Mirjana Sumarac-Dumanovic

Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis

Abstract.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 G0/G1 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.

In vitro and in vivo anti-melanoma action of metformin

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.

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.

Prolonged psychological stress suppresses cortisol secretion

objective  Response to acute psychological stress is characterized by activation of the hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic nervous system. However, response to the prolonged psychological stress is less well known.

Leptin levels and insulin sensitivity in obese and non-obese patients with polycystic ovary syndrome

The study was conducted to assess leptin levels and insulin sensitivity in obese and non-obese patients with polycystic ovary syndrome (PCOS). Twenty-two women with PCOS and 19 control healthy women were included in the study, divided into obese and non-obese groups. Leptin was determined using Linco Research radio-immunoassay while insulin sensitivity was calculated from intravenous glucose tolerance tests with frequent blood sampling using MINMOD analysis. Significantly higher basal leptin levels were found in obese compared to non-obese PCOS (31.76 +/- 3.06 vs. 10.42 +/- 2.31 ng/ml; p < 0.05) as well as in obese in comparison to non-obese controls (29.16 +/- 5.06 vs 8.51 +/- 0.88 ng/ml; p < 0.05). A negative correlation was found between insulin sensitivity and leptin levels in both obese (r = -0.2480; p > 0.05) and non-obese PCOS groups (r = -0.4620; p > 0.05). In conclusion, high serum leptin, insulin and testosterone levels together with reduced insulin sensitivity were found in obese PCOS women, suggesting that high leptin levels could be a characteristic of the obese PCOS phenotype.

Therapeutic improvement of glucoregulation in newly diagnosed type 2 diabetes patients is associated with a reduction of IL-17 levels.

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>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.

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 LC3β 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-dependent autophagic response might sensitize glioma cells to statin-induced apoptotic death.

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-1β, IFN-γ) 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-1β, IL-6, IFN-γ, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-β remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 μg/day) for five consecutive days significantly reduced TNF, IL-1β and IFN-γ 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-γ, 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.

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 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) 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, S6K, 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 S6K 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.

Total ghrelin levels during acute insulin infusion in patients with polycystic ovary syndrome

Controversial data were reported concerning fasting ghrelin (decreased, normal or elevated) in polycystic ovary syndrome (PCOS). The aim of our study was to clarify ghrelin levels in non-obese, overweight, and obese PCOS patients; to investigate the effect of acute insulin infusion on ghrelin in PCOS as a chronic insulin-resistant state, with and without the impact of obesity, and to examine ghrelin-androgen interaction. In that order, we evaluated 1) ghrelin levels among 8 non-obese patients with PCOS [body mass index (BMI): 20.52±1.31 kg/m2], 8 overweight and obese patients with PCOS (BMI: 34.36±6.53 kg/m2) and their respective controls, 2) ghrelin suppression during euglycemic hyperinsulinemic clamp, and 3) ghrelin-androgen interrelationship. After overnight fast, 2-h euglycemic hyperinsulinemic clamp, was performed in all investigated women. Fasting ghrelin was significantly lower in non-obese PCOS than in controls (64.74±25.69 vs 108.36±52.60; p<0.05) as well as in overweight and obese PCOS in comparison with controls (38.71 ± 14.18 vs 98.77± 40.49; p<0.05). Insulin infusion significantly suppressed ghrelin in all subgroups of investigated women. Analysis of variance for repeatable measures confirmed that there was no significant difference in pattern of response between PCOS and controls. In conclusion, women with PCOS had lower fasting ghrelin and decreased insulin sensitivity independently of their BMI, compared to the controls. In addition, there were no differences between fasting ghrelin levels among non-obese, overweight, and obese women with PCOS. During euglycemic hyperinsulinemic clamp, ghrelin decreased in all studied groups to a similar extent, implying that, compared to chronic hyperinsulinemia, acute hyperinsulinemia reduces ghrelin levels independently of the degree of insulin resistance.