The in vivo performance of bioartificial pancreas in bone marrow cavity: A case report of a spontaneous diabetic feline

Recent studies reported that bone marrow cavity offers a widely distributed and well-vascularized microenvironment which is a considerable implantation site for bioartificial pancreas (BAP). In this study, the in vivo performance of BAPs in bone marrow was further demonstrated in a spontaneous diabetes animal. Mouse insulinoma cells encapsulating in agarose gel were enclosed in a calcium phosphate cement chamber to create a BAP. Ten BAPs were implanted into the femur bone marrow cavity of a diabetic feline. The preprandial blood glucose level, 2 h glucose curve, serum C-peptide level and physiological conditions of the recipient were recorded perioperatively. Results showed that the cat still suffered from hyperglycemia postoperatively. However, the physiological conditions of feline were improved with an increase of serum C-peptide level. The peak point of 2 h glucose curve decreased from 400 to 165-290 mg/dl. The efficiency of exogenous insulin extended from 2 to 10-14 h postoperatively which reveals that the implanted BAPs had partial function. This case report revealed that BAPs implanted in the bone marrow cavity for the spontaneous diabetic is effective. The implanted BAPs provided therapeutic benefit despite sustained hyperglycemia. Further study shall be considered to improve the outcomes of BAPs transplantation.     

Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.     

C-peptide, insulin and proinsulinlike components in diabetic and nondiabetic human pancreas.

The contents of insulin and C-peptide extractable with acid alcohol from the tail of the pancreas and insulinoma were investigated, using gel filtration in seven nondiabetics including two patients with insulinoma and eight diabetics. The gel filtration patterns of both C-peptide and insulin in pancreatic extract were fairly stable even after the pancreas had been left for 14 hrs in the room temperature. In nondiabetics except cases of insulinoma the content of insulin in pancreas ranged from 1.42 to 4.56 U per gram and that of C-peptide from 8.76 to 25.63 microgram per gram wet pancreas. The proportion of proinsulinlike components (PLC) ranged from 0.01 to 2.04% of insulin plus PLC. In diabetics insulin content was low and ranged from 0 to 1.68 U per gram and that of C-peptide from 0 to 14.48 microgram per gram wet pancreas. In insulinoma, both insulin and C-peptide increased and PLC occupied 5.48 and 5.96%, respectively.     

What are the relevant parameters for the geometrical optimization of an implantable bioartificial pancreas?

A sphere within a cylinder representing the islet encapsulated in a hollow fiber can model an implantable bioartificial pancreas. Based on a finite element model for insulin response to a glucose load in the presence of various oxygen supplies, the present study aimed at pointing out the major parameters influencing this secretion. The computational results treated with the Taguchi method clearly demonstrated that geometrical parameters (fiber length and islet density) should be precisely optimized for an enhanced insulin response. This requires the collection of more relevant experimental data concerning the islet oxygen consumption. Moreover, the relative errors on glucose consumption or insulin secretion by the islets do not seem to affect the whole optimization process, which should focus on the oxygen supply to islets.     

A novel model of solute transport in a hollow-fiber bioartificial pancreas based on a finite element method

Extravascular bioartificial pancreas based on hollow fiber seems to be a promising treatment of diabetes mellitus. However, solutes mass-transport limitations in such a device could explain its lack of success. To determine critical device parameters, we have developed a novel tridimensional model based on finite element method for glucose, insulin, and oxygen diffusion around an islet of Langerhans encapsulated in a hollow-fiber section. A glucose ramp stimulation was applied outside the fiber and diffused to the islet. Concomitantly, a stationary oxygen partial pressure was applied outside the fiber, and determined local oxygen partial pressure on the islet environment. An insulin secretion model stimulated by a glucose concentration ramp and corrected by the local oxygen partial pressure was also implemented. Insulin secretion by the islet was thus computed as a response to glucose signal. The model predictions notably showed that the fiber radius had to be small enough to favor a fast response for insulin secretion and to ensure a maximal oxygen partial pressure in the islet environment. Besides the effect of fiber radius, a better islet oxygenation could be achieved by adjustments on the islet density, i.e., on the fiber length dedicated to a single islet. These hints should allow the future proposal of an optimal design for an implantable bioartificial pancreas.     

Theoretical analysis of the effect of convective flow on solute transport and insulin release in a hollow fiber bioartificial pancreas

The bioartificial pancreas, in which transplanted pancreatic tissue or isolated cells are cultured on a hollow fiber membrane, is an attractive approach to restore physiologic insulin delivery in the treatment of diabetes. Insulin response in prototype devices has been unacceptable due to the large mass transport limitations associated with the membrane and the surrounding shell region. Although available theoretical analyses provide some insight into the combined effects of transport and reaction in the bioartificial pancreas, they cannot quantitatively account for the effects of convective recirculation flow, complex intrinsic insulin secretory kinetics, and non-uniform distribution of pancreatic cells. We have developed a detailed model for glucose and insulin transport and insulin secretion in the hollow fiber bioartificial pancreas based on the solution of the mass and momentum conservation equations describing flow and transport in the lumen, matrix, and shell. Model predictions are in good agreement with literature data obtained in a hollow fiber device with minimal radial convective flow. Although no quantitative data are available for a device with significant radial convection, model simulations demonstrate that convective recirculation flow can dramatically improve insulin response, allowing the device to accurately capture the bi-phasic insulin secretion characteristic of the normal physiologic response. Results provide fundamental insights into the coupling between kinetics and transport in the hollow fiber system and a rational basis for the design of clinical devices.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

Alginate/aminopropyl-silicate/alginate membrane immunoisolatability and insulin secretion of encapsulated islets

We utilized the sol-gel reaction to prepare an immunoisolatable membrane for a microcapsule-shaped bioartificial pancreas. The membrane, derived from two precursors, 3-(aminopropyl)trimethoxysilane (APTrMOS) and tetramethoxysilane (TMOS), was formed onto calcium-alginate gel beads via electrostatic interaction. The molecular weight cutoff point of less than 150 000 required for immunoisolation was achieved at molar ratios ([APTrMOS]/[TMOS]) ranging from 0.60 to 2.40 with the amount of APTrMOS fixed at 3.40 mmol/(10 mL of calcium-alginate). When encapsulated in a membrane prepared at the molar ratio of 0.60, the islets contracted in volume and showed no response to stimulation by a high glucose concentration. However, islets in a membrane prepared at the molar ratio of 2.40 showed no contraction and responded to the glucose stimulation at almost the same level as free islets. These results demonstrated that the molar ratio of the precursors was a dominant factor affecting membrane permeability and the insulin secretion activity of the encapsulated islets.     

RNA molecular weight determination by agarose gel electrophoresis using formaldehyde as denaturant: comparison of RNA and DNA molecular weight markers

Reliable molecular weight measurements of RNA molecules as large as 4.0 X 10(6) dalton can be made on agarose gels containing 2.2 M formaldehyde as denaturant (Lehrach et al., 1977). Both eucaryotic and procaryotic ribosomal RNAs have generally been used as molecular weight markers. However, Maniatis et al. (1982) have suggested the use of restriction fragments of DNA as convenient molecular weight markers for RNA samples run in formaldehyde/agarose gels. This communication compares RNA and DNA molecular weight markers run under identical conditions.     

Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-I, and basic fibroblast growth factor by PLGA/PEG microspheres

The present investigation evaluates the effects of long-term, local delivery of insulin, insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF) on fat-graft survival using a poly (lactic-co-glycolic-acid)-polyethylene glycol (PLGA/PEG) microsphere delivery system. Twelve-micrometer PLGA/PEG microspheres incorporated separately with insulin, IGF-1, and bFGF were manufactured using a double-emulsion solvent-extraction technique. Inguinal fat from Sprague Dawley rats was harvested, diced, washed, and mixed with (1) insulin microspheres, (2) insulin-like growth factor-1 microspheres, (3) basic fibroblast growth factor microspheres, (4) a combination of the insulin and IGF-1 microspheres, and (5) a combination of insulin, IGF-1, and bFGF microspheres. The treated fat grafts were implanted autologously into subdermal pockets in six animals for each group. Animals receiving untreated fat grafts and fat grafts treated with blank microspheres constituted two external control groups (six animals per external control group). At 12 weeks, all fat-graft groups were compared on the basis of weight maintenance and a histomorphometric analysis of adipocyte area percentage, indices of volume retention and cell composition, respectively. Weight maintenance was defined as the final graft weight as a percent of the implanted graft weight. All growth factor treatments significantly increased fat-graft weight maintenance objectively, and volume maintenance grossly, in comparison with the untreated and blank microsphere-treated controls. Treatment with insulin and IGF-1, alone or in combination, was found to increase the adipocyte area percentage in comparison with fat grafts treated with bFGF alone or in combination with other growth factors. In conclusion, the findings of this study indicate that long-term, local delivery of growth factors with PLGA/PEG microspheres has the potential to increase fat-graft survival rates. Further, the type of growth factor delivered may influence the cellular/stromal composition of the grafted tissue.     

De novo adipose tissue generation through long-term, local delivery of insulin and insulin-like growth factor-1 by PLGA/PEG microspheres in an in vivo rat model: a novel concept and capability

This study was undertaken to characterize the duration of long-term growth factor delivery by poly(lactic-co-glycolic-acid)-polyethylene glycol (PLGA/PEG) microspheres and to evaluate the potential of long-term delivery of insulin and insulin-like growth factor-1 (IGF-1) for the de novo generation of adipose tissue in vivo. PLGA/PEG microspheres containing insulin and IGF-1, separately, were produced by a double-emulsion solvent-extraction technique. In the first phase of the experiment, the in vitro release kinetics of the microspheres were evaluated for the optical density and polyacrylamide gel electrophoresis of solutions incubated with insulin-containing microspheres for four different periods of time (n = 1). The finding of increased concentrations of soluble insulin with increased incubation time confirmed continual protein release. In the second stage of the experiment, 16 rats were divided equally into four study groups (insulin, IGF-1, insulin + IGF-1, and blank microspheres) (n = 4). Insulin and IGF-1 containing microspheres were administered directly to the deep muscular fascia of the rat abdominal wall to evaluate the potential for de novo adipose tissue generation via adipogenic differentiation from native nonadipocyte cell pools in vivo. Animals treated with blank microspheres served as an external control group. At the 4-week harvest period, multiple ectopic islands of adipose tissue were observed on the abdominal wall of the animals treated with insulin, IGF-1, and insulin + IGF-1 microspheres. Such islands were not seen in the blank microsphere group. Hematoxylin and eosin-stained sections of the growth factor groups demonstrated mature adipocytes interspersed with fibrous tissue superficial to the abdominal wall musculature and continuous with the fascia. Oil-Red-O stained sections demonstrated that these cells contained lipid. Computer-aided image analysis of histologic sections confirmed that there were statistically significant increases in the amount of "ectopic" adipose neotissue developed on the abdominal wall of animals treated with growth factor microspheres. In conclusion, this study confirms the long-term release of proteins from PLGA/PEG microspheres up to 4 weeks and demonstrates the potential of long-term local insulin and IGF-1 to induce adipogenic differentiation to mature lipid-containing adipocytes from nonadipocyte cell pools in vivo at 4 weeks.     

Insulin degradation in insulinoma: evidence for the occurrence of an inactive form of glutathione-insulin transhydrogenase and for the absence of insulin A and B chains degrading protease(s)

Islet cell tumors (insulinomas) have been found to contain insulin-degrading activity. Apparent K_m values for insulin obtained with tumor extracts were similar to those found for other tissues and for purified glutatione-insulin transhydrogenase (GIT). In Ouchterlony double diffusion experiments with antibody to purified human liver GIT, each tumor extract gave a single precipitation band of identity with purified human liver GIT. Examination by chromatography on Sephadex G-75 of the products formed from ¹²⁵I-insulin upon incubation with tumor extracts showed the same products (A chain, and B chain rich-A chain aggregate) as previously found with purified GIT; however, there was no further degradation (i.e., proteolysis) of A chain to low molecular weight components. These results indicate that the insulin-degrading activity present in the islet tumor is, in fact, GIT and that the protease(s) that further catabolizes the insulin A and B chains is apparently missing in the insulinoma. These data could be interpreted to indicate that the function of GIT in this tissue is to promote the biosynthesis of proinsulin and insulin rather than their degradation. Data are also presented which indicate that in insulinoma GIT is present in an inactive state as a divalent metal ion complex since it could be activated with EDTA and/or GSH.     

A synthetic polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-stearyl methacrylate), stimulates insulin release from RINm5F insulinoma cells

A water-soluble phospholipid-like polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-stearyl methacrylate) (PMC(18), average molecular weight = 4.3 x 10(4)), at a concentration (0.5-5 mg/ml) showing no inhibition of cell proliferation, stimulated insulin release from RINm5F rat insulinoma cells in a concentration- and time-related manner. But poly(2-methacryloyloxyethyl phosphorylcholine) and other synthetic phospholipid-like polymers failed to stimulate insulin release.     

Renal uptake and degradation of trapped-label insulin

In order to study the kinetics of insulin degradation in the kidneys and liver, insulin was labelled by a trapped-label procedure and injected into rats. In contrast to conventional 125I-insulin, the trapped-label preparation allows quantitative measurements of the extent of degradation in vivo because the final degradation products do not leave the cells. One hour after injection, the amount of radioactivity in the kidneys from a trace dose of trapped-label insulin was 10 times higher that from conventionally labelled insulin; over 80% of the increase was due to low molecular weight degradation products which were retained in the kidneys. The amount of acid-precipitable radioactivity in the blood was the same for both labelled preparations, indicating that their rates of clearance were similar. In the kidney, we detected no degradation products of molecular weight intermediate between intact insulin and the end products of proteolysis. After 2 h, 33% of the injected dose remained in the kidneys and only 13% in the liver. Over 80% of the renal radioactivity was sedimentable in an isotonic density gradient, indicating that intact insulin, as well as degradation products in the cells, were enclosed within membrane-bound vesicles.     

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Identification of a calcium-regulated insulinoma cell phosphoprotein as an islet cell keratin

This report describes the cytoskeleton nature of a 60,000-mol-wt protein, P60, previously shown to undergo Ca2+ influx-induced phosphorylation concomitant with insulin release in hamster insulinoma cells. Four lines of evidence suggest that P60 is an intermediate filament protein of the keratin class. (a) As previously described (Schubart, U.K., 1982, J. Biol. Chem. 257:12231-12238), Triton X-100-insoluble cytoskeletons are enriched for P60; (b) these cytoskeletons contain 7-11-nm filaments as determined by negative staining; (c) immunoblot analysis revealed that all proteins detected in the insulinoma cell cytoskeletons are recognized by a monoclonal antibody that interacts with a common determinant in all intermediate filament proteins; and (d) P60 was shown, by its identical migration on two-dimensional electrophoresis and by its immunologic relatedness, to be analogous to a known keratin present in HeLa cells. An antibody specific for P60, as judged by immunoblotting, was developed in a rabbit. In indirect immunofluorescence studies on insulinoma cells, this anti-P60 antibody produced a filamentous staining pattern. The antibody also permitted the identification of P60 in normal pancreatic islets as determined both by immunoblotting of hamster islet proteins resolved by two-dimensional electrophoresis and by indirect immunofluorescence microscopy on cryostat sections of hamster pancreas. In addition, the antibody recognized an antigen in the epithelial layer of pancreatic exocrine ducts, as determined by indirect immunofluorescence. The data have implications for the embryonic origin of pancreatic islets. Together with the phosphorylation data, these findings suggest that this islet cell cytokeratin may be involved in the regulation of insulin release.     

Giant Pancreatic Tumor With Clinical Characteristics of Insulinoma But Without Common Pathologic Features

ObjectiveTo report a case of a large pancreatic tumor that had clinical characteristics of an insulinoma without classic pathologic features.MethodsWe describe a 58-year-old woman who presented with a 3-month history of symptomatic hypoglycemic episodes, which were characterized by confusion. The laboratory, imaging, and pathologic findings are summarized, the current literature on giant insulinomas is reviewed, and the distinction between clinical and pathologic diagnosis of neuroendocrine tumors is discussed.ResultsThe biochemical diagnosis of insulinoma was established with concomitant low fasting blood glucose concentrations and inappropriately high insulin levels. An abdominal computed tomographic scan revealed a mass (10 by 11.7 by 9.7 cm) in the head and body of the pancreas, which was resected. Pathologic examination revealed a massive neuroendocrine tumor (13.5 by 11 by 8 cm) without immunohistochemical evidence of insulin expression. Nevertheless, tumor resection resulted in decreased blood insulin levels and resolution of the patient’s hypoglycemia.ConclusionAlthough more than 95% of insulinomas are smaller than 3 cm, this case is unique in that the extremely large pancreatic tumor had clinical characteristics of an insulinoma but did not have the classic pathologic findings. Because of the extensive pancreatic resection, the patient is dependent on both insulin and orally administered pancreatic enzymes but remained free of symptoms and disease recurrence at 1-year follow-up. (Endocr Pract. 2011;17:e12-e16)