Amyloid fibril formation is progressive and correlates with beta-cell secretion in transgenic mouse isolated islets.
OBJECTIVE
Amyloid fibrils are formed in islets isolated from transgenic mice expressing the gene for human islet amyloid polypeptide (IAPP) by an unknown mechanism. This model of islet amyloidosis in Type II (non-insulin-dependent) diabetes mellitus has been used to investigate the temporal and glucose dependency of fibril formation.


METHODS
To determine the time course and nature of amyloid-like accumulations and the role of glucose, transgenic mouse islets were cultured for 2-12 days in medium containing glucose (4.2 mmol/l, 11.1 mmol/l or 16.7 mmol/l) or 3.3 mmol/l glucose plus non-glucose secretagogues, 10 mmol/l leucine, 10 mmol/l leucine + 0.1 mmol/l tolbutamide, 10 mmol/l alpha-ketoisocaproic acid + 10 mmol/l glutamine. The extent of fibril formation was determined by quantitative immuno-electron microscopy. Insulin and islet amyloid polypeptide secretion into the media was measured by radioimmunoassay.


RESULTS
Extracellular amyloid fibrils immunoreactive for islet amyloid polypeptide were visible initially after 6 days of culture in 11.1 mmol/l glucose and formed 2.3 +/- 0.8 % of the islet area after 12 days; small accumulations of intracellular fibrils and amorphous extracellular islet amyloid polypeptide-immunoreactive material were present at 6-12 days. Beta-cell secretion was increased significantly by 16.7 mmol/l glucose and by alpha-ketoisocaproic acid + glutamine. The proportion of fibrillar amyloid (amyloid area/islet area%) correlated with the amount of insulin (r = 0.55, p < 0.05) and IAPP (r = 0.5, p < 0.05) in the culture media. Evidence of cellular damage was present in less than 10 % cells and correlated with the degree of fibril deposition (r = 0.8, p < 0.0001).


CONCLUSIONS
These data suggest that islet amyloid polypeptide amyloid is formed primarily at extracellular sites in isolated transgenic mouse islets and progressive fibril formation correlates with beta-cell secretion. [Diabetologia (1999) 42: 1219-1227]
MacArthur
D L
DL
Department of Human Anatomy and Genetics, University of Oxford, UK.
de Koning
E J
EJ
Verbeek
J S
JS
Morris
J F
JF
Clark
A
A
D000682:Amyloid / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D005947:Glucose / Q000494:pharmacology; D005973:Glutamine / Q000494:pharmacology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000096:biosynthesis; D058228:Islet Amyloid Polypeptide; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007651:Keto Acids / Q000494:pharmacology; D007930:Leucine / Q000494:pharmacology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D013997:Time Factors; D014044:Tolbutamide / Q000494:pharmacology
D000682:Amyloid; D017077:Culture Media, Conditioned; D007004:Hypoglycemic Agents; D007328:Insulin; D058228:Islet Amyloid Polypeptide; D007651:Keto Acids; D005973:Glutamine; C013082:alpha-ketoisocaproic acid; D014044:Tolbutamide; D007930:Leucine; D005947:Glucose