title: Molecular mechanisms of lipoapoptosis and metformin protection in GLP-1 secreting cells.
abstract: BACKGROUND
  Evidence is emerging that elevated serum free fatty acids (hyperlipidemia) contribute to the pathogenesis of type-2-diabetes, and lipotoxicity is observed in many cell types. We recently published data indicating lipotoxic effects of simulated hyperlipidemia also in GLP-1-secreting cells, where the antidiabetic drug metformin conferred protection from lipoapoptosis. The aim of the present study was to identify mechanisms involved in mediating lipotoxicity and metformin lipoprotection in GLP-1 secreting cells. These signaling events triggered by simulated hyperlipidemia may underlie reduced GLP-1 secretion in diabetic subjects, and metformin lipoprotection by metformin could explain elevated plasma GLP-1 levels in diabetic patients on chronic metformin therapy. The present study may thus identify potential molecular targets for increasing endogenous GLP-1 secretion through enhanced viability of GLP-1 secreting cells in diabetic hyperlipidemia and obesity.
  
  
  METHODS
  We have studied molecular mechanisms mediating lipotoxicity and metformin-induced lipoprotection in GLP-1-secreting L-cells in vitro, using the murine GLUTag cell line as a model. Diabetic hyperlipidemia was simulated in this cell system by addition of the fatty acid palmitate. Caspase-3 activity was used as a measure of GLUTag cell apoptosis. ROS production was determined using a fluorescent probe, and the activation of intracellular signaling pathways was assessed by Western blotting.
  
  
  RESULTS
  Palmitate increased ROS production in GLP-1 secreting cells, and the lipotoxic effects of palmitate were abolished in the presence of the antioxidant Trolox. Further, palmitate phosphorylated p38 and inhibition of p38 using the p38 inhibitor SB203580 significantly reduced palmitate-induced caspase-3 activity. Pre-incubation of palmitate with metformin further increased palmitate induced ROS production, while significantly reducing the expression of p38.
  
  
  CONCLUSIONS
  This study demonstrates that palmitate induces ROS production and that the palmitate induced lipotoxicity is the result of increased ROS production, where the ROS sensitive MKK3/6-p38 pathway mediates lipoapoptosis of GLP-1-secreting cells. Further, in the presence of simulated hyperlipidemia, metformin increases ROS production. However, metformin significantly decreases the expression of p38, indicating that metformin mediated lipoprotection involves reduced activity of the p38 signaling pathway.
authors.affiliation: Karolinska Institutet, Department of Clinical Science and Education, Unit for Diabetes Research, Södersjukhuset, SE-118 83 Stockholm, Sweden. camilla.kappe@ki.se
authors.forename: Camilla
authors.initials: C
authors.lastname: Kappe
authors.forename: Jens J
authors.initials: JJ
authors.lastname: Holst
authors.forename: Qimin
authors.initials: Q
authors.lastname: Zhang
authors.forename: Ake
authors.initials: A
authors.lastname: Sjöholm
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002839:Chromans / Q000494:pharmacology; D019610:Cytoprotection; D005230:Fatty Acids, Nonesterified / Q000633:toxicity*; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism*; D006949:Hyperlipidemias / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D048371:MAP Kinase Kinase 3 / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D048669:MAP Kinase Kinase 6 / Q000378:metabolism; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D008822:Mice, Transgenic; D010168:Palmitates / Q000633:toxicity; D017382:Reactive Oxygen Species; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D000975:Antioxidants; D002839:Chromans; D005230:Fatty Acids, Nonesterified; D007004:Hypoglycemic Agents; D010168:Palmitates; D017382:Reactive Oxygen Species; D052216:Glucagon-Like Peptide 1; D008687:Metformin; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D048670:MAP Kinase Kinase 4; D048669:MAP Kinase Kinase 6; D053148:Caspase 3; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid