Papers by Darko Stevanovic
PLOS ONE, 2014
<p>POMC and c-Fos immunoreactivity was identified in sections of the hypothalamus using mul... more <p>POMC and c-Fos immunoreactivity was identified in sections of the hypothalamus using multi-label immunofluorescence immunohistochemistry. Separate nuclear (DAPI, blue), POMC (green), c-Fos (red) and composite (merge) confocal laser-scanning microscope images are shown from a representative section. The arrowhead indicates a neuron that contains both POMC and c-Fos immunoreactivity. The scale bar represents 20 µm.</p
Cell Journal (Yakhteh), 2016
Objective The close relationship between energy metabolism, nutritional state, and reproductive p... more Objective The close relationship between energy metabolism, nutritional state, and reproductive physiology suggests that nutritional and metabolic disorders can disrupt normal reproductive function and fertility. Considering the importance of leptin and ghrelin effects in regulation of the hypothalamic-pituitary-gonadal axis, the objective of this study was to investigate the influence of obesity and centrally applied ghrelin on immunohistochemical appearance and quantitative morphology of the pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) producing cells in adult male rats. Materials and Methods In this experimental study, animals were given two differ- ent diets: normal-fat (NF) and high-fat (HF), for 4 weeks, corresponding to normal and positive energy balance (n=2×14), respectively. Each group was subsequently divided into two subgroups (n=7) receiving intracerebroventricular (ICV) injections of either ghrelin [G, 1 µg/5 µL phosphate buffered saline (P...
Metabolic diseases such as diabetes and obesity are a growing healthcare concern, and their incre... more Metabolic diseases such as diabetes and obesity are a growing healthcare concern, and their increasing rates are attributed to increased consumption of carbohydrate-rich diets and sugar-sweetened beverages. Fibroblast growth factor 21 (FGF21) is a complex metabolic regulator, and there is significant evidence that it may play a role in fructose metabolism, driving relative aversion to sweet taste. As such, we examined the relationship between FGF21 and the preferential intake of simple carbohydrates in mice, both as liquid solutions and as dietary additives. Genetic deletion of FGF21 or its obligate co-receptor β-klotho (KLB) had no impact on preference for sugar sweetened solutions. FGF21 overexpression, however, substantially suppressed preference for fructose solutions, but had no effect on glucose or sucrose preference. Infusions of FGF21 also suppressed fructose preference specifically, an effect that was dependent on expression of KLB in the CNS. These results demonstrate that...
Molecular Metabolism, 2017
, et al. 2017. "Fibroblast growth factor 21 (FGF21) is robustly induced by ethanol and has a prot... more , et al. 2017. "Fibroblast growth factor 21 (FGF21) is robustly induced by ethanol and has a protective role in ethanol associated liver injury." Molecular Metabolism 6 (11): 1395-1406.
Molecular and Cellular Endocrinology, 2017
Autophagy, a process of controlled cellular self-digestion, could be involved in cyclic remodelin... more Autophagy, a process of controlled cellular self-digestion, could be involved in cyclic remodeling of the human endometrium. We investigated endometrial mRNA expression of 23 autophagy-related (ATG) genes and transcription factors in healthy controls (n = 12) and anovulatory polycystic ovary syndrome (PCOS) patients (n = 24), as well as in their subgroup (n = 12) before and after metformin treatment. The mRNA levels of transcription factor forkhead box protein O1 (FOXO1) and several molecules involved in autophagosome formation (ATG13, RB1-inducible coiled-coil 1), autophagosome nucleation (ATG14, beclin 1, SH3-domain GRB2-like endophilin B1), autophagosome elongation (ATG3, ATG5, γ-aminobutyric acid receptor-associated protein - GABARAP), and delivery of ubiquitinated proteins to autophagosomes (sequestosome 1), were significantly reduced in anovulatory PCOS compared to healthy endometrium. Free androgen index, but not free estrogen index, insulin levels, or body mass index, negatively correlated with the endometrial expression of ATG3, ATG14, and GABARAP in PCOS patients. Treatment of PCOS patients with metformin (2 g/day for 3 months) significantly increased the endometrial mRNA levels of FOXO1, ATG3, and UV radiation resistance-associated gene. These data suggest that increased androgen availability in PCOS is associated with metformin-sensitive transcriptional downregulation of endometrial autophagy.
Endocrinology, 2015
Fibroblast growth factor 21 (FGF21) has multiple metabolic actions, including the induction of br... more Fibroblast growth factor 21 (FGF21) has multiple metabolic actions, including the induction of browning in white adipose tissue. Although FGF21 stimulated browning results from a direct interaction between FGF21 and the adipocyte, browning is typically associated with activation of the sympathetic nervous system through cold exposure. We tested the hypothesis that FGF21 can act via the brain, to increase sympathetic activity and induce browning, independent of cell-autonomous actions. We administered FGF21 into the central nervous system via lateral ventricle infusion into male mice and found that the central treatment increased norepinephrine turnover in target tissues that include the inguinal white adipose tissue and brown adipose tissue. Central FGF21 stimulated browning as assessed by histology, expression of uncoupling protein 1, and the induction of gene expression associated with browning. These effects were markedly attenuated when mice were treated with a β-blocker. Additi...
Endocrine Abstracts, 2013
Endocrine Abstracts, 2013
International Journal of Obesity, 2010
In a comment to our paper reporting an increase in the activity of the interleukin (IL)-23/IL-17 ... more In a comment to our paper reporting an increase in the activity of the interleukin (IL)-23/IL-17 (Th17) cytokine axis in obese women,1 Chen2 suggests that IL-17 might have a role in the development of obesity-related cancer. This indeed seems to be a plausible assumption in light of the recent experimental evidence supporting the involvement of IL-17 signalling in carcinogenesis.2 In relation to this problem, one should also bear in mind the ability of IL-17 to stimulate production of nitric oxide (NO), a pleiotropic free radical with a complex role in tumour progression.3 In contrast to the anticancer effects of high NO amounts generated by activated macrophages or NO-releasing compounds, low levels of NO produced by tumour cells and/or surrounding stroma apparently promote tumour growth through vasodilatation and angiogenesis.3 Others and we have recently demonstrated that IL-17 can trigger the expression of inducible NO synthase and subsequent NO production in various cell types through Janus kinase/signal transducers and activators of transcription/interferon regulatory factor-1, mitogen-activated protein kinase/activator protein-1 and tumour necrosis factor receptor-associated factor/nuclear factor-κB signalling pathways.4 Interestingly, although readily activating inducible NO synthase in endothelial cells, fibroblasts, astrocytes and some tumour cell lines, IL-17 was completely unable to stimulate NO production in macrophages.4 In contrast, the activity of the IL-12/interferon-γ (Th1) cytokine axis, which apparently was not increased in obese women,1 has been regarded as crucial for stimulation of NO-dependent macrophage anticancer responses.4, 5 Therefore, it is conceivable to assume that a shift of balance between Th1 and Th17 responses towards the latter, as seen in obesity,1 could contribute to cancer progression through selective low-level inducible NO synthase activation in tumour and/or surrounding stromal cells. Such an assumption is consistent with the ability of IL-17 to promote tumour growth via stimulation of endothelial cell migration and up-regulation of pro-angiogenic factors in fibroblasts and cancer cells.6 It should be noted, however, that IL-17 has also been found to activate tumour-specific cytotoxic CD8+ T cells, thus restricting tumour growth in vivo.7 Although this suggests a complex, context-dependent role of IL-17 in cancer, its possible involvement in development and progression of obesity-related cancer, as suggested by Chen,2 seems worthy of further investigation.
General physiology and biophysics, 2012
Ghrelin, the endogenous ligand of growth hormone secretagogue receptor type 1a (GHS-R1a), has eme... more Ghrelin, the endogenous ligand of growth hormone secretagogue receptor type 1a (GHS-R1a), has emerged as pleiotropic modulator of diverse biological functions, including energy homeostasis and recently, reproduction. The influence of intracerebroventricularly (ICV) administered ghrelin (1 μg/day/rat for 5 days) to rats of different ages, i.e., peripubertal (38 days), adult (60 days) and middle-aged (180 days) on the ventral prostate size and morphology, serum testosterone levels and testis weight was examined. Ghrelin treatment significantly increased (p < 0.05) absolute ventral prostate weight in peripubertal and middle-aged rats, by 27% and 37% respectively, due to enhancement of epithelial and/or luminal compartment of the gland. In adult rats, both absolute and relative volumes of the acinar lumen were significantly decreased (p < 0.05), by 38% and 44% respectively, which was associated with significant increases (p < 0.05) in relative and absolute volumes of interacinar stroma, whereas ventral prostate weigh was unchanged. Irrespective of animal age, ghrelin did not affect serum testosterone levels. These are the first results of ghrelin treatment effects on healthy prostate appearance, which allow us to conclude that the rat ventral prostate response to ghrelin depends on the developmental stage of animals. Our results merit further investigations and may have clinical implications, especially in the light of data on possible role of ghrelin in prostate hypertrophy and adenomas.
Molecular and Cellular Endocrinology, 2013
Signaling through the mammalian target of rapamycin complex 1 (mTORC1) and its effectors the S6-k... more Signaling through the mammalian target of rapamycin complex 1 (mTORC1) and its effectors the S6-kinases (S6K) in the hypothalamus is thought to be involved in nutrient sensing and control of food intake. Given the anatomical proximity of this pathway to circuits for the hormone ghrelin, we investigated the potential role of the mTORC1/S6K pathway in mediating the metabolic effects of ghrelin. We found that ghrelin promoted phosphorylation of S6K1 in the mouse hypothalamic cell line N-41 and in the rat hypothalamus after intracerebroventricular administration. Rapamycin, an inhibitor of mTORC1, suppressed ghrelin-induced phosphorylation of hypothalamic S6K1 and increased food intake and insulin in rats. Chronic peripheral administration of ghrelin induced a significant increase in body weight, fat mass and food efficiency in wild-type and S6K2-knockout but not in S6K1-knockout mice. We therefore propose that ghrelin-induced hyperphagia, adiposity and insulin secretion are controlled by a central nervous system involving the mTORC1/S6K1 pathway.
Microscopy and Microanalysis, 2013
Recent studies have shown that ghrelin increases pancreatic exocrine secretion. However, the pote... more Recent studies have shown that ghrelin increases pancreatic exocrine secretion. However, the potential effects of ghrelin on the morphology of exocrine pancreas (EP) remain unknown. In this work, using fractal analysis, we demonstrate that centrally administered ghrelin increases structural complexity and tissue disorder in rat EP. The study was carried out on a total of 40 male Wistar rats divided into four groups (n = 10): ghrelin-treated animals (average age, 1.5 months), ghrelin-treated animals (8.5 months), and controls (1.5 and 8.5 months). The pancreas tissue sections were stained with hematoxylin/eosin and visualized by light microscopy. For each animal, the average values of tissue fractal dimension, lacunarity, as well as parameters of co-occurrence matrix texture, were determined using tissue digital micrographs. The results indicate that ghrelin administration increases EP fractal dimension and textural entropy, and decreases lacunarity, regardless of the age. To our kno...
Immunobiology, 2013
We explored the effect of therapeutic glucoregulation on the blood levels of proinflammatory T he... more We explored the effect of therapeutic glucoregulation on the blood levels of proinflammatory T helper (Th)17 cytokines interleukin (IL)-17 and IL-23, and Th1 cytokines interferon (IFN)-γ and IL-12 in newly diagnosed type 2 diabetes patients. The investigated group consisted of 23 subjects (17 men and 6 women, age 26-64). The cytokine serum levels, glycated hemoglobin (HbA1c) as a marker of glucoregulation, homeostasis model assessment index as a measure of insulin resistance (HOMA-IR), and body mass index (BMI) were determined before and after 12 weeks of therapy consisting of standard lifestyle modification and metformin (1000 mg b.i.d.). The levels of Th17 and Th1 cytokines before treatment did not correlate with age, BMI or HOMA-IR. The patients with poor glucoregulation (HbA1c&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;7%, n=12), compared to those with good glucoregulation (HbA1c≤7%, n=11), had higher serum levels of Th17 and Th1 cytokines, but only the differences in IL-17 (median 21.2 pg/ml vs. 4.8 pg/ml) and IFN-γ 5 (0.6 pg/ml vs. 27.7 pg/ml) reached statistical significance (p=0.003 and p=0.012, respectively). The reduction of HbA1c values (from 8.6 to 5.9%, p=0.000) observed upon treatment in patients with poor glucoregulation was associated with a significant decrease in the concentration of IL-17 (from 21.2 to 12.9 pg/ml, p=0.020), but not IFN-γ (50.6 vs. 52.3, p=0.349). These data indicate that therapeutic improvement of glucoregulation might contribute to a reduction of IL-17 levels in newly diagnosed type 2 diabetes patients.
European Journal of Pharmacology, 2011
The in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated usi... more The in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated using B16 mouse melanoma cell line. Metformin caused a G 2 /M cell cycle arrest associated with apoptotic death of melanoma cells, as confirmed by the flow cytometric analysis of cell cycle/DNA fragmentation, phosphatidylserine exposure and caspase activation. Metformin-mediated apoptosis of melanoma cells was preceded by induction of oxidative stress and mitochondrial membrane depolarization, measured by flow cytometry in cells stained with appropriate fluorescent reporter dyes. The expression of tumor suppressor protein p53 was increased, while the mRNA levels of anti-apoptotic Bcl-2 were reduced by metformin, as revealed by cell-based ELISA and real-time RT-PCR, respectively. Treatment with metformin did not stimulate expression of the cycle blocker p21, indicating that p21 was dispensable for the observed cell cycle arrest. The activation of AMP-activated protein kinase (AMPK) was not required for the anti-melanoma action of metformin, as AMPK inhibitor compound C completely failed to restore viability of metformin-treated B16 cells. Metformin induced autophagy in B16 cells, as demonstrated by flow cytometry-detected increase in intracellular acidification and immunoblot-confirmed upregulation of autophagosome-associated LC3-II. Autophagy inhibitors ammonium chloride and wortmannin partly restored the viability of metformin-treated melanoma cells. Finally, oral administration of metformin led to a significant reduction in tumor size in a B16 mouse melanoma model. These data suggest that anti-melanoma effects of metformin are mediated through p21-and AMPK-independent cell cycle arrest, apoptosis and autophagy associated with p53/Bcl-2 modulation, mitochondrial damage and oxidative stress.
Endocrine …, 2008
In most obese patients, obesity is associated with a low-grade inflammation of white adipose tiss... more In most obese patients, obesity is associated with a low-grade inflammation of white adipose tissue resulting from chronic activation of the innate immune system and which can subsequently lead to insulin resistance, impaired glucose tolerance and even diabetes. It is still not ...
Cellular and Molecular Life Sciences, 2007
The present study reports for the first time a dual antiglioma effect of the well-known antidiabe... more The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G(0)/G(1) phase without inducing significant cell death. In confluent C6 cultures, on the other hand, metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251, while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin.
Brain, Behavior, and Immunity, 2012
We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbala... more We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbalanceinduced inflammation. Rats were subjected for four weeks to three different dietary regimes: normal (standard food), high-fat (standard food with 30% lard) or food-restricted (70%, 50%, 40% and 40% of the expected food intake in 1st, 2nd, 3rd and 4th week, respectively). Compared to normal-weight controls, starved, but not obese rats had significantly higher levels of proinflammatory cytokines (TNF, IL-1b, IFN-c) in the blood. When compared to normally fed animals, the hearts of starved and obese animals expressed higher levels of mRNAs encoding proinflammatory mediators (TNF, IL-1b, IL-6, IFN-c, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-b remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 lg/day) for five consecutive days significantly reduced TNF, IL-1b and IFN-c levels in the blood of starved rats, as well as TNF, IL-17 and IL-12p40 mRNA expression in the hearts of obese rats. Conversely, ICV ghrelin increased the levels of IFN-c, IL-17, IL-12p35 and IL-12p40 mRNA in the heart tissue of food-restricted animals. This was associated with an increase of immunosuppressive ACTH/corticosterone production in starved animals and a decrease of the immunostimulatory adipokine leptin both in food-restricted and high-fat groups. Ghrelin activated the energy sensor AMP-activated protein kinase (AMPK) in the hypothalamus and inhibited extracellular signal-regulated kinase (ERK) in the hearts of obese, but not starved rats. Therefore, central ghrelin may play a complex role in energy imbalance-induced inflammation by modulating HPA axis, leptin and AMPK/ERK signaling pathways.
Endocrine …, 2011
... Milosevic 2 , Dejan Nesic 1 , Dragan Micic 3 , Mirjana Sumarac-Dumanovic 3 , Vera Popovic 3 &... more ... Milosevic 2 , Dejan Nesic 1 , Dragan Micic 3 , Mirjana Sumarac-Dumanovic 3 , Vera Popovic 3 &amp; Vesna Starcevic 1. 1 Institute of Medical Physiology, School of Medicine, University of Belgrade, Belgrade, Serbia; 2 Institute for Biological Research Sinisa Stankovic, University of ...
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Papers by Darko Stevanovic