Inositol Addition Research Articles

Inositol as a new enhancer for improving lipid production and accumulation in Schizochytrium sp. SR21.

Lipids produced from agricultural and industrial residues using oleaginous microorganisms for use as biofuels are attracting the attention of researchers due to their environmental benefits. However, low efficiencies and high costs limit their application to a certain extent. The present study is the first to use inositol as an enhancer to improve the production and accumulation of lipids during fermentation by the microalga Schizochytrium sp. SR21. The study aimed to maximize the production of lipids and docosahexaenoic acid (DHA) by optimizing the conditions of inositol addition into the fermentation medium. The corresponding key enzyme and metabolite profiles of SR21 were evaluated. The results indicated that the addition of 120mgL-1 of inositol to the medium at 48h improved lipid and DHA production by 13.90 and 20.82%, resulting in total concentrations of 22.86 and 8.53g/L, respectively. Moreover, the ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) increased by 23.38% and is consistent with the results of the metabolomic analysis. The activity of enzymes (i.e., PC, G6PDH, NADPH-ME, and ACL) related to fatty acid synthesis in strain SR21 also increased significantly (43.38%, 28.68%, 37.47%, and 19.87%, respectively). Metabolomic analysis also showed that inositol promoted lipid synthesis in SR21 and significantly increased the relative proportion of UFAs by affecting the citrate cycle and SFA and UFA metabolic pathways. Thus, inositol is an ideal enhancer of lipid production and accumulation by oleaginous microorganisms. Graphical abstract.

Inositol Induces a Profound Alteration in the Pattern and Rate of Synthesis and Turnover of Membrane Lipids in Saccharomyces cerevisiae

The addition of inositol to actively growing yeast cultures causes a rapid increase in the rate of synthesis of phosphatidylinositol and, simultaneously, triggers changes in the expression of hundreds of genes. We now demonstrate that the addition of inositol to yeast cells growing in the presence of choline leads to a dramatic reprogramming of cellular lipid synthesis and turnover. The response to inositol includes a 5-6-fold increase in cellular phosphatidylinositol content within a period of 30 min. The increase in phosphatidylinositol content appears to be dependent upon fatty acid synthesis. Phosphatidylcholine turnover increased rapidly following inositol addition, a response that requires the participation of Nte1p, an endoplasmic reticulum-localized phospholipase B. Mass spectrometry revealed that the acyl species composition of phosphatidylinositol is relatively constant regardless of supplementation with inositol or choline, whereas phosphatidylcholine acyl species composition is influenced by both inositol and choline. In medium containing inositol, but lacking choline, high levels of dimyristoylphosphatidylcholine were detected. Within 60 min following the addition of inositol, dimyristoylphosphatidylcholine levels had decreased from approximately 40% of total phosphatidylcholine to a basal level of less than 5%. nte1Delta cells grown in the absence of inositol and in the presence of choline exhibited lower levels of dimyristoylphosphatidylcholine than wild type cells grown under these same conditions, but these levels remained largely constant after the addition of inositol. These results are discussed in relationship to transcriptional regulation known to be linked to lipid metabolism in yeast.

Multiple Endoplasmic Reticulum-to-Nucleus Signaling Pathways Coordinate Phospholipid Metabolism with Gene Expression by Distinct Mechanisms

In many organisms the coordinated synthesis of membrane lipids is controlled by feedback systems that regulate the transcription of target genes. However, a complete description of the transcriptional changes that accompany the remodeling of membrane phospholipids has not been reported. To identify metabolic signaling networks that coordinate phospholipid metabolism with gene expression, we profiled the sequential and temporal changes in genome-wide expression that accompany alterations in phospholipid metabolism induced by inositol supplementation in yeast. This analysis identified six distinct expression responses, which included phospholipid biosynthetic genes regulated by Opi1p, endoplasmic reticulum (ER) luminal protein folding chaperone and oxidoreductase genes regulated by the unfolded protein response pathway, lipid-remodeling genes regulated by Mga2p, as well as genes involved in ribosome biogenesis, cytosolic stress response, and purine and amino acid metabolism. We also report that the unfolded protein response pathway is rapidly inactivated by inositol supplementation and demonstrate that the response of the unfolded protein response pathway to inositol is separable from the response mediated by Opi1p. These data indicate that altering phospholipid metabolism produces signals that are relayed through numerous distinct ER-to-nucleus signaling pathways and, thereby, produce an integrated transcriptional response. We propose that these signals are generated in the ER by increased flux through the pathway of phosphatidylinositol synthesis.

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 microg ml(-1) in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 microg ml(-1) in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 microg ml(-1), repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 microg ml(-1) inositol than in those grown in the same medium with 1 microg ml(-1) inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 microg ml(-1) of inositol was higher than that of the yeast cells grown in the same medium with 1 microg ml(-1) of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.

Inositol and phosphatidylinositol-mediated invertase secretion in Schizosaccharomyces pombe

In this study, the effects of inositol addition on invertase secretion, biosynthesis of mRNA encoding secreted invertase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe were examined. We found that specific invertase activity was at its maximum when the concentration of added inositol reached 800 μg/100 ml in the synthetic medium containing 2.0% (w/v) sucrose. However, cell growth was steadily increased as inositol concentrations were increased from 0 to 1000 μg/100 ml. When the concentration of added inositol was 100 μg/100 ml in the synthetic medium, repression of invertase secretion occurred at sucrose concentration higher than 0.5% (w/v). However, when S. pombe was cultured in the synthetic medium containing 800 μg/100 ml, repression of invertase secretion occurred only at sucrose concentrations above 2.0% (w/v). Less residual reducing sugar was left in the 800 μg/100 ml inositol-containing medium than in the 100 μg/100 ml inositol-containing medium as sucrose concentration was increased. More mRNA encoding secreted invertase was detected in cells grown in the 800 μg/100 ml inositol-containing medium (2.0% sucrose) than in those grown in the 100 μg/100 ml inositol-containing medium. These results demonstrate that higher concentration of added inositol in the synthetic medium could derepress invertase secretion and INV + gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 800 μg/100 ml of inositol was higher than that of the yeast cells grown in the same medium with 100 μg/100 ml of inositol. This means that PI may be involved in derepression of invertase secretion.

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni–NTA agarose affinity chromatography. In mammalian sources, PrP C is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.

Increased Alcohol Acetyltransferase Activity by Inositol Limitation in Saccharomyces cerevisiae in Sake Mash

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest isoamyl acetate/isoamyl alcohol (E/A) ratio in sake was obtained, and inositol addition caused a decrease in E/A ratio. In several strains tested, inositol addition to the mash decreased isoamyl acetate content and E/A ratio in sake Inositol addition significantly decreased alcohol acetyltransferase (AATase) activity which is responsible for the synthesis of acetate esters from alcohols and acetyl coenzyme A. The results of Northern blot analysis and disruption of theOPII gene, an inositol/choline-mediated negative regulatory gene, showed that the decrease in AATase activity following inositol addition is not due to a transcriptional event. Inositol addition increased phosphatidylinositol (PI) content 3-fold in sake mash yeast cells, while it had no effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl-serine (PS) contents. When cell-free extracts prepared from sake mash yeast cells were treated with chloroform or phospholipase C to remove PI, no difference in AATase activity in sake mash between with (Ino+) and without (Ino−) inositol addition was observed. PI prepared from sake mash yeast cells inhibited AATase activity more strongly than PC and PE. Furthermore, when PI, PC, PE and PS at a ratio (1.0:1.28:0.70:0.09) corresponding to the phospholipid composition of Ino+ sake mash yeast cells were added to a reaction mixture, the AATase activity decreased to 26–55% that of yeast cells from the Ino- mash with a phospholipid composition of 0.34:1.28:0.7:0.09. Approximately all of the PI was recovered in the ammonium sulfate precipitate of the cell-free extract, while only half of the PC and PE was recovered. The acidic phospholipid, phosphatidylglycerol, as well as PI inhibited AATase activity more strongly than PC, despite its having the same fatty acid composition as PC. These results suggest that the strong inhibition of AATase activity by PI is due to its high adsorptive capacity for the AATase protein. Therefore, rice polishing can remove inositol from rice leading to an increase in AATase activity, and resulting in a high E/A ratio in sake.

Increased alcohol acetyltransferase activity by inositol limitation in Saccharomyces cerevisiae in sake mash

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest isoamyl acetate/isoamyl alcohol (E/A) ratio in sake was obtained, and inositol addition caused a decrease in E/A ratio. In several strains tested, inositol addition to the mash decreased isoamyl acetate content and E/A ratio in sake Inositol addition significantly decreased alcohol acetyltransferase (AATase) activity which is responsible for the synthesis of acetate esters from alcohols and acetyl coenzyme A. The results of Northern blot analysis and disruption of the OPII gene, an inositol/choline-mediated negative regulatory gene, showed that the decrease in AATase activity following inositol addition is not due to a transcriptional event. Inositol addition increased phosphatidylinositol (PI) content 3-fold in sake mash yeast cells, while it had no effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl-serine (PS) contents. When cell-free extracts prepared from sake mash yeast cells were treated with chloroform or phospholipase C to remove PI, no difference in AATase activity in sake mash between with (Ino+) and without (Ino−) inositol addition was observed. PI prepared from sake mash yeast cells inhibited AATase activity more strongly than PC and PE. Furthermore, when PI, PC, PE and PS at a ratio (1.0:1.28:0.70:0.09) corresponding to the phospholipid composition of Ino+ sake mash yeast cells were added to a reaction mixture, the AATase activity decreased to 26–55% that of yeast cells from the Ino- mash with a phospholipid composition of 0.34:1.28:0.7:0.09. Approximately all of the PI was recovered in the ammonium sulfate precipitate of the cell-free extract, while only half of the PC and PE was recovered. The acidic phospholipid, phosphatidylglycerol, as well as PI inhibited AATase activity more strongly than PC, despite its having the same fatty acid composition as PC. These results suggest that the strong inhibition of AATase activity by PI is due to its high adsorptive capacity for the AATase protein. Therefore, rice polishing can remove inositol from rice leading to an increase in AATase activity, and resulting in a high E/A ratio in sake.

Increased Ethyl Caproate Production by Inositol Limitation in Saccharomyces cerevisiae

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2 , ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2, ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

Combination of inositol and serotonin reuptake inhibitors in the treatment of depression

Background: Inositol has been reported to be an effective treatment in depression, and we hypothesized that inositol addition might enhance or speed up response to serotonin selective reuptake inhibitors (SSRI). Methods: Twenty-seven depressed patients completed a double-blind controlled 4-week trial of SSRI plus placebo or SSRI plus inositol. Hamilton Depression Rating Scale was used as an assessment tool at baseline, and 1, 2, 3, and 4 weeks. Results: No significant difference was found between the two treatment groups. Conclusions: Previous studies combining different effective antidepressant therapies similarly found no evidence for additive effects [e.g., monoamine oxidase inhibitors (MAOI) plus tricyclic antidepressants (TCA), TCA plus lithium]. By contrast, augmentation by lithium or MAOI after a failed course of antidepressant treatment is effective and should be studied with inositol.

Role of phosphatidylinositol (PI) in ethanol production and ethanol tolerance by a high ethanol producing yeast

The effect of inositol addition on phospholipids, cell growth, ethanol production and ethanol tolerance in a high ethanol producing Saccharomyces sp were studied. Addition of inositol greatly influenced major phospholipid synthesis. With inositol in the fermentation medium, phosphatidylinositol (PI) content was increased, while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were decreased. However, without inositol in the fermentation medium, PI content dropped down within 24 h, then increased, but was lower than in the presence of inositol. When yeast cells had a higher content of PI, they produced ethanol much more rapidly and tolerated higher concentrations of ethanol. During ethanol shock treatment at 18% (v/v) ethanol, yeast cells with a higher concentration of PI lost their viability much more slowly than those with a lower concentration of PI, indicating that the PI content in these yeast cells can play an important role in ethanol production and ethanol tolerance. Fatty acids and ergosterol were not responsible for high ethanol tolerance and high ethanol production in this yeast strain.

Inositol-mediated invertase secretion in Saccharomyces sp. W 4

In this study, the effects of inositol addition on invertase secretion in Saccharomyces sp. W4 were examined. We found that invertase secretion in this strain was repressed when the completely synthetic medium lacking exogenous inositol contains more than 0.2% (w/v) glucose; however, when the yeast cells were grown in the same medium plus inositol, repression of invertase secretion in this strain occurred only at glucose concentrations higher than 2.0% (w/v). Furthermore, when sucrose was used as carbon source, increased invertase activity was observed in the presence of inositol. Repression occurred only at sucrose concentrations above 4.0% (w/v). More cell growth occurred and less residual reducing sugar was left in the inositol-containing medium than in the inositol-deficient medium as glucose or sucrose concentration was increased. These results demonstrate that inositol supplementation could derepress invertase secretion and cell growth in this yeast strain to some extent and sucrose was more suitable for invertase production than glucose. When the repressed yeast cells were transferred to the synthetic medium with low concentration glucose or sucrose, they could synthesize more extracellular invertase in the presence of inositol than in the absence of inositol.

Phosphatidylcholine biosynthesis via the CDP-choline pathway in Saccharomyces cerevisiae. Multiple mechanisms of regulation.

Multiple mechanisms of regulation in the CDP-choline pathway for phosphatidylcholine (PC) synthesis were revealed by exploring the effects of choline and inositol on this pathway in Saccharomyces cerevisiae. At exogenous choline concentrations below 100 microM, phosphocholine cytidylyltransferase was rate-limiting; at higher choline concentrations the conversion of choline to phosphocholine by choline kinase became rate-limiting. Choline and inositol were found to regulate choline uptake; this established another regulatory mechanism by which PC synthesis is regulated in yeast. Inositol addition did not immediately affect labeled choline uptake or its incorporation into PC in actively dividing cells; however, preculturing the cells in the presence of choline decreased the rate of choline uptake, and this effect was amplified by the concomitant addition of inositol and choline. Additionally, a growth phase dependent effect of inositol supplementation was observed. Inositol addition to stationary phase cells resulted in an increase in choline uptake and subsequent PC production in these cells. This increase was shown to be due to an increase in the rate of choline transport into the cell. In the presence of inositol, choline transport is the main regulatory mechanism controlling flux through the CDP-choline pathway in S. cerevisiae. Inositol supplementation resulted in changes in the levels of enzyme activity detected in vitro. However, the effects observed in vivo correlated exclusively with changes in choline uptake. Choline transporter assays were consistent with these results. Since both the CPT1 and EPT1 gene products catalyze the cholinephosphotransferase reaction in vitro (Hjelmstad, R. H., and Bell, R. M. (1991) J. Biol. Chem. 266, 4357-4365), the effect of inositol on these two separate routes for PC biosynthesis was investigated. The data revealed that only cells harboring a functional CPT1 gene synthesized PC in vivo. These cells (ept1-delta 1::URA3) also displayed an identical mode of regulation in response to inositol as did cells containing an intact EPT1 gene (wild type) indicating there is no requirement for an alternate functional CDP-amino-alcohol pathway for inositol to regulate PC synthesis via the CDP-choline pathway.

Effect of Methionine Supplementation to a Low-Soybean Protein Diet on Liver Lipid Metabolism

Female Wistar rats were fed a low-soybean protein (8%) diet supplemented with different concentrations of DL-methionine (0-1.2%) during the lactation period. Diet supplementation with methionine caused a significant increase in rat body weight. The addition of 0.3% DL-methionine to the diet did not cause an increase in liver triacylglycerol concentration in 21-day-old rats, but an increase did occur after the addition of 0.6% DL-methionine to the diet. Supplementation of the diet with DL-methionine did not change liver phospholipid or protein concentrations, nor plasma triacylglycerol levels. Liver lipid synthesis from [1-14C]acetate in 21-day-old rats whose dams had been fed an 8% soybean protein diet supplemented with 1.2% DL-methionine was significantly higher than in the group receiving no DL-methionine supplementation. Hepatic production of CO2 and of water-soluble acid products from [1-14C]palmitate did not differ between groups. Inositol addition to a low-soybean protein diet supplemented with DL-methionine did not change triacylglycerol nor phospholipid levels in the liver.

Regulation and location of phosphatidylglycerol and phosphatidylinositol synthesis in type II cells isolated from fetal rat lung

1. 1. myo-Inositol decreases the synthesis of phosphatidylglycerol by type II cells isolated from fetal rat lung. Inositol addition also increases the synthesized amount of surfactant phosphatidylinositol. These observations indicate that at least part of the decreasing effect of inositol on phosphatidylglycerol formation is the result of competition between phosphatidylglycerol and phosphatidylinositol synthesis for a common pool of CDPdiacylglycerol. 2. 2. Studies on the subcellular localization of enzymes measured under optimal conditions suggested that the enzymic activity required for the formation of phosphatidylglycerol is located mainly in the mitochondria, but most likely also for a small part in the endoplasmic reticulum, while the enzymic activity required for phosphatidylinositol formation is located in the endoplasmic reticulum. 3. 3. Inositol was found to inhibit glycerolphosphate phosphatidyltransferase in the microsomal fraction but not in the mitochondrial fraction derived from the type II cells, indicating that the competition between phosphatidylglycerol and phosphatidylinositol synthesis for CDPdiacylglycerol takes place in the endoplasmic reticulum. 4. 4. This latter observation together with the observation of a switch-over from surfactant phosphatidylinositol to phosphatidylglycerol production around term indicate that the endoplasmic reticulum is the intracellular site of surfactant phosphatidylglycerol production.

Inter-relations between the phospholipids of rat pancreatic islets during glucose stimulation, and their response to medium inositol and tetracaine.

1. Isolated rat pancreatic islets subjected to an increase in glucose concentration from 0.5 to 3 mg/ml showed an increased insulin secretion and 32P-labelling of their phospholipids, especially of phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, and CDP diglyceride. Neither fructose or inositol produced a similar effect. 2. The enhanced labelling of CDP diglyceride and phosphatidylglycerol was suppressed by adding inositol (0.1 mg/ml), while the phosphatidylinositol labelling was increased still further. 3. Tetracaine (0.5 mM), an antagonist of insulin secretion, inhi-ited phosphatidylinositol synthesis while phosphatidylglycerol, CDP diglyceride and phosphatidic acid formation were markedly increased. These effects on phospholipid synthesis were substantially reversed by adding inositol to the medium. Tetracaine also prevented the catabolism of phosphatidylinositol in prelabelled islets but this inhibiton was not reversed by inositol. 4. Inositol addition did not affect insulin secretion in glucose-stimulated islets nor secretion in tetracaine-treated islets. This need not exclude a possible role for heightened phosphatidylinositol cleavage in stimulated insulin secretion.