Insulin effects on core neurotransmitter pathways involved in schizophrenia neurobiology: a meta-analysis of preclinical studies. Implications for the treatment.

Impairment of insulin action and metabolic dysregulation have traditionally been associated with schizophrenia, although the molecular basis of such association remains still elusive. The present meta-analysis aims to assess the impact of insulin action manipulations (i.e., hyperinsulinemia, hypoinsulinemia, systemic or brain insulin resistance) on glutamatergic, dopaminergic, γ-aminobutyric acid (GABA)ergic, and serotonergic pathways in the central nervous system. More than one hundred outcomes, including transcript or protein levels, kinetic parameters, and other components of the neurotransmitter pathways, were collected from cultured cells, animals, or humans, and meta-analyzed by applying a random-effects model and adopting Hedges'g to compare means. Two hundred fifteen studies met the inclusion criteria, of which 180 entered the quantitative synthesis. Significant impairments in key regulators of synaptic plasticity processes were detected as the result of insulin handlings. Specifically, protein levels of N-methyl-D-aspartate receptor (NMDAR) subunits including type 2A (NR2A) (Hedges' g = -0.95, 95%C.I. = -1.50, -0.39; p = 0.001; I2 = 47.46%) and 2B (NR2B) (Hedges'g = -0.69, 95%C.I. = -1.35, -0.02; p = 0.043; I2 = 62.09%), and Postsynaptic density protein 95 (PSD-95) (Hedges'g = -0.91, 95%C.I. = -1.51, -0.32; p = 0.003; I2 = 77.81%) were found reduced in insulin-resistant animal models. Moreover, insulin-resistant animals showed significantly impaired dopamine transporter activity, whereas the dopamine D2 receptor mRNA expression (Hedges'g = 3.259; 95%C.I. = 0.497, 6.020; p = 0.021; I2 = 90.61%) increased under insulin deficiency conditions. Insulin action modulated glutamate and GABA release, as well as several enzymes involved in GABA and serotonin synthesis. These results suggest that brain neurotransmitter systems are susceptible to insulin signaling abnormalities, resembling the discrete psychotic disorders' neurobiology and possibly contributing to the development of neurobiological hallmarks of treatment-resistant schizophrenia.

Glyoxalase 1 knockdown induces age-related ß-cell dysfunction and glucose intolerance in mice.

Tight control of glycemia is a major treatment goal for type 2 diabetes mellitus (T2DM). Clinical studies indicated that factors other than poor glycemic control may be important in fostering T2DM progression. Increased levels of methylglyoxal (MGO) associate with complications development, but its role in the early steps of T2DM pathogenesis has not been defined. Here, we show that MGO accumulation induces an age-dependent impairment of glucose tolerance and glucose-stimulated insulin secretion in mice knockdown for glyoxalase 1 (Glo1KD). This metabolic alteration associates with the presence of insular inflammatory infiltration (F4/80-positive staining), the islet expression of senescence markers, and higher levels of cytokines (MCP-1 and TNF-α), part of the senescence-activated secretory profile, in the pancreas from 10-month-old Glo1KD mice, compared with their WT littermates. In vitro exposure of INS832/13 ß-cells to MGO confirms its casual role on ß-cell dysfunction, which can be reverted by senolytic treatment. These data indicate that MGO is capable to induce early phenotypes typical of T2D progression, paving the way for novel prevention approaches to T2DM.

Resveratrol Improves Endothelial Function by A PREP1-Mediated Pathway in Mouse Aortic Endothelial Cells.

PREP1 is a homeodomain transcription factor that impairs metabolism and is involved in age-related aortic thickening. In this study, we evaluated the role of PREP1 on endothelial function. Mouse Aortic Endothelial Cells (MAECs) transiently transfected with a Prep1 cDNA showed a 1.5- and 1.6-fold increase in eNOSThr495 and PKCα phosphorylation, respectively. Proinflammatory cytokines Tnf-α and Il-6 increased by 3.5 and 2.3-fold, respectively, in the presence of Prep1, while the antioxidant genes Sod2 and Atf4 were significantly reduced. Bisindolylmaleimide reverted the effects induced by PREP1, suggesting PKCα to be a mediator of PREP1 action. Interestingly, resveratrol, a phenolic micronutrient compound, reduced the PREP1 levels, eNOSThr495, PKCα phosphorylation, and proinflammatory cytokines and increased Sod2 and Atf4 mRNA levels. The experiments performed on the aorta of 18-month-old Prep1 hypomorphic heterozygous mice (Prep1i/+) expressing low levels of this protein showed a 54 and 60% decrease in PKCα and eNOSThr495 phosphorylation and a 45% reduction in Tnf-α levels, with no change in Il-6, compared to same-age WT mice. However, a significant decrease in Sod2 and Atf4 was observed in Prep1i/+ old mice, indicating the lack of age-induced antioxidant response. These results suggest that Prep1 deficiency partially improved the endothelial function in aged mice and suggested PREP1 as a novel target of resveratrol.

Interleukin 6 reduces vascular smooth muscle cell apoptosis via Prep1 and is associated with aging.

Aging exacerbates neointimal formation by reducing apoptosis of vascular smooth muscle cells (VSMCs) and induces inflammation within vascular wall. Prep1 is a homeodomain transcription factor which stimulates the expression of proinflammatory cytokines in aortic endothelial cell models and plays a primary role in the regulation of apoptosis. In this study, we have investigated the role of Prep1 in aorta of Prep1 hypomorphic heterozygous mice (Prep1i/+ ) and in VSMCs, and its correlation with aging. Histological analysis from Prep1i/+ aortas revealed a 25% reduction in medial smooth muscle cell density compared to WT animals. This result paralleled higher apoptosis, caspase 3, caspase 9 and p53 levels in Prep1i/+ mice and lower Bcl-xL. Prep1 overexpression in VSMCs decreased apoptosis by 25% and caspase 3 and caspase 9 expression by 40% and 37%. In parallel, Bcl-xL inhibition by BH3I-1 and p53 induction by etoposide reverted the antiapoptotic effect of Prep1. Experiments performed in aorta from 18 months old WT mice showed a significant increase in Prep1, p16INK4 , p21Waf1 and interleukin 6 (IL-6) compared to youngest animals. Similar results have been observed in H2 O2 -induced senescent VSMCs. Interestingly, the synthetic Prep1 inhibitory peptide Prep1 (54-72) reduced the antiapoptotic effects mediated by IL-6, particularly in senescent VSMCs. These results indicate that IL-6-Prep1 signaling reduces apoptosis, by modulating Bcl-xL and p53 both in murine aorta and in VSMCs. In addition, age-dependent increase in IL-6 and Prep1 in senescent VSMCs and in old mice may be involved in the aging-related vascular dysfunction.

Adipocyte precursor cells from first degree relatives of type 2 diabetic patients feature changes in hsa-mir-23a-5p, -193a-5p, and -193b-5p and insulin-like growth factor 2 expression.

First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes in miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes in these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation was also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D.

Periprostatic adipose tissue promotes prostate cancer resistance to docetaxel by paracrine IGF-1 upregulation of TUBB2B beta-tubulin isoform.

Growing evidence supports the pivotal role played by periprostatic adipose tissue (PPAT) in prostate cancer (PCa) microenvironment. We investigated whether PPAT can affect response to Docetaxel (DCTX) and the mechanisms associated. Conditioned medium was collected from the in vitro differentiated adipocytes isolated from PPAT which was isolated from PCa patients, during radical prostatectomy. Drug efficacy was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide citotoxicity assay. Culture with CM of human PPAT (AdipoCM) promotes DCTX resistance in two different human prostate cancer cell lines (DU145 and PC3) and upregulated the expression of BCL-xL, BCL-2, and TUBB2B. AG1024, a well-known IGF-1 receptor inhibitor, counteracts the decreased response to DCTX observed in presence of AdipoCM and decreased TUBB2B expression, suggesting that a paracrine secretion of IGF-1 by PPAT affect DCTX response of PCa cell. Collectively, our study showed that factors secreted by PPAT elicits DCTX resistance through antiapoptotic proteins and TUBB2B upregulation in androgen independent PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte-released factors and IGF-1 axis to overcome DCTX resistance in patients with PCa.

In severe obesity, subcutaneous adipose tissue cell-derived cytokines are early markers of impaired glucose tolerance and are modulated by quercetin.

BACKGROUND: Excessive adiposity provides an inflammatory environment. However, in people with severe obesity, how systemic and local adipose tissue (AT)-derived cytokines contribute to worsening glucose tolerance is not clear. METHODS: Ninty-two severely obese (SO) individuals undergoing bariatric surgery were enrolled and subjected to detailed clinical phenotyping. Following an oral glucose tolerance test, participants were included in three groups, based on the presence of normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or type 2 diabetes (T2D). Serum and subcutaneous AT (SAT) biopsies were obtained and mesenchymal stem cells (MSCs) were isolated, characterized, and differentiated in adipocytes in vitro. TNFA and PPARG mRNA levels were determined by qRT-PCR. Circulating, adipocyte- and MSC-released cytokines, chemokines, and growth factors were assessed by multiplex ELISA. RESULTS: Serum levels of IL-9, IL-13, and MIP-1ß were increased in SO individuals with T2D, as compared with those with either IGT or NGT. At variance, SAT samples obtained from SO individuals with IGT displayed levels of TNFA which were threefold higher compared to those with NGT, but not different from those with T2D. Elevated levels of TNFα were also found in differentiated adipocytes, isolated from the SAT specimens of individuals with IGT and T2D, compared to those with NGT. Consistent with the pro-inflammatory milieu, IL-1ß and IP-10 secretion was significantly higher in adipocytes from individuals with IGT and T2D. Moreover, increased levels of TNFα, both mRNA and secreted protein were detected in MSCs obtained from IGT and T2D, compared to NGT SO individuals. Exposure of T2D and IGT-derived MSCs to the anti-inflammatory flavonoid quercetin reduced TNFα levels and was paralleled by a significant decrease of the secretion of inflammatory cytokines. CONCLUSION: In severe obesity, enhanced SAT-derived inflammatory phenotype is an early step in the progression toward T2D and maybe, at least in part, attenuated by quercetin.

DNA Methylation and Type 2 Diabetes: Novel Biomarkers for Risk Assessment?

Diabetes is a severe threat to global health. Almost 500 million people live with diabetes worldwide. Most of them have type 2 diabetes (T2D). T2D patients are at risk of developing severe and life-threatening complications, leading to an increased need for medical care and reduced quality of life. Improved care for people with T2D is essential. Actions aiming at identifying undiagnosed diabetes and at preventing diabetes in those at high risk are needed as well. To this end, biomarker discovery and validation of risk assessment for T2D are critical. Alterations of DNA methylation have recently helped to better understand T2D pathophysiology by explaining differences among endophenotypes of diabetic patients in tissues. Recent evidence further suggests that variations of DNA methylation might contribute to the risk of T2D even more significantly than genetic variability and might represent a valuable tool to predict T2D risk. In this review, we focus on recent information on the contribution of DNA methylation to the risk and the pathogenesis of T2D. We discuss the limitations of these studies and provide evidence supporting the potential for clinical application of DNA methylation marks to predict the risk and progression of T2D.

Diabetes and Cognitive Impairment: A Role for Glucotoxicity and Dopaminergic Dysfunction.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia, responsible for the onset of several long-term complications. Recent evidence suggests that cognitive dysfunction represents an emerging complication of DM, but the underlying molecular mechanisms are still obscure. Dopamine (DA), a neurotransmitter essentially known for its relevance in the regulation of behavior and movement, modulates cognitive function, too. Interestingly, alterations of the dopaminergic system have been observed in DM. This review aims to offer a comprehensive overview of the most relevant experimental results assessing DA's role in cognitive function, highlighting the presence of dopaminergic dysfunction in DM and supporting a role for glucotoxicity in DM-associated dopaminergic dysfunction and cognitive impairment. Several studies confirm a role for DA in cognition both in animal models and in humans. Similarly, significant alterations of the dopaminergic system have been observed in animal models of experimental diabetes and in diabetic patients, too. Evidence is accumulating that advanced glycation end products (AGEs) and their precursor methylglyoxal (MGO) are associated with cognitive impairment and alterations of the dopaminergic system. Further research is needed to clarify the molecular mechanisms linking DM-associated dopaminergic dysfunction and cognitive impairment and to assess the deleterious impact of glucotoxicity.

Prep1 regulates angiogenesis through a PGC-1α-mediated mechanism.

Angiogenesis depends on a delicate balance between the different transcription factors, and their control should be considered necessary for preventing or treating diseases. Pre-B-cell leukemia transcription factor regulating protein 1 (Prep1) is a homeodomain transcription factor that plays a primary role in organogenesis and metabolism. Observations performed in a Prep1 hypomorphic mouse model, expressing 3-5% of the protein, show an increase of embryonic lethality due, in part, to defects in angiogenesis. In this study, we provide evidence that overexpression of Prep1 in mouse aortic endothelial cells (MAECs) stimulates migration, proliferation, and tube formation. These effects are paralleled by an increase of several proangiogenic factors and by a decrease of the antiangiogenic gene neurogenic locus notch homolog protein 1 (Notch1). Prep1-mediated angiogenesis involves the activation of the p160 Myb-binding protein (p160)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway. Indeed, Prep1 overexpression increases its binding with p160 and induces a 4-fold increase of p160 and 70% reduction of PGC-1α compared with control cells. Incubation of MAECs with a synthetic Prep1(54-72) peptide, mimicking the Prep1 region involved in the interaction with p160, reverts the proangiogenic effects mediated by Prep1. In addition, Prep1 levels increase by 3.2-fold during the fibroblast growth factor ß (bFGF)-mediated endothelial colony-forming cells' activation, whereas Prep1(54-72) peptide reduces the capability of these cells to generate tubular-like structures in response to bFGF, suggesting a possible role of Prep1 both in angiogenesis from preexisting vessels and in postnatal vasculogenesis. Finally, Prep1 hypomorphic heterozygous mice, expressing low levels of Prep1, show attenuated placental angiogenesis and vessel formation within Matrigel plugs. All of these observations indicate that Prep1, complexing with p160, decreases PGC-1α and stimulates angiogenesis.-Cimmino, I., Margheri, F., Prisco, F., Perruolo, G., D'Esposito, V., Laurenzana, A., Fibbi, G., Paciello, O., Doti, N., Ruvo, M., Miele, C., Beguinot, F., Formisano, P., Oriente, F. Prep1 regulates angiogenesis through a PGC-1α-mediated mechanism.

Potential Mechanisms of Bisphenol A (BPA) Contributing to Human Disease.

Bisphenol A (BPA) is an organic synthetic compound serving as a monomer to produce polycarbonate plastic, widely used in the packaging for food and drinks, medical devices, thermal paper, and dental materials. BPA can contaminate food, beverage, air, and soil. It accumulates in several human tissues and organs and is potentially harmful to human health through different molecular mechanisms. Due to its hormone-like properties, BPA may bind to estrogen receptors, thereby affecting both body weight and tumorigenesis. BPA may also affect metabolism and cancer progression, by interacting with GPR30, and may impair male reproductive function, by binding to androgen receptors. Several transcription factors, including PPARγ, C/EBP, Nrf2, HOX, and HAND2, are involved in BPA action on fat and liver homeostasis, the cardiovascular system, and cancer. Finally, epigenetic changes, such as DNA methylation, histones modification, and changes in microRNAs expression contribute to BPA pathological effects. This review aims to provide an extensive and comprehensive analysis of the most recent evidence about the potential mechanisms by which BPA affects human health.

Role of the HIF-1α/Nur77 axis in the regulation of the tyrosine hydroxylase expression by insulin in PC12 cells.

Tyrosine hydroxylase (TH), catalyzing the conversion of tyrosine into l-DOPA, is the rate-limiting enzyme in dopamine synthesis. Defects in insulin action contribute to alterations of TH expression and/or activity in the brain and insulin increases TH levels in 1-methyl-4-phenylpyridinium (MPP+)-treated neuronal cells. However, the molecular mechanisms underlying the regulation of TH by insulin have not been elucidated yet. Using PC12 cells, we show for the first time that insulin increases TH expression in a biphasic manner, with a transient peak at 2 hr and a delayed response at 16 hr, which persists for up to 24 hr. The use of a dominant negative hypoxia-inducible factor 1-alpha (HIF-1α) and its pharmacological inhibitor chetomin, together with chromatin immunoprecipitation (ChIP) experiments for the specific binding to TH promoter, demonstrate the direct role of HIF-1α in the early phase. Moreover, ChIP experiments and transfection of a dominant negative of the nerve growth factor IB (Nur77) indicate the involvement of Nur77 in the late phase insulin response, which is mediated by HIF-1α. In conclusion, the present study shows that insulin regulates TH expression through HIF-1α and Nur77 in PC12 cells, supporting the critical role of insulin signaling in maintaining an appropriate dopaminergic tone by regulating TH expression in the central nervous system.

Adipose Tissue Dysfunction as Determinant of Obesity-Associated Metabolic Complications.

Obesity is a critical risk factor for the development of type 2 diabetes (T2D), and its prevalence is rising worldwide. White adipose tissue (WAT) has a crucial role in regulating systemic energy homeostasis. Adipose tissue expands by a combination of an increase in adipocyte size (hypertrophy) and number (hyperplasia). The recruitment and differentiation of adipose precursor cells in the subcutaneous adipose tissue (SAT), rather than merely inflating the cells, would be protective from the obesity-associated metabolic complications. In metabolically unhealthy obesity, the storage capacity of SAT, the largest WAT depot, is limited, and further caloric overload leads to the fat accumulation in ectopic tissues (e.g., liver, skeletal muscle, and heart) and in the visceral adipose depots, an event commonly defined as "lipotoxicity." Excessive ectopic lipid accumulation leads to local inflammation and insulin resistance (IR). Indeed, overnutrition triggers uncontrolled inflammatory responses in WAT, leading to chronic low-grade inflammation, therefore fostering the progression of IR. This review summarizes the current knowledge on WAT dysfunction in obesity and its associated metabolic abnormalities, such as IR. A better understanding of the mechanisms regulating adipose tissue expansion in obesity is required for the development of future therapeutic approaches in obesity-associated metabolic complications.

Nutritional Factors, DNA Methylation, and Risk of Type 2 Diabetes and Obesity: Perspectives and Challenges.

A healthy diet improves life expectancy and helps to prevent common chronic diseases such as type 2 diabetes (T2D) and obesity. The mechanisms driving these effects are not fully understood, but are likely to involve epigenetics. Epigenetic mechanisms control gene expression, maintaining the DNA sequence, and therefore the full genomic information inherited from our parents, unchanged. An interesting feature of epigenetic changes lies in their dynamic nature and reversibility. Accordingly, they are susceptible to correction through targeted interventions. Here we will review the evidence supporting a role for nutritional factors in mediating metabolic disease risk through DNA methylation changes. Special emphasis will be placed on the potential of using DNA methylation traits as biomarkers to predict risk of obesity and T2D as well as on their response to dietary and pharmacological (epi-drug) interventions.

Epigenetic modifications of the Zfp/ZNF423 gene control murine adipogenic commitment and are dysregulated in human hypertrophic obesity.

AIMS/HYPOTHESIS: Subcutaneous adipocyte hypertrophy is associated with insulin resistance and increased risk of type 2 diabetes, and predicts its future development independent of obesity. In humans, subcutaneous adipose tissue hypertrophy is a consequence of impaired adipocyte precursor cell recruitment into the adipogenic pathway rather than a lack of precursor cells. The zinc finger transcription factor known as zinc finger protein (ZFP) 423 has been identified as a major determinant of pre-adipocyte commitment and maintained white adipose cell function. Although its levels do not change during adipogenesis, ectopic expression of Zfp423 in non-adipogenic murine cells is sufficient to activate expression of the gene encoding peroxisome proliferator-activated receptor γ (Pparγ; also known as Pparg) and increase the adipogenic potential of these cells. We investigated whether the Zfp423 gene is under epigenetic regulation and whether this plays a role in the restricted adipogenesis associated with hypertrophic obesity. METHODS: Murine 3T3-L1 and NIH-3T3 cells were used as fibroblasts committed and uncommitted to the adipocyte lineage, respectively. Human pre-adipocytes were isolated from the stromal vascular fraction of subcutaneous adipose tissue of 20 lean non-diabetic individuals with a wide adipose cell size range. mRNA levels were measured by quantitative real-time PCR, while methylation levels were analysed by bisulphite sequencing. Chromatin structure was analysed by micrococcal nuclease protection assay, and DNA-methyltransferases were chemically inhibited by 5-azacytidine. Adipocyte differentiation rate was evaluated by Oil Red O staining. RESULTS: Comparison of uncommitted (NIH-3T3) and committed (3T3-L1) adipose precursor cells revealed that Zfp423 expression increased (p < 0.01) in parallel with the ability of the cells to differentiate into mature adipocytes owing to both decreased promoter DNA methylation (p < 0.001) and nucleosome occupancy (nucleosome [NUC] 1 p < 0.01; NUC2 p < 0.001) in the 3T3-L1 compared with NIH-3T3 cells. Interestingly, non-adipogenic epigenetic profiles can be reverted in NIH-3T3 cells as 5-azacytidine treatment increased Zfp423 mRNA levels (p < 0.01), reduced DNA methylation at a specific CpG site (p < 0.01), decreased nucleosome occupancy (NUC1, NUC2: p < 0.001) and induced adipocyte differentiation (p < 0.05). These epigenetic modifications can also be initiated in response to changes in the pre-adipose cell microenvironment, in which bone morphogenetic protein 4 (BMP4) plays a key role. We finally showed that, in human adipocyte precursor cells, impaired epigenetic regulation of zinc nuclear factor (ZNF)423 (the human orthologue of murine Zfp423) was associated with inappropriate subcutaneous adipose cell hypertrophy. As in NIH-3T3 cells, the normal ZNF423 epigenetic profile was rescued by 5-azacytidine exposure. CONCLUSIONS/INTERPRETATION: Our results show that epigenetic events regulate the ability of precursor cells to commit and differentiate into mature adipocytes by modulating ZNF423, and indicate that dysregulation of these mechanisms accompanies subcutaneous adipose tissue hypertrophy in humans.

Oleic acid promotes prostate cancer malignant phenotype via the G protein-coupled receptor FFA1/GPR40.

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men and the second leading cause of cancer-related death in industrialized countries. Epidemiologic evidence suggests that obesity promotes aggressive PCa. Recently, a family of Free Fatty Acid (FFA) receptors (FFARs) has been identified and reported to affect several crucial biological functions of tumor cells such as proliferation, invasiveness, and apoptosis. Here we report that oleic acid (OA), one of the most prevalent FFA in human plasma, increases proliferation of highly malignant PC3 and DU-145 PCa cells. Furthermore, docetaxel cytotoxic action, the first-line chemotherapeutic agent for the treatment of androgen-independent PCa, was significantly reduced in the presence of OA, when measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, suggesting that this FFA plays also a role in chemoresistance. OA induced intracellular calcium increase, in part due to the store operated calcium entry (SOCE), measured by a calcium imaging technique. Moreover, PI3K/Akt signaling pathway was enhanced, as revealed by increased Akt phosphorylation levels. Intriguingly, attenuating the expression of FFA1/GPR40, a receptor for long chain FFA including OA, prevented the OA-induced effects. Of relevance, we found that FFA1/GPR40 is significantly overexpressed in tissue specimens of PCa, compared to benign prostatic hyperplasia tissues, at both mRNA and protein expression level, analyzed by Real Time RT-PCR and immunofluorescence experiments, respectively. Our data suggest that OA promotes an aggressive phenotype in PCa cells via FFA1/GPR40, calcium and PI3K/Akt signaling. Thus, FFA1/GPR40, might represent a potential useful prognostic biomarker and therapeutic target for the treatment of advanced PCa.

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.

Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype.

The SGK1 inhibitor SI113 induces autophagy, apoptosis, and endoplasmic reticulum stress in endometrial cancer cells.

Endometrial cancer is often characterized by PI3K/AKT pathway deregulation. Recently it has been suggested that SGK1, a serine/threonine protein kinase that shares structural and functional similarities with the AKT family, might play a role in cancer, since its expression and/or activity has been found to be deregulated in different human tumors. However, the role of SGK1 in endometrial cancer has been poorly investigated. Here, we show that SGK1 expression is increased in tissue specimens from neoplastic endometrium. The SGK1 inhibitor SI113 induced a significant reduction of endometrial cancer cells viability, measured by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. This effect was associated to the increase of autophagy, as revealed by the increase of the markers LC3B-II and beclin I, detected by both immunofluorescence and western blot analysis. SI113 treatment caused also apoptosis of endometrial cancer cells, evidenced by the cleavage of the apoptotic markers PARP and Caspase-9. Intriguingly, these effects were associated to the induction of endoplasmic reticulum stress markers GRP78 and CHOP evaluated by both Real-Time RT-PCR and Western Blot analysis. Increased expression of SGK1 in endometrial cancer tissues suggest a role for SGK1 in this type of cancer, as reported for other malignancies. Moreover, the efficacy of SI113 in affecting endometrial cancer cells viability, possibly via endoplasmic reticulum stress activation, identifies SGK1 as an attractive molecular target for new tailored therapeutic intervention for the treatment of endometrial cancer.