White cell and platelet content affects the release of bioactive factors in different blood-derived scaffolds.

Platelet-derived factors are biomaterials that might accelerate healing process in oral, maxillofacial, and several other applications. Release of specific factors by platelet concentrates is critical to achieving a successful outcome. Here, we have shown that platelet-rich fibrin (PRF) clots were beneficial sources of leukocytes, which may directly affect the release of chemokines and growth factors. When compared with the standard leukocyte-PRF (L-PRF), the experimental low-force modified procedure [defined as advanced-PRF (A-PRF)] entrapped the same content of viable leukocytes, released a similar amount of inflammatory cytokines, but secreted 3-, 1.6-, 3-, and 1.2-fold higher levels of Eotaxin, CCL5, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), respectively. A leukocyte-free scaffold, such as plasma rich in growth factors (PRGF), released only platelet-specific factors and, in particular, the F3 fraction, the richest in growth factors, secreted higher amount of CCL5 and PDGF compared to F1 and F2 fractions. In conclusion, different procedures and leukocyte content affect cytokine, chemokines, and growth factor release from platelet derivatives, which may be helpful in different clinical settings.

Hoxa5 undergoes dynamic DNA methylation and transcriptional repression in the adipose tissue of mice exposed to high-fat diet.

BACKGROUND/OBJECTIVES: The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes. SUBJECTS/METHODS: We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox (Hox) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed. RESULTS: Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance (P<0.001) and insulin sensitivity (P<0.05). MeDIP-seq data analysis revealed a uniform distribution of differentially methylated regions (DMR) through the entire adipocyte genome, with a higher number of hypermethylated regions in HFD mice (P<0.005). This different methylation profile was accompanied by increased expression of the Dnmt3a DNA methyltransferase (Dnmt; P<0.05) and the methyl-CpG-binding domain protein Mbd3 (P<0.05) genes in HFD mice. Gene ontology analysis revealed that, in the HFD-treated mice, the Hox family of development genes was highly enriched in differentially methylated genes (P=0.008). To validate this finding, Hoxa5, which is implicated in fat tissue differentiation and remodeling, has been selected and analyzed by bisulphite sequencing, confirming hypermethylation in the adipose tissue from the HFD mice. Hoxa5 hypermethylation was associated with downregulation of Hoxa5 mRNA and protein expression. Feeding animals previously exposed to the HFD with a standard chow diet for two further months improved the metabolic phenotype of the animals, accompanied by return of Hoxa5 methylation and expression levels (P<0.05) to values similar to those of the control mice maintained under standard chow. CONCLUSIONS: HFD induces adipose tissue abnormalities accompanied by epigenetic changes at the Hoxa5 adipose tissue remodeling gene.

Bisphenol A environmental exposure and the detrimental effects on human metabolic health: is it necessary to revise the risk assessment in vulnerable population?

In the last decades, many reports have focused the attention on deleterious effects of novel environmental chemical compounds, including bisphenol A (BPA), on human health. BPA, a common and widely chemical contaminant acting as endocrine disruptor, accumulates in adipose tissue and may affect adipocyte metabolic and inflammatory functions. BPA, at low chronic doses, is now considered as an obesogen compound, and might contribute to the rise of metabolic syndrome, visceral adiposity and diabetes epidemics. The BPA worldwide presence in the environment is responsible for chronic exposure during vulnerable periods, such as foetal and neonatal life. The BPA source of contamination can occur via food, beverage, wastewater, air, dust and soil. BPA, as lipophilic compound, may accumulate into the adipose tissue already during foetal life and may affect adulthood health, through adverse effects on the growth and development of organs and tissues. Thus, based on several studies, it would be crucial to consider further actions aimed to refine risk assessment at least in vulnerable population, such as foetuses, infants and young children, to prevent metabolic diseases and obesity.

Growth-promoting action and growth factor release by different platelet derivatives.

Abstract Platelet derivatives are commonly used in wound healing and tissue regeneration. Different procedures of platelet preparation may differentially affect growth factor release and cell growth. Preparation of platelet-rich fibrin (PRF) is accompanied by release of growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor ß1 (TGFß1), and several cytokines. When compared with the standard procedure for platelet-rich plasma (PRP), PRF released 2-fold less PDGF, but >15-fold and >2-fold VEGF and TGFß1, respectively. Also, the release of several cytokines (IL-4, IL-6, IL-8, IL-10, IFNγ, MIP-1α, MIP-1ß and TNFα) was significantly increased in PRF-conditioned medium (CM), compared to PRP-CM. Incubation of both human skin fibroblasts and human umbilical vein endothelial cells (HUVECs) with PRF-derived membrane (mPRF) or with PRF-CM enhanced cell proliferation by >2-fold (p<0.05). Interestingly, PRP elicited fibroblast growth at a higher extent compared to PRF. At variance, PRF effect on HUVEC growth was significantly greater than that of PRP, consistent with a higher concentration of VEGF in the PRF-CM. Thus, the procedure of PRP preparation leads to a larger release of PDGF, as a possible result of platelet degranulation, while PRF enhances the release of proangiogenic factors.

Adipocyte-released insulin-like growth factor-1 is regulated by glucose and fatty acids and controls breast cancer cell growth in vitro.

AIMS/HYPOTHESIS: Type 2 diabetes and obesity are associated with increased risk of site-specific cancers. We have investigated whether metabolic alterations at the level of adipose-derived differentiating cells may affect specific phenotypes of breast cancer cells. METHODS: Growth profiles of breast cancer cell lines were evaluated in co-cultures with differentiated adipocytes or their precursor cells and upon treatment with adipocyte conditioned media. Production and release of cytokines and growth factors were assessed by real-time RT-PCR and multiplex-based ELISA assays. RESULTS: Co-cultures with either differentiated mouse 3T3-L1 or human mammary adipocytes increased viability of MCF-7 cells to a greater extent, when compared with their undifferentiated precursors. Adipocytes cultured in 25 mmol/l glucose were twofold more effective in promoting cell growth, compared with those grown in 5.5 mmol/l glucose, and activated mitogenic pathways in MCF-7 cells. Growth-promoting action was also enhanced when adipocytes were incubated in the presence of palmitate or oleate. Interestingly, 3T3-L1 and human adipocytes released higher amounts of keratinocyte-derived chemokine/IL-8, the protein 'regulated upon activation, normally T expressed, and secreted' (RANTES), and IGF-1, compared with their precursor cells. Their levels were reduced upon incubation with low glucose and enhanced by fatty acids. Moreover, both undifferentiated cells and differentiated adipocytes from obese individuals displayed about twofold higher IGF-1 release and MCF-7 cell growth induction than lean individuals. Finally, inhibition of the IGF-1 pathway almost completely prevented the growth-promoting effect of adipocytes on breast cancer cells. CONCLUSIONS/INTERPRETATION: IGF-1 release by adipocytes is regulated by glucose and fatty acids and may contribute to the control of cancer cell growth in obese individuals.

Risk of autoimmune diseases in patients with RASopathies: systematic study of humoral and cellular immunity.

BACKGROUND: Abnormalities of the immune system are rarely reported in patients affected by RASopathies. Aim of the current study was to investigate the prevalence of immune system dysfunction in a cohort of patients affected by RASopathies. STUDY DESIGN: A group of 69 patients was enrolled: 60 at the Federico II University, Naples, 7 at University Magna Graecia of Catanzaro, 2 at "Scuola Medica Salernitana", Salerno. An age- and sex-matched control group was also enrolled. Autoimmune disorders were investigated according to international consensus criteria. Immune framework was also evaluated by immunoglobulin levels, CD3, CD4, CD8, CD19, CD56 lymphocyte subpopulations, autoantibodies levels and panel of inflammatory molecules, in both patients and controls. RESULTS: Frequent upper respiratory tract infections were recorded in 2 patients; pneumonia, psoriasis and alopecia in single patients. Low IgA levels were detected in 8/44 patients (18.18%), low CD8 T cells in 13/35 patients (37.14%). Anti-tg and anti-TPO antibodies were detected in 3/24 patients (12.5%), anti r-TSH in 2 cases (8.33%), all in euthyroidism. Serum IgA and CD8 levels were significantly lower in patients than in controls (p 0.00685; p 0.000656 respectively). All tested patients showed increased inflammatory molecules compared to controls. These findings may anticipate the detection of overt autoimmune disease. CONCLUSIONS: Patients affected by RASopathies are at risk to develop autoimmune disorders. Routine screening for autoimmunity is recommended in patients with RASopathy.

Hepatocyte nuclear factor (HNF)-4alpha-driven epigenetic silencing of the human PED gene.

AIMS/HYPOTHESIS: Overexpression of PED (also known as PEA15) determines insulin resistance and impaired insulin secretion and may contribute to progression toward type 2 diabetes. Recently, we found that the transcription factor hepatocyte nuclear factor (HNF)-4alpha binds to PED promoter and represses its transcription. However, the molecular details responsible for regulation of PED gene remain unclear. METHODS: Here we used gain and loss of function approaches to investigate the hypothesis that HNF-4alpha controls chromatin remodelling at the PED promoter in human cell lines. RESULTS: HNF-4alpha production and binding induce chromatin remodelling at the -250 to 50 region of PED, indicating that remodelling is limited to two nucleosomes located at the proximal promoter. Chromatin immunoprecipitation assays also revealed concomitant HNF-4alpha-induced deacetylation of histone H3 at Lys9 and Lys14, and increased dimethylation of histone H3 at Lys9. The latter was followed by reduction of histone H3 Lys4 dimethylation. HNF-4alpha was also shown to target the histone deacetylase complex associated with silencing mediator of retinoic acid and thyroid hormone receptor, both at the PED promoter, and at GRB14 and USP21 regulatory regions, leading to a reduction of mRNA levels. Moreover, HNF-4alpha silencing and PED overexpression were accompanied by a significant reduction of hepatic glycogen content. CONCLUSIONS/INTERPRETATION: These results show that HNF-4alpha serves as a scaffold protein for histone deacetylase activities, thereby inhibiting liver expression of genes including PED. Dysregulation of these mechanisms may lead to upregulation of the PED gene in type 2 diabetes.

Glucosamine-induced endoplasmic reticulum stress affects GLUT4 expression via activating transcription factor 6 in rat and human skeletal muscle cells.

AIMS/HYPOTHESIS: Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells. METHODS: Real-time RT-PCR analysis, 2-deoxy-D: -glucose (2-DG) uptake and western blot analysis were carried out in rat and human muscle cell lines. RESULTS: In both rat and human myotubes, glucosamine treatment caused a significant increase in the expression of the ER stress markers immunoglobulin heavy chain-binding protein/glucose-regulated protein 78 kDa (BIP/GRP78 [also known as HSPA5]), X-box binding protein-1 (XBP1) and activating transcription factor 6 (ATF6). In addition, glucosamine impaired insulin-stimulated 2-DG uptake in both rat and human myotubes. Interestingly, pretreatment of both rat and human myotubes with the chemical chaperones 4-phenylbutyric acid (PBA) or tauroursodeoxycholic acid (TUDCA), completely prevented the effect of glucosamine on both ER stress induction and insulin-induced glucose uptake. In both rat and human myotubes, glucosamine treatment reduced mRNA and protein levels of the gene encoding GLUT4 and mRNA levels of the main regulators of the gene encoding GLUT4 (myocyte enhancer factor 2 a [MEF2A] and peroxisome proliferator-activated receptor-gamma coactivator 1alpha [PGC1alpha]). Again, PBA or TUDCA pretreatment prevented glucosamine-induced inhibition of GLUT4 (also known as SLC2A4), MEF2A and PGC1alpha (also known as PPARGC1A). Finally, we showed that overproduction of ATF6 is sufficient to inhibit the expression of genes GLUT4, MEF2A and PGC1alpha and that ATF6 silencing with a specific small interfering RNA is sufficient to completely prevent glucosamine-induced inhibition of GLUT4, MEF2A and PGC1alpha in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: In this work we show that glucosamine-induced ER stress causes insulin resistance in both human and rat myotubes and impairs GLUT4 production and insulin-induced glucose uptake via an ATF6-dependent decrease of the GLUT4 regulators MEF2A and PGC1alpha.

Preliminary data on effects of metformin on PED/PEA-15 cellular levels in obese women with polycystic ovary syndrome.

BACKGROUND: The cellular abundance of the phosphoprotein enriched in diabetes (PED/PEA-15), a 15 kDa protein related to insulin resistance (IR), is increased in women with polycystic ovary syndrome (PCOS). AIM: To investigate whether metformin (MET) has additive effects on PED/PEA-15 protein levels. MATERIAL/SUBJECTS AND METHODS: This is an open label, prospective clinical study over 6 months. Ten hyperandrogenic obese PCOS women [age: 24.6+/-1.6 yr; body mass index (BMI): 30.7+/-1.2 kg/m(2)] were treated with MET (1250 mg/day). Ten age- and BMI-matched normo-androgenic women were used as controls. Outcome measures are: PED/PEA-15 protein levels, fasting plasma glucose and insulin (FPI), reciprocal index of homeostasis model assessment of insulin resistance (1/HOMA-IR); quantitative insulin sensitivity check index (QUICKI); wholebody insulin sensitivity index (ISI); SHBG; total testosterone; free androgen index (FAI). RESULTS: At baseline FPI and PED/PEA- 15 protein levels were higher, while 1/HOMA-IR, QUICKI, and ISI were lower (p<0.001) in MET group than in controls. After treatment, independently of body weight and hyperandrogenism, FPI, and PED/PEA-15 protein levels decreased (p=0.001 and 0.004, respectively), while, 1/HOMA-IR, QUICKI, and ISI increased (p<0.001). PED/PEA-15 protein levels correlated significantly with ISI either before (r=0.636; p=0.048), and after treatment (r=0.758; p=0.011). CONCLUSIONS: PED/PEA-15 protein levels reduced after a short course of treatment with MET in a group hyperandrogenic obese PCOS women. This effect was independent of body weight and hyperandrogenism, and correlated with ISI, thus adding a further benefit to obese PCOS women.

Overproduction of phosphoprotein enriched in diabetes (PED) induces mesangial expansion and upregulates protein kinase C-beta activity and TGF-beta1 expression.

AIMS/HYPOTHESIS: Overproduction of phosphoprotein enriched in diabetes (PED, also known as phosphoprotein enriched in astrocytes-15 [PEA-15]) is a common feature of type 2 diabetes and impairs insulin action in cultured cells and in mice. Nevertheless, the potential role of PED in diabetic complications is still unknown. METHODS: We studied the effect of PED overproduction and depletion on kidney function in animal and cellular models. RESULTS: Transgenic mice overexpressing PED (PEDTg) featured age-dependent increases of plasma creatinine levels and urinary volume, accompanied by expansion of the mesangial area, compared with wild-type littermates. Serum and kidney levels of TGF-beta1 were also higher in 6- and 9-month-old PEDTg. Overexpression of PED in human kidney 2 cells significantly increased TGF-beta1 levels, SMAD family members (SMAD)2/3 phosphorylation and fibronectin production. Opposite results were obtained following genetic silencing of PED in human kidney 2 cells by antisense oligonucleotides. Inhibition of phospholipase D and protein kinase C-beta by 2-butanol and LY373196 respectively reduced TGF-beta1, SMAD2/3 phosphorylation and fibronectin production. Moreover, inhibition of TGF-beta1 receptor activity and SMAD2/3 production by SB431542 and antisense oligonucleotides respectively reduced fibronectin secretion by about 50%. TGF-beta1 circulating levels were significantly reduced in Ped knockout mice and positively correlated with PED content in peripheral blood leucocytes of type 2 diabetic patients. CONCLUSIONS/INTERPRETATION: These data indicate that PED regulates fibronectin production via phospholipase D/protein kinase C-beta and TGF-beta1/SMAD pathways in kidney cells. Raised PED levels may therefore contribute to the abnormal accumulation of extracellular matrix and renal dysfunction in diabetes.

Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells.

In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Calpha (PKCalpha) and beta activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCalpha and -beta activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCbeta with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCbeta block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCbeta. Finally, blocking PKCalpha expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCalpha and -beta. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCbeta plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.

Risk assessment for chemical pickling of metals contaminated by radioactive materials.

In recent years, many cases of contamination of metal scraps by unwanted radioactive materials have occurred. Moreover, international organisations are evaluating the possibility to re-use or to recycle metals coming from nuclear power plants. The metal recycling industry has started to worry about radiation exposure of workers that could be in contact with contaminated metals during each manufacturing phase. Risks are strongly dependent on the radiation source features. The aim of this study is to perform risk assessment for workers involved in chemical pickling of steel coils. Monte Carlo simulations have been performed, using the MCNP package and considering coils contaminated with (60)Co, (137)Cs, (241)Am and (226)Ra. Under the most conservative conditions (coil contaminated with 1.0 kBq g(-1) of (60)Co), the dose assessment results lower than the European dose limit for the population (1 mSv y(-1)), considering a maximum number of 10 contaminated coils handled per year. The only exception concerns the case of (241)Am, for which internal contamination could be non- negligible and should be verified in the specific cases. In every case, radiation exposure risk for people standing at 50 m from the coil is widely <1 mSv y(-1).

The insulin receptor substrate (IRS)-1 recruits phosphatidylinositol 3-kinase to Ret: evidence for a competition between Shc and IRS-1 for the binding to Ret.

Tyrosine 1062 of Ret, which represents an intracytoplasmic docking site for multiple signaling molecules, is essential for Ret-mediated activation of phosphatidylinositol 3-Kinase (PI3-K). PI3-K, in turn, has been implicated in inducing cell survival and neoplastic transformation mediated by Ret. We have examined the mechanisms by which Ret stimulates PI3-K. Here we show that the Insulin Receptor Substrate-1 (IRS-1) is tyrosine phosphorylated and associated with the p85 regulatory subunit of PI3-K in response to Ret activation. IRS-1 coimmunoprecipitates with Ret and co-expression of IRS-1 results in the potentiation of Ret-mediated activation of Akt(PKB), a bona fide effector of PI3-K. The association with the PTB domain of IRS-1 depends on the phosphorylation of tyrosine 1062 of Ret. The deletion of asparagine 1059 (delN1059) and the substitution of leucine 1061 (L1061P), two Ret mutations identified in families affected by congenital megacolon (Hirschsprung's disease), impair the binding of IRS-1 to Ret as well as Ret-mediated Akt(PKB) stimulation. Finally, we show that Shc, which was previously identified as another ligand of Y1062 of Ret, competes with IRS-1 for the binding to Ret pY1062. All together, these findings suggest that IRS-1 is a component of the signaling pathway which leads to Ret-mediated PI3-K activation, a pathway which can be targeted by Hirschsprung-associated Ret mutations. The alternative binding of Shc and IRS-1 to Ret pY1062 can be a system to modulate the activation of different intracellular signaling pathways and to elicit different biological responses following Ret activation.

PED/PEA-15: an anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis.

PED/PEA-15 is a recently cloned 15 kDa protein possessing a death effector domain (DED). In MCF-7 and HeLa cells, a fivefold overexpression of PED/PEA-15 blocked FasL and TNFalpha apoptotic effects. This effect of PED overexpression was blocked by inhibition of PKC activity. In MCF-7 and HeLa cell lysates, PED/PEA-15 co-precipitated with both FADD and FLICE. PED/PEA-15-FLICE association was inhibited by overexpression of the wild-type but not of a DED-deletion mutant of FADD. Simultaneous overexpression of PED/PEA-15 with FADD and FLICE inhibited FADD-FLICE co-precipitation by threefold. Based on cleavage of the FLICE substrate PARP, this inhibitory effect was paralleled by a threefold decline in FLICE activation in response to TNF-alpha. TNFalpha, in turn, reduces PED association with the endogenous FADD and FLICE of the cells. Thus, PED/PEA-15 is an endogenous protein inhibiting FAS and TNFR1-mediated apoptosis. At least in part, this function may involve displacement of FADD-FLICE binding through the death effector domain of PED/PEA-15.

A novel human insulin receptor gene mutation uniquely inhibits insulin binding without impairing posttranslational processing.

The precise nature of the insulin-binding site of the insulin receptor (IR) has not been determined, although the importance of several regions of the alpha-subunit in insulin binding has been demonstrated. A naturally occurring mutation in a patient with severe insulin resistance that changes the Ser323 codon in the alpha-subunit of the IR to a leucine codon is associated with markedly impaired insulin binding to cells from the patient and to transfected cells expressing the mutant receptor. However, unlike other IR alpha-subunit mutations associated with decreased insulin binding, this mutation does not lead to a defect in posttranslational processing or cell-surface expression of IRs. Thus, the defect in insulin binding associated with the Leu323 mutant IR is a direct result of an alteration in the insulin-binding site. No natural IR mutation described thus far is associated with both decreased insulin binding and normal cell-surface expression of the mutant receptor. This study demonstrates the critical role that Ser323 of the IR alpha-subunit plays in insulin binding, either by forming part of the binding site or by stabilizing its conformation.

Protein kinase C (PKC)-alpha activation inhibits PKC-zeta and mediates the action of PED/PEA-15 on glucose transport in the L6 skeletal muscle cells.

Overexpression of the PED/PEA-15 protein in muscle and adipose cells increases glucose transport and impairs further insulin induction. Like glucose transport, protein kinase C (PKC)-alpha and -beta are also constitutively activated and are not further stimulatable by insulin in L6 skeletal muscle cells overexpressing PED (L6(PED)). PKC-zeta features no basal change but completely loses insulin sensitivity in L6(PED). In these cells, blockage of PKC-alpha and -beta additively returns 2-deoxy-D-glucose (2-DG) uptake to the levels of cells expressing only endogenous PED (L6(WT)). Blockage of PKC-alpha and -beta also restores insulin activation of PKC-zeta in L6(PED) cells, with that of PKC-alpha sixfold more effective than PKC-beta. Similar effects on 2-DG uptake and PKC-zeta were also achieved by 50-fold overexpression of PKC-zeta in L6(PED). In L6(WT), fivefold overexpression of PKC-alpha or -beta increases basal 2-DG uptake and impairs further insulin induction with no effect on insulin receptor or insulin receptor substrate phosphorylation. In these cells, overexpression of PKC-alpha blocks insulin induction of PKC-zeta activity. PKC-beta is 10-fold less effective than PKC-alpha in inhibiting PKC-zeta stimulation. Expression of the dominant-negative K(281)-->W PKC-zeta mutant simultaneously inhibits insulin activation of PKC-zeta and 2-DG uptake in the L6(WT) cells. We conclude that activation of classic PKCs, mainly PKC-alpha, inhibits PKC-zeta and may mediate the action of PED on glucose uptake in L6 skeletal muscle cells.

The IR1152 mutant insulin receptor selectively impairs insulin action in skeletal muscle but not in liver.

In patients harboring the IR1152 mutant insulin receptor, hepatic glucose production was normally suppressed by insulin. Hepatocytes without the insulin receptor gene and expressing IR1152 (Hep(MUT)) also showed normal insulin suppression of glucose production and full insulin response of glycogen synthase. In contrast, expression of the IR1152 mutant in skeletal muscle maximally increased glucose uptake and storage, preventing further insulin stimulation. IRS-1 phosphorylation was normally stimulated by insulin in both intact Hep(MUT) and L6 skeletal muscle cells expressing the IR1152 mutant (L6(MUT)). At variance, IRS-2 phosphorylation exhibited high basal levels with no further insulin-dependent increase in L6(MUT) but almost normal phosphorylation, both basal and insulin-stimulated, in the Hep(MUT) cells. In vitro, IR1152 mutant preparations from both the L6(MUT) and the Hep(MUT) cells exhibited increased basal and no insulin-stimulated phosphorylation of IRS-2 immobilized from either muscle or liver cells. IR1152 internalization in liver and muscle cells closely paralleled the ability of this mutant to phosphorylate IRS-2 in vivo in these cells. Block of receptor internalization (wild-type and mutant) in the liver and muscle cells also inhibited IRS-2, but not IRS-1, phosphorylation. Thus, the mechanisms controlling insulin receptor internalization differ in liver and skeletal muscle cells and may enable IR1152 to control glucose metabolism selectively in liver. In both cell types, receptor internalization seems necessary for IRS-2 but not IRS-1 phosphorylation.

Alginate-hyaluronan composite hydrogels accelerate wound healing process.

In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.

Thyrotropin regulates autophosphorylation and kinase activity in both the insulin and the insulin-like growth factor-I receptors in FRTL5 cells.

TSH regulation of insulin and insulin-like growth factor-I (IGF-I) receptor kinases has been studied in FRTL5 cultured thyroid cells. Preincubation of intact cells with TSH increased by 2-fold insulin and IGF-I receptor autophosphorylation and phosphorylation of the p175 endogenous substrate for the receptors. Enhanced phosphorylations reached a maximum within 30 min, were maintained for 30 min more, and vanished after 120 min of TSH incubation. TSH dose-responses exhibited half-maximal and maximal effects at 1 and 10 pM, respectively. In vitro, insulin as well as IGF-I receptors purified from cells treated with 10 pM TSH also exhibited 2-fold enhanced receptor autophosphorylation and kinase activity toward the exogenous substrate poly(Glu,Tyr) (4:1). At variance with TSH, cell incubation with either 8-bromo-cAMP or the protein kinase-C activator 12-O-tetradecanoylphorbol-13-acetate inhibited insulin and IGF-I receptor kinases. In intact cells, TSH stimulation of insulin and IGF-I receptor kinases was accompanied by enhanced turnover of phosphate on autophosphorylated receptors, increased receptor tyrosine phosphorylation, and decreased receptor serine/threonine phosphorylation in response to insulin. Incubation of in vivo labeled insulin and IGF-I receptors with extracts from TSH-treated cells also decreased receptor phosphoserine and phosphothreonine content. Furthermore, preincubation of insulin and IGF-I receptors with extracts from TSH-treated cells enhanced in vitro autophosphorylation. The latter effect was inhibited by the serine/threonine phosphatase inhibitors fluoride and okadaic acid, but not by the tyrosine phosphatase inhibitor vanadate. The data suggest that in FRTL5 cells, TSH induces the activity of a Ser/Thr protein phosphatase, which dephosphorylates insulin and IGF-I receptors and enhances their endogenous kinases.

Antiphosphotyrosine immunoprecipitation of an insulin-stimulated receptor phosphatase activity from FRTL5 cells.

Phosphotyrosine-containing proteins (PYPs) from FRTL5 epithelial cells were immunoprecipitated with Sepharose-linked phosphotyrosine antibody and phosphorylated in vitro with insulin and insulin-like growth factor-I (IGF-I) receptor kinases. Insulin preactivation of the kinases resulted in increased phosphorylation of a single 175,000 mol wt PYP (p175) beside insulin and IGF-I receptors. Phosphorylation of both p175 and receptors exhibited almost identical time courses and insulin dose responses, suggesting that p175 may serve as a substrate for the insulin and IGF-I receptor kinases. Preincubation of autophosphorylated receptors with PYPs derived from insulin-stimulated cells decreased 32P-labeled receptor precipitation by antiphosphotyrosine-Sepharose by 50-70% compared to that obtained upon preincubation with PYPs from unstimulated cells. This effect was not the result of PYP-receptor competition, was observed on both in vivo and in vitro phosphorylated receptors, and was almost completely blocked by 100 microM sodium vanadate, suggesting PYP copurification of a phosphotyrosine phosphatase. No phosphatase was recovered in lysates from insulin-unstimulated cells. Maximum activity was detected after 2 min of insulin stimulation, decreasing by more than 50% after 30 min. Antiphosphotyrosine recovery of p175 from cell lysates exhibited almost identical insulin dependency. Complete recovery of both p175 and phosphatase activity was obtained after receptor immunodepletion of the extracts. Thus, a receptor phosphatase activity from insulin-stimulated FRTL5 cells is retained on an antiphosphotyrosine affinity matrix and correlates with phosphorylation of the p175 substrate for the insulin and IGF-I receptor kinases.