Temperature-sensitive random insulin granule diffusion is a prerequisite for recruiting granules for release.
Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance measurements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 degrees C to 24 degrees C caused a dramatic 81% drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of directed events (estimated energy of activation approximately 135 kJ/mol) than that of the granule velocities (approximately 22 kJ/mol) suggests that cooling-induced suppression of insulin granule movement is attributable to factors other than reduced motor protein adenosine 5'-triphosphatase activity. Indeed, cooling suppresses random granule diffusion by approximately 50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion.
Ivarsson
Rosita
R
The Diabetes Programme at Lund University, Department of Physiological Sciences, BMC B11, SE-221 84 Lund, Sweden.
Obermüller
Stefanie
S
Rutter
Guy A
GA
Galvanovskis
Juris
J
Renström
Erik
E
D000818:Animals; D004058:Diffusion; D004594:Electrophysiology; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D018613:Microscopy, Confocal; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured
D007328:Insulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins