Insulin and insulin-like growth factor I (IGF I) stimulate phosphorylation of a Mr 175,000 cytoskeleton-associated protein in intact FRTL5 cells.

Abstract FRTL5 rat thyroid cells possess separate high affinity receptors for insulin and insulin-like growth factor I (IGF I) that undergo beta-subunit phosphorylation upon interaction with the specific ligand. Phosphorylation is rapid and dose-dependent and occurs primarily on tyrosine residues. Within 2 min, both insulin and IGF I also give rise to a Mr 175,000 phosphoprotein (pp175) that can be immunoprecipitated by anti-phosphotyrosine antibody (alpha-Tyr(P]. Phosphorylation of pp175 occurs on serine and threonine as well as tyrosine residues. When FRTL5 cells are solubilized with 1% Triton X-100, alpha-Tyr(P) immunoprecipitates phosphorylated insulin and IGF I receptors but little pp175 from the Triton-soluble fraction. After treatment of the Triton-insoluble portion with 1% sodium dodecyl sulfate at 100 degrees C, pp175 can be identified by immunoprecipitation with alpha-Tyr(P). The fraction of FRTL5 cells that remains after extraction of an attached monolayer with 1% Triton for 5 min at 22 degrees C contains most of the cytoskeleton and also nuclei. Extraction of this 32P-labeled cytoskeleton preparation with sodium dodecyl sulfate followed by alpha-Tyr(P) immunoprecipitation results in almost complete recovery of the pp175 content of the cells. When a nuclear fraction was prepared from FRTL5 cells by differential centrifugation, pp175 was not found in the nuclear pellet from labeled cells, but greater than 80% of pp175 was recovered in the supernatant. We conclude that pp175 is a common substrate for insulin and IGF I receptor kinases which, in FRTL5 cells, is associated with the cytoskeleton. It is suggested that phosphorylation of proteins associated with cytoskeletal elements could be involved in insulin and IGF I action in cells

Thermal stability and aggregation of sulfolobus solfataricus b-glycosidase are dependent upon the N-e-methylation of specific lysyl residues: critical role of in vivo post-translational modifications.

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of β-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native β-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of β-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of β-glycosidase from S. solfataricu

447. AP20187-Inducible Insulin-Like Effects in Diabetic Muscle and Liver Transduced with AAV

Diabetes Mellitus, characterized by insulin deficiency (type I) or resistance (type II), derives from insulin action impairments in hormone target tissues: muscle, liver and adipocytes. Insulin regulates metabolism and glucose homeostasis through binding to a specific membrane receptor (IR) with tyrosine kinase activity. Induction of the insulin receptor signaling in hormone target cells may represent a tool to rescue glucose homeostasis in both insulin and insulin receptor deficiencies. Recently we have described that homodimerization of the chimeric insulin receptor LFv2IRE induced by the small dimerizer drug AP20187 results in insulin like actions in hepatocytes trasduced with adeno-associated viral vectors (AAV)

Internalization of the constitutively active arginine 1152-->glutamine insulin receptor occurs independently of insulin at an accelerated rate.

Signals controlling the insulin receptor endocytotic pathway have been investigated using the R1152Q insulin receptor mutant (M). This mutant receptor exhibits high levels of insulin-independent kinase activity, impaired autophosphorylation, and lack of an insulin stimulatory effect on both auto- and substrate phosphorylation. NIH-3T3 fibroblasts expressing M receptors displayed a 2.5-fold higher 125I-insulin internalization rate than wild type (WT) but lacked insulin-induced receptor internalization and down-regulation. Cell surface recycling of internalized receptors also occurred at a higher rate in M cells and was unaffected by insulin. Cell preincubation with 35 mM Tris, which inhibits the insulin receptor degradative route, elicited no effect on M receptor recycling but inhibited that of WT by 40%. In contrast, the energy depleter 2,4-dinitrophenol, which inhibits normal insulin receptor retroendocytosis, impaired M receptor recycling 4-fold more effectively than that of WT. The release of internalized intact 125I-insulin was 6-fold greater in M than in WT fibroblasts and was almost completely inhibited by dinitrophenol, whereas insulin degradation by M cells was 4-fold decreased as compared with WT. Thus, internalization and recycling of the constitutively active Gln1152 receptor kinase occur in the absence of autophosphorylation. However, tyrosine phosphorylation appears to be required for proper sorting of endocytosed insulin receptors

Activation and mitochondrial translocation of protein kinase Cδ are necessary for insulin stimulation of pyruvate dehydrogenase complex activity in muscle and liver cells

In L6 skeletal muscle cells and immortalized hepatocytes, insulin induced a 2-fold increase in the activity of the pyruvate dehydrogenase (PDH) complex. This effect was almost completely blocked by the protein kinase C (PKC) delta inhibitor Rottlerin and by PKCdelta antisense oligonucleotides. At variance, overexpression of wild-type PKCdelta or of an active PKCdelta mutant induced PDH complex activity in both L6 and liver cells. Insulin stimulation of the activity of the PDH complex was accompanied by a 2.5-fold increase in PDH phosphatases 1 and 2 (PDP1/2) activity with no change in the activity of PDH kinase. PKCdelta antisense blocked insulin activation of PDP1/2, the same as with PDH. In insulin-exposed cells, PDP1/2 activation was paralleled by activation and mitochondrial translocation of PKCdelta, as revealed by cell subfractionation and confocal microscopy studies. The mitochondrial translocation of PKCdelta, like its activation, was prevented by Rottlerin. In extracts from insulin-stimulated cells, PKCdelta co-precipitated with PDP1/2. PKCdelta also bound to PDP1/2 in overlay blots, suggesting that direct PKCdelta-PDP interaction may occur in vivo as well. In intact cells, insulin exposure determined PDP1/2 phosphorylation, which was specifically prevented by PKCdelta antisense. PKCdelta also phosphorylated PDP in vitro, followed by PDP1/2 activation. Thus, in muscle and liver cells, insulin causes activation and mitochondrial translocation of PKCdelta, accompanied by PDP phosphorylation and activation. These events are necessary for insulin activation of the PDH complex in these cells

Vitamin D deficiency is a risk factor for infections in patients affected by HCV-related liver cirrhosis

Objectives: To evaluate the prevalence of vitamin D deficiency and its impact on infections in HCV-related liver cirrhosis. Methods: We enrolled 291 patients affected by HCV-related liver cirrhosis. Serum vitamin D levels were dosed at enrolment. The presence of infection was assessed at baseline and during follow-up based on physical examination and laboratory analyses. Results: Vitamin D deficiency (15 (p = 0.003), Child-Pugh class B/C vs A (p < 0.001), and active hepatocellular carcinoma (HCC) (p < 0.001). At multivariate analysis, vitamin D deficiency (p < 0.01), HCC (p < 0.05), hospitalization (p < 0.001) and exposure to immunosuppressant agents (p < 0.05) were independent risk factors for infection at baseline. Conclusions: Vitamin D may play a role in the development of infections in patients affected by liver cirrhosis, and preventive strategies with vitamin D supplementation are to be evaluated in randomized controlled trials

Mutation in a conserved motif next to the insulin receptor key autophosphorylation sites de-regulates kinase activity and impairs insulin action.

We have recently reported two non-insulin-dependent diabetic patients exhibiting a heterozygous point mutation (R1152-Q) next to the key tyrosine autophosphorylation sites (Y1146, Y1150, Y1151) of the insulin receptor. In the present study, we demonstrate that the Q1152 mutation alters a previously unrecognized consensus sequence in the insulin receptor family of tyrosine kinases. To define the effect of this alteration on insulin receptor function, the mutant insulin receptor (Q1152) was constructed and overexpressed in NIH-3T3 cells. In spite of normal insulin binding, "in vivo" and "in vitro" autophosphorylation as well as transphosphorylation by the wild-type receptor (WT) were deficient in Q1152 as compared with the transfected WT receptors. Insulin-stimulated kinase activity toward poly(Glu, Tyr) 4:1 and the endogenous substrates p120 and p175 were also impaired in Q1152. However, insulin-independent kinase activity of Q1152 was 2-5-fold higher than that of WT. While insulin stimulated 2-deoxyglucose uptake and glycogen synthase activity in WT-transfected cells with a sensitivity proportional to receptor number, no insulin stimulation was observed in Q1152 cells. Similar to the kinase, insulin-independent glycogen synthase activity and 2-deoxyglucose uptake were 2-fold higher in Q1152 than in either WT or parental cells. We conclude that the Q1152 mutation deregulates insulin receptor kinase and generates insulin insensitivity in cells. Alterations in this highly conserved region of the insulin receptor may contribute to non-insulin dependent diabetes mellitin pathogenesis in humans

Protein Kinase C-α Regulates Insulin Action and Degradation by Interacting with Insulin Receptor Substrate-1 and 14-3-3ϵ

Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKC alpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3 epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKC alpha coprecipitation with IRS-1, but not with IRS-2, and with 14-3-3 epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3 epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKC alpha activity, without altering IRS-1/PKC alpha co-precipitation. 14-3-3 epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3 epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKC alpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKC alpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3 epsilon depletion on insulin signaling. Moreover, PKC alpha inhibition was accompanied by a > 2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3 epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKC alpha, and 14-3-3 epsilon. The presence of 14-3-3 epsilon in the complex is not necessary for IRS-1/PKC alpha interaction but modulates PKC alpha activity, thereby regulating insulin signaling and degradation

Glucose regulates insulin mitogenic effect by modulating SHP-2 activation and localization in JAr cells.

The glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6 mm glucose (LG), proliferation and thymidine incorporation were induced by serum, epidermal growth factor, and insulin-like growth factor 1 but not by insulin. In contrast, at 25 mm glucose (HG), proliferation and thymidine incorporation were stimulated by insulin, serum, epidermal growth factor, and insulin-like growth factor 1 to a comparable extent, whereas basal levels were 25% lower than those in LG. HG culturing also enhanced insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine phosphorylations while decreasing basal phosphorylations. These actions of glucose were accompanied by an increase in cellular tyrosine phosphatase activity. The activity of SHP-2 in HG-treated JAr cells was 400% of that measured in LG-treated cells. SHP-2 co-precipitation with IRS1 was also increased in HG-treated cells. SHP-2 was mainly cytosolic in LG-treated cells. However, HG culturing largely redistributed SHP-2 to the internal membrane compartment, where tyrosine-phosphorylated IRS1 predominantly localizes. Further exposure to insulin rescued SHP-2 cytosolic localization, thereby preventing its interaction with IRS1. Antisense inhibition of SHP-2 reverted the effect of HG on basal and insulin-stimulated insulin receptor and IRS1 phosphorylation as well as that on thymidine incorporation. Thus, in JAr cells, glucose modulates insulin mitogenic action by modulating SHP-2 activity and intracellular localization

Composite alginate-hyaluronan sponges for the delivery of tranexamic acid in post-extractive alveolar wounds

The management of wounds in patients on anticoagulant therapy who require oral surgical procedures is problematic and often results in a non-satisfactory healing process. Here we report a method to prepare an advanced dressing able to avoid uncontrolled bleeding by occluding the post-extractive alveolar wounds, and simultaneously, capable of a fast release of tranexamic acid (TA). Composite alginate/hyaluronan (ALG/HA) sponge dressings loaded with TA were prepared by a straightforward internal gelation method followed by a freeze-drying step. Both blank and drug-loaded sponges were soft, flexible, elegant in appearance and non-brittle in nature. SEM analysis confirmed the porous nature of these dressings. The integration of HA influenced the microstructure, reducing the porosity, modifying the water uptake kinetic and increasing the resistance to compression. TA release from ALG/HA sponges showed a controlled release up to 3h and it was faster in the presence of HA. Finally, an in vitro clotting test performed on human whole blood confirmed that the TA-loaded sponges significantly reduce the blood clotting index (BCI) by 30% compared to ALG/HA20 sponges. These results suggest that, if placed in a socket cavity, these dressings could give a relevant help to the blood hemostasis after dental extractions, especially in patients with coagulation disorders