Recent research suggests that islet allotransplantation can be an effective treatment of patients with an unstable form of type 1 diabetes (1–3). However, the current standard islet quality assays have a limited ability to predict transplantation outcomes (4–6).
Islet transplantation into the kidney capsule of immunodeficient mice with streptozotocin-induced diabetes is currently considered the most reliable assessment that correlates with the outcomes of clinical transplantation (7). However, this method can only be used retrospectively, because islet evaluation takes from several days to weeks. Therefore, there is a need to establish more rapid and reliable methods to predict the outcomes of clinical islet transplantation.
Measurement of intracellular ATP content has been proven as a simple and reliable indicator of cell viability, including the viability of islet cells (8, 9). The ATP content of transplanted tissue has been shown to correlate with graft function and survival (10–12). In addition, the ATP content of freshly isolated human islets showed large variability (13). For the above reasons, intraislet ATP content is regarded as a candidate for predicting in vivo islet function (13).
To our knowledge, however, there have been no reports demonstrating that the intraislet ATP content itself can be a reliable assay to predict islet transplantation outcomes. To address this topic, we compared the intraislet ATP content, glucose- or glucose plus theophylline-stimulated insulin release, and the loss of islets during the first 24 hr of culture to predict diabetes reversal in nude mice transplanted with porcine islets.
In this study, 49 mice were transplanted with porcine islets from 30 isolations from adult market pigs. The ATP content of 10 handpicked porcine islets from islet preparations that had reversed the hyperglycemia of diabetic athymic mice was significantly higher than in those that were unable to cure them (9.6±4.5 pmol vs. 4.2±2.2 pmol; P<0.001; Table 1). The total ATP content of the 1000 islet equivalent (IEQ; 1 IEQ=an average diameter of 150 μm) aliquots showed a significant difference between the cured and the noncured mice (320.1±214.6 pmol vs. 195.5±74.7 pmol; P=0.028; Table 1), which became more apparent after normalization for islet protein (1.8±0.7 nmol/mg protein vs. 1.0±0.8 nmol/mg protein; P=0.005; Table 1). However, there was no significant difference in the glucose- or glucose plus theophylline-stimulated insulin release and the loss of islets during the initial 24 hr of culture between the cured and the noncured mice (Table 1).
Next, we assessed the ability of the ATP content to predict transplantation outcomes using receiver-operator characteristic analysis. The ATP measurement using 10 handpicked islets was the best predictor of transplantation outcome in this study; with an area under the curve (AUC) of 0.867 (95% CI 0.744–0.989, P<0.001, Fig. 1A). At an ATP content cut-off value for the group of 10 handpicked islets of 5.40 pmol, the sensitivity and the specificity were 83.3% and 73.3%, respectively (Fig. 1A). In regards to transplant efficacy, the ATP content of the 1000 IEQ aliquots was less predictive than that of the 10 handpicked islets (73% correct classification at an ATP content cut-off value for 1000 IEQ aliquots of 194 pmol, with 77.3% sensitivity and 52.9% specificity, Fig. 1B). However, the predictive power was slightly increased after normalization for islet protein (78% correct classification at a cut-off value for the ATP content per protein of more than or equal to 1.20 nmol/mg protein, with 83.3% sensitivity and 61.5% specificity, Fig. 1C). The AUCs for glucose- or glucose plus theophylline-stimulated insulin release and the loss of islets during the initial 24 hr of culture were insignificant.
We found large variability in the ATP content of isolated porcine islets, which was also reported for human islets in a previous study (13). In addition, in this study, it was not the current standard quality assay but the intraislet ATP content that revealed significant correlation with the transplantation outcome in nude mice. These results coincide with previous reports that the current methods for evaluating islet quality cannot predict islet transplantation efficacy (4–6). Recently, the ADP/ATP assay using the ApoGlow kit (Cambrex Bio Science Nottingham Ltd., Nittingham, UK) was reported to correlate with in vivo nude mice transplantation outcomes (6). However, the assay kit could not discriminate the ATP/ADP ratio of the assay mixture using pure ADP and ATP (data not shown).
We used two sample types (10 handpicked islets with a diameter of 100–150 μm and the 1000 IEQ aliquots) to measure ATP content. Among them, the group of 10 handpicked islets had better predictive power on transplantation outcomes than the 1000 IEQ aliquot.
Measurement of the ATP content using 10 handpicked islets may have several advantages. First, handpicking islets within a narrow size distribution reduces the need to normalize islet mass (14, 15). We also found small variability in the DNA content of the 10 independent handpicked islet samples (13.06±1.27 ng/10 islets). Second, the handpicking procedure enables us to mainly select islets from the isolated islet preparation, probably eliminating contamination with nonislet ATP, which makes it possible to increase the predictive ability of the ATP assay. Third, handpicked islets with a diameter of 100 to 150 μm remove the effects of large islets, which are inferior to small islets in in vitro function and in in vivo transplantation outcomes (16, 17). Therefore, we suggest that the selection of small islets (100–150 μm) is reasonable for the measurement of ATP content, as they may be the most responsible for the function of the engrafted islet.
In conclusion, the ATP content of islets, especially when using handpicked islets with a diameter of 100–150 μm, could be a good predictor of graft efficacy in nude mice.
MATERIALS AND METHODS
Porcine Islet Isolation and Culture
All animal procedures applied were approved by the Institutional Animal Care and Use Committee of the Seoul National University Hospital. The porcine islet isolation and purification were performed using adult market pigs, as previously described (18). In brief, the harvested pancreas was distended intraductally with UW solution containing 0.15% Liberase PI (Roche Biochemicals, Basel, Switzerland) at a concentration of 1.7 mL of University of Wisconsin (UW) solution per gram of pancreatic tissue. The distended pancreas was digested in a modified Ricordi chamber. Liberated islets were separated from nonislet tissue on a continuous UW/OptiPrep density gradient (1.100 and 1.060 g/cm3) on a COBE 2991 cell separator (Gambro BCT Inc., Lakewood, CO) at 2200g for 5 min. The islets were then cultured free floating in standard culture medium (M199; GIBCO BRL, Grand Island, NY) supplemented with 10% pig serum.
In Vitro Test of Islet Function
Glucose-stimulated insulin release was measured and expressed as stimulation index (18), calculated as the ratio of stimulated (16.7 mM glucose or 16.7 mM glucose plus 5 mM theophylline) to basal (1.67 mM glucose) insulin release during static incubation in Krebs–Ringer bicarbonate HEPES buffer.
Two types of samples were used for the measurement of ATP concentration; one group consisted of 10 handpicked islets within a small size distribution (a diameter of 100–150 μm) and the other aliquots of 1000 IEQ. The ATP content was measured using the Bioluminescent Somatic Cell Assay Kit (Sigma Chemicals, St Louis, MO). In the aliquots of 1000 IEQ, the concentration of islet protein was quantified by the Bradford dye-binding protein assay to normalize the amount of islets (19). For each experiment, two samples were taken and each assay was performed in triplicate. To validate the variability of islet mass in the 10 handpicked islets, the DNA contents of 10 independent handpicked islet samples were measured using the PicoGreen dsDNA kit (Molecular Probes, Eugene, OR).
In Vivo Islet Potency
The in vitro tests of islet functions were assessed immediately before islet transplantation under the kidney capsule of athymic Balb/c nude mice (Orient Co. Ltd., Seoul, Korea), which were rendered diabetic with a single intraperitoneal injection of 230 mg/kg streptozotocin (Sigma Chemicals) 4 to 5 days before transplantation. Nonfasting blood glucose was measured with a glucose meter (One Touch Ultra Sensor, Lifescan, Milpitas, CA). Mice with sustained hyperglycemia (≥350 mg/dL) were used as islet graft recipients. Two thousand IEQ of porcine islets per recipient were transplanted under the kidney capsule. Blood samples were taken from the tail vein twice weekly to determine glucose levels. Islets were considered efficacious when two consecutive posttransplant blood glucose levels were below 200 mg/dL. Nephrectomies were performed to determine the graft dependence of diabetes reversal.
All values are expressed as mean±SD. Comparisons of data were carried out using Student's unpaired t test. The optimal cutoff value, the AUC, and the 95% confidence intervals were determined from receiver-operator characteristic curves. Correlations were analyzed using the Spearman rank test. P values less than 0.05 were considered statistically significant.
1.Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med
2006; 355: 1318.
2.Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes
2005; 54: 2060.
3.Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med
2000; 343: 230.
4.Gerling IC, Kotb M, Fraga D, et al. No correlation between in vitro and in vivo function of human islets. Transplant Proc
1998; 30: 587.
5.Eckhard M, Brandhorst D, Winter D, et al. The role of current product release criteria for identification of human islet preparations suitable for clinical transplantation. Transplant Proc
2004; 36: 1528.
6.Goto M, Holgersson J, Kumagai-Braesch M, et al. The ADP/ATP ratio: A novel predictive assay for quality assessment of isolated pancreatic islets. Am J Transplant
2006; 6: 2483.
7.Ichii H, Inverardi L, Pileggi A, et al. A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations. Am J Transplant
2005; 5: 1635.
8.Ishii S, Saito T, Ise K, et al. Evaluation of energy state of islet independent of size using a newly developed ATP bioluminescence assay. Transplant Proc
2005; 37: 3499.
9.Caraher EM, Conroy SJ, Newsholme P. Evidence for enhanced rates of complement activation in serum from patients with newly diagnosed insulin-dependent diabetes mellitus exposed to rat islet cells and complement-dependent induction of islet cell apoptosis. J Endocrinol
1999; 162: 143.
10.Fujino Y, Kuroda Y, Saitoh Y. Adenine nucleotides metabolism of the canine pancreas during preservation by the two-layer cold storage method. Kobe J Med Sci
1991; 37: 255.
11.Pegg DE, Foreman J, Hunt CJ, et al. The mechanism of action of retrograde oxygen persufflation in renal preservation. Transplantation
1989; 48: 210.
12.Higashi H, Takenaka K, Fukuzawa K, et al. Restoration of ATP contents in the transplanted liver closely relates to graft viability in dogs. Eur Surg Res
1989; 21: 76.
13.Brandhorst D, Brandhorst H, Hering BJ, et al. Large variability of the intracellular ATP content
of human islets isolated from different donors. J Mol Med
1999; 77: 93.
14.Sweet IR, Gilbert M, Scott S, et al. Glucose-stimulated increment in oxygen consumption rate as a standardized test of human islet quality
. Am J Transplant
2008; 8: 183.
15.Sweet IR, Gilbert M, Jensen R, et al. Glucose stmulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality
2005; 80: 1003.
16.Lehmann R, Zuellig RA, Kugelmeier P, et al. Superiority of small islets in human islet transplantation. Diabetes
2007; 56: 594.
17.MacGregor RR, Williams SJ, Tong PY, et al. Small rat islets are superior to large islets in in vitro function and in transplantation outcomes. Am J Physiol
2006; 290: E771.
18.Kim JH, Kim HI, Lee KW, et al. Influence of strain and age differences on the yields of porcine islet isolation: Extremely high islet yields from SPF CMS miniature pigs. Xenotransplantation
2007; 14: 60.
19.Marshall T, Williams KM. Phenol addition to the Bradford dye binding assay improves sensitivity and gives a characteristic response with different proteins. J Biochem Biophys Methods
1986; 13: 145.