Leptin triggers Ca(2+) imbalance in monocytes of overweight subjects.

Obesity is a major risk factor in numerous diseases, in which elevated intracellular Ca(2+) plays a major role in increased adiposity. We examined the difference between Ca(2+) signals in monocytes of lean and overweight subjects and the relationship between leptin induced NADPH oxidase activation and intracellular calcium concentration [Ca(2+)](i) homeostasis. Our results are as follows: (1) The basal level of [Ca(2+)](i) in resting monocytes of overweight subjects (OW monocytes) was higher than that in control cells, whereas the leptin-induced peak of the Ca(2+) signal was lower and the return to basal level was delayed. (2) Ca(2+) signals were more pronounced in OW monocytes than in control cells. (3) Using different inhibitors of cellular signaling, we found that in control cells the Ca(2+) signals originated from intracellular pools, whereas in OW cells they were generated predominantly by Ca(2+)-influx from medium. Finally, we found correlation between leptin induced superoxide anion generation and Ca(2+) signals. The disturbed [Ca(2+)](i) homeostasis in OW monocytes was fully restored in the presence of fluvastatin. Statins have pleiotropic effects involving the inhibition of free radical generation that may account for its beneficial effect on elevated [Ca(2+)](i) and consequently on the pathomechanism of obesity.

Obesity abrogates the concentration-dependent effect of leptin on endogenous cholesterol synthesis in human monocytes.

Leptin the cytokine-like hormone is involved not only in local inflammations, but it regulates cholesterol biosynthesis in human monocytes. Since, monocyte-membrane composition in obesity shows considerable difference from control cells, our aim was to elucidate the concentration dependence of the effect of leptin in OW monocytes, and the downstream signaling of high and low leptin concentrations. Control and OW monocytes were stimulated with leptin in the presence or absence of different inhibitors. Our results are as follows: a concentration-dependent biphasic effect could only be detected in control monocytes whereas in OW cells only elevated cholesterol synthesis was found. The signal pathway of 50 ng/mL leptin stimulation involves Ca(2+) signal, activation of PI3K, MAPK and HMG CoA reductase. In the 500 ng/mL leptin-stimulated control monocytes the suppression of cholesterol synthesis was dependent on the Ca(2+) signal, the H-7 sensitive cPKC and PI3K activation, whereas in OW monocytes only PI3K was involved in increased cholesterol synthesis. We conclude that leptin-signaling in OW monocytes is characterized by Ca(2+) influx, abrogation of H-7 sensitive cPKC activation, and by PI3K mediated PKC activation.

The concentration dependent biphasic effect of leptin on endogenous cholesterol synthesis in human monocytes.

In human monocytes 100 ng/mL leptin increased both statin-inhibitable free radical and cholesterol production in vitro. In our recent study, we aimed to elucidate the concentration dependence of observed leptin-effect. Following leptin stimulation cholesterol synthesis was measured in the presence of inhibitors to determine affected signal pathways. Leptin at low (10-100 ng/mL) concentrations increased [(14)C]acetate incorporation, whereas at 250 ng/mL and higher concentrations it suppressed cholesterol synthesis. HMG CoA reductase, phosphatidyl-3-kinase (PI3K) and mitogen activated protein kinase (MAPK) were involved in mediating leptin effects at low concentrations, whereas the cholesterol synthesis suppression was abolished by inhibitors of protein kinase C (PKC) and PI3K.

Leptin stimulates endogenous cholesterol synthesis in human monocytes: New role of an old player in atherosclerotic plaque formation. Leptin-induced increase in cholesterol synthesis.

The role of leptin in the pathomechanism of atherosclerosis, through its free radical generating ability is established. Its effect however, on the regulation of intracellular cholesterol synthesis has not been studied. The aim of the present study was to elucidate whether leptin influences endogenous cholesterol synthesis in monocytes. Furthermore, leptin signaling to HMG CoA reductase in control and hypercholesterolemic monocytes were compared. The in vitro effect of leptin was studied on freshly isolated human monocytes obtained from healthy control volunteers and patients with hypercholesterolemia. Our results can be summarized as follows: (1) Leptin is able to increase endogenous cholesterol synthesis in human monocytes in vitro. (2) The cholesterol synthesis increasing effect of the hormone is more pronounced in hypercholesterolemic monocytes with high basal cholesterol biosynthesis. (3) The leptin-induced Ca(2+) signal was involved in the enhancement of HMG CoA reductase activation in monocytes from both controls and hypercholesterolemic patients. (4) In control monocytes the Ca(2+) signal originated from intracellular pools, whereas in patients, Ca(2+)-influx and protein kinase C activation were found to be responsible for the leptin-effect. Mevalonate cycle inhibiting fluvastatin and 25-hydroxycholesterol decreased cholesterol production in leptin-stimulated monocytes. Our present study provides the first proof of the cholesterol synthesis enhancing effect of leptin through a statin-sensitive pathway in circulating monocytes. Furthermore our results suggest that leptin can be involved in the pathomechanism of atherosclerotic plaque formation also through its effect on cholesterol biosynthesis in monocytes.

Crosstalk of sterol-dependent and non-sterol-dependent signaling in human monocytes after in vitro addition of LDL.

The aim of the present study was to investigate low density lipoprotein (LDL)-induced, non-sterol-dependent signaling and its possible role in cholesterol balance. LDL in 10 microg ml(-1) concentration could induce inositol trisphosphate (IP3) and Ca2+ signal generation through a pertussis toxin (PT) sensitive G protein in human monocytes. The increase in [Ca2+]i was derived from the intracellular pools. LDL also induced activation and translocation of protein kinase C (PKC) into the cell membrane, by processes, which were significantly inhibited in the first 20 min by preincubation with PT and PKC-inhibitor H-7. The PKC-activating phorbol-12-myristate-13-acetate (PMA), differently from LDL, enhanced the LDL-receptor (LDL-R)-mediated binding and degradation of [125I]LDL, but inhibited endogenous cholesterol synthesis, and both effects were inhibited by H-7. The LDL-induced inhibition of binding and degradation of [125I]LDL was not affected by H-7, whereas decreased cholesterol synthesis was counteracted by H-7. These results suggest the existence of a non-sterol-dependent signal pathway of LDL-Rs, by which endogenous cholesterol synthesis, that is, the [14C]acetate incorporation, is regulated through PKC activation.

Neuropeptides induced a pronounced and statin-sensitive dysregulation of mevalonate cycle in human monocytes of patients with hypercholesterolemia.

Angiotensin II (Ang II) and leptin generate statin-inhibitable superoxide anion production that accounts for only part of the entire superoxide anion production. In our recent studies, we aimed at elucidating whether Ang II and leptin, affecting the intensity of the mevalonate cycle, are able to increase endogenous cholesterol synthesis. Furthermore, we compared the superoxide anion and cholesterol production capability of monocytes of healthy control volunteers and monocytes obtained from patients with hypercholesterolemia (HC). We also studied the differences of the produced statin-inhibitable superoxide anion and cholesterol synthesis in control and HC-monocytes, depending on the applied stimulating ligands. In control and HC-monocytes--stimulated by Ang II, leptin, fenyl-Me-Leu-Phe (FMLP), phorbol-12-myristate-13-acetate (PMA) and A23187--we determined the proportion of mevalonate cycle-dependent and -independent superoxide and cholesterol production, using lovastatin (Lov), and 25-hydroxycholesterol (25-HC). According to our results; (1) superoxide anion generation in HC-monocytes was elevated after Ang II, leptin and FMLP-stimulation, whereas PMA and A23187-stimulation had lower stimulating effect in HC than in control cells. (2) Cholesterol synthesis was increased only after stimulation with Ang II and leptin. (3) The Ang II and leptin-induced total superoxide anion generation and cholesterol synthesis were more elevated in HC than in control monocytes. (4) In contrast, the increase in Lov and 25-HC sensitive cholesterol synthesis were higher in resting, but lower in stimulated HC monocytes than in control cells. Summarizing our results, we concluded that Ang II and leptin are involved in enhancement of endogenous cholesterol synthesis through a statin-sensitive pathway.

The association between angiotensin II-induced free radical generation and membrane fluidity in neutrophils of patients with metabolic syndrome.

Angiotensin II (Ang II) is able to induce free radical generation in neutrophils, which is more elevated in neutrophils of patients with hypercholesterolemia (HC). In addition, the signal processing through angiotensin I (Ang I) receptors is altered. In present study, we compared the Ang II-triggered free radical generation of neutrophils obtained from patients with relatively isolated forms of metabolic syndrome (MS) with membrane-bound cholesterol content and membrane fluidity. We determined the enhancement of Ang II-induced superoxide anion and leukotriene C(4) (LTC(4)) generation, membrane fluidity and cell-bound cholesterol content of neutrophils obtained from 12 control subjects, 11 patients with obesity (Ob), 10 patients with type 2 diabetes mellitus (t2-DM) and 12 patients with HC. The alteration of signal processing was studied after preincubation with different inhibiting drugs. Superoxide anion, LTC(4) production and membrane rigidity were increased in the following order: control < Ob < t2-DM < HC. Both Ang II-induced superoxide anion and LTC(4) generation were decreased in control cells by pertussis toxin and fluvastatin (Flu), whereas in each patient group, mepacrin, verapamil and Flu were effective, suggesting alterations in signal pathways, which may be attributed to isoprenylation. The enhancement of superoxide anion and LTC(4) generation correlated significantly with membrane rigidity, independently from the experimental groups and membrane-bound cholesterol content. Membrane rigidity of neutrophils, obtained from patients with MS, plays a role in Ang II-induced free radical generation independent of intracellular cholesterol homeostasis.

Angiotensin II-induced oxidative burst is fluvastatin sensitive in neutrophils of patients with hypercholesterolemia.

The aim of this study was to investigate the effect of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor fluvastatin (Flu) on angiotensin II (AII)-stimulated neutrophils of patients with hypercholesterolemia. Results suggest that a 6-week-long Flu administration completely counteracted the AII-induced increase in superoxide anion and leukotriene C4 production of the neutrophils of patients with hypercholesterolemia. However, the failure of signal processing through pertussis toxin-sensitive G protein, the increase in [Ca2+]i in membrane-bound protein kinase C activity, and the increase in neutrophil-bound cholesterol content were only partially restored by Flu. In addition, Flu had no effect on the increased membrane rigidity of the neutrophils of patients with hypercholesterolemia. To sum it up, Flu administration had a beneficial effect on AII-triggered reactive oxygen species generation; it resulted in partial restoration of signaling processes and of membrane composition, but membrane fluidity remained unchanged.

Different anticancer effects of fluvastatin on primary hepatocellular tumors and metastases in rats.

Rats (FLF1) were pretreated with 2 and 20 mg/kg/day fluvastatin (Flu), and after 6 weeks, hepatocellular tumor cells were inoculated under the left renal capsule. At different times, growth and pyruvate kinase (PK) activity of the primary tumors and lymph node metastases were determined. Flu had a dose-dependent inhibitory effect on primary and metastatic tumors, and the inhibitory effect on growth and PK activity in metastases were higher than in primary tumors. Finally, Flu had an earlier inhibitory effect on the early appeared PK activity in metastases than in primary tumors.

HMG CoA reductase inhibitor fluvastatin arrests the development of implanted hepatocarcinoma in rats.

BACKGROUND: Fluvastain (Flu) is widely accepted to have anticancer effect although its in vivo chemopreventive ability in cancer has not been studied. The therapeutic and chemopreventive effects of Flu were compared in vivo in the present study. MATERIALS AND METHODS: Under the left renal capsule of FLF1 hybrid rats 10(6) hepatocarcinoma cells were implanted (He/De14) on sponge disc. The differences in net weight between the left and right kidneys were determined as tumor weights. Flu was administered per os in 0.5, 2 and 20 mg/kg/day doses. RESULTS: The 21-day pretreatment before tumor implantation with Flu had no effect on tumor development in the absence of Flu treatment after the implantation. The addition of Flu before and after tumor implantation demonstrated a more intensive anticancer effect than in the case of treatment given only after tumor implantation. CONCLUSION: Flu has in 0.5-20 mg/kg/day doses a therapeutic and also a preventive effect on hepatocarcinoma growth in rats depending on the type of administration.

Altered signal pathway in angiotensin II-stimulated neutrophils of patients with hypercholesterolaemia.

Angiotensin II (AII) in 1-10 nM concentrations has an in vivo immunostimulating effect on human neutrophils. The release of superoxide anions and leukotrienes (LTs) is significantly increased by 10 nM AII-stimulated neutrophils of patients with hypercholesterolaemia (HCH). These oxidizing agents may be involved in the damage of vessel walls, i.e., in atherosclerotic plaque formation. To clarify the receptor types and signal pathways in neutrophils of healthy controls and patients, inositol trisphosphate (IP(3)) production and Ca(2+) signalling were studied. Neutrophils were pretreated before AII stimulation with different inhibitory drugs. In control cells, the stimulation occurred predominantly through pertussis toxin-sensitive, type angiotensin 1 receptors. This induced IP(3) production and Ca(2+) signalling from intracellular pools. In neutrophils of hypercholesterolaemic patients, the enhanced release of oxidizing agents was dependent more on type angiotensin 2 than type angiotensin 1 receptors. After stimulation, there was no IP(3) production detected. The Ca(2+) signalling was lower than in control cells and was dependent on extracellular Ca(2+).