title: Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice.
abstract: OBJECTIVE
  Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR(-/-)) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca(2+) handling in these islets.
  
  
  METHODS
  Isolated islets from both LDLR(-/-) and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca(2+) level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0-10mmol/l) of methyl-beta-cyclodextrin (MβCD).
  
  
  RESULTS
  The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR(-/-) than in WT islets, paralleled by an impairment of Ca(2+) handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR(-/-) compared with WT islets. Removal of excess cholesterol from LDLR(-/-) islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca(2+) handling was also normalized in cholesterol-depleted LDLR(-/-) islets. Cholesterol removal from WT islets by 0.1 and 1.0mmol/l MβCD impaired both GSIS and Ca(2+) handling. In addition, at 10mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.
  
  
  CONCLUSIONS
  Abnormally high (LDLR(-/-) islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca(2+) handling. Normalization of cholesterol improves Ca(2+) handling and insulin secretion in LDLR(-/-) islets.
authors.affiliation: Department of Structural and Functional Biology, Institute of Biology, State University of Campinas, SP, Brazil.
authors.forename: J C
authors.initials: JC
authors.lastname: Souza
authors.forename: E C
authors.initials: EC
authors.lastname: Vanzela
authors.forename: R A
authors.initials: RA
authors.lastname: Ribeiro
authors.forename: L F
authors.initials: LF
authors.lastname: Rezende
authors.forename: C A
authors.initials: CA
authors.lastname: de Oliveira
authors.forename: E M
authors.initials: EM
authors.lastname: Carneiro
authors.forename: H C F
authors.initials: HC
authors.lastname: Oliveira
authors.forename: A C
authors.initials: AC
authors.lastname: Boschero
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism
chemical_list: D007328:Insulin; D011973:Receptors, LDL; D002784:Cholesterol; D005947:Glucose; D002118:Calcium