Short-term action of insulin on Aplysia neurons: generation of a possible novel modulator of ion channels.

In mollusks as in other animals, peptides can act as hormones, growth factors, and neurotransmitters. The presence of insulin in vertebrate brain as well as its actions on nerve cells led us to examine the electrophysiological effects of the mammalian hormone on Aplysia neurons. Application of insulin extracellularly causes hyperpolarization of L14 and L10, identified neurons of the abdominal ganglion. This hyperpolarization is associated with a decreased membrane conductance that reverses at -35 mV. We also injected inositol phosphate glycan (IPG) into the identified neurons. This complex sugar, which was purified from rat liver and which is a putative second messenger for insulin in nonneural vertebrate cells (Saltiel and Cuatrecasas, 1986; Saltiel, Osterman, and Darnell, 1988), causes hyperpolarization with decreased membrane conductance in L14 and L10 similar to the effects of insulin. Furthermore, exposure of isolated ganglia to insulin results in the generation of IPG with a compensating decrease in its glycosyl-phosphatidylinositol precursor. We suggest that, in addition to its other roles, insulin may function as a neuropeptide transmitter using IPG as a second messenger.     

Insulin-induced glycerolipid mediators and the stimulation of glucose transport in BC3H-1 myocytes

We have previously demonstrated that insulin stimulates glycerolipid synthesis and phospholipid hydrolysis in BC3H-1 myocytes, resulting in the generation of membrane diacylglycerol, a known cellular mediator. This led us to the original proposal that diacylglycerol may contribute to the mediation of insulin action, especially stimulation of glucose transport. The fact that agents such as phenylephrine and phorbol esters, which increase or act as membrane diacylglycerols, are fully active in stimulating glucose transport in this tissue lent further support to this proposal. In this paper, we demonstrate that the diacylglycerol analogues PMA (4 beta-phorbol 12-myristate 13-acetate) and mezerein (both possessing 12 beta- and 13 alpha-O-linked substituents as well as a 4 beta-hydroxyl group) each increase the Vmax of the glucose transporter as does insulin. Diacylglycerol generated by the addition of phospholipase C also stimulates glucose uptake to a maximum which is equal and nonadditive to that of insulin, while addition of the narrowly active phosphatidylinositol-specific phospholipase C which generates the putative phosphoinositol-glycan mediator of Saltiel et al. (Saltiel, A., Fox, J., She Lin, P., and Cutrecasas, P. (1986) Science 233, 967-972) stimulates pyruvate dehydrogenase in these cells without any effect on glucose uptake. Pretreatment of the myocytes with PMA resulted in desensitization of subsequent glucose uptake to stimulation by phenylephrine, but had no effect on stimulation of glucose uptake by phospholipase C or by insulin, indicating that PMA pretreatment primarily desensitizes agonist-induced polyphosphoinositide hydrolysis which, as we have previously shown, is not involved in the insulin-induced generation of diacylglycerol. This was confirmed by the absence of intracellular Ca2+ mobilization during insulin administration, as measured by the sensitive fluorescent probe fura-2 in attached monolayer BC3H-1 myocytes. Furthermore, we have shown that insulin-generated diacylglycerol satisfies several criteria for a mediator of insulin action, including the demonstration that insulin-stimulated endogenous diacylglycerol generation is antecedent to glucose transport and has an identical insulin dose-response curve and moreover that the magnitude and time course of subsequent stimulation of glucose transport is reproduced by the addition of the simple exogenous diacylglyerol, dioctanoylglycerol, in the complete absence of the hormone. These results establish a central role for insulin-induced glycerolipid metabolism in mediating insulin-stimulated glucose transport in BC3H-1 myocytes.     

Short-term action of insulin on Aplysia neurons: Generation of a possible novel modulator of ion channels

In mollusks as in other animals, peptides can act as hormones, growth factors, and neurotransmitters. The presence of insulin in vertebrate brain as well as its actions on nerve cells led us to examine the electrophysiological effects of the mammalian hormone on Aplysia neurons. Application of insulin extracellularly causes hyperpolarization of L14 and L10, identified neurons of the abdominal ganglion. This hyperpolarization is associated with a decreased membrane conductance that reverses at ?35 mV. We also injected inositol phosphate glycan (IPG) into the identified neurons. This complex sugar, which was purified from rat liver and which is a putative second messenger for insulin in nonneural vertebrate cells (Saltiel and Cuatrecasas, 1986; Saltiel, Osterman, and Darnell, 1988), causes hyperpolarization with decreased membrane conductance in L14 and L10 similar to the effects of insulin. Furthermore, exposure of isolated ganglia to insulin results in the generation of IPG with a compensating decrease in its glycosyl-phosphatidylinositol precursor. We suggest that, in addition to its other roles, insulin may function as a neuropeptide transmitter using IPG as a second messenger.     

Fasting Decreases the Content of D-Chiroinositol in Human Skeletal Muscle

Two classes of inositol phosphoglycans have been implicated as second messengers of insulin, one that activates pyruvate dehydrogenase and contains D-chiroinositol, and one that inhibits cyclic AMP–dependent protein kinase and contains myoinositol. We examined the effects of a 3-day fast on muscle contents of inositols in healthy humans. An oral glucose tolerance test was performed and a biopsy was obtained from the quadriceps femoris muscle after an overnight fast and after a 72-hour fast. The 72-hour fast significantly increased plasma glucose (1.5- to 2-fold) and insulin (2- to 4-fold) after glucose ingestion versus the values after the overnight fast, indicating the manifestation of peripheral insulin resistance. The 72-hour fast resulted in an ∼20% decrease in the muscle content of D-chiroinositol (P < 0.02), but no change in the myoinositol content. These data demonstrate that fasting specifically decreases the muscle content of D-chiroinositol in human muscle and this may contribute to the finding that insulin-mediated activation of pyruvate dehydrogenase is attenuated after short-term starvation.     

Wheat-germ agglutinin mimics metabolic effects of insulin without increasing receptor autophosphorylation

Expression of insulin metabolic effects can be obtained by anti-receptor antibodies without activation of the tyrosine kinase activity [O'Brien R. M., Soos M. A. and Siddle K. (1987) EMBO J. 6, 4003-4010; Forsayeth J. R., Caro J. F., Sinha M. K., Maddux B. A. and Goldfine I. D. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 34,448-34,514; Ponzio G., Contreres J. O., Debant A., Baron V., Gautier N., Dolais-Kitabgi J. and Rossi B. (1988) EMBO J. 7, 4111-4117; Hawley D. M., Maddux B. A., Patel R. G., Wong K. Y., Mamula P. W., Firestone G. L., Brunetti A., Verspohl E. and Goldfine I. D. (1989) J. biol. Chem. 264, 2438-2444; Soos M. A., O'Brien R. M., Brindle N. P. J., Stigter J. M., Okamoto A. K., Whittaker J. and Siddle K. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5217-5221.]. Recently, we have proposed that receptor cross-linking is sufficient in itself to stimulate glycogen synthesis, even if aggregation was performed on receptors mutated on Tyr 1162 and Tyr 1163 and thus devoid of tyrosine kinase activity [Debant A., Ponzio G., Clauser E., Contreres J. O. and Rossi B. (1989) Biochemistry 28, 14-17]. The aim of this study was to gain information on the involvement of receptor clustering in the expression of the different insulin biological effects. To this end, we studied the mimetic effects of wheat-germ agglutinin, which is likely to induce receptor aggregation without interacting with the receptor protein moiety. Wheat-germ agglutinin failed to promote DNA synthesis, whereas the lectin behaved as a potent mimicker of insulin on tyrosine aminotransferase activity and amino-acid transport. However, this stimulatory effect did not parallel the activation of receptor autophosphorylation. Our data reinforce the idea that the expression of the metabolic effects of insulin are not strictly dependent on a general tyrosine kinase activation.     

Dietary Myoinositol Results in Lower Urine Glucose and in Lower Postprandial Plasma Glucose in Obese Insulin Resistant Rhesus Monkeys.

In a previous study, D-chiroinositol added to a meal (0.5 g/kg) resulted in significantly lower postprandial plasma glucose concentrations without an increase in insulin concentrations in obese insulin-resistant monkeys. The present report describes the effects of another isomer of inositol, myoinositol, on postprandial plasma glucose and insulin concentrations and on urine glucose concentrations in 6 similarly insulin-resistant monkeys. The three 5 day study periods included a control period (liquid diet ad libitum) and 2 experimental periods (liquid diet ad libitum with either 1.5 g/kg/day myoinositol or D-chiroinositol added). Twenty-four hour urine samples were collected during each 5 day period. On the sixth day of each period the monkeys were anesthetized 110 min after completing either the control meal (15 ml/kg) or the experimental meals (1.5 g/kg myoinositol or D-chiroinositol) and plasma samples were obtained at 120, 150,180, 210, 240, 270 and 300 min. The plasma glucose concentration was lower after the meal with myoinositol compared to the control meal at 120, 150 and 180 min (p's<0.05). The plasma insulin concentration was lower after the meal with myoinositol compared to the control meal at 150 and 180 min (p's<0.05). In addition, 24 hour urine glucose concentrations were lower during the myoinositol diet compared to the control diet (p<0.001). The plasma glucose concentration was lower after the meal with D-chiroinositol compared to the control meal at 150, 240, 270 and 300 min (p's≥0.05). In obese insulin-resistant monkeys, myoinositol added to the diet lowers urine glucose concentrations and both myoinositol and D-chiroinositol added to a meal lower postprandial plasma glucose concentrations without increasing postprandial insulin concentrations. Therefore, myoinositol, like D-chiroinositol, may be a useful agent for reducing meal-induced hyperglycemia without inducing hyperinsulinemia in insulin-resistant subjects.     

Inositol 1,2-cyclic 4,5-trisphosphate is formed in the rat parotid gland on muscarinic stimulation

Hawkins et al. [Hawkins, P.T., Berrie, C.P., Morris, A.J., and Downes, C.P. (1987) Biochem J. 243, 211-218] were unable to find any formation of inositol 1,2-cyclic 4,5-trisphosphate on muscarinic stimulation of rat parotid slices, contrary to what has been found in mouse pancreas and in platelets. We have repeated the studies of Hawkins et al. using [3H]inositol-prelabelled rat parotid minilobules and our improved HPLC method for clearly separating the three inositol trisphosphates. Substantial amounts of inositol 1,2-cyclic 4,5-trisphosphate formed on muscarinic stimulation of rat parotid minilobules, amounting to 5% of inositol 1,4,5-trisphosphate at 10 sec and one third of inositol 1,4,5-trisphosphate at 5 min.     

Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium

Inositol 1,4,5-trisphosphate is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. We have recently identified high levels of specific binding sites for inositol 1,4,5-trisphosphate in brain membranes (Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161) and have now further characterized these sites. In cerebellar membranes, inositol 1,4,5-trisphosphate binding sites are abundant (20 pmol/mg protein) and display high affinity and selectivity for inositol 1,4,5-trisphosphate (KD approximately equal to 40 nM), whereas other inositol phosphates such as inositol 1,3,4,5-tetrakisphosphate (Ki approximately equal to 10 microM) and inositol 1,4-bisphosphate (Ki approximately equal to 10 microM) exhibit much lower affinity for this site. Submicromolar concentrations of calcium strongly inhibit inositol 1,4,5-trisphosphate binding (IC50 approximately equal to 300 nM). A sharp increase in binding occurs at slightly alkaline pH. These results suggest that actions of inositol 1,4,5-trisphosphate are regulated by physiological alterations in intracellular pH and calcium concentrations.     

Evidence for insulin-dependent activation of S6 and microtubule-associated protein-2 kinases via a human insulin receptor/v-ros hybrid

The abilities of a series of six mutants of the human insulin receptor, an insulin receptor/v-ros hybrid (IR-ros) and the P68gag-ros transforming protein to stimulate S6 protein kinase have been assessed. Insulin receptor mutants in which either 1 or 2 tyrosine residues have been replaced with phenylalanine (YF1, YF3) have lost some or all of the capacity to mediate the activation of S6 kinase in response to insulin. None of the four mutants that contain deletions (spBam, spBamYF3, iBgl, T-t) elicit an insulin-dependent stimulation of S6 kinase. A previous study of the IRros hybrid receptor demonstrated that it was unable to cause either insulin-stimulated thymidine incorporation or glucose uptake (Ellis, L., Morgan, D. O., Jong, S.-M., Wang, L.-H., Roth, R. A., and Rutter, W. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5101-5105). In contrast, the IRros chimera appears to mediate the activation of S6 protein kinase by insulin. In further evaluating the biological activities of the IRros hybrid, we have examined its effects on a microtubule-associated protein-2 (MAP2) kinase that is thought to be an early target in the cascade of reactions leading to increased S6 phosphorylation (Sturgill, T. W., Ray, L. B., Erickson, E., and Maller, J. L. (1988) Nature 334, 715-718). We find that the IRros receptor stimulates the MAP2 protein kinase from 3- to 6-fold in insulin-treated cells, conferring more than a 30-fold increase in the insulin sensitivity of MAP2 kinase activation.     

Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells

The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells.     

Linkage of organic phosphates to oxygen binding in human hemoglobin at high concentrations

We have performed high-precision oxygen binding studies on human hemoglobin tetramers in the presence of a series of limited, subsaturating amounts of the effector compounds 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP). The use of thin-layer optical methods enabled the use of high hemoglobin concentrations, preventing complications arising from the dissociation of the tetramer into dimers. Model-independent, simultaneous analysis of all data for each effector demonstrated that the intrinsic oxygen binding characteristics of the molecule are in agreement with those determined in earlier high-precision studies [e.g., Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry 26, 3995-4002] and that the affinity of the tetramer for the tightly binding effector IHP changes most markedly between the second and fourth oxygen binding steps, perhaps indicating a large conformational change. The data were then analyzed by using the truncated allosteric model [Di Cera, E., Robert, C. H., & Gill, S. J. (1987) Biochemistry 26, 4003-4008], which is based on the hypothesis that a quaternary conformational change occurs in the hemoglobin tetramer before the third and fourth oxygen molecules bind.     

Tyrosine kinase-activating growth factors potentiate thrombin- and AIF4- -induced phosphoinositide breakdown in hamster fibroblasts. Evidence for positive cross-talk between the two mitogenic signaling pathways

Basic fibroblast growth factor (FGF) and alpha-thrombin can stimulate DNA synthesis in Chinese hamster fibroblasts (CCL39) by two separate signaling pathways (Chambard, J.C., Paris, S., L'Allemain, G., and Pouysségur, J. (1987) Nature 326, 800-803) but can also act synergistically. We have examined whether this synergism might depend upon changes in inositol lipid metabolism. Indeed, FGF, which has no effect on its own on phosphoinositide hydrolysis, potentiates (by up to 2-fold) thrombin-induced formation of inositol phosphates. This enhancing effect is also observed upon direct activation by AIF4- of the GTP-binding protein coupled to phospholipase C, and is best revealed when phospholipase C is weakly stimulated. With low thrombin concentrations or with AIF4-, the formation of inositol phosphates is immediately increased with a marked reduction of the initial lag, whereas at high thrombin concentrations, the stimulation by FGF becomes pronounced only after desensitization of phospholipase C to thrombin. FGF-induced potentiation is not mimicked by calcium ionophores, but is likewise elicited by epidermal growth factor, platelet-derived growth factor, and to a lesser extent by insulin, other growth factors known to activate receptor tyrosine kinases. We therefore propose that the tyrosine kinase-activating growth factors enhance the coupling between GTP-binding protein and phospholipase C, presumably through the phosphorylation of one of these two proteins. Treatment of cells with pertussis toxin attenuates thrombin-induced phospholipase C activity but does not impede the potentiation by FGF. Comparison of the potentiating effects of FGF on inositol phosphate formation and on DNA synthesis suggests than an increased production of second messengers by the inositol lipid pathway in the first hours of stimulation might be, at least in part, responsible for the synergistic actions of FGF and thrombin on DNA synthesis.     

Galphaz negatively regulates insulin secretion and glucose clearance

Relatively little is known about the in vivo functions of the alpha subunit of the heterotrimeric G protein Gz (Galphaz). Clues to one potential function recently emerged with the finding that activation of Galphaz inhibits glucose-stimulated insulin secretion in an insulinoma cell line (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., Fields, T. A., and Casey, P. J. (2005) J. Biol. Chem. 280, 31708-31713). To extend this study in vivo, a Galphaz knock-out mouse model was utilized to determine whether Galphaz function plays a role in the inhibition of insulin secretion. No differences were discovered in the gross morphology of the pancreatic islets or in the islet DNA, protein, or insulin content between Galphaz-null and wild-type mice. There was also no difference between the insulin sensitivity of Galphaz-null mice and wild-type controls, as measured by insulin tolerance tests. Galphaz-null mice did, however, display increased plasma insulin concentrations and a corresponding increase in glucose clearance following intraperitoneal and oral glucose challenge as compared with wild-type controls. The increased plasma insulin observed in Galphaz-null mice is most likely a direct result of enhanced insulin secretion, since pancreatic islets isolated from Galphaz-null mice exhibited significantly higher glucose-stimulated insulin secretion than those of wild-type mice. Finally, the increased insulin secretion observed in Galphaz-null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in Galphaz-null islets in the absence of exogenous stimulation. These findings indicate that Galphaz may be a potential new target for therapeutics aimed at ameliorating beta-cell dysfunction in Type 2 diabetes.     

Effects of D-Chiroinositol Added to a Meal on Plasma Glucose and Insulin in Hyperinsulinemic Rhesus Monkeys

We have previously demonstrated that D-chiroinositol, administered intravenously to insulin-resistant monkeys, increases the rate of disappearance of plasma glucose and insulin. The purpose of the present study was to determine whether orally administered D-chiroinositol might also similarly improve the postprandial plasma glucose profile of hyperinsulinemic insulin-resistant monkeys. A complete liquid diet meal (15 ml/kg body weight) was ingested by each of six monkeys on two occasions separated by 10 days, with conditions identical except D-chiroinositol (500 mg/kg body weight) was added to the second meal. At 110 minutes following each meal, the monkeys were anesthetized and blood samples obtained at 120, 150, 180, 210, 240, 270 and 300 minutes. Plasma glucose and insulin concentrations were determined. The mean plasma glucose concentration (120 ? 300 minutes) was significantly lower after the meal containing D-chiroinositol compared to the control meal (7.1 ± 1.2 vs. 7.8 ± 1.2 mM) (p<0.05). Plasma insulin concentrations tended to be lower after the meal containing D-chiroinositol compared to the control meal (3930 ± 1068 vs. 4518 ± 1200 pM) (p<0.15, ns). We conclude that in hyperinsulinemic monkeys, D-chiroinositol added to a meal lowers postprandial plasma glucose without an increase in plasma insulin, and therefore may be a useful agent for reducing meal-induced hyperglycemia without inducing hyperinsulinemia.     

The metabolism of inositol 1,3,4-trisphosphate to inositol 1,3-bisphosphate

We previously demonstrated a pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) to inositol 3,4-bisphosphate (Ins(3,4)P2) in calf brain extracts. Inositol polyphosphate 1-phosphatase, a Mg2+-dependent, lithium ion-inhibited enzyme, specifically hydrolyzes Ins(1,3,4)P3 to Ins(3,4)P2 and Ins(1,4)P2 to Ins 4-P (Inhorn, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). Now we have found an alternative pathway for the metabolism of Ins(1,3,4)P3 in crude calf brain extracts. Along this pathway, Ins(1,3,4)P3 is first converted to Ins(1,3)P2 which is further hydrolyzed to Ins 1-P. This pathway involves a 4-phosphatase and a 3-phosphatase which do not require Mg2+ and are not inhibited by lithium ions. A similar 4-phosphatase also degrades Ins(3,4)P2 to Ins 3-P. Three different inositol bisphosphates formed from calf brain supernatant are each further metabolized by a separate enzyme. The three inositol monophosphates, i.e. Ins 1-P, Ins 3-P, and Ins 4-P, are converted to inositol by inositol monophosphate phosphatase (Ackermann, K. E., Gish, B. G., Honchar, M. P., and Sherman, W. R. (1987) Biochem. J. 242, 517-524).     

Calcium messenger system: Role of protein phosphorylation and inositol bisphospholipids

Poovaiah, B. W., Reddy, A. S. N. and McFadden, J. J. 1987. Calcium messenger system: Role of protein phosphorylation and inositol bisphospholipids.     

Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A., Nguyen, H., Galceran, J., Pessah, I. N., and Allen, P. D. (1998) J. Cell Biol. 140, 843-851), and C(2)C(12) cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immuno-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mm K(+), inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C(2)C(12) myotubes. However, 1B5 myotubes (as well as ryanodine-treated C(2)C(12) myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl borate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.     

Insulin-stimulated diacylglycerol production results from the hydrolysis of a novel phosphatidylinositol glycan

We recently described the insulin-dependent release of a carbohydrate substance from plasma membranes which regulated certain intracellular enzymes (Saltiel, A. R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5793-5797). This enzyme-modulating substance appeared to arise from the phosphodiesterase hydrolysis of a novel inositol-containing glycolipid. This is supported by observations that insulin stimulated the rapid generation of [3H]myristate-labeled diacylglycerol in cultured BC3Hl myocytes. Myristoyl diacylglycerol production in these cells was unaffected by epinephrine, although arachidonate-labeled diacylglycerol was rapidly produced in response to stimulation by this alpha-1 adrenergic agent. The production of distinct species of diacylglycerol was apparently due to hormonally specific hydrolysis of different precursors. A novel glycolipid was identified on silica TLC or high pressure liquid chromatography which served as a substrate for the insulin-stimulated phosphodiesterase reaction. This glycolipid was metabolically labeled with radioactive inositol, glucosamine, and myristic acid, suggesting a phosphatidylinositol (PI)-glycan structure. Treatment of this glycolipid with a PI-specific phospholipase C resulted in the generation of two products: an inositol phosphate-glycan which modulated the activity of the low Km cAMP phosphodiesterase and myristoyl diacylglycerol. Insulin caused the rapid hydrolysis of the PI-glycan, which was then apparently resynthesized. These data further suggest that insulin stimulates the activity of a phospholipase C which selectively hydrolyzes a novel PI-glycan, releasing a carbohydrate enzyme modulator as well as a unique species of diacylglycerol.     

Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+

We recently identified an enzyme which we have designated inositol polyphosphate 1-phosphatase that hydrolyzes both inositol 1,3,4-trisphosphate (Ins-1,3,4-P3) and inositol 1,4-bisphosphate (Ins-1,4-P2), yielding inositol 3,4-bisphosphate and inositol 4-phosphate, respectively, as products (Inhorn, R. C., Bansal, V.S., and Majerus, P.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). We have now purified the inositol polyphosphate 1-phosphatase 3600-fold from calf brain supernatant. The purified enzyme has an apparent molecular mass of 44,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphatase activities. The enzyme hydrolyzes Ins-1,4-P2 with an apparent Km of approximately 4-5 microM, while it degrades Ins-1,3,4-P3 with an apparent Km of approximately 20 microM. The enzyme hydrolyzes these substrates at approximately the same maximal velocity. Inositol polyphosphate 1-phosphatase shows a sigmoidal dependence upon magnesium ion, with 0.3 mM Mg2+ causing half-maximal stimulation. A Hill plot of the data is linear with a value of n = 1.9, suggesting that the enzyme binds magnesium cooperatively. Calcium and manganese inhibit enzyme activity, with 50% inhibition at approximately 6 microM. Lithium inhibits Ins-1,4-P2 hydrolysis uncompetitively with a Ki of approximately 6 mM. This mechanism of lithium inhibition is similar to that observed for the inositol monophosphate phosphatase (originally designated myo-inositol-1-phosphatase; Hallcher, L.M., and Sherman, W.R. (1980) J. Biol. Chem. 255, 10896-10901), suggesting that these two enzymes are related. Lithium also inhibits Ins-1,3,4-P3 hydrolysis with an estimated Ki of 0.5-1 mM.     

A model phosphatase 2C --> phosphatase 1 activation cascade via dual control of inhibitor-1 (INH-1) and DARPP-32 dephosphorylation by two inositol glycan putative insulin mediators from beef liver

Two inositol phosphoglycans (IPG) isolated from beef liver and designated as putative insulin mediators were demonstrated to reciprocally enhance the dephosphorylation of inhibitor-1 (INH-1) and DARPP-32, thus directly activating phosphatase 2C and disinhibiting phosphatase 1 in a potential protein phosphatase 2C --> phosphatase 1 cascade mechanism. One IPG termed pH 2.0, containing Dchiro-inositol and galactosamine, stimulated the dephosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range. A second, termed pH 1.3, containing myo-inositol glucosamine and mannose acted reciprocally to inhibit the cAMP-dependent protein kinase phosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range. These model experiments are discussed in terms of the observed dephosphorylation of INH-1 with insulin action documented in the literature and the activation of both phosphatase 1 and 2C described in intact cells and in vivo with insulin action.