c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells

2007 ◽  
Vol 293 (1) ◽  
pp. E48-E56 ◽  
Author(s):  
J. Jason Collier ◽  
Pili Zhang ◽  
Kim B. Pedersen ◽  
Susan J. Burke ◽  
John W. Haycock ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Heterologous Promoter ◽  
Kinase Gene ◽  
Glucose Response ◽  
Glucose Flux ◽  
Element Binding Protein ◽  
Regulated Gene Expression

Increased glucose flux generates metabolic signals that control transcriptional programs through poorly understood mechanisms. Previously, we demonstrated a necessity in hepatocytes for c-Myc in the regulation of a prototypical glucose-responsive gene, L-type pyruvate kinase (L-PK) (Collier JJ, Doan TT, Daniels MC, Schurr JR, Kolls JK, Scott DK. J Biol Chem 278: 6588–6595, 2003). Pancreatic β-cells have many features in common with hepatocytes with respect to glucose-regulated gene expression, and in the present study we determined whether c-Myc was required for the L-PK glucose response in insulin-secreting (INS-1)-derived 832/13 cells. Glucose increased c-Myc abundance and association with its heterodimer partner, Max. Manipulations that prevented the formation of a functional c-Myc/Max heterodimer reduced the expression of the L-PK gene. In addition, glucose augmented the binding of carbohydrate response element binding protein (ChREBP), c-Myc, and Max to the promoter of the L-PK gene in situ. The transactivation of ChREBP, but not of c-Myc, was dependent on high glucose concentrations in the contexts of either the L-PK promoter or a heterologous promoter. The glucose-mediated transactivation of ChREBP was independent of mutations that alter phosphorylation sites thought to regulate the cellular location of ChREBP. We conclude that maximal glucose-induced expression of the L-PK gene in INS-1-derived 832/13 cells involves increased c-Myc abundance, recruitment of c-Myc, Max, and ChREBP to the promoter, and a glucose-stimulated increase in ChREBP transactivation.

scholarly journals Long-Term Exposure of Pancreatic β-Cells to Palmitate Results in SREBP-1C-Dependent Decreases in GLP-1 Receptor Signaling via CREB and AKT and Insulin Secretory Response

Endocrinology ◽  
2016 ◽  
Vol 157 (6) ◽  
pp. 2243-2258 ◽  
Author(s):  
Annalisa Natalicchio ◽  
Giuseppina Biondi ◽  
Nicola Marrano ◽  
Rossella Labarbuta ◽  
Federica Tortosa ◽  
...  
Keyword(s):  
Protein Expression ◽  
Insulin Release ◽  
Binding Protein ◽  
Camp Response Element Binding ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Camp Response Element ◽  
Element Binding Protein ◽  
Viral Oncogene Homolog ◽  
Erk Kinase

The effects of prolonged exposure of pancreatic β-cells to high saturated fatty acids on glucagon-like peptide-1 (GLP-1) action were investigated. Murine islets, human pancreatic 1.1B4 cells, and rat INS-1E cells were exposed to palmitate for 24 hours. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting, respectively. Specific short interfering RNAs were used to knockdown expression of the GLP-1 receptor (Glp1r) and Srebf1. Insulin release was assessed with a specific ELISA. Exposure of murine islets, as well as of human and INS-1E β-cells, to palmitate reduced the ability of exendin-4 to augment insulin mRNA levels, protein content, and release. In addition, palmitate blocked exendin-4-stimulated cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, whereas phosphorylation of MAPK-ERK kinase-1/2 and ERK-1/2 was not altered. Similarly, RNA interference-mediated suppression of Glp1r expression prevented exendin-4-induced cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, but did not impair exendin-4 stimulation of MAPK-ERK kinase-1/2 and ERK-1/2. Both islets from mice fed a high fat diet and human and INS-1E β-cells exposed to palmitate showed reduced GLP-1 receptor and pancreatic duodenal homeobox-1 (PDX-1) and increased sterol regulatory element-binding protein (SREBP-1C) mRNA and protein levels. Furthermore, suppression of SREBP-1C protein expression prevented the reduction of PDX-1 and GLP-1 receptor levels and restored exendin-4 signaling and action. Finally, treatment of INS-1E cells with metformin for 24 h resulted in inhibition of SREBP-1C expression, increased PDX-1 and GLP-1 receptor levels, consequently, enhancement of exendin-4-induced insulin release. Palmitate impairs exendin-4 effects on β-cells by reducing PDX-1 and GLP-1 receptor expression and signaling in a SREBP-1C-dependent manner. Metformin counteracts the impairment of GLP-1 receptor signaling induced by palmitate.

scholarly journals Functional synergism in the carbohydrate-induced activation of liver-type pyruvate kinase gene expression

1995 ◽  
Vol 308 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Z Liu ◽  
H C Towle
Keyword(s):  
Pyruvate Kinase ◽  
Binding Sites ◽  
Transcriptional Level ◽  
Primary Hepatocytes ◽  
Kinase Gene ◽  
Glucose Response ◽  
Hepatic Expression ◽  
Elevated Glucose ◽  
Response Prompting ◽  
Late Transcription

Hepatic expression of the liver-type pyruvate kinase (L-PK) gene is induced at the transcriptional level by increased carbohydrate metabolism in the rat. The carbohydrate response of the L-PK gene requires sequences from -171 to -124, which encompass adjacent major late transcription factor (MLTF)-like and hepatic nuclear factor (HNF)-4 binding sites. Neither site alone is capable of conferring a response, prompting us to explore the mechanism of synergy between the MLTF-like factor and HNF-4. Spacing requirements between the two factor binding sites were tested by generating a series of mutations that altered the distance between these sites. Surprisingly, all of the constructs with spacing mutations were capable of responding to elevated glucose when introduced into primary hepatocytes. Thus the glucose response does not depend on the rigid phasing of the MLTF-like and HNF-4 factors, suggesting that the factors binding to these two sites do not interact directly with each other. Substitution or inversion of the PK HNF-4 site abrogated the response to glucose and also significantly suppressed the promoter activity under non-inducing conditions. We conclude that the MLTF-like factor and HNF-4 co-operate functionally to maintain the basal activity, as well as the carbohydrate responsiveness, of the L-PK gene. A mechanism other than co-operative DNA binding is responsible for the synergism.

Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring

2009 ◽  
Vol 296 (2) ◽  
pp. C306-C316 ◽  
Author(s):  
Omar M. Faruque ◽  
Dung Le-Nguyen ◽  
Anne-Dominique Lajoix ◽  
Eric Vives ◽  
Pierre Petit ◽  
...  
Keyword(s):  
Camp Response Element Binding ◽  
Subcellular Targeting ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Anchoring Proteins ◽  
Element Binding Protein ◽  
Cell Permeable Peptide ◽  
Subcellular Compartmentalization ◽  
The Impact

Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated “AKAPis” has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic β-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic β-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.

scholarly journals ChREBP binding to fatty acid synthase and L-type pyruvate kinase genes is stimulated by glucose in pancreatic β-cells

2006 ◽  
Vol 47 (11) ◽  
pp. 2482-2491 ◽  
Author(s):  
Gabriela da Silva Xavier ◽  
Guy A. Rutter ◽  
Frédérique Diraison ◽  
Chrysovalantis Andreolas ◽  
Isabelle Leclerc
Keyword(s):  
Fatty Acid ◽  
Pyruvate Kinase ◽  
Fatty Acid Synthase ◽  
Β Cells ◽  
Pancreatic Β Cells

scholarly journals ROCK1 regulates insulin secretion from β-cells

2021 ◽  
Author(s):  
Byung-Jun Sung ◽  
Sung-Bin Lim ◽  
Jae Hyeon Kim ◽  
Won-Mo Yang ◽  
Rohit N Kulkarni ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Pyruvate Kinase ◽  
Glucose Homeostasis ◽  
Isolated Islets ◽  
Whole Body ◽  
Proximity Ligation Assay ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Glucose Stimulated Insulin Secretion

Objective: The endocrine pancreatic β-cells play a pivotal role in the maintenance of whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate b-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homeostasis. Methods: Mice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1loxP/loxP, β-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. Insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1-/- mice or β-cell lines with knockdown of ROCK1 were also evaluated. Proximity ligation assay was performed to determine the physical interactions between PK and ROCK1. Results: Mice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1-/- mice displayed progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS was markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium levels, ATP levels, and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that ROCK1 binding to PK is greatly enhanced by glucose stimulation in β-cells. Conclusions: Our findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and maintenance of glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

scholarly journals Cytokine-mediated Down-regulation of the Transcription Factor cAMP-response Element-binding Protein in Pancreatic β-Cells

2003 ◽  
Vol 278 (25) ◽  
pp. 23055-23065 ◽  
Author(s):  
Purevsuren Jambal ◽  
Sara Masterson ◽  
Albina Nesterova ◽  
Ron Bouchard ◽  
Barbara Bergman ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Protein ◽  
Camp Response Element Binding ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Camp Response Element ◽  
Down Regulation ◽  
Response Element ◽  
Element Binding Protein

Expression of α2- and β-adrenoceptor subtypes in human islets of Langerhans

1996 ◽  
Vol 148 (3) ◽  
pp. 531-543 ◽  
Author(s):  
R J Lacey ◽  
S L F Chan ◽  
H C Cable ◽  
R F L James ◽  
C W Perret ◽  
...  
Keyword(s):  
Islets Of Langerhans ◽  
Human Islets ◽  
Receptor Subtype ◽  
Ribonuclease Protection Assay ◽  
Human Pancreas ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Mrna Species ◽  
Human Islets Of Langerhans

Abstract Sequences from cDNA molecules encoding α2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human α2C2-, α2C4- and α2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of α2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic β-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both α2C2 and α2C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding α2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each α2-adrenoceptor subtype in sections of human pancreas. All three subtypes of α2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffinembedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three α2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple α2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of α2-adrenoceptor subtype mRNA species in pancreatic β-cells was confirmed by Northern blotting of RNA extracted from the clonal β-cell line, HIT-T15. Transcripts encoding each of the three cloned α2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with β2-adrenoceptor mRNA revealed expression of this species in islet β-cells but not in the exocrine tissue of the pancreas. Journal of Endocrinology (1996) 148, 531–543

scholarly journals Upstream Stimulatory Factor Proteins Are Major Components of the Glucose Response Complex of the L-type Pyruvate Kinase Gene Promoter

1995 ◽  
Vol 270 (6) ◽  
pp. 2640-2643 ◽  
Author(s):  
Anne-Marie Lefran¸ois-Martinez ◽  
Antoine Martinez ◽  
Bénédicte Antoine ◽  
Michel Raymondjean ◽  
Axel Kahn
Keyword(s):  
Pyruvate Kinase ◽  
Gene Promoter ◽  
Upstream Stimulatory Factor ◽  
Kinase Gene ◽  
Glucose Response ◽  
Stimulatory Factor ◽  
Response Complex
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