Susan J. Tudhope

Fructose 2,6-bisphosphate is essential for glucose-regulated gene transcription of glucose-6-phosphatase and other ChREBP target genes in hepatocytes.

Glucose metabolism in the liver activates the transcription of various genes encoding enzymes of glycolysis and lipogenesis and also G6pc (glucose-6-phosphatase). Allosteric mechanisms involving glucose 6-phosphate or xylulose 5-phosphate and covalent modification of ChREBP (carbohydrate-response element-binding protein) have been implicated in this mechanism. However, evidence supporting an essential role for a specific metabolite or pathway in hepatocytes remains equivocal. By using diverse substrates and inhibitors and a kinase-deficient bisphosphatase-active variant of the bifunctional enzyme PFK2/FBP2 (6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase), we demonstrate an essential role for fructose 2,6-bisphosphate in the induction of G6pc and other ChREBP target genes by glucose. Selective depletion of fructose 2,6-bisphosphate inhibits glucose-induced recruitment of ChREBP to the G6pc promoter and also induction of G6pc by xylitol and gluconeogenic precursors. The requirement for fructose 2,6-bisphosphate for ChREBP recruitment to the promoter does not exclude the involvement of additional metabolites acting either co-ordinately or at downstream sites. Glucose raises fructose 2,6-bisphosphate levels in hepatocytes by reversing the phosphorylation of PFK2/FBP2 at Ser32, but also independently of Ser32 dephosphorylation. This supports a role for the bifunctional enzyme as the phosphometabolite sensor and for its product, fructose 2,6-bisphosphate, as the metabolic signal for substrate-regulated ChREBP-mediated expression of G6pc and other ChREBP target genes.

Elevated Glucose Represses Liver Glucokinase and Induces Its Regulatory Protein to Safeguard Hepatic Phosphate Homeostasis

OBJECTIVE The induction of hepatic glucose 6-phosphatase (G6pc) by glucose presents a paradox of glucose-induced glucose intolerance. We tested whether glucose regulation of liver gene expression is geared toward intracellular homeostasis. RESEARCH DESIGN AND METHODS The effect of glucose-induced accumulation of phosphorylated intermediates on expression of glucokinase (Gck) and its regulator Gckr was determined in hepatocytes. Cell ATP and uric acid production were measured as indices of cell phosphate homeostasis. RESULTS Accumulation of phosphorylated intermediates in hepatocytes incubated at elevated glucose induced rapid and inverse changes in Gck (repression) and Gckr (induction) mRNA concomitantly with induction of G6pc, but had slower effects on the Gckr-to-Gck protein ratio. Dynamic metabolic labeling in mice and liver proteome analysis confirmed that Gckr and Gck are low-turnover proteins. Involvement of Max-like protein X in glucose-mediated Gck-repression was confirmed by chromatin immunoprecipitation analysis. Elevation of the Gck-to-Gckr ratio in hepatocytes was associated with glucose-dependent ATP depletion and elevated urate production confirming compromised phosphate homeostasis. CONCLUSIONS The lowering by glucose of the Gck-to-Gckr ratio provides a potential explanation for the impaired hepatic glucose uptake in diabetes. Elevated uric acid production at an elevated Gck-to-Gckr ratio supports a role for glucose regulation of gene expression in hepatic phosphate homeostasis.

Regulation of p53 and Rb links the alternative NF-κB pathway to EZH2 expression and cell senescence.

There are two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-κB2(p52)/RelB containing complexes, and is dependent on IKKα and NF-κB inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-κB pathway subunits NF-κB2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-κB2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-κB target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-κB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-κB also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-κB can inhibit tumor suppressor function and promote tumorigenesis.

A Novel Mechanism for Regulating Hepatic Glycogen Synthesis Involving Serotonin and Cyclin-Dependent Kinase-5

Hepatic autonomic nerves regulate postprandial hepatic glucose uptake, but the signaling pathways remain unknown. We tested the hypothesis that serotonin (5-hydroxytryptamine [5-HT]) exerts stimulatory and inhibitory effects on hepatic glucose disposal. Ligands of diverse 5-HT receptors were used to identify signaling pathway(s) regulating glucose metabolism in hepatocytes. 5-HT had stimulatory and inhibitory effects on glycogen synthesis in hepatocytes mediated by 5-HT1/2A and 5-HT2B receptors, respectively. Agonists of 5-HT1/2A receptors lowered blood glucose and increased hepatic glycogen after oral glucose loading and also stimulated glycogen synthesis in freshly isolated hepatocytes with greater efficacy than 5-HT. This effect was blocked by olanzapine, an antagonist of 5-HT1/2A receptors. It was mediated by activation of phosphorylase phosphatase, inactivation of glycogen phosphorylase, and activation of glycogen synthase. Unlike insulin action, it was not associated with stimulation of glycolysis and was counteracted by cyclin-dependent kinase (cdk) inhibitors. A role for cdk5 was supported by adaptive changes in the coactivator protein p35 and by elevated glycogen synthesis during overexpression of p35/cdk5. These results support a novel mechanism for serotonin stimulation of hepatic glycogenesis involving cdk5. The opposing effects of serotonin, mediated by distinct 5-HT receptors, could explain why drugs targeting serotonin function can cause either diabetes or hypoglycemia in humans.

Profound invariant natural killer T-cell deficiency in inflammatory arthritis

Objectives Data from rodent models indicate that invariant natural killer T (iNKT) cells are key regulators of many immune responses including autoimmune arthritis, but their role in human diseases is unclear. The aims of this study are to determine whether iNKT cell frequency and function are altered in patients with rheumatoid arthritis (RA), and the clinical significance of such iNKT cell abnormalities. Methods Peripheral blood iNKT cell frequency and proliferative response to an iNKT cell-specific agonist, α-galactosylceramide were measured in 46 RA patients (including 23 untreated, newly diagnosed patients), 22 healthy controls and 27 patients presenting with recent-onset joint pain. The relationship between iNKT cell frequency and clinical characteristics and the effects of immunosuppressive treatment was examined. Results Compared with healthy controls, RA patients had a decreased frequency of peripheral blood iNKT cells (median 0.001% vs 0.021%, p<0.001) and the proliferative response of this subset to α-galactosylceramide was also diminished in the patient group (median fold-expansion 31 vs 121, p=0.037). These abnormalities preceded the initiation of disease-modifying or immunosuppressive therapy, whose effect was to increase the circulating iNKT cell frequency (p=0.037). Furthermore, iNKT cell frequency correlated inversely with the systemic inflammatory marker, C-reactive protein (p=0.008). Finally, in patients presenting with recent-onset joint symptoms, normal peripheral blood iNKT cell frequency predicted a non-inflammatory cause of joint pain. Conclusion iNKT cell deficiency is present in patients with RA and other inflammatory arthropathy. Normal iNKT cell frequency predicts non-inflammatory causes of joint pain.

Glucose Induces Protein Targeting to Glycogen in Hepatocytes by Fructose 2,6-Bisphosphate-Mediated Recruitment of MondoA to the Promoter

ABSTRACT In the liver, a high glucose concentration activates transcription of genes encoding glucose 6-phosphatase and enzymes for glycolysis and lipogenesis by elevation in phosphorylated intermediates and recruitment of the transcription factor ChREBP (carbohydrate response element binding protein) and its partner, Mlx, to gene promoters. A proposed function for this mechanism is intracellular phosphate homeostasis. In extrahepatic tissues, MondoA, the paralog of ChREBP, partners with Mlx in transcriptional induction by glucose. We tested for glucose induction of regulatory proteins of the glycogenic pathway in hepatocytes and identified the glycogen-targeting proteins, GL and PTG (protein targeting to glycogen), as being encoded by Mlx-dependent glucose-inducible genes. PTG induction by glucose was MondoA dependent but ChREBP independent and was enhanced by forced elevation of fructose 2,6-bisphosphate and by additional xylitol-derived metabolites. It was counteracted by selective depletion of fructose 2,6-bisphosphate with a bisphosphatase-active kinase-deficient variant of phosphofructokinase 2/fructosebisphosphatase 2, which prevented translocation of MondoA to the nucleus and recruitment to the PTG promoter. We identify a novel role for MondoA in the liver and demonstrate that elevated fructose 2,6-bisphosphate is essential for recruitment of MondoA to the PTG promoter. Phosphometabolite activation of MondoA and ChREBP and their recruitment to target genes is consistent with a mechanism for gene regulation to maintain intracellular phosphate homeostasis.

Cyclin-Dependent Kinase (CDK) Inhibitors: Structure-Activity Relationships and Insights into the CDK-2 Selectivity of 6-Substituted 2-Arylaminopurines.

Purines and related heterocycles substituted at C-2 with 4′-sulfamoylanilino and at C-6 with a variety of groups have been synthesized with the aim of achieving selectivity of binding to CDK2 over CDK1. 6-Substituents that favor competitive inhibition at the ATP binding site of CDK2 were identified and typically exhibited 10–80-fold greater inhibition of CDK2 compared to CDK1. Most impressive was 4-((6-([1,1′-biphenyl]-3-yl)-9H-purin-2-yl)amino) benzenesulfonamide (73) that exhibited high potency toward CDK2 (IC50 0.044 μM) but was ∼2000-fold less active toward CDK1 (IC50 86 μM). This compound is therefore a useful tool for studies of cell cycle regulation. Crystal structures of inhibitor–kinase complexes showed that the inhibitor stabilizes a glycine-rich loop conformation that shapes the ATP ribose binding pocket and that is preferred in CDK2 but has not been observed in CDK1. This aspect of the active site may be exploited for the design of inhibitors that distinguish between CDK1 and CDK2.

PARP1 expression, activity and ex vivo sensitivity to the PARP inhibitor, talazoparib (BMN 673), in chronic lymphocytic leukaemia

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors. We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 – 190052 pmol PAR/106 cells) compared to that seen in healthy volunteer lymphocytes (2451 – 7519 pmol PAR/106 cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2′-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function. PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib.

Invariant natural killer T (iNKT) cell deficiency in chronic mucocutaneous candidiasis--a consequence or a cause?

Chronic mucocutaneous candidiasis (CMC) is a group of heterogeneous disorders characterised by primary selective susceptibility to chronic, recurrent Candida infections. The genetic defect of one subgroup of CMC patients have been identified as mutations of the autoimmune regulator (AIRE) gene. Recent data implicated the AIRE gene in iNKT cell development, raising the possibility that iNKT cells may be important in defending against Candida infections. In this study, we enumerated the circulating iNKT frequency in 22 CMC patients (9 with AIRE gene mutations) and 25 healthy controls. We also examined the effect of Candida stimulation on iNKT cells in vitro. Our data demonstrated that peripheral iNKT cell frequency is significantly reduced in CMC patients compared to healthy controls, regardless of their AIRE gene mutation status. Direct stimulation with heat-inactivated whole Candida did not induce iNKT cell proliferation. Furthermore, circulating iNKT cell frequencies in some healthy controls were comparable to CMC patients. These observations suggest that iNKT cell deficiency is part of the CMC disease phenotype irrespective of the presence of AIRE gene mutations but does not appear to confer susceptibility to chronic Candida infections. We postulate that the reduced circulating iNKT cell frequency in CMC is a consequence rather than a cause of chronic Candida infections.

Opposite effects of a glucokinase activator and metformin on glucose-regulated gene expression in hepatocytes.

Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti‐hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials.