The assessment of functional potency of islets prior to transplantation is important to achieve success in islet transplantation. In vitro
assessment methods of islets, including viability using membrane permeable fluorescence dye (21
), dynamic glucose stimulated insulin release assay using a perifusion system (23
), oxygen consumption assay (25
), assessment of ATP/ADP ratio and reactive oxygen species (27
), islet cell composition and β cell apoptosis (28
) are currently utilized by various islet transplant centers to evaluate the overall quality of isolated human islets. However, none of current assays measures insulin synthesis in β cells, including the transcriptional response of insulin gene to glucose stimulation. In this study, we developed an assay that measures glucose-induced newly synthesized premature INS
mRNA using a set of single human islets. We have shown that the SI of premature INS
mRNA synthesis and insulin release in high glucose medium over 16 hours from the same set of islets predicted blood glucose levels of diabetic NODscid
mice 7 and 21 days after islet transplantation, while the conventional quality assessment results, such as SI of perifusion assay and percent β cell apoptosis, did not.
It is generally believed that glucose regulated preproinsulin mRNA transcription is controlled by long-term glucose stimulation balanced by both transcription and mRNA degradation rates (5
). However, transcriptional regulation coupled to short-term glucose stimulation is also reported in rodent (31
), as well as human islets (16
). Evans-Molina et al. showed an increase of premature INS
mRNA after 8 hours of high-glucose stimulation (7 mmol/L and 25 mmol/L) as compared to basal glucose incubation (2.5 mmol/L) in human islets (16
). Our study showed no significant transcriptional change in 4 and 8 hours stimulations, but a significant increase in premature INS
mRNA after culturing for 16 hours in 17 mmol/L glucose medium compared to those cultured in 3.3 mmol/L glucose. The difference between these two results may be due to the different glucose concentrations used. We plan to evaluate glucose concentration and incubation time to further shorten the assay period in order to obtain the results before islet transplantation takes place.
The quantitation of basal INS mRNA and glucose induced newly synthesized INS mRNA in an islet reflects the number of functional β cells, as well as the viability of β cells existing in an islet. The production of glucose induced INS is reduced in damaged islets, as confirmed by attenuated INS synthesis caused by cellular damage induced by heat treatment (). The values obtained by mature or premature INS mRNA normalized by ACTB mRNA reflect the β cell content in an islet, as well as the actual function/viability of existing β cells. The normalization of premature INS mRNA by mature plus premature INS mRNA would represent the transcriptional capacity of INS in existing β cells better than normalization by ACTB mRNA. Since mature INS mRNA did not increase during 16 hour- high glucose stimulation, it is reasonable to use the mature INS mRNA as an indicator of existing β cell quantity. However, the fold difference (premature INS mRNA normalized by mature INS mRNA) will increase when the quantities of mature INS are reduced significantly due to cellular damage as seen in . This is the rationale for using SI to compare the transcriptional response of β cells between islet preparations.
The amount of premature and mature INS
mRNA was not necessary proportional to the size of the islets. Islets, by their nature, have a varying number of beta cells. This natural variation, as well as variations in beta cell viability may be caused by donor factors, or islet isolation itself. Large islets tend to have central necrosis due to limited diffusion of oxygen and nutrients (33
), which negatively affects glucose induced INS
synthesis. The quality of large and medium sized islets is considered more important in assessing the overall quality of an islet preparation, as the factors to convert islet number to IEQ favor larger islets (35
). Our assay was sensitive enough to detect a difference in Ct of the housekeeping gene ACTB
among large, medium, and small islet samples (). Overall, small to medium size islets responded better than large islets in our study which agrees with results previously described by Lehmann et al.(32
). However, we found that size-dependent glucose-induced INS
mRNA synthesis was not consistent from one islet preparation to another. Even if large islets do not respond to high glucose, when the number of large islets in the preparation is small, it will have less influence on the overall islet quality and function. In two islet preparations shown in , the adjusted SIs were similar to the SI of medium size of the corresponding islets, since more than 50% of total IEQ were from medium size islets in both cases. The size distribution similar to these two cases was confirmed in additional 10 islet preparations (data not shown). Therefore, it is appropriate to use medium size islets for this assay as representative islets in most of islet preparations.
It is reasonable to speculate that islets losing glucose-induced INS mRNA transcription capacity also fail to secrete insulin over a long period, thus depleting the available insulin. Interestingly, insulin secretion by short-term glucose stimulation also correlated to 16 hours premature INS mRNA synthesis. Glucose induced premature INS mRNA synthesis and insulin released in 16-hour high glucose stimulation were shown to be good indicators to predict islet transplant outcome in diabetic mice. Negative and positive predictive value for SI_INS mRNA and SI_16 hr insulin are to be examined after the results of more cases are collected. Since mice were not treated with exogenous insulin after transplantation, the ability of insulin production by transplanted islets under hyperglycemic condition is a key factor to reverse diabetes. However, clinical islet transplantation recipients are treated with exogenous insulin to control blood glucose levels to avoid the exposure of islets to high glucose. We also plan to determine if a correlation exists between premature INS synthesis in islets and clinical transplantation outcome.
In summary, the measurement of glucose induced premature INS mRNA normalized by mature INS mRNA can be analyzed using a set of single islets. The combination of SI_INS mRNA and SI_16h insulin from the same set of islets significantly predicted human islet function transplanted into diabetic NODscid mice. These two new factors were shown to be more predictable than the results obtained from conventional islet quality assessment methods. This microassay may be a useful method to predict islet function after transplantation in patients with type 1 diabetes.