| 1. |
- Aili Fagerholm, Siri, 1980-
(författare)
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Insulin signaling in primary adipocytes in insulin sensitive and insulin resistant states
- 2013
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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.
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| 2. |
- Loinder, Kristina, 1963-
(författare)
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Nuclear receptor corepressor N-CoR : role in transcriptional repression
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human body consists of a multitude of cells of varying appearance and function. With a few exceptions they are genetically identical, and the key to their divergence lies in their different specific patterns of gene expression. Gene expression may be regulated at the level of transcription, in two opposing directions; either activation or repression. Gene transcription is controlled by transcription factors, which bind to regulatory DNA sequences, and direct gene expression in concert with auxiliary proteins. Among these the nuclear receptor corepressor N-CoR holds a central position. It serves as a docking unit between many different DNA-bound transcription factors, such as nuclear receptors, and large complexes of repressor proteins. Many repressor complexes of distinct compositions have been shown to contain N-CoR.N-CoR plays a vital part in normal fetal development, and its involvement has been implicated in several pathological conditions. It has been shown to interact with unliganded nuclear receptors via CoRNR-box motifs in the C-terminal half of the protein. We have identified an NR-box motif, typical of coactivators, in the N-terminal part of N-CoR, which we have shown to be capable of interacting with the nuclear receptors RARα and TRß both in vitro and in vivo. A mutated NR-box motif did not interact accordingly. We discovered that the NR-box motif found in N-CoR displayed a ligand-dependent interaction with TRß in GST pulldown experiments, and that the immediate NR-box environment in N-CoR resembles NRbox environments in the coactivator CBP. We investigated a possible role for theN-CoR NRbox motif in regulation of the TSHß gene from a negative TR response element found in its promoter. In transient transfectiqns of GH3 cells, we found that both TRß3 and N-CoR are necessary for ligand-induced repression from this response element to occur. Mutating the NR-box abolished the repressive potential of N-CoR. The results were corroborated by results from transient transfections of HEK293/T cells, where siRNA-targeted degradation of endogenous N-CoR mRNA annihilated the ligand-induced repression, and where wild type mouse N-CoR but not mutated N-CoR restored the repression. In vitro binding assays also showed that TR, bound to its negative response element in the TSHß gene promoter, displayed an obligate ligand dependence in its interaction with N-CoR.In several different leukemias N-CoR holds a key role. Abberant transcription factors bind stronger toN-CoR than their normal counterparts, leading to constitutive repression of key genes in hematopoiesis. Retinoid signaling, mediated by RARs plays a central part in differentiation of myeloid cells. We therefore investigated the extent of N-CoR expression in the myeloid cell line THP-1 during differentiation. Analyses both at mRNA- and at protein level showed that N-CoR expression was down-regulated as the myeloid cells differentiated. Exploring the effects of this on genes controlled by retinoic acid, we found in transient transfections of THP-1 cells that N-CoR modulated the expression level both at basal and at ligand-activated level. Several reports by others have also emphasized the importance of relative levels of different coregulatory proteins for determining the amplitude of the transcriptional response.N-CoR binds both to transcription factors and to repressor complexes, but so far no report has been published regarding its possible DNA-binding capacity, anticipated by analysis of its amino acid sequence. Employing the selected and amplified binding sites (SAAB) assay we showed that N-CoR bound to DNA. Sequence determination resulted in the identification of a DNA sequence, ATNNTNCTC, which binds specifically toN-CoR. This finding adds another variable in the spectrum of N-CoR interactions.
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| 3. |
- Sauma, Lilian, 1976-
(författare)
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Transcriptional activity of PPARγ in primary human adipocytes
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The prevalence of obesity is increasing in most parts of the world and is a strong risk factor for the development of insulin resistance, type 2 diabetes and hypertension. Adipose tissue is mainly composed of adipocytes which store energy in the form of triglycerides and release it as free fatty acids. Adipose tissue is one of the major regulators of energy homeostasis in the body. Adipose tissue in different regions of the body has different characteristics and adipocytes in intra-abdominal fat depots are more associated with insulin resistance than adipocytes from subcutaneous fat depots.Research performed during the past several years has led to an explosion in the understanding of adipose tissue and the active role that it plays in aspects of physiology and pathophysiology. One important discovery has been identification of the nuclear hormone receptor called peroxisome proliferator-activated receptor γ (PPARγ). Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor, which is highly expressed in adipocytes. PPARγ has been shown to affect several genes of importance for lipid metabolism, differentiation of fat cells and insulin sensitivity. The PPARγ receptor can be activated by thiazolidinediones (TZD), a class of insulinsensitising drugs, which promote fatty acid storage in fat depots and decrease glucose levels in plasma, thus, demonstrating the importance of PPARγ activity in insulin resistance and metabolic syndrome.This thesis has investigated the transcriptional activity of PPARγ in a clinically relevant cell type for insulin resistance and type 2 diabetes; the primary human adipocyte. For this purpose, a method for transfection of primary human adipocytes by electroporation and for measurement of the activity of PPARγ has been developed and optimised. This method has been used to study the effect of saturated and unsaturated fatty acids on the transcriptional activity of PPARγ. Interestingly, it was been found that saturated fatty acids can activate PPARγ, thus promoting a protection against diabetes. The strongest activator was the monounsaturated palmitoleic acid. The transcriptional activity of PPARγ in primary human adipocytes from intra-abdominal and subcutaneous adipose tissues was also examined. It was found that PPARγ activity is considerably lower in adipocytes from visceral compared with subcutaneous fat from the same subject. Another reason for using human tissue to reach clinical relevance shown here was that the same difference in PPARγ activity could not be found between intra-abdominal and subcutaneous fat tissues in mice. This finding may serve as the basis of why excess intraabdominal fat tissue is associated with high risk for development of type 2 diabetes and cardiovascular diseases.The blood pressure regulating renin-angiotensin system (RAS) in human adipose tissue and in isolated adipocytes was examined and related to PPARγ. It was found that the production of angiotensin II, which is an important hormone for increasing the blood pressure, can be produced by isolated adipocytes and that the production is higher in adipocytes coming from omental than subcutaneous fat tissue. Further, it was shown that angiotensin II inhibits PPARγ activity in omental adipocytes, thus reducing the insulin sensitivity. Therefore, this study connects two of the major risk factors in obesity; diabetes and hypertension, and may also explain how drugs, which inhibit the RAS, can also be protective against diabetes. In conclusion, the findings in this thesis give new knowledge about regulating mechanisms of fat cells and its importance in diabetes and cardiovascular disease.
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| 4. |
- Strid, Tobias, 1982-
(författare)
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The enzymatic machinery of leukotriene biosynthesis : Studies on ontogenic expression, interactions and function
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Leukotrienes (LTs) are biologically active arachidonic acid (AA) derivatives generated by the 5-lipoxygenase (5-LO) pathway. They are produced by myeloid cells. 5-LO converts AA to LTA4 in cooperation with 5-LO activating protein (FLAP). LTA4 is converted to LTB4, by LTA4-hydrolase (LTA4H) or to LTC4 by LTC4-synthase (LTC4S). LTs act on cells through plasma membrane bound G-protein coupled receptors found on leukocytes, smooth muscle and endothelial cells. We report here protein-protein interactions of proteins involved in LTC4 synthesis. 5-LO interacts with cytosolic domains of the integral membrane proteins FLAP and LTC4S at the nuclear envelope, in addition LTC4S interacts with FLAP through its hydrophobic membrane spanning regions. We constructed an LTC4S promoter controlled GFP reporter vector, displaying cell specific expression and sensitivity to agents known to affect LTC4S expression. The vector was used to create transgenic mice expressing GFP as a reporter for LTC4S. Ontogenic mouse expression studies revealed that the complete LT biosynthesis machinery was present at e11.5 primarily in the hematopoietic cells colonizing the liver. Although mature myeloid cells were the main contributors, a substantial amount of FLAP message was also detected in hematopoietic stem and progenitor cells, indicating possible functions for FLAP in hematopoietic regulation. Functional analyses using FLAP knockout mice suggested fine-tuning roles for LTs during differentiation, primarily along the B-lymphocyte differentiation path.
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| 5. |
- Svartz, Jesper, 1972-
(författare)
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Leukotriene C4 synthase : studies on oligomerization and subcellular localization
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Leukotrienes (LTs) are polyunsaturated fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 can be hydrolyzed to LTB4, or be conjugated with glutathione forming LTC4. LTC4 together with its metabolites LTD4 and LTE4, formed by amino acid removal from the glutathione moiety, constitute the cysteinyl LTs that are the active compounds of "slow reacting substance of anaphylaxis" (SRS-A). LTC4 and LTD4 are involved in several inflammatory conditions, e.g. asthma and allergic rhinitis. The conversion of LTA4 to LTC4 is catalyzed by an integral membrane protein, LTC4 synthase (LTC4S), localized on the endoplasmic reticulum (ER) and nuclear envelope. This 150 amino acid protein has four transmembrane helices and two hydrophilic loops oriented to the lumen side of the ER membrane. LTC4S belongs to a family of proteins called membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG).We have shown that LTC4S and another MAPEG member, microsomal glutathione S-transferase (MGST)-1, interact and colocalize in transiently transfected cells. Coexpression decreased their catalytic activities indicating functional significance of the interaction between LTC4S and MGST1. LTC4S was demonstrated to form homo-oligomers in cell free assays using GST pulldown assays, as well as in living cells using bioluminescence resonance energy transfer (BRET) technique. When testing various truncated variants of LTC4S in BRET assays two hydrophobic regions were mapped as interaction domains: amino acids 6-27 and 114-135. GFP-fusion proteins containing the latter sequence also showed distinct ER/nuclear envelope localization and a minimal ER/nuclear envelope localization sequence was mapped to amino acids 117-132. In cell free assays we also demonstrated interactions between 5-LO, fivelipoxygenase activating protein (FLAP) and LTC4S. The second hydrophilic loop of LTC4S was found to be important for interaction with 5-LO, whereas the N-terminal part of LTC4S gave the strongest interaction with FLAP. LTC4 diminished the interaction between 5-LO and FLAP suggesting a feed-back regulatory mechanism. Our results concerning LTC4S oligomer formation and mapping of interaction domains may provide novel means to rational design of LTC4S inhibitors.
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