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| 2. |
- 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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| 3. |
- Ramesh, Rashmi, 1984-, et al.
(författare)
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Dietary Sugar Shifts Mitochondrial Metabolism and Small RNA Biogenesis in Sperm
- 2023
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Ingår i: Antioxidants and Redox Signaling. - : MARY ANN LIEBERT, INC. - 1523-0864 .- 1557-7716. ; 38:16-18, s. 1167-1183
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Tidskriftsartikel (refereegranskat)abstract
- Aims: Increasing concentrations of dietary sugar results in a linear accumulation of triglycerides in male Drosophila, while inducing a U-shaped obesity response in their offspring. Here, using a combination of proteomics and small RNA (sRNA) sequencing, we aimed at understanding the molecular underpinning in sperm for such plasticity.Results: Proteomic analysis of seminal vesicles revealed that increasing concentrations of dietary sugar resulted in a bell-shaped induction of proteins involved in metabolic/redox regulation. Using stains and in vivo redox reporter flies, this pattern could be explained by changes in sperm production of reactive oxygen species (ROS), more exactly mitochondria-derived H2O2. By quenching ROS with the antioxidant N-acetyl cysteine and performing sRNA-seq on sperm, we found that sperm miRNA is increased in response to ROS. Moreover, we found sperm mitosRNA to be increased in high-sugar diet conditions (independent of ROS). Reanalyzing our previously published data revealed a similar global upregulation of human sperm mitosRNA in response to a high-sugar diet, suggesting evolutionary conserved mechanisms.Innovation: This work highlights a fast response to dietary sugar in mitochondria-produced H2O2 in Drosophila sperm and identifies redox-sensitive miRNA downstream of this event.Conclusions: Our data support a model where changes in the sperm mitochondria in response to dietary sugar are the primary event, and changes in redox homoeostasis are secondary to mitochondrial ROS production. These data provide multiple candidates for paternal intergenerational metabolic responses as well as potential biomarkers for human male fertility.
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| 4. |
- Toia, Beatrice, 1993-
(författare)
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Molecular characterization of the interplay between DNA-PK and TRF2 in telomere protection
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genetic material of mammalian cells is organized as linear chromosomes inside their nucleus. In order to preserve genomic stability, mammalian cells have developed several mechanisms to detect and repair DNA lesions that can occasionally occur in the genome. Nevertheless, in certain circumstances, these pathways can promote aberrant repair that can result in genomic instability and tumorigenesis. One such case are the ends of linear chromosomes, which are particularly susceptible since they can be mistakenly recognized as DNA lesions by the DNA repair machinery, which seeks to repair the damage by joining the two DNA ends together and thus causing rearrangements and genome instability. Therefore, the linear ends of chromosomes need to be protected from being sensed as sites of damage, and this is promoted by the presence of specialized structures known as telomeres, with the aim of preventing determinantal consequences of having exposed DNA ends. Telomeres are formed by a repetitive DNA sequence that is bound by a protein complex named shelterin, which forms a protective structure at the end of chromosomes. Among the shelterin subunits, TRF2 plays a crucial role. Other factors that have been found essential for the protection of telomeres are Apollo and DNA-PK proteins which, strikingly, are also involved in DNA repair mechanisms.Understanding the underlying mechanism of telomere protection and the interplay between factors involved is of relevance since defects in telomere protection activate aberrant repair, which is associated with genome instability, a prime mechanism shaping cancer.In Paper I, we focused on how Apollo and the catalytic subunit of DNA-PK (DNA-PKcs) cooperate in preserving telomere homeostasis, and we shed light on the mechanistic function of DNA-PKcs in granting access to Apollo to the telomeric ends. We found that this function requires the kinase activity of DNA-PKcs to promote autophosphorylation, and the binding of Apollo to DNA-PKcs for optimal positioning at the DNA ends. This study shows an analogous mechanism of function of DNA-PK at sites of DNA lesions and at telomeres.In Paper II, we found that the localization of DNA-PK at telomeres post-replication is essential to block the nucleolytic erosion of the telomeric ends by any other nuclease than Apollo. This protective function is also independently fulfilled by the iDDR domain of TRF2, which specifically inhibits the endonucleolytic activity of MRN protein complex. This study reveals the high pressure for cells to keep tight control of resection at telomeres.In Paper III, we expanded our studies by investigating the role of Apollo and DNA-PKcs in ALT cancerous cells, which utilize a recombination-mediated mechanism to elongate telomeres. Here, we show that DNA-PKcs and Apollo have a conserved role in promoting G-overhang formation, but in the absence of Apollo and/or DNA-PKcs kinase activity, telomeres are insensitive to fusion. Moreover, we found that Apollo promotes telomere recombination events at ALT telomeres. This study opens the possibility for different mechanisms of telomere maintenance in ALT cells.
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| 5. |
- Örkenby, Lovisa, 1992-
(författare)
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Small non-coding RNA in early fly development : plasticity, interactions and improved bioinformatic tools
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- At fertilization, the male and female pronuclei undergo a transformation from germline to pluripotency as they fuse, marking the beginning of Drosophila embryogenesis. As the parental contributions decrease, the zygote takes control of its genome in a process called the maternal-to-zygotic transition (MZT). Several small non-coding RNAs (sncRNAs), a very large and diverse group of RNAs, have regulatory roles during this transition. This includes for example microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs). Regulation by miRNAs mainly occurs through mediating maternal mRNA degradation, while piRNAs operate by repressing transposable elements (TEs) and regulating the nanos-induced embryonic body axis determination.In this thesis, the complex and dynamic field of early Drosophila embryogenesis and sncRNAs are put in relation to the included papers. In Paper I, I explored the most stress-sensitive embryonic period and found that stress before the midblastula transition retains maternal miRNAs. These miRNAs impact zygotic gene activation by modulating the boundary factor Elba1, leading to compromised transcription control. Paper III examines the piRNA population during MZT. I find differences of unique piRNA sequences in embryos of different ages but not in target preferences, potentially highlighting the importance of constant repression of certain TEs. Paper II addresses specific difficulties with sncRNA seq data analysis and presents a bioinformatic framework to improve these analyses using a sequence-based strategy.This thesis highlights the intricate interplay of sncRNAs in the critical period of early Drosophila embryogenesis and offers insights into their regulatory roles.
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| 6. |
- Örtegren Kugelberg, Unn, 1975-, et al.
(författare)
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Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes : segregation of specific proteins and functions
- 2006
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 273:14, s. 3381-3392
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Tidskriftsartikel (refereegranskat)abstract
- Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.
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| 7. |
- Örtegren, Unn, 1975-, et al.
(författare)
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A new role for caveolae as metabolic platforms
- 2007
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Ingår i: Trends in endocrinology and metabolism. - : Elsevier BV. - 1043-2760 .- 1879-3061. ; 18:9, s. 344-349
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane of cells functions as a barrier to the environment. Caveolae are minute invaginations of the membrane that selectively carry out the exchange of information and materials with the environment, by functioning as organizers of signal transduction and through endocytosis. Recent findings of uptake of different metabolites and of lipid metabolism occurring in caveolae, point to a new general function of caveolae. As gateways for the uptake of nutrients across the plasma membrane, and as platforms for the metabolic conversion of nutrients, especially in adipocytes, caveolae are now emerging as active centers for many aspects of intermediary metabolism, with implications for our understanding of obesity, diabetes and other metabolic disorders.
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| 8. |
- Öst, Anita, 1965-, et al.
(författare)
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Attenuated mTOR signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes
- 2010
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Ingår i: Molecular Medicine. - : Feinstein Institute for Medical Research. - 1076-1551 .- 1528-3658. ; 16:07-Aug, s. 235-246
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Tidskriftsartikel (refereegranskat)abstract
- The protein kinase mammalian target of rapamycin (mTOR) mediates insulin control ofprotein synthesis, autophagy, mitochondrial function, and, through feedback signaling tophosphorylation of IRS1 at serine residues, mTOR directly controls insulin signaling. Weshow that in adipocytes from patients with type 2 diabetes (T2D) insulin activation of mTORis attenuated and that the resultant phenotype is compatible with, and can be mimicked by,loss of mTOR activation. In T2D adipocytes mitochondrial function is impaired andautophagy strongly upregulated, with concomitant increased autophagic destruction ofmitochondria and lipofuscin particles, and a dependence on autophagy for ATP production.Conversely, mitochondrial dysfunction attenuates insulin activation of mTOR, enhancesautophagy and attenuates feedback to IRS1. Our findings put mTOR in the driver´s seat of aninsulin resistance that in adipocytes can be fuelled by mitochondrial dysfunction,inflammation, ER-stress, or hypoxia.
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| 9. |
- Öst, Anita, 1965-
(författare)
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Lipid Metabolism and Insulin Signalling in Adipocytes : enhanced autophagy in type 2 diabetes
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Energy storage in the adipose tissue, to an extent leading to obesity, is associated with local as well assystemic insulin resistance. When insulin-producing beta-cells in the pancreas gradually fail tocompensate, plasma levels of glucose rise and overt type 2 diabetes is diagnosed. Adipocytes are largecells, mostly consisting of one big central lipid droplet, with the surrounding plasma membrane full ofsmall invaginations called caveolae. As caveolae contain the insulin receptor and several other insulinsignallingproteins, we have investigated several aspects of caveolae. We have also mapped mechanismsand defects in the insulin-signalling network in adipocytes from type 2 diabetic patients.In paper I, we show that a subtype of caveolae has the capability to synthesize triglycerides from fattyacids and glycerol-3-phosphate. The triglyceride-synthesizing caveolae subtype also contains perilipin,suggesting the existence of a mechanism to protect newly made triglycerides from hydrolysis.In paper II, we demonstrate that adipocytes from patients with type 2 diabetes have an attenuated insulinstimulatedphosphorylation of IRS-1 at Ser-307 (human sequence), which correlates with reduced insulinstimulatedphosphorylation of IRS-1 at tyrosine residues. Insulin-stimulated phosphorylation of IRS-1 atSer-307 is dependent on the nutrient sensor TORC1. This finding indicates that adipocytes from type 2diabetic patients have reduced TORC1 activity.In paper III, we focus on the mechanisms for RBP4-induced insulin resistance. We also continue ourmapping of insulin-resistance in adipocytes from type 2 diabetes. These cells exhibit, in addition toimpaired insulin-stimulated glucose uptake and the defects presented in paper I, impaired insulinstimulatedphosphorylation of ERK. We do, however, not see any defects in PKB signalling. Neither dowe se any enhanced insulin-stimulated phosphorylation of IRS-1 at Ser-312 (human sequence), a site thatin mice is hyper-stimulated in response to high-fat feeding. Incubation with RBP4 recapitulates all defectswe so far have seen in type 2 diabetes except reduced insulin-stimulated glucose uptake. These results aremirrored by blockade of endogenously produced RBP4 in the incubations with adipocytes from type 2diabetic patients. In other words, RBP4-blocking antibodies restore all insulin-signalling defects we havefound in adipocytes from type 2 diabetic patients, except insulin-stimulated glucose uptake.In paper IV we show by several approaches that TORC1 activation is down-regulated in adipocytes fromtype 2 diabetic patients. The main finding is that there is enhanced autophagy in those adipocytes.Interestingly, autophagy may be a mechanism to enhance the breakdown of stored triglycerides in theadipocyte.In conclusion, our data suggest that caveolae, in addition to being micro-domains for insulin-signallingare metabolic platforms. We describe defects in insulin-signalling in adipocytes from type 2 diabeticpatients where the main finding is enhanced autophagy in these obese patients. The perceived starvationin adipose tissue might via secretion of adipokines, such as RBP4, have implications for local as well assystemic insulin-resistance.
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| 10. |
- Öst, Anita, 1965-, et al.
(författare)
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Retinol-binding protein-4 attenuates insulin-induced phosphorylation of IRS1 and ERK1/2 in primary human adipocytes
- 2007
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Ingår i: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. - : Wiley. - 1530-6860. ; 21:13, s. 3696-3704
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Tidskriftsartikel (refereegranskat)abstract
- Reduced sensitivity to insulin in adipose, muscle, and liver tissues is a hallmark of type 2 diabetes. Animal models and patients with type 2 diabetes exhibit elevated levels of circulating retinol-binding protein (RBP4), and RBP4 can induce insulin resistance in mice. However, little is known about how RBP4 affects insulin signaling. We examined the mechanisms of action of RBP4 in primary human adipocytes. RBP4-treated adipocytes exhibited the same molecular defects in insulin signaling, via IRS1 to MAP kinase, as in adipocytes from patients with type 2 diabetes. Without affecting autophosphorylation of the insulin receptor, RBP4 blocked the insulin-stimulated phosphorylation of IRS1 at serine (307) [corresponding to serine (302) in the murine sequence] and concomitantly increased the EC50 (from 0.5 to 2 nM) for insulin stimulation of IRS1 phosphorylation at tyrosine. The phosphorylation of IRS1 at serine (312) [corresponding to serine (307) in the murine sequence] was not affected in cells from diabetic patients and was also not affected by RBP4. The EC50 for insulin stimulation of downstream phosphorylation of MAP kinase ERK1/2 was increased (from 0.2 to 0.8 nM) by RBP4. We show that ERK1/2 phosphorylation is similarly impaired in adipocytes from patients with type 2 diabetes. However, the sensitivity to insulin for downstream signaling to control of protein kinase B and glucose uptake was not affected by RBP4. When insulin-resistant adipocytes from patients with type 2 diabetes were incubated with antibodies against RBP4, insulin-induced phosphorylation of IRS1 at serine (307) was normalized and the EC50 for insulin stimulation of ERK1/2 phosphorylation was reduced. Endogenous levels of RBP4 were markedly reduced in adipocytes from obese or type 2 diabetic subjects, whereas expression levels of RBP4 mRNA were unaffected. These findings indicate that RBP4 may be released from diabetic adipocytes and act locally to inhibit phosphorylation of IRS1 at serine (307), a phosphorylation site that may integrate nutrient sensing with insulin signaling.
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