Influence of cryopreservation on the sensitivity of human islets to tumor necrosis factor.
Tumor necrosis factor (TNF alpha) has been shown to inhibit insulin release and it has been postulated to-be an important effector in islet rejection. We studied the effect of cryopreservation on glucose oxidation 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 absence of TNF alpha, looking for changes that could explain a different susceptibility to rejection for cryopreserved islets. Islets were isolated from multiple organ donor pancreata by collagenase digestion. The islets were then cultured for 7 days, cryopreserved (-0.25 degrees C/min), and stored in liquid N2. After 24 h of culture, thawed islets were cultured for an other 24 h in the presence or absence of TNF alpha. Islets were then washed to remove the cytokine and incubated in Krebs-Ringer bicarbonate (5 or 20 mM glucose), and both the cGMP content of the islets and the hormone concentration in the medium were determined by radio-immunoassay. GOR was measured as the production of 14CO2 from 5 or 20 mM D-[U-14C]glucose, and de novo lipid synthesis was determined as D-[U-14C]glucose incorporation into different lipidic fractions. Cryopreservation did not significantly modify the hormone response to glucose but it partially reversed the TNF alpha-induced inhibitory effect on insulin release in the presence of 20 mM glucose. In addition, the inhibitory effect of TNF alpha on phosphatidylcholine labeling was attenuated in cryopreserved islets compared with noncryopreserved islets. TNF alpha significantly stimulated islet nitrite production and cGMP accumulation, both effects being of a similar magnitude in cryopreserved and noncryopreserved islets. Our results suggest that cryopreservation can modify the metabolic and hormone response of human islets to TNF alpha. This effect is not mediated by changes in the TNF alpha-induced islet nitric oxide production or cGMP accumulation.
Vara
E
E
Department of Biochemistry, Facultad de Medicina, Universidad Complutense, Madrid, Spain.
Arias-Díaz
J
J
Garcia
C
C
Balibrea
J L
JL
D000293:Adolescent; D000328:Adult; D015925:Cryopreservation; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004351:Drug Resistance; D005947:Glucose / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D016381:Islets of Langerhans Transplantation; D008055:Lipids / Q000096:biosynthesis; D008875:Middle Aged; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D013004:Somatostatin / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D007328:Insulin; D008055:Lipids; D009573:Nitrites; D014409:Tumor Necrosis Factor-alpha; D013004:Somatostatin; D000242:Cyclic AMP; D006152:Cyclic GMP; D005947:Glucose