Glucose Stimulation Test Research Articles

QUALITY CONTROL OF PORCINE ISLETS AND SERTOLI CELL ISOLATION FOR USE IN XENOTRANSPLANTATION.

A297 Aims: One of the main constraints in cell processing for xenotransplantation is a lack of validated specialized techniques for the isolation and culture of donor tissue, as well as assuring the quality and function of the xenotransplant. In this study, we have developed modifications to previously described techniques for the isolation of neonatal porcine islets and Sertoli cells in order to improve their quality, function and the reproducibility of the isolation process. In addition we describe techniques for measuring yield, function, viability, and purity of the cell isolates. Methods: Sertoli cell purity was assessed using anti Mullerian inhibiting substance immunohistochemistry (IHC), and for islet purity, dithizone and anti insulin IHC. Viability and degree of apoptosis were measured by flow cytometry for propidium iodide, trypan blue, and annexin. Islet function was measured by static glucose stimulation test, and Sertoli cell function was determined by measuring the level of Clusterin production. Results: Our techniques reproducibly give yields of 140 thousand islet equivalents per gram of pancreas with a viability and purity of greater than 90%, and glucose stimulation indices higher than 10. The yield per gram of testes was 25 – 30 million Sertoli cells, as well as high levels of Clusterin production. Conclusions: These combined isolation and measurement techniques assure better quality control of islet and Sertoli cell processing.

Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells.

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family with a high degree of specificity for epithelial cells in vitro and in vivo. Our aim was to study the effect of KGF on beta-cell growth and differentiation on islet-like cell clusters derived from human fetal pancreas. We investigated the effects of KGF, in vitro, on beta-cell differentiation from undifferentiated pancreatic precursor cells and in vivo after transplantating human fetal pancreatic cells into athymic rats treated with KGF. Treatment of islet-like cell clusters with KGF in vitro did not change the number of insulin producing cells, as measured by the measurement of insulin content or DNA. The in vivo treatment of recipient rats with KGF increased the number of beta cells within the grafts 8 weeks after transplantation. At this time, glucose-stimulated insulin secretion was evaluated by glucose stimulation tests in rats bearing the transplants. Measurements of human C-peptide concentrations after glucose challenge showed that the newly differentiated beta cells in the KGF-treated group were functionally competent as opposed to the control group, where the graft failed to release insulin appropriately. These findings suggest that in vivo, KGF is capable of inducing human fetal beta-cell expansion. The growth promoting effect of KGF on beta cells occurred mainly through the activation of ductal cell proliferation and their subsequent differentiation into beta cells.

An audit of 500 subcutaneous glucagon stimulation tests to assess growth hormone and ACTH secretion in patients with hypothalamic-pituitary disease.

The insulin tolerance test (ITT) is usually regarded as the 'gold standard' for the assessment of the hypothalamic-pituitary axis (growth hormone (GH) and ACTH) but must be used with caution and is contra-indicated in certain groups of patients. The glucagon stimulation test (GST) has previously been shown to be a good alternative when the ITT is contra-indicated and like the ITT stimulates both GH and ACTH secretion. There is however limited data on the use of the GST in patients with hypothalamic-pituitary disease. An audit of 500 GST was performed in 374 patients with hypothalamic-pituitary disease. Glucagon was administered via the subcutaneous route and bloods were taken at times 0 90 120 150 180 210 and 240 minutes. In the vast majority peak GH (84.4%) and cortisol (93%) responses occurred between 120 and 180 minutes Little information was obtained from the 240 minute sample. The medical supervision required was minimal and the side-effects encountered during this test were mild; 20% of the tests were associated with nausea occasionally with vomiting sweating or headaches. Four patients fainted but recovered quickly. This large audit has shown that the glucose stimulation test is well tolerated and can easily be performed in an out-patient setting with minimal medical supervision. The 240 minute sample added little additional information and could be omitted.

University of wisconsin solution with trypsin inhibitor pefabloc improves survival of viable human and primate impure islets during storage.

Background. Recent studies suggest that impure islets (islets which have not been isolated from exocrine tissue and other parts of the pancreas) have great potential for successful transplantation. The evidence that supports this view includes findings that embedded islets (islets surrounded by exocrine tissue) undergo less apoptosis, peripancreatic lymph nodes prevent recurrence of IDDM (insulin dependent diabetes mellitus), and that islet yields and insulin content decrease during the purification process. Improved protocols have also been developed to prevent allorejection of impure islets. Despite these promising results, the storage of impure islets remains difficult, and was a method sought to decrease storage losses.Methods. Storage methods of impure human and non-human primate islets were compared, using either culture media or University of Wisconsin solution (UW). The effects of trypsin inhibition using Pefabloc (Roche Molecular Biochemicals, Indianapolis, IN) during storage period were also examined.Results. Low temperature and inhibition of trypsin activity during storage of impure islets improved both islet yield and viability. It was found that using UW solution and trypsin inhibition allowed perfect preservation of viable impure islets up to 48 h. A functional assay by glucose stimulation test showed these impure islet responded to glucose stimulation after 24 h.Conclusion. The benefits of storing impure islets using UW solution and Pefabloc at low temperature have been established. This improved method of preserving impure islets makes this model of transplantation even more viable.

Entrapment of islets into reversible disulfide hydrogels.

Currently, there are three types of devices for a bioartificial pancreas; microencapsulation, an extravascular diffusion chamber, and an intravascular diffusion chamber. The purpose of the present study was to provide a new extracellular matrix hydrogel for the devices of extra- and intravascular diffusion chamber types. As the sol-gel transition of this hydrogel is reversible, refilling of islets in vivo will be possible without a severe traumatic procedure. The hydrogel was produced from a polyacrylamide derivative carrying thiol groups synthesized by radical copolymerization of acrylamide and N,N'-bis-acrylcystamine, followed by reduction of the disulfide bonds in the copolymer. This water-soluble copolymer was used to entrap hamster islets by re-formation of disulfide bonds on the copolymer to produce a hydrogel. The formed hydrogel was easily reliquefied by reduction of the disulfide crosslinks to thiols. Insulin release from the islet-entrapped hydrogel continued for more than 1 month when examined in vitro. A static glucose stimulation test for the entrapped islets exhibited an increased insulin release.

Glutamate decarboxylase antibody levels predict rate of β-cell decline in adult-onset diabetes

Glutamate decarboxylase autoantibodies (GAD65Ab) and β-cell function were evaluated at and 3 years after diabetes onset in consecutive subjects over 15 years of age. At onset, 21 32 (66%) insulin-treated patients (mean age 43, range 16–79 years) had GAD65Ab; all GAD65Ab persisted 3 years later. At onset, 20 82 (24%) non-insulin-treated patients (mean age 56, range 20–79 years) had GAD65Ab. Of those with persistent GAD65Ab, 8 non-insulin-treated and 11 insulin-treated patients consented to follow-up glucose and glucagon stimulation tests. For non-insulin-treated patients, quantitative GAD65Ab index at onset correlated inversely with 1+3 min C-peptide response to glucose ( r = −0.68, P < 0.05) and to glucagon ( r = −0.79, P < 0.05) 3 years later. Those with high (> 0.50) initial GAD65Ab index had lower C-peptide (fasting, 1+3 min after glucose and after glucagon) 3 years later, versus those with low (<0.50) initial GAD65Ab index ( P < 0.05). In conclusion, not only did GAD65Ab presence predict future insulin dependence, but higher GAD65Ab levels may mark more rapid decline in β-cell function in apparent non-insulin-dependent diabetes.

Comparison of TRF, Propranolol-Glucagon, Insulin and Glucose Stimulation Tests in Acromegaly

In 7 acromegalic patients growth hormone responses were studied following administration of synthetic TRF, propranolol-glucagon, insulin, and glucose p.o. Except for the glucose tolerance test, a good reproducibility of the STH response was observed. In 5 out of the 7 patients, there was a distinct rise in the plasma STH level after TRF. All patients with a positive insulin tolerance test responded to TRF, as did the two late responders to glucagon; the early responder to the latter test did not respond to TRF. It has been suggested (Liuzzi et al. 1974a) that TRF might be used as a screening test for detecting hypothalamic dependency of the acromegaly. This study suggests that further study is required before accepting this hypothesis and that a response to a combination of tests (TRF, glucagon, insulin) might be a better screening method.