Isolation of viable porcine islets by selective osmotic shock without enzymatic digestion.

Islet transplantation is a potential cure for type 1 diabetes, but clinical results have been disappointing. Currently, islet isolation is by enzymatic digestion of the pancreas which has significant pitfalls: warm ischemia exposure, collagenase-induced damage to the islet mass and viability, poor reproducibility, high cost, a relatively low number of islets obtained per whole pancreas, and selection of islets for collagenase resistance rather than for glucose responsiveness. In the present study we performed a series of experiments in a porcine model to demonstrate the feasibility of a new isolation method based on selective osmotic shock (SOS) using very high glucose solutions, doubling or tripling physiological osmotic strength. The SOS method can be carried out at room temperature or in the cold eliminating warm ischemia time which damages the islets. The SOS method does not depend on the texture of the pancreas so all pancreases can be processed identically and the process can be fully automated. The SOS method isolates all the islets of the pancreas regardless of size and shape allowing a greater number of islets to be harvested. The SOS method avoids exposure to toxins in collagenase solutions, is inexpensive and selects for islets with high concentrations of Glut 2 transporters, representing the best glucose responding islets. The SOS method showed a comparable recovery of islets from young pig pancreas and the islets showed improved viability. We conclude that the selective osmotic shock (SOS) method of separating islets from the pancreatic tissue is superior to the collagenase method.

Effect of glycemic index on whole-body substrate oxidation in obese women.

BACKGROUND: Glycemic index is hypothesized to determine fuel partitioning through serum plasma insulin modifications induced by dietary carbohydrates, thereby modulating fat accretion or oxidation. OBJECTIVE: To assess the glycemic effects on postprandial fuel oxidation and blood response. DESIGN: In all, 12 obese women were fed on a randomized crossover design with two test meals (breakfast+lunch). High- or low-glycemic meals were provided on separate days. Energy intake on high-glycemic meal was 7758+/-148 kJ and for low-glycemic meal was 7806+/-179 kJ. Carbohydrates supplied were 273+/-5 and 275+/-6 g, respectively. Macronutrient distribution was 55% carbohydrates, 30% fat and 15% protein. Fuel oxidation was measured continuously in a respiratory chamber for 10 h. Serum glucose, free fatty acids (FFA), insulin and glucagon samples were taken for 5 h after breakfast. RESULTS: Glucose AUC changed significantly in response to different glycemic breakfast. Low- vs high-glycemic breakfast was 211+/-84 and 379+/-164 mmol/l (P<0.05). Similarly, insulin changed from 94+/-37 and 170+/-87 nmol/l (P<0.05), respectively. The rate of increment for serum glucose and insulin reached by the high- vs low-glycemic meal was 1.8 times more with the high-glycemic breakfast. Serum FFA were similarly suppressed by both meal types by 3 h after meal intake, but then raised significantly more with the low-glycemic meal by the fourth and fifth hour (P<0.05). Plasma glucagon did not show a significant variation with glycemic index. Carbohydrate and fat oxidation was not modified by glycemic meal characteristics, being virtually the same for low- vs high-glycemic comparisons in the 5 h following breakfast and lunch (P=NS). CONCLUSION: This study demonstrates that dietary glycemic characteristics were unable to modify fuel partitioning in sedentary obese women.

Proinsulin serum concentrations in women with polycystic ovary syndrome: a marker of beta-cell dysfunction?

BACKGROUND: The aim of this study was to establish the effect of polycystic ovary syndrome (PCOS) adjusted for adiposity on proinsulin concentrations. METHODS: Ninety-one women with PCOS and 72 normal cycling (NC) women were recruited. A 2 h, 75 g oral glucose tolerance test was performed. Glucose and insulin were measured in each sample. Proinsulin and C-peptide were determined at 0 and 30 min and the fasting proinsulin/insulin ratio (PI/I) was calculated. Insulin sensitivity was estimated by insulin sensitivity index (ISI) composite, and beta-cell function was estimated by insulinogenic index. RESULTS: Insulin, proinsulin and C-peptide concentrations were higher in women with PCOS than in NC women (P < 0.05). PI/I and insulinogenic index were similar in both groups. Proinsulin concentrations increased with body mass index (P < 0.05) only in women with PCOS; therefore, proinsulin concentrations were higher in obese PCOS patients compared with obese control women (P < 0.05). Moreover, a positive association between proinsulin concentrations and waist diameter adjusted for C-peptide (P < 0.05) and a negative association between proinsulin concentrations and ISI composite values were observed in PCOS patients (P < 0.05). CONCLUSIONS: Data suggest that in PCOS patients an elevated proinsulin concentration could reflect insulin resistance more than beta-cell dysfunction. However, the elevated concentration of proinsulin in these patients could also result from impaired beta-cell function resulting from intra-abdominal obesity independently of insulin resistance.

Evaluation of insulin resistance in two kinds of South American camelids: llamas and alpacas.

Insulin resistance was evaluated in South American camelids, llamas and alpacas, by use of the minimal model test and the insulin tolerance test. Animals were catheterized for long-term studies and tamed to minimize stress during evaluation. Results indicated a low insulin sensitivity index (SI) = 0 to 0.97, median = 0.39 x 10(-4) min/uIU x ml, about a fifth the value in other mammals and humans. The KITT was between 1.43 and 3.19 %/min, also significantly lower than that reported for humans. Glycosylated hemoglobin concentration was 6%, and HbAlc concentration was 5.5%; red blood cell lifetime, as measured by use of the 51Cr method, was 120 days, similar to the value in humans. We concluded that llamas and alpacas have naturally higher blood glucose concentration than do humans and other mammals during the glucose tolerance test. Using the same mathematical tools to evaluate glucose metabolism as those used in people, South American camelids appear to be resistant to insulin. Thus, the South American camelid may be a useful new animal model for the study of sugar metabolism and various facets of diabetes mellitus, especially protection from the deleterious effects of glycosylation.

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca(2+) signaling, and insulin secretion in the anx7(+/-) knockout mouse.

The mammalian anx7 gene codes for a Ca(2+)-activated GTPase, which supports Ca(2+)/GTP-dependent secretion events and Ca(2+) channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca(2+) signaling in secreting pancreatic beta cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the beta cells. The nullizygous anx7 (-/-) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/-) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/-) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca(2+) channel functions are normal. However, electrooptical recordings indicate that the (+/-) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP(3))-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP(3) receptor expression and function in pancreatic islets. The profound increase in islets, beta cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic beta cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca(2+) signaling through IP(3)-sensitive Ca(2+) stores.

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium.

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.

Glucagon induces suppression of ATP-sensitive K+ channel activity through a Ca2+/calmodulin-dependent pathway in mouse pancreatic beta-cells.

Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion in pancreatic beta-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of the hypothesis that changes in beta-cells ATP-sensitive K+ (K(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 x 10(-7) m) in the presence of 11.1 mm glucose caused closure of K(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy K(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K(ATP) channel activity until Ca2+ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects were seen when db-cAMP was used instead of glucagon. These results support the view that glucagon and other cAMP-generating agonists enhance glucose-induced beta-cell electrical activity through a Ca2+/calmodulin dependent-closure of K(ATP) channels.

Evidence that calcium release-activated current mediates the biphasic electrical activity of mouse pancreatic beta-cells.

The electrical response of pancreatic beta-cells to step increases in glucose concentration is biphasic, consisting of a prolonged depolarization with action potentials (Phase 1) followed by membrane potential oscillations known as bursts. We have proposed that the Phase 1 response results from the combined depolarizing influences of potassium channel closure and an inward, nonselective cation current (ICRAN) that activates as intracellular calcium stores empty during exposure to basal glucose (Bertram et al., 1995). The stores refill during Phase 1, deactivating ICRAN and allowing steady-state bursting to commence. We support this hypothesis with additional simulations and experimental results indicating that Phase 1 duration is sensitive to the filling state of intracellular calcium stores. First, the duration of the Phase 1 transient increases with duration of prior exposure to basal (2.8 mM) glucose, reflecting the increased time required to fill calcium stores that have been emptying for longer periods. Second, Phase 1 duration is reduced when islets are exposed to elevated K+ to refill calcium stores in the presence of basal glucose. Third, when extracellular calcium is removed during the basal glucose exposure to reduce calcium influx into the stores, Phase 1 duration increases. Finally, no Phase 1 is observed following hyperpolarization of the beta-cell membrane with diazoxide in the continued presence of 11 mm glucose, a condition in which intracellular calcium stores remain full. Application of carbachol to empty calcium stores during basal glucose exposure did not increase Phase 1 duration as the model predicts. Despite this discrepancy, the good agreement between most of the experimental results and the model predictions provides evidence that a calcium release-activated current mediates the Phase 1 electrical response of the pancreatic beta-cell.

Ionic mechanisms involved in the regulation of insulin secretion by muscarinic agonists.

The effects of the muscarinic agonist oxotremorine-m (oxo-m) on insulin secretion, K(+)-permeability and electrical activity from isolated mouse pancreatic islets were studied. Oxo-m potentiated glucose-induced insulin secretion in a dose-dependent manner, saturating at ca. 10 microM. At 11.2 mM glucose, oxo-m (0.1 and 10 microM) had two distinct effects on beta-cell electrical activity. Both concentrations increased the steady-state burst frequency, however, at 10 microM an initial and transient polarization was measured, and the subsequent activity was accompanied by a slight depolarization. The polarizing effect of oxo-m was almost completely suppressed by charybdotoxin (ChTX), a blocker of the large conductance (maxi) [Ca2+]i-activated potassium channel (K(Ca)). In the presence of 11.2 mM glucose, oxo-m (50 microM) provoked a significant and transient increase in the 86Rb efflux from perifused islets. This effect was inhibited by ChTX. ChTX also potentiated oxo-m stimulated insulin secretion in the presence of glucose. Finally, the balance between the polarizing and depolarizing effects of oxo-m was variable in different islets and depended on glucose concentration. Insulin secretion stimulated by oxo-m in the presence of glucose was more closely correlated to the agonist induced increase in burst frequency than to an increase in plateau fraction. We conclude that muscarinic stimulation has at least two effects on beta-cell electrical activity, an initial hyperpolarization, owing to activation of K(Ca) channels, followed by depolarization and high-frequency bursts, proposed to reflect the activation of a current sensitive to the depletion of intracellular Ca2+ stores (CRAC).

Magnitude and modulation of pancreatic beta-cell gap junction electrical conductance in situ.

The parallel gap junction electrical conductance between a beta-cell and its nearest neighbors was measured by using an intracellular microelectrode to clamp the voltage of a beta-cell within a bursting islet of Langerhans. The holding current records consisted of bursts of inward current due to the synchronized oscillations in membrane potential of the surrounding cells. The membrane potential record of the impaled cell, obtained in current clamp mode, was used to estimate the behavior of the surrounding cells during voltage clamp, and the coupling conductance was calculated by dividing the magnitude of the current bursts by that of the voltage bursts. The histogram of coupling conductance magnitude from 26 cells was bimodal with peaks at 2.5 and 3.5 nS, indicating heterogeneity in extent of electrical communication within the islet of Langerhans. Gap junction conductance reversibly decreased when the temperature was lowered from 37 to 30 degrees C and when the extracellular calcium concentration was raised from 2.56 to 7.56 mM. The coupling conductance decreased slightly during the active phase of the burst. Activation of adenylate cyclase with forskolin (10 microM) resulted in an increase in cell-to-cell electrical coupling. We conclude that beta-cell gap junction conductance can be measured in situ under near physiological conditions. Furthermore, the magnitude and physiological regulation of beta-cell gap junction conductance suggest that intercellular electrical communication plays an important role in the function of the endocrine pancreas.

A role for calcium release-activated current (CRAC) in cholinergic modulation of electrical activity in pancreatic beta-cells.

S. Bordin and colleagues have proposed that the depolarizing effects of acetylcholine and other muscarinic agonists on pancreatic beta-cells are mediated by a calcium release-activated current (CRAC). We support this hypothesis with additional data, and present a theoretical model which accounts for most known data on muscarinic effects. Additional phenomena, such as the biphasic responses of beta-cells to changes in glucose concentration and the depolarizing effects of the sarco-endoplasmic reticulum calcium ATPase pump poison thapsigargin, are also accounted for by our model. The ability of this single hypothesis, that CRAC is present in beta-cells, to explain so many phenomena motivates a more complete characterization of this current.

Modulatory mechanism of ACTH on insulin secretion: effect on cytosolic Ca2+, membrane potential and Ca(2+-ATPase activity.

The aim of this work was to get some insight into the mechanism by which ACTH produces its enhancing effect on glucose-induced insulin secretion. For this purpose we have determined: a) the release of insulin by isolated rat islets incubated with 3.3 or 16.6 mM glucose with or without the addition of 500 pg/ml ACTH, together with the changes induced by ACTH on b) cytosolic [Ca2+] of isolated B cells, c) islet plasma membrane Ca(2+)-ATPase activity and d) changes in membrane potential of single mouse islets. ACTH significantly enhanced the release of insulin elicited by either 3.3 or 16.6 mM glucose. This hormone concentration also induced a significant increase in the cytosolic [Ca2+] in isolated B cells. ACTH did not produce B cell membrane depolarization. Conversely, ACTH produced a significant decrease in islet plasma membrane Ca(2+)-ATPase activity. These results suggest that ACTH in concentrations similar to those attained by the endogenous peptide at the islet interstitium exerts its positive modulation on glucose-induced secretion of insulin, at least partly through its increasing effect on cytosolic [Ca2+] of B cells. The latter might be the consequence of the decreasing effect of ACTH on Ca(2+)-ATPase activity rather than to stimulation of voltage-dependent Ca(2+)-channels.

Oxotremorine-m potentiation of glucose-induced insulin release from rat islets involves M3 muscarinic receptors.

cDNAs encoding for M1 and M3 muscarinic acetylcholine (ACh) receptors were detected in rat pancreatic islet cells by polymerase chain reaction (PCR) amplification techniques. A new cholinergic agonist, oxotremorine-m (oxo-m), in the presence of glucose (5.6 mM), produced a dose-dependent potentiation of insulin secretion saturating at approximately 5 microM. This effect was suppressed by the L-type Ca2+ channel blocker nifedipine. Higher doses of oxo-m (50 microM) induced a biphasic insulin response both at low (5.6 mM) or high (16.7 mM) glucose concentrations. In a Ca(2+)-deficient medium containing glucose (5.6 mM), oxo-m evoked only a reduced first phase of insulin secretion. The potentiating effects of oxo-m were inhibited by the muscarinic receptor antagonists 4-diphenylacetoxy-N-methylpiperidine methiodide (M3), hexahydro-sila-difenidol hydrochloride, p-fluoro analogue (M3 > M1 > M2), and pirenzepine (M1) in a dose-dependent manner; half-maximal inhibitory concentration values were approximately 5, 20, and 340 nM, respectively. The PCR results demonstrate the presence of M1 and M3 muscarinic ACh receptors in the islet tissue, and the secretion data strongly suggest that the potentiation of glucose-induced insulin release evoked by oxo-m depends on the activation of a muscarinic M3-subtype receptor present in the beta-cell membrane.

Single-microelectrode voltage clamp measurements of pancreatic beta-cell membrane ionic currents in situ.

A conventional patch clamp amplifier was used to test the feasibility of measuring whole-cell ionic currents under voltage clamp conditions from beta-cells in intact mouse islets of Langerhans perifused with bicarbonate Krebs buffer at 37 degrees C. Cells impaled with a high resistance microelectrode (ca. 0.150 G omega) were identified as beta-cells by the characteristic burst pattern of electrical activity induced by 11 mM glucose. Voltage-dependent outward K+ currents were enhanced by glucose both in the presence and absence of physiological bicarbonate buffer and also by bicarbonate regardless of the presence or absence of glucose. For comparison with the usual patch clamp protocol, similar measurements were made from single rat beta-cells at room temperature; glucose did not enhance the outward currents in these cells. Voltage-dependent inward currents were recorded in the presence of tetraethylammonium (TEA), an effective blocker of the K+ channels known to be present in the beta-cell membrane. Inward currents exhibited a fast component with activation-inactivation kinetics and a delayed component with a rather slow inactivation; inward currents were dependent on Ca2+ in the extracellular solution. These results suggest the presence of either two types of voltage-gated Ca2+ channels or a single type with fast and slow inactivation. We conclude that it is feasible to use a single intracellular microelectrode to measure voltage-gated membrane currents in the beta-cell within the intact islet at 37 degrees C, under conditions that support normal glucose-induced insulin secretion and that glucose enhances an as yet unidentified voltage-dependent outward K+ current.

The diabetogenic agent alloxan increases K+ permeability by a mechanism involving activation of ATP-sensitive K(+)-channels in mouse pancreatic beta-cells.

The effects of the diabetogenic agent, alloxan, on membrane potential, input resistance and electrical activity of normal mouse pancreatic beta-cells were studied. Tetraethylammonium (TEA), quinine and Glyburide were used to block K(+)-channels and to elucidate the mechanisms underlying alloxan's effects on beta-cell membrane potential. Exposure of the islet to alloxan (75-100 microM) in the presence of glucose (11 mM), produced a rapid (15 sec), transient inhibition of electrical activity, often accompanied by hyperpolarization of the membrane, and this was followed by recovery of the burst pattern. This early effect of alloxan was followed after approximately 15 min by a complete inhibition of electrical activity and hyperpolarization. The inhibition accompanied by hyperpolarization was associated with a decrease in input resistance, indicating increased K(+)-conductance. Both the transient and delayed effects of alloxan were blocked by glucose (33 mM), quinine and glyburide but not by other conditions which induce continuous electrical activity such as elevated external [K+] (10 mM), ouabain, K+ removal, or TEA (20 mM). The transient inhibition induced by alloxan may be due to a direct competition with glucose transport/metabolism since it did not occur when alpha-keto isocaproic acid (KIC) was used to induce electrical activity. The delayed inhibition may reflect indirect effects of accumulation of this agent or its metabolites within the cell. Since both effects of alloxan are blocked by glyburide they appear to involve activation of the ATP-sensitive K(+)-channel (K-ATP).

Prolactin treatment increases GLUT2 but not the G protein subunit content in cell membranes from cultured neonatal rat islets.

Neonatal rat islets exhibit a reduced secretory response to glucose, compared to adult rat islets. The maturation of the secretory response is stimulated by prolactin (PRL). We show here by immunoblot analysis that PRL increases the beta-cell/liver glucose transporter GLUT2 in membrane fractions from cultured neonatal rat islets. This increase (+86%) may explain, at least in part, the development of a mature glucose response. G proteins modulate insulin secretion from pancreatic beta-cells. We show here by immunoblot analysis that, in contrast to the effect on GLUT2, PRL treatment does not modify the G protein subunits alpha i2, alpha i3, alpha o, alpha s, alpha q and beta 35 and beta 36, in cultured neonatal islets.