| 1. |
- Ahmad, Faiyaz, et al.
(författare)
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Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the beta(3)-adrenergic receptor agonist CL316243: effects of caveolin-1 knockdown on formation/maintenance of macromolecular signalling complexes
- 2009
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Ingår i: BIOCHEMICAL JOURNAL. - 0264-6021. ; 424:3, s. 399-410
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Tidskriftsartikel (refereegranskat)abstract
- In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta(3)-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KID of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained P-32-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta(3)-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KID. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.
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| 2. |
- Ahmad, Faiyaz, et al.
(författare)
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Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB
- 2007
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Ingår i: Biochemical Journal. - 0264-6021. ; 404, s. 257-268
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Tidskriftsartikel (refereegranskat)abstract
- Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phospho-inosifide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3B Delta 604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their coimmunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-Delta PKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.
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| 3. |
- Berger, Karin, et al.
(författare)
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Phosphodiesterase 3B is localized in caveolae and smooth ER in mouse hepatocytes and is important in the regulation of glucose and lipid metabolism.
- 2009
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:3
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Tidskriftsartikel (refereegranskat)abstract
- Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction processes mediated by cAMP and cGMP. One PDE family member, PDE3B, plays an important role in the regulation of a variety of metabolic processes such as lipolysis and insulin secretion. In this study, the cellular localization and the role of PDE3B in the regulation of triglyceride, cholesterol and glucose metabolism in hepatocytes were investigated. PDE3B was identified in caveolae, specific regions in the plasma membrane, and smooth endoplasmic reticulum. In caveolin-1 knock out mice, which lack caveolae, the amount of PDE3B protein and activity were reduced indicating a role of caveolin-1/caveolae in the stabilization of enzyme protein. Hepatocytes from PDE3B knock out mice displayed increased glucose, triglyceride and cholesterol levels, which was associated with increased expression of gluconeogenic and lipogenic genes/enzymes including, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor gamma, sterol regulatory element-binding protein 1c and hydroxyl-3-methylglutaryl coenzyme A reductase. In conclusion, hepatocyte PDE3B is localized in caveolae and smooth endoplasmic reticulum and plays important roles in the regulation of glucose, triglyceride and cholesterol metabolism. Dysregulation of PDE3B could have a role in the development of fatty liver, a condition highly relevant in the context of type 2 diabetes.
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| 4. |
- Bramsved, Rebecka, 1982, et al.
(författare)
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Birth Weight, Childhood and Young Adult Overweight, and the Risk of Coronary Heart Disease in Men.
- 2024
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Ingår i: Arteriosclerosis, thrombosis, and vascular biology. - 1524-4636. ; 44:1, s. 314-321
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Tidskriftsartikel (refereegranskat)abstract
- Low birth weight is a known risk factor for adult coronary heart disease (CHD), but the additional effect of weight development during childhood and early adult life has not been studied.We included 35659 men born 1945 to 1961 from the population-based BMI Epidemiology Study Gothenburg, with data available on birthweight, BMI in childhood (8 years), and BMI in young adulthood (20 years). Information on CHD diagnoses was retrieved from national registers. We used Cox proportional hazards regression to estimate hazard ratios and 95% CIs for the risk of early and late CHD (before and after 58.4 years of age, respectively).During follow-up, a total of 3380 cases of CHD (fatal and nonfatal) were registered. Birth weight was inversely associated with the risk of both early (hazard ratio, 0.88 per SD increase [95% CI, 0.84-0.92]) and late (hazard ratio, 0.94 per SD increase [95% CI, 0.90-0.98]) CHD, independently of BMI at 8 years and BMI change during puberty. In a model including birth weight (below or above the median) together with overweight at 8 and 20 years, only birth weight and young adult overweight, but not overweight in childhood, were significantly associated with the risk of CHD. A birth weight below the median, followed by overweight at 20 years of age was associated with a more than doubled risk of early CHD (hazard ratio, 2.29 [95% CI, 1.86-2.81]), compared with the reference (birth weight above the median and normal weight at 20 years of age). This excess risk was even more pronounced for a birthweight below 2.5 kg.We demonstrate that low birth weight and young adult overweight are important developmental markers of risk for adult CHD. These findings motivate a life course perspective for prevention and risk assessment of adult CHD.
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| 5. |
- Bramsved, Rebecka, 1982, et al.
(författare)
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Impact of BMI and smoking in adolescence and at the start of pregnancy on birth weight
- 2023
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Ingår i: BMC Pregnancy and Childbirth. - : Springer Science and Business Media LLC. - 1471-2393. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundBirth weight is an indicator of intra-uterine conditions but also a determinant for future health. The importance of preconception health for a healthy birth weight has been emphasized, but evidence is lacking on how modifiable factors in adolescence, such as body mass index (BMI) and smoking, affect future pregnancy outcome. We evaluated associations between BMI and smoking in adolescence and at the start of pregnancy and birth weight of the first-born child.MethodsThis longitudinal study included 1256 mothers, born 1962-1992, and their first-born children, born between 1982-2016. Self-reported questionnaire information on weight, height and smoking at age 19 was cross-linked with national register data obtained at the start of pregnancy and with the birth weights of the children. Univariable and multivariable linear regressions were performed to determine the impact of maternal factors at 19 years of age and at the start of the pregnancy respectively, and the importance of BMI status at these points of time for the birth weight of the first child.ResultsBMI and smoking at the start of the pregnancy displayed strong associations with birth weight in a multivariable analysis, BMI with a positive association of 14.9 g per BMI unit (95% CI 6.0; 23.8 p = 0.001) and smoking with a negative association of 180.5 g (95% CI -275.7; -85.4) p = 0.0002). Smoking and BMI at 19 years of age did not show this association. Maternal birth weight showed significant associations in models at both time-points. Becoming overweight between age 19 and the start of the pregnancy was associated with a significantly higher birth weight (144.6 (95% CI 70.7;218.5) p = 0.0002) compared to mothers with normal weight at both time points.ConclusionsOur findings indicate that the time period between adolescence and first pregnancy could be a window of opportunity for targeted health promotion to prevent intergenerational transmission of obesity.
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| 6. |
- Lindh, Rebecka
(författare)
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Hormonal regulation of phosphodiesterase 3B in adipocytes
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Insulin induced activation of the cAMP degrading enzyme phosphodiesterase (PDE) 3B in adipocytes is important for insulin mediated inhibition of lipolysis. The aim of this thesis was to contribute with knowledge on the acute activation of PDE3B in response to insulin and catecholamines in adipocytes. The activation of PDE3B was investigated from three different perspectives namely; phosphorylation of PDE3B, localization of PDE3B and PDE3B interactions with other proteins. As models to study activation of PDE3B we used primary rat and mouse adipocytes, 3T3-L1 adipocytes and caveolin-1 knock out mouse adipocytes. The study on PDE3B phosphorylation shows that PDE3B is multisite phosphorylated in response to both insulin and catecholamines. We were able to identify six phosphorylation sites on PDE3B namely; 273, S296, S421, S424/5, S474 and S536. From the studies on PDE3B localization we conclude that PDE3B is localized to caveolae and endoplasmic reticulum (ER) in adipocytes and that caveolae localized PDE3B is specifically activated by catecholamines while PDE3B in ER is specifically activated by insulin. Finally, the studies on PDE3B interactions demonstrate that insulin and catecholamines stimulate the recruitment of PDE3B into large macromolecular complexes in adipocytes. The recruitment is dependent on caveolin-1 especially in response to insulin and the constituents of the PDE3B macromolecular complexes are dependent on the stimuli used. After insulin stimulation insulin signaling molecules such as protein kinase B (PKB) are associated with PDE3B in the large complex while after catecholamine stimulation adrenergic signaling molecules such as protein kinase A (PKA) are associated with PDE3B.
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| 7. |
- Lindh, Rebecka, et al.
(författare)
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Multisite phosphorylation of adipocyte and hepatocyte phosphodiesterase 3B.
- 2007
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Ingår i: Biochimica et Biophysica Acta: Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1773:4, s. 584-592
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Tidskriftsartikel (refereegranskat)abstract
- Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.
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