Efekti suplementacije probiotikom na morfologiju krvnih pločica gojaznih žena i žena sa prokomernom težinom

Obesity could be associated with enhanced platelet (PL) function and thrombosis. Also, changes in gut microbiota composition were observed in obese patients, and probiotics supplementation has been shown to improve some of the obesity-associated metabolic parameters. This study aimed to determine the possible effects of probiotic supplementation on platelet morphology in overweight/obese premenopausal women.Twenty overweight (BMI = 25.0-29.9 kg/m2) and obese (BMI ≥ 30.0 kg/m2) women participated in double- blind, randomized, placebo-controlled study. They were randomly assigned to receive one capsule daily of probiotics (7x1010 CFU Lactobacillus plantarum 299v (DSM9843), 5x109 CFU Saccharomyces cerevisiae var. boulardii and 40mg octacosanol; N=10) or placebo (N=10) for 3 months. To determine changes in the parameters of platelet activation - the platelet count (PC), platelet distribution width (PDW), Feret diameter, and hypogranular appearance, blood samples were obtained for an automated hematology analyser and peripheral smears staining. Although difference in PC was not found between groups, supplementation with probiotics decreased PDW compared to the placebo group (p<0.05). Regarding platelets diameter, there is an decrease in group supplemented with probiotics compared to placebo, however, a statistical difference has not been reached. Further, the proportion of hypogranular PL was increased in a probiotic-supplemented group compared to the placebo group (p<0.01). In conclusion, probiotic supplementation achieved improvements in platelets morphology and hematological indices in overweight/obese premenopausal women.Gojaznost je često povezana sa povećanom funkcijom krvnih pločica i trombozom. Takođe, istraživanja ukazuju na promenjen sastav mikrobioma creva kod gojaznih osoba, kao i da suplementacija određenim probioticima može imati pozitivan uticaj na telesnu masu. Cilj rada je bio da se ispita uticaj suplementacije probiotskog preparata na morfološke karakteristike krvnih pločica u randomizovanoj, dvostruko-slepoj, placebo-kontrolisanoj studiji. U tu svrhu, regrutovano je 20 žena sa prekomernom težinom (BMI ≥ 25,0 kg/m2) koje su podeljene u dve grupe. Prva grupa je pila dnevno jednu kapsulu probiotskog preparata tokom tri meseca (kombinaciju soja Lactobacillus plantarum 299v (DSM9843), Saccharomyces cerevisiae var. boulardii i 40 mg oktakozanola; N = 10), dok je druga grupa bila placebo grupa. Određen je broj krvnih pločica, i njihova raspodela po volumenu (eng. platelet distribution width, PDW), a zatim je posmatrana morfologija krvnih pločica i izmeren njihov dijametar iz krvnih razmaza. Analiza je pokazala da su krvne pločice kod gojaznih/žena sa prekomernom težinom uglavnom krupne, što je jedan od pokazatelja njihove aktivnosti. U interventoj grupi nije zapažena promena u broju krvnih pločica, ali je značajno smanjen PDW u poređenju sa placebo grupom (p < 0,05). Dalje, pokazano je da je udeo hipogranularnih krvnih pločica bio povećan u poređenju sa placebo grupom (p < 0,01). Ovi rezultati ukazuju na pozitivan efekat probiotika na morfološke karakteristike krvnih pločica i mogućnost primene probiotika u regulaciji njihove aktivnosti u patološkim stanjima.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Intracerebroventricular Administration of Metformin Inhibits Ghrelin-Induced Hypothalamic AMP-Kinase Signalling and Food Intake

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 mu g), metformin (50, 100 or 200 mu g), 5-amino-imidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR, 25 mu g) and L-Ieucine (1 mu g) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, 56K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting 56K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus. Copyright (c) 2012 S. Karger AG, BaselMinistry of Science and Technological Development of the Republic of Serbia [41025, 175067

Intracerebroventricular Administration of Metformin Inhibits Ghrelin-Induced Hypothalamic AMP-Kinase Signalling and Food Intake

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 mu g), metformin (50, 100 or 200 mu g), 5-amino-imidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR, 25 mu g) and L-Ieucine (1 mu g) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, 56K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting 56K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus. Copyright (c) 2012 S. Karger AG, BaselMinistry of Science and Technological Development of the Republic of Serbia [41025, 175067

Inhibition of AMPK-dependent autophagy enhances in vitro antiglioma effect of simvastatin

The role of autophagy, a process in which the cell self-digests its own components, was investigated in glioma cell death induced by the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting drug simvastatin. Induction of autophagy and activation of autophagy-regulating signalling pathways were analyzed by immunoblotting. Flow cytometry/fluorescent microscopy was used to assess autophagy-associated intracellular acidification and apoptotic markers (phosphatidylserine exposure, DNA fragmentation and caspase activation). Cell viability was determined by crystal violet, MTT or LDH release assay. Simvastatin treatment of U251 and C6 glioma cell lines caused the appearance of autophagolysosome-like intracytoplasmic acidic vesicles. The induction of autophagy in U251 cells was confirmed by the upregulation of autophagosome-associated LC3-II and pro-autophagic beclin-1, as well as by the downregulation of the selective autophagic target p62. Simvastatin induced the activation of AMP-activated protein kinase (AMPK) and its target Raptor, while simultaneously downregulating activation of Akt. Mammalian target of rapamycin (mTOR). a major AMPK/Akt downstream target and a major negative autophagy regulator, and its substrate p70 S6 kinase 1 were also inhibited by simvastatin. Mevalonate, the product of HMG-CoA reductase enzymatic activity, AMPK siRNA or pharmacological inactivation of AMPK with compound C suppressed, while the inhibitors of Akt (10-DEBC hydrochloride) and mTOR (rapamycin) mimicked autophagy induction by simvastatin. Inhibition of autophagy with bafilomycin A1, 3-methyladenine and LC3 beta shRNA, as well as AMPK inhibition with compound C or AMPK siRNA, markedly increased apoptotic death of simvastatin-treated U251 cells. These data suggest that inhibition of AMPK-depenclent autophagic response might sensitize glioma cells to statin-induced apoptotic death. (C) 2011 Elsevier Ltd. All rights reserved.Ministry of Science and Technological Development of the Republic of Serbia [41025, 173053

AMP-activated protein kinase-dependent and -independent mechanisms underlying in vitro antiglioma action of compound C

We investigated the effect of compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), on proliferation and viability of human U251 and rat C6 glioma cell lines. Compound C caused G(2)/M cell cycle block, accompanied by apoptotic glioma cell death characterized by caspase activation, phosphatidylserine exposure and DNA fragmentation. The mechanisms underlying the pro-apoptotic action of compound C involved induction of oxidative stress and downregulation of antiapoptotic molecule Bcl-2, while no alteration of pro-apoptotic Bax was observed. Compound C diminished AMPK phosphorylation and enzymatic activity, resulting in reduced phosphorylation of its target acetyl CoA carboxylase. AMPK activators metformin and AICAR partly prevented the cell cycle block, oxidative stress and apoptosis induced by compound C. The small interfering RNA (siRNA) targeting of human AMPK mimicked compound C-induced G(2)/M cell cycle arrest, but failed to induce oxidative stress and apoptosis in U251 glioma cells. In conclusion, our data indicate that AMPK inhibition is required, but not sufficient for compound C-mediated apoptotic death of glioma cells. (c) 2009 Elsevier Inc. All rights reserved.Ministry of Science of the Republic of Serbia [145073, 145067, 143030

Inhibition of AMPK-dependent autophagy enhances in vitro antiglioma effect of simvastatin

The role of autophagy, a process in which the cell self-digests its own components, was investigated in glioma cell death induced by the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting drug simvastatin. Induction of autophagy and activation of autophagy-regulating signalling pathways were analyzed by immunoblotting. Flow cytometry/fluorescent microscopy was used to assess autophagy-associated intracellular acidification and apoptotic markers (phosphatidylserine exposure, DNA fragmentation and caspase activation). Cell viability was determined by crystal violet, MTT or LDH release assay. Simvastatin treatment of U251 and C6 glioma cell lines caused the appearance of autophagolysosome-like intracytoplasmic acidic vesicles. The induction of autophagy in U251 cells was confirmed by the upregulation of autophagosome-associated LC3-II and pro-autophagic beclin-1, as well as by the downregulation of the selective autophagic target p62. Simvastatin induced the activation of AMP-activated protein kinase (AMPK) and its target Raptor, while simultaneously downregulating activation of Akt. Mammalian target of rapamycin (mTOR). a major AMPK/Akt downstream target and a major negative autophagy regulator, and its substrate p70 S6 kinase 1 were also inhibited by simvastatin. Mevalonate, the product of HMG-CoA reductase enzymatic activity, AMPK siRNA or pharmacological inactivation of AMPK with compound C suppressed, while the inhibitors of Akt (10-DEBC hydrochloride) and mTOR (rapamycin) mimicked autophagy induction by simvastatin. Inhibition of autophagy with bafilomycin A1, 3-methyladenine and LC3 beta shRNA, as well as AMPK inhibition with compound C or AMPK siRNA, markedly increased apoptotic death of simvastatin-treated U251 cells. These data suggest that inhibition of AMPK-depenclent autophagic response might sensitize glioma cells to statin-induced apoptotic death. (C) 2011 Elsevier Ltd. All rights reserved.Ministry of Science and Technological Development of the Republic of Serbia [41025, 173053

Immunomodulatory actions of central ghrelin in diet-induced energy imbalance

We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbalance-induced 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-1 beta, IFN-gamma) 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-1 beta, IL-6, IFN-gamma, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-beta remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 mu g/day) for five consecutive days significantly reduced TNF, IL-1 beta and IFN-gamma 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 1FN-gamma, IL-17,1L-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. (C) 2011 Elsevier Inc. All rights reserved.Ministry of Science and Technological Development of the Republic of Serbia [41025, 175067

Immunomodulatory actions of central ghrelin in diet-induced energy imbalance

We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbalance-induced 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-1 beta, IFN-gamma) 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-1 beta, IL-6, IFN-gamma, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-beta remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 mu g/day) for five consecutive days significantly reduced TNF, IL-1 beta and IFN-gamma 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 1FN-gamma, IL-17,1L-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. (C) 2011 Elsevier Inc. All rights reserved.Ministry of Science and Technological Development of the Republic of Serbia [41025, 175067