scholarly journals Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression

2003 ◽  
Vol 371 (3) ◽  
pp. 761-774 ◽  
Author(s):  
Gabriela da SILVA XAVIER ◽  
Isabelle LECLERC ◽  
Aniko VARADI ◽  
Takashi TSUBOI ◽  
S. Kelly MOULE ◽  
...  
Keyword(s):  
Gene Expression ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Active Form ◽  
Dominant Negative ◽  
Β Cells ◽  
Dominant Negative Form ◽  
Ampk Activity ◽  
Glucose Stimulated Insulin Secretion ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) has recently been implicated in the control of preproinsulin gene expression in pancreatic islet β-cells [da Silva Xavier, Leclerc, Salt, Doiron, Hardie, Kahn and Rutter (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4023–4028]. Using pharmacological and molecular strategies to regulate AMPK activity in rat islets and clonal MIN6 β-cells, we show here that the effects of AMPK are exerted largely upstream of insulin release. Thus forced increases in AMPK activity achieved pharmacologically with 5-amino-4-imidazolecarboxamide riboside (AICAR), or by adenoviral overexpression of a truncated, constitutively active form of the enzyme (AMPKα1.T172D), blocked glucose-stimulated insulin secretion. In MIN6 cells, activation of AMPK suppressed glucose metabolism, as assessed by changes in total, cytosolic or mitochondrial [ATP] and NAD(P)H, and reduced increases in intracellular [Ca2+] caused by either glucose or tolbutamide. By contrast, inactivation of AMPK by expression of a dominant-negative form of the enzyme mutated in the catalytic site (AMPKα1.D157A) did not affect glucose-stimulated increases in [ATP], NAD(P)H or intracellular [Ca2+], but led to the unregulated release of insulin. These results indicate that inhibition of AMPK by glucose is essential for the activation of insulin secretion by the sugar, and may contribute to the transcriptional stimulation of the preproinsulin gene. Modulation of AMPK activity in the β-cell may thus represent a novel therapeutic strategy for the treatment of type 2 diabetes mellitus.

scholarly journals Decreased Insulin Secretion and Accumulation of Triglyceride in β Cells Overexpressing a Dominant-negative Form of AMP-activated Protein Kinase

2010 ◽  
Vol 57 (2) ◽  
pp. 141-152 ◽  
Author(s):  
Yukiko OKAZAKI ◽  
Kazuhiro ETO ◽  
Tokuyuki YAMASHITA ◽  
Masayuki OKAMOTO ◽  
Mitsuru OHSUGI ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Dominant Negative ◽  
Β Cells ◽  
Dominant Negative Form ◽  
Amp Activated Protein Kinase ◽  
Negative Form

scholarly journals Characterization of the Role of AMP-Activated Protein Kinase in the Regulation of Glucose-Activated Gene Expression Using Constitutively Active and Dominant Negative Forms of the Kinase

2000 ◽  
Vol 20 (18) ◽  
pp. 6704-6711 ◽  
Author(s):  
Angela Woods ◽  
Dalila Azzout-Marniche ◽  
Marc Foretz ◽  
Silvie C. Stein ◽  
Patricia Lemarchand ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Fatty Acid Synthase ◽  
Physiological Role ◽  
Rat Hepatocytes ◽  
Dominant Negative ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Constitutively Active

ABSTRACT In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (α1312) acts as a constitutively active kinase, while the other (α1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of α1312 increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of α1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion

2004 ◽  
Vol 286 (6) ◽  
pp. E1023-E1031 ◽  
Author(s):  
Isabelle Leclerc ◽  
Wolfram W. Woltersdorf ◽  
Gabriela da Silva Xavier ◽  
Rebecca L. Rowe ◽  
Sarah E. Cross ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Pancreatic Islets ◽  
Human Islets ◽  
Β Cells ◽  
Min6 Cells ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently been shown to act on skeletal muscle and liver in part through the activation of AMP-activated protein kinase (AMPK). Whether metformin or the satiety factor leptin, which also stimulates AMPK in muscle, regulates this enzyme in pancreatic islets is unknown. We have recently shown that forced increases in AMPK activity inhibit insulin secretion from MIN6 cells (da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Biochem J 371: 761–774, 2003). Here, we explore whether 1) glucose, metformin, or leptin regulates AMPK activity in isolated islets from rodent and human and 2) whether changes in AMPK activity modulate insulin secretion from human islets. Increases in glucose concentration from 0 to 3 and from 3 to 17 mM inhibited AMPK activity in primary islets from mouse, rat, and human, confirming previous findings in insulinoma cells. Incubation with metformin (0.2–1 mM) activated AMPK in both human islets and MIN6 β-cells in parallel with an inhibition of insulin secretion, whereas leptin (10–100 nM) was without effect in MIN6 cells. These studies demonstrate that AMPK activity is subject to regulation by both glucose and metformin in pancreatic islets and clonal β-cells. The inhibitory effects of metformin on insulin secretion may therefore need to be considered with respect to the use of this drug for the treatment of type 2 diabetes.

Imaging Glucose-Regulated Insulin Secretion and Gene Expression in Single Islet β-Cells: Control by AMP-Activated Protein Kinase

2004 ◽  
Vol 40 (3S) ◽  
pp. 179-190 ◽  
Author(s):  
Guy A. Rutter ◽  
Isabelle Leclerc ◽  
Takashi Tsuboi ◽  
Gabriela da Silva Xavier ◽  
Frédérique Diraison ◽  
...  
Keyword(s):  
Gene Expression ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Β Cells ◽  
Amp Activated Protein Kinase

scholarly journals Activation of the AMP-activated protein kinase enhances glucose-stimulated insulin secretion in mouse β-cells

Islets ◽  
2010 ◽  
Vol 2 (3) ◽  
pp. 156-163 ◽  
Author(s):  
Martina Düfer ◽  
Katja Noack ◽  
Peter Krippeit-Drews ◽  
Gisela Drews
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Β Cells ◽  
Glucose Stimulated Insulin Secretion ◽  
Amp Activated Protein Kinase

Glucose regulates the cortical actin network through modulation of Cdc42 cycling to stimulate insulin secretion

2003 ◽  
Vol 285 (3) ◽  
pp. C698-C710 ◽  
Author(s):  
Angela K. Nevins ◽  
Debbie C. Thurmond
Keyword(s):  
Insulin Secretion ◽  
Active Form ◽  
Dominant Negative ◽  
Actin Network ◽  
Cortical Actin ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Actin Remodeling ◽  
Glucose Stimulated Insulin Secretion ◽  
Constitutively Active

Glucose-stimulated insulin granule exocytosis in pancreatic β-cells involves cortical actin remodeling that results in the transient disruption of the interaction between polymerized actin with the plasma membrane t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. To examine the mechanism underlying the initiation of cortical actin remodeling, we have used the actin nucleating/stabilizing agent jasplakinolide to show that remodeling is initiated at a step proximal to the ATP-sensitive K+ channels in the stimulus-secretion pathway. Confocal immunofluorescent microscopy revealed that cortical actin remodeling was required for glucose-stimulated insulin secretion. Furthermore, glucose was found to mediate the endogenous activation state of the Rho family GTPase Cdc42, a positive proximal effector of actin polymerization, resulting in a net decrease of Cdc42-GTP within 5 min of stimulation. Intriguingly, glucose stimulation resulted in the rapid and reversible glucosylation of Cdc42, suggesting that glucose inactivated Cdc42 by selective glucosylation to induce cortical actin rearrangement. Moreover, expression of the constitutively active form of Cdc42 (Q61L) inhibited glucose-stimulated insulin secretion, whereas the dominant negative form (T17N) was without effect, suggesting that glucose-stimulated insulin secretion requires Cdc42 cycling to the GDP-bound state. In contrast, KCl-stimulated insulin secretion was unaffected by the expression of dominant negative or constitutively active Cdc42 and ceased to modulate endogenous Cdc42 activation, consistent with glucose-dependent cortical actin remodeling. These findings reveal that glucose regulates the cortical actin network through modulation of Cdc42 cycling to induce insulin secretion in pancreatic β-cells.

Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells

2003 ◽  
Vol 284 (5) ◽  
pp. C1297-C1308 ◽  
Author(s):  
Kenneth R. Hallows ◽  
Gary P. Kobinger ◽  
James M. Wilson ◽  
Lee A. Witters ◽  
J. Kevin Foskett
Keyword(s):  
Protein Kinase ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Dominant Negative ◽  
T84 Cells ◽  
Physiological Relevance ◽  
Polarized Epithelia ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-gated Cl− channel and cellular conductance regulator, but the detailed mechanisms of CFTR regulation and its regulation of other transport proteins remain obscure. We previously identified the metabolic sensor AMP-activated protein kinase (AMPK) as a novel protein interacting with CFTR and found that AMPK phosphorylated CFTR and inhibited CFTR-dependent whole cell conductances when coexpressed with CFTR in Xenopus oocytes. To address the physiological relevance of the CFTR-AMPK interaction, we have now studied polarized epithelia and have evaluated the localization of endogenous AMPK and CFTR and measured CFTR activity with modulation of AMPK activity. By immunofluorescent imaging, AMPK and CFTR share an overlapping apical distribution in several rat epithelial tissues, including nasopharynx, submandibular gland, pancreas, and ileum. CFTR-dependent short-circuit currents ( Isc ) were measured in polarized T84 cells grown on permeable supports, and several independent methods were used to modulate endogenous AMPK activity. Activation of endogenous AMPK with the cell-permeant adenosine analog 5-amino-4-imidazolecarboxamide-1-β-d-ribofuranoside (AICAR) inhibited forskolin-stimulated CFTR-dependent I sc in nonpermeabilized monolayers and monolayers with nystatin permeabilization of the basolateral membrane. Raising intracellular AMP concentration in monolayers with basolateral membranes permeabilized with α-toxin also inhibited CFTR, an effect that was unrelated to adenosine receptors. Finally, overexpression of a kinase-dead mutant AMPK-α1 subunit (α1-K45R) enhanced forskolin-stimulated I sc in polarized T84 monolayers, consistent with a dominant-negative reduction in the inhibition of CFTR by endogenous AMPK. These results indicate that AMPK plays a physiological role in modulating CFTR activity in polarized epithelia and suggest a novel paradigm for the coupling of ion transport to cellular metabolism.

scholarly journals AMP-activated protein kinase as regulator of P2Y6 receptor-induced insulin secretion in mouse pancreatic β-cells

2013 ◽  
Vol 85 (7) ◽  
pp. 991-998 ◽  
Author(s):  
Ramachandran Balasubramanian ◽  
Hiroshi Maruoka ◽  
P. Suresh Jayasekara ◽  
Zhan-Guo Gao ◽  
Kenneth A. Jacobson
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
P2y6 Receptor ◽  
Amp Activated Protein Kinase

Activation of phosphatidylinositol 3-kinase is required for transcriptional activity of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: assessment of the role of protein kinase B and p70 S6 kinase

2000 ◽  
Vol 349 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Silvia FERNÁNDEZ DE MATTOS ◽  
Elisabet DE LOS PINOS ◽  
Manel JOAQUIN ◽  
Albert TAULER
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Transcriptional Activity ◽  
Protein Kinase B ◽  
Dominant Negative ◽  
Phosphatidylinositol 3 Kinase ◽  
S6 Kinase ◽  
Dominant Negative Form ◽  
Negative Form ◽  
Phosphatidylinositol 3

Previous studies have demonstrated that the F isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase(6PF2K/Fru-2,6-BPase) is transcriptionally regulated by growth factors. The aim of this study was to investigate the importance of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway in the regulation of 6PF2K/Fru-2,6-BPase gene expression. We have completed studies using chemical inhibitors and expression vectors for the proteins involved in this signalling cascade. Treatment of cells with LY 294002, an inhibitor of PI 3-kinase, blocked the epidermal growth factor (EGF)-dependent stimulation of 6PF2K/Fru-2,6-BPase gene transcription. Transient transfection of a constitutively active PI 3-kinase was sufficient to activate transcription from the F-type 6PF2K/Fru-2,6-BPase promoter. In contrast, co-transfection with a dominant-negative form of PI 3-kinase completely abrogated the stimulation by EGF, and down-regulated the basal promoter activity. In an attempt to determine downstream proteins that lie between PI 3-kinase and 6PF2K/Fru-2,6-BPase gene expression, the overexpression of a constitutively active form of protein kinase B (PKB) was sufficient to activate 6PF2K/Fru-2,6-BPase gene expression, even in the presence of either a dominant-negative form of PI 3-kinase or LY 294002. The over-expression of p70/p85 ribosomal S6 kinase or the treatment with its inhibitor rapamycin did not affect 6PF2K/Fru-2,6-BPase transcription. We conclude that PI 3-kinase is necessary for the transcriptional activity of F-type 6PF2K/Fru-2,6-BPase, and that PKB is a downstream effector of PI 3-kinase directly involved in the regulation of 6PF2K/Fru-2,6-BPase gene expression.

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