| 31. |
- Aguiló, Francesca, et al.
(author)
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Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARgamma.
- 2010
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 427:2
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Journal article (peer-reviewed)abstract
- In the cytosol of lipogenic tissue, ketone bodies are activated by AACS (acetoacetyl-CoA synthetase) and incorporated into cholesterol and fatty acids. AACS gene expression is particularly abundant in white adipose tissue, as it is induced during adipocyte differentiation. In order to elucidate the mechanism controlling the gene expression of human AACS and to clarify its physiological role, we isolated the human promoter, characterized the elements required to initiate transcription and analysed the expression of the gene in response to PPARgamma (peroxisome-proliferator-activated receptor gamma), an inducer of adipogenesis. We show that the human AACS promoter is a PPARgamma target gene and that this nuclear receptor is recruited to the AACS promoter by direct interaction with Sp1 (stimulating protein-1).
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| 32. |
- Ahmad, Faiyaz, et al.
(author)
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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
-
In: BIOCHEMICAL JOURNAL. - 0264-6021. ; 424:3, s. 399-410
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Journal article (peer-reviewed)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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| 33. |
- Ahmad, Faiyaz, et al.
(author)
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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
-
In: Biochemical Journal. - 0264-6021. ; 404, s. 257-268
-
Journal article (peer-reviewed)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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| 34. |
- Ahwazi, Danial, et al.
(author)
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Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965
- 2021
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In: Biochemical Journal. - 0264-6021. ; 478:15, s. 2977-2997
-
Journal article (peer-reviewed)abstract
- SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/ Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 μM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.
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| 35. |
- Albertsson, Per-Åke, et al.
(author)
-
Chloroplast membranes retard fat digestion and induce satiety: effect of biological membranes on pancreatic lipase/co-lipase
- 2007
-
In: Biochemical Journal. - 0264-6021. ; 401, s. 727-733
-
Journal article (peer-reviewed)abstract
- Human obesity is a global epidemic, which causes a rapidly increased frequency of diabetes and cardiovascular disease. One reason for obesity is the ready availability of refined food products with high caloric density, an evolutionarily new event, which makes over-consumption of food inevitable. Fat is a food product with high caloric density. The mechanism for regulation of fat intake has therefore been studied to a great extent. Such studies have shown that, as long as fat stays in the intestine, satiety is promoted. This occurs through the fat-released peptide hormones, the best known being CCK (cholecystokinin), which is released by fatty acids. Hence, retarded fat digestion with prolonged time for delivery of fatty acids promotes satiety. Pancreatic lipase, together with its protein cofactor, co-lipase, is the main enzymatic system responsible for intestinal fat digestion. We found that biological membranes, isolated from plants, animals or bacteria, inhibit the lipase/co-lipase-catalysed hydrolysis of triacylglycerols even in the presence of bile salt. We propose that the inhibition is due to binding of lipase/co-lipase to the membranes and adsorption of the membranes to the aqueous/triacylglycerol interface, thereby hindering lipase/co-lipase from acting on its lipid substrate. We also found that chloroplast membranes (thylakoids), when added to refined food, suppressed food intake in rats, lowered blood lipids and raised the satiety hormones, CCK and enterostatin. Consequently, the mechanism for satiety seems to be retardation of fat digestion allowing the fat products to stay longer in the intestine.
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| 36. |
- Anders, Emma, et al.
(author)
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Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1 : a novel mechanism for regulation of MCP-1 production and function
- 2018
-
In: Biochemical Journal. - 0264-6021. ; 475:4, s. 775-786
-
Journal article (peer-reviewed)abstract
- The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/ p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.
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| 37. |
- Andersson, C, et al.
(author)
-
Activation and inhibition of microsomal glutathione transferase from mouse liver.
- 1988
-
In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 249:3, s. 819-23
-
Journal article (peer-reviewed)abstract
- Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.
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| 38. |
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| 39. |
- Araya, Zufan, et al.
(author)
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Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1.
- 2003
-
In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 372:2, s. 529-534
-
Journal article (peer-reviewed)abstract
- The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Bj¨orkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5_-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.
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| 40. |
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