Pancreatic islets were isolated from 16 nondiabetic organ donors and, after culture for ϳ2 days in 5 mmol/l glucose, were perifused to characterize nutrient-induced insulin secretion in human islets. Stepwise increases from 0 to 30 mmol/l glucose (eight 30-min steps) evoked concentrationdependent insulin secretion with a threshold at 3-4 mmol/l glucose, K m at 6.5 mmol/l glucose, and V max at 15 mmol/l glucose. An increase from 1 to 15 mmol/l glucose induced biphasic insulin secretion with a prominent first phase (peak increase of ϳ18-fold) and a sustained, flat second phase (ϳ10-fold increase), which were both potentiated by forskolin. The central role of ATP-sensitive K ؉ channels in the response to glucose was established by abrogation of insulin secretion by diazoxide and reversible restoration by tolbutamide. Depolarization with tolbutamide or KCl (plus diazoxide) triggered rapid insulin secretion in 1 mmol/l glucose. Subsequent application of 15 mmol/l glucose further increased insulin secretion, showing that the amplifying pathway is operative. In control medium, glutamine alone was ineffective, but its combination with leucine or nonmetabolized 2-amino-bicyclo [2,2,1]-heptane-2-carboxylic acid (BCH) evoked rapid insulin secretion. The effect of BCH was larger in low glucose than in high glucose. In contrast, the insulin secretion response to arginine or a mixture of four amino acids was potentiated by glucose or tolbutamide. Palmitate slightly augmented insulin secretion only at the supraphysiological palmitateto-albumin ratio of 5. Inosine and membrane-permeant analogs of pyruvate, glutamate, or succinate increased insulin secretion in 3 and 10 mmol/l glucose, whereas lactate and pyruvate had no effect. In conclusion, nutrientinduced insulin secretion in normal human islets is larger than often reported. Its characteristics are globally similar to those of insulin secretion by rodent islets, with both triggering and amplifying pathways. The pattern of the biphasic response to glucose is superimposable on that in mouse islets, but the concentration-response curve is shifted to the left, and various nutrients, in particular amino acids, influence insulin secretion within the physiological range of glucose concentrations. Diabetes 55: 3470 -3477, 2006

Pig islets encapsulated in a subcutaneous macrodevice can control diabetes up to 6 months without immunosuppression.

Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.

-The biphasic pattern of glucose-induced insulin secretion is altered in type 2 diabetes. Impairment of the first phase is an early sign of ␤-cell dysfunction, but the underlying mechanisms are still unknown. Their identification through in vitro comparisons of islets from diabetic and control subjects requires characterization and quantification of the dynamics of insulin secretion by normal islets. When perifused normal human islets were stimulated with 15 mmol/l glucose (G15), the proinsulin/insulin ratio in secretory products rapidly and reversibly decreased (ϳ50%) and did not reaugment with time. Switching from prestimulatory G3 to G6 -G30 induced biphasic insulin secretion with flat but sustained (2 h) second phases. Stimulation index reached 6.7-and 3.6-fold for the first and second phases induced by G10. Concentration dependency was similar for both phases, with halfmaximal and maximal responses at G6.5 and G15, respectively. First-phase response to G15-G30 was diminished by short (30 -60 min) prestimulation in G6 (vs. G3) and abolished by prestimulation in G8, whereas the second phase was unaffected. After 1-2 days of culture in G8 (instead of G5), islets were virtually unresponsive to G15. In both settings, a brief return to G3-G5 or transient omission of CaCl2 restored biphasic insulin secretion. Strikingly, tolbutamide and arginine evoked immediate insulin secretion in islets refractory to glucose. In conclusion, we quantitatively characterized the dynamics of glucose-induced insulin secretion in normal human islets and showed that slight elevation of prestimulatory glucose reversibly impairs the first phase, which supports the view that the similar impairment in type 2 diabetic patients might partially be a secondary phenomenon. human islets; insulin secretion; biphasic pattern; type 2 diabetes; proinsulin GLUCOSE HOMEOSTASIS depends on a precise quantitative and temporal regulation of insulin secretion by pancreatic ␤-cells. When the concentration of glucose is rapidly and steadily increased, insulin secretion displays a biphasic time course. This peculiar dynamic was suspected from measurements of insulin in peripheral and portal blood in humans (4, 10) and convincingly established by in vitro experiments using the perfused rat pancreas and perifused islets (12, 41). Experimental studies in rodents have variably attributed the biphasic pattern to the release of distinct pools of insulin granules or to the time course of intracellular signals, in particular free cytosolic Ca 2ϩ , in ␤-cells (see reviews in Refs. 28,51,60,62,and 63).Although blood glucose concentrations never increase rapidly enough to induce biphasic insulin secretion in daily life, the pattern is a most sensitive expression of adequate ␤-cell functioning (9). Pioneer studies measuring changes in plasma insulin after intravenous bolus administration of glucose and more recent ones calculating the insulin secretion rate by deconvolution of C-peptide concentrations during hyperglycemic clamps have established that impairment of the f...

Background-A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non-human primates. The detrimental instant bloodmediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss; this reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen.

Bone nonunion is a pathological condition in which all bone healing processes have stopped, resulting in abnormal mobility between 2 bone segments. The incidence of bone-related injuries will increase in an aging population, leading to such injuries reaching epidemic proportions. Tissue engineering and cell therapy using mesenchymal stem cells (MSCs) have raised the possibility of implanting living tissue for bone reconstruction. Bone marrow was first proposed as the source of stem cells for bone regeneration. However, as the quantity of MSCs in the bone marrow decreases, the capacity of osteogenic differentiation of bone marrow stem cells is also impaired by the donor’s age in terms of reduced MSC replicative capacity; an increased number of apoptotic cells; formation of colonies positive for alkaline phosphatase; and decreases in the availability, growth potential, and temporal mobilization of MSCs for bone formation in case of fracture. Adipose-derived stem cells (ASCs) demonstrate several advantages over those from bone marrow, including a less invasive harvesting procedure, a higher number of stem cell progenitors from an equivalent amount of tissue harvested, increased proliferation and differentiation capacities, and better angiogenic and osteogenic properties in vivo. Subcutaneous native adipose tissue was not affected by the donor’s age in terms of cellular senescence and yield of ASC isolation. In addition, a constant mRNA level of osteocalcin and alkaline phosphatase with a similar level of matrix mineralization of ASCs remained unaffected by donor age after osteogenic differentiation. The secretome of ASCs was also unaffected by age when aiming to promote angiogenesis by vascular endothelial growth factor (VEGF) release in hypoxic conditions. Therefore, the use of adipose cells for bone tissue engineering is not limited by the donor’s age from the isolation of stem cells up to the manufacturing of a complex osteogenic graft.

These results demonstrated that STZ might be used to induce irreversible diabetes in rats and primates. In contrast, the low STZ sensitivity in pigs related to a low expression of GLUT2, higher number of immature beta cells and compensatory beta-cell hypertrophy, renders STZ-induced diabetes inappropriate for studying islet allografts in swine.

Morphological screening of the pig pancreas before isolation, optimal blood exsanguination, WIT <10 min, and an endotoxin content <30 EU/mg in Liberase PI batches determine successful pig islet isolation for xenotransplantation in primates.