Effect of Donor Age on Function of Isolated Human Islets
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糖尿病学杂志 2006年第5期
the Diabetes Institute for Immunology and Transplantation, Department of Surgery, University of Minnesota, Minneapolis, Minnesota
AIR, acute insulin response; AUC, area under the curve; CIT, cold ischemia time; CPI, C-peptide increment; GSIR, glucose-stimulated insulin release; HPLC, high-performance liquid chromatography; IRI, immunoreactive insulin; IVGTT, intravenous glucose tolerance test; OGTT, oral glucose tolerance test
ABSTRACT
This study intended to evaluate the impact of donor age on the function of isolated islets. Analysis of human islets from cadaveric donors (age 16eC70 years) was performed using glucose-stimulated insulin release (GSIR) (n = 93), islet ATP content (n = 27), diabetic nude mouse bioassay (n = 72), and the insulin secretory function after single-donor clinical islet allotransplantation (n = 7). The GSIR index was significantly higher in younger donors (age 40 years) than in older donors and negatively correlated with the donor age (r = eC0.535). Islet ATP was higher in younger donors (115.7 ± 17.7 vs. 75.7 ± 6.6 pmol/e蘥 DNA). The diabetes reversal rate of mice with 2,000 IE was significantly higher in younger donors (96 vs. 68%). C-peptide increment to glucose during intravenous glucose tolerance test at days 90eC120 after clinical transplantation showed negative correlation with donor age (r = eC0.872) and positive correlation with the islet mass (r = 0.832). On the other hand, acute insulin response to arginine only showed correlation with the islet mass and not with donor age. These results show that insulin secretory response to glucose deteriorates with increasing age and that it may be related to changes in ATP generation in -cells.
Aging is associated with an increased incidence of diabetes and impaired glucose tolerance (1). The decline in glucose tolerance is known to begin in the third or fourth decade of life and is progressive throughout the life span (2). Age-related change in insulin secretory function may contribute to the alterations in glucose metabolism with age. Many studies of the effect of aging on insulin secretion suggest that there is a relative insulin secretory defect in light of increased insulin resistance with aging (3). However, the results of human in vivo studies vary widely due to multiple confounding factors associated with aging such as decreased lean body mass and decreased physical activity. In rats, both in vitro and in vivo syngeneic islet transplantation studies have demonstrated that -cell function is negatively influenced by age (4eC9). One in vitro study with human islets has shown that islets from donors >50 years of age had lower insulin secretory responses to glucose than those from the juvenile donors (10). Clinical islet transplantation studies have not yet identified the effect of donor age on islet function posttransplant.
The success rate of allogeneic islet transplants for selected patients with type 1 diabetes has increased markedly by transplanting a large islet mass prepared from two to four donor pancreata and using a glucocorticoid-free immunosuppressive protocol (11eC13). However, despite improvements in islet isolation techniques, human islet transplants from a single donor have not been widely successful. Considering the shortage of donor pancreases, the ability to reliably obtain large numbers of high-quality islets from a single human pancreas would be of great benefit to human islet allotransplantation. Through studies on the factors influencing the recoverability of viable islets, donor characteristics including age have been shown to be possible determinants of the islet yield and function (10,14,15). The observation that pancreas grafts from donors >45 years of age were associated with an increased risk of developing poor glycemic control and premature loss of pancreas function (16,17) strongly suggests that donor age might also affect the outcome of human islet transplantation.
Therefore, this study was performed to determine whether there is any functional difference in isolated human islets according to donor age. We show that metabolic function of islets deteriorates with advancing donor age using the in vitro glucose-stimulated insulin release (GSIR) assay, nude mouse transplant bioassay, and the assessment of insulin secretory function after successful single-donor clinical islet allotransplantation. The possible mechanisms and the implications for human islet transplantation are discussed.
RESEARCH DESIGN AND METHODS
1 January 2000 to 4 December 2002, a single team at our institution processed 125 consecutive human cadaver donor pancreata. A total of 15 pancreata were excluded from our analysis for the following reasons: incomplete data (n = 4), technical failure in islet isolation (n = 1), prolonged (>18 h) cold ischemia time (CIT) (n = 5), and islet isolation from partial pancreata (n = 5). We analyzed the remaining 110 islet isolations by dividing the pancreata into two groups according to donor age: group 1, n = 41, age 40 years; group 2, n = 69, age >40 years. We compared the results of islet isolation, in vitro assays for islet quality, and diabetic nude mouse transplant bioassay between the two groups. We also analyzed the insulin secretory function of seven successful single-donor islet allografts with regard to donor age.
Islet isolation.
After informed consent had been obtained, cadaveric pancreata were removed from brain-dead donors as part of multiorgan procurement and were preserved either in cold University of Wisconsin solution (n = 72) or using a two-layer (University of Wisconsin/perfluorochemical) cold-storage method (n = 38) (18,19). On arrival at our laboratory, the preserved pancreas was perfused with a cold Liberase enzyme (Roche, Indianapolis, IN) through the pancreatic duct. The distended pancreas was dissociated using the automated method of Ricordi et al. (20), and islets were purified on gradients of iodixanol (Nycomed, Oslo, Norway) or Ficoll-diatrizoic acid (Biochrom, Berlin, Germany) using a Cobe 2991 cell separator (Gambro, Lakewood, CO). Islet yields were quantified by dithizone staining in duplicate using a standard islet diameter of 150 e蘭 as one islet equivalent (IE). Purified islets were cultured in CMRL (Connaught Medical Research Laboratories) 1066 medium with 0.5% human serum albumin or 10% fetal bovine serum before in vitro assessment or nude mouse transplant bioassay.
GSIR in vitro.
After overnight culture, aliquots of islets were incubated in RPMI medium containing 1.7 mmol/l glucose at 37°C for 30 min. Five hand-picked islets were incubated in RPMI containing either 1.7 or 16.7 mmol/l glucose at 37°C for 1 h in replicates of five. After incubation, supernatants were collected for immunoreactive insulin (IRI) measurement by enzyme immunoassay. Released insulin was expressed as e蘒 IRI · ng DNAeC1 · heC1. DNA content was measured using PicoGreen (Molecular Probes, Eugene, OR). The mean amount of insulin released in response to 16.7 mmol/l glucose was divided by the mean amount released by 1.7 mmol/l glucose to yield the GSIR index.
Insulin content.
After 48-h culture in CMRL 1066 medium containing 5.6 mmol/l glucose, triplicate aliquots of islets were washed with PBS. The islet pellets were suspended in 2 mmol/l acetic acid containing 0.25% BSA and shaken at 4°C for 18 h. After centrifugation, the supernatant was collected for insulin measurement by enzyme immunoassay and the pellets saved for DNA quantification. Insulin content of islets was expressed as microunits of IRI per nanogram of DNA.
Islet ATP content.
After isolation, nucleotides were immediately extracted from aliquots of 1,000 IE by adding 200 e蘬 ice-cold 10% trichloroacetic acid. After neutralization with 0.5 mol/l tri-n-octylamine (Sigma, St. Louis, MO) in 1,1,2-trichlorotrifluoroethane (freon) (Sigma), the phases were separated by centrifugation. The top neutral aqueous layer was collected and stored at eC80°C until high-performance liquid chromatography (HPLC) analysis, and the pellet was stored for DNA determination. HPLC was performed using a 3 e蘭, 15 cm x 4.6 mm Supelcosil LC-18-T column (Supelco, Bellefonte, PA) (21). ATP content of islets was expressed as picomoles of ATP per microgram DNA.
Nude mouse transplant bioassay.
Male athymic nude mice (National Cancer Institute, Hartford, CT) were used in compliance with the guidelines from the Institutional Animal Care Committee at the University of Minnesota. Diabetes was induced by intravenous injection of 240 mg/kg streptozotocin. Mice were considered diabetic when their blood glucose levels were 400 mg/dl for 2 consecutive days. After 48 h of culture, 1,000 and 2,000 IE were transplanted as pellets under the left kidney capsule of diabetic nude mice. After transplantation, random blood glucose levels were measured every day for the first week, then 3 days per week. Cure was defined as two consecutive blood glucose levels <200 mg/dl. At 30 days posttransplant, left nephrectomy was performed in the cured mice to confirm a return to hyperglycemia.
Assessment of insulin secretory function after clinical transplantation.
In eight C-peptideeCnegative, nonuremic, type 1 diabetic patients with hypoglycemia unawareness, we transplanted islets isolated from single donors with two-layer pancreas preservation purified on iodixanol density gradients and cultured for 48 h pretransplant (22). Institutional review board approval and informed consent had been obtained. After establishing access to the portal vein via mini-laparotomy or percutaneous transhepatic catheterization, 7,271 ± 366 IE/kg recipient body wt were infused. All participants received modified induction immunosuppression with antithymocyte globulin, daclizumab, and etanercept and modified maintenance immunosuppression with mycophenolate mofetil, sirolimus, and either no or ultraeClow-dose tacrolimus. All eight recipients achieved insulin independence 23eC112 days after transplantation. We performed intravenous glucose tolerance tests (IVGTTs), arginine stimulation tests, and oral glucose tolerance tests (OGTTs) 90eC120 days after transplantation in seven recipients who were insulin independent at the time of tests.
For IVGTTs, after two baseline samplings (eC10 and 0 min) for glucose, C-peptide, and insulin, 300 mg glucose/kg body wt was infused over 1 min in the fasting state. Consecutive samples were taken at 3, 4, 5, 7, 10, 15, 20, 25, and 30 min. On a separate day, 5 g arginine HCl were infused intravenously over 0.5 min in the fasting state, and C-peptide and insulin levels were measured at eC10, 0, 2, 3, 4, 5, 7, and 10 min. The C-peptide increment (CPI) to glucose and the acute insulin response (AIR) to glucose were calculated as the mean of the C-peptide or insulin level at 3, 4, and 5 min after the infusion minus the mean basal levels. The areas under the curve (AUCs) for C-peptide and insulin were calculated as the AUC above baseline over 30 min postinfusion. The CPI and AIR to arginine were calculated as the mean of the three highest values for 2, 3, 4, and 5 min postinfusion minus the mean basal values. Serum C-peptide and insulin were measured using a commercial enzyme immunoassay (Mercodia, Uppsala, Sweden) in duplicate. The OGTT was performed with 75 g oral glucose.
Statistics.
All statistical and regression analyses were performed using the MedCalc program (Mariakerke, Belgium). All data were expressed as means ± SE or otherwise as indicated. Several variables of glucose-stimulated insulins released in vitro and insulin contents did not show a normal distribution (Kolmogorov-Smirnov test), and natural logarithmic transformation was used to render the distribution of these variables normal (ln). The differences between groups 1 and 2 were considered significant if the P value was <0.05 using an unpaired Student’s t test, Wilcoxon’s signed-rank test, or a 2 test.
RESULTS
Islet donor characteristics.
There were no significant differences in BMI, CIT, or pancreas procurement team between groups 1 and 2 (Table 1). However, group 1 (age 40 years) had significantly higher proportions of males, trauma as a cause of death, and two layer as a preservation method (Table 1).
Islet isolation yield.
In multiple regression analysis with donor age, sex, cause of death, and preservation method as independent variables, both donor age and preservation method were found to significantly influence the islet yields. Thus, we analyzed the islet yield variables versus donor age according to the preservation method (Table 2). Regardless of the preservation method, postdigest islet yields (islet equivalents per pancreas and islet equivalents per gram) were significantly higher in group 1 (age 40 years) than in group 2. Postpurification islet yields were also higher in group 1 (age 40 years) than in group 2 with either preservation method, although not statistically significant. There was no significant difference in islet equivalents per islet counts between two groups. The percentage of embedded islets was significantly higher in group 1 (age 40 years) than in group 2 in University of Wisconsin preservation cases.
In vitro quality assessment of isolated islets.
In multiple regression analysis with donor age, sex, cause of death, and preservation method as independent variables, only the donor age was found to significantly influence the GSIR index. The GSIR index was significantly higher in group 1 (age 40 years) than in group 2 (Table 3). The insulin release to low glucose concentration (1.7 mmol/l) was significantly lower in group 1 (age 40 years), but the insulin release to high glucose concentration (16.7 mmol/l) was not significantly different between two groups (Table 3). In correlation analysis, the log-transformed GSIR index was negatively correlated with the donor age (Fig. 1A; r = eC0.535, P < 0.001). The log-transformed amount of insulin released to low glucose concentration also correlated positively with the donor age (Fig. 1B; r = 0.445, P < 0.001). There was no significant difference in insulin content between the two groups (Table 3). Islet ATP content was higher in group 1 (age 40 years) than in group 2 (115.7 ± 17.7 vs. 75.7 ± 6.6 pmol/e蘥 DNA, P = 0.030) and negatively correlated with the donor age (Fig. 2; r = eC0.536, P = 0.004).
Nude mouse transplant bioassay.
The diabetes reversal rate and days to diabetes reversal for transplanted diabetic mice with 1,000 and 2,000 IE are shown in Fig. 3A and B). The diabetes reversal rate at 4 weeks with 2,000 IE was significantly higher in group 1 (age 40 years) than in group 2 (96 vs. 68%, P = 0.023). Although the diabetes reversal rate at 1 week with 1,000 IE was also significantly higher in group 1 (age 40 years) than in group 2 (58 vs. 24%, P = 0.025), at 4 weeks it was not significantly different between two groups (58 vs. 41%). In multiple logistic regression analysis with donor age, sex, cause of death, and preservation method as independent variables, only the donor age was significantly related to transplantation outcome with 2,000 IE. The donor age of 2,000 IE grafts, which reversed diabetes in nude mice, was significantly lower than that of grafts that failed to reverse diabetes (Fig. 3C).
Assessment of insulin secretory function after clinical transplantation.
CPI to glucose as derived from IVGTT performed at days 90eC120 after clinical transplantation showed significant negative correlation with donor age (Fig. 4A) and positive correlation with the number of islets transplanted (Table 4). In multiple regression analysis both donor age and the number of islets transplanted were significant independent variables for CPI to glucose. AUC for C-peptide also showed significant negative correlation with donor age. AIR to glucose showed the same correlation trend with either donor age or the number of islets transplanted, although their P values were 0.074 and 0.080, respectively. CPI to glucose and AUC for C-peptide showed stronger positive correlation with islet equivalents transplanted/donor age than with islet equivalents transplanted alone (Table 4), and CPI to glucose/islet equivalents transplanted also showed significant negative correlation with donor age (Fig. 4B). On the other hand, CPI and AIR to argine as derived from intravenous arginine stimulation test performed on days 90eC120 after clinical transplantation showed positive correlation only with the number of islets transplanted and did not correlate with donor age (Table 4).
In an analysis of the relationship of measures of insulin secretion and glycemic control, only CPI to glucose was significantly correlated with fasting plasma glucose (r = eC0.742, P = 0.050) and 2-h glucose after oral glucose load (r = eC0.763, P = 0.046). Both fasting plasma glucose and 2-h postload glucose also showed negative correlation with the number of islets transplanted (r = eC0.859, P = 0.013 and r = eC0.828, P = 0.021, respectively), but no significant relationship was observed between measures of glycemia and donor age in these seven insulin independent recipients.
DISCUSSION
Deterioration in -cell function is an independent predictor for the development of impaired glucose tolerance and type 2 diabetes (23,24). An impact on -cell function by aging has been assessed in many in vivo studies with widely varying results due to multiple confounding factors associated with aging such as obesity and decreased physical activity (3,25eC27). Our study clearly demonstrates that GSIR of isolated human islets deteriorates with advancing donor age. This loss of insulin secretory function with age was independent of donor BMI, sex, cause of death, or other procurement and isolation variables, suggesting that it could be a direct consequence of the process of aging. This finding supports that the decline in -cell function with aging can contribute to the increasing development of impaired glucose tolerance and type 2 diabetes and also to the progressive nature of the disease irrespective of an accompanying insulin resistance and/or hyperglycemia. Although we excluded the donors with the history of type 1, type 2, and gestational diabetes, we could not measure HbA1c in all the donors of this study and thus could not rule out that some donors, especially in the older group, might have had undiagnosed type 2 diabetes. Considering the age distribution of the older donors included in Fig. 1A (40eC49 years old, n = 29; 50eC59 years old, n = 23; 60eC64 years old, n = 5) and the percentage of diabetes in this age population (1,3), we may presume that the number of donors with undiagnosed diabetes, if any, was quite a few and did not skew the results.
Our results with a nude mouse transplant bioassay show a negative impact of aging on isolated human islet function posttransplant. Similar findings have been reported in syngeneic rat islet transplants; islets from 42-week-old donor rats failed to cure diabetes in streptozotocin-induced diabetic rats, while an identical number of islets from 10-week-old rats restored normoglycemia in the majority of recipients (8). Our observation that the diabetes reversal rate with the 2,000 IE graft from donors >40 years of age is not lower than that with the 1,000 IE graft from donors 40 years of age shows that the number of islets can be the major determinant for the immediate correction of hyperglycemia. However, the previous report that the use of older donor rats shortened the period of posttransplant normoglycemia after the correction of hyperglycemia suggested that the older islets may progressively deteriorate, leading to a shorter life span and/or to a loss of their physiologic responses (9). Thus, donor age might influence not only the cure rate depending on the graft mass but also the duration of metabolic normalization. Whether the transplantation of sufficient islet mass can overcome the negative effect of donor age on the duration of insulin independence needs to be evaluated in human islet transplantation.
In IVGTTs and arginine stimulation tests performed at 90eC120 days after transplantation in our seven insulin-independent recipients, insulin secretory response to either glucose or arginine correlated with the islet mass transplanted. This result is in accord with the observation from the Edmonton group that all the measures of insulin reserve in the recipients correlated with the islet mass transplanted at 3 months (13). Islet grafts from older donors may be less efficient in maintaining the -cell mass in the implant. It is known that the regenerative capacity of -cells decreases with age (28,29). Recently, this decreased formation of new islets with aging has also been reported with pancreatic tissue from 124 autopsies (30). Our result that insulin secretory responses to either glucose or arginine correlated with the islet mass transplanted irrespective of donor age suggests that, at least for 90eC120 days posttransplant, the older islets do not decrease in -cell mass faster than younger islets to a detectable degree.
In our clinical transplants, all eight recipients initially achieved insulin independence after the transplantation of 7,271 ± 366 IE/kg regardless of donor age. IVGTTs at 90eC120 days posttransplant in seven insulin independent recipients revealed significant negative correlation between insulin secretion parameters to glucose and donor age of islets, which was independent of the number of islets transplanted. The mechanism underlying this age-related deterioration of insulin secretory -cell function is not known. One study in rats indicated that glucose signal recognition and/or stimulus-secretion coupling may be the locus of impairment in the process of insulin secretion in older animals (6). Several in vivo studies have revealed an increased disorderliness of basal insulin release and an impairment of proinsulin conversion to insulin in older individuals (31eC33). Our in vitro finding of higher insulin release to low concentration of glucose (1.7 mmol/l) and lower GSIR index strongly suggests that the insulin secretory regulation of -cells by glucose is less efficient in older islets.
The mechanisms of insulinotropic action of glucose and arginine are different. While glucose stimulates insulin release by increasing intracellular ATP, arginine shows insulinotropic action by direct depolarization of plasma membrane (34) without involving changes in mitochondrial oxidative phosphorylation and production of ATP. The observation that patients with maternally inherited diabetes and deafness caused by mitochondrial tRNALEU(UUR) gene mutation showed impaired insulin secretory response to glucose but normal insulin secretion to arginine (35,36) supports the idea that the arginine stimulation test could be helpful in locating the defects in insulin secretion. In our seven recipients, insulin secretory responses to glucose but not to arginine showed negative correlation with donor age. This finding suggests that aging may affect the process related to increasing intracellular ATP in -cells. Our in vitro result that islet ATP content/DNA negatively correlates with donor age also lends support to the hypothesis that the intracellular ATP metabolism or mitochondrial function may be a target of -cell aging.
Although no study has reported the direct measurement of mitochondrial ATP synthesis in -cells with age, several mitochondrial functions have been observed to decline with age (37). Oxidative mitochondrial DNA lesions and mutations are found to increase markedly with age (38). It is plausible that the accumulation of such mutations leads to decreased gene expression, a decline in oxidative phosphorylation, and inefficient electron transport. Age-dependent decline of the mitochondria DNA content of tissues (39,40) and mitochondrial membrane potentials (41) have been reported. The report that insulin secretion evoked by glucose but not by plasma membrane depolarization was markedly blunted with decline in mitochondrial membrane potential following exposure to H2O2 also supported the hypothesis that mitochondria may be a target of oxidative damage in -cells during aging (42).
Among the various donor parameters, we found that donor age is an important determinant of islet function in terms of insulin secretion. Noteworthy is that both GSIR index and nude mouse transplant bioassay, which have been widely used to assess the functional quality of isolated islets, should be interpreted with the consideration of donor age. We observed a stronger correlation between islet equivalents transplanted/donor age and CPI to glucose than between islet equivalents transplanted and CPI to glucose in an attempt to take into account the donor age for a given islet preparation, as the Edmonton group did for the CIT index (12,13). This C-peptide response to intravenous glucose also correlated with the measures of glycemia (fasting plasma glucose and 2-h glucose during OGTT). Taken together, it seems highly likely that the higher the donor age the larger the islet mass required for better islet transplantation outcomes. Up to 90eC120 days after transplantation, however, we could not find a relationship between donor age and the measures of glycemia in seven recipients who were not taking exogenous insulin, although there was a significant negative correlation between the number of islets transplanted and the fasting plasma glucose or the 2-h postload glucose level. It may take longer follow-up in a larger number of recipients to determine the effect of donor age on clinical outcomes.
In contrast to the previous reports showing that successful islet isolations increased with increased donor age (10,43), our analysis showed that postdigest islet yields were higher in younger donors (age 40 years) than in older donors regardless of preservation methods and that postpurification yields were not significantly different between the two groups. However, postpurification recovery rates of islets tended to be lower in younger donors. Islets from juvenile donors are known to be difficult to separate from the acinar tissue without causing excessive fragmentation during digestion (10). Our observation that among younger donors embedded islets are relatively low when using two-layer preservation suggests that the increased use of this preservation method may further improve islet yields from younger donors. Thus, younger donors may be advantageous not only in terms of insulin secretory function but also with regard to islet isolation yield.
Donors >45eC60 years of age have been traditionally considered less than ideal for organ transplantation. For example, kidneys from donors >55 or 60 years of age overall have reduced functional reserve, which has an adverse effect on long-term graft survival rates (44eC46). The pancreas grafts from the >45-year-old donors also had significantly lower 1- and 5-year graft survival rates after primary simultaneous pancreas-kidney transplantations (16,17). However, considering the shortage of cadaveric donors, there is a strong need to maximally utilize the potential donor pool. Toward this goal, the transplantation of a higher mass of older islets prepared from two to four pancreata may have some role in human islet transplantation.
In summary, this study shows that insulin secretory regulation by glucose deteriorates with advancing age and that it may be related to ATP generation change in -cells with aging. These findings suggest that islet preparations from younger donors may improve the success rate of single-donoreCalone transplantation and that the higher the donor age the larger islet mass might be required to restore insulin independence posttransplant. Whether transplanting a sufficient islet mass from multiple pancreata can overcome the negative effect of donor age needs to be evaluated to fully utilize the available relatively small donor pool in human islet transplantation.
ACKNOWLEDGMENTS
The study was supported by grants from the National Center for Research Resources, National Institutes of Health (MO1-RR00400 and U42 RR016598-01), the Juvenile Diabetes Research Foundation (JDRF 4-1999-841), Roche Laboratories (RO49272), and the Hallym Academy of Sciences, Hallym University, Korea (to S.-H.I.).
We are indebted to Dr. Raja Kandaswamy, Dr. David Hunter, Jamen Parkey, Kathy Duderstadt, Kathy Hodges, Carrie Gibson, and the staff members of the University of Minnesota General Clinical Research Center for excellent patient care; to Jeremy Oberbroeckling for his technical expertise in the islet isolation laboratory; and to Dr. Robin Jevne and Dylan Zylla for data compilation and analysis.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
S.-H.I. is currently affiliated with Hallym University Sacred Heart Hospital, Anyang, Kyonggi-do, Korea
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AIR, acute insulin response; AUC, area under the curve; CIT, cold ischemia time; CPI, C-peptide increment; GSIR, glucose-stimulated insulin release; HPLC, high-performance liquid chromatography; IRI, immunoreactive insulin; IVGTT, intravenous glucose tolerance test; OGTT, oral glucose tolerance test
ABSTRACT
This study intended to evaluate the impact of donor age on the function of isolated islets. Analysis of human islets from cadaveric donors (age 16eC70 years) was performed using glucose-stimulated insulin release (GSIR) (n = 93), islet ATP content (n = 27), diabetic nude mouse bioassay (n = 72), and the insulin secretory function after single-donor clinical islet allotransplantation (n = 7). The GSIR index was significantly higher in younger donors (age 40 years) than in older donors and negatively correlated with the donor age (r = eC0.535). Islet ATP was higher in younger donors (115.7 ± 17.7 vs. 75.7 ± 6.6 pmol/e蘥 DNA). The diabetes reversal rate of mice with 2,000 IE was significantly higher in younger donors (96 vs. 68%). C-peptide increment to glucose during intravenous glucose tolerance test at days 90eC120 after clinical transplantation showed negative correlation with donor age (r = eC0.872) and positive correlation with the islet mass (r = 0.832). On the other hand, acute insulin response to arginine only showed correlation with the islet mass and not with donor age. These results show that insulin secretory response to glucose deteriorates with increasing age and that it may be related to changes in ATP generation in -cells.
Aging is associated with an increased incidence of diabetes and impaired glucose tolerance (1). The decline in glucose tolerance is known to begin in the third or fourth decade of life and is progressive throughout the life span (2). Age-related change in insulin secretory function may contribute to the alterations in glucose metabolism with age. Many studies of the effect of aging on insulin secretion suggest that there is a relative insulin secretory defect in light of increased insulin resistance with aging (3). However, the results of human in vivo studies vary widely due to multiple confounding factors associated with aging such as decreased lean body mass and decreased physical activity. In rats, both in vitro and in vivo syngeneic islet transplantation studies have demonstrated that -cell function is negatively influenced by age (4eC9). One in vitro study with human islets has shown that islets from donors >50 years of age had lower insulin secretory responses to glucose than those from the juvenile donors (10). Clinical islet transplantation studies have not yet identified the effect of donor age on islet function posttransplant.
The success rate of allogeneic islet transplants for selected patients with type 1 diabetes has increased markedly by transplanting a large islet mass prepared from two to four donor pancreata and using a glucocorticoid-free immunosuppressive protocol (11eC13). However, despite improvements in islet isolation techniques, human islet transplants from a single donor have not been widely successful. Considering the shortage of donor pancreases, the ability to reliably obtain large numbers of high-quality islets from a single human pancreas would be of great benefit to human islet allotransplantation. Through studies on the factors influencing the recoverability of viable islets, donor characteristics including age have been shown to be possible determinants of the islet yield and function (10,14,15). The observation that pancreas grafts from donors >45 years of age were associated with an increased risk of developing poor glycemic control and premature loss of pancreas function (16,17) strongly suggests that donor age might also affect the outcome of human islet transplantation.
Therefore, this study was performed to determine whether there is any functional difference in isolated human islets according to donor age. We show that metabolic function of islets deteriorates with advancing donor age using the in vitro glucose-stimulated insulin release (GSIR) assay, nude mouse transplant bioassay, and the assessment of insulin secretory function after successful single-donor clinical islet allotransplantation. The possible mechanisms and the implications for human islet transplantation are discussed.
RESEARCH DESIGN AND METHODS
1 January 2000 to 4 December 2002, a single team at our institution processed 125 consecutive human cadaver donor pancreata. A total of 15 pancreata were excluded from our analysis for the following reasons: incomplete data (n = 4), technical failure in islet isolation (n = 1), prolonged (>18 h) cold ischemia time (CIT) (n = 5), and islet isolation from partial pancreata (n = 5). We analyzed the remaining 110 islet isolations by dividing the pancreata into two groups according to donor age: group 1, n = 41, age 40 years; group 2, n = 69, age >40 years. We compared the results of islet isolation, in vitro assays for islet quality, and diabetic nude mouse transplant bioassay between the two groups. We also analyzed the insulin secretory function of seven successful single-donor islet allografts with regard to donor age.
Islet isolation.
After informed consent had been obtained, cadaveric pancreata were removed from brain-dead donors as part of multiorgan procurement and were preserved either in cold University of Wisconsin solution (n = 72) or using a two-layer (University of Wisconsin/perfluorochemical) cold-storage method (n = 38) (18,19). On arrival at our laboratory, the preserved pancreas was perfused with a cold Liberase enzyme (Roche, Indianapolis, IN) through the pancreatic duct. The distended pancreas was dissociated using the automated method of Ricordi et al. (20), and islets were purified on gradients of iodixanol (Nycomed, Oslo, Norway) or Ficoll-diatrizoic acid (Biochrom, Berlin, Germany) using a Cobe 2991 cell separator (Gambro, Lakewood, CO). Islet yields were quantified by dithizone staining in duplicate using a standard islet diameter of 150 e蘭 as one islet equivalent (IE). Purified islets were cultured in CMRL (Connaught Medical Research Laboratories) 1066 medium with 0.5% human serum albumin or 10% fetal bovine serum before in vitro assessment or nude mouse transplant bioassay.
GSIR in vitro.
After overnight culture, aliquots of islets were incubated in RPMI medium containing 1.7 mmol/l glucose at 37°C for 30 min. Five hand-picked islets were incubated in RPMI containing either 1.7 or 16.7 mmol/l glucose at 37°C for 1 h in replicates of five. After incubation, supernatants were collected for immunoreactive insulin (IRI) measurement by enzyme immunoassay. Released insulin was expressed as e蘒 IRI · ng DNAeC1 · heC1. DNA content was measured using PicoGreen (Molecular Probes, Eugene, OR). The mean amount of insulin released in response to 16.7 mmol/l glucose was divided by the mean amount released by 1.7 mmol/l glucose to yield the GSIR index.
Insulin content.
After 48-h culture in CMRL 1066 medium containing 5.6 mmol/l glucose, triplicate aliquots of islets were washed with PBS. The islet pellets were suspended in 2 mmol/l acetic acid containing 0.25% BSA and shaken at 4°C for 18 h. After centrifugation, the supernatant was collected for insulin measurement by enzyme immunoassay and the pellets saved for DNA quantification. Insulin content of islets was expressed as microunits of IRI per nanogram of DNA.
Islet ATP content.
After isolation, nucleotides were immediately extracted from aliquots of 1,000 IE by adding 200 e蘬 ice-cold 10% trichloroacetic acid. After neutralization with 0.5 mol/l tri-n-octylamine (Sigma, St. Louis, MO) in 1,1,2-trichlorotrifluoroethane (freon) (Sigma), the phases were separated by centrifugation. The top neutral aqueous layer was collected and stored at eC80°C until high-performance liquid chromatography (HPLC) analysis, and the pellet was stored for DNA determination. HPLC was performed using a 3 e蘭, 15 cm x 4.6 mm Supelcosil LC-18-T column (Supelco, Bellefonte, PA) (21). ATP content of islets was expressed as picomoles of ATP per microgram DNA.
Nude mouse transplant bioassay.
Male athymic nude mice (National Cancer Institute, Hartford, CT) were used in compliance with the guidelines from the Institutional Animal Care Committee at the University of Minnesota. Diabetes was induced by intravenous injection of 240 mg/kg streptozotocin. Mice were considered diabetic when their blood glucose levels were 400 mg/dl for 2 consecutive days. After 48 h of culture, 1,000 and 2,000 IE were transplanted as pellets under the left kidney capsule of diabetic nude mice. After transplantation, random blood glucose levels were measured every day for the first week, then 3 days per week. Cure was defined as two consecutive blood glucose levels <200 mg/dl. At 30 days posttransplant, left nephrectomy was performed in the cured mice to confirm a return to hyperglycemia.
Assessment of insulin secretory function after clinical transplantation.
In eight C-peptideeCnegative, nonuremic, type 1 diabetic patients with hypoglycemia unawareness, we transplanted islets isolated from single donors with two-layer pancreas preservation purified on iodixanol density gradients and cultured for 48 h pretransplant (22). Institutional review board approval and informed consent had been obtained. After establishing access to the portal vein via mini-laparotomy or percutaneous transhepatic catheterization, 7,271 ± 366 IE/kg recipient body wt were infused. All participants received modified induction immunosuppression with antithymocyte globulin, daclizumab, and etanercept and modified maintenance immunosuppression with mycophenolate mofetil, sirolimus, and either no or ultraeClow-dose tacrolimus. All eight recipients achieved insulin independence 23eC112 days after transplantation. We performed intravenous glucose tolerance tests (IVGTTs), arginine stimulation tests, and oral glucose tolerance tests (OGTTs) 90eC120 days after transplantation in seven recipients who were insulin independent at the time of tests.
For IVGTTs, after two baseline samplings (eC10 and 0 min) for glucose, C-peptide, and insulin, 300 mg glucose/kg body wt was infused over 1 min in the fasting state. Consecutive samples were taken at 3, 4, 5, 7, 10, 15, 20, 25, and 30 min. On a separate day, 5 g arginine HCl were infused intravenously over 0.5 min in the fasting state, and C-peptide and insulin levels were measured at eC10, 0, 2, 3, 4, 5, 7, and 10 min. The C-peptide increment (CPI) to glucose and the acute insulin response (AIR) to glucose were calculated as the mean of the C-peptide or insulin level at 3, 4, and 5 min after the infusion minus the mean basal levels. The areas under the curve (AUCs) for C-peptide and insulin were calculated as the AUC above baseline over 30 min postinfusion. The CPI and AIR to arginine were calculated as the mean of the three highest values for 2, 3, 4, and 5 min postinfusion minus the mean basal values. Serum C-peptide and insulin were measured using a commercial enzyme immunoassay (Mercodia, Uppsala, Sweden) in duplicate. The OGTT was performed with 75 g oral glucose.
Statistics.
All statistical and regression analyses were performed using the MedCalc program (Mariakerke, Belgium). All data were expressed as means ± SE or otherwise as indicated. Several variables of glucose-stimulated insulins released in vitro and insulin contents did not show a normal distribution (Kolmogorov-Smirnov test), and natural logarithmic transformation was used to render the distribution of these variables normal (ln). The differences between groups 1 and 2 were considered significant if the P value was <0.05 using an unpaired Student’s t test, Wilcoxon’s signed-rank test, or a 2 test.
RESULTS
Islet donor characteristics.
There were no significant differences in BMI, CIT, or pancreas procurement team between groups 1 and 2 (Table 1). However, group 1 (age 40 years) had significantly higher proportions of males, trauma as a cause of death, and two layer as a preservation method (Table 1).
Islet isolation yield.
In multiple regression analysis with donor age, sex, cause of death, and preservation method as independent variables, both donor age and preservation method were found to significantly influence the islet yields. Thus, we analyzed the islet yield variables versus donor age according to the preservation method (Table 2). Regardless of the preservation method, postdigest islet yields (islet equivalents per pancreas and islet equivalents per gram) were significantly higher in group 1 (age 40 years) than in group 2. Postpurification islet yields were also higher in group 1 (age 40 years) than in group 2 with either preservation method, although not statistically significant. There was no significant difference in islet equivalents per islet counts between two groups. The percentage of embedded islets was significantly higher in group 1 (age 40 years) than in group 2 in University of Wisconsin preservation cases.
In vitro quality assessment of isolated islets.
In multiple regression analysis with donor age, sex, cause of death, and preservation method as independent variables, only the donor age was found to significantly influence the GSIR index. The GSIR index was significantly higher in group 1 (age 40 years) than in group 2 (Table 3). The insulin release to low glucose concentration (1.7 mmol/l) was significantly lower in group 1 (age 40 years), but the insulin release to high glucose concentration (16.7 mmol/l) was not significantly different between two groups (Table 3). In correlation analysis, the log-transformed GSIR index was negatively correlated with the donor age (Fig. 1A; r = eC0.535, P < 0.001). The log-transformed amount of insulin released to low glucose concentration also correlated positively with the donor age (Fig. 1B; r = 0.445, P < 0.001). There was no significant difference in insulin content between the two groups (Table 3). Islet ATP content was higher in group 1 (age 40 years) than in group 2 (115.7 ± 17.7 vs. 75.7 ± 6.6 pmol/e蘥 DNA, P = 0.030) and negatively correlated with the donor age (Fig. 2; r = eC0.536, P = 0.004).
Nude mouse transplant bioassay.
The diabetes reversal rate and days to diabetes reversal for transplanted diabetic mice with 1,000 and 2,000 IE are shown in Fig. 3A and B). The diabetes reversal rate at 4 weeks with 2,000 IE was significantly higher in group 1 (age 40 years) than in group 2 (96 vs. 68%, P = 0.023). Although the diabetes reversal rate at 1 week with 1,000 IE was also significantly higher in group 1 (age 40 years) than in group 2 (58 vs. 24%, P = 0.025), at 4 weeks it was not significantly different between two groups (58 vs. 41%). In multiple logistic regression analysis with donor age, sex, cause of death, and preservation method as independent variables, only the donor age was significantly related to transplantation outcome with 2,000 IE. The donor age of 2,000 IE grafts, which reversed diabetes in nude mice, was significantly lower than that of grafts that failed to reverse diabetes (Fig. 3C).
Assessment of insulin secretory function after clinical transplantation.
CPI to glucose as derived from IVGTT performed at days 90eC120 after clinical transplantation showed significant negative correlation with donor age (Fig. 4A) and positive correlation with the number of islets transplanted (Table 4). In multiple regression analysis both donor age and the number of islets transplanted were significant independent variables for CPI to glucose. AUC for C-peptide also showed significant negative correlation with donor age. AIR to glucose showed the same correlation trend with either donor age or the number of islets transplanted, although their P values were 0.074 and 0.080, respectively. CPI to glucose and AUC for C-peptide showed stronger positive correlation with islet equivalents transplanted/donor age than with islet equivalents transplanted alone (Table 4), and CPI to glucose/islet equivalents transplanted also showed significant negative correlation with donor age (Fig. 4B). On the other hand, CPI and AIR to argine as derived from intravenous arginine stimulation test performed on days 90eC120 after clinical transplantation showed positive correlation only with the number of islets transplanted and did not correlate with donor age (Table 4).
In an analysis of the relationship of measures of insulin secretion and glycemic control, only CPI to glucose was significantly correlated with fasting plasma glucose (r = eC0.742, P = 0.050) and 2-h glucose after oral glucose load (r = eC0.763, P = 0.046). Both fasting plasma glucose and 2-h postload glucose also showed negative correlation with the number of islets transplanted (r = eC0.859, P = 0.013 and r = eC0.828, P = 0.021, respectively), but no significant relationship was observed between measures of glycemia and donor age in these seven insulin independent recipients.
DISCUSSION
Deterioration in -cell function is an independent predictor for the development of impaired glucose tolerance and type 2 diabetes (23,24). An impact on -cell function by aging has been assessed in many in vivo studies with widely varying results due to multiple confounding factors associated with aging such as obesity and decreased physical activity (3,25eC27). Our study clearly demonstrates that GSIR of isolated human islets deteriorates with advancing donor age. This loss of insulin secretory function with age was independent of donor BMI, sex, cause of death, or other procurement and isolation variables, suggesting that it could be a direct consequence of the process of aging. This finding supports that the decline in -cell function with aging can contribute to the increasing development of impaired glucose tolerance and type 2 diabetes and also to the progressive nature of the disease irrespective of an accompanying insulin resistance and/or hyperglycemia. Although we excluded the donors with the history of type 1, type 2, and gestational diabetes, we could not measure HbA1c in all the donors of this study and thus could not rule out that some donors, especially in the older group, might have had undiagnosed type 2 diabetes. Considering the age distribution of the older donors included in Fig. 1A (40eC49 years old, n = 29; 50eC59 years old, n = 23; 60eC64 years old, n = 5) and the percentage of diabetes in this age population (1,3), we may presume that the number of donors with undiagnosed diabetes, if any, was quite a few and did not skew the results.
Our results with a nude mouse transplant bioassay show a negative impact of aging on isolated human islet function posttransplant. Similar findings have been reported in syngeneic rat islet transplants; islets from 42-week-old donor rats failed to cure diabetes in streptozotocin-induced diabetic rats, while an identical number of islets from 10-week-old rats restored normoglycemia in the majority of recipients (8). Our observation that the diabetes reversal rate with the 2,000 IE graft from donors >40 years of age is not lower than that with the 1,000 IE graft from donors 40 years of age shows that the number of islets can be the major determinant for the immediate correction of hyperglycemia. However, the previous report that the use of older donor rats shortened the period of posttransplant normoglycemia after the correction of hyperglycemia suggested that the older islets may progressively deteriorate, leading to a shorter life span and/or to a loss of their physiologic responses (9). Thus, donor age might influence not only the cure rate depending on the graft mass but also the duration of metabolic normalization. Whether the transplantation of sufficient islet mass can overcome the negative effect of donor age on the duration of insulin independence needs to be evaluated in human islet transplantation.
In IVGTTs and arginine stimulation tests performed at 90eC120 days after transplantation in our seven insulin-independent recipients, insulin secretory response to either glucose or arginine correlated with the islet mass transplanted. This result is in accord with the observation from the Edmonton group that all the measures of insulin reserve in the recipients correlated with the islet mass transplanted at 3 months (13). Islet grafts from older donors may be less efficient in maintaining the -cell mass in the implant. It is known that the regenerative capacity of -cells decreases with age (28,29). Recently, this decreased formation of new islets with aging has also been reported with pancreatic tissue from 124 autopsies (30). Our result that insulin secretory responses to either glucose or arginine correlated with the islet mass transplanted irrespective of donor age suggests that, at least for 90eC120 days posttransplant, the older islets do not decrease in -cell mass faster than younger islets to a detectable degree.
In our clinical transplants, all eight recipients initially achieved insulin independence after the transplantation of 7,271 ± 366 IE/kg regardless of donor age. IVGTTs at 90eC120 days posttransplant in seven insulin independent recipients revealed significant negative correlation between insulin secretion parameters to glucose and donor age of islets, which was independent of the number of islets transplanted. The mechanism underlying this age-related deterioration of insulin secretory -cell function is not known. One study in rats indicated that glucose signal recognition and/or stimulus-secretion coupling may be the locus of impairment in the process of insulin secretion in older animals (6). Several in vivo studies have revealed an increased disorderliness of basal insulin release and an impairment of proinsulin conversion to insulin in older individuals (31eC33). Our in vitro finding of higher insulin release to low concentration of glucose (1.7 mmol/l) and lower GSIR index strongly suggests that the insulin secretory regulation of -cells by glucose is less efficient in older islets.
The mechanisms of insulinotropic action of glucose and arginine are different. While glucose stimulates insulin release by increasing intracellular ATP, arginine shows insulinotropic action by direct depolarization of plasma membrane (34) without involving changes in mitochondrial oxidative phosphorylation and production of ATP. The observation that patients with maternally inherited diabetes and deafness caused by mitochondrial tRNALEU(UUR) gene mutation showed impaired insulin secretory response to glucose but normal insulin secretion to arginine (35,36) supports the idea that the arginine stimulation test could be helpful in locating the defects in insulin secretion. In our seven recipients, insulin secretory responses to glucose but not to arginine showed negative correlation with donor age. This finding suggests that aging may affect the process related to increasing intracellular ATP in -cells. Our in vitro result that islet ATP content/DNA negatively correlates with donor age also lends support to the hypothesis that the intracellular ATP metabolism or mitochondrial function may be a target of -cell aging.
Although no study has reported the direct measurement of mitochondrial ATP synthesis in -cells with age, several mitochondrial functions have been observed to decline with age (37). Oxidative mitochondrial DNA lesions and mutations are found to increase markedly with age (38). It is plausible that the accumulation of such mutations leads to decreased gene expression, a decline in oxidative phosphorylation, and inefficient electron transport. Age-dependent decline of the mitochondria DNA content of tissues (39,40) and mitochondrial membrane potentials (41) have been reported. The report that insulin secretion evoked by glucose but not by plasma membrane depolarization was markedly blunted with decline in mitochondrial membrane potential following exposure to H2O2 also supported the hypothesis that mitochondria may be a target of oxidative damage in -cells during aging (42).
Among the various donor parameters, we found that donor age is an important determinant of islet function in terms of insulin secretion. Noteworthy is that both GSIR index and nude mouse transplant bioassay, which have been widely used to assess the functional quality of isolated islets, should be interpreted with the consideration of donor age. We observed a stronger correlation between islet equivalents transplanted/donor age and CPI to glucose than between islet equivalents transplanted and CPI to glucose in an attempt to take into account the donor age for a given islet preparation, as the Edmonton group did for the CIT index (12,13). This C-peptide response to intravenous glucose also correlated with the measures of glycemia (fasting plasma glucose and 2-h glucose during OGTT). Taken together, it seems highly likely that the higher the donor age the larger the islet mass required for better islet transplantation outcomes. Up to 90eC120 days after transplantation, however, we could not find a relationship between donor age and the measures of glycemia in seven recipients who were not taking exogenous insulin, although there was a significant negative correlation between the number of islets transplanted and the fasting plasma glucose or the 2-h postload glucose level. It may take longer follow-up in a larger number of recipients to determine the effect of donor age on clinical outcomes.
In contrast to the previous reports showing that successful islet isolations increased with increased donor age (10,43), our analysis showed that postdigest islet yields were higher in younger donors (age 40 years) than in older donors regardless of preservation methods and that postpurification yields were not significantly different between the two groups. However, postpurification recovery rates of islets tended to be lower in younger donors. Islets from juvenile donors are known to be difficult to separate from the acinar tissue without causing excessive fragmentation during digestion (10). Our observation that among younger donors embedded islets are relatively low when using two-layer preservation suggests that the increased use of this preservation method may further improve islet yields from younger donors. Thus, younger donors may be advantageous not only in terms of insulin secretory function but also with regard to islet isolation yield.
Donors >45eC60 years of age have been traditionally considered less than ideal for organ transplantation. For example, kidneys from donors >55 or 60 years of age overall have reduced functional reserve, which has an adverse effect on long-term graft survival rates (44eC46). The pancreas grafts from the >45-year-old donors also had significantly lower 1- and 5-year graft survival rates after primary simultaneous pancreas-kidney transplantations (16,17). However, considering the shortage of cadaveric donors, there is a strong need to maximally utilize the potential donor pool. Toward this goal, the transplantation of a higher mass of older islets prepared from two to four pancreata may have some role in human islet transplantation.
In summary, this study shows that insulin secretory regulation by glucose deteriorates with advancing age and that it may be related to ATP generation change in -cells with aging. These findings suggest that islet preparations from younger donors may improve the success rate of single-donoreCalone transplantation and that the higher the donor age the larger islet mass might be required to restore insulin independence posttransplant. Whether transplanting a sufficient islet mass from multiple pancreata can overcome the negative effect of donor age needs to be evaluated to fully utilize the available relatively small donor pool in human islet transplantation.
ACKNOWLEDGMENTS
The study was supported by grants from the National Center for Research Resources, National Institutes of Health (MO1-RR00400 and U42 RR016598-01), the Juvenile Diabetes Research Foundation (JDRF 4-1999-841), Roche Laboratories (RO49272), and the Hallym Academy of Sciences, Hallym University, Korea (to S.-H.I.).
We are indebted to Dr. Raja Kandaswamy, Dr. David Hunter, Jamen Parkey, Kathy Duderstadt, Kathy Hodges, Carrie Gibson, and the staff members of the University of Minnesota General Clinical Research Center for excellent patient care; to Jeremy Oberbroeckling for his technical expertise in the islet isolation laboratory; and to Dr. Robin Jevne and Dylan Zylla for data compilation and analysis.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
S.-H.I. is currently affiliated with Hallym University Sacred Heart Hospital, Anyang, Kyonggi-do, Korea
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