ArticlePDF Available

The Effect of Quercetin towards Adipocytes Count in Toxoplasma gondii Profilin - exposed Adipocytes In Vitro

Authors:

Abstract

Toxoplasma gondii is one of the protozoan causes of chronic infection that allegedly causes obese (infectobesity). Some previous studies have showed that profilin Toxoplasma gondii has a role in inflammation by promoting interleukin-12 (IL - 12) which induce adipocyte dysfunction through the hyperplasia and hyperproliferation of adipocyte cells. Those processes lead to metabolic syndrome which increase adipocytes count through reducing insulin receptor’s sensitivity. On the other hand, Toxoplasma gondii , as an obligate intracellular parasite, can also damage the pancreatic beta cells. In response to inflammation, adipocytes produce Reactive Oxygen Species (ROS). To scavenge ROS antioxidants are required. Quercetin, an exogenous antioxidant, can be widely found in natural products that might be a promising candidate for development of antioxidant treatment interventions to prevent adipocytopathy. This research aims to explore the effects of quercetin towards Adipocytes Count stimulated from T. gondii profilin-exposed adipocytes. This research using visceral adipocyte rat that was cultured in Dulbecco’s Modified Eagle Medium (DMEM). After 70% confluency, adipocytes were exposed to 20 μΜ T. gondii profilin and treated with four doses of quercetin; 31.25, 62.5, 125, and 250 μΜ that incubated 48 hours. After incubation period, adipocytes were observed using inverted microscope and were captured in high power field magnification using camera. Adipocytes were counted from each captured photo and all groups were analyzed using Analysis of variance (ANOVA) test. The results showed that quercetin significantly reduced adipocyte cell count T. gondii profilin-exposed adipocytes compared to untreated cells (ANOVA p = 0,00). The effective dose to lower adipocyte cell count was 31,25 μΜ. This study implies that quercetin has a potent antioxidant that can prevent toxoplasmosis-mediated adipocytopathy.
Journal of Physics: Conference Series
PAPER • OPEN ACCESS
The Effect of Quercetin towards Adipocytes Count in Toxoplasma gondii
Profilin - exposed Adipocytes In Vitro
To cite this article: Y D Setia et al 2020 J. Phys.: Conf. Ser. 1430 012028
View the article online for updates and enhancements.
This content was downloaded from IP address 45.40.126.59 on 08/01/2020 at 00:47
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
IOP Publishing
doi:10.1088/1742-6596/1430/1/012028
1
The Effect of Quercetin towards Adipocytes Count in
Toxoplasma gondii Profilin - exposed Adipocytes In Vitro
Y D Setia
1*
, S Poeranto
1
, T E Hernowati
2
, A Iskandar
1
, Sudjari
1
1
Parasitology Department, Faculty of Medicine, Universitas Brawijaya, Veteran Street number
1 Malang, Indonesia
2
Clinical Pathology Department, Faculty of Medicine, Universitas Brawijaya, Veteran Street
number 1 Malang, Indonesia
*Corresponding author : dr.yulia.fk@ub.ac.id
Abstract. Toxoplasma gondii is one of the protozoan causes of chronic infection that allegedly
causes obese (infectobesity). Some previous studies have showed that profilin Toxoplasma
gondii has a role in inflammation by promoting interleukin-12 (IL – 12) which induce adipocyte
dysfunction through the hyperplasia and hyperproliferation of adipocyte cells. Those processes
lead to metabolic syndrome which increase adipocytes count through reducing insulin receptor’s
sensitivity. On the other hand, Toxoplasma gondii, as an obligate intracellullar parasite, can also
damage the pancreatic beta cells. In response to inflammation, adipocytes produce Reactive
Oxygen Species (ROS). To scavenge ROS antioxidants are required. Quercetin, an exogenous
antioxidant, can be widely found in natural products that might be a promising candidate for
development of antioxidant treatment interventions to prevent adipocytopathy. This research
aims to explore the effects of quercetin towards Adipocytes Count stimulated from T.
gondii profilin-exposed adipocytes. This research using visceral adipocyte rat that was cultured
in Dulbecco's Modified Eagle Medium (DMEM). After 70% confluency, adipocytes were
exposed to 20 µM T. gondii profilin and treated with four doses of quercetin; 31.25, 62.5, 125,
and 250 µM that incubated 48 hours. After incubation period, adipocytes were observed using
inverted microscope and were captured in high power field magnification using camera.
Adipocytes were counted from each captured photo and all groups were analyzed using Analysis
of variance (ANOVA) test. The results showed that quercetin significantly reduced adipocyte
cell count T. gondii profilin-exposed adipocytes compared to untreated cells (ANOVA p = 0,00).
The effective dose to lower adipocyte cell count was 31,25 µM. This study implies that quercetin
has a potent antioxidant that can prevent toxoplasmosis-mediated adipocytopathy.
Key word : quercetin, adipocyte count, Toxoplasma gondii profilin
1. INTRODUCTION
Due to its high prevalence, obesity is a major global health issue, including in developing countries and
children (Ng M, et al. 2014). The increasing number of obese patients demands more research about
pathogenesis of obesity to identify more effective novel intervention targets. Life style interventions and
body weight reduction alone have shown unsatisfying results, especially because of the problems of
discontinuity (Reever GM, et al. 2013). Several studies described infection through inflammatory
pathways as a potential cause of obesity, further defined as infectobesity (Vasilakopoulou A and Roux
C W 2007; Hedge V and Dhurandar NV 2013). The revelation of infectious agents playing roles in
obesity leads to the idea of specific treatment modalities for obese people due to infection (Hedge V and
Dhurandar NV 2013).
The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
IOP Publishing
doi:10.1088/1742-6596/1430/1/012028
2
One of the potential pathogens involved in obesity pathogenesis is Toxoplasma gondii (T. gondii). This
apicomplexan protozoa infected 30% of population around the globe (Reever GM, et al. 2013). As an
intracellular parasite, T. gondii can infect all nucleated cells, including adipocytes (Toulah, Fawzia H,
et al. 2011). As other members of the apicomplexan phylum, the actin cytoskeleton change during
gliding movement is essential for T. gondii during host cell invasion. Profilin is an important component
for actin polymerisation during actin-dependent gliding movement of T. Gondii (Plattner, Fabienne et
al. 2008). Profilin-like protein of T. gondii is also an immunogenic element that stimulates inflammatory
pathway through its recognition by the endosomal pattern recognition receptor (PRR), Toll-like receptor
11 (TLR 11) (Andrade WA. et al. 2013; Susanto, et al. 2014; Yarovinsky F 2014; Iskandar A et al.
2016).
As a response to inflammation, cells will generate of reactive oxygen species (ROS). ROS production
leads to random and unregulated intracellular oxidation, which in turn triggers oxidation of iron,
intracellular lipids, proteins, and DNA, resulting in vast intracellular molecular damage. Many diseases
potentially arise as the consequence, such as neurodegenerative diseases, atherosclerosis, aging process,
and metabolic syndrome (Holmström, Kira M and Toren F. 2014).
To scavenge ROS and intracellular damage, antioxidants are needed. Body cells are equipped with
endogenous enzimatic antioxidant. Glutathione (GSH), a cystein protein contain tripeptide, has an
important role in cellular redox (Holmström, Kira M and Toren F. 2014).
Synthetic antioxidants are commercially available, but the safety and toxicity risks of synthetic
antioxidants are higher than natural antioxidant (Ebrahimzadeh, et. al. 2008). Flavonoid is one of the
most well recognized exogenous natural antioxidants, which is produced outside the body. Quercetin, a
type of flavonoid, can be widely found in natural products, such as onion, cherry, tomato, broccoli,
apple, green tea, black tea, grape, or blueberry (Fazel S et al. 2015) ; Ratnawati R and Hernowati TE.
2015). Previous studies showed that quercetin inhibits proliferation and differentiation of pre-adipocyte
culture by decreasing the expression of adipocytokines, such as CCAAT/enhancer binding protein alpha
(C/EBPα) and sterol regulatory element binding protein 1c (SBREP-1c) (Ratnawati R and Hernowati
TE. 2015).
This study aims to explore the potency of quercetin to scavenge ROS free radicals and to stimulate GSH
endogenous antioxidant by exposure of T. gondii profilin-exposed adipocytes. T. gondii profilin can be
recognised by TLR 11 and stimulate inflammatory pathway that has causal relationship with
adipocytopathy. The results of this study could be implied in development of potential antioxidant
treatment interventions to prevent toxoplasmosis-mediated adipocytopathy.
2. MATERIAL AND METHOD
2.1. Experimental design
This research used true experimental study using adipocyte culture that exposed to profilin T.
gondii and treated by quercetin. Samples were divided into 6 groups. Each group contains four
replication samples each, namely : Negative control (maturated only); Positive control (maturated and
20 µM T. gondii profilin exposed); Q 31.25 (maturated, 20 µM T. gondii profilin and Quercetin 31.25
µM exposed); Q 62.5 (maturated, 20 µM T. gondii profilin and Quercetin 62.5 µM exposed); Q 125
(maturated, 20 µM T. gondii profilin and Quercetin 125 µM exposed), and Q 250 (maturated, 20 µM T.
gondii profilin and Quercetin 250 µM exposed).
Adipocyte culture
Adipocyte culture was developed from adipose tissue of 1 month old wistar rats. Adipose tissue was
collected from peritoneal and retroperitoneal regions. The tissue was munched mechanically by scalpel
or scissors and digested enzimatically by type 1 collagenase (Worthington).
The obtained cells were maintained in a culture flask and nourished using Dulbecco’s Modified Eagle
Medium (DMEM) (Gibco©) containing sodium bicarbonate, L-glutamine, antibiotics (100U/ml
The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
IOP Publishing
doi:10.1088/1742-6596/1430/1/012028
3
Pennicillin and 100 mg/ml Streptomycin (MP Biomedicals, LCC)), and supplemented with 10% of heat-
inactivated fetal bovine serum (Gibco©).
Adipocyte culture were kept at 37° C, 5% CO
2
environment. Culture media were changed every 48 hours
until confluency was achieved (Ratnawati R and Hernowati TE. 2015; Zhu S et al 2010). After the cells
in the culture flask were confluent, the researchers then subcultured the cells into 12-well culture plates.
The cells in the culture plates achieve the same treatment as in the culture flask.
2.2. Profilin and quercetin exposure
All of the confluent groups of pre-adipocyte culture were maturated by 0,1 µM dexamethasone, 0,5 mM
isobutylmethylxanthine, and 0,1 µM insulin (Ratnawati R and Hernowati TE. 2015). Simultaneously
with maturation process, some of the cell cultures were exposed to 20 µM T. gondii profilin
(MyBioSource) (positive control and four quercetin treatment groups) and quercetin (Sigma) (four
quercetin treatment groups) dissolved in DMEM then incubated for 48 hours (Ratnawati R and
Hernowati TE. 2015; Mochamad R, et al. 2013).
2.3. Adipocyte count measurement
After 48 hours of incubation, adipocytes were observed using inverted microscope and were captured
in high power field magnification using camera.
2.4. Statistical analysis
The number of adipocytes in each groups were presented as mean and standard error of the mean (SEM)
of four independent replications. Homogen and normal data (p>0,05) were analysed by ANOVA test.
2.5. Ethical statement
All procedures involving animals were in accordance with the ethical standards of Faculty of
Medicine, Brawijaya University (No 99/EC/KEPK-PSPD/03/2017).
3. Result
The results showed that the mean of adipocyte number in positive control group (95) was higher
compared to negative control (81.33). The decline in ROS levels were observed in all quercetin
treatment groups (Q 31.25= 84.67, Q 62.5 = 71.67, Q 125 = 73.67 and Q 250 = 57.33) compared to
negative or positive control groups (Figure 1).
The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
IOP Publishing
doi:10.1088/1742-6596/1430/1/012028
4
Figure 1.
Mean number of adipocyte in each
groups
From statistical analysis using ANOVA, the results showed that quercetin
significantly reduced adipocyte cell count T. gondii profilin-exposed adipocytes compared to untreated
cells (ANOVA p = 0,00).
4. DISCUSSION
During T. gondii infection, profilin acts as a pathogen associated molecular pattern (PAMP)
recognised by the endosomal Toll-like receptor, TLR-11 (Yarovinsky F 2014). This recognition further
stimulates the inflammatory pathway, eventually leading to the production of ROS (Furukawa S, et al.
2004).
The results showed there was significant difference between negative and positive control, which
corresponds with the theoretical basis that profilin of T. gondiigenerates intracellular oxidative stress.
The excessive ROS production stimulated by T. gondii profilin was unable to be neutralized by
endogenous antioxidants alone (Marı M, et al. 2009), thus it stimulates intracellular molecular damage
of lipids, proteins, and DNA. These damages in turn becomes the basic mechanisms of diseases,
including hiperplasia of adipocyte that can lead to obesity and metabolic syndrome (Furukawa S, et al.
2004); Holmström, Kira M and Toren F. 2014).
The results here showed significant differences between control and all doses of quercetin groups.
These results confirmed the theorical basis that quercetin acts as an antioxidant in T. gondii profilin-
exposed by significantly decreasing ROS levels (Fazel S, et al. 2015; Mochamad R, et al. 2013).
Consistent with this results, a previous study by Lee et al. in 2013 observed quercetin effectivity as a
ROS scavenger using H2DCFDA staining in fibrosarcoma culture in vitro. The study revealed that
quercetin treatment was able to scavenge ROS level at doses of 5, 10, or 50 50 µg/ml (Lee DE, et al.
2013). In this study, the researchers found no significant difference among three different doses (31,25,
62,5 and 125 µM) of quercetin groups. This indicated that all doses employed in this study have similar
effectivity as antioxidant, where the minimum dose of 31.25µM showed similar effectivity to those of
higher doses (62.5 and 125 µM).
81.33
95.00
84.67
71.67 73.67
57.33
0.00
20.00
40.00
60.00
80.00
100.00
120.00
negative
control
positive
control
Q 31,25 Q 62,5 Q 125 Q 250
Adipocyte count
The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
IOP Publishing
doi:10.1088/1742-6596/1430/1/012028
5
5. Conclusion
This study implies that quercetin has a potent antioxidant that can prevent toxoplasmosis-mediated
adipocytopathy.
Acknowledgements
This research was supported by BPPM of Faculty of Medicine, Universitas Brawijaya, and Indonesia’s
Ministry of Research and Technology and High Education.
References
[1] Andrade, W. A. et al., 2013. Combined action of nucleic acid-sensing toll-like receptors and
TLR11/TLR12 heterodimers imparts resistance to toxoplasma gondii in mice. Cell Host and
Microbe, 13(1), pp. 42-53.
[2] EBRAHIMZADEH, M. A., POURMORAD, F. & HAFEZI, S., 2008. Antioxidant Activities of
Iranian Corn Silk. Turk J Biol, Volume 32, pp. 43-49.
[3] Fazel, S., Luigi, G., Daglia, M. & Mohammad, S., 2015. Role of quercetin as an alternative for
obesity treatment : You are what you eat !. FOOD CHEMISTRY, 179(February), pp. 305-310.
[4] Furukawa, S. et al., 2004. Increased oxidative stress in obesity and its impact on metabolic
syndrome. The journal of clinical investigation, 114(12), pp. 1752-1761.
[5] Hedge, V. & Dhurandar, N. V., 2013. Microbes and obesity - interrelationship between infection,
adipose tissue, and the immune system. Clinical Microbiology and Infection, Volume 19, pp.
314 - 320.
[6] Holmström, K. M. & Finkel, T., 2014. Cellular mechanisms and physiological consequences of
redox-dependent signalling. Nature reviews. Molecular cell biology, 15(6), pp. 411-421.
[7] Indra, M. R., Karyono, S., Ratnawati, R. & Malik, S. G., 2013. Quercetin suppresses inflammation
by reducing ERK1/2 phosphorylation and NF kappa B activation in Leptin-induced Human
Umbilical Vein Endothelial Cells (HUVECs).. BMC research notes, 6(1), pp. 275-282.
[8] Iskandar, A. et al., 2016. The levels of Toxoplasma gondii profilin and adiponectin in obese
patients complicated with or without metabolic syndrome as compared to non-obese patients.
Asian Pacific Journal of Tropical Disease, 6(4), pp. 265-268.
[9] Lee, D. E. et al., 2013. Quercetin Suppresses Intracellular ROS Formation, MMP Activation, and
Cell Motility in Human Fibrosarcoma Cells. Journal of Food Science, 78(9), pp. 1464-1469.
[10] Li, C., Zhang, W.-J., Choi, J. & Frei, B., 2016. Quercetin affects glutathione levels and redox
ratio in human aortic endothelial cells not through oxidation but formation and cellular export
of quercetin-glutathione conjugates and upregulation of glutamate-cystein ligase. Redox
Biology, Volume 9, pp. 220-228.
[11] Marı, M. et al., 2009. Mitochondrial Glutathione, a Key Survival Antioxidant. ANTIOXIDANTS
& REDOX SIGNALING, 11(11), pp. 2685-2701.
[12] Ng, M. et al., 2014. Global, regional, and national prevalence of overweight and obesity in
children and adults during 1980 - 2013: a systematic analysis for the Global Burden of Disease
Study 2013. Lancet, 384(9945), pp. 766-781.
[13] Plattner, F. et al., 2008. Toxoplasma Profilin Is Essential for Host Cell Invasion and TLR11-
Dependent Induction of an Interleukin-12 Response. Cell Host and Microbe, 3(February), pp.
77-87.
[14] Ratnawati, R. & Hernowati, T. E., 2015. Respon Proliferasi , Diferensiasi dan Ekspresi C / Ebp
Akibat Paparan Quercetin Pada Kultur Preadiposit Tikus ( Rattus Norvegicus ) Strain Wistar
Secara In Vitro. Research Journal of Life science, 02(01), pp. 23-33.
[15] Reeves, G. M. et al., 2013. A positive association between T. gondii seropositivity and obesity.
Frontiers in public health, 1(73), pp. 1 - 6.
The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
IOP Publishing
doi:10.1088/1742-6596/1430/1/012028
6
[16] Susanto, H., Indra, R. & Sudjari, 2014. Studi Adiposopati In Vitro Efek Induksi Profilin Terhadap
Ekspresi Il-6 Dan Tnf- Sebagai Kandidat Prediktor Disfungsi Adiposit Akibat Infeksi
Toxoplasma Gondii pada Kultur Adiposit Subkutan. Research Journal of Life science, 01(02),
pp. 85-92.
[17] Toulah, F. H., Al-Ahl, S. A. S., Amin, D. M. & Hamouda, M. H., 2011. Toxoplasma gondii:
Ultrastructure study of the entry of tachyzoites into mammalian cells. Saudi Journal of
Biological Sciences, Volume 18, pp. 151-156.
[18] Vasilakopoulou, A. & le Roux, C. W., 2007. Could a virus contribute to weight gain?.
International journal of obesity, 31(9), pp. 1350-1356.
[19] Wang, X. & Roper, M. G., 2014. Measurement of DCF fluorescence as a measure of reactive
oxygen species in murine islets of Langerhans. Anal Method, 6(9), pp. 3019-3024.
[20] Yarovinsky, F., 2014. Innate immunity to Toxoplasma gondii infection. Nature Publishing
Group, 14(2), pp. 109-121.
[21] Zhu, S. et al., 2010. Toxicity of derivates from semicarbazide-sensitive amine oxidase-mediated
deamination of methylamine against Toxoplas
ResearchGate has not been able to resolve any citations for this publication.
Full-text available
Article
Introduction. Obesity is a metabolic syndrome as risk factor to the cardiovascular disease. Obesity occurs caused by adipocyte massive that is started by proliferation and differentiation of preadipocytes that involves transcription factors such as C/EBPα ,PPARγ and SREBP-1. Is known the TNF α and [Ca²⁺]i Increase when adiponectin decrease at the end of adipocyte differentiation. Therefore it is a deemed treat the obesity through the inhibition of adipocyte proliferation and differentiation using natural resources. Quercetin in due of the flavonoid found in apple,vegetables etc. The aim of this study is to prove the affectivity of quercetin to inhibit proliferation and differentiation of preadipocyte through inhibited of C/EBPα in rat preadipocytes culture method. Methods. Their study was a laboratory experimental. Quercetin was exposed to the preadipocytes human culture, after it was induced by differentiation stimulator the doses quercetin was grouped in: control, 50 µM (Q50), 125 µM (Q125) and 625 µM (Q625) groups. The amount of proliferation and differentiation was descriptive analyzed. The expression of C/EBPα, were identified by Immunocytochemistry and western blotting. Result. Quercetin 100 inhibited the rate of proliferation cell, while quersetin 625 inhibited differentiation in preadipocytes culture. There was a dose dependent to quersetin towards the decrease of PPARγ expression and C/EBPα expression in this preadipocytes culture. Conclusion. It is conclusion that quersetin inhibits proliferation and differentiation preadipocytes culture through the decrease of the expresion of C/EBP α. Keyword : quercetin, preadipocyte, differentiation
Full-text available
Article
Endothelial dysfunction due to vascular inflammation and oxidative stress critically contributes to the etiology of atherosclerosis. The intracellular redox environment plays a key role in regulating endothelial cell function and is intimately linked to cellular thiol status, including and foremost glutathione (GSH). In the present study we investigated whether and how the dietary flavonoid, quercetin, affects GSH status of human aortic endothelial cells (HAEC) and their response to oxidative stress. We found that treating cells with buthionine sulfoximine to deplete cellular GSH levels significantly reduced the capacity of quercetin to inhibit lipopolysaccharide (LPS)-induced oxidant production. Furthermore, incubation of HAEC with quercetin caused a transient decrease and then full recovery of cellular GSH concentrations. The initial decline in GSH was not accompanied by a corresponding increase in glutathione disulfide (GSSG). To the contrary, GSSG levels, which were less than 0.5% of GSH levels at baseline (0.26 ± 0.01 vs. 64.7 ± 1.9 nmol/mg protein, respectively), decreased by about 25% during incubation with quercetin. As a result, the GSH:GSSG ratio increased by about 70%, from 253 ± 7 to 372 ± 23. These quercetin-induced changes in GSH and GSSG levels were not affected by treating HAEC with 500µM ascorbic acid phosphate for 24h to increase intracellular ascorbate levels. Incubation of HAEC with quercetin also led to the appearance of extracellular quercetin-glutathione conjugates, which was paralleled by upregulation of the multidrug resistance protein 1 (MRP1). Furthermore, quercetin slightly but significantly increased mRNA and protein levels of glutamate-cysteine ligase (GCL) catalytic and modifier subunits. Taken together, our results suggest that quercetin causes loss of GSH in HAEC, not because of oxidation but due to formation and cellular export of quercetin-glutathione conjugates. Induction by quercetin of GCL subsequently restores GSH levels, thereby suppressing LPS-induced oxidant production.
Full-text available
Article
Objective: To find out the levels of Toxoplasma gondii (T. gondii) profilin and adiponectin in obese patients complicated with or without metabolic syndrome as compared to non-obese patients. Methods: This study was an observational analytic study using cross sectional design. After interview, the subjects were performed with a anthropometric test and then a metabolic syndrome panel. The levels of profilin and adiponectin were detected by using ELISA method. Results: There was a significant difference of T. gondii profilin between the obese complicated with metabolic syndrome group and the non-obese group (P = 0.00; α = 0.05), as well as the metabolically healthy obese, in which the level of profilin was significantly higher in the group as compared to the non-obese group (P = 0.001; α = 0.05). Adiponectin level of the obese complicated with metabolic syndrome group was significantly lower as compared to the metabolically healthy obese and non-obese group (P = 0.001; α = 0.05). Conclusions: The level of T. gondii profilin in obese patients was higher than that in the nonobese, whereas the level of adiponectin in obese patients complicated with metabolic syndrome was lower than that in the obese without metabolic syndrome and non-obese patients.
Full-text available
Article
In islets of Langerhans, oxidative stress induced by reactive oxygen species (ROS) is thought to be critically involved in β-cell dysfunction during the development of diabetes. However, ROS have also been hypothesized to play a role in cellular signalling. To aid in delineating the effects of ROS in living islets of Langerhans, the endocrine portion of the pancreas that contain β-cells, we sought to develop a robust and reproducible protocol to measure these species using the fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). The protocol that was developed minimized photobleaching and leakage of H2DCF from murineislets and utilized a normalization procedure to further reduce experimental variability. The method allowed for ~25 min of DCF measurement in living islets. We used the developed protocol to compare DCF fluorescence from batches of islets incubated in varying glucose concentrations and observed ~1.5-fold higher fluorescence signals in 3 vs. 20 mM glucose. The effects of diazoxide, which clamps open K(+) ATP channels reducing intracellular [Ca(2+)] ([Ca(2+)]i) without affecting glucose metabolism, were also investigated. The presence of diazoxide increased DCF fluorescence at all glucose concentrations tested while addition of 30 mM K(+) to increase [Ca(2+)]i reduced the fluorescence by ~15%. With the developed protocol, all experimental methods tested to increase [Ca(2+)]i resulted in a decrease in DCF fluorescence, potentially indicating involvement of ROS in intracellular signalling cascades.
Full-text available
Article
Background: In 2010, overweight and obesity were estimated to cause 3·4 million deaths, 3·9% of years of life lost, and 3·8% of disability-adjusted life-years (DALYs) worldwide. The rise in obesity has led to widespread calls for regular monitoring of changes in overweight and obesity prevalence in all populations. Comparable, up-to-date information about levels and trends is essential to quantify population health effects and to prompt decision makers to prioritise action. We estimate the global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013. Methods: We systematically identified surveys, reports, and published studies (n=1769) that included data for height and weight, both through physical measurements and self-reports. We used mixed effects linear regression to correct for bias in self-reports. We obtained data for prevalence of obesity and overweight by age, sex, country, and year (n=19,244) with a spatiotemporal Gaussian process regression model to estimate prevalence with 95% uncertainty intervals (UIs). Findings: Worldwide, the proportion of adults with a body-mass index (BMI) of 25 kg/m(2) or greater increased between 1980 and 2013 from 28·8% (95% UI 28·4-29·3) to 36·9% (36·3-37·4) in men, and from 29·8% (29·3-30·2) to 38·0% (37·5-38·5) in women. Prevalence has increased substantially in children and adolescents in developed countries; 23·8% (22·9-24·7) of boys and 22·6% (21·7-23·6) of girls were overweight or obese in 2013. The prevalence of overweight and obesity has also increased in children and adolescents in developing countries, from 8·1% (7·7-8·6) to 12·9% (12·3-13·5) in 2013 for boys and from 8·4% (8·1-8·8) to 13·4% (13·0-13·9) in girls. In adults, estimated prevalence of obesity exceeded 50% in men in Tonga and in women in Kuwait, Kiribati, Federated States of Micronesia, Libya, Qatar, Tonga, and Samoa. Since 2006, the increase in adult obesity in developed countries has slowed down. Interpretation: Because of the established health risks and substantial increases in prevalence, obesity has become a major global health challenge. Not only is obesity increasing, but no national success stories have been reported in the past 33 years. Urgent global action and leadership is needed to help countries to more effectively intervene. Funding: Bill & Melinda Gates Foundation.
Article
Traditionally corn silk (CS) has been used as diuretic, antilithiasic, uricosuric, and antiseptic. It is used for the treatment of edema as well as for cystitis, gout, kidney stones, nephritis, and prostatitis. In the present study, the antioxidant properties of ethanol-water extract from CS were estimated by different methods. Also phenol and flavonoid content of the extract were measured by Folin Ciocalteu and AlCl3 assays. CS extract contained a significant amount of phenol and flavonoids. The percentage of DPPH radical scavenged by CS extract was 92.6 at a concentration of 1.6 mg ml -1. IC50 of the extract and the standard compounds butylated hydroxytoluene (BHA) and quercetin was 0.59, 0.053, and 0.025 mg ml-1, respectively. Iron chelating activity of the extract was less than the standard compounds. CS extract showed nitric oxide-scavenging effect less than the reference agent (quercetin). The extract showed a high reducing ability. According to ferric thiocyanate (FTC) method, the extract showed more than 88% inhibition of linoleic acid peroxidation. It might be concluded that some of the properties of CS in traditional medicine is due to its antioxidant ability.
Article
Reactive oxygen species (ROS), which were originally characterized in terms of their harmful effects on cells and invading microorganisms, are increasingly implicated in various cell fate decisions and signal transduction pathways. The mechanism involved in ROS-dependent signalling involves the reversible oxidation and reduction of specific amino acids, with crucial reactive Cys residues being the most frequent target. In this Review, we discuss the sources of ROS within cells and what is known regarding how intracellular oxidant levels are regulated. We further discuss the recent observations that reduction-oxidation (redox)-dependent regulation has a crucial role in an ever-widening range of biological activities - from immune function to stem cell self-renewal, and from tumorigenesis to ageing.