| 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. |
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| 3. |
- Alkhori, Liza, et al.
(författare)
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The corepressor Atrophin specifies odorant receptor expression in Drosophila
- 2014
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Ingår i: The FASEB Journal. - : Federation of American Societies for Experimental Biology. - 0892-6638 .- 1530-6860. ; 28:3, s. 1355-1364
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Tidskriftsartikel (refereegranskat)abstract
- In both insects and vertebrates, each olfactory sensory neuron (OSN) expresses one odorant receptor (OR) from a large genomic repertoire. How a receptor is specified is a tantalizing question addressing fundamental aspects of cell differentiation. Here, we demonstrate that the corepressor Atrophin (Atro) segregates OR gene expression between OSN classes in Drosophila. We show that the knockdown of Atro result in either loss or gain of a broad set of ORs. Each OR phenotypic group correlated with one of two opposing Notch fates, Notch responding, Nba (N(on)), and nonresponding, Nab (N(off)) OSNs. Our data show that Atro segregates ORs expressed in the Nba OSN classes and helps establish the Nab fate during OSN development. Consistent with a role in recruiting histone deacetylates, immunohistochemistry revealed that Atro regulates global histone 3 acetylation (H3ac) in OSNs and requires Hdac3 to segregate OR gene expression. We further found that Nba OSN classes exhibit variable but higher H3ac levels than the Nab OSNs. Together, these data suggest that Atro determines the level of H3ac, which ensures correct OR gene expression within the Nba OSNs. We propose a mechanism by which a single corepressor can specify a large number of neuron classes.-Alkhori, L., Öst, A., Alenius, M. The corepressor Atrophin specifies odorant receptor expression in Drosophila.
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| 4. |
- Ceder, Mikaela, 1991-
(författare)
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Characterization of Novel Solute Carriers in Humans, Mice and Flies : Solute Carriers in a Broad and Narrow Perspective
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The solute carrier family is the largest family of membrane-bound transporters in humans, with 430 members divided into 65 subfamilies. They transport various substrates across lipid barriers and are vital for absorption, distribution, metabolism and excretion in all cell types in the body. Despite being involved in vital functions, and their effect on both physiology and pathophysiology, many transporters are not characterized. The aim of this thesis was to study newly identified putative solute carriers of which little is known. In Paper I, the relationship of solute carriers in humans and fruit flies was studied. The study revealed that 54 of the 65 subfamilies in humans have one or more orthologues in fruit flies, and a total of 381 orthologues were identified in fruit flies. In Paper II, a comprehensive study of the putative solute carriers and their response to different sugar concentrations were performed. Several, but not all, putative solute carriers were altered in cell cultures maintained in media containing low or no glucose, and the expression normalized upon refeeding with glucose. Similar results were observed in fruit flies subjected to complete starvation or diets with varying sugar concentrations. Last, in Paper III and IV, characterization of one putative solute carrier, UNC93A, was performed. The studies revealed that UNC93A was a conserved protein with an abundant expression in the body of mice but with a restricted expression in fruit flies. The protein was found to possibly be expressed at, or close to, the plasma membrane of cells and to co-localize with Twik-Acid sensitive potassium channels. UNC93A was found to be important for the renal function in fruit flies and to affect survival and membrane potentials in cells. The findings of this thesis establish a high conservation of several putative solute carriers and that they have a highly dynamic regulation during fluctuating energy and glucose availability. Further, while several clear biological aspects of UNC93A was identified, the exact function of transporter proteins is cumbersome to find and more research about these transporters is needed to fully understand their mechanistic role and their association and/or involvement in health and sickness.
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| 5. |
- Danielsson, Anna, et al.
(författare)
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Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes
- 2005
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Ingår i: Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 280:41, s. 34389-3492
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Tidskriftsartikel (refereegranskat)abstract
- Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC50 for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t1/2 of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.
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| 6. |
- Danielsson, Anna, et al.
(författare)
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Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes
- 2005
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 272:1, s. 141-151
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Tidskriftsartikel (refereegranskat)abstract
- Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO2 at 37 °C. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1/2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulin-stimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgery-induced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.
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| 7. |
- Danielsson, Anna, et al.
(författare)
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Short-Term Overeating Induces Insulin Resistance in Fat Cells in Lean Human Subjects
- 2009
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Ingår i: Molecular Medicine. - : Springer Science and Business Media LLC. - 1076-1551 .- 1528-3658. ; 15:7-8, s. 228-234
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Tidskriftsartikel (refereegranskat)abstract
- Insulin resistance and type 2 diabetes (T2D) are closely linked to obesity. Numerous prospective studies have reported on weight gain, insulin resistance, and insulin signaling in experimental animals, but not in humans. We examined insulin signaling in adipocytes from lean volunteers, before and at the end of a 4-wk period of consuming a fast-food, high-calorie diet that led to weight gain. We also examined adipocytes from patients with T2D. During the high-calorie diet, subjects gained 10% body weight and 19% total body fat, but stayed lean (body mass index = 24.3 kg/m2) and developed moderate systemic insulin resistance. Similarly to the situation in T2D subjects, in subjects on the high-calorie diet, the amount of insulin receptors was reduced and phosphorylation of IRS1 at tyrosine and at serine-307 (human sequence, corresponding to murine serine-302) were impaired. The amount of insulin receptor substrate protein-1 (IRS1) and the phosphorylation of IRS1 at serine-312 (human sequence, corresponding to murine serine-307) were unaffected by the diet. Unlike the T2D subjects, in subjects on the high-calorie diet, likely owing to the ongoing weight-gain, phosphorylation of MAP-kinases ERK1/2 became hyperresponsive to insulin. To our knowledge this study is the first to investigate insulin signaling during overeating in humans, and it demonstrates that T2D effects on intracellular insulin signaling already occur after 4 wks of a high-calorie diet and that the effects in humans differ from those in laboratory animals.
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| 8. |
- Franck, Niclas, et al.
(författare)
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Insulin-induced GLUT4 translocation to the plasma membrane is blunted in large compared with small primary fat cells isolated from the same individual
- 2007
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 50:8, s. 1716-1722
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Tidskriftsartikel (refereegranskat)abstract
- Aims/hypothesis: Several studies have suggested that large fat cells are less responsive to insulin than small fat cells. However, in these studies, large fat cells from obese individuals were compared with smaller fat cells from leaner participants, in effect making it impossible to draw conclusions about whether there is a causal relationship between fat cell size and insulin sensitivity. We hypothesised that small fat cells might be more insulin-responsive than large adipocytes when obtained from the same individual. Materials and methods: We developed a method of sorting isolated primary human fat cells by using nylon filters of two different pore sizes. The cells were stained to visualise DNA, which allowed discrimination from artefacts such as lipid droplets. The sorted cells were left to recover overnight, since we had previously demonstrated that this is necessary for correct assessment of insulin response. Results: We found similar amounts of the insulin receptor (IR), IRS-1 and GLUT4 when we compared small and large adipocytes from the same volunteer by immunoblotting experiments using the same total cell volume from both cell populations. Activation of IR, IRS-1 and Akt1 (also known as protein kinase B) by insulin was similar in the two cell populations. However, immunofluorescence confocal microscopy of plasma membrane sheets did not reveal any increase in the amount of GLUT4 in the plasma membrane following insulin stimulation in the large fat cells, whereas we saw a twofold increase in the amount of GLUT4 in the small fat cells. Conclusions/interpretation: Our results support a causal relationship between the accumulation of large fat cells in obese individuals and reduced insulin responsiveness.
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| 9. |
- Karlsson, Margareta, 1942-, et al.
(författare)
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Colocalization of insulin receptor and insulin receptor substrate-1 to caveolae in primary human adipocytes
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:12, s. 2471-2479
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Tidskriftsartikel (refereegranskat)abstract
- Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated signalling. Here we report that in primary human subcutaneous adipocytes the insulin receptor was localized to caveolae by electron microscopy/immunogold detection and by isolating caveolae from plasma membranes. Part of insulin receptor substrate 1 (IRS1), the immediate downstream signal mediator, was colocalized with the insulin receptor in the plasma membrane and caveolae, as demonstrated by immunofluorescence microscopy, immunogold electron microscopy, and immunogold electron microscopy of transfected recombinant HA-IRS1. In contrast, rat epididymal adipocytes lacked IRS1 at the plasma membrane. Depletion of cholesterol from the cells using β-cyclodextrin blocked insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulin stimulation of protein kinase B and Map-kinases extracellular signal-related kinase (ERK)1/2 phosphorylation. Insulin-stimulated phosphorylation of the insulin receptor and IRS1 was not affected, indicating that caveolae integrity is required downstream of IRS1. In conclusion we show that insulin receptor and IRS1 are both caveolar proteins and that caveolae are required for both metabolic and mitogenic control in human adipocytes. Our results establish caveolae as foci of insulin action and stress the importance of examining human cells in addition to animal cells and cell lines.
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| 10. |
- Nätt, Daniel, et al.
(författare)
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Human sperm displays rapid responses to diet
- 2019
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Ingår i: PLoS biology. - San Francisco, CA United States : PUBLIC LIBRARY SCIENCE. - 1544-9173 .- 1545-7885. ; 17:12
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Tidskriftsartikel (refereegranskat)abstract
- The global rise in obesity and steady decline in sperm quality are two alarming trends that have emerged during recent decades. In parallel, evidence from model organisms shows that paternal diet can affect offspring metabolic health in a process involving sperm tRNA-derived small RNA (tsRNA). Here, we report that human sperm are acutely sensitive to nutrient flux, both in terms of sperm motility and changes in sperm tsRNA. Over the course of a 2-week diet intervention, in which we first introduced a healthy diet followed by a diet rich in sugar, sperm motility increased and stabilized at high levels. Small RNA-seq on repeatedly sampled sperm from the same individuals revealed that tsRNAs were up-regulated by eating a high-sugar diet for just 1 week. Unsupervised clustering identified two independent pathways for the biogenesis of these tsRNAs: one involving a novel class of fragments with specific cleavage in the T-loop of mature nuclear tRNAs and the other exclusively involving mitochondrial tsRNAs. Mitochondrial involvement was further supported by a similar up-regulation of mitochondrial rRNA-derived small RNA (rsRNA). Notably, the changes in sugar-sensitive tsRNA were positively associated with simultaneous changes in sperm motility and negatively associated with obesity in an independent clinical cohort. This rapid response to a dietary intervention on tsRNA in human sperm is attuned with the paternal intergenerational metabolic responses found in model organisms. More importantly, our findings suggest shared diet-sensitive mechanisms between sperm motility and the biogenesis of tsRNA, which provide novel insights about the interplay between nutrition and male reproductive health.
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