FTO Gene Polymorphisms at the Crossroads of Metabolic Pathways of Obesity and Epigenetic Influences.

In this review, we summarize the current state of knowledge on the fat mass and obesity-associated (FTO) gene and its role in obesity. The FTO-encoded protein is involved in multiple molecular pathways contributing to obesity as well as other metabolic complexities. This review emphasizes the epigenetic influence on the FTO gene as a new approach in the treatment and management of obesity. Several known substances have a positive effect on reducing FTO expression. Depending on which variant of the single nucleotide polymorphism (SNP) is present, the profile and level of gene expression changes. Implementation of environmental change measures could lead to reduced phenotypic manifestation of FTO expression. Treating obesity through FTO gene regulation will have to include various complex signal pathways in which FTO takes part. Identification of FTO gene polymorphisms may be useful for the development of individual obesity management strategies, including the recommendation of taking certain foods and supplements.

Antioxidant and Antiproliferative Activities of Kale (Brassica oleracea L. Var. acephala DC.) and Wild Cabbage (Brassica incana Ten.) Polyphenolic Extracts.

Brassicaceae are rich in healthy phytochemicals that have a positive impact on human health. The aim of this study was to analyze the phenolic compounds and antioxidant and anticancer potential of traditional Croatian kale (Brassica oleracea L. var. acephala DC.) and wild cabbage (Brassica incana Ten.) extracts. The phenolic groups and antioxidant activity were determined by spectrophotometry, selected phenolic compounds (ferulic acid, sinapic acid, salicylic acid, kaempferol, and quercetin) were analyzed by LC-MS/MS, and anticancer potential was evaluated in vitro using HeLa cells. The extracts of both plant species are rich in phenolic compounds and showed significant antioxidant activity at similar levels. LC-MS/MS detected sinapic acid as the most abundant phenolic acid, followed by ferulic acid, while salicylic acid was present at lower concentrations. A comparative analysis showed that wild cabbage contained significantly more sinapic acid, while kale contained more kaempferol and quercetin. Both Brassica extracts at a concentration of 50 µg mL-1 showed an antiproliferative effect on HeLa cells, while they did not affect the proliferation of normal human skin fibroblasts. Wild cabbage extract also showed an antiproliferative effect on HeLa cells at a lower applied concentration of 10 µg mL-1 of extracts. The clonogenic analysis also revealed the inhibitory effect of the extracts on HeLa colony growth.

In Vivo Skin Regeneration and Wound Healing Using Cell Micro-Transplantation.

Background: The accumulation of senescent cells in tissues alters tissue homeostasis and affects wound healing. It is also considered to be the main contributing factor to aging. In addition to losing their ability to divide, senescent cells exert detrimental effects on surrounding tissues through their senescence-associated secretory phenotype (SASP). They also affect stem cells and their niche, reducing their capacity to divide which increasingly reduces tissue regenerative capacity over time. The aim of our study was to restore aged skin by increasing the fraction of young cells in vivo using a young cell micro-transplantation technique on Fischer 344 rats. Employing the same technique, we also used wild-type skin fibroblasts and stem cells in order to heal Dominant Dystrophic Epidermolysis Bulosa (DDEB) wounds and skin blistering. Results: We demonstrate that implantation of young fibroblasts restores cell density, revitalizes cell proliferation in the dermis and epidermis, rejuvenates collagen I and III matrices, and boosts epidermal stem cell proliferation in rats with advancing age. We were also able to reduce blistering in DDEB rats by transplantation of skin stem cells but not skin fibroblasts. Conclusions: Our intervention proves that a local increase of young cells in the dermis changes tissue homeostasis well enough to revitalize the stem cell niche, ensuring overall skin restoration and rejuvenation as well as healing DDEB skin. Our method has great potential for clinical applications in skin aging, as well as for the treatment of various skin diseases.

Long-term effects of melatonin and resveratrol on aging rats: A multi-biomarker approach.

Aging-related impaired body structure and functions may be, at least partially, caused by elevated oxidative stress. Melatonin (MEL) and resveratrol (RSV) may act as antioxidant and anti-aging compounds, but these actions in experimental animals and humans are controversial. Herein, a rat model of aging was used to study the long-term sex-related effects of MEL and RSV treatment on body mass and blood/plasma parameters of DNA damage, oxidative status (glutathione and malondialdehyde levels), and concentrations of sex hormones. Starting from the age of 3mo, for the next 9mo or 21mo male and female Wistar rats (n = 4-7 per group) were given water to drink (controls) or 0.1 % ethanol in water (vehicle), or MEL or RSV (each 10 mg/L vehicle). DNA damage in whole blood cells was tested by comet assay, whereas in plasma, glutathione, malondialdehyde, and sex hormones were determined by established methods. Using statistical analysis of data by ANOVA/Scheffe post hoc, we observed a similar sex- and aging-dependent rise of body mass in both sexes and drop of plasma testosterone in control and vehicle-treated male rats, whose pattern remained unaffected by MEL and RSV treatment. Compared with controls, all other parameters remained largely unchanged in aging and differently treated male and female rats. We concluded that the sex- and aging-related pattern of growth and various blood parameters in rats were not affected by the long-term treatment with MEL and RSV at the estimated daily doses (300-400 µg/kg b.m.) that exceed usual moderate consumption in humans.

Epilipidomics of Senescent Dermal Fibroblasts Identify Lysophosphatidylcholines as Pleiotropic Senescence-Associated Secretory Phenotype (SASP) Factors.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging.

Sex-dependent expression of metallothioneins MT1 and MT2 and concentrations of trace elements in rat liver and kidney tissues: Effect of gonadectomy.

Metallothioneins (MTs) exhibit binding affinity for several essential and toxic trace elements. Previous studies in rodents indicated sex differences in the hepatic and renal expression of MTs and concentrations of various elements. The mechanism responsible for these differences has not been resolved. Here, in the liver and kidney tissues of sham-operated and gonadectomized male and female rats we determined the expression of MT1 and MT2 (MT1&2) mRNA by RT-PCR, abundance of MT1&2 proteins by Western blotting and immunocytochemistry, concentrations of essential (Fe, Zn, Cu, Co) and toxic (Cd, Hg, Pb) elements by ICP-MS, and oxidative status parameters (SOD, GPx, MDA, GSH) by biochemical methods. In both organs, the expression of MT1&2 mRNA and MT1&2 proteins was female-dominant, upregulated by castration, and downregulated by ovariectomy. Concentrations of Fe in the liver and Co in the kidneys followed the same pattern. Most other elements (Zn, Cu, Cd, Hg) exhibited female- or male-dominant sex differences, affected by gonadectomy in one or both organs. Pb was sex- and gonadectomy-unaffected. GPx and MDA were elevated and associated with the highest concentrations of Fe only in the female liver. We conclude that the sex-dependent expression of MT1&2 mRNA and proteins in the rat liver and kidneys may include different mechanisms. In the liver, the female-dominant tissue concentrations of Fe may generate oxidative stress which is a potent enhancer of MTs production, whereas in kidneys, the female-dominant expression of MTs may be unrelated to Fe-mediated oxidative stress.

Mathematical model and computer simulations of telomere loss.

The molecular mechanisms that control the limited number of human cell divisions has occupied researchers ever since its first description in 1961. There is evidence that this limited growth capacity, referred to as cellular or replicative senescence, is the basis for organismal ageing. Numerous studies point to the molecular mechanisms of telomere involvement in this phenomenon. A hallmark of cell senescence is high stochasticity where individual cells enter senescence in a completely random and stochastic fashion. Therefore, mathematical modelling and computational simulations of telomere dynamics are often used to explain this stochastic nature of cell ageing. Models published thus far were based on the molecular mechanisms of telomere biology and how they dictate the dynamics of cell culture proliferation. In the present work we propose an advanced model of telomere controlled cell senescence based on abrupt telomere shortening, thus explaining some important but thus far overlooked aspects of cell senescence. We test our theory by simulating the proliferative potential and two-sister experiment originally conducted by Smith and Whitney in 1980.

Mechanisms of fetal epigenetics that determine telomere dynamics and health span in adulthood.

Advances in epigenetics now enable us to better understand environmental influences on the genetic background of human diseases. This refers especially to fetal development where an adverse intrauterine environment impacts oxygen and nutrient supply to the fetus. Recently, differences in telomere length and telomere loss dynamics among individuals born with intrauterine growth restriction compared to normal controls have been described. In this paper we propose possible molecular mechanisms that (pre)program telomere epigenetics during pregnancy. This programming sets differences in telomere lengths and dynamics of telomere shortening in adulthood and therefore dictates the dynamics of aging and morbidity in later life.

Mutual interactions between telomere heterogeneity and cell culture growth dynamics shape stochasticity of cell aging.

Mathematical modeling and computational simulations are often used to explain the stochastic nature of cell aging. The models published thus far are based on the molecular mechanisms of telomere biology and how they dictate the dynamics of cell culture proliferation. However, the influence of cell growth conditions on telomere dynamics has been widely overlooked. These conditions include interactions with surrounding cells through contact inhibition, gradual accumulation of non-dividing cells, culture propagation and other cell culture maintenance factors. In order to follow the intrinsic growth dynamics of normal human fibroblasts we employed the fluorescent dye DiI and FACS analysis which can distinguish cells that undergo different numbers of divisions within culture. We observed rapid generation of cell subpopulations undergoing from 0 to 9 divisions within growing cultures at each passage analyzed. These large differences in number of divisions among individual cells guarantee a strong impact on generation of telomere length heterogeneity in normal cell cultures and suggest that culture conditions should be included in future modeling of cell senescence.

Telomeres, Nutrition, and Longevity: Can We Really Navigate Our Aging?

Telomeres are dynamic chromosome-end structures that serve as guardians of genome stability. They are known to be one of the major determinants of aging and longevity in higher mammals. Studies have demonstrated a direct correlation between telomere length and life expectancy, stress, DNA damage, and onset of aging-related diseases. This review discusses the most important factors that influence our telomeres. Various genetic and environmental factors such as diet, physical activity, obesity, and stress are known to influence health and longevity as well as telomere dynamics. Individuals currently have the opportunity to modulate the dynamics of their aging and health span, monitor these processes, and even make future projections by following their telomere dynamics. As telomeres react to positive as well as negative health factors, we should be able to directly influence our telomere metabolism, slow their deterioration, and diminish our aging and perhaps extend our life and health span.

Higher prevalence of FTO gene risk genotypes AA rs9939609, CC rs1421085, and GG rs17817449 and saliva containing Staphylococcus aureus in obese women in Croatia.

Obesity is composed of multifunctional interactions of eating habits, behaviors, microbiota, genetics, and other unknown factors. We hypothesize that correlations occur between the fat mass and obesity-associated single nucleotide polymorphisms (FTO SNPs), the composition of microorganisms in the saliva, and life habits in obese women from Zagreb County. Our results of the analysis of 3 FTO SNPs showed a statistically significant positive correlation among the frequencies of the high-risk genotypes AA rs9939609 (P = .0367), CC rs1421085 (P = .0367), and GG rs17817449 (P = .0065) of the FTO gene in obese cases. Interestingly, 39.13% of obese women were triple homozygous for all 3 risk alleles. Furthermore, the composition of the oral microbiota in the obese group showed a higher occurrence of a major human pathogen, bacterium Staphylococcus aureus, but a significantly low presence of bacteria Streptococcus oralis, Streptococcus mitis, and Serratia ureilytica compared with the control group. The investigation also revealed that obese women prefer to consume candy and snacks and more meat and meat-derived products, sleep less than 6 hours per day, and had higher hypertension in comparison with the control group. These results support the hypothesis that female obesity is strongly related to all 3 variants of the FTO gene and perhaps a specific composition of microbiota in saliva due to dietary habits. Considering the bimodal distribution of the SNPs and bacterial content of saliva in obese women taken together are factors to consider for risk of obesity.

Reduced telomere length is not associated with early signs of vascular aging in young men born after intrauterine growth restriction: a paradox?

OBJECTIVE: The mechanisms that increase cardiovascular risk in individuals born small for gestational age (SGA) are not well understood. Telomere shortening has been suggested to be a predictor of disease onset. Our aim was to determine whether impaired intrauterine growth is associated with early signs of vascular aging and whether telomere length could be a biomarker of this pathway. METHODS: One hundred and fourteen healthy young men born SGA or after normal pregnancy [appropriate for gestational age (AGA)] were enrolled. Patient data were gathered from questionnaires and clinical exams, including blood pressure (BP) measurement routine laboratory analyses, and carotid intima-media thickness (cIMT). Leukocyte telomere length (LTL) was assessed by quantitative PCR. Birth data were obtained from medical records. RESULTS: The SGA group had significantly higher pulse pressure and cIMT, and a trend to increased SBP and heart rate in comparison to the AGA group. Interestingly, SGA men exhibited a 42% longer LTL than the AGA group. LTL was inversely associated with age, BMI, BP and birth parameters. In multiple regression analysis, BMI was the key determinant of SBP and cIMT. CONCLUSION: Young men born SGA show early signs of vascular aging. Unexpectedly, in our cohort, the SGA group had longer telomeres than the normal controls. Although longer telomeres are predictive of better health in the future, our findings could indicate a faster telomere attrition rate and probable early onset of cardiovascular risk in SGA participants. Follow-up of this cohort will clarify hypothesis and validate telomere dynamics as indicators of future health risks.

Telomere Q-PNA-FISH--reliable results from stochastic signals.

Structural and functional analysis of telomeres is very important for understanding basic biological functions such as genome stability, cell growth control, senescence and aging. Recently, serious concerns have been raised regarding the reliability of current telomere measurement methods such as Southern blot and quantitative polymerase chain reaction. Since telomere length is associated with age related pathologies, including cardiovascular disease and cancer, both at the individual and population level, accurate interpretation of measured results is a necessity. The telomere Q-PNA-FISH technique has been widely used in these studies as well as in commercial analysis for the general population. A hallmark of telomere Q-PNA-FISH is the wide variation among telomere signals which has a major impact on obtained results. In the present study we introduce a specific mathematical and statistical analysis of sister telomere signals during cell culture senescence which enabled us to identify high regularity in their variations. This phenomenon explains the reproducibility of results observed in numerous telomere studies when the Q-PNA-FISH technique is used. In addition, we discuss the molecular mechanisms which probably underlie the observed telomere behavior.

The two faces of Cdk8, a positive/negative regulator of transcription.

Three cyclin dependent kinases, Cdk7, Cdk8 and Cdk9 are intimately connected with the processes of RNA polymerase II dependent transcription initiation and elongation in eukaryotic cells. Each of these kinases is part of a larger multisubunit complex, TFIIH, Mediator and p-TEFb respectively. Of the three kinases, Cdk8 is the most complex given that it has been associated with both positive and negative effects on transcription via mechanisms that include regulation of transcription factor turnover, regulation of CTD phosphorylation and regulation of activator or repressor function. Furthermore, Cdk8 has emerged as a key regulator of multiple transcriptional programs linked to nutrient/growth factor sensing and differentiation control. As such Cdk8 represents a potentially interesting therapeutic drug target. In this review we summarize the current state of knowledge on Cdk8 function both in yeast and higher eukaryotes as well as discussing the effects of Cdk8 null mutations at the organismal level.

Tebrophen--an old polyphenol drug with anticancer potential.

In vitro high-throughput screening was carried out in order to detect new activities for old drugs and to select compounds for the drug development process comprising new indications. Tebrophen, a known antiviral drug, was found to inhibit activities on inflammation and cancer related targets. In primary screening this semisynthetic halogenated polyphenol was identified to inhibit the activities of kinases ZAP-70 and Lck (IC50 0.34 µM and 16 µM, respectively), as well as hydrolase DPPIV (at 80 µM 41% inhibition). Next, it showed no cytotoxic effects on standard cell lines within 24 h. However, tebrophen slowed propagation of breast cancer (MDA-MB-231), osteosarcoma (U2OS) and cervical carcinoma (HeLa), through at least 35 population doublings in a dose-dependent manner. It completely stopped the division of the prostate cancer (PC3) cell line at 50 µM concentration and the cells entered massive cell death in less than 20 days. On the other hand, tebrophen did not influence the growth of normal fibroblasts. According to the measured oxidative burst and estimated in silico parameters its direct antioxidative ability is limited. The obtained results indicate that tebrophen can be considered a promising lead molecule for generating more soluble derivatives with specific anticancer efficacy.

Human Xp/Yp telomere analysis by Southern-STELA.

Telomeres are specialized structures designed to protect the ends of linear chromosomes. They are dynamic structures such that in normal somatic cells they constantly shorten as cell division progresses. There is compelling evidence that telomere shortening leads to cell senescence, a process perceived as the main cause of aging in higher mammals. Therefore, the features of telomere shortening are of great importance in understanding cell senescence and aging in general. By identifying unique subtelomeric regions, large enough to produce strong chemiluminescent signals, we have provided a new tool for Southern blot analysis of individual human Xp/Yp telomeres. We extend these results with quantitative fluorescence in situ hybridization using peptide nucleic acid probe (PNA Q-FISH) analysis of telomeres on the Y chromosome. Our results demonstrates unequal shortening dynamics between the p and q telomeres.

The load of short telomeres, estimated by a new method, Universal STELA, correlates with number of senescent cells.

Short telomeres are thought to trigger senescence, most likely through a single - or a group of few - critically shortened telomeres. Such short telomeres are thought to result from a combination of gradual linear shortening resulting from the end replication problem, reflecting the division history of the cell, superimposed by a more stochastic mechanism, suddenly causing a significant shortening of a single telomere. Previously, studies that have tried to explore the role of critically shortened telomeres have been hampered by methodological problems. With the method presented here, Universal STELA, we have a tool that can directly investigate the relationship between senescence and the load of short telomeres. The method is a variant of the chromosome-specific STELA method but has the advantage that it can demonstrate short telomeres regardless of chromosome. With Universal STELA, we find a strong correlation between the load of short telomeres and cellular senescence. Further we show that the load of short telomeres is higher in senescent cells compared to proliferating cells at the same passage, offering an explanation of premature cell senescence. This new method, Universal STELA, offers some advantages compared to existing methods and can be used to explore many of the unanswered questions in telomere biology including the role that telomeres play in cancer and aging.

Abrupt telomere shortening in normal human fibroblasts.

Aging is one of the most basic properties of living organisms. Abundant evidence supports the idea that cell senescence underlies organismal aging in higher mammals. Therefore, examining the molecular mechanisms that control cell and replicative senescence is of great interest for biology and medicine. Several discoveries strongly support telomere shortening as the main molecular mechanism that limits the growth of normal cells. Although cultures gradually approach their growth limit, appearance of individual senescent cells is sudden and stochastic. A theoretical model of abrupt telomere shortening has been proposed in order to explain this phenomenon, but until now there was no reliable experimental evidence supporting this idea. Here, we have employed novel methodology to provide evidence for the generation of extrachromosomal circular telomeric DNA as a result of abrupt telomere shortening in normal human fibroblasts. This mechanism ensures heterogeneity in growth potential among individual cells, which is crucial for gradual progression of the aging process.

Telomerase activity in HeLa cervical carcinoma cell line proliferation.

Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate (3)H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0-1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-beta-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.

Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin.

Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT), which is responsible for cell killing. In this report, we describe the construction of DT-resistant HeLa cell lines by engineering the toxin-resistant form of its specific substrate, protein elongation factor-2. Using site-specific mutagenesis of the histidine precursor of diphthamide, the histidine residue of codon 715 in human EF-2 cDNA was substituted with one of four amino acid residue codons: leucine, methionine, asparagine or glutamine. Mutant EF-2s were subcloned into a pCMVexSVneo expression vector, transfected into HeLa cells, and DT-resistant cell clones were isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin (5-20 ng/mL) was demonstrated by: (1) the normal morphological appearance of the cells; (2) their unaffected or slightly slower growth rates; (3) their undisturbed electrophoretic DNA profiles whose integrity was virtually preserved. Mutant cell strains showed also considerable levels of resistance to very high concentrations of DT, in that they maintained slower but consistent rates of cell growth. It was hence concluded that despite its strict conservation and unique modification, the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones should prove valuable hosts for various DT gene-containing vectors that express the toxin intracellularly.