| 1. |
- Bestas, Burcu, et al.
(författare)
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A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo
- 2023
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Ingår i: NATURE COMMUNICATIONS. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes. SpCas9 unintended editing is a major concern. Here the authors report an off-target method using Duplex Sequencing with increased sensitivity for Cas9 mutation detection; they also identify a Cas9 variant of the II-B subfamily with intrinsic high fidelity (PsCas9) and see improved specificity.
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| 2. |
- Carobbio, Stefania, et al.
(författare)
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Adaptive changes of the Insig1/SREBP1/SCD1 set point help adipose tissue to cope with increased storage demands of obesity
- 2013
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Ingår i: Diabetes. - : Cell Press. - 0012-1797 .- 1939-327X. ; 62:11, s. 3697-3708
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Tidskriftsartikel (refereegranskat)abstract
- The epidemic of obesity imposes unprecedented challenges on human adipose tissue (WAT) storage capacity that may benefit from adaptive mechanisms to maintain adipocyte functionality. Here, we demonstrate that changes in the regulatory feedback set point control of Insig1/SREBP1 represent an adaptive response that preserves WAT lipid homeostasis in obese and insulin-resistant states. In our experiments, we show that Insig1 mRNA expression decreases in WAT from mice with obesity-associated insulin resistance and from morbidly obese humans and in in vitro models of adipocyte insulin resistance. Insig1 downregulation is part of an adaptive response that promotes the maintenance of SREBP1 maturation and facilitates lipogenesis and availability of appropriate levels of fatty acid unsaturation, partially compensating the antilipogenic effect associated with insulin resistance. We describe for the first time the existence of this adaptive mechanism in WAT, which involves Insig1/SREBP1 and preserves the degree of lipid unsaturation under conditions of obesity-induced insulin resistance. These adaptive mechanisms contribute to maintain lipid desaturation through preferential SCD1 regulation and facilitate fat storage in WAT, despite on-going metabolic stress.
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| 3. |
- Hu, Lijuan, et al.
(författare)
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Over-expression of Adamts1 in mice alters bone mineral density
- 2012
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Ingår i: Journal of Bone and Mineral Metabolism. - : Springer Science and Business Media LLC. - 0914-8779 .- 1435-5604. ; 30:3, s. 304-311
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Tidskriftsartikel (refereegranskat)abstract
- ADAMTS1, a secreted multifunctional metalloproteinase with disintegrin and thrombospondin motifs, is an early response gene of parathyroid hormone (PTH) in osteoblasts. Mice engineered to lack Adamts1 are smaller compared to wild-type (WT) mice and ADAMTS1 metalloproteinase activity has been shown to increase osteoblastic growth in collagen gels. However, there are no reports investigating the consequence of Adamts1 over-expression on bone tissue in vivo. Here, we analyze bones of female and male transgenic (TG) mice over-expressing mouse Adamts1 using peripheral quantitative computed tomography to evaluate its effect on bone shape and mineral density. Western blotting of protein extracts and immunohistochemistry of bone sections reveal increased presence of Adamts1 protein in TG bones compared to WT bones. Phenotypic analyses of femur show that female TG mice have reduced metaphyseal total density, trabecular bone mineral density and trabecular mineral content. In contrast, male TG mice which were without changes in the metaphysis showed increased total density and cortical density at the mid-diaphysis cortical site. Female TG mice showed no significant changes at the cortical site compared to WT mice. Furthermore, diaphyseal endosteal compartment was only affected in male TG mice. Along these lines, Adamts1 increased blood levels of PTH only in females whereas it reduced osteocalcin levels only in males. These results reveal that Adamts1 has an impact on bone mineral density and thus further confirm Adamts1 as a potent regulator of bone remodeling.
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| 4. |
- Lelliott, Christopher J., et al.
(författare)
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Ablation of PGC-1beta results in defective mitochondrial activity, thermogenesis, hepatic function, and cardiac performance
- 2006
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 4:11
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Tidskriftsartikel (refereegranskat)abstract
- The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta) has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.
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| 5. |
- Lindgren, Anna, et al.
(författare)
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Adiponectin receptor 2 deficiency results in reduced atherosclerosis in the brachiocephalic artery in apolipoprotein e deficient mice.
- 2013
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 8:11
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Tidskriftsartikel (refereegranskat)abstract
- Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2. The original aim of this study was to investigate the effect of combined AdipoR1 and AdipoR2 deficiency (AdipoR1(-/-)AdipoR2(-/-)) on atherosclerosis. However, we made the interesting observation that AdipoR1 (-/-) AdipoR2 (-/-) leads to embryonic lethality demonstrating the critical importance of the adiponectin signalling system during development. We then investigated the effect of AdipoR2-ablation on the progression of atherosclerosis in apolipoprotein E deficient (ApoE (-/-) ) mice. AdipoR2(-/-)ApoE(-/-) mice fed an atherogenic diet had decreased plaque area in the brachiocephalic artery compared with AdipoR2 (+/+) ApoE(-/-) littermate controls as visualized in vivo using an ultrasound biomicroscope and confirmed by histological analyses. The decreased plaque area in the brachiocephalic artery could not be explained by plasma cholesterol levels or inflammatory status. However, accumulation of neutral lipids was decreased in peritoneal macrophages from AdipoR2(-/-)ApoE(-/-) mice after incubation with oxidized LDL. This effect was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages from AdipoR2(-/-)ApoE(-/-) mice compared with AdipoR2(+/+)ApoE(-/-) controls after incubation with oxidized LDL. In summary, we show that adiponectin receptors are crucial during embryonic development and that AdipoR2-deficiency slows down the progression of atherosclerosis in the brachiocephalic artery of ApoE-deficient mice.
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| 6. |
- Palmgren, Henrik, et al.
(författare)
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Elevated Adipocyte Membrane Phospholipid Saturation Does Not Compromise Insulin Signaling
- 2023
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Ingår i: DIABETES. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 72:10, s. 1350-1363
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Tidskriftsartikel (refereegranskat)abstract
- Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.
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| 7. |
- Rodriguez-Cuenca, Sergio, et al.
(författare)
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Allostatic hypermetabolic response in PGC1α/β heterozygote mouse despite mitochondrial defects
- 2021
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Ingår i: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 35:9
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Tidskriftsartikel (refereegranskat)abstract
- Aging, obesity, and insulin resistance are associated with low levels of PGC1α and PGC1β coactivators and defective mitochondrial function. We studied mice deficient for PGC1α and PGC1β [double heterozygous (DH)] to investigate their combined pathogenic contribution. Contrary to our hypothesis, DH mice were leaner, had increased energy dissipation, a pro-thermogenic profile in BAT and WAT, and improved carbohydrate metabolism compared to wild types. WAT showed upregulation of mitochondriogenesis/oxphos machinery upon allelic compensation of PGC1α4 from the remaining allele. However, DH mice had decreased mitochondrial OXPHOS and biogenesis transcriptomes in mitochondria-rich organs. Despite being metabolically healthy, mitochondrial defects in DH mice impaired muscle fiber remodeling and caused qualitative changes in the hepatic lipidome. Our data evidence first the existence of organ-specific compensatory allostatic mechanisms are robust enough to drive an unexpected phenotype. Second, optimization of adipose tissue bioenergetics is sufficient to maintain a healthy metabolic phenotype despite a broad severe mitochondrial dysfunction in other relevant metabolic organs. Third, the decrease in PGC1s in adipose tissue of obese and diabetic patients is in contrast with the robustness of the compensatory upregulation in the adipose of the DH mice.
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| 8. |
- Ruiz, Mario, 1984, et al.
(författare)
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Sphingosine 1-phosphate mediates adiponectin receptor signaling essential for lipid homeostasis and embryogenesis.
- 2022
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Cells and organisms require proper membrane composition to function and develop. Phospholipids are the major component of membranes and are primarily acquired through the diet. Given great variability in diet composition, cells must be able to deploy mechanisms that correct deviations from optimal membrane composition and properties. Here, using lipidomics and unbiased proteomics, we found that the embryonic lethality in mice lacking the fluidity regulators Adiponectin Receptors 1 and 2 (AdipoR1/2) is associated with aberrant high saturation of the membrane phospholipids. Using mouse embryonic fibroblasts (MEFs) derived from AdipoR1/2-KO embryos, human cell lines and the model organism C. elegans we found that, mechanistically, AdipoR1/2-derived sphingosine 1-phosphate (S1P) signals in parallel through S1PR3-SREBP1 and PPARγ to sustain the expression of the fatty acid desaturase SCD and maintain membrane properties. Thus, our work identifies an evolutionary conserved pathway by which cells and organisms achieve membrane homeostasis and adapt to a variable environment.
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| 9. |
- Vorkapic, Emina, et al.
(författare)
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ADAMTS-1 in abdominal aortic aneurysm
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 12:6
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Extracellular matrix degradation is a hallmark of abdominal aortic aneurysm (AAA). Among proteases that are capable of degrading extracellular matrix are a disintegrin and metalloproteases with thrombospondin motifs (ADAMTS). Pathogenesis of these proteases in AAA has not been investigated until date.METHODS AND RESULTS: Human aneurysmal and control aortas were collected and analyzed with RT-PCR measuring the ADAMTS-1, 4,5,6,8,9,10,13,17 and ADAMTSL-1. Expression of a majority of the investigated ADAMTS members on mRNA level was decreased in aneurysm compared to control aorta. ADAMTS-1 was one of the members that was reduced most. Protein analysis using immunohistochemistry and western blot for localization and expression of ADAMTS-1 revealed that ADAMTS-1 was present predominantly in areas of SMCs and macrophages in aneurysmal aorta and higher expressed in AAA compared to control aortas. The role of ADAMTS-1 in AAA disease was further examined using ADAMTS-1 transgenic/apoE-/- mice with the experimental angiotensin II induced aneurysmal model. Transgenic mice overexpressing ADAMTS-1 showed to be similar to ADAMTS-1 wild type mice pertaining collagen, elastin content and aortic diameter.CONCLUSION: Several of the ADAMTS members, and especially ADAMTS-1, are down regulated at mRNA level in AAA, due to unknown mechanisms, at the same time ADAMTS-1 protein is induced. The cleavage of its substrates, don't seem to be crucial for the pathogenesis of AAA but rather more important in the development of thoracic aortic aneurysm and atherosclerosis as shown in previous studies.
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