Palmitic acid stimulates glucose incorporation in the adipocyte by a mechanism likely involving intracellular calcium

The effect of palmitic acid on basal and insulin-stimulated incorporation of glucose into rat adipocytes was studied. Palmitic acid (2.40 mM) stimulated basal as well as insulin-stimulated glucose incorporation in rat adipocytes three and twofold, respectively. Similar degrees of stimulation of basal glucose oxidation by palmitate were also observed. The ability of palmitic acid to stimulate glucose uptake was additive with respect to the stimulation induced by insulin and was proportional to the palmitic acid concentration between 0.15 mM and 2.40 mM. Stimulation of glucose incorporation by palmitic acid was inhibited by preincubating the cells with quin2-AM, which accumulates intracellularly yielding the trapped chelator form. quin2, which binds intracellular Ca2+.The concentration of quin2-AM required for half-maximal inhibition of palmitic acid stimulated glucose incorporation was 3.8 +/- 1.2 microM (mean +/- SEM). The inhibition of palmitic acid-stimulated glucose incorporation by quin2-AM (10 microM) was overcome by incubating cells with the Ca2+ ionophore, A23187, in the presence of extracellular Ca2+ (2.6 mM). Chelation of extracellular Ca2+ with EGTA did not significantly affect the magnitude of palmitic acid-stimulated glucose incorporation. Dantrolene (12.5-100 microM) failed to affect basal or palmitic acid-stimulated glucose incorporation. These findings suggest that palmitic acid stimulates incorporation of glucose in the adipocyte by a mechanism dependent upon intracellular but not extracellular Ca2+.     

Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action

It has been proposed that an elevation in cytosolic free Ca2+ may play a role in either mediating or antagonizing the ability of insulin to stimulate glucose uptake in adipocytes. This question has been addressed in the present studies using isolated fura-2-loaded rat adipocytes stimulated with a variety of agonists. The effects of insulin, oxytocin, norepinephrine, ATP, and ionomycin on cytosolic free Ca2+ levels were assessed and compared with their effects on transport-limited glucose oxidation. Oxytocin and ionomycin at concentrations which caused 3-5-fold increases in cytosolic Ca2+, by releasing Ca2+ from internal stores, had no effect on insulin-stimulated glucose oxidation. ATP and norepinephrine which caused more modest increases in Ca2+, by mechanisms at least partially dependent on external stores, inhibited insulin-stimulated glucose oxidation. Insulin had no effect on basal Ca2+ levels nor did it modulate the Ca2+ elevation caused by other agonists. These data suggest that insulin-stimulated glucose transport is not associated with an increase in cytosolic Ca2+. In addition, although there appears to be a correlation between inhibition of insulin-stimulated glucose transport and the effect of certain agonists to promote Ca2+ influx, there is not a general obligatory relationship between an elevation in cytosolic Ca2+ and antagonism of this insulin action.     

Significance of Ca2+, Rb+ fluxes, of cAMP and cGMP for the CCK8-modulated insulin release

In rat pancreatic islets the effects of cholecystokinin-8 (CCK8) on glucose-mediated insulin release, 45Ca2+ net uptake, 45Ca2+ efflux, 86Rb+ efflux, cAMP- and cGMP levels were studied. In the presence of a substimulatory glucose concentration (3 mM) CCK8 concentrations of up to 1 microM had no effect on insulin release, but CCK8 at 10 nM potentiated the stimulatory effect of glucose (11.1 mM). 10 nM CCK8 enhanced glucose-stimulated 45Ca2+ net uptake but was ineffective at substimulatory glucose levels. CCK8 had no effect on cAMP and cGMP levels in the presence of 11.1 mM glucose, CCK8 increased 86Rb+ (a measure of K+) in the presence of both 3 and 11.1 mM glucose. This effect was abolished when Ca2+ was omitted from the perifusion medium. CCK8 did not alter glucose (11.1 mM)-stimulated 45Ca2+ efflux rate. These data indicate that (1) CCK8 potentiates glucose-stimulated insulin secretion possibly via an effect on Ca2+ uptake, 2) by affecting Ca2+ uptake, CCK8 enhances K+ efflux, and 3) CCK8 does not mediate its effect via cAMP or cGMP. With respect to 86Rb+ efflux the mechanism of CCK8 action appears to be different from that of glucose. When the mechanism of CCK action on islets is compared with that on exocrine pancreas (data from others) there are similarities (importance of Ca2+ uptake and non-importance of cAMP and cGMP).     

Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells.

Insulin stimulation of hydrogen peroxide production by rat epididymal fat cells was investigated by studying the oxidation of formate to CO2 by endogenous catalase. Under optimal concentrations of formate (0.1 to 1 mM) and glucose (0.275 mM), insulin stimulated formate oxidation 1.5- to 2.0-fold. Inhibitors of catalase activity, including nitrite and azide, inhibited both basal and insulin-stimulated formate oxidation at concentrations that did not interfere with insulin effects on glucose C-1 oxidation or glucose H-3 incorporation into lipids. The addition of exogenous catalase increased formate oxidation only slightly, while exogenous H2O2 (0.5 mM) stimulated formate oxidation by endogenous catalase strongly. These data indicate that the insulin-stimulated H2O2 production was intracellular. Insulin dose-response curves for formate oxidation were identical with those for glucose H-3 incorporation into lipids. The dependence of relative insulin effects on the logarithm of the glucose concentration was bell-shaped for formate oxidation and correlated highly with the coresponding dependences of glucose C-1 oxidation and glucose H-3 incorporation into lipids. This suggests that insulin stimulation of intracellular H2O2 production is linked to glucose metabolism. Since it is known that extracellular H2O2 can mimic insulin in several respects, these observations suggest that H2O2 may act as a "second messenger" for the observed effects of insulin.     

Modulation of glucose uptake in adipose tissue by nitric oxide-generating compounds

There is increasing evidence that endogenous nitric oxide (NO) influences adipogenesis, lipolysis and insulin-stimulated glucose uptake. We investigated the effect of NO released from S-nitrosoglutathione (GSNO) and S-nitroso N-acetylpenicillamine (SNAP) on basal and insulin-stimulated glucose uptake in adipocytes of normoglycaemic and streptozotocin (STZ)-induced diabetic rats. GSNO and SNAP at 0.2, 0.5, and 1 mM brought about a concentration-dependent increase in basal and insulin-stimulated 2-deoxyglucose uptake in adipocytes of normoglycaemic and STZ-induced diabetic rats. SNAP at 1.0 mM significantly elevated basal 2-deoxyglucose uptake (115.8 ± 10.4%) compared with GSNO at the same concentration (116.1 ± 9.4%;P 0.05) in STZ-induced diabetic rats. Conversely, SNAP at concentrations of 10 mM and 20 mM significantly decreased basal 2-deoxyglucose uptake by 50.0 ± 4.5% and 61.5 ± 7.2% respectively in adipocytes of STZ-induced diabetic rats (P 0.05). GSNO at concentrations of 10 mM and 20 mM also significantly decreased basal 2-deoxyglucose uptake by 50.8 ± 6.4% and 55.2 ± 7.8% respectively in adipocytes of STZ-induced diabetic rats (P 0.05). These observations indicate that NO released from GSNO and SNAP at 1 mM or less stimulates basal and insulin-stimulated glucose uptake, and at concentrations of 10 mM and 20 mM inhibits basal glucose uptake. The additive effect of GSNO or SNAP, and insulin observed in this study could be due to different mechanisms and warrants further investigation.     

Maintenance of intracellular calcium in Escherichia coli

Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells. Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli. Cells of E. coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid. The concentration of free [Ca2+]i in unstarved cells was maintained at 90 +/- 10 nM, irrespective of the Ca2+ concentration in the extracellular medium. Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium. In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration. In starved cells [Ca2+]i varied from 0.2 to 0.7 microM when the loading Ca2+ concentration varied from 10 microM to 10 mM. Addition of glucose lowered the Ca2+ levels to 90 nM. Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux. Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool. These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free [Ca2+]i is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.     

Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic beta-cells

Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2'-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.     

The effect of fetal calf serum on intracellular calcium in GH3 cells

We have investigated the mechanism of action of fetal calf serum (FCS) on GH3 pituitary tumour cells by measuring intracellular free calcium levels. On the addition of FCS (1%) there was a transient increase in intracellular Ca2+ levels which was attenuated in conditions of reduced extracellular calcium concentrations. The Ca2+ response was abolished by the prior addition of lanthanum chloride (1mM). In contrast, the elevation of cytosolic calcium levels by TRH (100nM), an agonist which causes the mobilisation of calcium from the endoplasmic reticulum, was attenuated but not abolished by lanthanum chloride (1mM). We suggest that FCS (1%) causes the release of calcium from the plasma membrane and the influx of calcium from the extracellular milieu, but does not mobilise calcium from the endoplasmic reticulum.     

Glucose 6-phosphate regulates Ca2+ steady state in endoplasmic reticulum of islets. A possible link in glucose-induced insulin secretion

Glucose stimulation of islets is coupled with the rapid intracellular release of myo-inositol 1,4,5-trisphosphate (IP3) and arachidonic acid which in turn mobilize Ca2+ stored in the endoplasmic reticulum (ER). The metabolism of glucose is required for insulin secretion although the link between glucose metabolism and the cellular events resulting in insulin release is unknown. In digitonin-permeabilized islets, glucose 6-phosphate (0.5-4 mM) increased significantly the ATP-dependent Ca2+ content of the ER at a free Ca2+ concentration of 1 microM. At 0.2 microM free Ca2+, glucose 6-phosphate (2-10 mM) had a smaller effect. Glucose, phosphate, mannose 6-phosphate, and fructose 1,6-diphosphate had no effect on the ATP-dependent Ca2+ content of the ER. Glucose 1-phosphate and fructose 6-phosphate also increased ATP-dependent Ca2+ content of the ER, presumably due to conversion to glucose 6-phosphate by islet phosphoglucomutase and phosphoglucoisomerase, respectively. The glucose 6-phosphate increase in the ATP-dependent Ca2+ content of the ER was shown to be mediated by glucose 6-phosphatase localized to the ER. Both arachidonic acid (10 microM) and the Ca2+ ionophore A23187 (2 microM) mobilized Ca2+ stored in the ER by glucose 6-phosphate. However, IP3-induced (10 microM) Ca2+ release from the ER was abolished in the presence of glucose 6-phosphate (0.5-10 mM). We propose that glucose 6-phosphate could provide a regulatory link between glucose metabolism and intracellular Ca2+ regulation by augmenting Ca2+ sequestered in the ER as well as attenuating IP3-induced Ca2+ release. Thus, glucose 6-phosphate would serve as an "off" signal leading to a decrease in intracellular Ca2+ when both the free Ca2+ and glucose 6-phosphate concentrations have increased following glucose stimulus.     

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.     

The role of P(i) in glycolytic inhibition of calcium ion uptake by ELD ascites tumor cells

In contrast to previous investigations at 25 degrees C, glucose was shown to support 45Ca2+ uptake at 37 degrees C in intact ELD ascites tumor cells. Intact ascites tumor cells in vitro accumulated up to 5.0 micromol of 45Ca2+ per g cells dry wt. within 20 min. In the presence of 10.0 mM glucose, intracellular P(i) levels fell from 40.0 micromol x g(-1) cells dry wt. to 20.0 micromol x g(-1) cells dry wt. in 5 min. Intracellular P(i) levels were maintained by 20.0 mM extracellular Tris-P(i). 45Ca2+ uptake was inhibited in P(i)-depleted cells, even though the metabolic rate (as measured by Q(lactate)) and energy state (as measured by ATP levels) were at acceptable levels. Evidence has been presented suggesting that previous reports of glucose inhibition of calcium uptake can be attributed to a competition for available intracellular P(i) between glycolytic processes and the mitochondrial calcium uptake mechanism.     

Testosterone rapidly stimulates insulin release from isolated pancreatic islets through a non-genomic dependent mechanism.

The action of testosterone on the 45Ca2+ uptake and insulin secretion was studied in short-term experiments using isolated pancreatic islets of Langerhans. Testosterone (1 microM) stimulated 45Ca2+ uptake within 60 seconds of incubation on similar proportion than tolbutamide. Also, the hormone rapidly increased insulin release (34%; 180 seconds) on the presence of non-stimulatory concentrations of glucose (3 mM). Impermeant testosterone-BSA significantly stimulated the secretion of insulin to a lower percentage (10%). The action of the hormone is specific--neither 17beta-E2 nor progesterone stimulated insulin secretion in the presence of 3 mM glucose. The action of testosterone on insulin secretion was dose-dependent, and at rat plasma physiological concentrations (25 nM), stimulus was 17% (p < 0.05). In conclusion, in isolated pancreatic islets experiments, physiological concentration of testosterone rapidly stimulate insulin secretion and 45Ca2+ uptake through a membrane bound mechanism.     

Postprandial changes in cytosolic free calcium and glucose uptake in adipocytes in obesity and non-insulin-dependent diabetes mellitus

We evaluated the possible relationship between [Ca2+]i and glucose uptake in the postabsorptive state and postprandially in adipocytes obtained from normal and obese subjects, as well as from patients with non-insulin-dependent diabetes mellitus (NIDDM). Adipocytes isolated from overnight-fasted obese and NIDDM patients revealed high levels of [Ca2+]i (p less than 0.05 vs. control) in association with a decreased insulin-stimulated glucose uptake (p less than 0.05 vs. controls). In obese and NIDDM patients treated with oral hypoglycemic agents, the overnight fasting levels of [Ca2+]i were increased postprandially (p less than 0.05), concomitantly with a further decrease in insulin-stimulated 2-deoxyglucose uptake. Although the precise nature of the relationship between [Ca2+]i in specific insulin target tissues and diminished insulin action remains unknown, it is clear that high levels of [Ca2+]i may contribute to the development of insulin resistance.     

Role of calcium in the secretion of leptin from white adipocytes

The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B, and W-13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus calcium appears to be specifically required for glucose utilization. On the other hand, (45)Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A-23187, and ATP) increased (45)Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A-23187, calcium, and thapsigargin inhibited ISLS, even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS, mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake, and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits ISLS at a level independent of glucose metabolism. Thus calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.     

The effects of alpha- and beta-adrenergic agents, Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 microM) and 1 microM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 microM), 50 microM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 microM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 microM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 microM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 microM) and epinephrine (1 microM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 microM but maximal concentrations of epinephrine (e.g., 1 microM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 microM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 microM) was added. Similarly, removal of 0.1 microM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either alpha or beta receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic beta-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in beta-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba2+ was not inhibited by D-glucose. Ba2+ served as a substitute for Ca2+ both in maintaining the glucose metabolism after removal of extracellular Ca2+ and making it possible for glucose to stimulate insulin release. Furthermore, Ba2+ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the beta-cell content of cyclic AMP. It is likely that the observed actions of Ba2+ are mediated by Ca2+, since Ca2+ -dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba2+ specifically.     

Diversity in the effects of extracellular ATP and adenosine on the cellular processing and physiologic actions of insulin in rat adipocytes

ATP or adenosine (1 mM) added to extracellular buffer abolished both chloroquine- and monensin-dependent accumulation of [125I]iodoinsulin in isolated rat adipocytes. The effects of ATP were not secondary to its conversion to adenosine and were mimicked by beta, gamma-methyleneadenosine 5'-triphosphate. ATP, but not adenosine, partially inhibited the binding of insulin to the cellular receptor. Neither ATP nor adenosine had any significant effect on both internalization of cell-bound insulin and externalization of the internalized hormone. The degradation of cell-bound insulin was reduced to a considerable extent by both 0.1 mM chloroquine and 5 mM ATP, to a lesser degree by 1 mM ATP, and not significantly by 1 or 5 mM adenosine. Physiologically, (a) 1 mM ATP had a strong, while 1 mM adenosine had a mild inhibitory effect on the insulin-stimulated glucose transport without affecting its basal activity, (b) both ATP and adenosine moderately stimulated basal as well as insulin-stimulated glycogen synthase, and (c) ATP, but not adenosine, transiently stimulated basal cAMP phosphodiesterase without affecting the insulin-stimulated enzyme. Phosphodiesterase in cells that had been exposed to ATP for 30 min was refractory to ATP added afresh, but not to insulin. These data suggest that (a) extracellular ATP may block the degradative pathway of insulin processing, (b) adenosine might render the ordinarily irreversible intracellular traffic of insulin reversible or modulate a pathway which is yet to be identified, (c) the previously reported effect of ATP on glycogen synthase may not involve phosphorylation, (d) ATP stimulates cAMP phosphodiesterase by a mechanism which is distinct from that of insulin, and (e) the degradative pathway of insulin processing may not be involved in the physiologic actions of the hormone on glycogen synthase and phosphodiesterase.     

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 M) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.     

Effect of glucose on endothelin-1-induced calcium transients in cultured bovine retinal pericytes.

Published work has shown that endothelin-1-induced contractility of bovine retinal pericytes is reduced after culture in high concentrations of glucose. The purpose of the present study was to establish the profile of endothelin-1-induced calcium transients in pericytes and to identify changes occurring after culture in high concentrations of glucose. Glucose had no effect on basal levels of cytosolic calcium or on endothelin-1-induced calcium release from intracellular stores. However, influx of calcium from the extracellular medium after endothelin-1 stimulation was reduced in pericytes that had been cultured in 25 mM D-glucose. L-type Ca(2+) currents were identified by patch clamping. The L-type Ca(2+) channel agonist, (-)-Bay K8644, caused less influx of calcium from the extracellular medium in pericytes that had been cultured in 25 mM D-glucose than in those cultured with 5 mM D-glucose. However, 3-O-methylglucose, a nonmetabolizable analogue of glucose which can cause glycation, had similar effects to those of high concentrations of glucose. The results suggest that reduced function of the L-type Ca(2+) channel that occurs in bovine retinal pericytes after culture in high concentrations of D-glucose is probably due to glycation of a channel protein.