| 1. |
- Jones, Helena, et al.
(författare)
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beta-cell PDE3B regulates Ca(2+)-stimulated exocytosis of insulin.
- 2007
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Ingår i: Cellular Signalling. - : Elsevier BV. - 1873-3913 .- 0898-6568. ; 19:Feb 12, s. 1505-1513
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Tidskriftsartikel (refereegranskat)abstract
- cAMP signaling is important for the regulation of insulin secretion in pancreatic beta-cells. The level of intracellular cAMP is controlled through its production by adenylyl cyclases and its breakdown by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE3B is involved in the regulation of nutrient-stimulated insulin secretion. Here, aiming at getting deeper functional insights, we have examined the role of PDE3B in the two phases of insulin secretion as well as its localization in the beta-cell. Depolarization-induced insulin secretion was assessed and in models where PDE3B was overexpressed [islets from transgenic RIP-PDE3B/7 mice and adenovirally (AdPDE3B) infected INS-I (832/13) cells], the first phase of insulin secretion, occurring in response to stimulation with high K+ for 5 min, was significantly reduced (similar to 25% compared to controls). In contrast, in islets from PDE3B(-/-) mice the response to high K+ was increased. Further, stimulation of isolated beta-cells from RIP-PDE3B/7 islets, using successive trains of voltage-clamped depolarizations, resulted in reduced Ca2+-triggered first phase exocytotic response as well as reduced granule mobilization-dependent second phase, compared to wild-type beta-cells. Using sub-cellular fractionation, confocal microscopy and transmission electron microscopy of isolated mouse islets and INS-1 (832/13) cells, we show that endogenous and overexpressed PDE3B is localized to insulin granules and plasma membrane. We conclude that PDE3B, through hydrolysis of cAMP in pools regulated by Ca2+, plays a regulatory role in depolarization-induced insulin secretion and that the enzyme is associated with the exocytotic machinery in beta-cells. (c) 2007 Elsevier Inc. All rights reserved.
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| 2. |
- Ahmad, Faiyaz, et al.
(författare)
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Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B
- 2005
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New Jersey : Humana Press. - 1940-6029 .- 1064-3745. ; 307, s. 93-107
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.
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| 3. |
- Ahmad, F, et al.
(författare)
-
Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells
- 2000
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Ingår i: Journal of Immunology. - 1550-6606. ; 164:9, s. 4678-4688
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Tidskriftsartikel (refereegranskat)abstract
- Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba-, F/Bb-, F/Bc-) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of insulin-like growth factor-1 (IGF-1), activities of PKB, cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) cells, and F/B and F/B- cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities approximately 2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity approximately 10-fold and PDE3B phosphorylation and activity ( approximately 4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased ( approximately 10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B- cells, IGF-1 activated PDE4, not PDE3B, suggesting that kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells.
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| 4. |
- Choi, Young Hun, et al.
(författare)
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Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B-null mice
- 2006
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Ingår i: Journal of Clinical Investigation. - 0021-9738. ; 116:12, s. 3240-3251
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Tidskriftsartikel (refereegranskat)abstract
- Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated hpogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.
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| 5. |
- Degerman, Eva, et al.
(författare)
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From PDE3B to the regulation of energy homeostasis.
- 2011
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Ingår i: Current Opinion in Pharmacology. - : Elsevier BV. - 1471-4973 .- 1471-4892. ; 11, s. 676-682
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Tidskriftsartikel (refereegranskat)abstract
- The incidence of obesity in the developed world is increasing at an alarming rate. Concurrent with the increase in the incidence of obesity is an increase in the incidence of type 2 diabetes. Cyclic AMP (cAMP) and cGMP are key second messengers in all cells; for example, when it comes to processes of relevance for the regulation of energy metabolism, cAMP is a key mediator in the regulation of lipolysis, glycogenolysis, gluconeogenesis and pancreatic β cell insulin secretion. PDE3B, one of several enzymes which hydrolyze cAMP and cGMP, is expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, hypothalamic cells and β cells. It has been shown, using PDE3 inhibitors and gene targeting approaches in cells and animals, that altered levels of PDE3B result in a number of changes in the regulation of glucose and lipid metabolism and in overall energy homeostasis. This article highlights the complexity involved in the regulation of PDE3B by hormones, and in the regulation of downstream metabolic effects by PDE3B in several interacting tissues.
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| 6. |
- Degerman, Eva, et al.
(författare)
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Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B
- 1998
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 14:1, s. 43-53
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Tidskriftsartikel (refereegranskat)abstract
- Phosphodiesterases (PDEs) include a large group of structurally related enzymes that belong to at least seven related gene families (PDEs 1-7) that differ in their primary structure, affinity for cAMP and cGMP, response to specific effectors, sensitivity to specific inhibitors, and regulatory mechanism. One characteristic of PDE3s involves their phosphorylation and activation in response to insulin as well as to agents that increase cAMP in adipocytes, hepatocytes, and platelets and in response to insulin-like growth factor 1 in pancreatic beta cells. In adipocytes, activation of the membrane-associated PDE3B is the major mechanism whereby insulin antagonizes catecholamine-induced lipolysis. PDE3B activation results in increased degradation of cAMP and, thereby, a lowering of the activity of cAMP-dependent protein kinase (PKA). The reduced activity of PKA leads to a net dephosphorylation and decreased activity of hormone-sensitive lipase and reduced hydrolysis of triglycerides. Activation of the rat adipocyte PDE3B by insulin is associated with phosphorylation of serine-302. The mechanism whereby insulin stimulation leads to phosphorylation/activation of PDE3B is only partly understood. In rat adipocytes, lipolytic hormones and other agents that increase cAMP, including isoproterenol, also induce rapid phosphorylation, presumably catalyzed by PKA, of serine-302 of PDE3B. The phosphorylation is associated with activation of the enzyme, most likely representing "feedback" regulation of cAMP, presumably allowing close coupling of the regulation of steady-state concentrations of both cAMP and PKA and, thereby, control of lipolysis. In the review we describe methods and strategies used in the authors' laboratories to study phosphorylation and activation of PDE3B in adipocytes and in vitro.
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| 7. |
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| 8. |
- Heimann, Emilia, et al.
(författare)
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Expression and regulation of cyclic nucleotide phosphodiesterases in human and rat pancreatic islets.
- 2010
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:12
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Tidskriftsartikel (refereegranskat)abstract
- As shown by transgenic mouse models and by using phosphodiesterase 3 (PDE3) inhibitors, PDE3B has an important role in the regulation of insulin secretion in pancreatic β-cells. However, very little is known about the regulation of the enzyme. Here, we show that PDE3B is activated in response to high glucose, insulin and cAMP elevation in rat pancreatic islets and INS-1 (832/13) cells. Activation by glucose was not affected by the presence of diazoxide. PDE3B activation was coupled to an increase as well as a decrease in total phosphorylation of the enzyme. In addition to PDE3B, several other PDEs were detected in human pancreatic islets: PDE1, PDE3, PDE4C, PDE7A, PDE8A and PDE10A. We conclude that PDE3B is activated in response to agents relevant for β-cell function and that activation is linked to increased as well as decreased phosphorylation of the enzyme. Moreover, we conclude that several PDEs are present in human pancreatic islets.
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| 9. |
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| 10. |
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| 11. |
- Härndahl, Linda, et al.
(författare)
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Beta-cell-targeted overexpression of phosphodiesterase 3B in mice causes impaired insulin secretion, glucose intolerance, and deranged islet morphology.
- 2004
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Ingår i: The Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 279:15, s. 15214-22
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Tidskriftsartikel (refereegranskat)abstract
- The second messenger cAMP mediates potentiation of glucose-stimulated insulin release. Use of inhibitors of cAMP-hydrolyzing phosphodiesterase (PDE) 3 and overexpression of PDE3B in vitro have demonstrated a regulatory role for this enzyme in insulin secretion. In this work, the physiological significance of PDE3B-mediated degradation of cAMP for the regulation of insulin secretion in vivo and glucose homeostasis was investigated in transgenic mice overexpressing PDE3B in pancreatic beta-cells. A 2-fold overexpression of PDE3B protein and activity blunted the insulin response to intravenous glucose, resulting in reduced glucose disposal. The effects were "dose"-dependent because mice overexpressing PDE3B 7-fold failed to increase insulin in response to glucose and hence exhibited pronounced glucose intolerance. Also, the insulin secretory response to intravenous glucagon-like peptide 1 was reduced in vivo. Similarly, islets stimulated in vitro exhibited reduced insulin secretory capacity in response to glucose and glucagon-like peptide 1. Perifusion experiments revealed that the reduction specifically affected the first phase of glucose-stimulated insulin secretion. Furthermore, morphological examinations demonstrated deranged islet cytoarchitecture. In conclusion, these results are consistent with an essential role for PDE3B in cAMP-mediated regulation of insulin release and glucose homeostasis.
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| 12. |
- Härndahl, Linda, et al.
(författare)
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Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic beta -cell exocytosis and release of insulin.
- 2002
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:40, s. 37446-37455
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Tidskriftsartikel (refereegranskat)abstract
- Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic b-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on b-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6-8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13), and insulin secretion in response to stimulation with high glucose (11.1 mM) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nM) to augment glucose-stimulated insulin secretion was inhibited by approximately 30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single b-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 mM) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.
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| 13. |
- Jones, Helena, et al.
(författare)
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Early and rapid development of insulin resistance, islet dysfunction and glucose intolerance after high-fat feeding in mice overexpressing phosphodiesterase 3B.
- 2006
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Ingår i: Journal of Endocrinology. - : Bioscientifica. - 1479-6805 .- 0022-0795. ; 189:3, s. 629-641
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Tidskriftsartikel (refereegranskat)abstract
- Inadequate islet adaptation to insulin resistance leads to glucose intolerance and type 2 diabetes. Here we investigate whether β-cell cAMP is crucial for islet adaptation and prevention of glucose intolerance in mice. Mice with a β-cell-specific, 2-fold overexpression of the cAMP-degrading enzyme phosphodiesterase 3B (RIP-PDE3B/2 mice) were metabolically challenged with a high-fat diet. We found that RIP-PDE3B/2 mice early and rapidly develop glucose intolerance and insulin resistance, as compared with wild-type littermates, after 2 months of high-fat feeding. This was evident from advanced fasting hyperinsulinemia and early development of hyper-glycemia, in spite of hyperinsulinemia, as well as impaired capacity of insulin to suppress plasma glucose in an insulin tolerance test. In vitro analyses of insulin-stimulated lipogenesis in adipocytes and glucose uptake in skeletal muscle did not reveal reduced insulin sensitivity in these tissues. Significant steatosis was noted in livers from high-fat-fed wild-type and RIP-PDE3B/2 mice and liver triacyl-glycerol content was 3-fold higher than in wild-type mice fed a control diet. Histochemical analysis revealed severe islet perturbations, such as centrally located α-cells and reduced immunostaining for insulin and GLUT2 in islets from RIP-PDE3B/2 mice. Additionally, in vitro experiments revealed that the insulin secretory response to glucagon-like peptide-1 stimulation was markedly reduced in islets from high-fat-fed RIP-PDE3B/2 mice. We conclude that accurate regulation of β-cell cAMP is necessary for adequate islet adaptation to a perturbed metabolic environment and protective for the development of glucose intolerance and insulin resistance.
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| 14. |
- Langin, Dominique, et al.
(författare)
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Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium
- 1993
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Ingår i: Proceedings of the National Academy of Sciences. - 1091-6490. ; 90:11, s. 4897-4901
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Tidskriftsartikel (refereegranskat)abstract
- The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa). It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region. The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively. Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase. Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control. Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium. The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C. The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators). The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis.
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| 15. |
- Laurell, H, et al.
(författare)
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The hormone-sensitive lipase (LIPE) gene located on chromosome 19q13.1-->13.2 is not duplicated on 19p13.3
- 1995
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Ingår i: International Journal of Obesity. - 1476-5497. ; 19:8, s. 590-592
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Tidskriftsartikel (refereegranskat)abstract
- The existence of a DNA polymorphism at the hormone-sensitive lipase locus could be of great interest for genetic analysis of obesity and related disorders since hormone-sensitive lipase is the rate-limiting enzyme of adipose tissue lipolysis and therefore plays a key role in energy metabolism. The polymorphic dinucleotide repeat D19S120 was identified within a human genomic clone selected with a rat hormone-sensitive lipase cDNA. This marker was subsequently localized to the short arm of chromosome 19 (p13.3) whereas human hormone-sensitive lipase (LIPE) had been mapped to the long arm of chromosome 19 (q13.1-->13.2). A duplication of the hormone-sensitive lipase gene or the presence of a pseudogene could explain the discrepancy. Cosmids from the two regions were analyzed in Southern blot experiments. A human adipose tissue hormone-sensitive lipase full-length cDNA probe hybridized only to cosmids from the 19q13.1-->13.2 region whereas the D19S120 amplicon probe hybridized only to cosmids from the p13.3 region. These data show that the occurrence of gene duplication or the presence of a pseudogene on the short arm of chromosome 19 is very unlikely and that D19S120 is unrelated to the hormone-sensitive lipase gene.
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| 16. |
- Mairal, A, et al.
(författare)
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Characterization of a novel testicular form of human hormone-sensitive lipase
- 2002
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 291:2, s. 286-290
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5' end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells.
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| 17. |
- Mei, Jie, et al.
(författare)
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C(2)-ceramide influences the expression and insulin-mediated regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 adipocytes.
- 2002
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Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 51:3, s. 631-637
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Tidskriftsartikel (refereegranskat)abstract
- Cyclic nucleotide phosphodiesterase (PDE) 3B plays an important role in the antilipolytic action of insulin and, thereby, the release of fatty acids from adipocytes. Increased concentrations of circulating fatty acids as a result of elevated or unrestrained lipolysis cause insulin resistance. The lipolytic action of tumor necrosis factor (TNF)-alpha is thought to be one of the mechanisms by which TNF-alpha induces insulin resistance. Ceramide is the suggested second messenger of TNF-alpha action, and in this study, we used 3T3-L1 adipocytes to investigate the effects of C(2)-ceramide (a short-chain ceramide analog) on the expression and regulation of PDE3B and lipolysis. Incubation of adipocytes with 100 micromol/l C(2)-ceramide (N-acetyl-sphingosine) resulted in a time-dependent decrease of PDE3B activity, accompanied by decreased PDE3B protein expression. C(2)-ceramide, in a time- and dose-dependent manner, stimulated lipolysis, an effect that was blocked by H-89, an inhibitor of protein kinase A. These ceramide effects were prevented by 20 micromol/l troglitazone, an antidiabetic drug. In addition to downregulation of PDE3B, the antilipolytic action of insulin was decreased by ceramide treatment. These results, together with data from other studies on PDE3B and lipolysis in diabetic humans and animals, suggest a novel pathway by which ceramide induces insulin resistance. Furthermore, PDE3B is demonstrated to be a target for troglitazone action in adipocytes.
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| 18. |
- Reinbothe, Thomas, 1981, et al.
(författare)
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Glutaredoxin-1 mediates NADPH-dependent stimulation of calcium-dependent insulin secretion
- 2009
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Ingår i: Mol Endocrinol. - : The Endocrine Society. - 1944-9917 .- 0888-8809. ; 23:6, s. 893-900
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Tidskriftsartikel (refereegranskat)abstract
- Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca(2+)-induced exocytosis in pancreatic beta-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated beta-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat beta-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca(2+)-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca(2+) currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca(2+) currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic beta-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates beta-cell exocytosis and, in turn, insulin secretion.
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| 19. |
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| 20. |
- Sonesson, Anders, et al.
(författare)
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Research and education form competing activity systems in externally funded doctoral education
- 2023
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Ingår i: Nordic Journal of Studies in Educational Policy. - 2002-0317. ; 9:2, s. 173-190
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Tidskriftsartikel (refereegranskat)abstract
- Several authors have described how the formalization of recent decades has steered doctoral education towards structured curricula, more managerial control and new models for supervision. Largely absent from these accounts, however, is if and how doctoral education has been affected by the concurrent changes in research governance, in particular by the ‘projectification’ of research. For this study, we were interested in the convergence of educational formalization with research projectification around doctoral education in the context of highly competitive, externally funded research in medicine and health sciences in Sweden. Using Cultural-historical activity theory and constructing activity systems for education and research, respectively, we were able to identify several contradictions and tensions, both within and between systems, that were consequences of adaptations to the abovementioned formalization and research policy changes. The contradictions were manifested in the tying of doctoral students, and their education, to their supervisors’ research projects, grants and future prospects, and in students being deprived of opportunities for learning and developing independence. Supervisors were torn between supervision and project management while doctoral students had to balance being students and project members. Our analysis provides a system level explanation to previously reported pedagogical and ethical challenges in STEM doctoral education.
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| 21. |
- Stenson, Lena
(författare)
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Hormone-Sensitive Lipase and Protein Kinase B; Molecular characterization in testis, adipose tissue and pancreatic beta cells.
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hormone-sensitive lipase (HSL) and protein kinase B (PKB) are enzymes that in different ways are associated with lipid metabolism. While HSL is the well-known lipolytic enzyme responsible for hydrolysis of stored triacylglycerols in adipose tissue, PKB is a recently identified serine/threonine protein kinase, ubiquitously expressed and involved in signal transduction pathways induced by insulin and growth factors. Here, with focus on lipid-containing tissues from which information on their presence was previously scarce or lacking, expression of HSL in testis and expression/regulation of PKB in adipocytes and pancreatic beta cells are reported. A testicular isoform of HSL (HSLtes) is formed through addition of a unique N-terminal domain, comprising 300 amino acids, to the 768/775 amino acids, respectively, of rat and human HSLadi, i.e., the adipocyte form. The testis-specific part is more hydrophilic than HSLadi. In man, this addition occurs through alternative splicing of a testis-specific exon located 16 kb upstream of the first exon encoding HSLadi. HSLtes expression occurs primarily in spermatozoa and maturing sperm and not in the endocrine Leydig cells. HSLtes appears to exert a lipase/cholesterol esterase activity similar to that of HSLadi but its exact role is yet unknown. PKB isoforms (alfa, beta and gamma) are expressed in rat adipocytes and beta cells of pancreatic islets. Insulin stimulation of rat adipocytes leads to rapid and reversible phosphorylation and activation of PKB. Activation of PKB by the insulin-mimetic compound peroxovanadate is associated with translocation of PKB from the cytosol to the membrane fraction of rat adipocytes. In clonal beta cells, PKB is phosphorylated and activated upon stimulation with IGF-I. In both cells, the activation of PKB is abolished through inhibition of phosphatidylinositol-3 kinase (PI3K), suggesting a position for PKB down-stream of PI3K in signalling by insulin/IGF-I in these cells.
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| 22. |
- Stenson, Lena, et al.
(författare)
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Localization of hormone-sensitive lipase to rat Sertoli cells and its expression in developing and degenerating testes
- 1994
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 355:2, s. 125-130
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Tidskriftsartikel (refereegranskat)abstract
- Using in situ hybridization, hormone-sensitive lipase was found to be expressed in a stage-dependent manner in Sertoli cells of rat testis. No expression was found in Leydig cells but expression in spermatids could not be excluded. These results suggest a role for hormone-sensitive lipase in the metabolism of lipid droplets in Sertoli cells, in contrast to its previously proposed function in steroid biosynthesis. The expression of testicular hormone-sensitive lipase mRNA and protein, both larger in size compared to other tissues, coincided with the onset of spermatogenesis and was dependent on scrotal localization of the testis, suggesting a temperature-dependent, pretranslational regulation of expression.
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| 23. |
- Stenson, Lena, et al.
(författare)
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Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase
- 1996
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Ingår i: Genomics. - : Elsevier BV. - 1089-8646 .- 0888-7543. ; 35:3, s. 441-447
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Tidskriftsartikel (refereegranskat)abstract
- By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSLtes. Due to an addition of amino acids at the NH2-termini, rat and human HSLtes consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSLadi). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSLadi. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSLadi sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M(r) approximately 120,000) that exhibited catalytic activity similar to that of HSLadi. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells.
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| 24. |
- Stenson, Lena, et al.
(författare)
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Protein kinase B is expressed in pancreatic beta cells and activated upon stimulation with insulin-like growth factor I
- 1998
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 250:1, s. 181-186
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Tidskriftsartikel (refereegranskat)abstract
- Protein kinase B (PKB) is involved in signaling to a multitude of important cellular events and is activated by insulin and growth factors, including insulin-like growth factor I (IGF-I). We show here expression of PKB in pancreatic islets and in the beta cell lines HIT-T15, INS-1, and RINm5F. Expression of PKB mRNA and the presence of PKB isoforms (alpha, beta, and gamma) were assessed by Northern blot analysis and RT-PCR, respectively. Antibodies recognizing different parts of PKB isoforms were employed to demonstrate PKB protein expression by immunoblot analysis. By use of immunohistochemistry in rat and mouse pancreatic tissue sections, PKB was localized to predominantly beta cells. Regulation of PKB was examined in INS-1 and RINm5F cells; upon stimulation with IGF-I (5-10 min), PKB was phosphorylated and activated (approximately 3-fold) by a wortmannin-sensitive mechanism, indicating involvement of phosphatidylinositol-3 kinase. The possible participation of PKB in signal transduction pathways modulating cAMP-dependent insulin secretion and in proliferation of beta cells is discussed.
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| 25. |
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| 26. |
- Sågetorp, Jenny, 1980-
(författare)
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Cyclic AMP Oscillations in Insulin-Secreting Cells
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cyclic AMP is an intracellular messenger that regulates numerous processes in various types of cells. In pancreatic β-cells, cAMP potentiates the secretion of insulin by promoting Ca2+ signals and by amplifying Ca2+-triggered exocytosis. Whereas Ca2+ signals have been extensively characterized, little is known about the kinetics of cAMP signals. To enable measurements of the cAMP concentration beneath the plasma membrane ([cAMP]pm) of individual cells, a translocation biosensor was created based on fluorescent-protein-tagged subunits of protein kinase A (PKA). Evanescent wave microscopy imaging of biosensor-expressing clonal β-cells revealed that the insulinotropic hormones glucagon and GLP-1 triggered pronounced oscillations in [cAMP]pm. Simultaneous measurements of the intracellular Ca2+ concentration showed that cAMP and Ca2+ oscillations were synchronized and interdependent. [cAMP]pm oscillations were also triggered in clonal and primary mouse β-cells by an elevation of the glucose concentration from 3 to 11 mM. These oscillations were preceded and enhanced by elevations of Ca2+. However, conditions raising cytoplasmic ATP could trigger cAMP elevations also without accompanying Ca2+ changes, indicating that adenylyl cyclase activity may be directly controlled by the substrate concentration. Experiments with 3-isobutylmethylxanthine (IBMX) and various family-selective phosphodiesterase (PDE) inhibitors indicated that [cAMP]pm oscillations are generated by periodic formation of the messenger by adenylyl cyclases. PDE1 and PDE3 as well as IBMX-insensitive mechanisms shape [cAMP]pm, but no single PDE isoform was required for glucose generation of [cAMP]pm oscillations. Recordings of single-cell insulin secretion kinetics with a fluorescent biosensor that reports formation of the phospholipid PIP3 in the plasma membrane in response to autocrine insulin receptor activation showed that [cAMP]pm oscillations were paralleled by pulsatile insulin release. Whereas adenylyl cyclase inhibition suppressed both [cAMP]pm oscillations and pulsatile insulin release, elevation of [cAMP]pm enhanced secretion. Investigation of the effects of different temporal patterns of [cAMP]pm showed that brief [cAMP]pm elevation is sufficient to trigger cytoplasmic responses, whereas sustained elevation is required to induce translocation of the PKA catalytic subunit into the nucleus. In conclusion, these studies demonstrate for the first time in mammalian cells that [cAMP]pm oscillates in response to physiological stimuli. The glucose-induced [cAMP]pm oscillations are generated by periodic cAMP production mediated by interplay between ATP and Ca2+ in the sub-membrane space, and may contribute to both triggering and amplifying pathways of insulin secretion. Apart from regulating the precise kinetics of insulin exocytosis, temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.
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| 27. |
- Wijkander, Jonny, et al.
(författare)
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Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate
- 1997
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 272:34, s. 21520-21526
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Tidskriftsartikel (refereegranskat)abstract
- Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.
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| 28. |
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| 29. |
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