title: Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation.
abstract: BACKGROUND
  The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation.
  
  
  METHODS
  Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC.
  
  
  RESULTS
  Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation.
  
  
  CONCLUSIONS
  This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification.
authors.affiliation: Department of Internal Medicine (DIMI), University of Genoa, 16132 Genoa, Italy. odetti@unige.it
authors.forename: P
authors.initials: P
authors.lastname: Odetti
authors.forename: I
authors.initials: I
authors.lastname: Aragno
authors.forename: R
authors.initials: R
authors.lastname: Rolandi
authors.forename: S
authors.initials: S
authors.lastname: Garibaldi
authors.forename: S
authors.initials: S
authors.lastname: Valentini
authors.forename: L
authors.initials: L
authors.lastname: Cosso
authors.forename: N
authors.initials: N
authors.lastname: Traverso
authors.forename: D
authors.initials: D
authors.lastname: Cottalasso
authors.forename: M A
authors.initials: MA
authors.lastname: Pronzato
authors.forename: U M
authors.initials: UM
authors.lastname: Marinari
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000032:analysis; D003094:Collagen / Q000737:chemistry* / Q000648:ultrastructure*; D003922:Diabetes Mellitus, Type 1 / Q000473:pathology* / Q000503:physiopathology; D019270:Fructosamine / Q000032:analysis; D017127:Glycation End Products, Advanced / Q000032:analysis; D006031:Glycosylation; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D008297:Male; D018625:Microscopy, Atomic Force / Q000379:methods; D051381:Rats; D011913:Rats, Inbred BB; D012016:Reference Values; D013710:Tendons / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D017127:Glycation End Products, Advanced; D019270:Fructosamine; D003094:Collagen; D001120:Arginine; C062187:pentosidine; D008239:Lysine