Corinna Dickel

Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001

Location of the sulphonylurea receptor at the cytoplasmic face of the β‐cell membrane

1 In insulin‐secreting cells the location of the sulphonylurea receptor was examined by use of a sulphonylurea derivative representing the glibenclamide molecule devoid of its cyclohexyl moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH7.4 compound IV is only present in charged form. 2 Lipid solubility declined in the order tolbutamide > compound III > compound IV. 3 The dissociation constant (KD) for binding of compound IV to the sulphonylurea receptor in HIT‐cells (pancreatic β‐cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III. 4 In mouse pancreatic β‐cells, drug concentrations inhibiting adenosine 5′‐triphosphate‐sensitive K+ channels (KATP‐channels) half‐maximally (EC50) were determined by use of the patch‐clamp technique. When the drugs were applied to the extracellular side of outside‐out or the intracellular side of inside‐out membrane patches, the ratio of extracellular to intracellular EC50 values was 281 for compound IV, 25.5 for compound III and 1.2 for tolbutamide. 5 In mouse pancreatic β‐cells, measurement of KATP‐channel activity in cell‐attached patches and recording of insulin release displayed much higher EC50 values for compound IV than inside‐out patch experiments. A corresponding, but less pronounced difference in EC50 values was observed for compound III, whereas the EC50 values for tolbutamide did not differ significantly. 6 It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the β‐cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.

Diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel in mouse pancreatic β-cells

1 . In mouse pancreatic β‐cells the regulation of the diazoxide‐sensitivity of the adenosine 5′‐triphosphate‐dependent K+ channel (K‐ATP‐channel) was examined by use of the patch‐clamp technique. 2 . In intact β‐cells incubated at 37°C in the presence of 3 mm d‐glucose, diazoxide did not affect the single channel conductance but stimulated channel‐opening activity. Diazoxide produced half‐maximal effects at 82 μm and 13 fold activation at maximally effective concentrations (300–400 μm). The response to diazoxide (300 μm) was not completely suppressed by saturating tolbutamide concentrations (1 or 5 mm). 3 . Inside‐out patch‐clamp experiments were carried out using an experimental protocol favouring phosphorylation of membrane proteins. Under these conditions diazoxide was ineffective in the absence of any nucleotides, weakly effective in the presence of MgATP (26 or 87 μm) and strongly effective in the presence of the Mg complexes of adenosine 5′‐diphosphate, 2′‐deoxyadenosine 5′‐diphosphate or guanosine 5′diphosphate (MgADP, MgdADP or MgGDP). 4 . In inside‐out patches exposed to nucleotide‐free solutions, saturating concentrations of tolbutamide did not cause complete block of K‐ATP‐channels. When the channels were activated by MgdADP (48 μm), tolbutamide was even less effective. Sensitization of MgdADP‐induced channel activation by diazoxide further weakened the effects of tolbutamide. 5 . Diazoxide (50 or 300 μm) prevented the complete channel block induced by saturating tolbutamide concentrations in the presence of Mg2+ and ADP (1 mm). 6. In the presence of Mg2+, the K‐ATP‐channel‐blocking potency of cytosolic ATP decreased in the order inside‐out > outside‐out > whole‐cell configuration of the patch‐clamp technique. 7. It is concluded that the K‐ATP‐channel is controlled via four separate binding sites for inhibitory nucleotides (e.g. free ATP and ADP), stimulatory nucleotides (MgADP, MgdADP, MgGDP), sulphonylureas and diazoxide. Strong inhibition of the channel openings by sulphonylureas results from occupation of both sites for nucleotides. Diazoxide is only effective when the site for stimulatory nucleotides is occupied.

Interaction of tolbutamide and cytosolic nucleotides in controlling the ATP-sensitive K+ channel in mouse β-cells

1 In mouse pancreatic β‐cells the role of cytosolic nucleotides in the regulation of the sulphonylurea sensitivity of the adenosine 5′‐triphosphate‐sensitive K+ channel (KATP‐channel) was examined. Patch‐clamp experiments with excised inside‐out membrane patches were carried out using an experimental protocol favouring phosphorylation of membrane proteins. 2 In the absence of Mg2+, the KATP‐channel‐inhibiting potency of cytosolic nucleotides decreased in the order ATP = adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS) > adenosine 5′‐diphosphate (ADP) > adenosine 5′‐O‐(2‐thiodiphosphate) (ADPβS) = adenylyl‐imidodiphosphate (AMP‐PNP) > 2′‐deoxyadenosine 5′‐triphosphate (dATP) > uridine 5′‐triphosphate (UTP) > 2′‐deoxyadenosine 5′‐diphosphate (dADP) > guanosine 5′‐triphosphate (GTP) > guanosine 5′‐diphosphate (GDP) > uridine 5′‐diphosphate (UDP). 3 In the presence of Mg2+, the inhibitory potency of cytosolic nucleotides decreased in the order ATPγS > ATP > AMP‐PNP > ADPβS > dATP > UTP. In the presence of Mg2+, the KATP‐channels were activated by dADP, GTP, GDP and UDP. 4 Tolbutamide inhibited the KATP‐channels not only in the presence but also in the prolonged absence of Mg2+. In nucleotide‐free solutions, the potency of tolbutamide was very low. When about half of the KATP‐channel activity was inhibited by ATP, AMP‐PNP, ADPβS or ADP (absence of Mg2+), the potency of tolbutamide was increased. 5 Tolbutamide (100 μm) slightly enhanced the channel‐inhibiting potency of AMP‐PNP and inhibited the channel‐activating effect of MgGDP in a non‐competitive manner. 6 Channel activation by MgGDP (0.5 mM) competitively antagonized the inhibitory responses to AMP‐PNP (1 μm‐1 mM). This effect of GDP was neutralized by tolbutamide (100 μm). 7 The stimulatory effect of 0.5 mM MgGDP was neutralized by 200 μm AMP‐PNP. Under these conditions the potency of tolbutamide was much higher than in the presence of 0.5 mM MgGDP alone or in the absence of any nucleotides. 8 dADP (0.3–1 mM) increased the potency of tolbutamide. Additional application of 200 μm AMP‐PNP caused a further increase in the potency of tolbutamide. 9 In conclusion, in the simultaneous presence of inhibitory and stimulatory nucleotides, binding of sulphonylureas to their receptor causes direct inhibition of channel activity, non‐competitive inhibition of the action of stimulatory nucleotides and interruption of the competitive interaction between stimulatory and inhibitory nucleotides. The latter effect increases the proportion of KATP‐channels staying in the nucleotide‐blocked state. In addition, this state potentiates the direct effect of sulphonylureas.

A Peroxisome Proliferator-Activated Receptor γ-Retinoid X Receptor Heterodimer Physically Interacts with the Transcriptional Activator PAX6 to Inhibit Glucagon Gene Transcription

The peptide hormone glucagon stimulates hepatic glucose output, and its levels in the blood are elevated in type 2 diabetes mellitus. The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has essential roles in glucose homeostasis, and thiazolidinedione PPARγ agonists are clinically important antidiabetic drugs. As part of their antidiabetic effect, thiazolidinediones such as rosiglitazone have been shown to inhibit glucagon gene transcription through binding to PPARγ and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene. However, how thiazolidinediones and PPARγ inhibit PAX6 activity at the glucagon promoter remained unknown. After transient transfection of a glucagon promoter-reporter fusion gene into a glucagon-producing pancreatic islet α-cell line, ligand-bound PPARγ was found in the present study to inhibit glucagon gene transcription also after deletion of its DNA-binding domain. Like PPARγ ligands, also retinoid X receptor (RXR) agonists inhibited glucagon gene transcription in a PPARγ-dependent manner. In glutathione transferase pull-down assays, the ligand-bound PPARγ-RXR heterodimer bound to the transactivation domain of PAX6. This interaction depended on the presence of the ligand and RXR, but it was independent of the PPARγ DNA-binding domain. Chromatin immunoprecipitation experiments showed that PPARγ is recruited to the PAX6-binding proximal glucagon promoter. Taken together, the results of the present study support a model in which a ligand-bound PPARγ-RXR heterodimer physically interacts with promoter-bound PAX6 to inhibit glucagon gene transcription. These data define PAX6 as a novel physical target of PPARγ-RXR.

Loss of insulin-induced inhibition of glucagon gene transcription in hamster pancreatic islet alpha cells by long-term insulin exposure

Aims/hypothesisDiabetes mellitus type 2 is characterised by hyperglucagonaemia, resulting in hepatic glucose production and hyperglycaemia. Considering that insulin inhibits glucagon secretion and gene transcription, hyperglucagonaemia in the face of hyperinsulinaemia in diabetes mellitus type 2 suggests that there is insulin resistance also at the glucagon-producing pancreatic islet alpha cells. However, the molecular mechanism of alpha cell insulin resistance is unknown. Therefore, the effect of molecules implicated in conferring insulin resistance in some other tissues was investigated on insulin-induced inhibition of glucagon gene transcription in alpha cells.MethodsReporter gene assays and biochemical techniques were used in the glucagon-producing hamster pancreatic islet alpha cell line InR1-G9.ResultsFrom among 16 agents tested, chronic insulin treatment was found to abolish insulin-induced inhibition of glucagon gene transcription. Overproduction of constitutively active protein kinase B (PKB) still inhibited glucagon gene transcription after chronic insulin treatment; together with a markedly reduced insulin-induced phosphorylation and, thus, activation of PKB, this indicates that targets upstream of PKB within the insulin signalling pathway are affected. Indeed, chronic insulin treatment markedly reduced IRS-1 phosphorylation, insulin receptor (IR) autophosphorylation and IR content. Cycloheximide and in vivo labelling experiments attributed IR downregulation to enhanced degradation.Conclusions/interpretationThese results show that an extended exposure of alpha cells to insulin induces IR downregulation and loss of insulin-induced inhibition of glucagon gene transcription. They suggest that hyperinsulinaemia, through IR downregulation, may confer insulin resistance to pancreatic islet alpha cells in diabetes mellitus type 2.

Characterization of a novel Foxa (hepatocyte nuclear factor-3) site in the glucagon promoter that is conserved between rodents and humans.

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the Foxa [Fox (forkhead box) subclass A; also known as HNF-3 (hepatocyte nuclear factor-3)] family are required for cell-specific activation of the glucagon gene in pancreatic islet alpha-cells. However, their action on the glucagon gene is poorly understood. In the present study, comparative sequence analysis and molecular characterization using protein-DNA binding and transient transfection assays revealed that the well-characterized Foxa-binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. A novel Foxa site was identified that is conserved in rats, mice and humans. It mediates activation of the glucagon gene by Foxa proteins and confers cell-specific promoter activity in glucagon-producing pancreatic islet alpha-cell lines. In contrast with previously identified Foxa-binding sites in the glucagon promoter, which bind nuclear Foxa2, the novel Foxa site was found to bind preferentially Foxa1 in nuclear extracts of a glucagon-producing pancreatic islet alpha-cell line, offering a mechanism that explains the decrease in glucagon gene expression in Foxa1-deficient mice. This site is located just upstream of the TATA box (between -30 and -50), suggesting a role for Foxa proteins in addition to direct transcriptional activation, such as a role in opening the chromatin at the start site of transcription of the glucagon gene.

Inhibition of human insulin gene transcription by peroxisome proliferator-activated receptor γ and thiazolidinedione oral antidiabetic drugs

Background and purpose:  The transcription factor peroxisome proliferator‐activated receptor γ (PPARγ) is essential for glucose homeostasis. PPARγ ligands reducing insulin levels in vivo are used as drugs to treat type 2 diabetes mellitus. Genes regulated by PPARγ have been found in several tissues including insulin‐producing pancreatic islet β‐cells. However, the role of PPARγ at the insulin gene was unknown. Therefore, the effect of PPARγ and PPARγ ligands like rosiglitazone on insulin gene transcription was investigated.

Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine. II. Electrophysiological and fluorimetric studies.

Prolonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K(+) channels (K(ATP) channels), such as sulfonylureas, imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al., pages 1685-1694, this issue). To elucidate the underlying mechanisms, K(ATP) channel activity, plasma membrane potential and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 microM each) K(ATP) channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 microM) induced a marked reduction of K(ATP) channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 microM) or tolbutamide (500 microM) had no lasting effect on K(ATP) channel activity. Correspondingly, phentolamine-, alinidine-, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca(2+)](i) normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds, whereas the [Ca(2+)](i) increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca(2+)](i) elicited by a K(+) depolarization was diminished in secretagogue-pretreated B-cells, the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K(ATP) channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca(2+)](i) regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis, which responds only weakly to substantial increases in [Ca(2+)](i) (in the case of tolbutamide).