No 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.The 3-phosphoinositide-dependent protein kinase 1 (PDK1) functions as an upstream activator of a group of AGC family protein kinases that are stimulated by insulin, growth factors, and numerous other agonists (42). These include isoforms of protein kinase B (PKB), also known as Akt (23), p70 ribosomal S6 kinase (S6K) (17), serum and glucocorticoid-induced protein kinase (SGK) (32), and p90 ribosomal S6 kinase (RSK) (27). Activation of PKB and other AGC kinases plays crucial roles in regulating diverse effects of extracellular agonists on cells by phosphorylating regulatory proteins that control metabolism, growth, proliferation, and survival (21). Many if not all of the cellular effects of insulin are mediated through activation of PKB (18, 58). PKB also stimulates the activation of S6K1, which plays an important role in regulating protein synthesis and cell growth (17). Genetic analysis of the PDK1-signaling pathway in Drosophila melanogaster and mice also suggests that this pathway plays an important role in regulating organism size. For example, Drosophila organisms with reduced levels of PDK1 (51), PKB (56), or S6K (40) are all small, possessing cells with reduced volume. Similarly, mice with decreased levels of PDK1 (33) and mice lacking PKB␣ (18) or S6K isoforms (48) also display small-organism and -cell phenotypes.PDK1 activates at least 23 AGC kinases by phosphorylating a specific Thr or Ser residue located within the T-loop of the kinase domain (42). Maximal activation also necessitates phosphorylation of a Ser/Thr residue located C-terminal to the catalytic domain within a region known as the hydrophobic motif. Recent work has established that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 phosphorylate the hydrophobic motif of S6K1 and PKB (52,61). In the case of RSK, a se...

Metformin is the first-line antidiabetic drug with over 100 million users worldwide, yet its mechanism of action remains unclear1. 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 in haemoglobin A1c (HbA1c) in 10,577 participants of European ancestry. rs8192675 is 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 550mg of metformin, suggesting rs8192675 as a potential biomarker for stratified medicine.

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.

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.

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 PKB␣ and PKB␤. Complete inhibition of PKB activity is achieved in liver cells incubated with 1-10 mol/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. Diabetes 56:2218-2227, 2007 P rotein kinase B (PKB) is a member of the AGC family of protein kinases (1-3). In mammals, there are three isoforms (PKB␣, PKB␤, and PKB␥) (1). PKB is activated following induction of phosphatidylinositol 3 (PI3) kinase activity and the resultant generation of the lipid second messengers PI 3,4,5 trisphosphate and PI 3,4 bisphosphate (4). These lipids bind to the PH domain of PKB, altering its conformation and permitting access to upstream protein kinases (5). Phosphoinositide-dependent protein kinase-1 phosphorylates PKB at Thr 308 (6), and a second phosphorylation (at Ser 473 ) occurs through the action of an alternative kinase, such as the rapamycin-insensitive mTOR complex 2 (TORC2) (7). Therefore, most growth factors, including platelet-derived growth factor, epidermal growth factor, and insulin, which are potent activators of PI3 kinase, also strongly induce PKB in cells.One of the first substrates of PKB to be characterized was GSK3, as part of the insulin signaling pathway that regulates glycogen metabolism (8). Since then, multiple potential substrates of PKB have been proposed including the proapoptotic protein Bad (9,10), the tuberous sclerosis complex (TSC)2 gene product (11), the Rab-GAP AS160 (12), proline-rich Akt substrate of 40 kDa (PRAS40) (13), and the key forkhead transcription ...

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. Diabetes 53:842-846, 2004 T ype 2 diabetes is characterized by peripheral insulin resistance, increased hepatic gluconeogenesis, loss of glucose-induced insulin secretion and, in most patients, obesity (1). Recent data suggest that dysregulated fatty acid metabolism might be a key unifying mediator of these disparate phenomena (2-4).Sterol regulatory element binding proteins (SREBPs) are transcription factors crucial in the regulation of fatty acid and cholesterol metabolism. To date, three SREBP isoforms have been identified: SREBP-1a and SREBP-1c, derived from a single gene (SREBF1) through alternative promoter usage, and SREBP-2, encoded by a separate gene (SREBF2). The aminoterminal segment of the SREBPs contains an acidic transactivation domain and a basic helix-loop-helix leucine zipper (bHLH-Zip) region that mediates protein dimerization and DNA binding. SREBPs are embedded in the membrane of the endoplasmic reticulum as 120-kDa precursor proteins. Following fatty acid or cholesterol depletion, 68-kDa aminoterminal fragments (mature SREBP) are cleaved proteolytically from these precursor proteins and migrate into the nucleus (5). There they activate different target genes, encoding key enzymes of fatty acid and cholesterol metabolism. Of the two isoforms, SREBP-1c is the predominant transcript in most organs, including liver and adipose tissue, of adult animals (6). The mouse isoform of SREBP-1c, also known as adipocyte differentiation and d...

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2617-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.