Effects of a phorbol ester and clomiphene on protein phosphorylation and insulin secretion in rat pancreatic islets

The potentiation of glucose-stimulated insulin release induced by 100 nM-12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by clomiphene, an inhibitor of protein kinase C (PK C), in a dose-dependent manner. Clomiphene at concentrations up to 50 microM had a modest inhibitory action (27%) on insulin release stimulated by 10 mM-glucose alone, but had no effect on the potentiation of insulin release induced by forskolin. Islet PK C activity, associated with a particulate fraction, was stimulated maximally by 100 nM-TPA. This stimulation was blocked by clomiphene in a dose-dependent manner, with 50% inhibition at 30 microM. Incubation of intact islets with TPA after preincubation with [32P]Pi and 10 mM-glucose to label intracellular ATP resulted primarily in enhanced phosphorylation of a 37 kDa protein (mean value, +/- S.E.M., 36,700 +/- 600 Da; n = 7). This increased phosphorylation was blocked by the simultaneous inclusion of clomiphene. Subcellular fractionation revealed the presence of the 37 kDa phosphoprotein in a 24,000 g particulate fraction of islet homogenates. Neither clomiphene nor TPA affected the rate of glucose oxidation by islets. These results show that the phosphorylation state of a 37 kDa membrane protein parallels the modulation of insulin release induced by TPA and clomiphene and support a role for PK C in the insulin-secretory mechanism.

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Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2

Acta Endocrinologica ◽

10.1530/eje.0.1310510 ◽

1994 ◽

Vol 131 (5) ◽

pp. 510-515 ◽

Cited By ~ 5

Author(s):

Osamu Kozawa ◽

Haruhiko Tokuda ◽

Atsushi Suzuki ◽

Jun Kotoyori ◽

Yoshiaki Ito ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase A2 ◽

Phospholipase C ◽

Phospholipase A ◽

Phosphoinositide Hydrolysis ◽

Prostaglandin E ◽

Dependent Manner ◽

Kinase C ◽

Dose Dependent

Kozawa O, Tokuda H, Suzuki A, Kotoyori J, Ito Y, Oiso Y. Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2. Eur J Endocrinol 1994;131:510–15. ISSN 0804–4643 It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2α stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2α-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2α in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2α- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2α-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2α-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells. Osamu Kozawa, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan

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Abstract 653: Muscle Ring Finger-1 (MuRF1) Expression Shifts Cardiomyocyte Substrate Utilization from Fatty Acids to Glucose

Circulation ◽

10.1161/circ.116.suppl_16.ii_120-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Monte S Willis ◽

Jon Schisler ◽

Holly McDonough ◽

Cam Patterson

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Oxidation ◽

Glucose Oxidation ◽

Relative Proportion ◽

Substrate Utilization ◽

Acid Oxidation ◽

Protective Mechanism ◽

Dependent Manner ◽

Dose Dependent

Previous work has suggested that MuRF1, a cardiac-specific protein, regulates metabolism by its interactions with proteins that regulate ATP transport, glycolysis, and the electron transport chain. We recently identified that MuRF1 is cardioprotective in ischemia reperfusion injury. In the current study, we investigated the effects of MuRF1 expression on metabolic substrate utilization and found that MuRF1 shifts substrate utilization from fatty acids to glucose in a dose-dependent manner. Isolated neonatal ventricular cardiomyocytes were treated with an adenovirus expressing MuRF1 (Ad.MuRF1) or GFP (Ad.GFP) at a range of 0–25 MOI (Multiplicity Of Infection). 14C-Oleate or 14C-glucose were added to cells for 1 hour and 14C-CO2 release was determined using the CO2-trapping method. Trapped 14CO2 and acid soluble metabolites were used to calculate total fatty acid oxidation. Cardiomyocytes treated with 5–25 MOI Ad.MuRF1 demonstrated a dose dependent decrease in fatty acid oxidation of 10.5 +/− 2.3(5 MOI), 8.5 +/− 1.9 (10 MOI), 6.6 +/− 1.6 (15 MOI), and 5.1 +/− 1.3 (25 MOI) nmol oleate/mg protein/h. Compared with control cardiomyocytes treated with 5–25 MOI Ad.GFP (average of 5–25 MOI=13.5 +/− 0.7 nmol oleate/mg protein/h), this represents a 22.2%– 62.2% decrease in fatty acid oxidation. Inversely, glucose oxidation increased with increasing MuRF1 expression. Cardiomyocytes infected with 25 MOI Ad.MuRF1 oxidized 184% more glucose (28.9 +/− 4.6 nmol glucose/mg protein/h) compared to control cells treated with 25 MOI Ad.GFP (15.7 +/− 1.3 nmol glucose/mg protein/h). Increasing MuRF1 expression resulted in no net gain or loss of calculated ATP production (1699 +/− 157 vs. 1480 +/− 188 nmol ATP/mg protein/h). The co-utilization of glucose and fatty acids as substrates for the production of ATP allows the heart to adapt to both environmental stress and disease. Increasing the relative proportion of glucose oxidation in relationship to fatty acids is a known protective mechanism during cardiac stress, and may represent one mechanism by which MuRF1 is cardioprotective.

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Effect of galanin on glucose-, arginine-, or potassium-stimulated insulin release

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1989.256.5.e619 ◽

1989 ◽

Vol 256 (5) ◽

pp. E619-E623

Author(s):

T. Yoshimura ◽

J. Ishizuka ◽

G. H. Greeley ◽

J. C. Thompson

Keyword(s):

Insulin Release ◽

High Glucose ◽

Second Phase ◽

Dependent Manner ◽

Perfused Pancreas ◽

Isolated Perfused Pancreas ◽

Second Phases ◽

High Concentrations ◽

Dose Dependent ◽

Stimulation Of

We have examined the effect of galanin infusion on glucose-stimulated release of insulin from the isolated perfused pancreas of the rat to better characterize the effect of galanin on the first and second phases of insulin release. The effects of galanin on insulin release stimulated by L-arginine or high concentrations of potassium were also examined. When perfusion of galanin was started 4 min before the start of perfusion of high glucose (16.7 mM), galanin (10(-8)-10(-11) M) inhibited both the first and second phases of insulin release in a dose-dependent manner. When perfusion of galanin (10(-8) or 10(-9) M) was started simultaneously with high glucose (16.7 mM), only the second phase of insulin release was suppressed (P less than 0.05). Galanin (10(-9) M) failed to inhibit insulin release stimulated by L-arginine (10 and 5 mM) or potassium (25 and 20 mM). These findings suggest that the inhibitory action of galanin on glucose-stimulated insulin release is exerted on early intracellular events that occur during the stimulation of insulin release and that are common to both phases. Because galanin does not inhibit insulin release stimulated by L-arginine or potassium, galanin may inhibit glucose-stimulated closure of potassium channels.

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Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v89.9.3378 ◽

1997 ◽

Vol 89 (9) ◽

pp. 3378-3384 ◽

Cited By ~ 71

Author(s):

Beatriz Bellosillo ◽

Mireia Dalmau ◽

Dolors Colomer ◽

Joan Gil

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Interleukin 4 ◽

Leukemia Cells ◽

Parp Cleavage ◽

Dependent Manner ◽

Kinase C ◽

Dose Dependent ◽

Pkc Activation

Abstract B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived B lymphocytes that express high levels of Bcl-2. We examined the involvement of CED-3/ICE-like proteases in the apoptosis of B-CLL cells. One of the substrates of these proteases is poly(ADP [adenosine 5′-diphosphate]-ribose) polymerase (PARP). The effect of different factors that induce the apoptosis of B-CLL cells on the proteolytic cleavage of PARP has been studied. Treatment of B-CLL cells with different concentrations of dexamethasone (1 to 1,000 μmol/L) induced in a dose-dependent manner the cleavage of PARP. Dexamethasone induced PARP cleavage after 12 hours of incubation, which was almost complete at 48 hours. PARP cleavage during apoptosis of B-CLL cells was studied in cells from eight patients and a correlation was found between cell viability and the degree of PARP cleavage. Incubation in vitro of B-CLL cells with fludarabine for 48 hours induced PARP cleavage in all the cases studied. Protein kinase C (PKC) activation with 100 nmol/L TPA (12-O-tetradecanoylphorbol 13-acetate) or incubation with interleukin-4 (10 ng/mL) prevented either dexamethasone- or fludarabine-induced proteolysis of PARP. Incubation of B-CLL cells with the CED-3/ICE–like protease inhibitor Z-VAD.fmk inhibited spontaneous and dexamethasone-induced PARP cleavage and DNA fragmentation in a dose-dependent manner. Furthermore, Z-VAD.fmk prevented the cytotoxic effect of dexamethasone. These results indicate that CED-3/ICE–like proteases play an important role in the apoptosis of B-CLL cells.

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Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Molecular Biology of the Cell ◽

10.1091/mbc.9.5.1209 ◽

1998 ◽

Vol 9 (5) ◽

pp. 1209-1220 ◽

Cited By ~ 68

Author(s):

Alexander V. Chibalin ◽

Juleen R. Zierath ◽

Adrian I. Katz ◽

Per-Olof Berggren ◽

Alejandro M. Bertorello

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

Α Subunit ◽

Kinase C ◽

Dose Dependent ◽

Α Subunits ◽

K Activity ◽

Phosphatidylinositol 3

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

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Interaction between perchlorate and nifedipine on insulin secretion from mouse pancreastic islets

Bioscience Reports ◽

10.1007/bf01145963 ◽

1993 ◽

Vol 13 (2) ◽

pp. 107-117 ◽

Cited By ~ 6

Author(s):

Gerd Larsson-Nyrén ◽

Janove Sehlin

Keyword(s):

Insulin Secretion ◽

Insulin Release ◽

Specific Effect ◽

Maximum Effect ◽

Dependent Manner ◽

Inhibitory Effects ◽

Maximum Inhibition ◽

Second Phases ◽

Dose Dependent ◽

Higher Sensitivity

In order to elucidate the mechanisms responsible for the stimulatory effect of perchlorate (ClO4−) on insulin secretion, we have investigated the interaction between this chaotropic anion and the organic calcium antagonist nifedipine. This drug, known as a blocker of L-type calcium channels, was chosen as a tool to test the idea that ClO4− acts on insulin secretion by stimulating the gating of voltage-controlled Ca2+ channels. ClO4− amplified the stimulatory effect of D-glucose on insulin release from perfused pancreas (first and second phases) as well as from isolated islets incubated in static incubations for 60 min. This indicates that ClO4− amplifies physiologically regulated insulin secretion. Nifedipine reduced D-glucose-induced (20 mM) insulin release in a dose-dependent manner with half-maximum effect at about 0.8 μM and apparent maximum effect at 5 μM nifedipine. In the presence of 20 mM D-glucose, the inhibitory effects of 0.5, 1 or 5 μM nifedipine were only slightly, if at all, counteracted by perchlorate. When 12 mM ClO4− and 20 mM D-glucose were combined, calculation of the specific effect of ClO4− revealed that nifedipine produced almost maximum inhibition already at 0.05 μM. Thus, the perchlorate-induced amplification of D-glucose-stimulated insulin release shows higher sensitivity to nifedipine than the D-glucose-effect as such. This supports the hypothesis that perchlorate primarily affects the voltage-sensitive L-type calcium channel in the β-cell.

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Regulation of prostaglandin synthesis by protein kinase C in mouse peritoneal macrophages

Biochemical Journal ◽

10.1042/bj2600471 ◽

1989 ◽

Vol 260 (2) ◽

pp. 471-478 ◽

Cited By ~ 41

Author(s):

H J Pfannkuche ◽

V Kaever ◽

D Gemsa ◽

K Resch

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Peritoneal Macrophages ◽

Dependent Manner ◽

Membrane Phospholipids ◽

Dependent Protein Kinase ◽

Kinase C ◽

Dependent Protein ◽

Dose Dependent ◽

Mouse Peritoneal Macrophages

Resident mouse peritoneal macrophages synthesized and released prostaglandins (PGs) when challenged with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (DiC8). Both stimuli were found to activate Ca2+/phospholipid-dependent protein kinase C (PKC). 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (‘H-7’) and D-sphingosine, known to inhibit PKC by different mechanisms, were able to decrease the PKC activity of macrophages in a dose-dependent manner. Addition of either PKC inhibitor decreased PG synthesis and also the release of arachidonic acid (AA) from phospholipids induced by TPA or DiC8. Simultaneously TPA or DiC8 also decreased incorporation of free AA into membrane phospholipids of macrophages. AA incorporation could be restored, however, by pretreatment with the PKC inhibitors. Our results demonstrate an involvement of PKC in the regulation of PG synthesis in mouse peritoneal macrophages and provide further evidence that reacylation of released fatty acids may be an important regulatory step.

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Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000430120 ◽

2015 ◽

Vol 36 (2) ◽

pp. 555-568 ◽

Cited By ~ 13

Author(s):

Jiaoqian Ying ◽

Yuan Zhang ◽

Shan Gong ◽

Zhigang Chang ◽

Xiaofeng Zhou ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Ventricular Myocytes ◽

Dependent Manner ◽

The Novel ◽

Inotropic Effects ◽

Kinase C ◽

Melanocortin 4 Receptor ◽

Dose Dependent ◽

Ca2 Channels

Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca2+ currents (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased ICa,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-β-S or anti-Gβ antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca2+-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKCθ), and PKCθ inhibition abolished the decrease in ICa,L induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca2+ channels via the MC4-R that couples sequentially to the βγ subunits of Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes.

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Parathyroid Hormone (1-34) Counteracts the Suppression of Interleukin-11 Expression by Glucocorticoid in Murine Osteoblasts: A Possible Mechanism for Stimulating Osteoblast Differentiation Against Glucocorticoid Excess

Endocrinology ◽

10.1210/en.2013-1915 ◽

2013 ◽

Vol 154 (3) ◽

pp. 1156-1167 ◽

Cited By ~ 7

Author(s):

Rika Kuriwaka-Kido ◽

Shinsuke Kido ◽

Yuka Miyatani ◽

Yuji Ito ◽

Takeshi Kondo ◽

...

Keyword(s):

Bone Formation ◽

Gene Transcription ◽

Osteoblast Differentiation ◽

Mechanical Strain ◽

Dependent Manner ◽

Rapid Induction ◽

Kinase C ◽

High Doses ◽

Interfering Rna ◽

Dose Dependent

Abstract Glucocorticoid (GC) excess causes a rapid loss of bone with a reduction in bone formation. Intermittent PTH (1-34) administration stimulates bone formation and counteracts the inhibition of bone formation by GC excess. We have previously demonstrated that mechanical strain enhances interleukin (IL)-11 gene transcription by a rapid induction of ΔFosB expression and protein kinase C (PKC)-δ-mediated phosphorylation of phosphorylated mothers against decapentaplegic (Smad)-1. Because IL-11 suppresses the expression of dickkopf-1 and -2 and stimulates Wnt signaling, IL-11 appears to mediate at least a part of the effect of mechanical strain on osteoblast differentiation and bone formation. The present study was undertaken to examine the effect of PTH(1-34) and GCs on IL-11 expression in murine primary osteoblasts (mPOBs). PTH(1-34) treatment of mPOBs enhanced IL-11 expression in a time- and dose-dependent manner. PTH(1-34) also stimulated ΔFosB expression and Smad1 phosphorylation, which cooperatively stimulated IL-11 gene transcription. PTH(1-34)-induced Smad1 phosphorylation was mediated via PKCδ and was abrogated in mPOBs from PKCδ knockout mice. Dexamethasone suppressed IL-11 gene transcription enhanced by PTH(1-34) without affecting ΔFosB expression or Smad1 phosphorylation, and dexamethasone-GC receptor complex was bound to JunD, which forms heterodimers with ΔFosB. High doses of PTH(1-34) counteracted the effect of dexamethasone on apoptosis of mPOBs, which was blunted by neutralizing anti-IL-11 antibody or IL-11 small interfering RNA. These results demonstrate that PTH(1-34) and GCs interact to regulate IL-11 expression in parallel with osteoblast differentiation and apoptosis and suggest that PTH(1-34) and dexamethasone may regulate osteoblast differentiation and apoptosis via their effect on IL-11 expression.

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Ganglioside GQ1b-induced terminal differentiation in cultured mouse keratinocytes. Phosphoinositide turnover forms the onset signal

Biochemical Journal ◽

10.1042/bj2790665 ◽

1991 ◽

Vol 279 (3) ◽

pp. 665-670 ◽

Cited By ~ 20

Author(s):

Y Yada ◽

Y Okano ◽

Y Nozawa

Keyword(s):

Terminal Differentiation ◽

Keratinocyte Differentiation ◽

Dependent Manner ◽

Transglutaminase Activity ◽

Phosphoinositide Turnover ◽

Kinase C ◽

Pre Treatment ◽

Mouse Keratinocytes ◽

Dose Dependent ◽

Pkc Activation

Investigations were undertaken to see whether mouse keratinocyte differentiation was elicited by gangliosides. Among the gangliosides tested only GQ1b, a tetrasialoganglioside containing two disialosyl residues, induced keratinocyte differentiation, as indicated by the formation of cornified envelopes, enhancement of transglutaminase activity and suppression of DNA synthesis. Upon stimulation with GQ1b the mass content of Ins(1,4,5)P3 and the intracellular Ca2+ levels were markedly enhanced in a time- and dose-dependent manner, whereas no significant changes were observed with other gangliosides, thereby indicating activation of phospholipase C for the onset of keratinocyte differentiation. Furthermore, only GQ1b promoted the translocation of protein kinase C (PKC) from cytosol to membrane. Inhibition of PKC with H-7 or down-regulation of the enzyme by prolonged pre-treatment with phorbol 12,13-dibutyrate greatly suppressed transglutaminase activity and formation of cornified envelopes induced by GQ1b. These results demonstrate that the tetrasialoganglioside GQ1b generates the initial differentiation signal in mouse keratinocytes through phosphoinositide turnover, and also suggest that PKC activation may act at certain, as yet unidentified, stages of differentiation processes.

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