Serena Cabaro

Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.

Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.

Prep1 Controls Insulin Glucoregulatory Function in Liver by Transcriptional Targeting of SHP1 Tyrosine Phosphatase

OBJECTIVE We investigated the function of the Prep1 gene in insulin-dependent glucose homeostasis in liver. RESEARCH DESIGN AND METHODS Prep1 action on insulin glucoregulatory function has been analyzed in liver of Prep1-hypomorphic mice (Prep1i/i), which express 2–3% of Prep1 mRNA. RESULTS Based on euglycemic hyperinsulinemic clamp studies and measurement of glycogen content, livers from Prep1i/i mice feature increased sensitivity to insulin. Tyrosine phosphorylation of both insulin receptor (IR) and insulin receptor substrate (IRS)1/2 was significantly enhanced in Prep1i/i livers accompanied by a specific downregulation of the SYP and SHP1 tyrosine phosphatases. Prep1 overexpression in HepG2 liver cells upregulated SYP and SHP1 and inhibited insulin-induced IR and IRS1/2 phosphorylation and was accompanied by reduced glycogen content. Consistently, overexpression of the Prep1 partner Pbx1, but not of p160MBP, mimicked Prep1 effects on tyrosine phosphorylations, glycogen content, and on SYP and SHP1 expression. In Prep1 overexpressing cells, antisense silencing of SHP1, but not that of SYP, rescued insulin-dependent IR phosphorylation and glycogen accumulation. Both Prep1 and Pbx1 bind SHP1 promoter at a site located between nucleotides −2,113 and −1,778. This fragment features enhancer activity and induces luciferase function by 7-, 6-, and 30-fold, respectively, in response to Prep1, Pbx1, or both. CONCLUSIONS SHP1, a known silencer of insulin signal, is a transcriptional target of Prep1. In liver, transcriptional activation of SHP1 gene by Prep1 attenuates insulin signal transduction and reduces glucose storage.

PED/PEA-15 induces autophagy and mediates TGF-beta1 effect on muscle cell differentiation

TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.

PREP1 deficiency downregulates hepatic lipogenesis and attenuates steatohepatitis in mice

Aims/hypothesisThe aim of this study was to investigate the function of Prep1 (also known as Pknox1) in hepatic lipogenesis.MethodsThe hepatic lipogenesis pathway was evaluated by real-time RT-PCR and Western blot. Biochemical variables were assessed using a clinical chemistry analyser.ResultsSerum triacylglycerols and liver expression of fatty acid synthase (FAS) were significantly decreased in Prep1 hypomorphic heterozygous (Prep1i/+) mice compared with their non-hypomorphic littermates. Upstream FAS expression, phosphorylation of protein kinase C (PKC)ζ, liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) increased in Prep1i/+ mice, while protein and mRNA levels of the lipid phosphatase inhibitor of PKCζ, SH2-containing inositol 5′-phosphatase 2 (SHIP2), was more than 60% reduced. Consistent with these findings, HepG2 cells transfected with Prep1 cDNA exhibited increased triacylglycerol accumulation and FAS expression, with strongly reduced PKCζ, LKB1, AMPK and ACC phosphorylation. Further experiments revealed the presence of both Prep1 and its major partner Pbx1 at the Ship2 (also known as Inppl1) promoter. PBX-regulating protein 1 (PREP1) and pre-B cell leukaemia transcription factor 1 (PBX1) enhanced Ship2 transcription. The PREP1HR mutant, which is unable to bind PBX1, exhibited no effect on Ship2 function, indicating transcriptional activation of Ship2 by the PREP1/PBX1 complex. Treatment with a methionine- and choline-deficient diet (MCDD) induced steatosis in both Prep1i/+ and non-hypomorphic control mice. However, alanine aminotransferase increase, intracellular triacylglycerol content and histological evidence of liver steatosis, inflammation and necrosis were significantly less evident in Prep1i/+ mice, indicating that Prep1 silencing protects mice from MCDD-induced steatohepatitis.Conclusions/interpretationOur results indicate that Prep1 silencing reduces lipotoxicity by increasing PKCζ/LKB1/AMPK/ACC signalling, while levels of PREP1 expression may determine the risk of steatohepatitis and its progression.

A peptide antagonist of Prep1-p160 interaction improves ceramide-induced insulin resistance in skeletal muscle cells

Prep1 is a homeodomain transcription factor belonging to the TALE protein family. Its overexpression affects glucose metabolism in several tissues. In particular, in skeletal muscle tissue the interaction of Prep1 with its cofactor p160 impairs GLUT4 expression and glucose uptake.In this study, we show that ceramides (C2cer), a class of lipids antagonizing insulin signalling, increase the levels of Prep1 and p160 in a dose and time-dependent fashion in L6 cells and induce their association by 80%. We find that C2cer exposure inhibits insulin receptor, IRS1 and Akt phosphorylation and reduces insulin-stimulated glycogen content and glucose uptake by 1.3- and 2.1-fold, respectively. The synthetic Prep1(54-72) peptide, mimicking the Prep1 region involved in the interaction with p160, reduces in vitro Prep1-p160 binding in a dose-dependent way (IC50 = 0.20μM). In C2cer-treated L6 cells, 10μM Prep1(54-72) restores insulin signalling impaired by ceramide treatment. Prep1 overexpressing L6 cells display similar metabolic alterations observed in ceramide-treated L6 cells and the presence of Prep1(54-72) mitigates these events. All these findings suggest that disruption of the Prep1/p160 molecular interaction enhances insulin sensitivity impaired by ceramides in skeletal muscle cells and indicate this complex as an important target for type 2 diabetes.Prep1 is a homeodomain transcription factor belonging to the TALE protein family. Its overexpression affects glucose metabolism in several tissues. In particular, in skeletal muscle tissue the interaction of Prep1 with its cofactor p160 impairs GLUT4 expression and glucose uptake. In this study, we show that ceramides (C2cer), a class of lipids antagonizing insulin signalling, increase the levels of Prep1 and p160 in a dose and time-dependent fashion in L6 cells and induce their association by 80%. We find that C2cer exposure inhibits insulin receptor, IRS1 and Akt phosphorylation and reduces insulin-stimulated glycogen content and glucose uptake by 1.3- and 2.1-fold, respectively. The synthetic Prep1(54-72) peptide, mimicking the Prep1 region involved in the interaction with p160, reduces in vitro Prep1-p160 binding in a dose-dependent way (IC50 = 0.20μM). In C2cer-treated L6 cells, 10μM Prep1(54-72) restores insulin signalling impaired by ceramide treatment. Prep1 overexpressing L6 cells display similar metabolic alterations observed in ceramide-treated L6 cells and the presence of Prep1(54-72) mitigates these events. All these findings suggest that disruption of the Prep1/p160 molecular interaction enhances insulin sensitivity impaired by ceramides in skeletal muscle cells and indicate this complex as an important target for type 2 diabetes.

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

Abstract 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.

Prep1 Deficiency Affects Olfactory Perception and Feeding Behavior by Impairing BDNF-TrkB Mediated Neurotrophic Signaling

Prep1 is a homeodomain transcription factor which has an important role in hindbrain development. Prep1 expression is also kept in adult mouse brain and in particular within the olfactory bulbs. Moreover, many Prep1 neurons co-localize with Calbindin-positive periglomerular interneurons in olfactory glomerular layer. However, Prep1 function in this brain region is still unknown. In this study, we show that Prep1 hypomorphic heterozygous (Prep1i/+) mice express low levels of protein and feature a 30% reduction of olfactory bulb area, compared to WT mice. In addition, Prep1i/+ mice olfactory bulb histological analysis indicated a 20% lower cytochrome C oxidase activity within the glomerular layer, accompanied by a reduced number of periglomerular interneurons, compared to the WT littermates. Consistently, olfactory perception test highlighted that Prep1 hypomorphic heterozygous mice display a scant ability to distinguish odors, which significantly impacts on feeding behavior, as Prep1i/+ mice revealed a reduced preference for high-fat food. Analysis of BDNF signaling, which represents the main molecular mediator of olfactory plasticity, showed that Prep1i/+ mouse olfactory bulbs feature a 30% reduction of TrkB receptor levels and a decreased activation of ERK1/2. Similarly, overexpression of Prep1 in mouse neuronal cells (N2A) caused an increase of TrkB expression levels, BDNF-induced ERK phosphorylation, and cell viability, compared to control cells. We conclude that Prep1 deficiency alters olfactory morpho-functional integrity and olfaction-mediated eating behavior by affecting BDNF-TrkB signaling. Prep1 could, therefore, play a crucial role in behavioral dysfunctions associated to impaired responsiveness to BDNF.

Prep1 deficiency improves metabolic response in white adipose tissue

Prep1 is a gene encoding for a homeodomain transcription factor which induces hepatic and muscular insulin resistance. In this study, we show that Prep1 hypomorphic heterozygous (Prep1i/+) mice, expressing low levels of protein, featured a 23% and a 25% reduction of total body lipid content and epididymal fat, respectively. The percentage of the small adipocytes (25-75 μm) was 30% higher in Prep1i/+ animals than in the WT, with a reciprocal difference in the large adipose cells (100-150 and >150 μm). Insulin-stimulated insulin receptor tyrosine and Akt serine phosphorylation markedly increased in Prep1i/+ mice, paralleled by 3-fold higher glucose uptake and a significant increase of proadipogenic genes such as C/EBPα, GLUT4, and FABP4. Moreover, T cells infiltration and TNF-α, IFNγ and leptin expression were reduced in adipose tissue from Prep1i/+ mice, while adiponectin levels were 2-fold higher. Furthermore, Prep1i/+ mature adipocytes released lower amounts of pro-inflammatory cytokines and higher amount of adiponectin compared to WT cells. Incubation of murine liver cell line (NMuLi) with conditioned media (CM) from mature adipocytes of Prep1i/+ mice improved glucose metabolism, while those from WT mice had no effect. Consistent with these data, Prep1 overexpression in 3T3-L1 adipocytes impaired adipogenesis and insulin signaling, and increased proinflammatory cytokine secretion. All these findings suggest that Prep1 silencing reduces inflammatory response and increases insulin sensitivity in adipose tissue. In addition, CM from mature adipocytes of Prep1i/+ mice improve metabolism in hepatic cells.

miR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal

Evidence has been provided linking microRNAs (miRNAs) and diabetic complications, by the regulation of molecular pathways, including insulin-signaling, involved in the pathophysiology of vascular dysfunction. Methylglyoxal (MGO) accumulates in diabetes and is associated with cardiovascular complications. This study aims to analyze the contribution of miRNAs in the MGO-induced damaging effect on insulin responsiveness in mouse aortic endothelial cells (MAECs). miRNA modulation was performed by transfection of specific miRNA mimics and inhibitors in MAECs, treated or not with MGO. miRNA-target protein levels were evaluated by Western blot. PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulation by miR-214 was tested by luciferase assays and by the use of a target protector specific for miR-214 on PHLPP2-3′UTR. This study reveals a 4-fold increase of PHLPP2 in MGO-treated MAECs. PHLPP2 levels inversely correlate with miR-214 modulation. Moreover, miR-214 overexpression is able to reduce PHLPP2 levels in MGO-treated MAECs. Interestingly, a direct regulation of PHLPP2 is proved to be dependent by miR-214. Finally, the inhibition of miR-214 impairs the insulin-dependent Akt activation, while its overexpression rescues the insulin effect on Akt activation in MGO-treated MAECs. In conclusion, this study shows that PHLPP2 is a target of miR-214 in MAECs, and identifies miR-214 downregulation as a contributing factor to MGO-induced endothelial insulin-resistance.