Low levels of C-peptide have clinical significance for established Type 1 diabetes.

AIM: To determine whether the low C-peptide levels (< 50 pmol/l) produced by the pancreas for decades after onset of Type 1 diabetes have clinical significance. METHODS: We evaluated fasting C-peptide levels, duration of disease and age of onset in a large cross-sectional series (n = 1272) of people with Type 1 diabetes. We then expanded the scope of the study to include the relationship between C-peptide and HbA1c control (n = 1273), as well as diabetic complications (n = 324) and presence of hypoglycaemia (n = 323). The full range of C-peptide levels was also compared with 1,5-Anhydroglucitol, a glucose responsive marker. RESULTS: C-peptide levels declined for decades after diagnosis, and the rate of decline was significantly related to age of onset (P < 0.0001), after adjusting for disease duration. C-peptide levels > 10 pmol/l were associated with protection from complications (e.g. nephropathy, neuropathy, foot ulcers and retinopathy; P = 0.03). Low C-peptide levels were associated with poor metabolic control measured by HbA1c (P < 0.0001). Severe hypoglycaemia was associated with the lowest C-peptide levels compared with mild (P = 0.049) or moderate (P = 0.04) hypoglycaemia. All levels of measurable C-peptide were responsive to acute fluctuations in blood glucose levels as assessed by 1,5-Anhydroglucitol (P < 0.0001). CONCLUSIONS: Low C-peptide levels have clinical significance and appear helpful in characterizing groups at-risk for faster C-peptide decline, complications, poorer metabolic control and severe hypoglycaemia. Low C-peptide levels may be a biomarker for characterizing at-risk patients with Type 1 diabetes.

Islet isolation from human pancreas with extended cold ischemia time.

The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 +/- 0.7 hours). Donors averaged 50.8 +/- 2.6 (standard error of the mean) years old with a body mass index of 28.6 +/- 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 +/- 41 KIEQ postpurification, 359 +/- 29 KIEQ (purification loss 13.5%); and postculture 317 +/- 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 +/- 5 g). Most islets were recovered in the purest fraction (purity 79.7% +/- 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% +/- 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 +/- 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.

Central role of defective apoptosis in autoimmunity.

Lymphocyte development, selection and education represent tightly controlled immune processes that normally prevent autoimmunity. Lymphocyte development likely induces cellular selection through apoptosis to remove potentially autoreactive cells. Dysregulated apoptosis, both interrupted as well as accelerated apoptosis, are now demonstrated as central defects in diverse murine autoimmune disease. In murine models of autoimmune lupus, mutations in cell death receptor Fas (CD95) and its ligand, FasL (CD95 L), have been identified. These errors create a lymphoid system resistant to apoptosis. In contrast, select lymphoid subpopulations of maturing autoimmune prone non-obese diabetic mice have identifiable and pathogenic T cells with both in vivo and in vitro heightened apoptosis after drug interventions. In part, these defects are due to faulty activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) that normally protect against apoptotic death. The genetic basis of interrupted NF-kappaB in pathogenic memory T cells in diabetes is attributable to a developmentally controlled gene defect in an essential subunit of the proteasome. No specific gene in most common forms of human autoimmune disease has yet been identified. Functional assays from diverse laboratories repeatedly demonstrate heightened apoptosis in multiple cellular signaling pathways for cell death, suggesting a common theme in disease causality.

Transplantation of islets using microencapsulation: studies in diabetic rodents and dogs.

Studies involving the transplantation of human islets in Type I diabetics have been of significant value both in documenting the potential importance of islet transplantation as a therapeutic modality, and in defining some of the problems which must be overcome before this approach can be used in large numbers of patients. The currently limited supply of adult human pancreatic glands, and the fact that chronic immunosuppression is required to successfully transplant islets into patients, indicate that techniques must be further developed and refined for allo- and xenografting of isolated islets from human and animal sources to diabetic patients. An increasing body of evidence using microencapsulation techniques strongly suggests that this will be achieved during the next few years. Data from our laboratory in rodents and dogs indicate that these systems can function for extended periods of time. In one study, insulin independence was achieved in spontaneously diabetic dogs by islet microencapsulation inside uncoated alginate gel spheres (Mr exclusion >600 kD). No synthetic materials or membrane coatings were employed in this study. Spheres containing canine islets were implanted into the peritoneum of 4 diabetic dogs. The animals received low-dose CsA (levels below readable limits by HPLC at 3 weeks). Implantation of these spheres completely supplanted exogenous insulin therapy in the dogs for 60 to >175 days. Blood glucose concentration averaged 122+/-4 mg/dl for these animals during the first 2 months. The glycosylated hemoglobin (HbAIC) levels during this period dropped from 6.7+/-0.5% to 4.2+/-0.2% (P<0.001). IVGTT K-values at 1 and 2 months postimplantation were 1.6+/-0.1 (P<0.002) and 1.9+/-0.1 (P<0.001), respectively compared with 0.71+/-0.3 before implantation. In a second group of studies, bovine islets were immobilized inside a new type of selectively permeable "microreactor" (Mr exclusion <150 kD) and implanted into the peritoneum of 33 STZ-induced diabetic rats without any immunosuppression. Diabetes was promptly reversed, and normoglycemia maintained for periods of several weeks to months. Immunohistochemical staining of microreactors recovered from these animals revealed well-granulated beta-cells consistent with functionally active insulin synthesis and secretion. To test further the secretory function of the islets, some of the explanted microreactors were incubated in media containing either basal or stimulatory concentrations of glucose. The islets responded with an approximately 3- to 5-fold average increase above basal insulin secretion. These results are encouraging, and may have important implications in assessing the potential role of these microencapsulation systems as therapy for human insulin-dependent diabetes.

Xenotransplantation of porcine and bovine islets without immunosuppression using uncoated alginate microspheres.

Uncoated spherical hydrogel microspheres (calcium alginate, nominal M(r) exclusion of > 600 kD) 800-900 microns in diameter were employed to prevent immune rejection of discordant islet xenografts isolated from pigs and cows. The islets were immobilized in the microspheres and injected into the peritoneum of 14 nonimmunosuppressed streptozotocin (STZ)-induced diabetic C57BL/6J mice. Four recipients received islet grafts from bovine calves, and 10 received islet grafts from pigs. In the control group of 15 diabetic mice implanted with nonencapsulated islets, 6 received i.p. porcine islets and 5 received i.p. bovine islets, whereas remaining 4 received porcine islets under the kidney capsule. Plasma glucose concentrations in recipients of the alginate-encapsulated islets promptly dropped from a preimplantation value of 498 +/- 47 (mean +/- SEM) to 142 +/- 6 (bovine) and 178 +/- 7 mg/dl (porcine) during the first wk. All the animals sustained these levels for at least 1 mo. Two mice implanted with bovine islets subsequently reverted to diabetes (plasma glucose > 250 mg/dl) at 43 days postimplantation. The remaining grafts maintained function for > 10 wk. In contrast, nonencapsulated islets failed to function, or sustained euglycemia for < 4 days. Mice receiving encapsulated islets showed a 23-38% gain in body weight during the first mo after implantation, compared with < 1% (P < 0.002) and 32% (P = 0.84) for the untreated diabetic (n = 6) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (> 10 wk) revealed viable islets, with well-granulated alpha, beta, and delta cells; the external surfaces of the microreactors were free of fibrotic overgrowth and exhibited only occasional host cell adherence. Uptake studies with IgG and thyroglobulin (M(r) of 669 kD) suggest that the microreactors were permeable to molecules with a molecular weight of up to > 600 kD (including the various proteins of the complement system, M(r) of 24-570 kD). Spheres implanted in the peritoneum after only 1 wk stained positive for both IgG and for the C3 component of complement. These findings suggest that prolonged survival of discordant xenografts of porcine and bovine islets in the STZ diabetic mouse model can be achieved with uncoated alginate microspheres that are permeable to IgG and complement. The question of whether similar results can be achieved with uncoated alginate microspheres in higher animals remains to be fully determined.

Encapsulation technologies.

Encapsulation systems have been developed in which cells are separated from the immune system of the host by permselective barriers. These systems do not require a life-long regimen of high dose immunosuppressive drugs to prevent immune rejection. Furthermore, they offer a solution to the problem of human cell procurement by permitting use of cells and tissues from animal sources. Three major types of immunoisolation devices have been studied by our group. These include perfusion devices anastomosed to the vascular system as atrioventricular (AV) shunts, tubular membrane diffusion chambers, and microreactors. This technology is applicable to treating a number of diseases by transplantation of cells that produce specific bioactive substances. In essence, this approach constitutes a living drug delivery and detoxification system. Our work has focused mainly on developing a new treatment for diabetes using encapsulated pancreatic islets. In the first type of system, canine and porcine islets were distributed in a chamber surrounding a permselective acrylic membrane (nominal M(r) exclusion of 80 kDa), and the devices implanted intraperitoneally as AV shunts into diabetic, totally pancreatectomized dogs without use of immunosuppression. In the second type of system, the islets were sealed within the acrylic membranes and the chambers implanted into the peritoneum of diabetic, pancreatectomized dogs (canine islets), streptozotocin (STZ)-induced diabetic rats (canine, bovine and porcine islets), and spontaneously diabetic BB/Wor rats (canine islets) without use of immunosuppression. In the third type of system (microreactors), the islets were implanted into the peritoneum of STZ-induced diabetic mice without use of immunosuppression, into STZ-induced diabetic rats (bovine and porcine islets) both with and without use of low dose immunosuppression, and into spontaneously diabetic dogs (canine islets) with low dose CsA. Results indicate that all three types of encapsulation systems significantly improve glucose homeostasis and can function for periods of several months to more than a year.

Secretory function of biohybrid pancreas devices containing isolated porcine islets.

Isolated porcine islets represent a potential source for discordant islet xenografts in diabetic patients. The authors therefore investigated insulin secretion from isolated porcine islets both in vitro and in vivo. For in vitro studies, islets were maintained in culture or placed in biohybrid perfusion devices consisting of a plastic housing containing a selectively permeable acrylic copolymer tubular membrane. Culture medium was circulated through the devices in a closed loop system. After 3 months the cultured islets secreted insulin at levels of 354 +/- 49 microU/equivalent islet number (EIN)/day (mean +/- standard error of the mean [SEM]; n = 10). They responded to glucose stimulation (5 to 16 mmol/L steps) with significant increases in insulin secretion. The biohybrid devices seeded with islets produced 23 +/- 2 (mean +/- SEM; n = 8) units insulin per day over periods of 83 +/- 8 days. For in vivo studies, islets were sealed within membrane chambers and implanted in the peritoneal cavity of streptozotocin induced diabetic Lewis rats. Chambers with a total of 2 x 10(4) islets per rat normalized the plasma glucose values of 10 rats, with the concentrations decreasing from 487 +/- 18 to 97 +/- 10 mg/dl during the first month. All grafts maintained normoglycemia for longer then 3 months. Histologic studies of long-term chamber implants in rats (1-20 months of age) showed viable islets, with varying degrees of beta cell granulation. These studies suggest the long-term functioning of porcine islets both in vitro and in vivo as discordant xenografts.

Relationship between insulin secretion and oxygen tension in hybrid diffusion chambers.

Oxygen tension is of potential importance in hybrid diffusion chambers, where both islet density and the site chosen for implantation can significantly affect the pO2 within the chambers. To investigate this, isolated islets were incubated at oxygen tensions of 38 mmHg ("low") and 154 mmHg ("ambient"). The mean (+/- SD) ratio between insulin secretion at low and at ambient oxygen tensions was 0.92 +/- 0.27 (n = 10) for porcine islets and 0.80 +/- 0.08 (n = 5) for canine islets. The pO2 was then determined in vivo in cylindrical diffusion chambers (inner diameter 4.5 mm) fabricated from acrylic copolymer, seeded with isolated porcine islets at densities of 0, 15, 30, and 45 islets/mm3, and implanted intraperitoneally in rats. The pO2 levels inside the chambers after 2 weeks were 57 +/- 6 (n = 12), 39 +/- 5 (n = 6), 38 +/- 6 (n = 6), and 43 +/- 3 (n = 6) mmHg, respectively. After 6 weeks, the results were 51 +/- 3 (n = 6), 26 +/- 8 (n = 3), 29 +/- 12 (n = 3), and 40 +/- 5 (n = 3) mmHg, respectively. In comparison, similar chambers containing 10 islets/mm3 cultured for 1 week at ambient oxygen had a pO2 of 120 +/- 9 (n = 4) mmHg immediately underneath the membrane and 67 +/- 7 (n = 4) mmHg at the axial center.(ABSTRACT TRUNCATED AT 250 WORDS)

Net calcium and acid release at fertilization in eggs of sea urchins and ascidians.

Sea urchin eggs lose about 10-30% of their total calcium content upon fertilization. We have investigated the mechanism of this calcium-loss with an ion-selective vibrating probe system. Upon fertilization of Arbacia punctulata and Lytechinus pictus eggs we could measure a calcium efflux signal with an average duration of 204 +/- 26 s and 146 +/- 46 s, respectively. Measurements of hydrogen ion signals in normal and in low sodium media showed that the release of cortical vesicle material from these eggs lasts for about 30 and 50 s, respectively. The data indicate that most of the calcium that is lost from sea urchin eggs originates from the cytosol in which it is released during fertilization and then pumped out through the plasma membrane. Calcium loss due to cortical granule release accounts for less than 14% of the total loss measured. We also measured a substantial post-fertilization calcium efflux in eggs of Phallusia mammilata, with an average duration of 265 +/- 18 s followed by smaller periodic effluxes that corresponded to oscillations in the [Ca2+]i during contractile waves in these eggs. These data, together with the lack of cortical granules in ascidian eggs, indicate that Phallusia eggs also pump out a substantial amount of calcium through the plasma membrane after fertilization.

Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7-37).

Non-insulin-dependent diabetes mellitus (NIDDM, type 2 diabetes) is a disorder of glucose homeostasis characterized by hyperglycaemia, peripheral insulin resistance, impaired hepatic glucose metabolism, and diminished glucose-dependent secretion of insulin from pancreatic beta-cells. Glucagon-like-peptide-1(7-37) (GLP-1) is an intestinally derived hormone that may be useful for the treatment of NIDDM because it acts in vivo to increase the level of circulating insulin, and thus lower the concentration of blood glucose. This therapeutic effect may result from the ability of GLP-1 to compensate for a defect in the glucose signalling pathway that regulates insulin secretion from beta-cells. In support of this concept we report here that GLP-1 confers glucose sensitivity to glucose-resistant beta-cells, a phenomenon we term glucose competence. Induction of glucose competence by GLP-1 results from its synergistic interaction with glucose to inhibit metabolically regulated potassium channels that are also targeted for inhibition by sulphonylurea drugs commonly used in the treatment of NIDDM. Glucose competence allows membrane depolarization, the generation of action potentials, and Ca2+ influx, events that are known to trigger insulin secretion.

Use of an extracellular, ion-selective, vibrating microelectrode system for the quantification of K(+), H (+), and Ca (2+) fluxes in maize roots and maize suspension cells.

An ion-selective vibrating-microelectrode system, which was originally used to measure extracellular Ca(2+) gradients generated by Ca(2+) currents, was used to study K(+), H(+) and Ca(2+) transport in intact maize (Zea mays L.) roots and individual maize suspension cells. Comparisons were made between the vibrating ion-selective microelectrode, and a technique using stationary ion-selective microelectrodes to measure ionic gradients in the unstirred layer at the surface of plant roots. The vibrating-microelectrode system was shown to be a major improvement over stationary ion-selective microelectrodes, in terms of sensitivity and temporal resolution. With the vibrating ion microelectrode, it was easy to monitor K(+) influxes into maize roots in a background K(+) concentration of 10 mM or more, while stationary K(+) electrodes were limited to measurements in a background K(+) concentration of 0.3 mM or less. Also, with this system it was possible to conduct a detailed study of root Ca(2+) transport, which was previously not possible because of the small fluxes involved. For example, we were able to investigate the effect of the excision of maize roots on Ca(2+) influx. When an intact maize root was excised from the seedling at a position 3 cm from the site of measurement of Ca(2+) transport, a rapid fourfold stimulation of Ca(2+) influx was observed followed by dramatic oscillations in Ca(2+) flux, oscillating between Ca(2+) influx and efflux. These results clearly demonstrate that wound or perturbation responses of plant organs involve transient alterations in Ca(2+) transport, which had previously been inferred by demonstrations of touch-induced changes in cytoplasmic calcium. The sensitivity of this system allows for the measurement of ion fluxes in individual plant cells. Using vibrating K(+) and H(+)electrodes, it was possible to measure H(+)efflux and both K(+) influx and efflux in individual maize suspension cells under different conditions. The availability of this technique will greatly improve our ability to study ion transport at the cellular level, in intact plant tissues and organs, and in specialized cells, such as root hairs or guard cells.