Gene Editing and Human iPSCs in Cardiovascular and Metabolic Diseases.

The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.

Mild exacerbation of obesity- and age-dependent liver disease progression by senolytic cocktail dasatinib + quercetin.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent and represents a growing challenge in terms of prevention and treatment. A minority of affected patients develops inflammation, subsequently fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCC is a leading cause of cancer-related death. An increased number of senescent cells correlate with age-related tissue degeneration during NAFLD-induced HCC. Senolytics are promising agents that target selectively senescent cells. Previous studies showed that whereas a combination of the senolytic drugs dasatinib and quercetin (D + Q) reduced NAFLD in mice, D + Q lacked efficacy in removing doxorubicin-induced ß-gal-positive senescent cells in human HCC xenografted mice. Whether D + Q has an effect on the age-associated spectrum of NAFLD-inflammation-HCC remains unknown. METHODS: Here, we utilized an established model of age- and obesity-associated HCC, the low dose diethylnitrosamine (DEN)/high fat diet (HFD), a regimen promoting liver inflammation and tumorigenesis over a long period of 9 months. Four groups of mice each were created: group 1 included control untreated mice; group 2 included mice treated with D + Q; group 3 included mice undergoing the DEN/HFD protocol; group 4 included mice undergoing the DEN/HFD protocol with the administration of D + Q. At the end of the chemical/dietary regimen, we analyzed liver damage and cell senescence by histopathology, qPCR and immunoblotting approaches. RESULTS: Unexpectedly, D + Q worsened liver disease progression in the DEN/HFD mouse model, slightly increasing histological damage and tumorigenesis, while having no effect on senescent cells removal. CONCLUSIONS: In summary, using an animal model that fully recapitulates NAFLD, we demonstrate that these compounds are ineffective against age-associated NAFLD-induced HCC. Video Abstract.

Bioactive Compounds from Lemon (Citrus limon) Extract Overcome TNF-α-Induced Insulin Resistance in Cultured Adipocytes.

The consumption of plant-based food is important for health promotion, especially regarding the prevention and management of chronic diseases such as diabetes. We investigated the effects of a lemon extract (LE), containing ≥20.0% total flavanones and ≥1.0% total hydroxycinnamic acids, on insulin signaling in murine 3T3-L1 adipocytes treated with TNF-α, which was used to mimic in vitro the insulin resistance condition that characterizes diabetes mellitus. Our results showed LE increased PPARγ, GLUT4 and DGAT-1 levels, demonstrating the potential of this lemon extract in the management of insulin resistance conditions associated with TNF-α pathway activation. LE treatment further decreased the release of interleukin 6 (IL-6) and restored triglyceride synthesis, which is the main feature of a healthy adipocyte.

Non-competitive heme oxygenase-1 activity inhibitor reduces non-small cell lung cancer glutathione content and regulates cell proliferation.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related death mainly due to its high metastatic rate. Impairment of redox homeostasis mechanisms has been previously described in NSCLC and is associated with the disease itself as well as with comorbidities such as smoking. The aim of the present in vitro study was to evaluate the effect of selective and non-competitive inhibition of heme oxygenase-1 (HO-1) on cancer redox homeostasis with particular regards to glutathione (GSH) metabolism related enzymes. NSCLC cell line (A549) was treated with the HO-1 activity inhibitor VP13/47 (10 µM) and we further evaluated cell viability, apoptosis, mitochondrial dysfunction and oxidative stress. Our results showed that VP13/47 significantly reduced HO-1 expression and total HO activity thus, resulting in a significant reduction of cell viability, proliferation and increased apoptosis, mitochondrial dysfunction and oxidative stress. Consistently with increased oxidative stress, we also showed that reduced GSH was significantly decreased and such effect was also accompanied by a significant downregulation of the enzymes involved in its biosynthesis. Taken all together our results show that selective HO-1 inhibition significantly impairs NSCLC progression and may represent a possible pharmacological strategy for new chemotherapy agents.

Epoxyeicosatrienoic intervention improves NAFLD in leptin receptor deficient mice by an increase in PGC1α-HO-1-PGC1α-mitochondrial signaling.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and is considered to be an inflammatory disorder characterized by fatty acid accumulation, oxidative stress, and lipotoxicity. We have previously reported that epoxyeicosatrienoic acid-agonist (EET-A) has multiple beneficial effects on cardiac, renal and adipose tissue function while exhibiting both anti-inflammatory and anti-oxidant activities. We hypothesized that EET-A intervention would play a central role in attenuation of obesity-induced steatosis and hepatic fibrosis that leads to NAFLD. METHODS: We studied the effect of EET-A on fatty liver using db/db mice as a model of obesity. Mice were fed a high fat diet (HFD) for 16 weeks and administered EET-A twice weekly for the final 8 weeks. RESULTS: db/db mice fed HFD significantly increased hepatic lipid accumulation as manifested by increases in NAS scores, hepatic fibrosis, insulin resistance, and inflammation, and decreases in mitochondrial mitofusin proteins (Mfn 1/2) and anti-obesity genes Fibroblast growth factor 21 (FGF21) and Cellular Repressor of E1A-Stimulated Genes 1 (CREG1). EET-A administration reversed the decrease in these genes and reduced liver fibrosis. Knockout of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in EET-A treated mice resulted in a reversal of the beneficial effects of EET-A administration. CONCLUSIONS: EET-A intervention diminishes fatty acid accumulation, fibrosis, and NFALD associated with an increase in HO-1-PGC1α and increased insulin receptor phosphorylation. A pharmacological strategy involving EETs may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NAFLD.

Cold Press Pomegranate Seed Oil Attenuates Dietary-Obesity Induced Hepatic Steatosis and Fibrosis through Antioxidant and Mitochondrial Pathways in Obese Mice.

AIM: Obesity is associated with metabolic syndrome, hypertension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), and type 2 diabetes. In this study, we investigated whether the dietary supplementation of pomegranate seed oil (PSO) exerted a protective effect on liver lipid uptake, fibrosis, and mitochondrial function in a mouse model of obesity and insulin resistance. METHOD: In this in vivo study, eight-week-old C57BL/6J male mice were fed with a high fat diet (HFD) for 24 weeks and then were divided into three groups as follows: group (1) Lean; group (n = 6) (2) HF diet; group (n = 6) (3) HF diet treated with PSO (40 mL/kg food) (n = 6) for eight additional weeks starting at 24 weeks. Physiological parameters, lipid droplet accumulation, inflammatory biomarkers, antioxidant biomarkers, mitochondrial biogenesis, insulin sensitivity, and hepatic fibrosis were determined to examine whether PSO intervention prevents obesity-associated metabolic syndrome. RESULTS: The PSO group displayed an increase in oxygen consumption, as well as a decrease in fasting glucose and blood pressure (p < 0.05) when compared to the HFD-fed mice group. PSO increased both the activity and expression of hepatic HO-1, downregulated inflammatory adipokines, and decreased hepatic fibrosis. PSO increased the levels of thermogenic genes, mitochondrial signaling, and lipid metabolism through increases in Mfn2, OPA-1, PRDM 16, and PGC1α. Furthermore, PSO upregulated obesity-mediated hepatic insulin receptor phosphorylation Tyr-972, p-IRB tyr1146, and pAMPK, thereby decreasing insulin resistance. CONCLUSIONS: These results indicated that PSO decreased obesity-mediated insulin resistance and the progression of hepatic fibrosis through an improved liver signaling, as manifested by increased insulin receptor phosphorylation and thermogenic genes. Furthermore, our findings indicate a potential therapeutic role for PSO in the prevention of obesity-associated NAFLD, NASH, and other metabolic disorders.

Mesenteric lymph nodes as alternative site for pancreatic islet transplantation in a diabetic rat model.

BACKGROUND: Islet transplantation has progressively become a safe alternative to pancreas transplantation for the treatment of type 1 diabetes. However, the long-term results of islet transplantation could be significantly increased by improving the quality of the islet isolation technique even exploring alternative islet transplantation sites to reduce the number of islets required to mitigate hyperglycemia. The goal of the study was to test the lymph node as a suitable anatomical location for islet engraftment in a rodent model. METHODS: Forty Lewis rats, 6-8 weeks old, body weight 250-300 g, have been used as islet donors and recipients in syngeneic islet transplantation experiments. Ten rats were rendered diabetic by one injection of 65 mg/Kg of streptozotocin. After pancreas retrieval from non diabetic donors, islet were isolated and transplanted in the mesenteric lymph nodes of 7 diabetic rats. Rats were followed for 30 days after islet transplantation. RESULTS: A total of 7 islet transplantations in mesenteric lymph nodes have been performed. Two rats died 24 and 36 h after transplantation due to complications. No transplanted rat acquired normal glucose blood levels and insulin independence after the transplantation. However, the mean blood levels of glycemia were significantly lower in transplanted rats compared with diabetic rats (470.4 mg/dl vs 605 mg/dl, p 0.04). Interestingly, transplanted rats have a significant weight increase after transplantation compared to diabetic rats (mean value 295 g in transplanted rats vs 245 g in diabetic rats, p < 0.05), with an overall improvement of social activities and health. Immunohistochemical analysis of the 5 mesenteric lymph nodes of transplanted rats demonstrated the presence of living islets in one lymph node. CONCLUSIONS: Although islet engraftment in lymph nodes is possible, islet transplantation in lymph nodes in rats resulted in few improvements of glucose parameters.

Heme Oxygenase-1 Inhibition Sensitizes Human Prostate Cancer Cells towards Glucose Deprivation and Metformin-Mediated Cell Death.

High levels of heme oxygenase (HO)-1 have been frequently reported in different human cancers, playing a major role in drug resistance and regulation of cancer cell redox homeostasis. Metformin (MET), a drug widely used for type 2 diabetes, has recently gained interest for treating several cancers. Recent studies indicated that the anti-proliferative effects of metformin in cancer cells are highly dependent on glucose concentration. The present work was directed to determine whether use of a specific inhibitor of HO-1 activity, alone or in combination with metformin, affected metastatic prostate cancer cell viability under different concentrations of glucose. MTT assay and the xCELLigence system were used to evaluate cell viability and cell proliferation in DU145 human prostate cancer cells. Cell apoptosis and reactive oxygen species were analyzed by flow cytometry. The activity of HO-1 was inhibited using a selective imidazole-based inhibitor; genes associated with antioxidant systems and cell death were evaluated by qRT-PCR. Our study demonstrates that metformin suppressed prostate cancer growth in vitro and increased oxidative stress. Disrupting the antioxidant HO-1 activity, especially under low glucose concentration, could be an attractive approach to potentiate metformin antineoplastic effects and could provide a biochemical basis for developing HO-1-targeting drugs against solid tumors.

Protective Effects of Caffeic Acid Phenethyl Ester (CAPE) and Novel Cape Analogue as Inducers of Heme Oxygenase-1 in Streptozotocin-Induced Type 1 Diabetic Rats.

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease resulting in the destruction of insulin producing ß-cells of the pancreas, with consequent insulin deficiency and excessive glucose production. Hyperglycemia results in increased levels of reactive oxygen species (ROS) and nitrogen species (RNS) with consequent oxidative/nitrosative stress and tissue damage. Oxidative damage of the pancreatic tissue may contribute to endothelial dysfunction associated with diabetes. The aim of the present study was to investigate if the potentially protective effects of phenethyl ester of caffeic acid (CAPE), a natural phenolic compound occurring in a variety of plants and derived from honeybee hive propolis, and of a novel CAPE analogue, as heme oxygenase-1 (HO-1) inducers, could reduce pancreatic oxidative damage induced by excessive amount of glucose, affecting the nitric oxide synthase/dimethylarginine dimethylaminohydrolase (NOS/DDAH) pathway in streptozotocin-induced type 1 diabetic rats. Our data demonstrated that inducible nitric oxide synthase/gamma-Glutamyl-cysteine ligase (iNOS/GGCL) and DDAH dysregulation may play a key role in high glucose mediated oxidative stress, whereas HO-1 inducers such as CAPE or its more potent derivatives may be useful in diabetes and other stress-induced pathological conditions.

Ablation of soluble epoxide hydrolase reprogram white fat to beige-like fat through an increase in mitochondrial integrity, HO-1-adiponectin in vitro and in vivo.

We have shown that epoxyeicosatrienoic acids (EETs), specifically 11,12- and 14,15-EETs, reduce adipogenesis in human mesenchymal stem cells and mouse preadipocytes (3T-3L1). In this study, we explore the effects of soluble epoxide hydrolase (sEH) deletion on various aspects of adipocyte-function, including programing for white vs. beige-like fat, and mitochondrial and thermogenic gene-expressions. We further hypothesize that EETs and heme-oxygenase 1 (HO-1) form a synergistic, functional module whose effects on adipocyte and vascular function is greater than the effects of sEH deletion alone. In in vitro studies, we examined the effect of sEH inhibitors on MSC-derived adipocytes. MSC-derived adipocytes exposed to AUDA, an inhibitor of sEH, exhibit an increased number of small and healthy adipocytes, an effect reproduced by siRNA for sEH. in vivo studies indicate that sEH deletion results in a significant decrease in adipocyte size, inflammatory adipokines NOV, TNFα, while increasing adiponectin (p < 0.05). These findings are associated with a decrease in body weight (p < 0.05), and visceral fat (p < 0.05). Importantly, sEH deletion was associated with a significant increase in Mfn1, COX 1, UCP1 and adiponectin (p < 0.03). sEH deletion was manifested by a significant increase in EETs isomers 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET and an increased EETs/DHETEs ratio. Notably, activation of HO-1 gene expression further increased the levels of EETs, suggesting that the antioxidant HO-1 system protects EETs from degradation by ROS. These results are novel in that sEH deletion, while increasing EET levels, resulted in reprograming of white fat to express mitochondrial and thermogenic genes, a phenotype characteristic of beige-fat. Thus, EETs agonist(s) and sEH inhibitors may have therapeutic potential in the treatment of metabolic syndrome and obesity.

Human centenarian-associated SIRT6 mutants modulate hepatocyte metabolism and collagen deposition in multilineage hepatic 3D spheroids.

Non-alcoholic fatty liver disease (NAFLD), encompassing fatty liver and its progression into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), is one of the rapidly rising health concerns worldwide. SIRT6 is an essential nuclear sirtuin that regulates numerous pathological processes including insulin resistance and inflammation, and recently it has been implicated in the amelioration of NAFLD progression. SIRT6 overexpression protects from formation of fibrotic lesions. However, the underlying molecular mechanisms are not fully delineated. Moreover, new allelic variants of SIRT6 (N308K/A313S) were recently associated with the longevity in Ashkenazi Jews by improving genome maintenance and DNA repair, suppressing transposons and killing cancer cells. Whether these new SIRT6 variants play different or enhanced roles in liver diseases is currently unknown. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect liver metabolism and associated diseases. We present evidence that overexpression of centenarian-associated SIRT6 variants dramatically altered the metabolomic and secretomic profiles of unchallenged immortalized human hepatocytes (IHH). Most amino acids were increased in the SIRT6 N308K/A313S overexpressing IHH when compared to IHH transfected with the SIRT6 wild-type sequence. Several unsaturated fatty acids and glycerophospholipids were increased, and ceramide tended to be decreased upon SIRT6 N308K/A313S overexpression. Furthermore, we found that overexpression of SIRT6 N308K/A313S in a 3D hepatic spheroid model formed by the co-culture of human immortalized hepatocytes (IHH) and hepatic stellate cells (LX2) inhibited collagen deposition and fibrotic gene expression in absence of metabolic or dietary challenges. Hence, our findings suggest that novel longevity associated SIRT6 N308K/A313S variants could favor the prevention of NASH by altering hepatocyte proteome and lipidome.

Detection of cell-free histones in the cerebrospinal fluid of pediatric central nervous system malignancies by imaging flow cytometry.

Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

The costs and benefits of senotherapeutics for human health.

Cellular senescence is a major contributor to age-related diseases in humans; however, it also has a beneficial role in physiological and pathological processes, including wound healing, host immunity, and tumour suppression. Reducing the burden of cell senescence in animal models of cardiometabolic disorders, inflammatory conditions, neurodegenerative diseases, and cancer using pharmaceutical approaches that selectively target senescent cells (ie, senolytics) or that suppress senescence-associated secretory phenotype (ie, senomorphics) holds great promise for the management of chronic age-associated conditions. Although studies have provided evidence that senolytics or senomorphics are effective at decreasing the number of senescent cells in humans, the short-term and long-term side-effects of these therapies are largely unknown. In this Review, we systematically discuss the senolytics and senomorphics that have been investigated in clinical trials or have been used off-label, presenting their various adverse effects. Despite the potential of senotherapeutics to transform anti-ageing medicine, a cautionary approach regarding unwanted dose-dependent side-effects should be adopted.

Adipocyte-Specific Expression of PGC1α Promotes Adipocyte Browning and Alleviates Obesity-Induced Metabolic Dysfunction in an HO-1-Dependent Fashion.

Recent studies suggest that PGC1-α plays a crucial role in mitochondrial and vascular function, yet the physiological significance of PGC1α and HO expression in adipose tissues in the context of obesity-linked vascular dysfunction remains unclear. We studied three groups of six-week-old C57BL/6J male mice: (1) mice fed a normal chow diet; (2) mice fed a high-fat diet (H.F.D.) for 28 weeks, and (3) mice fed a high-fat diet (H.F.D.) for 28 weeks, treated with adipose-specific overexpression of PGC-1α (transgenic-adipocyte-PGC-1α) at week 20, and continued on H.F.D. for weeks 20-28. R.N.A. arrays examined 88 genes involved in adipocyte proliferation and maturation. Blood pressure, tissue fibrosis, fasting glucose, and oxygen consumption were measured, as well as liver steatosis, and the expression levels of metabolic and mitochondrial markers. Obese mice exhibited a marked reduction of PGC1α and developed adipocyte hypertrophy, fibrosis, hepatic steatosis, and decreased mitochondrial respiration. Mice with adipose-specific overexpression of PGC1-α exhibited improvement in HO-1, mitochondrial biogenesis and respiration, with a decrease in fasting glucose, reduced blood pressure and fibrosis, and increased oxygen consumption. PGC-1α led to the upregulated expression of processes associated with the browning of fat tissue, including UCP1, FGF21, and pAMPK signaling, with a reduction in inflammatory adipokines, NOV/CCN3 expression, and TGFß. These changes required HO-1 expression. The R.N.A. array analysis identified subgroups of genes positively correlated with contributions to the browning of adipose tissue, all dependent on HO-1. Our observations reveal a positive impact of adipose-PGC1-α on distal organ systems, with beneficial effects on HO-1 levels, reversing obesity-linked cardiometabolic disturbances.

Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: A pilot study.

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-ß (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

Nociceptin/orphanin FQ opioid receptor (NOP) selective ligand MCOPPB links anxiolytic and senolytic effects.

Accumulation of senescent cells may drive age-associated alterations and pathologies. Senolytics are promising therapeutics that can preferentially eliminate senescent cells. Here, we performed a high-throughput automatized screening (HTS) of the commercial LOPAC®Pfizer library on aphidicolin-induced senescent human fibroblasts, to identify novel senolytics. We discovered the nociceptin receptor FQ opioid receptor (NOP) selective ligand 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole (MCOPPB, a compound previously studied as potential anxiolytic) as the best scoring hit. The ability of MCOPPB to eliminate senescent cells in in vitro models was further tested in mice and in C. elegans. MCOPPB reduced the senescence cell burden in peripheral tissues but not in the central nervous system. Mice and worms exposed to MCOPPB also exhibited locomotion and lipid storage changes. Mechanistically, MCOPPB treatment activated transcriptional networks involved in the immune responses to external stressors, implicating Toll-like receptors (TLRs). Our study uncovers MCOPPB as a NOP ligand that, apart from anxiolytic effects, also shows tissue-specific senolytic effects.

GDF11 inhibits adipogenesis and improves mature adipocytes metabolic function via WNT/ß-catenin and ALK5/SMAD2/3 pathways.

OBJECTIVE: GDF11 is a member of the TGF-ß superfamily that was recently implicated as potential "rejuvenating" factor, which can ameliorate metabolic disorders. The main objective of the presented study was to closely characterize the role of GDF11 signaling in the glucose homeostasis and in the differentiation of white adipose tissue. METHODS: We performed microscopy imaging, biochemical and transcriptomic analyses of adipose tissues of 9 weeks old ob/ob mice and murine and human pre-adipocyte cell lines. RESULTS: Our in vivo experiments employing GDF11 treatment in ob/ob mice showed improved glucose/insulin homeostasis, decreased weight gain and white adipocyte size. Furthermore, GDF11 treatment inhibited adipogenesis in pre-adipocytes by ALK5-SMAD2/3 activation in cooperation with the WNT/ß-catenin pathway, whose inhibition resulted in adipogenic differentiation. Lastly, we observed significantly elevated levels of the adipokine hormone adiponectin and increased glucose uptake by mature adipocytes upon GDF11 exposure. CONCLUSION: We show evidence that link GDF11 to adipogenic differentiation, glucose, and insulin homeostasis, which are pointing towards potential beneficial effects of GDF11-based "anti-obesity" therapy.

Redox and Epigenetics in Human Pluripotent Stem Cells Differentiation.

Significance: Since their discovery, induced pluripotent stem cells (iPSCs) had generated considerable interest in the scientific community for their great potential in regenerative medicine, disease modeling, and cell-based therapeutic approach, due to their unique characteristics of self-renewal and pluripotency. Recent Advances: Technological advances in iPSC genome-wide epigenetic profiling led to the elucidation of the epigenetic control of cellular identity during nuclear reprogramming. Moreover, iPSC physiology and metabolism are tightly regulated by oxidation-reduction events that mainly occur during the respiratory chain. In theory, iPSC-derived differentiated cells would be ideal for stem cell transplantation as autologous cells from donors, as the risks of rejection are minimal. Critical Issues: However, iPSCs experience high oxidative stress that, in turn, confers a high risk of increased genomic instability, which is most often linked to DNA repair deficiencies. Genomic instability has to be assessed before iPSCs can be used in therapeutic designs. Future Directions: This review will particularly focus on the links between redox balance and epigenetic modifications-in particular based on the histone variant macroH2A1-that determine DNA damage response in iPSCs and derived differentiated cells, and that might be exploited to decrease the teratogenic potential on iPSC transplantation. Antioxid. Redox Signal. 34, 335-349.

In vitro effects of bioflavonoids rich lemon extract on pre-adipocyte differentiation.

Lemon fruit is a source of bioactive compounds, which has many beneficial effects on health. Obesity is characterized by over-accumulation of adipose tissue as a result of increased adipocyte size and number. Adipogenesis is mediated and assisted by various transcription factors that induce lipid-metabolizing enzymes followed by an increase of perilipin expression and lipid droplets generation. Here, we evaluate the effect of lemon extract (LE) as radical scavenger and the consequent regulation of adipocyte differentiation and lipid accumulation. 3T3-L1 murine pre-adipocytes were differentiated and treated with different LE concentrations. The high percentages of flavonoid contained in LE led to a significant inhibition of DPPH radical and reactive oxygen species, demonstrating a strong radical scavenger activity. Treatment of 3T3-cells with LE showed a significant decrease of perilipin expression, subsequently confirmed by the reduction of lipid droplet accumulation, resulting from Oil Red O Staining and by the downregulation of PPARγ and DGAT-1mRNA.

Senescence-like phenotype in post-mitotic cells of mice entering middle age.

Staining mice tissues for ß-galactosidase activity is a fundamental tool to detect age- or disease-associated cellular senescence. However, reported analyses of positivity for senescence-associated ß-galactosidase activity or for other markers of senescence in post-mitotic cells of healthy murine tissues have been fragmentary or inconclusive. Here, we attempted to independently deepen this knowledge using multiple senescence markers within the same cells of wild type mice entering middle age (9 months of age). A histochemistry protocol for the pH-dependent detection of ß-galactosidase activity in several tissues was used. At pH 6, routinely utilized to detect senescence-associated ß-galactosidase activity, only specific cellular populations in the mouse body (including Purkinje cells and choroid plexus in the central nervous system) were detected as strongly positive for ß-galactosidase activity. These post-mitotic cells were also positive for other established markers of senescence (p16, p21 and DPP4), detected by immunofluorescence, confirming a potential senescent phenotype. These data might contribute to understanding the functional relation between the senescence-associated ß-galactosidase activity and senescence markers in post-mitotic cells in absence of disease or advanced aging.