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1.	Klionsky, Daniel J., et al. (författare) Guidelines for the use and interpretation of assays for monitoring autophagy 2012 Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544 Forskningsöversikt (refereegranskat)abstract In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
2.	Brännmark, Cecilia, 1983- (författare) Insulin Signaling in Human Adipocytes a Systems Biology Approach 2012 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Obesity and a sedentary life style are associated with type 2 diabetes, a disease starting with insulin resistance in the adipose tissue, which spreads to the whole body. Despite large research efforts to understand the insulin signaling system, there is little knowledge of the mechanisms behind insulin resistance and type 2 diabetes developments. We have herein focused on the insulin signaling in adipocytes, elucidating mechanisms for early signaling. We have also modeled isolated adipocytes and data from the in vivo, whole bodysituation, concurrently. We also mapped and quantitatively described differences in the insulin signaling of adipocytes from type 2 diabetics and non-diabetics.In paper I we show that neither insulin degradation, receptor internalization, nor feedback signals can as separate explanations cause the overshoot in tyrosine phosphorylation of IRS1, while an endocytosis-dependent feedback mechanism explains all available data.In paper II we show that it is not possible to scale up the experimentally determined glucose uptake by isolated human adipocytes to match the glucose uptake profile of the whole adipose tissue in vivo. Other insulin effects need to be accounted for.In paper III we show that attenuation of the positive feedback to serine 307 phosphorylation of IRS1 can explain the insulin resistance in the insulin signaling in adipocytes seen in type 2 diabetes. However, to fully explain both the signaling and the glucose uptake, a reduction in the amount of Glut4 is also needed.
3.	Lindberg, Jan, et al. (författare) Synthesis of inositol phosphoglycans containing thiol-terminated spacers for efficient coupling to maleimide functionalized solid phases or proteins 2002 Ingår i: Tetrahedron. - 0040-4020 .- 1464-5416. ; 58:21, s. 4245-4254 Tidskriftsartikel (refereegranskat)abstract The synthesis of inositol phosphoglycans (IPGs), analogous to second messengers of insulin, to provide a small targeted library of compounds is described. These derivatives contain the glucosamine(a1-6)myo-inositol 1,2-cyclic phosphate motif. A thiol-terminated spacer was introduced, for their immobilization, by a radical elongation of an allyl ether with benzyl mercaptane. ⌐ 2002 Elsevier Science Ltd. All rights reserved.
4.	Nyman, Elin, et al. (författare) Requirements for multi-level systems pharmacology models to reach end-usage : the case of type 2 diabetes 2016 Ingår i: Interface Focus. - London, UK : The Royal Society. - 2042-8898 .- 2042-8901. ; 6:2 Forskningsöversikt (refereegranskat)abstract We are currently in the middle of a major shift in biomedical research: unprecedented and rapidly growing amounts of data may be obtained today, from in vitro, in vivo and clinical studies, at molecular, physiological and clinical levels. To make use of these large-scale, multi-level datasets, corresponding multi-level mathematical models are needed, i.e. models that simultaneously capture multiple layers of the biological, physiological and disease-level organization (also referred to as quantitative systems pharmacology-QSP-models). However, today's multi-level models are not yet embedded in end-usage applications, neither in drug research and development nor in the clinic. Given the expectations and claims made historically, this seemingly slow adoption may seem surprising. Therefore, we herein consider a specific example-type 2 diabetes-and critically review the current status and identify key remaining steps for these models to become mainstream in the future. This overview reveals how, today, we may use models to ask scientific questions concerning, e.g., the cellular origin of insulin resistance, and how this translates to the whole-body level and short-term meal responses. However, before these multi-level models can become truly useful, they need to be linked with the capabilities of other important existing models, in order to make them 'personalized' (e.g. specific to certain patient phenotypes) and capable of describing long-term disease progression. To be useful in drug development, it is also critical that the developed models and their underlying data and assumptions are easily accessible. For clinical end-usage, in addition, model links to decisionsupport systems combined with the engagement of other disciplines are needed to create user-friendly and cost-efficient software packages.
5.	Stenkula, Karin, 1973- (författare) A molecular approach to insulin signalling and caveolae in primary adipocytes 2007 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear.Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell.The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects.The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane.In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.
6.	Aboulaich, Nabila, et al. (författare) Association and insulin regulated translocation of hormone-sensitive lipase with PTRF 2006 Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 350:3, s. 657-661 Tidskriftsartikel (refereegranskat)abstract Polymerase I and transcript release factor (PTRF) is in human adipocytes mainly localized at the plasma membrane. This localization was under control of insulin, which translocated PTRF to the cytosol and nucleus, indicating a novel role for PTRF in insulin transcriptional control. In the plasma membrane PTRF was specifically bound to a triacylglycerol-metabolizing subclass of caveolae containing hormone-sensitive lipase (HSL). In response to insulin PTRF was translocated to the cytosol in parallel with HSL. PTRF and HSL were quantitatively immunoprecipitated from the cytosol by antibodies against either PTRF or HSL. The findings indicate also a novel extranuclear function for PTRF in the control of lipolysis.
7.	Aboulaich, Nabila, 1976- (författare) Expanding role of caveolae in control of adipocyte metabolism : proteomics of caveolae 2006 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The primary function of adipose tissue is to store energy in the form of triacylglycerol, which is hydrolyzed to fatty acids to supply other tissues with energy. While insulin promotes the storage of triacylglycerol, catecholamines stimulate its hydrolysis. The development of type II diabetes is strongly associated with obesity, indicating a role of triacylglycerol metabolism in the pathogenesis of diabetes. Caveolae are plasma membrane invaginations found in most cells but are highly abundant in adipocytes. Insulin receptors are localized in caveolae and their function depends on intact caveolae structures. In the present thesis work, mass spectrometry-based methodology allowed identification of a number of new proteins and their posttranslational modifications in caveolae of human adipocytes. Variable N-terminal acetylation and phosphorylation of caveolin-1α and caveolin-1β were identified, which might regulate the function of caveolae. The transcription regulator protein PTRF was identified as the major caveolae associated protein. Specific proteolytic modifications of PTRF at the cytosolic surface of caveolae and phosphorylation on nine serine and one threonine residues were identified. Moreover, insulin induced translocation of PTRF from the plasma membrane to the nucleus. PTRF was previously shown to regulate the activity of both RNA polymerase I and polymerase II, thus a role of PTRF in mediating the anabolic action of insulin on protein synthesis and gene transcription is proposed.PTRF was also involved in an extranuclear function in the hormonal regulation of triacylglycerol metabolism in caveolae. PTRF was colocalized with the triacylglycerol regulator proteins perilipin and hormone-sensitive lipase (HSL) in the triacylglycerol-synthesizing caveolae subclass. We showed that, while perilipin was translocated to the plasma membrane, both PTRF and HSL were translocated from the plasma membrane to the cytosol as a complex in response to insulin. The perilipin recruited to the plasma membrane was highly threonine phosphorylated. By mass spectrometry, three phosphorylated threonine residues were identified and were located in an acidic domain in the lipid droplet targeting domain of perilipin. The insulin-induced recruitment of perilipin to the plasma membrane might, therefore be phosphorylation-dependent. Isoproterenol, which stimulates hydrolysis of triacylglycerol, induced a complete depletion of perilipin B from the plasma membrane, suggesting a function of perilipin B to protect newly synthesized triacylglycerol in caveolae from being hydrolyzed by HSL. The location of PTRF and HSL was not affected by isoproterenol, indicating that insulin is acting against a default presence of PTRF and HSL in caveolae.Taken together, this thesis expands our knowledge about caveolae and provided valuable information about their involvement in novel roles, particularly in the hormonal regulation of triacylglycerol metabolism.
8.	Aboulaich, Nabila, et al. (författare) Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes 2006 Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:17, s. 11446-11449 Tidskriftsartikel (refereegranskat)abstract In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.
9.	Aboulaich, Nabila, et al. (författare) Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes 2004 Ingår i: The Biochemical journal. - 1470-8728. ; 383:Pt 2, s. 237-248 Tidskriftsartikel (refereegranskat)abstract Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.
10.	Ahmad, Faiyaz, et al. (författare) 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 Ingår i: BIOCHEMICAL JOURNAL. - 0264-6021. ; 424:3, s. 399-410 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.
11.	Aili Fagerholm, Siri, 1980- (författare) Insulin signaling in primary adipocytes in insulin sensitive and insulin resistant states 2013 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Increasing numbers of people world-wide develops the disease type 2 diabetes. Development of type 2 diabetes is characterized by a shift from an insulin sensitive state to an insulin resistant state in peripheral insulin responding organs, which originates from the development of insulin resistance in the adipose tissue. Insulin resistance in combination with reduced pancreatic insulin secretion lead to overt type 2 diabetes.In this thesis, the insulin signaling network in primary adipocytes was analyzed. Key proteins and mechanisms were studied to gain deeper knowledge of signaling both in the insulin sensitive state and in the insulin resistant state produced by rapid weight gain as well as in type 2 diabetes.The surface of the adipocyte is dotted with invaginations in the cell membrane called caveolae that act as important metabolic and signaling platforms in adipocytes, and also harbor the insulin receptor. In paper I we show that insulin stimulation of primary adipocytes results in a rapid phosphorylation of the insulin receptor and caveolin-1, and that internalization of the proteins is mediated by endocytosis of caveolae.Weight gain due to overfeeding and obesity has been associated with the development of insulin resistance in insulin sensitive tissues such as the adipose tissue. In paper II we show that short-term overfeeding for one month of lean subjects results in an insulin resistant state. At the end of the study, the subjects had developed a mild systemic insulin resistance. Moreover, in isolated subcutaneous adipocytes we found several alterations of the insulin signaling pathway that mimicked alterations found in isolated subcutaneous adipocytes from subjects with type 2 diabetes.In paper III we present a first dynamic mathematical model of the insulin signaling network in human adipocytes that are based on experimental data acquired in a consistent fashion. The model takes account of insulin signaling in both the healthy, insulin sensitive state and in the insulin resistant state of type 2 diabetes. We show that attenuated mTORC1-mediated positive feedback to control of phosphorylation of IRS1 at Ser307 is an essential component of the insulin resistant state of type 2 diabetes. A future application of the model is the identification and evaluation of drug targets for the treatment of insulin resistance and type 2 diabetes.In paper IV we examine the protein kinase that catalyzes the insulin stimulated mTORC1- mediated feedback to IRS1. We find that the phosphorylation of IRS1 at Ser307 is not likely to be catalyzed by the kinases S6K1, mTOR or PKB. However, a catalyzing protein kinase for the in vitro phosphorylation of IRS1 at Ser307 was found to be associated with the complex mTORC1.In conclusion, this thesis provide new insights and characterize mechanisms of the intrinsically complex insulin signaling network of primary adipocytes, both in insulin sensitive and insulin resistant states.
12.	Al-Hilli, Safaa, 1965, et al. (författare) ZnO nanorods as an intracellular pH sensor 2007 Ingår i: European NanoSystems,2007. ; , s. 38-43 Konferensbidrag (refereegranskat)
13.	Al-Hilli, Safaa, 1965, et al. (författare) ZnO nanorods as an intracellular sensor for pH measurements 2007 Ingår i: JOURNAL OF APPLIED PHYSICS. - : AIP Publishing. - 0021-8979 .- 1089-7550. ; 102:8 Tidskriftsartikel (refereegranskat)
14.	Asif, Muhammad H, et al. (författare) Functionalised ZnO-nanorod-based selective electrochemical sensor for intracellular glucose 2010 Ingår i: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 25:10, s. 2205-2211 Tidskriftsartikel (refereegranskat)abstract In this article, we report a functionalised ZnO-nanorod-based selective electrochemical sensor for intracellular glucose. To adjust the sensor for intracellular glucose measurements, we grew hexagonal ZnO nanorods on the tip of a silver-covered borosilicate glass capillary (0.7 mu m diameter) and coated them with the enzyme glucose oxidase. The enzyme-coated ZnO nanorods exhibited a glucose-dependent electrochemical potential difference versus an Ag/AgCl reference microelectrode. The potential difference was linear over the concentration range of interest (0.5-1000 mu M). The measured glucose concentration in human adipocytes or frog oocytes using our ZnO-nanorod sensor was consistent with values of glucose concentration reported in the literature; furthermore, the sensor was able to show that insulin increased the intracellular glucose concentration. This nanoelectrode device demonstrates a simple technique to measure intracellular glucose concentration. (C) 2010 Elsevier B.V. All rights reserved.
15.	Asif, Muhammad H, 1978-, et al. (författare) Functionalized zinc oxide nanorod with ionophore-membrane coatingas an intracellular Ca2+ selective sensor 2009 Ingår i: Applied Physics Letters. - : EBSCO/American Institute of Physics. - 0003-6951 .- 1077-3118. ; 95:2, s. 23703- Tidskriftsartikel (refereegranskat)abstract The tip of a borosilicate glass capillary with functionalized hexagonal ZnO nanorods was used to make a sensitive electrochemical intracellular Ca2+ sensor. To adjust the sensor for Ca2+ measurements with sufficient selectivity and stability, polyvinyl chloride (PVC) membrane containing Ca2+ ionophores were coated on the surface. The membrane covered ZnO nanorods exhibited a Ca2+-dependent electrochemical potential difference versus an Ag/AgCl reference electrode. The potential difference was linear over a large concentration range (100 nM to 10 mM). The measurements of Ca2+ concentrations using our ZnO nanorods sensor in human fat cells or in frog egg cells were consistent with values of Ca2+ concentrations reported in the literature. This nanoelectrode device paves the way to measurements of intracellular biochemical species in specific locations within single living cells.
16.	Asif, Muhammad H., et al. (författare) Growth and Structure of ZnO Nanorods on a Sub-Micrometer Glass Pipette and Their Application as Intracellular Potentiometric Selective Ion Sensors 2010 Ingår i: Materials. - : MDPI AG. - 1996-1944. ; 3, s. 4657-4667 Tidskriftsartikel (refereegranskat)abstract This paper presents the growth and structure of ZnO nanorods on a sub-micrometer glass pipette and their application as an intracellular selective ion sensor. Highly oriented, vertical and aligned ZnO nanorods were grown on the tip of a borosilicate glass capillary (0.7 μm in diameter) by the low temperature aqueous chemical growth (ACG) technique. The relatively large surface-to-volume ratio of ZnO nanorods makes them attractive for electrochemical sensing. Transmission electron microscopy studies show that ZnO nanorods are single crystals and grow along the crystal’s c-axis. The ZnO nanorods were functionalized with a polymeric membrane for selective intracellular measurements of Na +. The membrane-coated ZnO nanorods exhibited a Na+-dependent electrochemical potential difference versus an Ag/AgCl reference micro-electrode within a wide concentration range from 0.5 mM to 100 mM. The fabrication of functionalized ZnO nanorods paves the way to sense a wide range of biochemical species at the intracellular level.
17.	Asif, Muhammad H., et al. (författare) Zinc Oxide Nanorods and their Application to Intracellular Glucose Measurements 2012 Ingår i: Nanotechnology and Nanomedicine in Diabetes. - : CRC Press. - 9781578087297 ; , s. 126-146 Bokkapitel (övrigt vetenskapligt/konstnärligt)
18.	Bauzá-Thorbrügge, Marco, et al. (författare) Adipocyte-specific ablation of the Ca2+ pump SERCA2 impairs whole-body metabolic function and reveals the diverse metabolic flexibility of white and brown adipose tissue. 2022 Ingår i: Molecular metabolism. - : Elsevier BV. - 2212-8778. ; 63 Tidskriftsartikel (refereegranskat)abstract Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ from the cytosol into the endoplasmic retitculum (ER) and is essential for appropriate regulation of intracellular Ca2+ homeostasis. The objective of this study was to test the hypothesis that SERCA pumps are involved in the regulation of white adipocyte hormone secretion and other aspects of adipose tissue function and that this control is disturbed in obesity-induced type-2 diabetes.SERCA expression was measured in isolated human and mouse adipocytes as well as in whole mouse adipose tissue by Western blot and RT-qPCR. To test the significance of SERCA2 in adipocyte functionality and whole-body metabolism, we generated adipocyte-specific SERCA2 knockout mice. The mice were metabolically phenotyped by glucose tolerance and tracer studies, histological analyses, measurements of glucose-stimulated insulin release in isolated islets, and gene/protein expression analyses. We also tested the effect of pharmacological SERCA inhibition and genetic SERCA2 ablation in cultured adipocytes. Intracellular and mitochondrial Ca2+ levels were recorded with dual-wavelength ratio imaging and mitochondrial function was assessed by Seahorse technology.We demonstrate that SERCA2 is downregulated in white adipocytes from patients with obesity and type-2 diabetes as well as in adipocytes from diet-induced obese mice. SERCA2-ablated adipocytes display disturbed Ca2+ homeostasis associated with upregulated ER stress markers and impaired hormone release. These adipocyte alterations are linked to mild lipodystrophy, reduced adiponectin levels, and impaired glucose tolerance. Interestingly, adipocyte-specific SERCA2 ablation leads to increased glucose uptake in white adipose tissue while the glucose uptake is reduced in brown adipose tissue. This dichotomous effect on glucose uptake is due to differently regulated mitochondrial function. In white adipocytes, SERCA2 deficiency triggers an adaptive increase in fibroblast growth factor 21 (FGF21), increased mitochondrial uncoupling protein 1 (UCP1) levels, and increased oxygen consumption rate (OCR). In contrast, brown SERCA2 null adipocytes display reduced OCR despite increased mitochondrial content and UCP1 levels compared to wild type controls.Our data suggest causal links between reduced white adipocyte SERCA2 levels, deranged adipocyte Ca2+ homeostasis, adipose tissue dysfunction and type-2 diabetes.
19.	Brannmark, Cecilia, et al. (författare) Adiponectin is secreted via caveolin 1-dependent mechanisms in white adipocytes 2020 Ingår i: Journal of Endocrinology. - : BIOSCIENTIFICA LTD. - 0022-0795 .- 1479-6805. ; 247:1, s. 25-38 Tidskriftsartikel (refereegranskat)abstract Here we have investigated the role of the protein caveolin 1 (Cav1) and caveolae in the secretion of the white adipocyte hormone adiponectin. Using mouse primary subcutaneous adipocytes genetically depleted of Cav1, we show that the adiponectin secretion, stimulated either adrenergically or by insulin, is abrogated while basal (unstimulated) release of adiponectin is elevated. Adiponectin secretion is similarly affected in wildtype mouse and human adipocytes where the caveolae structure was chemically disrupted. The altered ex vivo secretion in adipocytes isolated from Cav1 null mice is accompanied by lowered serum levels of the high-molecular weight (HMW) form of adiponectin, whereas the total concentration of adiponectin is unaltered. Interestingly, levels of HMW adiponectin are maintained in adipose tissue from Cav1-depleted mice, signifying that a secretory defect is present. The gene expression of key regulatory proteins known to be involved in cAMP/adrenergically triggered adiponectin exocytosis (the beta-3-adrenergic receptor and exchange protein directly activated by cAMP) remains intact in Cav1 null adipocytes. Microscopy and fractionation studies indicate that adiponectin vesicles do not co-localise with Cav1 but that some vesicles are associated with a specific fraction of caveolae. Our studies propose that Cav1 has an important role in secretion of HMW adiponectin, even though adiponectin-containing vesicles are not obviously associated with this protein. We suggest that Cav1, and/or the caveolae domain, is essential for the organisation of signalling pathways involved in the regulation of HMW adiponectin exocytosis, a function that is disrupted in Cav1/caveolae-depleted adipocytes.
20.	Brannmark, Cecilia, et al. (författare) Mass and Information Feedbacks through Receptor Endocytosis Govern Insulin Signaling as Revealed Using a Parameter-free Modeling Framework 2010 Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 285:26, s. 20171-20179 Tidskriftsartikel (refereegranskat)abstract Insulin and other hormones control target cells through a network of signal-mediating molecules. Such networks are extremely complex due to multiple feedback loops in combination with redundancy, shared signal mediators, and cross-talk between signal pathways. We present a novel framework that integrates experimental work and mathematical modeling to quantitatively characterize the role and relation between coexisting submechanisms in complex signaling networks. The approach is independent of knowing or uniquely estimating model parameters because it only relies on (i) rejections and (ii) core predictions (uniquely identified properties in unidentifiable models). The power of our approach is demonstrated through numerous iterations between experiments, model-based data analyses, and theoretical predictions to characterize the relative role of co-existing feedbacks governing insulin signaling. We examined phosphorylation of the insulin receptor and insulin receptor substrate-1 and endocytosis of the receptor in response to various different experimental perturbations in primary human adipocytes. The analysis revealed that receptor endocytosis is necessary for two identified feedback mechanisms involving mass and information transfer, respectively. Experimental findings indicate that interfering with the feedback may substantially increase overall signaling strength, suggesting novel therapeutic targets for insulin resistance and type 2 diabetes. Because the central observations are present in other signaling networks, our results may indicate a general mechanism in hormonal control.
21.	Bronnikov, Gennady, 1950-, et al. (författare) Acute effects of insulin on the activity of mitochondrial GPAT1 in primary adipocytes 2008 Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 367:1, s. 201-207 Tidskriftsartikel (refereegranskat)abstract The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing VMAX and KM for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1. These phosphorylation sites correspond to casein kinase-2 consensus sequences and are highly conserved in chordate animal, but not fly, fungal or plant, mtGPAT1. © 2007 Elsevier Inc. All rights reserved.
22.	Brännmark, Cecilia, et al. (författare) Insulin Signaling in Type 2 Diabetes : Experimental and Modeling Analyses Reveal Mechanisms of Insulin Resistance in Human Adipocytes 2013 Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:14, s. 9867-9880 Tidskriftsartikel (refereegranskat)abstract Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems-level mechanistic understanding of insulin resistance in humans. We developed a dynamic mathematical model of insulin signaling – normally and in diabetes – based on quantitative steady-state and dynamic time-course data on signaling intermediaries in human mature adipocytes. At the core of insulin resistance is attenuation of a positive feedback from mammalian target of rapamycin in complex with raptor (mTORC1) to the insulin receptor substrate-1 (IRS1), which explains reduced sensitivity and signal strength throughout the signaling network. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.
23.	Bäck, Karolina, et al. (författare) Differential effects of IGF-I, IGF-II and insulin in human preadipocytes and adipocytes - Role of insulin and IGF-I receptors 2011 Ingår i: Molecular and Cellular Endocrinology. - : Elsevier Science B.V., Amsterdam.. - 0303-7207 .- 1872-8057. ; 339:02-jan, s. 130-135 Tidskriftsartikel (refereegranskat)abstract We compared insulin and IGF effects in adipocytes expressing IR (insulin receptors), and preadipocytes expressing IR and IGF-IR (IGF-I receptors). Treatment of adipocytes with insulin, IGF-II or IGF-I resulted in phosphorylation of IR. Order of potency was insulin greater thanIGF-IIgreater than IGF-I. In preadipocytes IR, IGF-IR and insulin/IGF-I hybrid receptors (HR) were detected. Treatment of preadipocytes with IGF-I and IGF-II 10(-8) M resulted in activation of IGF-IR and IR whereas insulin was more potent in activating IR, with no effect on IGF-IR. In adipocytes glucose transport was 100-fold more sensitive to insulin than to IGFs and the maximal effect was higher with insulin. In preadipocytes glucose accumulation and DNA synthesis was equally sensitive to insulin and IGFs but the maximal effect was higher with IGF-I. In conclusion, insulin and IGF-I activate their cognate receptors and IGF-I also HR. IGF-II activates IR, IGF-IR and HR. Insulin and IGF-I are partial agonists to each others receptors.
24.	Cedersund, Gunnar, et al. (författare) Core-box modeling in the biosimulation of drug action 2008 Ingår i: Biosimulation in drug development. - Weinheim, Germany : Wiley-VCH Verlagsgesellschaft. - 9783527622672 - 9783527316991 ; , s. 115-139 Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract Biosimulation has the potential to drastically improve future drug development: mathematical models may be used to obtain maximal information from experimental data, and can be used to pin-point potential drug targets, in a much more systematic manner than is the case today. However, for this potential to be fulfilled, the developed models must themselves fulfill a number of requirements. For instance, the models must be trustworthy, and contain the necessary degree of mechanistic details. These two requirements are difficult to fulfill simultaneously, since a mechanistically detailed model typically becomes unidentifiable (i.e., over-parametrized with respect to existing data), which means that the model predictions are the result of arbitrary choices on a parameter manifold, and thus much less trustworthy. In this chapter, we review a recently developed modeling framework-core-box modeling-which has been developed to be able to fulfill both of the above requirements. A core-box model is a combination of two models: a core model and a gray-box model. The gray-box model is a mechanistically detailed description of the relevant biochemical processes, while the core model is a simplified version of the same processes, where all aspects that cannot uniquely be estimated from the data have been eliminated. The core-box model consists of both these models, and a translation between them. The translation allows the two sub-models to be considered as two degrees of zooming of the same core-box model, where changes in, for instance, parameters are preserved upon the zooming. A path is suggested for the development of a core-box model, including identifiability analysis, model reduction, system identification, and back-translation. All of these steps are reviewed, and the steps and advantages of the modeling framework are demonstrated on a core-box model for insulin signaling.
25.	Cedersund, Gunnar, et al. (författare) Model-Based Hypothesis Testing of Key Mechanisms in Initial Phase of Insulin Signaling 2008 Ingår i: PloS Computational Biology. - : Public Library of Science. - 1553-734X .- 1553-7358. ; 4:6 Tidskriftsartikel (refereegranskat)abstract Type 2 diabetes is characterized by insulin resistance of target organs, which is due to impaired insulin signal transduction. The skeleton of signaling mediators that provide for normal insulin action has been established. However, the detailed kinetics, and their mechanistic generation, remain incompletely understood. We measured time-courses in primary human adipocytes for the short-term phosphorylation dynamics of the insulin receptor (IR) and the IR substrate-1 in response to a step increase in insulin concentration. Both proteins exhibited a rapid transient overshoot in tyrosine phosphorylation, reaching maximum within 1 min, followed by an intermediate steady-state level after approximately 10 min. We used model-based hypothesis testing to evaluate three mechanistic explanations for this behavior: (A) phosphorylation and dephosphorylation of IR at the plasma membrane only, (B) the additional possibility for IR endocytosis, (C) the alternative additional possibility of feedback signals to IR from downstream intermediates. We concluded that (A) is not a satisfactory explanation, that (B) may serve as an explanation only if both internalization, dephosphorylation, and subsequent recycling are permitted, and that (C) is acceptable. These mechanistic insights cannot be obtained by mere inspection of the datasets, and they are rejections and thus stronger and more final conclusions than ordinary model predictions.
26.	Cedersund, Gunnar, et al. (författare) Putting the pieces together in diabetes research : Towards a hierarchical model of whole-body glucose homeostasis 2009 Ingår i: European Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0928-0987 .- 1879-0720. ; 36:1, s. 91-104 Tidskriftsartikel (refereegranskat)abstract Type 2 diabetes is one of the most widespread and rapidly spreading diseases world-wide and has been subject of extensive research efforts. However, understanding the molecular basis of the disease is increasing piecemeal and a consensus regarding the overall picture of normal metabolic regulation and malfunction in diabetes has not emerged. A systems biology approach, combining mathematical modelling with simultaneous high-throughput measurements, can be of considerable help. On the whole-body level, this has been done in pharmacokinetic and pharmacodynamic models, which recently have started to mature into more physiologically realistic organ-based models. At the other end of the spectrum, detailed models for crucial cellular processes are starting to mature into complete modules that potentially can be fitted into such whole-body organ-based models. The result of such a merge is a multi-level hierarchical model, which is a model type that has been common in technical systems. In this review, we report and exemplify some of the recent progress that has been made to achieve such a hierarchical model, and we argue why it is only through such a model that a Complete picture of diabetes mellitus can be obtained.
27.	Danielsson, Anna, et al. (författare) Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes 2005 Ingår i: Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 280:41, s. 34389-3492 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.
28.	Danielsson, Anna, et al. (författare) 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 Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 272:1, s. 141-151 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.
29.	Danielsson, Anna, 1973- (författare) Insulin signalling in human adipocytes : mechanisms of insulin resistance in type 2 diabetes 2007 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Prevalensen av fetma ökar drastiskt i stora delar av världen och utgör en stor riskfaktor för att utveckla insulinresistens och typ 2 diabetes. Fettväven kan bli mycket stor om för mycket energi tas upp av kroppen. Vid extrem övervikt är fettväven i kroppen i ett stresstillstånd, vilket gör att risken för att utveckla metabola sjukdomar som t.ex. typ 2 diabetes ökar. Fett lagras i olika fettdepåer i kroppen. Inlagringen i djupare kroppsdelar, runt och i inre organ s.k. visceralt fett, skiljer sig från fettväven som lagras direkt under huden s.k. subkutant fett. Nyare rön visar att mer visceral fettväv ökar risken för att utveckla insulinresistens och typ 2 diabetes.Fettcellen är tillsammans med muskel- och leverceller de viktigaste för glukosmetabolismen. Fettcellen är en stor cell, som man lätt kan se med blotta ögat. Storleken på ellerna varierar dock kraftigt i en och samma fettvävnad. Upptag av glukos från maten vi äter regleras av hormonet insulin. Insulinresistens är ett tillstånd då cellerna svarar dåligt på insulin, vilket gör att glukoshalten i blodet ökar. Detta förekommer vid typ 2 diabetes, men även vid andra tillstånd där cellerna blir stressade, t.ex. kirurgiska ingrepp. Insulinsignaleringen i fettcellen är komplex och signalöverföringen inne i cellen sker främst via en kaskad av fosforyleringar, där olika proteiner i en signalkedja fosforyleras eller defosforyleras. Slutligen leder denna fosforyleringskaskad till insulinets sluteffekter som t.ex. upptag av glukos, proteinsyntes och celltillväxt. Efter att insulin bundit till och fosforylerat/aktiverat insulinreceptorn delas signalen upp inne i cellen i två huvudvägar; den metabola signalvägen och den mitogena signalvägen. Insulinreceptorsubstrat 1, IRS1, är ett stort protein som insulinreceptorn verkar direkt på. Fosforylering av aminosyran tyrosin på IRS1 är mycket viktigt för fortsatt insulinsignalering i fettcellen. IRS1 fosforyleras även på aminosyran serin som svar på bl.a. insulin. Serinfosforyleringen av IRS1 hämmar eller stimulerar insulinsignaleringen, ofta genom återkoppling av insulinsignalen.Syftet med den här avhandlingen är att beskriva möjliga cellulära mekanismer i insulinsignaleringen vid insulinresistens som resultat av kirurgisk stress eller vid typ 2 diabetes i fettceller från människa.Häri har upptaget av glukos analyserats och jämförts i fettceller från olika fettdepåer. Viscerala fettceller har högre basalt och insulinstimulerat glukosupptag och mer glucostransportörprotein än subkutana fettceller. Däremot är det ingen skillnad i insulinkänslighet angående glukosupptaget i de olika typerna av fettceller.Vidare fann vi att den kirurgiskt orsakade insulinresistensen hos subkutana fettceller från människa återgår till det normala efter övernattinkubering av cellerna i odlingsmedium. Insulinresistensen vid typ 2 diabetes är däremot permanent och har en annan mekanism än den reversibla, stress-relaterade insulinresistensen. Insulinresistansen vid typ 2 diabetes beror på att signalöverföringen mellan olika proteiner i cellen är defekt. Insulinreceptorns förmåga att fosforylera IRS1 på aminosyran tyrosin är nedsatt hos patienter med typ 2 diabetes. Fosforyleringen av IRS1 på serin 307 (i den humana sekvensen) ökar snabbt hos icke-diabetiska fettceller som svar på insulin. Denna serinfosforylering verkar behövas för att IRS1 effektivt ska tyrosinfosforyleras och därmed leda insulinsignalen vidare inne i cellen. Fosforyleringen av IRS1 på serin 307 är kraftigt nedsatt hos subkutana fettceller från patienter med typ 2 diabetes. Fosforyleringen av IRS1 på serin 312 är däremot liknande i fettceller från icke-diabetiker och diabetiker (Öst et.al. (2007) Faseb.J. doi: 10.1096/fj.07-8173com). Fosforyleringen av IRS1 på serin 312 är mest involverad i insulinsignaleringens negativa återkoppling. Fosforyleringen av serin 307 sker snabbt och vid låga insulinkoncentrationer, medan fosforyleringen på serin 312 sker först efter lång inkubering och vid höga insulinkoncentrationer.Detta är en ny mekanism på cellulär nivå som möjligen kan beskriva insulinresistansen i fettceller från människa. Tillsammans styrs återkopplingen via den stimulerande fosforyleringen (serin 307) eller den hämmande fosforyleringen (serin 312) och kontrollerar insulinsignaleringen i cellen. Fosforyleringarna sker möjligen via samma proteinkinas och/eller proteinfosfatas och kan bli mål för terapeutiska läkemedel mot typ 2 diabetes i framtiden.
30.	Danielsson, Anna, et al. (författare) Phosphorylation of IRS1 at serine 307 and serine 312 in response to insulin in human adipocytes 2006 Ingår i: Biochemical and biophysical research communications. - : Elsevier BV. - 0006-291X. ; 342:4, s. 1183-1187 Tidskriftsartikel (refereegranskat)abstract Feedback control in insulin signaling involves serine phosphorylation of insulin receptor substrate-1 (IRS1). By analyzing the insulin-induced phosphorylation of IRS1 at serine 307, serine 312, and tyrosine in the same primary human adipocytes, we now report that negative feedback phosphorylation of serine 312 (corresponding to murine serine 307) required relatively high concentrations of insulin (EC50 = 3 nM) for a long time (t1/2 ca. 30 min) and reduced the steady-state tyrosine phosphorylation, without affecting the cellular concentration, of IRS1. In contrast, positive feedback phosphorylation of serine 307 was a rapid (t1/2 ca. 2 min) event at physiological concentrations of insulin (EC50 = 0.2 nM).
31.	Danielsson, Anna, et al. (författare) Short-Term Overeating Induces Insulin Resistance in Fat Cells in Lean Human Subjects 2009 Ingår i: Molecular Medicine. - : Springer Science and Business Media LLC. - 1076-1551 .- 1528-3658. ; 15:7-8, s. 228-234 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.
32.	Fagerholm, Siri, et al. (författare) Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes 2009 Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:6, s. e5985- Tidskriftsartikel (refereegranskat)abstract Background: The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized. Methodology/Principal Findings: We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t1/2 less than3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited. Conclusion: It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.
33.	Franck, Niclas, et al. (författare) Insulin-induced GLUT4 translocation to the plasma membrane is blunted in large compared with small primary fat cells isolated from the same individual 2007 Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 50:8, s. 1716-1722 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.
34.	Fulati, Alimujiang, 1981-, et al. (författare) An intracellular glucose biosensor based on nanoflake ZnO 2010 Ingår i: Sensors and actuators. B, Chemical. - : Elsevier. - 0925-4005 .- 1873-3077. ; 150:2, s. 673-680 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract In this study, an improved potentiometric intracellular glucose biosensor was fabricated with immobilization of glucose oxidase on a ZnO nanoporous material. The ZnO nanoporous material with a wall thickness around 200 nm was grown on the tip of a borosilicate glass capillary and used as a selective intracellular glucose sensor for the measurement of glucose concentrations in human adipocytes and frog oocytes. The results showed a fast response within 4 s and a linear glucosedependent electrochemical response over a wide range of glucose concentration (500 nM-10 mM). The measurements of intracellular glucose concentrations with our biosensor were consistent with the values of intracellular glucose concentrations reported in the literature. The sensor also demonstrated its capability by detecting an increase in the intracellular glucose concentration induced by insulin. We found that the ZnO nanoporous material provides sensitivity as high as 1.8 times higher than that obtained using ZnO nanorods under the same conditions. Moreover, the fabrication method in our experiment is simple and the excellent performance of the developed nanosensor in sensitivity, stability, selectivity, reproducibility and anti-interference was achieved. All these advantageous features of this intracellular glucose biosensor based on functionalised ZnO nanoporous material compared to ZnO nanorods demonstrate a promising way of enhancing glucose biosensor performance to measure reliable intracellular glucose concentrations within single living cells.
35.	Gustavsson, Johanna, 1956-, et al. (författare) Localization of the insulin receptor in caveolae of adipocyte plasma membrane 1999 Ingår i: The FASEB Journal. - 0892-6638 .- 1530-6860. ; 13:14, s. 1961-1971 Tidskriftsartikel (refereegranskat)abstract The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with β-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. β-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.
36.	Johansson, Rickard, et al. (författare) A two-dimensional bootstrap approach to model discrimination Annan publikation (övrigt vetenskapligt/konstnärligt)
37.	Johansson, Rikard, et al. (författare) Combining test statistics and models in bootstrapped model rejection : it is a balancing act 2014 Ingår i: BMC Systems Biology. - : BioMed Central (BMC). - 1752-0509. ; 8:46 Tidskriftsartikel (refereegranskat)abstract Background: Model rejections lie at the heart of systems biology, since they provide conclusive statements: that the corresponding mechanistic assumptions do not serve as valid explanations for the experimental data. Rejections are usually done usinge.g. the chi-square test (χ2) or the Durbin-Watson test (DW). Analytical formulas for the corresponding distributions rely on assumptions that typically are not fulfilled. This problem is partly alleviated by the usage of bootstrapping, a computationally heavy approach to calculate an empirical distribution. Bootstrapping also allows for a natural extension to estimation of joint distributions, but this feature has so far been little exploited.Results: We herein show that simplistic combinations of bootstrapped tests, like the max or min of the individual p-values, give inconsistent, i.e. overly conservative or liberal, results. A new two-dimensional (2D) approach based on parametric bootstrapping, on the other hand, is found both consistent and with a higher power than the individual tests, when tested on static and dynamic examples where the truth is known. In the same examples, the most superior test is a 2D χ2 vs χ2, where the second χ2-value comes from an additional help model, and its ability to describe bootstraps from the tested model. This superiority is lost if the help model is too simple, or too flexible. If a useful help model is found, the most powerful approach is the bootstrapped log-likelihood ratio (LHR). We show that this is because the LHR is one-dimensional, because the second dimension comes at a cost, and because LHR has retained most of the crucial information in the 2D distribution. These approaches statistically resolve a previously published rejection example for the first time.Conclusions: We have shown how to, and how not to, combine tests in a bootstrap setting, when the combinatio is advantageous, and when it is advantageous to include a second model. These results also provide a deeper insight into the original motivation for formulating the LHR, for the more general setting of nonlinear and non-nested models. These insights are valuable in cases when accuracy and power, rather than computational speed, are prioritized.
38.	Johansson, Rikard, 1982-, et al. (författare) Elucidating mechanisms of early insulin signaling in primary adipocytes and hepatocytes : a joint systems biology effort 2009 Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Type II diabetes is one of the most common diseases afflicting people today. Understanding how this disease works, not only on a cellular level and between different organs and tissues, but also how it affects whole body level homeostasis is crucial for enhancement of its treatment. We use model-bases analysis as a tool for distinguishing different biological hypothesis on the system behavior.The Insulin Receptor (IR), is located in the cell membrane as a dimer, and thus has the potential two bind two different insulin molecules. It can also undergo a series of phosphorylations, as well as having the ability to become internalized, and thus be removed from the cell’s censing area. However, it can then be recycled back to the membrane again. The major target of IR is the Insulin Receptor Substrate 1 (IRS1). IRS1 in turn mediates the signal further downstream through Protein Kinase B (PBK) and mammalian Target of Rapamycin (mTOR). In adipocytes the end result is the translocation of internal vesicles containing Glucose Transporters (GLUT4) to the membrane, thus increasing the uptake of glucose. The liver, on the other hand, responds by down regulating the endogenous glucose production.The activity of IRS1 is determined by its phospho-tyrosine composition. This in turn is regulated by at least two serine-phosphorylations, on ser307 and ser312. The serine levels of this protein are regulated by downstream kinases, of which only one is known, S6K. The ser307 phosphorylation appears to allow for a short term positive feedback while the ser312 phosphorylation has the dynamics of a more long term negative feedback.The overall dynamics of the IRS1 tyrosine phosphorylation is a mirror of that of the Insulin Receptor. They both have a quick response to insulin within minutes, manifested as a high overshoot before declining to a steady state level. The overshoot behavior of this system can be explained either by a downstream negative feedback, or by having an advanced internalization and recycling model. Several hypotheses of the negative feedback mechanisms necessary to allow for the receptor to adopt such a behavior have previously been rejected by us. So has the hypothesis of internalization (unpublished data). The internalized Insulin Receptors can account for only a small fraction of the total amount of receptors, it however seems to be necessary for its own down regulation, since without it the overshoot behavior disappears.The complexity of this system is immense and hence we keep to as minimal models as possible, only considering adding complexity to the system when data indicates so, or when a simpler model structure has been rejected. We model the system with a series of Ordinary Differential Equations (ODEs), optimize and estimate the parameters of a given model structure with the Systems Biology Toolbox (SBTB) and reject, or fail to reject, models based on their statistical agreement with our data. We search the entire approximated parameter space for a sample of all acceptable parameter values for any given parameter. We then look for commonalities shared between model simulations of all parameter sets in the sample. That is, a behavior of e.g. a state in the model that has to be above a certain threshold for it to be able to explain the data, while other states might be of arbitrary sizes. If we find such a commonality, we call it a core prediction. Assuming your data is correct and your analysis thorough, a Core Prediction has the same strength as a model rejection. The common aspect, shared between all acceptable parameter etc, is something that has to be true, no matter how much more data you acquire. One such core prediction, which led to the rejection of the internalization hypothesis, was that the amount of internalized IR had to be above 80% of the total receptor pool. We subsequently rejected this experimentally.
39.	Johansson, Rikard, 1982- (författare) Model-Based Hypothesis Testing in Biomedicine : How Systems Biology Can Drive the Growth of Scientific Knowledge 2017 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The utilization of mathematical tools within biology and medicine has traditionally been less widespread compared to other hard sciences, such as physics and chemistry. However, an increased need for tools such as data processing, bioinformatics, statistics, and mathematical modeling, have emerged due to advancements during the last decades. These advancements are partly due to the development of high-throughput experimental procedures and techniques, which produce ever increasing amounts of data. For all aspects of biology and medicine, these data reveal a high level of inter-connectivity between components, which operate on many levels of control, and with multiple feedbacks both between and within each level of control. However, the availability of these large-scale data is not synonymous to a detailed mechanistic understanding of the underlying system. Rather, a mechanistic understanding is gained first when we construct a hypothesis, and test its predictions experimentally. Identifying interesting predictions that are quantitative in nature, generally requires mathematical modeling. This, in turn, requires that the studied system can be formulated into a mathematical model, such as a series of ordinary differential equations, where different hypotheses can be expressed as precise mathematical expressions that influence the output of the model.Within specific sub-domains of biology, the utilization of mathematical models have had a long tradition, such as the modeling done on electrophysiology by Hodgkin and Huxley in the 1950s. However, it is only in recent years, with the arrival of the field known as systems biology that mathematical modeling has become more commonplace. The somewhat slow adaptation of mathematical modeling in biology is partly due to historical differences in training and terminology, as well as in a lack of awareness of showcases illustrating how modeling can make a difference, or even be required, for a correct analysis of the experimental data.In this work, I provide such showcases by demonstrating the universality and applicability of mathematical modeling and hypothesis testing in three disparate biological systems. In Paper II, we demonstrate how mathematical modeling is necessary for the correct interpretation and analysis of dominant negative inhibition data in insulin signaling in primary human adipocytes. In Paper III, we use modeling to determine transport rates across the nuclear membrane in yeast cells, and we show how this technique is superior to traditional curve-fitting methods. We also demonstrate the issue of population heterogeneity and the need to account for individual differences between cells and the population at large. In Paper IV, we use mathematical modeling to reject three hypotheses concerning the phenomenon of facilitation in pyramidal nerve cells in rats and mice. We also show how one surviving hypothesis can explain all data and adequately describe independent validation data. Finally, in Paper I, we develop a method for model selection and discrimination using parametric bootstrapping and the combination of several different empirical distributions of traditional statistical tests. We show how the empirical log-likelihood ratio test is the best combination of two tests and how this can be used, not only for model selection, but also for model discrimination.In conclusion, mathematical modeling is a valuable tool for analyzing data and testing biological hypotheses, regardless of the underlying biological system. Further development of modeling methods and applications are therefore important since these will in all likelihood play a crucial role in all future aspects of biology and medicine, especially in dealing with the burden of increasing amounts of data that is made available with new experimental techniques.
40.	Jufvas, Åsa, et al. (författare) Global differences in specific histone H3 methylation are associated with overweight and type 2 diabetes. 2013 Ingår i: Clinical Epigenetics. - : BioMed Central. - 1868-7083 .- 1868-7075. ; 5:1 Tidskriftsartikel (refereegranskat)abstract BACKGROUND: Epidemiological evidence indicates yet unknown epigenetic mechanisms underlying a propensity for overweight and type 2 diabetes. We analyzed the extent of methylation at lysine 4 and lysine 9 of histone H3 in primary human adipocytes from 43 subjects using modification-specific antibodies.RESULTS: The level of lysine 9 dimethylation was stable, while adipocytes from type 2 diabetic and non-diabetic overweight subjects exhibited about 40% lower levels of lysine 4 dimethylation compared with cells from normal-weight subjects. In contrast, trimethylation at lysine 4 was 40% higher in adipocytes from overweight diabetic subjects compared with normal-weight and overweight non-diabetic subjects. There was no association between level of modification and age of subjects.CONCLUSIONS: The findings define genome-wide molecular modifications of histones in adipocytes that are directly associated with overweight and diabetes, and thus suggest a molecular basis for existing epidemiological evidence of epigenetic inheritance.
41.	Jufvas, Åsa, et al. (författare) Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells 2011 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:1 Tidskriftsartikel (refereegranskat)abstract Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 mu g of protein with histone content of 60%-70% from 10 mL of fat cells. Differential proteolysis of the protein extracts by trypsin or ArgC-protease followed by nanoLC/MS/MS with alternating CID/ETD peptide sequencing identified 19 histone variants. Four variants were found at the protein level for the first time; particularly HIST2H4B was identified besides the only H4 isoform earlier known to be expressed in humans. Three of the found H2A potentially organize small nucleosomes in transcriptionally active chromatin, while two H2AFY variants inactivate X chromosome in female cells. HIST1H2BA and three of the identified H1 variants had earlier been described only as oocyte or testis specific histones. H2AFX and H2AFY revealed differential and variable N-terminal processing. Out of 78 histone modifications by acetylation/trimethylation, methylation, dimethylation, phosphorylation and ubiquitination, identified from six subjects, 68 were found for the first time. Only 23 of these modifications were detected in two or more subjects, while all the others were individual specific. The direct acid extraction of adipocytes allows for personal epigenetic analyses of human fat tissue, for profiling of histone modifications related to obesity, diabetes and metabolic syndrome, as well as for selection of individual medical treatments.
42.	Jufvas, Åsa, 1982- (författare) Human Adipocytes : Proteomic Approaches 2016 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Type 2 diabetes is characterized by increased levels of glucose in the blood originating from insulin resistance in insulin sensitive tissues and from reduced pancreatic insulin production. Around 400 million people in the world are diagnosed with type 2 diabetes and the correlation with obesity is strong. In addition to life style induction of obesity and type 2 diabetes, there are indications of genetic and epigenetic influences. This thesis has focused on the characterization of primary human adipocytes, who play a crucial role in the development of type 2 diabetes.Histones are important proteins in chromatin dynamics and may be one of the factors behind epigenetic inheritance. In paper I, we characterized histone variants and posttranslational modifications in human adipocytes. Several of the specific posttranslational histone modifications we identified have been characterized in other cell types, but the majority was not previously known. Moreover, we identified a variant of histone H4 on protein level for the first time.In paper II, we studied specific histone H3 methylations in the adipocytes. We found that overweight is correlated with a reduction of H3K4me2 while type 2 diabetes is associated with an increase of H3K4me3. This shows a genome-wide difference in important chromatin modifications that could help explain the epidemiologically shown association between epigenetics and metabolic health.Caveolae is a plasma membrane structure involved in the initial and important steps of insulin signaling. In paper III we characterized the IQGAP1 interactome in human adipocytes and suggest that IQGAP1 is a link between caveolae and the cytoskeleton. Moreover, the amount of IQGAP1 is drastically lower in adipocytes from type 2 diabetic subjects compared with controls implying a potential role for IQGAP1 in insulin resistance.In conclusion, this thesis provides new insights into the insulin signaling frameworks and the histone variants and modifications of human adipocytes.
43.	Jufvas, Åsa, et al. (författare) Scaffolding protein IQGAP1: an insulin-dependent link between caveolae and the cytoskeleton in primary human adipocytes? 2016 Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 473:19, s. 3177-3188 Tidskriftsartikel (refereegranskat)abstract The ubiquitously expressed IQ motif-containing GTPase activating protein-1 (IQGAP1) is a scaffolding protein implicated in an array of cellular functions, in particular by binding to cytoskeletal elements and signaling proteins. A role of IQGAP1 in adipocytes has not been reported. We therefore investigated the cellular IQGAP1 interactome in primary human adipocytes. Immunoprecipitation and quantitative mass spectrometry identified caveolae and caveolae-associated proteins as the major IQGAP1 interactors alongside cytoskeletal proteins. We confirmed co-localization of IQGAP1 with the defining caveolar marker protein caveolin-1 by confocal microscopy and proximity ligation assay. Most interestingly, insulin enhanced the number of IQGAP1 interactions with caveolin-1 by five-fold. Moreover, we found a significantly reduced abundance of IQGAP1 in adipocytes from patients with type 2 diabetes compared with cells from nondiabetic control subjects. Both the abundance of IQGAP1 protein and mRNA were reduced, indicating a transcriptional defect in diabetes. Our findings suggest a novel role of IQGAP1 in insulin-regulated interaction between caveolae and cytoskeletal elements of the adipocyte, and that this is quelled in the diabetic state.
44.	Jullesson, David, 1987, et al. (författare) Dominant negative inhibition data should be analyzed using mathematical modeling - Re-interpreting data from insulin signaling 2015 Ingår i: FEBS Journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 282:4, s. 788- Tidskriftsartikel (refereegranskat)abstract As our ability to measure the complexity of intracellular networks has evolved, it has become increasingly clear that we need new methods for data analysis: methods involving mathematical modeling. Nevertheless, it is still uncontroversial to publish and interpret experimental results without a model-based proof that the reasoning is correct. In the present study, we argue that this attitude probably needs to change in the future. We illustrate this need for modeling by considering the common experimental technique of using dominant-negative constructs. More specifically, we consider published time-series and dose-response data which previously have been used to argue that the protein S6 kinase does not phosphorylate insulin receptor substrate-1 at a specific serine residue. Using a presented general approach to interpret such data, we now demonstrate that the given dominant-negative data are not conclusive (i.e. that in the absence of other proofs, S6 kinase still may be the kinase). Using simulations with uncertainty analysis and analytical solutions, we show that an alternative explanation is centered around depletion of substrate, which can be tested experimentally. This analysis thus illustrates both the necessity and the benefits of using mathematical modeling to fully understand the implications of biological data, even for a small system and relatively simple data. Dominant negative inhibition data is commonly used to experimentally unravel signaling systems. We show that the traditional interpretation of such data is incomplete, and propose an improved model-based approach. The improvements are demonstrated on real data from insulin signaling. These results demonstrate the need for mathematical modeling as a tool for data analysis, even for small systems and simple data.
45.	Jönsson, Cecilia, et al. (författare) Insulin and beta-adrenergic receptors mediate lipolytic and anti-lipolytic signalling that is not altered by type 2 diabetes in human adipocytes 2019 Ingår i: Biochemical Journal. - : PORTLAND PRESS LTD. - 0264-6021 .- 1470-8728. ; 476, s. 2883-2908 Tidskriftsartikel (refereegranskat)abstract Control of fatty acid storage and release in adipose tissue is fundamental in energy homeostasis and the development of obesity and type 2 diabetes. We here take the whole signalling network into account to identify how insulin and beta-adrenergic stimulation in concert controls lipolysis in mature subcutaneous adipocytes obtained from non-diabetic and, in parallel, type 2 diabetic women. We report that, and show how, the anti-lipolytic effect of insulin can be fully explained by protein kinase B (PKB/Akt)-dependent activation of the phosphodiesterase PDE3B. Through the same PKB-dependent pathway beta-adrenergic receptor signalling, via cAMP and PI3K alpha, is anti-lipolytic and inhibits its own stimulation of lipolysis by 50%. Through this pathway both insulin and beta-adrenergic signalling control phosphorylation of FOXO1. The dose-response of lipolysis is bell-shaped, such that insulin is anti-lipolytic at low concentrations, but at higher concentrations of insulin lipolysis was increasingly restored due to inhibition of PDE3B. The control of lipolysis was not altered in adipocytes from diabetic individuals. However, the release of fatty acids was increased by 50% in diabetes due to reduced reesterification of lipolytically liberated fatty acids. In conclusion, our results reveal mechanisms of control by insulin and beta-adrenergic stimulation - in human adipocytes - that define a network of checks and balances ensuring robust control to secure uninterrupted supply of fatty acids without reaching concentrations that put cellular integrity at risk. Moreover, our results define how selective insulin resistance leave lipolytic control by insulin unaltered in diabetes, while the fatty acid release is substantially increased.
46.	Karlsson, Cecilia, 1983- (författare) Insulin Signalling in Human Adipocytes and its Interplay with beta-Adrenergic Control of Lipolysis 2018 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The prevalence of obesity has over the last 40 years nearly tripled and obesity is one of the major risk factors of developing type 2 diabetes. Type 2 diabetes was formerly called adultonset diabetes but today, probably due to the rise in childhood obesity, it is also seen in children and adolescents. Type 2 diabetes is diagnosed when the body no longer can control the glucose levels in the blood. This is due to an insulin resistant state in the insulin responding tissues, liver, adipose and muscle and insufficient production of insulin in the pancreas. However, in spite of extensive research the mechanisms behind insulin resistance is still not known.The adipose tissue is believed to play a major role in the development of whole body insulin resistance. Adipocytes are the most important sites for storage of the high energy containing triacylglycerols. Insulin stimulation causes the adipocyte to increase the uptake of glucose and to reduce lipolysis: the hydrolysis of triacylglycerol and release of glycerol and fatty acids. The insulin signalling network is complex with numerous proteins involved. These signaling proteins not only transmit the insulin signal but also create negative and positive feedbackloops and induce cross talk between different parts of the network and with the signalling of other hormones. One important positive feedback in insulin signalling is the mTORC1 mediated feedback to phosphorylation of IRS1 at serine 307. In paper I we found that in human adipocytes this feedback is not likely catalysed by the assumed kinase S6K1. However we find an immunoprecipitate of mTOR to contain a ser307 phosphorylating kinase.Scaffolding proteins serve as docking sites for several proteins to promote protein-protein interactions that facilitate signal transduction. In paper II we demonstrate the existence of the scaffolding protein IQGAP1 in human adipocytes and that the expression of IQGAP1 is downregulated in type 2 diabetes. We reveal that IQGAP1 co-localises with caveolae, invaginations of the plasma membrane where the insulin receptor is situated, and that this interaction is increased upon insulin stimulation.In paper III we focus on the control of lipolysis, and sought to understand the interplay between insulin and beta-adrenergic stimulation. We demonstrated that the re-esterification of fatty acids is downregulated in type 2 diabetes causing an increased release of fatty acids from the cells. We showed that beta-adrenergic stimulation with isoproterenol induced a negative feedback via PKA/Epac1 -> PI3K -> PKB -> PDE3B that reduced the cAMP levels and thereby also reduced lipolysis. We also showed that insulin, in addition to its well-known anti-lipolytic effect, at high concentrations had a positive effect on lipolysis. In conclusion we reveal an intricate control of the stimulation as well as the inhibition of lipolysis induced by both isoproterenol and insulin.
47.	Karlsson, Margareta, 1942-, et al. (författare) Colocalization of insulin receptor and insulin receptor substrate-1 to caveolae in primary human adipocytes 2004 Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:12, s. 2471-2479 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.
48.	Karlsson, Margareta, et al. (författare) In human adipocytes the insulin receptor and IRS1 are localized in caveolae, and caveolae destruction makes cells resistant to insulin signaling for metabolic and mitogenic control Annan publikation (övrigt vetenskapligt/konstnärligt)abstract Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated sigoaling. Here we show that in human adipocytes the iosulin receptor is localized in caveolae: by electron microscopy and immunogold detection and by isolating caveolae from plasma membranes. We similarly demonstrate that significant part of the immediate downstream signal mediator IRS1 is localized at the plasma membrane and caveolae. A detailed image shows the caveola as a bulb, protroding into the cell interior, with a neck attaching it to the plasma membrane. The caveolar structural protein caveolin is localized in the neck aod not in the bulb of the caveola. The receptor is active in caveolae since insulin stimulation caused tyrosine specific phosphorylation of the receptor recovered in isolated caveolae. Caveolae contain a major part of the free cholesterol in the plasma membrane and cholesterol is a stroctural component of caveolae. Depletion of cholesterol from the cells using B-cyclodextrio blocks insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulio stimulation of protein kinase B and Map-kinases ERK1/2 phosphorylation- in effect making the human adipocytes insulin resistant. The insulin-stimulated phosphorylation of the insulin receptor and IRS1 are, however, not affected, indicating that caveolae integrity is required downstream of IRS1, consistent with its colocalization with the insulin receptor io caveolae in human adipocytes.
49.	Karlsson, Margareta, 1942-, et al. (författare) Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes 2002 Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 16:2, s. 249-251 Tidskriftsartikel (refereegranskat)abstract Insulin-stimulated glucose uptake in muscle and adipose tissue is the result of translocation of insulin-regulated glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane. Here we report that GLUT4 in the plasma membrane of 3T3-L1 adipocytes were located predominantly in caveolae invaginations: by immunogold electron microscopy of plasma membranes, 88% of GLUT4 were localized to caveolae structures and this distribution within the plasma membrane was not affected by insulin. By immunofluorescence microscopy, a major part of GLUT 4 was colocalized with caveolin. The total amount of GLUT4 in the plasma membrane increased 2.2-fold in response to insulin as determined by immunogold electron or immunofluorescence microscopy. GLUT4 were enriched in caveolae fractions isolated without detergents from plasma membranes of rat adipocytes. In these fractions, GLUT4 were largely confined to caveolin-containing membranes of the caveolae preparation isolated from insulin-stimulated cells, determined by electron microscopy. Insulin increased the amount of GLUT4 2.7-fold in this caveolae fraction. Caveolae were purified further by immunoisolation with antibodies against caveolin. The amount of GLUT4 increased to the same extent in the immunopurified caveolae as in the cruder caveolae fractions from insulin-stimulated cells. We conclude that insulin induces translocation of GLUT4 to caveolae.
50.	Karlsson, Margareta, et al. (författare) Lipid composition of caveolae and of surrounding plasma membrane in rat adipocytes Annan publikation (övrigt vetenskapligt/konstnärligt)abstract Caveolae are invaginations of the plasma membrane that may arise from so called rafts in the presence of the structural protein caveolin. We have isolated caveolae from purified plasma membrane of primary rat adipocytes using ultrasonication to disrupt the membrane followed by density gradient ultracentrifugation. This caveolae fraction was further purified by adsorption to antibodies against caveolin. As a comparison we also isolated a detergent-insoluble fraction of the plasma membrane, utilizing the detergent insolubility of caveolae and rafts. Caveolae were strongly enriched in cholesterol and sphingomyelin, the concentration was 3.5 and 2.8-fold, respectively, higher in the caveolar membrane than in the surrounding plasma membrane. Phosphoacylglycerols were also concentrated in caveolae, while proteins were depleted compared to the surrounding plasma membrane. We have calculated that an average adipocyte caveola contains 18000 molecules of cholesterol, 6000 of sphingomyelin, 18000 of phosphoacylglycerol, 350 protein molecules, and about I 00 glycolipid molecules.We analyzed for a range of glycolipids and especially gangliosides. Of these GM3 and GD3 are the most prevalent and both were enriched in caveolae, together with GM1 and GDla. GDlb and GTib were present in the plasma membrane at low levels, while GM2, GD2, GQ1b, sulphatide, and lactosylceramide sulphate were not detected. None of them were detected in caveolae. As a first comprehensive and quantitative analysis of purified caveolae from primary cells, our results provide a firm basis for the examination of caveolae formation using artificial membranes.

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