Dorothy Sears Worrall

Toll-Like Receptor 4–Dependent and –Independent Cytokine Secretion Induced by Minimally Oxidized Low-Density Lipoprotein in Macrophages

Objective—Innate immune responses to oxidized low-density lipoprotein LDL (LDL) regulate the development of atherosclerosis. We demonstrated previously that an early form of oxidized LDL, minimally modified LDL (mmLDL), triggers cytoskeletal rearrangements in macrophages via CD14 and Toll-like receptor 4 (TLR4)/MD-2. Because lipopolysaccharide (LPS) activation of TLR4 leads to proinflammatory gene expression, in this study, we asked whether mmLDL also induced proinflammatory signaling. Methods and Results—We studied cytokine secretion and signaling in J774 and primary peritoneal macrophages stimulated with mmLDL, which was prepared by incubating LDL with cells expressing human 15-lipoxygenase. MmLDL stimulated robust phosphoinositide 3-kinase (PI3K) activation, and Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which exceeded that induced by LPS. On the other hand, although mmLDL induced nuclear factor &kgr;B (NF-&kgr;B) p65 translocation to the nucleus, there was no detectable NF-&kgr;B activation. However, mmLDL induced early mRNA and protein expression of the cytokines MIP-2, MCP-1, tumor necrosis factor-α, and interleukin-6. Chemokine MIP-2 but not MCP-1 secretion depended on TLR4/MyD88, ERK1/2, and PI3K signaling. In turn, TLR4 regulated phosphorylation of ERK1/2 but not of Akt, suggesting that mmLDL-induced PI3K activation is TLR4 independent. Conclusions—In macrophages, mmLDL activates TLR4-dependent and -independent signaling pathways, resulting in secretion of proinflammatory cytokines. These results provide new insights into the inflammatory origins of atherosclerosis.

Variance-modeled posterior inference of microarray data: detecting gene-expression changes in 3T3-L1 adipocytes

MOTIVATION Microarrays are becoming an increasingly common tool for observing changes in gene expression over a large cross section of the genome. This experimental tool is particularly valuable for understanding the genome-wide changes in gene transcription in response to thiazolidinedione (TZD) treatment. The TZD class of drugs is known to improve insulin-sensitivity in diabetic patients, and is clinically used in treatment regimens. In cells, TZDs bind to and activate the transcriptional activity of peroxisome proliferator-activated receptor gamma (PPAR-gamma). Large-scale array analyses will provide some insight into the mechanisms of TZD-mediated insulin sensitization. Unfortunately, a theoretical basis for analyzing array data has not kept pace with the rapid adoption of this tool. The methods that are commonly used, particularly the fold-change approach and the standard t-test, either lack statistical rigor or resort to generalized statistical models that do not accurately estimate variability at low replicate numbers. RESULTS We introduce a statistical framework that models the dependence of measurement variance on the level of gene expression in the context of a Bayesian hierarchical model. We compare several methods of parameter estimation and subsequently apply these to determine a set of genes in 3T3-L1 adipocytes that are differentially regulated in response to TZD treatment. When the number of experimental replicates is low (n = 2-3), this approach appears to qualitatively preserve an equivalent degree of specificity, while vastly improving sensitivity over other comparable methods. In addition, the statistical framework developed here can be readily applied to understand the implicit assumptions made in traditional fold-change approaches to array analysis.

Insulin Induces Heterologous Desensitization of G Protein-Coupled Receptor and Insulin-Like Growth Factor I Signaling by Downregulating β-Arrestin-1

ABSTRACT β-Arrestin-1 mediates agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCRs) and is also essential for GPCR mitogenic signaling. In addition, insulin-like growth factor I receptor (IGF-IR) endocytosis is facilitated by β-arrestin-1, and internalization is necessary for IGF-I-stimulated mitogen-activated protein (MAP) kinase activation. Here, we report that treatment of cells for 12 h with insulin (100 ng/ml) induces an ∼50% decrease in cellular β-arrestin-1 content due to ubiquitination of β-arrestin-1 and proteosome-mediated degradation. This insulin-induced decrease in β-arrestin-1 content was blocked by inhibition of phosphatidylinositol-3 kinase (PI-3 kinase) and MEK with wortmannin and PD98059, respectively. We also found a marked decrease in the association of β-arrestin-1 with the IGF-IR and a 55% inhibition of IGF-I-stimulated MAP kinase phosphorylation. In insulin-treated, β-arrestin-1-downregulated cells, there was complete inhibition of lysophosphatidic acid (LPA) or isoproterenol (ISO)-stimulated MAP kinase phosphorylation. This was associated with a decrease in β-arrestin-1 association with the β2-AR as well as a decrease in β-arrestin-1-Src and Src-β2-AR association. Ectopic expression of wild-type β-arrestin-1 in insulin-treated cells in which endogenous β-arrestin-1 had been downregulated rescued IGF-I- and LPA-stimulated MAP kinase phosphorylation. In conclusion, we found the following. (i) Chronic insulin treatment leads to enhanced β-arrestin-1 degradation. (ii) This downregulation of endogenous β-arrestin-1 is associated with decreased IGF-I-, LPA-, and ISO-mediated MAP kinase signaling, which can be rescued by ectopic expression of wild-type β-arrestin-1. (iii) Finally, these results describe a novel mechanism for heterologous desensitization, whereby insulin treatment can impair GPCR signaling, and highlight the importance of β-arrestin-1 as a target molecule for this desensitization mechanism.

Synthesis of an organoinsulin molecule that can be activated by antibody catalysis

We have developed a methodology of prodrug delivery by using a modified insulin species whose biological activity potentially can be regulated in vivo. Native insulin was derivatized with aldol-terminated chemical modifications that can be selectively removed by the catalytic aldolase antibody 38C2 under physiologic conditions. The derivatized organoinsulin (insulinD) was defective with respect to receptor binding and stimulation of glucose transport. The affinity of insulinD for the insulin receptor was reduced by 90% in binding studies using intact cells. The ability of insulinD to stimulate glucose transport was reduced by 96% in 3T3-L1 adipocytes and by 55% in conscious rats. Incubation of insulinD with the catalytic aldolase antibody 38C2 cleaved all of the aldol-terminated modifications, restoring native insulin. Treatment of insulinD with 38C2 also restored insulinDs receptor binding and glucose transport-stimulating activities in vitro, as well as its ability to lower glucose levels in animals in vivo. We propose that these results are the foundation for an in vivo regulated system of insulin activation using the prohormone insulinD and catalytic antibody 38C2 with potential therapeutic application.

Insulin-Mediated Cellular Insulin Resistance Decreases Osmotic Shock-Induced Glucose Transport in 3T3-L1 Adipocytes*

Similar to insulin, osmotic shock treatment of 3T3-L1 adipocytes causes translocation of GLUT4 protein to the plasma membrane and an increase in glucose transport activity. In our study, we evaluated the effect of chronic insulin treatment on the osmotic shock signaling pathway leading to GLUT4 translocation and glucose uptake. We found that chronic administration of insulin to the adipocytes induced cellular resistance to osmotic shock-stimulated GLUT4 translocation and glucose transport. We found that chronic insulin treatment attenuated shock-induced Gab-1 tyrosine phosphorylation. Furthermore, chronic insulin exposure led to a marked impairment in the ability of Gab-1 to associate with p85 subunit of PI 3-kinase in response to acute shock and insulin stimulation. Cells that were chronically treated with insulin showed a 70% and a 61% decrease in Gab-1 associated PI 3-kinase activity in shock- vs. insulin-treated cells, respectively. In addition, we found that chronic insulin treatment inhibited both insulin- and osmotic shock-induced membrane ruffling, indicating that two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling are decreased in chronically insulin-treated cells. The results described above clearly demonstrate that chronic insulin treatment induces a state of cellular resistance to osmotic shock signal transduction.

Insulin-Mediated Cellular Insulin Resistance Decreases Osmotic Shock-Induced Glucose Transport in 3T3-L1 Adipocytes*This work was supported by NIH Grant DK-33651 and the Veterans Administration Medical Research Service. Andrej Janez was supported by a grant from Slovenian Ministry of Science and Technology (sklad za mlade raziskovalce).

Similar to insulin, osmotic shock treatment of 3T3-L1 adipocytes causes translocation of GLUT4 protein to the plasma membrane and an increase in glucose transport activity. In our study, we evaluated the effect of chronic insulin treatment on the osmotic shock signaling pathway leading to GLUT4 translocation and glucose uptake. We found that chronic administration of insulin to the adipocytes induced cellular resistance to osmotic shock-stimulated GLUT4 translocation and glucose transport. We found that chronic insulin treatment attenuated shock-induced Gab-1 tyrosine phosphorylation. Furthermore, chronic insulin exposure led to a marked impairment in the ability of Gab-1 to associate with p85 subunit of PI 3-kinase in response to acute shock and insulin stimulation. Cells that were chronically treated with insulin showed a 70% and a 61% decrease in Gab-1 associated PI 3-kinase activity in shock- vs. insulin-treated cells, respectively. In addition, we found that chronic insulin treatment inhibited both insulin- and osmotic shock-induced membrane ruffling, indicating that two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling are decreased in chronically insulin-treated cells. The results described above clearly demonstrate that chronic insulin treatment induces a state of cellular resistance to osmotic shock signal transduction.

Insulin-Mediated Cellular Insulin Resistance Decreases Osmotic Shock-Induced Glucose Transport in 3T3-L1 Adipocytes1

Similar to insulin, osmotic shock treatment of 3T3-L1 adipocytes causes translocation of GLUT4 protein to the plasma membrane and an increase in glucose transport activity. In our study, we evaluated the effect of chronic insulin treatment on the osmotic shock signaling pathway leading to GLUT4 translocation and glucose uptake. We found that chronic administration of insulin to the adipocytes induced cellular resistance to osmotic shock-stimulated GLUT4 translocation and glucose transport. We found that chronic insulin treatment attenuated shock-induced Gab-1 tyrosine phosphorylation. Furthermore, chronic insulin exposure led to a marked impairment in the ability of Gab-1 to associate with p85 subunit of PI 3-kinase in response to acute shock and insulin stimulation. Cells that were chronically treated with insulin showed a 70% and a 61% decrease in Gab-1 associated PI 3-kinase activity in shock- vs. insulin-treated cells, respectively. In addition, we found that chronic insulin treatment inhibited both insulin- and osmotic shock-induced membrane ruffling, indicating that two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling are decreased in chronically insulin-treated cells. The results described above clearly demonstrate that chronic insulin treatment induces a state of cellular resistance to osmotic shock signal transduction.