Integrative GWAS and co-localisation analysis suggests novel genes associated with age-related multimorbidity.

Advancing age is the greatest risk factor for developing multiple age-related diseases. Therapeutic approaches targeting the underlying pathways of ageing, rather than individual diseases, may be an effective way to treat and prevent age-related morbidity while reducing the burden of polypharmacy. We harness the Open Targets Genetics Portal to perform a systematic analysis of nearly 1,400 genome-wide association studies (GWAS) mapped to 34 age-related diseases and traits, identifying genetic signals that are shared between two or more of these traits. Using locus-to-gene (L2G) mapping, we identify 995 targets with shared genetic links to age-related diseases and traits, which are enriched in mechanisms of ageing and include known ageing and longevity-related genes. Of these 995 genes, 128 are the target of an approved or investigational drug, 526 have experimental evidence of binding pockets or are predicted to be tractable, and 341 have no existing tractability evidence, representing underexplored genes which may reveal novel biological insights and therapeutic opportunities. We present these candidate targets for exploration and prioritisation in a web application.

Regulation of hepatic glucose production and AMPK by AICAR but not by metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1).

AIM: Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate that, in addition to activating protein kinase A, 8CPT-modified cAMP analogues suppress the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter ENT1. We aimed to exploit 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR, which is highly selective for a high-affinity binding-site on ENT1, to investigate the role of ENT1 in the liver-specific glucose-lowering properties of AICAR and metformin. METHODS: Primary mouse hepatocytes were incubated with AICAR and metformin in combination with cAMP analogues, glucagon, forskolin and NBMPR. Hepatocyte glucose production (HGP) and AMPK signalling were measured, and a uridine uptake assay with supporting LC-MS was used to investigate nucleoside depletion from medium by cells. RESULTS: AICAR and metformin increased AMPK pathway phosphorylation and decreased HGP induced by dibutyryl cAMP and glucagon. HGP was also induced by 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR; however, in each case this was resistant to suppression by AICAR but not by metformin. Cross-validation of tracer and mass spectrometry studies indicates that 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR inhibited the effects of AICAR, at least in part, by impeding its uptake into hepatocytes. CONCLUSIONS: We report for the first time that suppression of ENT1 induces HGP. ENT1 inhibition also impedes uptake and the effects of AICAR, but not metformin, on HGP. Further investigation of nucleoside transport may illuminate a better understanding of how metformin and AICAR each regulate HGP.

Metformin selectively targets redox control of complex I energy transduction.

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled.

Rab-GTPase binding effector protein 2 (RABEP2) is a primed substrate for Glycogen Synthase kinase-3 (GSK3).

Glycogen synthase kinase-3 (GSK3) regulates many physiological processes through phosphorylation of a diverse array of substrates. Inhibitors of GSK3 have been generated as potential therapies in several diseases, however the vital role GSK3 plays in cell biology makes the clinical use of GSK3 inhibitors potentially problematic. A clearer understanding of true physiological and pathophysiological substrates of GSK3 should provide opportunities for more selective, disease specific, manipulation of GSK3. To identify kinetically favourable substrates we performed a GSK3 substrate screen in heart tissue. Rab-GTPase binding effector protein 2 (RABEP2) was identified as a novel GSK3 substrate and GSK3 phosphorylation of RABEP2 at Ser200 was enhanced by prior phosphorylation at Ser204, fitting the known consensus sequence for GSK3 substrates. Both residues are phosphorylated in cells while only Ser200 phosphorylation is reduced following inhibition of GSK3. RABEP2 function was originally identified as a Rab5 binding protein. We did not observe co-localisation of RABEP2 and Rab5 in cells, while ectopic expression of RABEP2 had no effect on endosomal recycling. The work presented identifies RABEP2 as a novel primed substrate of GSK3, and thus a potential biomarker for GSK3 activity, but understanding how phosphorylation regulates RABEP2 function requires more information on physiological roles of RABEP2.

Variation in the glucose transporter gene SLC2A2 is associated with glycemic response to metformin.

Metformin is the first-line antidiabetic drug with over 100 million users worldwide, yet its mechanism of action remains unclear. Here the Metformin Genetics (MetGen) Consortium reports a three-stage genome-wide association study (GWAS), consisting of 13,123 participants of different ancestries. The C allele of rs8192675 in the intron of SLC2A2, which encodes the facilitated glucose transporter GLUT2, was associated with a 0.17% (P = 6.6 × 10(-14)) greater metformin-induced reduction in hemoglobin A1c (HbA1c) in 10,577 participants of European ancestry. rs8192675 was the top cis expression quantitative trait locus (cis-eQTL) for SLC2A2 in 1,226 human liver samples, suggesting a key role for hepatic GLUT2 in regulation of metformin action. Among obese individuals, C-allele homozygotes at rs8192675 had a 0.33% (3.6 mmol/mol) greater absolute HbA1c reduction than T-allele homozygotes. This was about half the effect seen with the addition of a DPP-4 inhibitor, and equated to a dose difference of 550 mg of metformin, suggesting rs8192675 as a potential biomarker for stratified medicine.

Investigation of salicylate hepatic responses in comparison with chemical analogues of the drug.

Anti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK. Salicylate also reduced mTOR signalling, but this property was observed widely among the analogues. In a sub-panel of analogues, salicylate alone reduced promoter activity of the key gluconeogenic enzyme glucose 6-phosphatase and suppressed basal glucose production in mouse primary hepatocytes. Both salicylate and 2,6 dihydroxybenzoic acid suppressed TNFα-induced IκB degradation, and in genetic knockout experiments, we found that the effect of salicylate on IκB degradation was AMPK-independent. Previous data also identified AMPK-independent regulation of glucose but we found that direct inhibition of neither NF-κB nor mTOR signalling suppressed glucose production, suggesting that other factors besides these cell signalling pathways may need to be considered to account for this response to salicylate. We found, for example, that H4IIE cells were exquisitely sensitive to uncoupling with modest doses of salicylate, which occurred on a similar time course to another anti-hyperglycaemic uncoupling agent 2,4-dinitrophenol, while there was no discernible effect at all of two salicylate analogues which are not anti-hyperglycaemic. This finding supports much earlier literature suggesting that salicylates exert anti-hyperglycaemic effects at least in part through uncoupling.

Cellular responses to the metal-binding properties of metformin.

In recent decades, the antihyperglycemic biguanide metformin has been used extensively in the treatment of type 2 diabetes, despite continuing uncertainty over its direct target. In this article, using two independent approaches, we demonstrate that cellular actions of metformin are disrupted by interference with its metal-binding properties, which have been known for over a century but little studied by biologists. We demonstrate that copper sequestration opposes known actions of metformin not only on AMP-activated protein kinase (AMPK)-dependent signaling, but also on S6 protein phosphorylation. Biguanide/metal interactions are stabilized by extensive π-electron delocalization and by investigating analogs of metformin; we provide evidence that this intrinsic property enables biguanides to regulate AMPK, glucose production, gluconeogenic gene expression, mitochondrial respiration, and mitochondrial copper binding. In contrast, regulation of S6 phosphorylation is prevented only by direct modification of the metal-liganding groups of the biguanide structure, supporting recent data that AMPK and S6 phosphorylation are regulated independently by biguanides. Additional studies with pioglitazone suggest that mitochondrial copper is targeted by both of these clinically important drugs. Together, these results suggest that cellular effects of biguanides depend on their metal-binding properties. This link may illuminate a better understanding of the molecular mechanisms enabling antihyperglycemic drug action.

The anti-neurodegenerative agent clioquinol regulates the transcription factor FOXO1a.

Many diseases of aging including AD (Alzheimer's disease) and T2D (Type 2 diabetes) are strongly associated with common risk factors, suggesting that there may be shared aging mechanisms underlying these diseases, with the scope to identify common cellular targets for therapy. In the present study we have examined the insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as CQ (clioquinol). The IIS [insulin/IGF-1 (insulin-like growth factor-1) signalling] kinase Akt/PKB (protein kinase B) inhibits the transcription factor FOXO1a (forkhead box O1a) by phosphorylating it on residues that trigger its exit from the nucleus. In HEK (human embryonic kidney)-293 cells, we found that CQ treatment induces similar responses. A key transcriptional response to IIS is the inhibition of hepatic gluconeogenic gene expression, and, in rat liver cells, CQ represses expression of the key gluconeogenic regulatory enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). The effects on FOXO1a and gluconeogenic gene expression require the presence of Zn2+ ions, reminiscent of much earlier studies examining diabetogenic properties of 8-hydroxyquinolines. Comparative investigation of the signalling properties of a panel of these compounds demonstrates that CQ alone exhibits FOXO1a regulation without diabetogenicity. Our results suggest that Zn2+-dependent regulation of FOXOs and gluconeogenesis may contribute to the therapeutic properties of this drug. Further investigation of this signalling response might illuminate novel pharmacological strategies for the treatment of age-related diseases.

Generation, validation and humanisation of a novel insulin resistant cell model.

Insulin resistance is a characteristic of type 2 diabetes and is a major independent risk factor for progression to the disease. In particular, insulin resistance associates with increased body fat and almost certainly contributes to the dramatic increase in risk of type 2 diabetes associated with obesity. Therefore, in order to design truly effective insulin sensitising agents, targeted at the mechanism of disease development, we aimed to generate an obesity-related insulin resistant cell model. Rat hepatoma cells were grown in the presence of serum isolated from obese rodents or obese human volunteers, and the insulin sensitivity of the cells monitored over time by measuring a well-characterised insulin regulated gene promoter. Higher insulin concentrations were required to fully repress the gene in the cells grown in obese rodent serum compared with those grown in serum from lean rodents (almost a 10-fold shift in insulin sensitivity). This was reversed by restoration of normal growth medium, while the insulin resistance was prevented by pioglitazone or metformin. Meanwhile, growth of cells in serum collected from obese human volunteers with diabetes also reduced the insulin sensitivity of the rat cells. No clinical marker predicted the degree of insulin resistance that was generated by the human serum. We have developed a novel insulin resistant cell model for the study of the molecular development of obesity-linked insulin resistance, screen for compounds to overcome obesity-related insulin resistance and potentially search for novel serum biomarkers of insulin resistance.

Insulin resistance in polycystic ovary syndrome is associated with defective regulation of ERK1/2 by insulin in skeletal muscle in vivo.

Insulin resistance is a recognized feature of PCOS (polycystic ovary syndrome). However, the molecular reason(s) underlying this reduced cellular insulin sensitivity is not clear. The present study compares the major insulin signalling pathways in skeletal muscle isolated from PCOS and controls. We measured whole-body insulin sensitivity and insulin signalling in skeletal muscle biopsies taken before and after acute exposure to hyperinsulinaemia in nine women diagnosed with PCOS and seven controls. We examined the expression, basal activity and response to in vivo insulin stimulation of three signalling molecules within these human muscle samples, namely IRS-1 (insulin receptor substrate-1), PKB (protein kinase B) and ERK (extracellular-signal-regulated kinase) 1/2. There was no significant difference in the expression, basal activity or activation of IRS-1 or PKB between PCOS and control subjects. However, there was a severe attenuation of insulin stimulation of the ERK pathway in muscle from all but two of the women with PCOS (the two most obese), and an accompanying trend towards higher basal phosphorylation of ERK1/2 in PCOS. These results are striking in that the metabolic actions of insulin are widely believed to require the IRS-1/PKB pathway rather than ERK, and the former has been reported as defective in some previous PCOS studies. Most importantly, the molecular defect identified was independent of adiposity. The altered response of ERK to insulin in PCOS was the most obvious signalling defect associated with insulin resistance in muscle from these patients.

Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance.

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.

Characterization of a protein kinase B inhibitor in vitro and in insulin-treated liver cells.

OBJECTIVE: Abnormal expression of the hepatic gluconeogenic genes (glucose-6-phosphatase [G6Pase] and PEPCK) contributes to hyperglycemia. These genes are repressed by insulin, but this process is defective in diabetic subjects. Protein kinase B (PKB) is implicated in this action of insulin. An inhibitor of PKB, Akt inhibitor (Akti)-1/2, was recently reported; however, the specificity and efficacy against insulin-induced PKB was not reported. Our aim was to characterize the specificity and efficacy of Akti-1/2 in cells exposed to insulin and then establish whether inhibition of PKB is sufficient to prevent regulation of hepatic gene expression by insulin. RESEARCH DESIGN AND METHODS: Akti-1/2 was assayed against 70 kinases in vitro and its ability to block PKB activation in cells exposed to insulin fully characterized. RESULTS: Akti-1/2 exhibits high selectivity toward PKBalpha and PKBbeta. Complete inhibition of PKB activity is achieved in liver cells incubated with 1-10 mumol/l Akti-1/2, and this blocks insulin regulation of PEPCK and G6Pase expression. Our data demonstrate that only 5-10% of maximal insulin-induced PKB is required to fully repress PEPCK and G6Pase expression. Finally, we demonstrate reduced insulin sensitivity of these gene promoters in cells exposed to submaximal concentrations of Akti-1/2; however, full repression of the genes can still be achieved by high concentrations of insulin. CONCLUSIONS: This work establishes the requirement for PKB activity in the insulin regulation of PEPCK, G6Pase, and a third insulin-regulated gene, IGF-binding protein-1 (IGFBP1); suggests a high degree of functional reserve; and identifies Akti-1/2 as a useful tool to delineate PKB function in the liver.

Genetic variants in human sterol regulatory element binding protein-1c in syndromes of severe insulin resistance and type 2 diabetes.

The transcription factor sterol regulatory element binding protein (SREBP)-1c is intimately involved in the regulation of lipid and glucose metabolism. To investigate whether mutations in this gene might contribute to insulin resistance, we screened the exons encoding the aminoterminal transcriptional activation domain in a cohort of 85 unrelated human subjects with severe insulin resistance. Two missense mutations (P87L and P416A) were found in single affected patients but not in 47 control subjects. However, these variants were indistinguishable from the wild-type in their ability to bind DNA or to transactivate an SREBP-1 responsive promoter construct. We also identified a common intronic single nucleotide polymorphism (C/T) located between exon 18c and 19c. In a case-control study of 517 U.K. Caucasian case subjects and 517 age- and sex-matched control subjects, the T-allele at this locus was significantly associated with type 2 diabetes in men (odds ratio = 1.42 [1.11-1.82], P = 0.005) but not women. In a separate population-based study of 1,100 Caucasians, carriers of the T-allele showed significantly higher levels of total and LDL cholesterol (P < 0.05) compared with wild-type individuals. In summary, we have conducted the first study of the SREBP-1c gene as a candidate for human insulin resistance. Although the rare mutations identified were functionally silent in the assays used, we obtained some evidence, which requires conformation in other populations, that a common variant in the SREBP-1c gene might influence diabetes risk and plasma cholesterol level.

Lamin expression in human adipose cells in relation to anatomical site and differentiation state.

Familial partial lipodystrophy-Dunnigan variety (FPLD) is an autosomal dominant form of lipodystrophy resulting in a loss of sc fat from the trunk and limbs with retention of fat in the visceral depots, face, and neck. Specific point mutations in the gene encoding the nuclear lamina proteins, lamins A and C, have been established to cause this syndrome. We undertook studies to determine which members of the lamin family were expressed in human fat cells, to examine the effect of differentiation state on lamin A and C expression in human preadipocytes, and to test the hypothesis that regional variation in lamin A/C expression might underlie the stereotyped anatomical pattern of FPLD. Lamins A, C, and B1, but not B2, were expressed in sc mature human adipocytes. Subcutaneous preadipocytes expressed all four lamins, with lamin A and C expression increasing with ex vivo differentiation. Consistent with previously reported resistance to ex vivo differentiation, omental preadipocytes did not show an increase in lamin A or C mRNA under these conditions. Lamin A/C mRNA levels were similar in isolated mature adipocytes and preadipocytes from omental, sc, and neck sites. However, lamin C was consistently lower, and the ratio of lamin A/C mRNA was higher in sc mature adipocytes compared with omental mature adipocytes. We conclude that the depot-specific pattern of lamin A/C expression does not provide clues to the mechanism of FPLD. Nonetheless, these studies provide new information regarding the expression of lamin isoforms in normal human adipose cells, which will inform future studies of the molecular pathogenesis of FPLD.