Lisa Logie

Mutation of the PDK1 PH Domain Inhibits Protein Kinase B/Akt, Leading to Small Size and Insulin Resistance

ABSTRACT 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 PKBα and PKBβ. Complete inhibition of PKB activity is achieved in liver cells incubated with 1–10 μmol/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.

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.

Mouse hypothalamic GT1-7 cells demonstrate AMPK-dependent intrinsic glucose-sensing behaviour

Aims/hypothesisHypothalamic glucose-excited (GE) neurons contribute to whole-body glucose homeostasis and participate in the detection of hypoglycaemia. This system appears defective in type 1 diabetes, in which hypoglycaemia commonly occurs. Unfortunately, it is at present unclear which molecular components required for glucose sensing are produced in individual neurons and how these are functionally linked. We used the GT1-7 mouse hypothalamic cell line to address these issues.MethodsElectrophysiological recordings, coupled with measurements of gene expression and protein levels and activity, were made from unmodified GT1-7 cells and cells in which AMP-activated protein kinase (AMPK) catalytic subunit gene expression and activity were reduced.ResultsHypothalamic GT1-7 neurons express the genes encoding glucokinase and ATP-sensitive K+ channel (KATP) subunits Kir6.2 and Sur1 and exhibit GE-type glucose-sensing behaviour. Lowered extracellular glucose concentration hyperpolarised the cells in a concentration-dependent manner, an outcome that was reversed by tolbutamide. Inhibition of glucose uptake or metabolism hyperpolarised cells, showing that energy metabolism is required to maintain their resting membrane potential. Short hairpin (sh)RNA directed to Ampkα2 (also known as Prkaa2) reduced GT1-7 cell AMPKα2, but not AMPKα1, activity and lowered the threshold for hypoglycaemia-induced hyperpolarisation. shAmpkα1 (also known as Prkaa1) had no effect on glucose-sensing or AMPKα2 activity. Decreased uncoupling protein 2 (Ucp2) mRNA was detected in AMPKα2-reduced cells, suggesting that AMPKα2 regulates UCP2 levels.Conclusions/interpretationWe have demonstrated that GT1-7 cells closely mimic GE neuron glucose-sensing behaviour, and reducing AMPKα2 blunts their responsiveness to hypoglycaemic challenge, possibly by altering UCP2 activity. These results show that suppression of AMPKα2 activity inhibits normal glucose-sensing behaviour and may contribute to defective detection of hypoglycaemia.

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.

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.

The anti-neurodegenerative agent clioquinol regulates the transcription factor FOXO1a

Many diseases of aging including AD (Alzheimers 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.

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.

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.

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

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.