dorsomorphin and Insulinoma

The function of β-cells is regulated by nutrient uptake and metabolism. The cells' metabolic state can be expressed as concentration ratios of AMP, ADP and ATP. Relative changes in these ratios regulate insulin release. An increase in the intracellular ATP concentration causes closure of K(ATP) channels and cell membrane depolarization, which triggers stimulus-secretion coupling (SSC). In addition to K(ATP) channels, the AMP-dependent protein kinase (AMPK), a major cellular fuel sensor in a variety of cells and tissues, also affects insulin secretion and β-cell survival. In a previous study we found that the widely used AMPK inhibitor compound C retards proliferation and induces apoptosis in the rat β-cell line INS-1E. We therefore tested the effects of AMPK activators (AICAR and metformin), and compound C on AMPK phosphorylation, insulin secretion, K(ATP) channel currents, cell membrane potential, intracellular calcium concentration, apoptosis and cell cycle distribution of INS-1E cells under standard cell culture conditions (11 mM glucose).. Western blotting, ELISA, patch-clamp, calcium imaging and flow cytometry.. We found that basal AMPK phosphorylation is enhanced by AICAR (1 mM) and metformin (1 mM) but remained unaffected by compound C (10 μM). Both AICAR and compound C stimulated basal insulin secretion whereas metformin had no effect. Pre-incubation with AICAR (1 mM) caused an inhibition of K(ATP) currents but did not significantly alter the average cell membrane potential (Vm) or the threshold potential of electrical activity. Acute administration of AICAR (300 μM) led to a depolarization of Vm, which was not due to an inhibition of the basal- or glucose-induced chloride conductance, and was not accompanied by elevations of intracellular calcium (Ca(i)). AICAR had no additive blocking effect on K(ATP) currents when applied together with tolbutamide. Compound C applied over 24 hours induced an increase in the percentage of cells positive for caspase activity, whereas AICAR (1 mM) applied for 48 hours was without effect. Medium glucose concentration <3 mM caused cell cycle arrest, caspase activation and an increase of cell granularity.. We conclude that under standard cell culture conditions the AMPK modulators AICAR and compound C, but not metformin, stimulate insulin secretion by AMPK-independent mechanisms.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Apoptosis; Calcium; Cell Line, Tumor; Cell Proliferation; Glucose; Hypoglycemic Agents; Insulin; Insulin Secretion; Insulinoma; KATP Channels; Membrane Potentials; Metformin; Phosphorylation; Pyrazoles; Pyrimidines; Rats; Ribonucleotides

To investigate whether AMP-activated protein kinase (AMPK) regulates the expression of pancreatic duodenal homeobox-1 (PDX-1), a beta-cell-specific transcription factor and whether PPARalpha/gamma is involved in the regulation of pancreatic beta-cell lines after acute stimulation.. Rat insulinoma cell line INS-1 was treated with an activator (AICAR) or inhibitor (Compound C) of AMPK as well as inhibitors of PPARs (MK886 to PPARalpha and BADGE to PPARgamma). The mRNA levels of PDX-1, PPARalpha and PPARgamma were measured using real-time RT-PCR, and Western blotting was used to detect the protein expression of these factors.. Activation of AMPK by AICAR induced significantly increased the expression of PDX-1, and this increase was abrogated when AMPK was inactivated by Compound C. Similarly, the expression of PPARalpha and PPARgamma was also increased by AICAR or decreased by Compound C. However AMPK activation did not increase nuclear PDX-1 protein levels when PPARalpha was inhibited. In contrast, AMPK activation still up-regulated PDX-1 protein levels during PPARgamma inhibition. Additionally, PPARalpha activation induced by fenofibrate significantly enhanced nuclear PDX-1 protein expression.. AMPK regulates the expression of PDX-1 at both the transcriptional and protein levels, and PPARalpha may be acutely involved in the regulation of INS-1 cells.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Blotting, Western; Cell Line, Tumor; Gene Expression Regulation; Homeodomain Proteins; Insulinoma; PPAR alpha; PPAR gamma; Pyrazoles; Pyrimidines; Rats; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleotides; RNA, Messenger; Trans-Activators