Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

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, t 1/2,3 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 cocaptured 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 pitmediated 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 primar

The impact of flooding on the value of residential property in the UK

Flooding of residential property is a real and growing phenomenon in the UK causing short and long-term detriment of various kinds to its victims. The issue of potential decrease in value of those properties which are located on the floodplain, though much discussed in the media, has received scant attention in the UK research literature. An extensive literature survey has revealed a need for methodological innovation in the field of temporal impact of flooding and the inadequacy of the current paradigms for inclusion of insurance into flood modelling. A wide-ranging review of data sources, including discussion with industry experts, has identified the requirement to generate primary data on the availability and cost of flood insurance. A novel framework has been developed for this research. This framework is an extension of the recent research in flood modelling and incorporates ideas from the wider house price analysis literature. Data collected via a questionnaire survey of householders has been combined with secondary data on property prices and flood designation in order to attribute any loss in property value to the correct vector of underlying flood status. The output from this study makes a contribution to the understanding of the impact of flooding on house prices, allowing for better valuation advice. Empirical findings are that the understandable concerns of residential property owners at risk of flooding regarding long term loss of property value are largely unfounded. Price discounts are observed for some recently flooded areas but they are temporary Improved appreciation of the impact of claims and flood risk on the cost of insurance has also emerged. The insurance market was not found to be instrumental in reducing the price of property. The output from the study also makes a methodological contribution in extending concepts relating to the relationship between flooding, insurance and house prices. This development is anticipated to facilitate refinement and updating of the empirical findings with reduced effort in the light of future events.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Insulin signaling in primary adipocytes in insulin sensitive and insulin resistant states

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

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-21319 Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocyte

response to insulin

Mechanistic explanations for counter-intuitive phosphorylation dynamics of the insulin receptor and insulin receptor substrate-1 i

Getting the conclusive lead with investigative genetic genealogy : A successful case study of a 16 year old double murder in Sweden

On the morning of October 19, 2004, an eight-year-old boy and a 56-year-old woman were stabbed to death on an open street in the city of Linko center dot ping, Sweden. The perpetrator left his DNA at the crime scene, and after 15 years of various investigation efforts, including more than 9000 interrogations and mass DNA screening of more than 6000 men, there were still no clues about the identity of the unknown murderer. The successful application of investigative genetic genealogy (IGG) in the US raised the interest for this tool within the Swedish Police Authority. After legal consultations it was decided that IGG could be applied in this double murder case as a pilot case study. From extensive DNA analysis, including whole-genome sequencing and genotype imputation, DNA data sets were established and searched within both GEDmatch and FamilyTree DNA genealogy databases. A number of fairly distant relatives were found from which family trees were created. The genealogy work resulted in two candidates, two brothers, one of whom matched the crime scene samples by routine STR profiling. The suspect confessed the murders at the initial police hearing and was later convicted of the murders. In this paper we describe the successful application of an emerging technology. We disclose details of the DNA analyses which, due to the poor quality and low quantity of the DNA, required reiterative sequencing and genotype imputation efforts. The successful application of IGG in this double murder case exemplifies its applicability not only in the US but also in Europe. The pressure is now high on the involved authorities to establish IGG as a tool for cold case criminal investigations and for missing person identifications. There is, however, a continuous need to accommodate legal, social and ethical aspects as well.Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation</p

Phosphorylation of IRS1 at Serine 307 in Response to Insulin in Human Adipocytes Is Not Likely to be Catalyzed by p70 Ribosomal S6 Kinase

The insulin receptor substrate-1 (IRS1) is phosphorylated on serine 307 (human sequence, corresponding to murine serine 302) in response to insulin as part of a feedback loop that controls IRS1 phosphorylation on tyrosine residues by the insulin receptor. This in turn directly affects downstream signaling and is in human adipocytes implicated in the pathogenesis of insulin resistance and type 2 diabetes. The phosphorylation is inhibited by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). The mTORC1-downstream p70 ribosomal protein S6 kinase (S6K1), which is activated by insulin, can phosphorylate IRS1 at serine 307 in vitro and is considered the physiological protein kinase. Because the IRS1 serine 307-kinase catalyzes a critical step in the control of insulin signaling and constitutes a potential target for treatment of insulin resistance, it is important to know whether S6K1 is the physiological serine 307-kinase or not. We report that, by several criteria, S6K1 does not phosphorylate IRS1 at serine 307 in response to insulin in intact human primary adipocytes: (i) The time-courses for phosphorylation of S6K1 and its phosphorylation of S6 are not compatible with the phosphorylation of IRS1 at serine 307; (ii) A dominant-negative construct of S6K1 inhibits the phosphorylation of S6, without effect on the phosphorylation of IRS1 at serine 307; (iii) The specific inhibitor of S6K1 PF-4708671 inhibits the phosphorylation of S6, without effect on phosphorylation of IRS1 at serine 307. mTOR-immunoprecipitates from insulin-stimulated adipocytes contains an unidentified protein kinase specific for phosphorylation of IRS1 at serine 307, but it is not mTOR or S6K1.Funding Agencies|Swedish Diabetes Fund||Novo Nordic Foundation||University of Linkoping||Swedish Research Council||</p

Cholesterol depletion of the cells blocks insulin receptor endocytosis.

<p>Isolated adipocytes were incubated with or without 10 mM β-cyclodextrin (β−CD) for 50 min, to deplete the plasma membrane of about 50% of its cholesterol, which completely destroys caveolae while leaving the cells intact <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005985#pone.0005985-Parpal1" target="_blank">[23]</a>. Insulin at 100 nM was added for another 5 min. Cells were then homogenized and plasma membrane (pm) and endosomal (endo) fractions isolated. A. Equal amounts of protein were subjected to SDS-PAGE and immunoblotting with (from the top) antibodies against insulin receptor β-subunit (IR) or phosphotyrosine (indicated is insulin receptor, IR-TyrP). B. Quantitation of three experiments. Presented as amount of IR or IR-TyrP in the presence of β-cyclodextrin as % of controls without β-cyclodextrin, mean±SE. *, p<0.05 by Student’s t-test.</p

Localization of insulin receptor and caveolin-1 in endosomes by immuno-gold electron microscopy.

<p>Isolated adipocytes were incubated with insulin at 100 nM for 10 min. Cells were then homogenized and the endosomal fraction isolated. Endosome vesicles were attached to grids, immunogold-labeled against caveolin-1 (6 nm gold particles) and the insulin receptor (15 nm gold particles), lyophilized and sputtered with a 2-nm tungsten film before examination by transmission electron microscopy. C and D are blow-ups from B; arrowheads indicate patches of caveolin-1 labeling; arrows indicate insulin receptor labeling. One experiment of three with similar results is illustrated.</p