Modulation of JNK and p38 stress activated protein kinases in isolated islets of Langerhans: insulin as an autocrine survival signal.
OBJECTIVE
The objective of this study was to determine the effects of islet isolation and cytokine exposure on e-JUN NH2 terminal kinase (JNK) and p38 activation and whether insulin or the p38 inhibitor PD169316 could modify the response.


BACKGROUND
Islet transplantation exposes the cells of the graft to a variety of stressful stimuli that could promote beta-cell death and lead to graft failure.


METHODS
Islets from canine (n = 12) and cadaveric human (n = 6) pancreata were isolated and purified. Islets were cultured in CMRL 1066 with and without 100 ng/ml insulin. The response to cytokine stimulation with tumor necrosis factor (TNF)alpha and IL-1 beta and the p38 inhibitor PD169316 was also observed. Islet lysates were analyzed by Western blotting for total and phosphorylated JNK and p38 content. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL) assay and by a specific cell death enzyme-linked immunosorbant assay (ELISA).


RESULTS
In unstimulated islets, JNK activity was highest immediately following isolation, declining over 3 days to a low baseline level. The activity of p38 was lowest immediately after isolation, increasing progressively with time. The addition of insulin resulted in a more rapid decline in JNK activity, as opposed to p38, which showed no decrease in phosphorylation in response to insulin. In the cytokine stimulation studies, IL-1 beta stimulated p38 activation in a dose dependent manner, while JNK was relatively unaffected. PD169316 (100 microg/ml) was able to inhibit p38 activation in response to the isolation procedure as well as cytokine stimulation. Apoptotic activity was highest 24 hours after isolation, and was significantly reduced when islets were maintained in insulin-supplemented medium.


CONCLUSIONS
Inhibition of the stress-activated protein kinase (SAPK) pathways may be important for the maintenance of islet cell survival following islet isolation for transplantation. This study supports an autocrine role of insulin in this process.
Paraskevas
S
S
Departments of Surgery, the Montreal General Hospital Research Institute and McGill University, Montreal, Quebec, Canada.
Aikin
R
R
Maysinger
D
D
Lakey
J R
JR
Cavanagh
T J
TJ
Agapitos
D
D
Wang
R
R
Rosenberg
L
L
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002102:Cadaver; D002352:Carrier Proteins / Q000378:metabolism*; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007328:Insulin / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology; D007515:Islets of Langerhans / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007328:Insulin; D007375:Interleukin-1; C485750:MAPK8IP3 protein, human; D009419:Nerve Tissue Proteins; D014409:Tumor Necrosis Factor-alpha; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C408604:2-(4-nitrophenyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazole