A computer model of pancreatic islet glycolysis

We have modeled an experiment with perifused pancreatic islet cells using our BIOSSIM language. The experiment and the resulting model are concerned with glucose uptake and glycolysis by the beta-cells of pancreatic islets. Although glycolysis appears to be involved in insulin release, we do not have enough information to represent insulin release in detail. The rapid entry of glucose into the beta-cell is promoted by a carrier having a very high tissue capacity. Phosphorylation of glucose by the low affinity enzyme glucokinase appears to be limiting for glycolysis. The effects of several hexose diphosphate activators of phosphofructokinase are modeled. Model behavior is described. The kinetic parameters of the enzyme submodels are given. Because of the difficulties of preparing large amounts of experimental material, information on pancreatic islet metabolism is limited. This model is a plausible explanation of the experimental results. Recent work on the genetically engineered glucose transporter and glucokinase is discussed.     

Development and characterization of a tissue engineered pancreatic substitute based on recombinant intestinal endocrine L‐cells

A tissue engineered pancreatic substitute (TEPS) consisting of insulin‐producing cells appropriately designed and encapsulated to support cellular function and prevent interaction with the host may provide physiological blood glucose regulation for the treatment of insulin dependent diabetes (IDD). The performance of agarose‐based constructs which contained either a single cell suspension of GLUTag‐INS cells, a suspension of pre‐aggregated GLUTag‐INS spheroids, or GLUTag‐INS cells on small intestinal submucosa (SIS), was evaluated in vitro for total cell number, weekly glucose consumption and insulin secretion rates (GCR and ISR), and induced insulin secretion function. The three types of TEPS studied displayed similar number of cells, GCR, and ISR throughout 4 weeks of culture. However, the TEPS, which incorporated SIS as a substrate for the GLUTag‐INS cells, was the only type of TEPS tested which was able to retain the induced insulin secretion function of non‐encapsulated GLUTag‐INS cells. Though improvements in the expression level of GLUTag‐INS cells and/or the number of viable cells contained within the TEPS are needed for successful treatment of a murine model of IDD, this study has revealed a potential method for promoting proper cellular function of recombinant L‐cells upon incorporation into an implantable three‐dimensional TEPS. Biotechnol. Bioeng. 2009;103: 828–834. © 2009 Wiley Periodicals, Inc.     

Development of embryonic chick insulin cells in culture: Beneficial effects of serum-free medium, raised nutrients, and biomatrix

Summary A previous finding that insulin cells do not survive or differentiate in explants of embryonic avian pancreas cultured in collagen gel with a serum-containing medium has provided a model system for identification of conditions favorable for development of these cells. To this end, we here modify the substrate and the medium. The epithelial component of dorsal pancreatic buds of 5-d chick embryos was cultured for 7 d on Matrigel in serum-containing and in serum-free medium, the latter incorporating insulin, transferrin, and selenium, Endocrine cell types were distinguished by immunocytochemistry; insulin cell counts were expressed as a proportion of insulin plus glucagon cells. With serum-containing medium, Matrigel stimulated a significant increase in this proportion as compared with collagen gel—3.1% as against 0.2%; the serum-free medium further increased this proportion to 17.3%. Raising the level of essential amino acids approximately fivefold increased the latter figure somewhat (to 18.9%), but it was more than doubled (to 37.4%) by raising the glucose concentration from 10 mM to 20 mM. Raising the levels of amino acids and glucose simultaneously yielded a lesser increase (to 31.8%). Some cultures grown in collagen gel and serum-containing medium for 7 d were transferred to Matrigel and serum-free medium for a further 7 d. Insulin cell development recovered, indicating that progenitor cells had survived and were stimulated to develop by the improved conditions. This study indicates that components of the biomatrix and the medium (in particular, a raised glucose concentration) are important for the survival and differentiation of embryonic insulin cells.     

Cortisone-induced islet cell hyperplasia in hamsters

Pancreatic islet cell hyperplasia was studied in hamsters during one to eight weeks of cortisone treatment. Measurement of serum glucose and insulin; pancreatic insulin, glucagon, somatostatin, pancreatic polypeptide as well as islet tissue morphometry were performed. Serum glucose was highest at week 2, followed by mild to moderate hyperglycemia. Serum insulin was increasingly higher from week 1 to week 8. Pancreatic insulin was maximal at week 5 then declined through week 8 in the presence of beta cell neurosis in markedly hyperplastic islets. Pancreatic concentration of somatostatin and pancreatic polypeptide moderately increased more than the control levels; however, compared with the controls, glucagon was reduced by cortisone treatment. Effect of cortisone in the four types of islet cells is discussed, particularly on beta cell hyperplasia, which appears to be a response to decreased insulin binding to the target organs with no changes in receptor concentration.     

Identification of the Ubiquitin-like Domain of Midnolin as a New Glucokinase Interaction Partner

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.     

The insulin sensitizing effect of homoisoflavone-enriched fraction in Liriope platyphylla Wang et Tang via PI3-kinase pathway

In the present study, we screened candidates for enhancing insulin action, using glucose uptake as an indicator, from Liriope platyphylla Wang et Tang (LPWT) extract, Liliaceae, in 3T3-L1 adipocytes. The mechanism of insulin sensitizing action in the fractions was also investigated. LPWT extract with 70% MeOH was sequentially separated with Diaion HP-20 and silica gel column chromatography. The 9:1 fraction from silica gel column chromatography increased glucose uptake with 1 ng/mL up to glucose uptake with 50 ng/mL insulin. The 9:1 fraction, determined as homoisoflavone-enriched fraction, worked as an insulin sensitizer. It increased insulin stimulated glucose uptake in 3T3-L1 adipocytes, insulin responsive cells, through increased glucose transporter 4 (GLUT4) contents in the plasma membrane. GLUT4 translocation was increased through insulin receptor substrate 1 (IRS1)-PI3 kinase-Akt signaling mechanism. Thus, homoisoflavone-enriched fraction in LPWT extract played an important role as an insulin sensitizer in adipocytes.     

Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms

Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in?vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.     

Glucose regulates the cortical actin network through modulation of Cdc42 cycling to stimulate insulin secretion

Glucose-stimulated insulin granule exocytosis in pancreatic beta-cells involves cortical actin remodeling that results in the transient disruption of the interaction between polymerized actin with the plasma membrane t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. To examine the mechanism underlying the initiation of cortical actin remodeling, we have used the actin nucleating/stabilizing agent jasplakinolide to show that remodeling is initiated at a step proximal to the ATP-sensitive K+ channels in the stimulus-secretion pathway. Confocal immunofluorescent microscopy revealed that cortical actin remodeling was required for glucose-stimulated insulin secretion. Furthermore, glucose was found to mediate the endogenous activation state of the Rho family GTPase Cdc42, a positive proximal effector of actin polymerization, resulting in a net decrease of Cdc42-GTP within 5 min of stimulation. Intriguingly, glucose stimulation resulted in the rapid and reversible glucosylation of Cdc42, suggesting that glucose inactivated Cdc42 by selective glucosylation to induce cortical actin rearrangement. Moreover, expression of the constitutively active form of Cdc42 (Q61L) inhibited glucose-stimulated insulin secretion, whereas the dominant negative form (T17N) was without effect, suggesting that glucose-stimulated insulin secretion requires Cdc42 cycling to the GDP-bound state. In contrast, KCl-stimulated insulin secretion was unaffected by the expression of dominant negative or constitutively active Cdc42 and ceased to modulate endogenous Cdc42 activation, consistent with glucose-dependent cortical actin remodeling. These findings reveal that glucose regulates the cortical actin network through modulation of Cdc42 cycling to induce insulin secretion in pancreatic beta-cells.     

Generation of glucose-responsive functional islets with a three-dimensional structure from mouse fetal pancreatic cells and iPS cells in vitro

Islets of Langerhans are a pancreatic endocrine compartment consisting of insulin-producing β cells together with several other hormone-producing cells. While some insulin-producing cells or immature pancreatic cells have been generated in vitro from ES and iPS cells, islets with proper functions and a three-dimensional (3D) structure have never been successfully produced. To test whether islets can be formed in vitro, we first examined the potential of mouse fetal pancreatic cells. We found that E16.5 pancreatic cells, just before forming islets, were able to develop cell aggregates consisting of β cells surrounded by glucagon-producing α cells, a structure similar to murine adult islets. Moreover, the transplantation of these cells improved blood glucose levels in hyperglycemic mice. These results indicate that functional islets are formed in vitro from fetal pancreatic cells at a specific developmental stage. By adopting these culture conditions to the differentiation of mouse iPS cells, we developed a two-step system to generate islets, i.e. immature pancreatic cells were first produced from iPS cells, and then transferred to culture conditions that allowed the formation of islets from fetal pancreatic cells. The islets exhibited distinct 3D structural features similar to adult pancreatic islets and secreted insulin in response to glucose concentrations. Transplantation of the islets improved blood glucose levels in hyperglycemic mice. In conclusion, the two-step culture system allows the generation of functional islets with a 3D structure from iPS cells.     

Inositol hexakisphosphate and sulfonylureas regulate beta-cell protein phosphatases

In human type 2 diabetes, loss of glucose-stimulated insulin exocytosis from the pancreatic beta-cell is an early pathogenetic event. Mechanisms controlling insulin exocytosis are, however, not fully understood. We show here that inositol hexakisphosphate (InsP(6)), whose concentration transiently increases upon glucose stimulation, dose-dependently and differentially inhibits enzyme activities of ser/thr protein phosphatases in physiologically relevant concentrations. None of the hypoglycemic sulfonylureas tested affected protein phosphatase-1 or -2A activity at clinically relevant concentrations in these cells. Thus, an increase in cellular phosphorylation state, through inhibition of protein dephosphorylation by InsP(6), may be a novel regulatory mechanism linking glucose-stimulated polyphosphoinositide formation to insulin exocytosis in insulin-secreting cells.     

Nutrient–secretion coupling in the pancreatic islet β-cell: recent advances

Insulin secretion from pancreatic islet β-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total β-cell mass is essentially unaltered, but β-cells become progressively “glucose blind” and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K⁺(K_ATP) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca²⁺, then prompt the “first phase” on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K_ATP channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.     

Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships

The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.     

Screen-printed electrodes modified with glucose oxidase immobilized in hybrid organosilicon sol-gel matrix

A new polymer composition was developed based on hybrid sol-gel material for the immobilization of enzymes on the surface of screen-printed carbon electrodes modified with Prussian blue. The existing and developed methods for the glucose oxidase (GO) immobilization are compared. Highly stable bioelectrodes were shown to be obtained by enzyme immobilization on a hybrid composition consisting of solgel/poly vinyl alcohol (PVA) (up to 60 sequential chains) and agar gel (up to 45 sequential chains). The range of glucose concentrations detected during enzyme immobilization in a hybrid sol-gel/PVA composition or agar gel without dilution of a sample was 1.0–5.9 μM and 3.6–6.3 μM, respectively. An analysis of wine products was conducted. The results obtained using the proposed biosensors were shown to differ insignificantly from those obtained by high-performance liquid chromatography (the correlation coefficient was 0.9998).     

Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells

One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies.     

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.     

Portal vein insulin response of glucose intolerant Yucatan miniature swine to common secretogogues

A group of glucose intolerant miniature swine exhibiting an impaired portal vein insulin response to an IVGTT were examined with respect to their portal vein insulin response to the secretogogues: isoproterenol, arginine and leucine. Equivalent insulin responses to isoproterenol and leucine were noted on the part of the glucose intolerant animals when compared to control subjects. An impaired portal vein insulin response was evident during an infusion of 0.5 g/kg arginine and again when a pulse injection of .25 g/kg glucose was administered in the presence of isoproterenol (.05 microgram/kg . min). The close agreement of these results with those reported for human diabetics suggests that a similar pancreatic defect, most probably associated with the glucoreceptor, is present in this group of glucose intolerant miniature swine.