Effect of sodium fluoride on cyclic AMP production in rat hepatocytes.

The effect of NaF on cAMP production was studied in hepatocytes isolated from fed and fasted rats. A four-six fold increase in hepatocyte cAMP production was observed in the presence of 10-20 mM NaF in cells isolated from either fed or fasted rats. The maximal stimulation of cAMP production was observed after a 10 min incubation in the presence of 1 mM theophylline. However, as little as 0.05-0.15 mM NaF induced a significant increase in cAMP production. It was also found that NaF would alter the production of glucose in isolated rat hepatocytes. When hepatocytes from fed rats were incubated with 0.05-5 mM NaF there was an increase in amount of glucose released from endogenous sources. Also NaF resulted in a decrease in lactate and pyruvate production. Similarly NaF stimulated glucose production in hepatocytes from fasted rats. The maximal stimulation was observed with about 0.15-0.25 mM NaF. At NaF concentrations greater than 1.5 mM a decrease in glucose production was observed. It is concluded that NaF increases the level of cAMP and alters glucose metabolism in intact hepatocytes.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Substrate-dependent effect of 1-34 human parathyroid hormone fragment, dibutyryl cAMP and cAMP on gluconeogenesis in rabbit renal tubules

In the presence of 0.5 mM extracellular Ca2+ concentration both 1-34 human parathyroid hormone fragment (0.5 micrograms/ml) as well as 0.1 mM dibutyryl cAMP stimulated gluconeogenesis from lactate in renal tubules isolated from fed rabbits. However, these two compounds did not affect glucose synthesis from pyruvate as substrate. When 2.5 mM Ca2+ was present the stimulatory effect of the hormone fragment on gluconeogenesis from lactate was not detected but dibutyryl cAMP increased markedly the rate of glucose formation from lactate, dihydroxyacetone and glutamate, and inhibited this process from pyruvate and malate. Moreover, dibutyryl cAMP was ineffective in the presence of either 2-oxoglutarate or fructose as substrate. Similar changes in glucose formation were caused by 0.1 mM cAMP. As concluded from the 'crossover' plot the stimulatory effect of dibutyryl cAMP on glucose formation from lactate may result from an acceleration of pyruvate carboxylation due to an increase of intramitochondrial acetyl-CoA, while an inhibition by this compound of gluconeogenesis from pyruvate is likely due to an elevation of mitochondrial NADH/NAD+ ratio, resulting in a decrease of generation of oxaloacetate, the substrate of phosphoenolpyruvate carboxykinase. Dibutyryl cAMP decreased the conversion of fracture 1,6-bisphosphate to fructose 6-phosphate in the presence of both substrates which may be secondary to an inhibition of fructose 1,6-bisphosphatase.     

Substrate-dependent effect of 1–34 human parathyroid hormone fragment,dibutyryl cAMP and cAMP on gluconeogenesis in rabbit renal tubules

In the presence of 0.5 mM extracellular Ca²⁺ concentration both 1–34 human parathyroid hormone fragment (0.5 μg/ml) as well as 0.1 mM dibutyryl cAMP stimulated gluconeogenesis from lactate in renal tubules isolated from fed rabbits. However, these two compounds did not affect glucose synthesis from pyruvate as substrate. When 2.5 mM Ca²⁺ was present the stimulatory effect of the hormone fragment on gluconeogenesis from lactate was not detected but dibutyryl cAMP increased markedly the rate of glucose formation from lactate, dihydroxyacetone and glutamate, and inhibited this process from pyruvate and malate. Moreover, dibutyryl cAMP was ineffective in the presence of either 2-oxoglutarate or fructose as substrate. Similar changes in glucose formation were caused by 0.1 mM cAMP. As concluded from the ‘crossover’ plot the stimulatory effect of dibutyryl cAMP on glucose formation from lactate may result from an acceleration of pyruvate carboxylation due to an increase of intramitochondrial acetyl-CoA, while an inhibition by this compound of gluconeogenesis from pyruvate is likely due to an elevation of mitochondrial NADH/NAD⁺ ratio, resulting in a decrease of generation of oxaloacetate, the substrate of phosphoenolpyruvate carboxykinase. Dibutyryl cAMP decreased the conversion of fracture 1,6-bisphosphate to fructose 6-phosphate in the presence of both substrates which may be secondary to an inhibition of fructose 1,6-bisphosphatase.     

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells.

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.     

Glucose regulation of guinea-pig sperm motility.

Washed guinea-pig spermatozoa from the vas deferens re-acquired progressive motility within 1-2 min of incubation in minimal culture medium containing pyruvate and lactate. When glucose was added, either at the beginning or after 15 min of incubation, the cells showed stimulated motility (increased straight-line velocity, linearity and beat-cross frequency, P less than 0.01). Re-acquisition of progressive motility was preceded by a significant (P less than 0.005) transient increase in sperm concentration of cyclic adenosine 5'-phosphate (cAMP) with or without glucose in the medium. Papaverine caused another large significant (P less than 0.001) increase in cAMP concentration; and 5.56mM glucose with papaverine caused a further stimulation in cAMP beyond that with papaverine alone (P less than 0.005). Although 0.05 or 5.56mM glucose plus alpha-chlorohydrin stimulated sperm motility, they did not further stimulate the increase in cAMP after 30 s of incubation. Thus, there was no apparent correlation between the glucose-stimulating effect on sperm motility and the enhancement of cAMP at 30 s. However, there was a close correlation between glucose-stimulated motility and enhancement of ATP (P less than 0.05) by glucose even under incubation conditions in which glucose caused the Crabtree effect (decrease in respiration rate).     

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).     

Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.     

Ubiquitination is involved in glucose-mediated downregulation of GIP receptors in islets

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that has a potent stimulatory effect on insulin release under conditions of normal glucose tolerance. However, its insulinotropic effect is reduced or even absent entirely in type 2 diabetic patients. In this study, we addressed the role of glucose concentration in the diabetic range of >or=11 mM, i.e., hyperglycemia per se, as a cause of the lack of response to GIP. Culturing rat and human pancreatic islets in >or=11 mM glucose for up to 24 h resulted in prevention of GIP-mediated intracellular cAMP increase compared with culturing in 5 mM glucose. Western blot analysis revealed a selective 67 +/- 2% (rat) and 60 +/- 8% (human) decrease of GIP-R expression in islets exposed to >or=11 mM glucose compared with 5 mM glucose (P < 0.001). We further immunoprecipitated GIP-R from islets and found that GIP-R was targeted for ubiquitination in a glucose- and time-dependent manner. Downregulation of GIP-R was rescued by treating isolated islets with proteasomal inhibitors lactacystin and MG-132, and the islets were once again capable of increasing intracellular cAMP levels in response to GIP. These results suggest that the GIP-R is ubiquitated, resulting in downregulation of the actions of GIP.     

Effects of cyclic AMP on DNA replication and protein biosynthesis in fetal rat islets of Langerhans maintained in tissue culture

The regulatory role of cyclic AMP (cAMP) in the growth and insulin production of the islet organ in vitro has been investigated. The effects of dibutyryl cyclic AMP (dbcAMP), theophylline , and 3-isobutyl-1-methylxanthine (IBMX) on DNA replication and on the biosynthesis of RNA and insulin in fetal rat islets of Langerhans maintained in tissue culture have been studied. Raising the glucose concentration from 2.7 mM to 16.7 mM caused a two-fold increase in DNA replication. Both dbcAMP and theophylline markedly inhibited the DNA replication at all glucose Concentrations studied. Low concentrations of IBMX stimulated DNA synthesis. However, at higher concentrations of this drug, known to considerably increase the islet cAMP levels , a marked inhibition of islet DNA replication was observed. Both (pro)insulin and total protein biosynthesis were stimulated by glucose, whereas dbcAMP stimulated only the (pro)insulin biosynthesis. Since glucose is known to raise islet intracellular levels of cAMP, which is known to be an inhibitor of cellular proliferation, the observed glucose stimulation of both islet-cell DNA replication and insulin production appeared conflicting. It is suggested that this dual effect of glucose may depend on a stimulation of proliferation in a limited pool of islet cells which may not exhibit an increase in cAMP.     

Inhibition of dibutyryl cyclic AMP-induced insulin release by alpha-2 adrenergic stimulation

Effects of alpha adrenergic agonists and antagonists on dibutyryl cAMP (Bt2cAMP)-induced insulin release were investigated with isolated rat pancreatic islets. Bt2cAMP (4 mM) produced 2-fold stimulation of insulin release in all the concentrations of glucose examined (3.3-16.7 mM). Clonidine but not phenylephrine inhibited the Bt2cAMP-stimulated insulin release in a concentration-dependent manner with approximate EC50 of nanomolar range. Yohimbine but not prazosin antagonized the inhibitory effect of clonidine on the Bt2-cAMP-induced insulin release. These results suggest that alpha-2 adrenergic mechanisms are involved in a step distal to cAMP generation.     

High glucose inhibits fructose uptake in renal proximal tubule cells: involvement of cAMP, PLC/PKC, p44/42 MAPK, and cPLA2

The precise signal that regulates fructose transport in renal proximal tubule cells (PTCs) under high glucose conditions is not yet known although fructose has been recommended as a substitute for glucose in the diets of diabetic people. Thus, we investigated that effect of high glucose on fructose uptake and its signaling pathways in primary cultured rabbit renal PTCs. Glucose inhibited the fructose uptake in a time- and dose-dependent manner. A maximal inhibitory effect of glucose on fructose uptake was observed at 25 mM glucose after 48 h, while 25 mM mannitol and l-glucose did not affect fructose uptake. Indeed, 25 mM glucose for 48 h decreased GLUT5 protein level. Thus, the treatment of 25 mM glucose for 48 h was used for this study. Glucose-induced (25 mM) inhibition of fructose uptake was blocked by pertussis toxin (PTX), SQ-22536 (an adenylate cyclase inhibitor), and myristoylated amide 14-22 (a protein kinase A inhibitor). Indeed, 25 mM glucose increased the intracellular cAMP content. Furthermore, 25 mM glucose-induced inhibition of fructose uptake was prevented by neomycin or U-73122 (phospholipase C inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase C inhibitors). In fact, 25 mM glucose increased the total PKC activity and translocation of PKC from the cytosolic to membrane fraction. In addition, PD 98059 (a p44/42 mitogen-activated protein kinase (MAPK) inhibitor) but not SB 203580 (a p38 MAPK inhibitor) and mepacrine or AACOCF3 (phospholipase A2 inhibitors) blocked 25 mM glucose-induced inhibition of fructose uptake. Results of Western blotting using the p44/42 MAPK and GLUT5 antibodies were consistent with the results of uptake experiments. In conclusion, high glucose inhibits the fructose uptake through cAMP, PLC/PKC, p44/42 MAPK, and cytosolic phospholipase A2 (cPLA2) pathways in the PTCs.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

Maintenance of pancreatic endocrine cells of the neonatal rat: IV. The effect of 3-amino-3-deoxyglucose

Monolayer islet cells prepared from neonatal rat pancreases were cultured in media with 5.5 mM glucose alone or further supplemented with 5 mM 3-amino-3-deoxyglucose (3A3dG) for a total of 7 days. After culture for 7 days, 3A3dG-supplementation maintained the recovery of insulin released into the medium during the last 2 days of a 7-day culture at a level 2.9 fold higher that at day 0. Similarly, the insulin content of the cells was significantly higher than the initial level at day 0 (2.8-fold) and that of the cells grown in medium with glucose alone (4.5-fold). The maximum secretory responses to glucose (2.8-16.7 mM), leucine (2.5-10 mM) and 2-ketoisocaproate (2.5-10 mM) were several times as high as the initial. Further, 3A3dG caused a selective deletion of fibroblasts mostly consisting of endocrine cells. In these monolayer cells, either the cAMP response to glucose or the cellular cAMP content were significant. In conclusion, it is suggested that the beneficial effect of 3A3dG may be associated with an increase in either the oxidative catabolism of amino acids or the activity of adenylate cyclase in the B cell.     

The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets.

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

IIb group metal ions (Zn2+, Cd2+, Hg2+) stimulate glucose transport activity by post-insulin receptor kinase mechanism in rat adipocytes

Zn2+ (1 mM), Cd2+ (1 mM), and Hg2+ (0.1 mM) belonging to the IIb group in the periodic table stimulated glucose transport activity and cAMP phosphodiesterase in rat adipocytes. The stimulation of glucose transport was due to the translocation of glucose transporters from the intracellular site to the plasma membrane. However, in intact adipocytes none of these ions stimulated insulin receptor kinase activity or phosphorylation of the 95-kDa subunit of insulin receptor or 170- or 60-kDa proteins at the tyrosyl residues. These proteins were markedly phosphorylated by addition of 0.3 nM insulin which stimulated glucose transport activity as effectively as these metal ions. These results indicate that Zn2+, Cd2+, and Hg2+ mimic insulin action by a post-receptor/kinase mechanism.