Myc response in this setting. DNA methylation analysis shows hypomethylation of cell cycle genes that are Myc targets in islets from young mice fed with a short-term HFD. Importantly, DNA hypomethylation of Myc response elements does not occur in islets from 1-year-old mice fed with a short-term HFD, impairing both Myc recruitment to cell cycle regulatory genes and ß-cell replication. We conclude that Myc is required for metabolic stress-mediated ß-cell expansion in young mice, but with aging, Myc upregulation is not sufficient to induce ß-cell replication by, at least partially, an epigenetically mediated resistance to Myc action.

Activation of Nrf2 Is Required for Normal and ChREBPα-Augmented Glucose-Stimulated ß-Cell Proliferation.

Patients with both major forms of diabetes would benefit from therapies that increase ß-cell mass. Glucose, a natural mitogen, drives adaptive expansion of ß-cell mass by promoting ß-cell proliferation. We previously demonstrated that a carbohydrate response element-binding protein (ChREBPα) is required for glucose-stimulated ß-cell proliferation and that overexpression of ChREBPα amplifies the proliferative effect of glucose. Here we found that ChREBPα reprogrammed anabolic metabolism to promote proliferation. ChREBPα increased mitochondrial biogenesis, oxygen consumption rates, and ATP production. Proliferation augmentation by ChREBPα required the presence of ChREBPß. ChREBPα increased the expression and activity of Nrf2, initiating antioxidant and mitochondrial biogenic programs. The induction of Nrf2 was required for ChREBPα-mediated mitochondrial biogenesis and for glucose-stimulated and ChREBPα-augmented ß-cell proliferation. Overexpression of Nrf2 was sufficient to drive human ß-cell proliferation in vitro; this confirms the importance of this pathway. Our results reveal a novel pathway necessary for ß-cell proliferation that may be exploited for therapeutic ß-cell regeneration.

T3 and Glucose Coordinately Stimulate ChREBP-Mediated Ucp1 Expression in Brown Adipocytes From Male Mice.

Increasing brown adipose tissue (BAT) activity is regarded as a potential treatment of obese, hyperglycemic patients with metabolic syndrome. Triiodothyronine (T3) is known to stimulate BAT activity by increasing mitochondrial uncoupling protein 1 (Ucp1) gene transcription, leading to increased thermogenesis and decreased body weight. Here we report our studies on the effects of T3 and glucose in two mouse models and in mouse immortalized brown preadipocytes in culture. We identified carbohydrate response element binding protein (ChREBP) as a T3 target gene in BAT by RNA sequencing and studied its effects in brown adipocytes. We found that ChREBP was upregulated by T3 in BAT in both hyperglycemic mouse models. In brown preadipocytes, T3 and glucose synergistically and dose dependently upregulated Ucp1 messenger RNA 1000-fold compared with low glucose concentrations. Additionally, we observed increased ChREBP and Ucp1 protein 11.7- and 19.9-fold, respectively, along with concomitant induction of a hypermetabolic state. Moreover, downregulation of ChREBP inhibited T3 and glucose upregulation of Ucp1 100-fold, whereas overexpression of ChREBP upregulated Ucp1 5.2-fold. We conclude that T3 and glucose signaling pathways coordinately regulate the metabolic state of BAT and suggest that ChREBP is a target for therapeutic regulation of BAT activity.

MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect.

Rapid cellular proliferation in early development and cancer depends on glucose metabolism to fuel macromolecule biosynthesis. Metabolic enzymes are presumed regulators of this glycolysis-driven metabolic program, known as the Warburg effect; however, few have been identified. We uncover a previously unappreciated role for Mannose phosphate isomerase (MPI) as a metabolic enzyme required to maintain Warburg metabolism in zebrafish embryos and in both primary and malignant mammalian cells. The functional consequences of MPI loss are striking: glycolysis is blocked and cells die. These phenotypes are caused by induction of p53 and accumulation of the glycolytic intermediate fructose 6-phosphate, leading to engagement of the hexosamine biosynthetic pathway (HBP), increased O-GlcNAcylation, and p53 stabilization. Inhibiting the HBP through genetic and chemical methods reverses p53 stabilization and rescues the Mpi-deficient phenotype. This work provides mechanistic evidence by which MPI loss induces p53, and identifies MPI as a novel regulator of p53 and Warburg metabolism.

DNA methylation alters transcriptional rates of differentially expressed genes and contributes to pathophysiology in mice fed a high fat diet.

OBJECTIVE: Overnutrition can alter gene expression patterns through epigenetic mechanisms that may persist through generations. However, it is less clear if overnutrition, for example a high fat diet, modifies epigenetic control of gene expression in adults, or by what molecular mechanisms, or if such mechanisms contribute to the pathology of the metabolic syndrome. Here we test the hypothesis that a high fat diet alters hepatic DNA methylation, transcription and gene expression patterns, and explore the contribution of such changes to the pathophysiology of obesity. METHODS: RNA-seq and targeted high-throughput bisulfite DNA sequencing were used to undertake a systematic analysis of the hepatic response to a high fat diet. RT-PCR, chromatin immunoprecipitation and in vivo knockdown of an identified driver gene, Phlda1, were used to validate the results. RESULTS: A high fat diet resulted in the hypermethylation and decreased transcription and expression of Phlda1 and several other genes. A subnetwork of genes associated with Phlda1 was identified from an existing Bayesian gene network that contained numerous hepatic regulatory genes involved in lipid and body weight homeostasis. Hepatic-specific depletion of Phlda1 in mice decreased expression of the genes in the subnetwork, and led to increased oil droplet size in standard chow-fed mice, an early indicator of steatosis, validating the contribution of this gene to the phenotype. CONCLUSIONS: We conclude that a high fat diet alters the epigenetics and transcriptional activity of key hepatic genes controlling lipid homeostasis, contributing to the pathophysiology of obesity.

Stress-induced activation of brown adipose tissue prevents obesity in conditions of low adaptive thermogenesis.

BACKGROUND: Stress-associated conditions such as psychoemotional reactivity and depression have been paradoxically linked to either weight gain or weight loss. This bi-directional effect of stress is not understood at the functional level. Here we tested the hypothesis that pre-stress level of adaptive thermogenesis and brown adipose tissue (BAT) functions explain the vulnerability or resilience to stress-induced obesity. METHODS: We used wt and triple ß1,ß2,ß3-Adrenergic Receptors knockout (ß-less) mice exposed to a model of chronic subordination stress (CSS) at either room temperature (22 °C) or murine thermoneutrality (30 °C). A combined behavioral, physiological, molecular, and immunohistochemical analysis was conducted to determine stress-induced modulation of energy balance and BAT structure and function. Immortalized brown adipocytes were used for in vitro assays. RESULTS: Departing from our initial observation that ßARs are dispensable for cold-induced BAT browning, we demonstrated that under physiological conditions promoting low adaptive thermogenesis and BAT activity (e.g. thermoneutrality or genetic deletion of the ßARs), exposure to CSS acted as a stimulus for BAT activation and thermogenesis, resulting in resistance to diet-induced obesity despite the presence of hyperphagia. Conversely, in wt mice acclimatized to room temperature, and therefore characterized by sustained BAT function, exposure to CSS increased vulnerability to obesity. Exposure to CSS enhanced the sympathetic innervation of BAT in wt acclimatized to thermoneutrality and in ß-less mice. Despite increased sympathetic innervation suggesting adrenergic-mediated browning, norepinephrine did not promote browning in ßARs knockout brown adipocytes, which led us to identify an alternative sympathetic/brown adipocytes purinergic pathway in the BAT. This pathway is downregulated under conditions of low adaptive thermogenesis requirements, is induced by stress, and elicits activation of UCP1 in wt and ß-less brown adipocytes. Importantly, this purinergic pathway is conserved in human BAT. CONCLUSION: Our findings demonstrate that thermogenesis and BAT function are determinant of the resilience or vulnerability to stress-induced obesity. Our data support a model in which adrenergic and purinergic pathways exert complementary/synergistic functions in BAT, thus suggesting an alternative to ßARs agonists for the activation of human BAT.

Induction of the ChREBPß Isoform Is Essential for Glucose-Stimulated ß-Cell Proliferation.

Carbohydrate-responsive element-binding protein (ChREBP) is a glucose-sensing transcription factor required for glucose-stimulated proliferation of pancreatic ß-cells in rodents and humans. The full-length isoform (ChREBPα) has a low glucose inhibitory domain (LID) that restrains the transactivation domain when glucose catabolism is minimal. A novel isoform of ChREBP (ChREBPß) was recently described that lacks the LID domain and is therefore constitutively and more potently active. ChREBPß has not been described in ß-cells nor has its role in glucose-stimulated proliferation been determined. We found that ChREBPß is highly expressed in response to glucose, particularly with prolonged culture in hyperglycemic conditions. In addition, small interfering RNAs that knocked down ChREBPß transcripts without affecting ChREBPα expression or activity decreased glucose-stimulated expression of carbohydrate response element-containing genes and glucose-stimulated proliferation in INS-1 cells and in isolated rat islets. Quantitative chromatin immunoprecipitation, electrophoretic mobility shift assays, and luciferase reporter assays were used to demonstrate that ChREBP binds to a newly identified powerful carbohydrate response element in ß-cells and hepatocytes, distinct from that in differentiated 3T3-L1 adipocytes. We conclude that ChREBPß contributes to glucose-stimulated gene expression and proliferation in ß-cells, with recruitment of ChREBPα to tissue-specific elements of the ChREBPß isoform promoter.