Kinetics Of Insulin Release Research Articles

Of mighty mice and diabetic men: animal models of islet dysfunction

Of mighty mice and diabetic men: animal models of islet dysfunction

Cx36-Mediated Coupling Reduces β-Cell Heterogeneity, Confines the Stimulating Glucose Concentration Range, and Affects Insulin Release Kinetics

We studied the effect of gap junctional coupling on the excitability of beta-cells in slices of pancreas, which provide a normal environment for islet cells. The electrophysiological properties of beta-cells from mice (C57Bl/6 background) lacking the gap junction protein connexin36 (Cx36(-/-)) were compared with heterozygous (Cx36(+/-)) and wild-type littermates (Cx36(+/+)) and with frequently used wild-type NMRI mice. Most electrophysiological characteristics of beta-cells were found to be unchanged after the knockout of Cx36, except the density of Ca(2+) channels, which was increased in uncoupled cells. With closed ATP-sensitive K(+) (K(ATP)) channels, the electrically coupled beta-cells of Cx36(+/+) and Cx36(+/-) mice were hyperpolarized by the membrane potential of adjacent, inactive cells. Additionally, the hyperpolarization of one beta-cell could attenuate or even stop the electrical activity of nearby coupled cells. In contrast, beta-cells of Cx36(-/-) littermates with blocked K(ATP) channels rapidly depolarized and exhibited a continuous electrical activity. Absence of electrical coupling modified the electrophysiological properties of beta-cells consistent with the reported increase in basal insulin release and altered the switch on/off response of beta-cells during an acute drop of the glucose concentration. Our data indicate an important role for Cx36-gap junctions in modulating stimulation threshold and kinetics of insulin release.

Glucose-stimulated Cdc42 Signaling Is Essential for the Second Phase of Insulin Secretion

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.

Protein Kinase Cδ Plays a Non-redundant Role in Insulin Secretion in Pancreatic β Cells

Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.

Preparation and Characterization of Insulin Nanoparticles Employing Chitosan and Poly(methylmethacrylate/methylmethacrylic acid) Copolymer

As most of polypeptides are marginally stable, a mild formulation procedure would be beneficial for the activities of these drugs. The objective of the present study was to develop a novel pH-sensitive nanoparticle system that was suitable for entrapment of hydrophilic insulin but without affecting its conformation. Chitosan was incorporated as a positively charged material, and one of the three poly(methylmethacrylate/methylmethacrylic acid) copolymers, consisting of Eudragit L100-55, L100, and S100, was used as a negatively charged polymer for preparation of three insulin nanoparticles, respectively. Three nanoparticles obtained were spherical. The mean diameters were in the range from 200 nm to 250 nm, and the entrapment efficiencies, from 50% to 70%. The surface analysis indicated that insulin was evenly distributed in the nanoparticles. Polymer ratio of chitosan to Eudragit was the factor which influenced the nanoparticles significantly. Characterization results showed that the electrostatic interactions existed, thus providing a mild formulation procedure which did not affect the chemical integrity and the conformation of insulin. In vitro release studies revealed that all three types of the nanoparticles exhibited a pH-dependant characteristic. The modeling data indicated that the release kinetics of insulin was nonlinear, and during the release process, the nanoparticles showed a polynomial swelling. On overall estimation, the insulin chitosan-Eudragit L100-55 nanoparticles may be better for the oral delivery. This new pH-sensitive nanoparticle formulation using chitosan and Eudragit L100-55 polymer may provide a useful approach for entrapment of hydrophilic polypeptides without affecting their conformation.

CaV2.3 calcium channels control second-phase insulin release

Concerted activation of different voltage-gated Ca2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type CaV2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in CaV2.3-knockout (CaV2.3–/–) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological CaV2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single CaV2.3–/– s cells. CaV2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the CaV2.3–/– mouse. We propose a specific role for CaV2.3 Ca2+ channels in second-phase insulin release, that of mediating the Ca2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation. (Less)

CaV2.3 calcium channels control second-phase insulin release

Concerted activation of different voltage-gated Ca( (2+) ) channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca(V)2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca(V)2.3-knockout (Ca(V)2.3(-/-)) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca(2+) channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca(V)2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca(2+) signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca(V)2.3(-/-) beta cells. Ca(V)2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca(V)2.3(-/-) mouse. We propose a specific role for Ca(V)2.3 Ca(2+) channels in second-phase insulin release, that of mediating the Ca(2+) entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.

Synthesis and characterisation of stimuli-responsive poly(N,N′-diethylacrylamide) hydrogels

Stimuli-responsive poly(N,N′-diethylacrylamide) gels were prepared by free radical polymerisation in aqueous solution, using N,N-methylenebisacrylamide as crosslinking agent. The gels were compared with the corresponding poly(N-isopropylacrylamide)-based gels. In particular, the swelling ratio of both gel types including the effect of the crosslinker content, their swelling and deswelling kinetics, their permeability and finally their drug (insulin) storage and controlled release ability were compared. In spite of the similarity in the monomer/crosslinker ratio, the deswelling kinetics and the critical temperatures (ca. 30–32°C in pure water), some differences could be observed. Compared to poly(N-isopropylacrylamide)-based gels, poly(N,N′-diethylacrylamide)-based gels show a broader phase transition temperature interval, a more pronounced dependency of the swelling ratio on the crosslinker content, slower reswelling kinetics, a higher ingress percentage for dextran standards ranging from 5 to 70kD, but lower ingress percentages for proteins (BSA, insulin) and much faster drug (insulin) release kinetics. While a non-linear release kinetic was observed in the case of the poly(N-isopropylacraylamide)-based gels both in water and in PBS (phosphate buffered saline), this was not the case for the poly(N,N′-diethylacrylamide)-based gels.

How cyclodextrin incorporation affects the properties of protein-loaded PLGA-based microspheres: the case of insulin/hydroxypropyl-β-cyclodextrin system

The aim of this work was to study the influence of cyclodextrin (CD) incorporation on the properties of protein-loaded poly(lactide-co-glycolide) (PLGA) microspheres, with particular regards to protein release kinetics. To this purpose, insulin-loaded microspheres were prepared by spray-drying emulsion or solution formulations, with or without hydroxypropyl-β-cyclodextrin (HPβCD), and fully characterized for encapsulation efficiency and release kinetics of both insulin and cyclodextrin. Homogeneous populations of spherical microparticles entrapping both insulin and HPβCD were obtained. In order to get an insight into insulin/HPβCD interactions occurring inside microspheres, Fourier transform infrared (FTIR) analysis in the Amide I region was performed. FTIR spectra of dried microspheres containing HPβCD showed a change in insulin secondary structure, attributed to the presence of insulin/HPβCD complexes within microspheres. Insulin release was affected by the presence of HPβCD depending on the initial formulation conditions. In the case of microspheres prepared from emulsion, cyclodextrin reduced only insulin burst, whereas in the case of microspheres obtained from solution, the overall insulin release rate was slowed down. Combining the release kinetics of HPβCD with the FTIR results on hydrated microspheres, it was concluded that the formation of insulin/HPβCD complexes inside microspheres is critical to decrease protein diffusivity in the polymer matrix and achieve an effective modulation of protein release rate.

Thermally modulated insulin release from microgel thin films.

We describe investigations of thermally triggered insulin release from poly(N-isopropylacrylamide-co-acrylic acid) microgel thin films prepared by layer-by-layer (LbL) polyelectrolyte assembly. The thermoresponsivity of these films was confirmed using light scattering techniques. Simultaneous monitoring of film collapse and insulin release kinetics shows that deswelling of the films is partially decoupled from macromolecule release and that release is mainly governed by partitioning effects. We hypothesize, however, that film thermoresponsivity plays an important role in that subjection to many thermal cycles enables the embedded peptide to solubilize and subsequently partition through film layers. Direct pulsatile and extended release studies confirm the capability of these films to release bursts of insulin over many cycles, and confirm that the magnitude of the release can be controlled based on film thickness. These insulin-impregnated films are extremely stable with the potential to release constant pulses of peptide for more than 1 month at a time.

Prolongation of islet allograft survival following ex vivo transduction with adenovirus encoding a soluble type 1 TNF receptor-Ig fusion decoy.

Islet transplantation is a viable long-term therapeutic alternative to daily insulin replacement for type I diabetes. The allogeneic nature of the transplants poses immunological challenges for routine clinical utility. Gene transfer of immunoregulatory molecules and those that improve insulin release kinetics provides rational approaches to facilitate allogeneic islet transplantation as a potential therapy. We have examined the efficacy of a soluble type 1 tumor necrosis factor receptor (TNFR) immunoglobulin-Fc fusion transgene (TNFR-Ig) to protect human islets from cytokine-induced apoptosis in culture, as well as in facilitating allogeneic islet transplants in diabetic mice. Cultured human islets were transduced with an adenoviral vector encoding human TNFR-Ig (Ad-TNFR-Ig). TNFR-Ig protein was secreted by cultured islets, as well as by transduced mouse islet transplants recovered from mouse recipients. Glucose-induced insulin release kinetics were comparable among untransduced, Ad-TNFR-Ig-infected human islets and vector-transduced islets exposed to cytokines. In parallel, Ad-TNFR-Ig-infected islets were protected from cytokine-induced apoptosis activation. Finally, diabetic mice transplanted with allogeneic islets expressing TNFR-Ig returned to and maintained normoglycemia significantly longer than untransduced islet recipients. These data support the potential utility of TNFR-Ig gene transfer to islets as a means of facilitating allogeneic islet transplantation.

Regulation of Insulin Exocytosis by Munc13-1

The slower kinetics of insulin release from pancreatic islet beta cells, as compared with other regulated secretory processes such as chromaffin granule secretion, can in part be explained by the small number of the insulin granules that are docked to the plasma membrane and readily releasable. In type-2 diabetes, the kinetics of insulin secretion become grossly distorted, and, to therapeutically correct this, it is imperative to elucidate the mechanisms that regulate priming and secretion of insulin secretory granules. Munc13-1, a synaptic protein that regulates SNARE complex assembly, is the major protein determining the priming of synaptic vesicles. Here, we demonstrate the presence of Munc13-1 in human, rat, and mouse pancreatic islet beta cells. Expression of Munc13-1, along with its cognate partners, syntaxin 1a and Munc18a, is reduced in the pancreatic islets of type-2 diabetes non-obese Goto-Kakizaki and obese Zucker fa/fa rats. In insulinoma cells, overexpressed Munc13-1-enhanced green fluorescent protein is translocated to the plasma membrane in a temperature-dependent manner. This, in turn, greatly amplifies insulin exocytosis as determined by patch clamp capacitance measurements and radioimmunoassay of the insulin released. The potentiation of exocytosis by Munc13-1 is dependent on endogenously produced diacylglycerol acting on the overexpressed Munc13-1 because it is blocked by a phospholipase C inhibitor (U73122) and abrogated when the diacylglycerol binding-deficient Munc13-1H567K mutant is expressed instead of the wild type protein. Our data demonstrate that Munc13-mediated vesicle priming is not restricted to neurotransmitter release but is also functional in insulin secretion, where it is subject to regulation by the diacylglycerol second messenger pathway. In view of our findings, Munc13-1 is a potential drug target for therapeutic optimization of insulin secretion in diabetes.

Macroencapsulation of rat islets without alteration of insulin secretion kinetics.

Transplantation of encapsulated islets may restore endogenous insulin secretion in type 1 diabetics with no need of lifetime immunosuppression of the recipient. A biomaterial should be developed which combined immunoisolation with rapid and efficient diffusion of glucose and insulin. Rat islets were macroencapsulated in capillaries (molecular cut off 50 kD) of differently modified polysulphone. Macroencapsulated islets were perifused to study the kinetics of glucose induced insulin secretion into the perifusion medium. Blending polysulphone (PSU) with poly vinyl pyrrolidone or sodium dodecyl sulphate was not suited for islet macroencapsulation since glucose induced insulin release was absent after encapsulation. Hydroxy methylation (CH2OH) of PSU improved the secretory behaviour of macroencapsulated islets depending on the degree of substitution (DS). At 0.8 DS glucose induced insulin secretion was delayed and inefficient. At maximal degrees of PSU-substitution (1.8) the kinetics of insulin release and the efficiency of insulin release were very similar to that observed of free floating islets. In conclusion, highly substituted hydroxy methylated polysulphone allows a rapid and efficient insulin release after macroencapsulation and is suited for the further development of a bioartificial pancreas.

In vitro test of new biomaterials for the development of a bioartificial pancreas.

The implantation of macroencapsulated islets has the potential to restore endogenous insulin secretion in type 1 diabetics, with no need for lifetime immunosuppression. To match the physiological fluctuations of blood glucose concentrations with appropriate insulin release, the macroencapsulation material must combine immunoprotection with optimal diffusion properties for glucose and insulin. The impact of chemical modifications of polysulphone (PSU) capillary polymers with a cutoff of 50 kD on glucose-induced insulin secretion of macroencapsulated rat islets was studied in perifusion experiments. The insulin release of free-floating islets showed the typical rapid response to glucose stimulation. Total insulin release (AUC between minute 30 and 120 of perifusion) reached 117+/-22 ng/ml. Blending PSU with polyvinylpyrrolidone or sodium-dodecyl-sulfate was not suitable for islet macroencapsulation, since glucose-induced insulin release was absent or disturbed. Hydroxy-methylation (CH2OH) of PSU improved the secretory behavior of macroencapsulated islets depending on the degree of PSU substitution (DS 0.8, AUC 62+/-15 ng/ml; DS 1.8, 111+/-24 ng/ml). In highly substituted PSU-capillaries the kinetics of glucose-induced insulin release was very similar to that observed in free-floating islets. Two consecutive glucose stimulations potentiated insulin release of free-floating islets during the second period of stimulation. Furthermore, freshly isolated macroencapsulated islets responded with more efficient insulin secretion after the initial priming. In conclusion, in vitro membrane screening identified highly substituted hydroxy-methylated PSU as the material of choice for islet encapsulation in a bioartificial pancreas.

Characterization of glucose-sensitive insulin release systems in simulated in vivo conditions

We studied the glucose-responsive insulin controlled release system based on the hydrogel poly(2-hydroxyethyl methacrylate-co- N, N-dimethylaminoethyl methacrylate), also called poly(HEMA-co-DMAEMA), with entrapped glucose oxidase, catalase and insulin. When exposed to physiological fluids, glucose diffuses into the hydrogel, glucose oxidase catalyzes the glucose conversion to gluconic acid, causing swelling of the pH-sensitive hydrogel and subsequently increased insulin release. The higher the glucose concentration in the medium, the higher and faster the swelling and release rates. The effects of polymer morphology and oxygen availability on hydrogel swelling and on insulin release kinetics were tested. Polymer morphology was modified by changing the crosslinking agent (tetraethylene glycol dimethacrylate) concentration (0–0.95 vol%). Oxygen availability was modified by changing the immobilized catalase concentration (0–15 units catalase per unit glucose oxidase) and by bubbling oxygen through the medium. The results indicated that: (i) Hydrogels without crosslinking agent were found to be stable in water, and their sensitivity to pH and glucose was higher than the chemically crosslinked hydrogels. (ii) Immobilization of catalase in addition to glucose oxidase in hydrogels prepared without crosslinking agent, resulted in enhanced swelling kinetic. In addition, we carried out primary in vivo experiments on rats, which demonstrated that at least some of the entrapped insulin retains its active form and is effective in reducing blood glucose levels. Moreover, no tissue encapsulation was observed around matrices implanted in the peritoneum. In conclusion, the pH-sensitive hydrogel poly(HEMA-co-DMAEMA) can be manipulated to produce glucose-responsive insulin release system that is effective in reducing blood glucose levels.

Improved vascularization of planar membrane diffusion devices following continuous infusion of vascular endothelial growth factor.

Improving blood vessel formation around an immunobarrier device should improve the survival of the encapsulated tissue. In the present study we investigated the formation of new blood vessels around a planar membrane diffusion device (the Baxter Theracyte System) undergoing a continuous infusion of vascular endothelial growth factor through the membranes and into the surrounding tissue. Each device (20 microl) had both an inner immunoisolation membrane and an outer vascularizing membrane. Human recombinant vascular endothelial growth factor-165 was infused at 100 ng/day (low dose: n = 6) and 500 ng/day (high dose: n = 7) for 10 days into devices implanted s.c. in Sprague-Dawley rats; noninfused devices transplanted for an identical period were used as controls (n = 5). Two days following the termination of VEGF infusion, devices were loaded with 20 microl of Lispro insulin (1 U/kg) and the kinetics of insulin release from the lumen of the device was assessed. Devices were then explanted and the number of blood vessels (capillary and noncapillary) was quantified using morphometry. High-dose vascular endothelial growth factor infusion resulted in two- to threefold more blood vessels around the device than that obtained with the noninfused devices and devices infused with low-dose vascular endothelial growth factor. This increase in the number of blood vessels was accompanied by a modest increase in insulin diffusion from the device in the high-dose vascular endothelial growth factor infusion group. We conclude that vascular endothelial growth factor can be used to improve blood vessel formation adjacent to planar membrane diffusion devices.

Dynamic measurements of oxygen tension in islets of Langerhans.

Oxidative metabolism plays a key role in glucose-induced insulin release (Ashcroft et al., 1973). With the development of a sensitive assay for insulin it was possible to characterize the dynamics of glucose-induced insulin release from the individual isolated islet of Langerhans (Bergsten and Hellman, 1993). To evaluate the role of the oxidative metabolism for the kinetics of insulin release a method for determining oxidative metabolism with a sensitivity comparable to that available for insulin is crucial. Glucose oxidation and oxygen consumption have been used to monitor glucose-induced metabolic changes (Ashcroft et al., 1973; Hutton and Malaisse, 1980; Longo et al., 1991). These studies indicate that insulin release and cellular metabolism, oxidation in particular, are tightly linked. However, the previously used techniques lack the sensitivity required to monitor the dynamics of metabolism from individual islets. Thus analysis of the coupling or synchronization between metabolic changes and secretion is hampered. To overcome this problem we have developed a new sensitive technique to monitor oxygen tension, which allows time-resolved measurements from individual islets, and characterized the responses in pO2 following exposure to glycolytic and mitochondrial substrates.KeywordsPancreatic IsletOxygen TensionInsulin ReleaseSynchronous OscillationMicroscope ChamberThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Impaired insulin secretion and excessive hepatic glucose production are both early events in the diabetic GK rat.

Adult Goto-Kakisaki Wistar (GK) rats exhibit a spontaneous non-insulin-dependent diabetes characterized by impaired glucose-induced insulin secretion, decreased beta-cell mass, hepatic glucose overproduction, and moderate insulin resistance in muscles and adipose tissues. To elucidate the pathogenesis of hyperglycemia in this animal model, we have studied insulin secretion and insulin action in 4-wk-old GK pups, just before weaning. In the postabsorptive state, their basal plasma glucose level was elevated (P < 0.001), and their tolerance to intravenous glucose was impaired. Their kinetics of insulin release in response to glucose was impaired, with a low acute phase of insulin release in vivo and in vitro (perfused pancreas). Basal glucose production was increased in the GK pups by 40% (P < 0.05). During euglycemic clamp performed at submaximal hyperinsulinemia, suppression of liver glucose production was less effective (P < 0.01) in the GK rats, whereas their overall glucose utilization was similar to that of the control group. This was correlated with a normal insulin-stimulated glucose utilization by epitrochlearis, soleus, and extensor digitorum longus muscles, diaphragm, and white adipose tissues. These data give body to the primacy of the beta-cell defects in the etiology of non-insulin-dependent diabetes mellitus in the GK rat. They also highlight a possible primary role of the liver defect. Peripheral insulin resistance does not contribute to the development of postnatal glucose intolerance in this diabetes model.

Insulin metabolism after relief of obstructive jaundice: Intravenous glucose tolerance test with portal blood sampling

Glucose intolerance and impaired insulin secretion are often associated with obstructive jaundice. Our objective was to determine whether such abnormalities would be ameliorated after jaundice was relieved by biliary drainage. Twenty-four patients with hepatobiliary malignancy prospectively underwent intravenous glucose tolerance test with femoral and portal blood sampling, and the kinetics of insulin release were determined. Sixteen patients had obstructive jaundice (group A) that had been completely relieved by percutaneous transhepatic biliary drainage by the time of intravenous glucose tolerance testing, and eight patients exhibited no jaundice (group B). Integrated immunoreactive insulin (sigmaIRI, 10 muU min/ml; mean +/- SD) and integrated C-peptide (sigmaCPR, 10 ng min/ml) in the portal blood in group A were significantly lower than those values in group B (sigmaIRI: group A, 436.0 +/- 260.6; group B, 714.3 +/-287.2; p< 0.01; sigmaCPR; group A, 26 +/- 10.1; group B 49.5 +/- 18.8; p<0.005). The hepatic insulin extraction ratio (portal-femoral difference of sigmaIRI divided by portal sigmaIRI) in group A was significantly higher than that in group B (group A, 0.75 +/- 0.06; group B, 0.55 +/- 0.05; p<0.001), whereas the hepatic CPR extraction ratio did not differ significantly between the two groups (group A, 0.37 +/- 0.10; group B, 0.39 +/- 0.05). The impaired insulin secretion caused by obstructive jaundice is not fully reversed after percutaneous transhepatic biliary drainage. The high hepatic extraction ratio of insulin in patients who had been treated with percutaneous transhepatic biliary drainage may compensate for the impaired insulin secretion, although its mechanism is still unclear.

Development of experimental insulin pumps with glucose-controlled release

In the present investigation, experimental insulin pumps have been developed in which the release of insulin is governed by the glucose concentration. The mechanical energy required for the glucose-dependent emptying of the insulin reservoir is provided by the carbon dioxide formed on the interaction of glucose with yeast cells. The developed pumps consist of a delivery unit containing an insulin reservoir and a reaction chamber in which 50 mg freeze-dried yeast and 150 μl glucose solution are incubated. Variable glucose-dependent insulin release could be obtained by using an exchangeable bioreactor, which is replaced at given intervals by a new bioreactor containing fresh supplies of freeze-dried yeast and glucose solution. The results with solutions of 100, 200, and 400 mg glucose per 100 ml show that insulin release kinetics are directly affected by the glucose concentration.