Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

The effect of sugars on (pro)insulin biosynthesis

Rates of incorporation of [4,5-(3)H]leucine into insulin plus proinsulin, designated ;(pro)insulin', and total protein in rat pancreatic islets were measured. Glucose stimulates rates of total protein and (pro)insulin biosynthesis, but (pro)insulin biosynthesis is stimulated preferentially. Mannose and N-acetylglucosamine also stimulate (pro)insulin and total protein biosynthesis; inosine and dihydroxyacetone stimulate (pro)insulin biosynthesis specifically. Fructose does not stimulate (pro)insulin biosynthesis when tested alone, but does so in the presence of low concentrations of glucose, mannose or N-acetylglucosamine. Many glucose analogues do not stimulate (pro)insulin biosynthesis. Mannoheptulose inhibits synthesis of (pro)insulin and total protein stimulated by glucose or mannose but not by dihydroxyacetone, inosine or N-acetylglucosamine; phloretin (9mum) inhibits N-acetylglucosamine-stimulated (pro)insulin biosynthesis preferentially. The data are in agreement with the view that the same glucose-sensor mechanism may control both insulin release and biosynthesis, and ;substrate-site' model is suggested. The threshold for stimulation of biosynthesis of (pro)insulin and total protein is lower than that found for glucose-stimulated insulin release; moreover the biosynthetic response to an elevation of glucose concentration is slower than that found for insulin release. The physiological implication of these findings is discussed. Caffeine and isobutylmethylxanthine, at concentrations known to increase islet 3':5'-cyclic AMP and potentiate glucose-induced insulin release, were without effect on rates of glucose-stimulated (pro)insulin biosynthesis.     

Kinetics of permeation and intracellular events associated with Me2SO permeation of rabbit kidneys during perfusion at 10 °C

The permeation kinetics of the cryoprotectant dimethylsulfoxide (Me₂SO) were investigated by the measure of total water, Me₂SO, and inulin spaces using radioactive tracers. Complete permeation of rabbit kidneys with a perfusate concentration of 3.0 M Me₂SO at 10 °C was obtained 35 min after reaching the maximum concentration when the cryoprotectant was introduced slowly. Equilibration was complete in the medulla before the cortex, probably due to the higher flow rates.Through study of the change in Me₂SO space and the inulin space, we were able to obtain evidence of cellular permeation of Me₂SO. In control kidneys, the inulin space decreased slightly during perfusion, an indication of cell swelling. Kidneys perfused with Me₂SO demonstrated a doubling of the inulin space, which did not decrease with time. The most likely explanation of this phenomenon is marked cell shrinkage, which was confirmed on histological examination and increased intraluminal tubular fluid. The picture is more complex since after intracellar equilibration, rehydration of the cell is not observed.     

Dimethyl sulfoxide maintains structure and function of cryopreserved equine endometrial explants

Availability of viable frozen-thawed endometrial tissues could facilitate detailed studies into physiologic and disease processes influencing the endometrium. This study was designed to investigate the cryosurvival of equine endometrial tissue. Previous studies in the human and horse have focused on cryopreservation of dissociated endometrial cells. To our knowledge, there are no studies on cryopreservation of endometrial explants. Our objectives were to 1) determine the influence of differing concentrations of the permeating cryoprotectant dimethyl sulfoxide (Me₂SO) on viability, structural integrity, and gene expression of cryopreserved equine endometrial tissues prior to and following a 5-day explant culture in vitro and 2) examine the influence of low (1000 mg/L dextrose) vs high (4500 mg/L dextrose) glucose medium during in vitro culture. Both 10% and 20% (v/v) concentrations of Me₂SO maintained viability following cryopreservation and in vitro culture. In addition, gene expression remained unaltered following cryopreservation with either 10% or 20% Me₂SO. However, tissue structural integrity was slightly reduced compared to the fresh control. Furthermore, there was no difference in structural integrity, cell viability, or gene expression between low and high glucose medium during in vitro culture. Although E-cadherin and Ki67 gene expression was not different among fresh, 10% Me₂SO, and 20% Me₂SO treatments prior to or following tissue culture, estrogen receptor-α and progesterone receptor gene expression were reduced in all groups after explant culture. This is the first report of successful cryopreservation of equine endometrial explants.     

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me₂SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME₂SO, 1M ME₂SO + 1M EG, and 1M ME₂SO + 0.5M EG). Next, fresh islets, 2M ME₂SO islets, and 1M ME₂SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME₂SO (1M ME₂SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME₂SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME₂SO + 0.5M EG), compared to islets preserved with 2M ME₂SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.     

Effect of interferon and double-stranded RNA on B-cell function in mouse islets of Langerhans.

The direct effects of alpha- and beta-interferons on isolated mouse pancreatic islets were investigated in vitro and found to be similar. After 7 h incubation with interferon concentrations above 350 units/ml, glucose-stimulated (pro)insulin biosynthesis was significantly inhibited, with only a slight inhibition of total protein biosynthesis. Inhibition could be abolished in the additional presence of an anti-interferon antibody. Interferon did not affect insulin release, total insulin content, or glucose oxidation of the islets. The stimulation of (pro)insulin biosynthesis by adenosine, D-glyceraldehyde, mannose, N-acetylglucosamine and leucine was also inhibited by interferon, with no effect on insulin release. At concentrations of dsRNA (double-stranded RNA) said to induce interferon (1-100 micrograms/ml), glucose-stimulated (pro)insulin biosynthesis was inhibited without significantly affecting insulin release. The dsRNA may itself inhibit stimulated (pro)insulin biosynthesis or may function indirectly by the induction of interferon.     

Function of rabbit kidneys in vitro at normothermia following equilibration with 3.0 M Me2SO and removal by hypertonic washout at 10 degrees C

In the present study, rabbit kidneys were assayed for function on a 37 °C in vitro perfusion system after perfusion on a 10 °C perfusion system which permits the slow introduction and removal of cryoprotectant. The final concentration of 3.0 M Me₂SO was introduced slowly at two different rates. The washout was achieved by perfusion with Me₂SO-free solutions made hypertonic with mannitol. Two regimens of washout were used: 800, 700, 600, 500, and 400 mOsm/kg; and 600, 500, and 400 mOsm/kg.During perfusion at 37 °C, the glomerular filtration rate was similar in all groups and this increased significantly in all groups with time. Protein leakage was minimal. All three Me₂SO groups showed a depressed Na reabsorption capacity, but the 800 mOsm group was the most severely affected. This was also found with glucose reabsorption. We concluded that rabbit kidneys will function well with the cryoprotectant Me₂SO up to 3 M concentration when introduced slowly and washed out with hypertonic mannitol beginning at 600 mOsm/kg. When 800 mOsm is used at the initial step, the proximal tubular function is severely affected.     

Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice

Background aimsWe recently showed that co-transplantation of mesenchymal stromal cells (MSCs) improves islet function and revascularization in vivo. Pre-transplant islet culture is associated with the loss of islet cells. MSCs may enhance islet cell survival or function by direct cell contact mechanisms and soluble mediators. We investigated the capacity of MSCs to improve islet cell survival or β-cell function in vitro using direct and indirect contact islet-MSC configurations. We also investigated whether pre-culturing islets with MSCs improves islet transplantation outcome.MethodsThe effect of pre-culturing islets with MSCs on islet function in vitro was investigated by measuring glucose-stimulated insulin secretion. The endothelial cell density of fresh islets and islets cultured with or without MSCs was determined by immunohistochemistry. The efficacy of transplanted islets was tested in vivo using a syngeneic streptozotocin-diabetic minimal islet mass model. Graft function was investigated by monitoring blood glucose concentrations.ResultsIndirect islet-MSC co-culture configurations did not improve islet function in vitro. Pre-culturing islets using a direct contact MSC monolayer configuration improved glucose-stimulated insulin secretion in vitro, which correlated with superior islet graft function in vivo. MSC pre-culture had no effect on islet endothelial cell number in vitro or in vivo.ConclusionsPre-culturing islets with MSCs using a direct contact configuration maintains functional β-cell mass in vitro and the capacity of cultured islets to reverse hyperglycemia in diabetic mice.     

A faster reconstitution of hematopoiesis after autologous transplantation of hematopoietic cells cryopreserved in 7.5% dimethyl sulfoxide if compared to 10% dimethyl sulfoxide containing medium

Our previous in vitro studies proved a higher clonogenic potential of peripheral blood progenitor cells cryopreserved in 7.5% dimethyl sulfoxide (Me₂SO) than in 10% Me₂SO containing medium. Based on this findings 7.5% Me₂SO cryopreservation medium was introduced to our protocol and both the hematopoietic recovery and infusion-related toxicity were compared with that obtained with standard 10% Me₂SO containing solution. Two cohorts of consecutive patients treated with autologous hematopoietic stem cell transplantation were included in the analysis: 56 patients with PBPCs cryopreserved in 7.5% Me₂SO solution and 52 patients who obtained cells cryopreserved in 10% Me₂SO. Both study groups did not differ significantly with regard to age, diagnosis, and the number of transplanted CD34⁺ cells. The time to leukocyte recovery was shorter for patients in the 7.5% Me₂SO treated group than in the 10% one. Reconstitution of platelets and the frequency of adverse events did not differ in both groups. Reduction of Me₂SO concentration from 10% to 7.5% in cryoprotective mixture has a beneficial impact on leukocyte recovery. These findings require verification in a prospective, randomized trial.     

Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity

Aim

To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity.

Methods

There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 °C. Dimethyl sulphoxide (Me₂SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks’ balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me₂SO in multi-well tissue culture plates at 1 °C/min to −80 °C. After storage overnight, the valve leaflets were warmed at approximately 11 °C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann’s solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth.

Results

After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me₂SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me₂SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks’ solution (Group 3) (χ², p = 0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (χ², p = 0.007). The rates of cell growth in Group 2 (two-step addition of Me₂SO) and Group 3 (one-step addition of Me₂SO with additional Hanks’ solution rinse) were similar and faster than the Group 4 (one-step addition of Me₂SO without the additional Hanks’ rinse).

Conclusion

Single-step addition of Me₂SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks’ solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me₂SO, and the 5% Me₂SO step in the two-step protocol, merely served to reduce this carryover.     

The significance of culture for successful cryopreservation of isolated pancreatic islets of Langerhans

It was the aim of the present study to investigate the significance of culture before and after freeze-thawing of isolated mouse pancreatic islets. To evaluate the impact of culture before freezing (5 degrees C/min; 2 M dimethyl sulfoxide), islets were frozen either directly after isolation or after 2, 4, or 7 days of culture in medium RPMI 1640. The culture period after thawing was 7 days. Islets immediately frozen exhibited virtually no (pro)insulin biosynthesis and also a severe inhibition of glucose-stimulated insulin release. The precultured (2-7 days), frozen islets synthesized and released insulin at rates comparable to those of nonfrozen, cultured islets. Studies of the effects of culture after freeze-thawing were performed after a 3-day culture period prior to freezing. The (pro)insulin biosynthetic rates did not differ between islets cultured for 0-7 days after thawing. There was an apparent increase of glucose-stimulated insulin release when the islets were cultured for more than 2 days after thawing. It may be that the decreased viability of islets frozen immediately after isolation was due to minor cell damage induced by the collagenase incubation. During culture the islets may recover and become more resistant to freeze-damage. The beneficial effect of culture after thawing may reflect the loss of damaged cells, which otherwise would influence the results of the viability tests.     

Permeability of the 17-day fetal rat pancreas to glycerol and dimethylsulfoxide

Experimentally induced diabetes in rats can be reversed by the transplantation of several fresh or frozen-thawed fetal pancreases. An important question to both the mechanistic and practical aspects of cryobiology is the role played by the permeation of protective additives during freezing, thawing, and subsequent dilution. Answers require knowledge of the kinetics of permeation of the specific additive into the cell or tissue. In this paper, we report isotopic measurements of the rate of permeation of 2 M glycerol and 1 and 2 M dimethylsulfoxide (Me₂SO) into 17-day fetal pancreases at 0 and 22 °C. In Me₂SO, equilibrium was achieved in about 10–15 min at 0 °C and in less than 10 min at 22 °C. In glycerol, equilibrium was attained in about 60 min at 22 °C; but at 0 °C permeation was only 65% complete after 180 min. In general, Me₂SO permeated 10–30 times more rapidly than glycerol at 0 °C, and glycerol permeated about 10 times more rapidly at 22 than at 0 °C.The kinetics of permeation were more characteristic of a two-compartment than a single-compartment system. In all probability, the two compartments are the intercellular space and the intracellular space. The permeability data suggest that each compartment occupies about half the total volume.     

Long-term treatment with atrial natriuretic peptide inhibits ATP production and insulin secretion in rat pancreatic islets

Atrial natriuretic peptide (ANP) levels correlate with hyperglycemia in diabetes mellitus, but ANP effects on pancreatic islet β-cell insulin secretion are controversial. ANP was investigated for short- and long-term effects on insulin secretion and mechanisms regulating secretion in isolated rat pancreatic islets. A 3-h incubation with ANP did not affect basal or glucose-stimulated islet insulin secretion. However, 7-day culture of islets with 5.5 mM glucose and ANP (1 nM - 1 μM) markedly inhibited subsequent glucose (11 mM)-stimulated insulin secretion; total islet insulin content was not affected. Following ANP removal for 24 h, the islet insulin-secretory response to glucose was restored. The insulin-secretory response to other insulin secretagogues, including α-ketoisocaproic acid, forskolin, potassium chloride, and ionomycin were also markedly inhibited by chronic exposure to ANP. However, the combination of potassium chloride and α-ketoisocaproic acid was sufficient to overcome the inhibitory effects of ANP on insulin secretion. The glucose-stimulated increases in islet ATP levels and the ATP/ADP ratio were completely inhibited in ANP 7-day-treated islets vs. control; removal of ANP for 24 h partially restored the glucose response. ANP did not affect islet glycolysis. ANP significantly increased levels of islet activated hormone-sensitive lipase and the expression of uncoupling protein-2 and peroxisome proliferator-activated receptor-δ and -α. Although islet ANP-binding natriuretic peptide receptor-A levels were reduced to 60% of control after 7-day culture with ANP, the ANP-stimulated cGMP levels remained similar to control islet levels. Thus, long-term exposure to ANP inhibits glucose-stimulated insulin secretion and ATP generation in isolated islets.     

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice

Background aimsCo-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation.MethodsMouse islets and kidney MSCs were co-encapsulated in alginate, and their function was assessed in vitro. A minimal mass of 350 syngeneic islets encapsulated alone or co-encapsulated with MSCs (islet+MSC) were transplanted intraperitoneally into diabetic mice, and blood glucose concentrations were monitored. Capsules were recovered 6 weeks after transplantation, and islet function was assessed.ResultsIslets co-encapsulated with MSCs in vitro had increased glucose-stimulated insulin secretion and content. The average blood glucose concentration of transplanted mice was significantly lower by 3 weeks in the islet+MSC group. By week 6, 71% of the co-encapsulated group were cured compared with 16% of the islet-alone group. Capsules recovered at 6 weeks had greater glucose-stimulated insulin secretion and insulin content in the islet+MSC group.ConclusionsMSCs improved the efficacy of microencapsulated islet transplantation. Using an isolated-graft model, we were able to eliminate the impact of MSC-mediated enhancement of revascularization and preservation of islet morphology and demonstrate that the improvement in insulin secretion and content is sustained in vivo and can significantly improve graft outcome.     

Regulation of RNA metabolism in relation to insulin production and oxidative metabolism in mouse pancreatic islets in vitro

This study was undertaken to investigate the long-term effects of different substrates, in particular glucose, on the regulation of islet RNA metabolism and the relationship of this regulation to the metabolism and insulin production of the islet B-cell. For this purpose collagenase-isolated mouse islets were used either in the fresh state or after culture for 2 or 5 days in RPMI 1640 plus 10% calf serum supplemented with various test compounds. Islets cultured with 16.7 mM glucose contained more RNA than those cultured with 3.3 mM glucose. Culture of islets in glucose at low concentrations inhibited glucose-stimulated RNA synthesis and this inhibitory effect was reversed by prolonged exposure to high glucose concentrations. Culture with 10 mM leucine and 3.3 mM glucose or with 10 mM 2-ketoisocaproate and 3.3 mM glucose increased the total RNA content of islets as compared to that of islets cultured with 3.3 mM glucose alone. Islets cultured with 5 mM theophylline maintained a high RNA content in the presence of 3.3 mM glucose. Theophylline also increased the islet RNA content when added together with 16.7 mM glucose, as compared to 16.7 mM glucose alone. Theophylline probably exerted this effect by decreasing the rate of RNA degradation. Changes in islet RNA metabolism showed a close correlation to changes in islet total protein biosynthesis, whereas islet (pro)insulin biosynthesis and insulin release exhibited different glucose-dependency patterns. The response of islet oxygen uptake to glucose was similar to that of islet RNA and protein biosynthesis. It is concluded that the RNA content of the pancreatic islets is controlled at the levels of both synthesis and degradation. Glucose stimulates the RNA synthesis and inhibits its degradation. Moreover, the results suggest that regulation of RNA synthesis may be mediated through islet metabolic fluxes and the cAMP system.     

Effects of phloretin and dextran-linked phloretin on pancreatic islet metabolism and insulin release

The effects of phloretin on islet metabolism and insulin release have been studied in isolated pancreatic islets of the rat. At a concentration of 0.18 mM phloretin inhibited insulin release stimulated by glucose or leucine but did not affect the oxidation rates of glucose or leucine, the rate of glucose utilization and the islet content of ATP. Higher concentrations of phloretin caused inhibition of the rate of glucose metabolism, but stimulation of insulin release. Insulin release stimulated by phloretin was inhibited by mannoheptulose but was independent of extracellular Ca²⁺ and was not potentiated by caffeine. Both inhibitory and stimulatory effects of dextran-linked phloretin on insulin release were also seen; a concentration of dextran-linked phloretin that did not inhibit islet metabolism inhibited glucose-stimulated insulin release, but not release stimulated by leucine or glyceraldehyde. Higher concentrations of dextran-linked phloretin inhibited glucose oxidation but stimulated insulin release. These data are discussed in terms of current models of the β-cell glucose-sensor mechanism.     

Optimization of cryoprotectant loading into murine and human oocytes

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me₂SO) into mouse oocytes at 23 °C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me₂SO exposure time, revealing that neither shrinkage nor Me₂SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me₂SO addition appears to result from interactions between the effects of Me₂SO toxicity and osmotic stress. We also investigated Me₂SO loading into mouse oocytes at 30 °C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me₂SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me₂SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.     

Effect of 2-deoxy-d-glucose on maintenance in culture of neonatal B cell of rat

Summary The effect of 2-deoxy-d-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-d-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-d-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.     

Glucose-dependent and Glucose-sensitizing Insulinotropic Effect of Nateglinide: Comparison to Sulfonylureas and Repaglinide

Nateglinide, a novel D-phenylalanine derivative, stimulates insulin release via closure of K_ATP channels in pancreatic β-cell, a primary mechanism of action it shares with sulfonylureas (SUs) and repaglinide. This study investigated (1) the influence of ambient glucose levels on the insulinotropic effects of nateglinide, glyburide and repaglinide, and (2) the influence of the antidiabetic agents on glucose-stimulated insulin secretion (GSIS) in vitro from isolated rat islets. The EC₅₀ of nateglinide to stimulate insulin secretion was 14 μM in the presence of 3mM glucose and was reduced by 6-fold in 8mM glucose and by 16-fold in 16mM glucose, indicating a glucose-dependent insulinotropic effect. The actions of glyburide and repaglinide failed to demonstrate such a glucose concentration-dependent sensitization. When tested at fixed and equipotent concentrations (~2x EC₅₀ in the presence of 8mM glucose) nateglinide and repaglinide shifted the EC₅₀s for GSIS to the left by 1.7mM suggesting an enhancement of islet glucose sensitivity, while glimepiride and glyburide caused, respectively, no change and a right shift of the EC₅₀. These data demonstrate that despite a common basic mechanism of action, the insulinotropic effects of different agents can be influenced differentially by ambient glucose and can differentially influence the islet responsiveness to glucose. Further, the present findings suggest that nateglinide may exert a more physiologic effect on insulin secretion than comparator agents and thereby have less propensity to elicit hypoglycemia in vivo.