INFLUENCE OF CRYOPRESERVATION ON THE SENSITIVITY OF HUMAN ISLETS TO TUMOR-NECROSIS-FACTOR

Tumor necrosis factor (TNF alpha) has been shown to inhibit insulin re lease and it has been postulated to be an important effector in islet rejection. We studied the effect of cryopreservation on glucose oxidat ion rate (GOR), lipid synthesis, hormone secretion (insulin, glucagon, somatostatin, thyrotropin-releasing hormone), and cyclic guanosine 3' ,5'-monophosphate (cGMP) content of human islets, in the presence or a bsence of TNF alpha, looking for changes that could explain a differen t susceptibility to rejection for cryopreserved islets. Islets were is olated from multiple organ donor pancreata by collagenase digestion. T he islets were then cultured for 7 days, cryopreserved (-0.25 degrees C/min), and stored in liquid N-2. After 24 h of culture, thawed islets were cultured for another 24 h in the presence or absence of TNF alph a. Islets were then washed to remove the cytokine and incubated in Kre bs-Ringer bicarbonate (5 or 20 mM glucose), and both the cGMP content of the islets and the hormone concentration in the medium were determi ned by radioimmunoassay. GOR was measured as the production of (CO2)-C -14 from 5 or 20 mM D-[U-C-14]glucose, and de novo lipid synthesis was determined as D-[U-C-14]glucose incorporation into different lipidic fractions. Cryopreservation did not significantly modify the hormone r esponse to glucose but it partially reversed the TNF alpha-induced inh ibitory effect on insulin release in the presence of 20 mM glucose. In addition, the inhibitory effect of TNF alpha on phosphatidylcholine l abeling was attenuated in cryopreserved islets compared with noncryopr eserved islets. TNF alpha significantly stimulated islet nitrite produ ction and cGMP accumulation, both effects being of a similar magnitude in cryopreserved and noncryopreserved islets. Our results suggest tha t cryopreservation can modify the metabolic and hormone response of hu man islets to TNF alpha. This effect is not mediated by changes in the TNF alpha-induced islet nitric oxide production or cGMP accumulation.