Effect of chronic coffee consumption on weight gain and glycaemia in a mouse model of obesity and type 2 diabetes.

OBJECTIVE: Epidemiological evidence shows that chronic coffee consumption in humans is correlated with a lower incidence of type 2 diabetes mellitus. For the experimental exploration of the underlying mechanisms, this effect needs to be replicated in an animal model of type 2 diabetes with a short lifespan. DESIGN: Male C57BL/6 mice consumed regular coffee or water ad libitum and the development of obesity and diabetes caused by high-fat diet (55% lipids, HFD) was observed from week 10 on for 35 weeks in comparison with mice feeding on a defined normal diet (9% lipids, ND). RESULTS: The massive weight gain in HFD mice was dose-dependently retarded (P=0.034), the moderate weight gain in ND mice was abolished (P<0.001) by coffee consumption, probably because of a lower feeding efficiency. The consumption of fluid (water or coffee) was significantly diminished by HFD (P<0.001), resulting in a higher coffee exposure of ND mice. On week 21 intraperitoneal glucose tolerance tests (IPGTT) showed a dose-dependent faster decline of elevated glucose levels in coffee-consuming HFD mice (P=0.016), but not in ND mice. Remarkably, a spontaneous decrease in non-fasting glycaemia occurred after week 21 in all treatment groups (P<0.001). On week 39 the IPGTT showed diminished peak of glucose levels in coffee-consuming HFD mice (P<0.05). HFD mice were hyperinsulinaemic and had significantly (P<0.001) enlarged islets. Coffee consumption did not affect islet size or parameters of beta-cell apoptosis, proliferation and insulin granule content. CONCLUSION: Coffee consumption retarded weight gain and improved glucose tolerance in a mouse model of type 2 diabetes and corresponding controls. This gives rise to the expectation that further insight into the mechanism of the diabetes-preventive effect of coffee consumption in humans may be gained by this approach.

Insulinotropic effect of high potassium concentration beyond plasma membrane depolarization.

The question whether K⁺ depolarization is an appropriate experimental substitute for the physiological nutrient-induced depolarization of the ß-cell plasma membrane was investigated using primary mouse ß-cells and islets. At basal glucose 40 mM K⁺ induced a massive monophasic response, whereas 15 mM K⁺ had only a minimal insulinotropic effect, even though the increase in the cytosolic Ca²âº concentration ([Ca²âº]i) was not inferior to that by 20 mM glucose. In voltage-clamp experiments, Ca²âº influx appeared as nifedipine-inhibitable inward action currents in the presence of sulfonylurea plus TEA to block compensatory outward K⁺ currents. Under these conditions, 15 mM K⁺ induced prolonged action currents and 40 mM K⁺ transformed the action current pattern into a continuous inward current. Correspondingly, 15 mM K⁺ led to an oscillatory increase and 40 mM K⁺ to a plateau of [Ca²âº]i superimposed on the [Ca²âº]i elevated by sulfonylurea plus TEA. Raising K⁺ to 15 or 40 mM in the presence of sulfonylurea (±TEA) led to a fast further increase of insulin secretion. This was reduced to basal levels by nifedipine or CoCl2. The effects of 15 mM K⁺ on depolarization, action currents, and insulin secretion were mimicked by adding 35 mM Cs⁺ and those of 40 mM K⁺ by adding 35 mM Rb⁺, in parallel with their ability to substitute for K⁺ as permeant cation. In conclusion, the alkali metals K⁺, Rb⁺, or Cs⁺ concentration-dependently transform the pattern of Ca²âº influx into the ß-cell and may thus generate stimuli of supraphysiological strength for insulin secretion.

Mechanisms of antihyperglycaemic action of efaroxan in mice: time for reappraisal of α2A-adrenergic antagonism in the treatment of type 2 diabetes?

AIMS/HYPOTHESIS: Inspired by recent speculation about the potential utility of α(2A)-antagonism in the treatment of type 2 diabetes, the study examined the contribution of α(2)-antagonism vs other mechanisms to the antihyperglycaemic activity of the imidazoline (±)-efaroxan. METHODS: Effects of the racemate and its pure enantiomers on isolated pancreatic islets and beta cells in vitro, as well as on hyperglycaemia in vivo, were investigated in a comparative manner in mice. RESULTS: In isolated perifused islets, the two enantiomers of efaroxan were equally potent in counteracting inhibition of insulin release by the ATP-dependent K(+) (K(ATP)) channel-opener diazoxide but (+)-efaroxan, the presumptive carrier of α(2)-antagonistic activity, was by far superior in counteracting inhibition of insulin release by the α(2)-agonist UK14,304. In vivo, (+)-efaroxan improved oral glucose tolerance at 100-fold lower doses than (-)-efaroxan and, in parallel with observations made in vitro, was more effective in counteracting UK14,304-induced than diazoxide-induced hyperglycaemia. The antihyperglycaemic activity of much higher doses of (-)-efaroxan was associated with an opposing pattern (i.e. with stronger counteraction of diazoxide-induced than UK14,304-induced hyperglycaemia), which implicates a different mechanism of action. CONCLUSIONS/INTERPRETATION: The antihyperglycaemic potency of (±)-efaroxan in mice is almost entirely due to α(2)-antagonism, but high doses can also lower blood glucose via another mechanism. Our findings call for reappraisal of the possible clinical utility of α(2A)-antagonistic compounds in recently identified subpopulations of patients in which a congenitally higher level of α(2A)-adrenergic activation contributes to the development and pathophysiology of type 2 diabetes.

Ca(2+)-dependent desensitization of insulin secretion by strong potassium depolarization.

Depolarization by a high K(+) concentration is a widely used experimental tool to stimulate insulin secretion. The effects occurring after the initial rise in secretion were investigated here. After the initial peak a fast decline occurred, which was followed by a slowly progressive decrease in secretion when a strong K(+) depolarization was used. At 40 mM KCl, but not at lower concentrations, the decrease continued when the glucose concentration was raised from 5 to 10 mM, suggesting an inhibitory effect of the K(+) depolarization. When tolbutamide was added instead of the glucose concentration being raised, a complete inhibition down to prestimulatory values was observed. Equimolar reduction of the NaCl concentration to preserve isoosmolarity enabled an increase in secretion in response to glucose. Unexpectedly, the same was true when the Na(+)-reduced media were made hyperosmolar by choline chloride or mannitol. The insulinotropic effect of tolbutamide was not rescued by the compensatory reduction of NaCl, suggesting a requirement for activated energy metabolism. These inhibitory effects could not be explained by a lack of depolarizing strength or by a diminished free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Rather, the complexation of extracellular Ca(2+) concomitant with the K(+) depolarization markedly diminished [Ca(2+)](i) and attenuated the inhibitory action of 40 mM KCl. This suggests that a strong but not a moderate depolarization by K(+) induces a [Ca(2+)](i)-dependent, slowly progressive desensitization of the secretory machinery. In contrast, the decline immediately following the initial peak of secretion may result from the inactivation of voltage-dependent Ca(2+) channels.

Do insulinotropic glucose-lowering drugs do more harm than good? The hypersecretion hypothesis revisited.

Significant progress has been made in recent years in the characterisation of the signal pathways of beta cell dysfunction and death in the pathogenesis of type 2 diabetes. Glucolipotoxicity acts as an exogenous factor whereas oxidative stress and endoplasmic reticulum stress may result from the processes of signal recognition and stimulated secretion within the beta cell. The pharmacological stimulation of secretion may thus appear to be a double-edged sword: it counteracts hyperglycaemia, but may do so at the expense of beta cell mass. So, in the long run, insulinotropic glucose-lowering drugs might do more harm than good. However, much of this logic is derived by analogy from the long-held assumption that beta cell hypersecretion imposed by insulin resistance causes the absolute secretion deficit in the later course of type 2 diabetes. In this concept the beta cell has a secondary role and loss of beta cell mass is necessary for the manifestation of type 2 diabetes. Recent studies have shown that a secretion deficit can exist well before insulin resistance and that major genetic risk factors concern beta cell function. Also, the evidence for a beta cell toxic effect of insulinotropic drugs is currently inconclusive. Assuming that the insulin secretion deficit is of pathogenetic importance in a network with insulin resistance as an aggravating factor, an insulinotropic glucose-lowering drug may do more good than harm if it relieves the beta cell from the stress of glucose overstimulation and does so without inducing hypoglycaemia.

The signalling role of action potential depolarization in insulin secretion: metabolism-dependent dissociation between action potential increase and secretion increase by TEA.

The K(+) channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse beta-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 microM glipizide) the nutrient secretagogue alpha-ketoisocaproic acid (KIC, 10mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 microM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca(2+)](i)) with an oscillatory characteristic. These effects depended on the activity of L-type Ca(2+) channels, even though the effect of TEA differed from that of 1 microM of the Ca(2+) channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of beta-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K(+) ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K(+) channels may be involved in this paradox effect of TEA.

Plasma membrane depolarization as a determinant of the first phase of insulin secretion.

The role of plasma membrane depolarization as a determinant of the initial phase of insulin secretion was investigated. NMRI mouse islets and beta-cells were used to measure the kinetics of insulin secretion, ATP and ADP content, membrane potential, and cytosolic free Ca(2+) concentration ([Ca(2+)](i)). The depolarization of metabolically intact beta-cells by KCl corresponded closely to the theoretical values. In contrast to physiological (glucose) or pharmacological (tolbutamide) ATP-sensitive K(+) (K(ATP)) channel block, KCl depolarization did not induce action potential spiking. The depolarization by 15 mM K(+) (21 mV) corresponded to the plateau depolarization by 50 or 500 microM tolbutamide; that by 40 mM K(+) (41 mV) corresponded to the action potential peaks. Nifedipine and diazoxide abolished action potentials but not KCl depolarization, suggesting that the depolarizing strength of 15, but not 40 mM K(+) corresponds to that of K(ATP) channel closure. K(+) (40 mM) induced a massive secretory response in the presence of 5 mM glucose, whereas 15 mM K(+), like 50 microM tolbutamide, was only slightly effective, even though a marked increase in [Ca(2+)](i) was produced. Raising glucose from 5 to 10 mM in the continued presence of 15 mM K(+) resulted in a strongly enhanced biphasic response. The depolarization pattern of this combination could be mimicked by combining basal glucose with 15 mM K(+) and 50 microM tolbutamide; however, the secretory response to these nonnutrients was much weaker. In conclusion, the initial secretory response to nutrient secretagogues is largely influenced by signaling mechanisms that do not involve depolarization.

Fuel-induced amplification of insulin secretion in mouse pancreatic islets exposed to a high sulfonylurea concentration: role of the NADPH/NADP+ ratio.

AIMS/HYPOTHESIS: The aim of this study was to examine whether the cytosolic NADPH/NADP+ ratio of beta cells serves as an amplifying signal in fuel-induced insulin secretion and whether such a function is mediated by cytosolic alpha-ketoglutarate. METHODS: Pancreatic islets and islet cells were isolated from albino mice by collagenase digestion. Insulin secretion of incubated or perifused islets was measured by ELISA. The NADPH and NADP+ content of incubated islets was determined by enzymatic cycling. The cytosolic Ca2+ concentration ([Ca2+]c) in islets was measured by microfluorimetry and the activity of ATP-sensitive K+ channels in islet cells by patch-clamping. RESULTS: Both 30 mmol/l glucose and 10 mmol/l alpha-ketoisocaproate stimulated insulin secretion and elevated the NADPH/NADP+ ratio of islets preincubated in the absence of fuel. The increase in the NADPH/NADP+ ratio was abolished in the presence of 2.7 micromol/l glipizide (closing all ATP-sensitive K+ channels). However, alpha-ketoisocaproate, but not glucose, still stimulated insulin secretion. That glipizide did not inhibit alpha-ketoisocaproate-induced insulin secretion was not the result of elevated [Ca2+]c, as glucose caused a more marked [Ca2+]c increase. Insulin release triggered by glipizide alone was moderately amplified by dimethyl alpha-ketoglutarate (which is cleaved to produce cytosolic alpha-ketoglutarate), but there was no indication of a signal function of cytosolic alpha-ketoglutarate. CONCLUSIONS/INTERPRETATION: The results strongly suggest that the NADPH/NADP+ ratio in the beta cell cytosol does not serve as an amplifying signal in fuel-induced insulin release. The study supports the view that amplification results from the intramitochondrial production of citrate by citrate synthase and from the associated export of citrate into the cytosol.

Essential role of the imidazoline moiety in the insulinotropic effect but not the KATP channel-blocking effect of imidazolines; a comparison of the effects of efaroxan and its imidazole analogue, KU14R.

AIMS/HYPOTHESIS: Imidazolines are a class of investigational antidiabetic drugs. It is still unclear whether the imidazoline ring is decisive for insulinotropic characteristics. MATERIALS AND METHODS: We studied the imidazoline efaroxan and its imidazole analogue, KU14R, which is currently classified as an imidazoline antagonist. The effects of both on stimulus secretion-coupling in normal mouse islets and beta cells were compared by measuring KATP channel activity, plasma membrane potential, cytosolic calcium concentration ([Ca2+]c) and dynamic insulin secretion. RESULTS: In the presence of 10 mmol/l but not of 5 mmol/l glucose, efaroxan (100 micromol/l) strongly enhanced insulin secretion by freshly isolated perifused islets, whereas KU14R (30, 100 or 300 micromol/l) was ineffective at both glucose concentrations. Surprisingly, the insulinotropic effect of efaroxan was not antagonised by KU14R. KATP channels were blocked by efaroxan (IC50 8.8 micromol/l, Hill slope -1.1) and by KU14R (IC50 31.9 micromol/l, Hill slope -1.5). Neither the KATP channel-blocking effect nor the depolarising effect of efaroxan was antagonised by KU14R. Rather, both compounds strongly depolarised the beta cell membrane potential and induced action potential spiking. However, KU14R was clearly less efficient than efaroxan in raising [Ca2+]c in single beta cells and whole islets at 5 mmol/l glucose. The increase in [Ca2+]c induced by 10 mmol/l glucose was affected neither by efaroxan nor by KU14R. Again, KU14R did not antagonise the effects of efaroxan. CONCLUSIONS/INTERPRETATION: The presence of an imidazole instead of an imidazoline ring leads to virtually complete loss of the insulinotropic effect in spite of a preserved ability to block KATP channels. The imidazole compound is less efficient in raising [Ca2+]c; in particular, it lacks the ability of the imidazoline to potentiate the enhancing effect of energy metabolism on Ca2+-induced insulin secretion.

Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures.

P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.

Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine. I. Secretory and morphological studies.

The desensitization of pancreatic B-cells against stimulation by insulin secretagogues that inhibit ATP-dependent K(+) channels (K(ATP) channels) was investigated by measuring insulin secretion of perifused pancreatic islets. Additionally, the islet insulin content and the number of secretory granules per B-cell were determined. Prior to the measurement of secretion, islets were cultured for 18 h in the presence or absence of the test agents in a cell-culture medium containing 5 mM glucose. The effects of three imidazolines, phentolamine, alinidine, and idazoxan (100 microM each) were compared with those of the well-characterized sulfonylurea, tolbutamide (500 microM), and those of the ion channel-blocking alkaloid, quinine (100 microM). Insulin secretion was strongly reduced upon re-exposure to phentolamine, alinidine, tolbutamide, and quinine, whereas idazoxan, which stimulated secretion only weakly, had no significant effect. The imidazoline secretagogues phentolamine and alinidine induced a cross-desensitization against the stimulatory effect of tolbutamide and quinine. A long-term depolarization with 40 mM KCl was also able to induce a significant reduction of the secretory response to all of the above secretagogues. The insulin content of cultured islets was moderately, but significantly reduced by alinidine, whereas the reduction by phentolamine, tolbutamide, and quinine was not significant. In contrast to these observations, the ultrastructural examination revealed that tolbutamide-treated B-cells had a high degree of degranulation, whereas the other test agents and 40 mM KCl produced only a partial degranulation, except for phentolamine, which produced no significant degranulation at all. These results suggest that the desensitization of insulin secretion is a common property of all agents that stimulate insulin secretion by depolarisation of the plasma membrane. Depending on the specific secretagogue, additional mechanisms, proximal and distal to Ca(2+) influx, appear to contribute to the desensitization (see Rustenbeck et al., pages 1695-1703, this issue).

Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine. II. Electrophysiological and fluorimetric studies.

Prolonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K(+) channels (K(ATP) channels), such as sulfonylureas, imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al., pages 1685-1694, this issue). To elucidate the underlying mechanisms, K(ATP) channel activity, plasma membrane potential and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 microM each) K(ATP) channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 microM) induced a marked reduction of K(ATP) channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 microM) or tolbutamide (500 microM) had no lasting effect on K(ATP) channel activity. Correspondingly, phentolamine-, alinidine-, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca(2+)](i) normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds, whereas the [Ca(2+)](i) increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca(2+)](i) elicited by a K(+) depolarization was diminished in secretagogue-pretreated B-cells, the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K(ATP) channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca(2+)](i) regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis, which responds only weakly to substantial increases in [Ca(2+)](i) (in the case of tolbutamide).

Mechanism of terfenadine block of ATP-sensitive K(+) channels.

The ATP-sensitive K(+) (K(ATP)) channel is a complex of a pore-forming inwardly rectifying K(+) channel (Kir6.2) and a sulphonylurea receptor (SUR). The aim of the present study was to gain further insight into the mechanism of block of K(ATP) channels by terfenadine. Channel activity was recorded both from native K(ATP) channels from the clonal insulinoma cell line RINm5F and from a C-terminal truncated form of Kir6.2 (Kir6.2Delta26), which - in contrast to Kir6.2 - expresses independently of SUR. Kir6.2Delta26 channels were expressed in COS-7 cells, and enhanced green fluorescent protein (EGFP) cDNA was used as a reporter gene. EGFP fluorescence was visualized by a laser scanning confocal microscope. Terfenadine applied to the cytoplasmic side of inside-out membrane patches concentration-dependently blocked both native K(ATP) channel and Kir6.2Delta26 channel activity, and the following values were calculated for IC(50) (the terfenadine concentration causing half-maximal inhibition) and n (the Hill coefficient): 1.2 microM and 0.7 for native K(ATP) channels, 3.0 microM and 1.0 for Kir6. 2Delta26 channels. Terfenadine had no effect on slope conductance of either native K(ATP) channels or Kir6.2Delta26 channels. Intraburst kinetics of Kir6.2Delta26 channels were not markedly affected by terfenadine and, therefore, terfenadine acts as a slow channel blocker on Kir6.2Delta26 channels. Terfenadine-induced block of Kir6. 2Delta26 channels demonstrated no marked voltage dependence, and lowering the intracellular pH to 6.5 potentiated the inhibition of Kir6.2Delta26 channels by terfenadine. These observations indicate that terfenadine blocks pancreatic B-cell K(ATP) channels via binding to the cytoplasmic side of the pore-forming subunit. The presence of the pancreatic SUR1 has a small, but significant enhancing effect on the potency of terfenadine.

Disopyramide block of K(ATP) channels is mediated by the pore-forming subunit.

The class Ia antiarrhythmic agent disopyramide blocks native ATP-sensitive K+ (K(ATP)) channels at micromolar concentrations. The K(ATP) channel is a complex of a pore-forming inwardly rectifying K+ channel (Kir6.2) and a sulfonylurea receptor (SUR). The aim of the present study was to further localize the site of action of disopyramide. We have used a C-terminal truncated form of Kir6.2 (Kir6.2delta26), which--in contrast to Kir6.2--expresses independently of SUR. Kir6.2delta26 channels were expressed in African green monkey kidney COS-7 cells, and enhanced green fluorescent protein (EGFP) cDNA was used as a reporter gene. EGFP fluorescence was visualized by a laser scanning confocal microscope. Disopyramide applied to the cytoplasmic membrane surface of inside-out patches inhibited Kir6.2delta26 channels half-maximally at 7.1 microM (at pH 7.15). Lowering the intracellular pH to 6.5 potentiated the inhibition of Kir6.2delta26 channels by disopyramide. These observations suggest that disopyramide directly blocks the pore-forming Kir6.2 subunit, in particular at reduced intracellular pH values that occur under cardiac ischaemia.

Imidazolines and the pancreatic B-cell. Actions and binding sites.

Stimulation of insulin secretion by imidazoline compounds displays variable characteristics. Phentolamine (10-100 microM) increased secretion of perifused mouse islets at nonstimulatory glucose concentrations (5 mM) and even in the absence of glucose. Idazoxan (20-100 microM) elicited a moderate increase in insulin secretion, which required the presence of a stimulatory glucose concentration (10 mM). Phentolamine is therefore a stimulator of secretion in its own right, whereas idazoxan may be termed an enhancer of secretion. Both compounds inhibited the activity of ATP-dependent K+ channels in inside-out patches from B-cells; however, idazoxan achieved only an incomplete block. Both compounds depolarized the B-cell plasma membrane to an extent that permitted the opening of voltage-dependent Ca2+ channels (-40 to -30 mV). An increase in cytoplasmic Ca2+ concentration was induced by phentolamine and much less so by idazoxan. Activation of protein kinase C, a possible mechanism to amplify Ca(2+)-induced secretion, could not be verified for phentolamine. It thus appears that stimulation of insulin secretion by phentolamine is due to its blocking effect on KATP channels, which may be the correlate of non-adrenergic imidazoline binding sites which were characterized in insulin-secreting HIT cells. Whether incomplete closure of KATP channels by idazoxan or additional effects are responsible for the requirement of high glucose to stimulate secretion remains to be clarified.

Heterogeneous characteristics of imidazoline-induced insulin secretion.

Imidazolines are regarded as a pharmacological group of insulin secretagogues with one uniform mechanism of action, namely closure of ATP-dependent K+ channels (K(ATP) channels) and, in consequence, depolarization of the plasma membrane, Ca2+ influx and stimulation of secretion. This assumption was investigated by measuring insulin secretion from perifused pancreatic islets in response to three imidazoline compounds and comparing the characteristics of secretion with changes in membrane potential and cytosolic Ca2+ concentration [Ca2+]i of single beta-cells. Phentolamine (32 microM) stimulated insulin secretion from perifused mouse islets in the presence of stimulatory (10 mM and 30 mM) and substimulatory (5 mM) glucose concentrations and even in the absence of glucose. Idazoxan in concentrations up to 500 microM was virtually ineffective in the presence of 5 mM glucose. At 10 mM glucose, there was a moderate but significant increase of secretion by idazoxan, 20 microM being nearly as effective as 100 microM. The effect of phentolamine was of slow onset and irreversible in the time frame of the experiments, while the effect of idazoxan was of fast onset and reversible. Alinidine also stimulated secretion in the presence of 10 mM glucose with fast and reversible kinetics, but in contrast to idazoxan, 100 microM was clearly more effective than 20 microM. These heterogeneous characteristics of secretion were reflected by changes of [Ca2+]i: the increase of [Ca2+]i by phentolamine was slow and only partially reversible, whereas idazoxan led to a smaller, but faster and reversible response. The increase of [Ca2+]i by phentolamine and idazoxan was abolished by the Ca2+ channel blocker D 600. Surprisingly, all three compounds depolarized the beta-cell plasma membrane from a resting potential of -71 mV to about -36 mV. Again, the effect of phentolamine was slow and that of idazoxan and alinidine fast. Thus, the characteristics of phentolamine-induced secretion appear to be attributable to the consequences of K(ATP) channel closure. It is unclear, however, why all three test compounds achieved the same degree of depolarization in spite of their known different efficiency to close K(ATP) channels. Apparently, there are additional mechanisms involved in the action of idazoxan and alinidine, which may contribute to the obvious differences in the characteristics of secretion.

Phosphatase inhibitors induce defective hormone secretion in insulin-secreting cells and entry into apoptosis.

A long-term (> or =24 h) exposure of insulin-secreting HIT T15 cells to the phosphatase inhibitor, okadaic acid (OA), at concentrations inhibiting serine/threonine phosphatases 1 (PP1) and 2A (PP2A) reduced proliferation and insulin secretion. The reduced proliferation was related to the induction of apoptosis as evidenced by morphological criteria and the occurrence of internucleosomal DNA fragmentation after 15 h in 50 nM OA. The compromised insulin secretion was not simply a consequence of a lowered hormone content and cell growth, but comprised also a complete suppression of secretion stimulated by K+ depolarisation and forskolin. K+ depolarisation of HIT cells cultured for 24 h in 50 nM OA resulted in a nearly unimpaired influx of Ca2+, but did not induce secretion. These observations suggest that the secretory defect may be localised distal to Ca2+ influx in stimulus secretion coupling of insulin-secreting cells.

Mitochondria present in excised patches from pancreatic B-cells may form microcompartments with ATP-dependent potassium channels.

Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K(ATP)) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or alpha-ketoisocaproic acid (10 mM) reduced the open probability of K(ATP) channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K(ATP) channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K(ATP) channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K(ATP) channels thus forming functional microcompartments.

No evidence for PKC activation in stimulation of insulin secretion by phentolamine.

The possible role of protein kinase C (PKC) activation in the course of insulin secretion induced by the imidazoline phentolamine was investigated by measuring the insulin secretion of perifused mouse islets and of insulin-secreting HIT cells and by measuring the PKC activity of HIT cells. When normal mouse islets were perifused with the imidazoline phentolamine (32 microM) or the sulfonylurea glibenclamide (1 microM), neither phentolamine nor glibenclamide could produce a stimulation of secretion which was stronger than that elicited by a strong depolarization. Under the same conditions, tetradecanoylphorbolacetate (TPA, 50 nM), a known activator of PKC activity in pancreatic islets, markedly enhanced the secretion induced by K+ depolarization. Phentolamine also stimulated insulin secretion of superfused HIT cells. When PKC activity in HIT cells was down-regulated to 15% of the initial value by overnight exposure to TPA (50 nM), the stimulatory effect of TPA on secretion was virtually abolished, while phentolamine was still able to elicit a monophasic secretion. TPA (50 nM) induced the typical redistribution of PKC activity in HIT cells: within 2 min, the share of membrane-bound PKC activity rose from 26% to 87% of the total PKC activity, which remained unchanged. In contrast, phentolamine (32 microM) had no effect on PKC distribution, did not down-regulate PKC and had no effect on PKC activity once it was down-regulated by TPA. Thus, the recent suggestion that the insulinotropic effect of imidazolines involves an activation of PKC could not be verified for phentolamine.

Polyamine modulation of mitochondrial calcium transport. I. Stimulatory and inhibitory effects of aliphatic polyamines, aminoglucosides and other polyamine analogues on mitochondrial calcium uptake.

In this study, the regulation of mitochondrial Ca2+ transport by polyamines structurally related to spermine and by analogous polycationic compounds was characterized. Similar to spermine, a number of amino groups containing cationic compounds exerted a dual effect on Ca2+ transport of isolated rat liver mitochondria: a decrease in Ca2+ uptake velocity and an enhancement of Ca2+ accumulation. In contrast to the effects of spermine and other aliphatic polyamines, however, the accumulation-enhancing effect of aminoglucosides, basic polypeptides, and metal-amine complexes turned into an inhibition of Ca2+ accumulation at higher concentrations. Within groups of structurally related compounds, the potency to decrease Ca2+ uptake velocity and to enhance Ca2+ accumulation correlated with the number of cationic charges. The presence of multiple, distributed cationic charges was a necessary, but not sufficient criterion for effects on mitochondrial Ca2+ transport, because cationic polyamines and basic oligopeptides which did not enhance mitochondrial Ca2+ accumulation could be identified. Spermine was not able to antagonize the blocking of Ca2+ uptake by ruthenium red, but rather showed an apparent synergism, which can be explained as a displacement of membrane-bound Ca2+ by spermine. The aminoglucosides, gentamicin and neomycin, but not the inactive polyamine bis(hexamethylene)-triamine, inhibited the binding of spermine to intact mitochondria. Apparently, the binding of spermine, gentamicin, and a number of polyamine analogues to low-affinity binding sites at mitochondria, which have low, but distinct structural requirements and which may correspond to phospholipid headgroups, indirectly influences the activity state of the mitochondrial Ca2+ uniporter. The ability of aminoglucosides to displace spermine from the mitochondria and to inhibit mitochondrial Ca2+ accumulation may contribute to the mitochondrial lesions, which are known to occur early in the course of aminoglucoside-induced nephrotoxicity.