Maciej Suski

Hypooxytocinaemia in obese Zucker rats relates to oxytocin degradation in liver and adipose tissue

The metabolic action of oxytocin has recently been intensively studied to assess the ability of the peptide to regulate energy homeostasis. Despite the obvious weight-reducing effect of oxytocin observed in experimental studies, plasma oxytocin levels were found to be unchanged or even elevated in human obesity. The aim of our study was to evaluate the changes in the oxytocin system in Zucker rats, an animal model closely mirroring morbid obesity in humans. Plasma oxytocin levels were measured in obese Zucker rats and lean controls by enzyme immunoassay after plasma extraction. The expression of oxytocin and oxytocin receptor (OXTR) was assessed at the mRNA and protein levels by quantitative real-time PCR and immunoblotting respectively. Plasma and tissue activity of oxytocinase, the main enzyme involved in oxytocin degradation, were measured by fluorometric assay using an arylamide derivate as the substrate. Obese Zucker rats displayed a marked reduction in plasma oxytocin levels. Elevated liver and adipose tissue oxytocinase activity was noticed in obese Zucker rats. Hypothalamic oxytocin gene expression was not altered by the obese phenotype. OXTR mRNA and protein levels were upregulated in the adipose tissue of obese animals in contrast to the reduced OXTR protein levels in skeletal muscle. Our results show that obesity is associated with reduced plasma oxytocin due to increased peptide degradation by liver and adipose tissue rather than changes in hormone synthesis. This study highlights the importance of the oxytocin system in the pathogenesis of obesity and suggests oxytocinase inhibition as a candidate approach in the therapy of obesity.

Maternal stress predicts altered biogenesis and the profile of mitochondrial proteins in the frontal cortex and hippocampus of adult offspring rats

Currently, much attention is focused on the influence of mitochondrial disturbances at the onset of depression. The goal of this study was to investigate the impact of prenatal stress (an animal model of depression) on the mitochondrial biogenesis proteins and mitoproteome profile in the frontal cortex and hippocampus of adult 3-month-old male rats following a prenatal stress procedure. Our results show that rats that were exposed to prenatal stress stimuli displayed depression-like behaviors based on the sucrose preference and elevated plus maze tests. It has been found that the level of the PGC-1α protein was reduced in the frontal cortex and hippocampus of the adult offspring after the prenatal stress procedure. Moreover, in the frontal cortex, the level of the pro-apoptotic protein Bax was up-regulated. Two-dimensional electrophoresis coupled with mass spectrometry showed the statistically significant down-regulation of the mitochondrial ribosomal protein L12 (Mrpl12) and mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 2 (NDUFV2) as well as the up-regulation of the Tubulin Polymerization Promoting Proteins (Tppp/p25) in the frontal cortex. In contrast, in the hippocampus, the mitochondrial pyruvate dehydrogenase E1 component subunit beta, the voltage-dependent anion-selective channel protein 2 (VDAC2), and the GTP-binding nuclear protein RAN (RAN) were down-regulated and the expression of phosphatidylethanolamine-binding protein 1 (PEBP-1) was enhanced. These findings provide new evidence that stress during pregnancy may lead not only to behavioral deficits, but also to disturbances in the brain mitoproteome profile in adult rat offspring.

Mitochondrial Aldehyde Dehydrogenase Activation by Alda‐1 Inhibits Atherosclerosis and Attenuates Hepatic Steatosis in Apolipoprotein E‐Knockout Mice

Background Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda‐1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE−/−) mice. Methods and Results Alda‐1 caused decrease of atherosclerotic lesions approximately 25% as estimated by “en face” and “cross‐section” methods without influence on plasma lipid profile, atherosclerosis‐related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda‐1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda‐1‐treated apoE−/− mice. Alda‐1 attenuated formation of 4‐hydroxy‐2‐nonenal (4‐HNE) protein adducts and decreased triglyceride content in liver tissue. Two‐dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda‐1 treatment in liver of apoE−/− mice, mostly proteins related to metabolism and oxidative stress. The most up‐regulated were the proteins that participated in beta oxidation of fatty acids. Conclusions Collectively, Alda‐1 inhibited atherosclerosis and attenuated NAFLD in apoE−/− mice. The pattern of changes suggests a beneficial effect of Alda‐1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda‐1 action require further investigation.

Beneficial effect of amantadine on postoperative pain reduction and consumption of morphine in patients subjected to elective spine surgery.

OBJECTIVE To analyze the effect of coadministration of morphine and amantadine on postoperative pain reduction and morphine consumption in patients after elective spine surgery. METHODS In double-blinded study, 60 patients (ASA physical status I-II) were randomized into two groups. Group A was given oral amantadine 50 or 100 mg 1 hour before surgery and 8, 20, 32 hours after operation. Group P received placebo at identical times. Pain was assessed using numerical rating scale before first administration of morphine and in 2, 3, 4, 6, 24, and 48 hours after operation. The amounts of morphine consumed were recorded up to 48 hours after surgery. Blood samples were taken twice in 2 hours after surgery and plasma levels of morphine and its main metabolites were measured. RESULTS As compared with placebo, amantadine significantly reduced intra-operative Fentanyl use and sensation of postoperative pain. Up to 48 hours after operation, the cumulative consumption of morphine was 25% lower in the amantadine group. Moreover, intensity of nausea and vomiting tended to be lower in A group. Starting from 12th hour after surgery, the level of postoperative sedation was lower in patients who received amantadine, as compared with placebo group. No significant differences in plasma levels of morphine ant its metabolites were observed between A and P groups. CONCLUSIONS Pre- and postoperative administration of amantadine significantly reduced fentanyl use during operation, as well as reduced the postoperative pain and decreased morphine consumption in young patients undergoing orthopedic surgery.

Deficient hippocampal insulin signaling and augmented Tau phosphorylation is related to obesity- and age-induced peripheral insulin resistance: a study in Zucker rats

BackgroundInsulin signaling and Tau protein phosphorylation in the hippocampi of young and old obese Zucker fa/fa rats and their lean controls were assessed to determine whether obesity-induced peripheral insulin resistance and aging are risk factors for central insulin resistance and whether central insulin resistance is related to the pathologic phosphorylation of the Tau protein.ResultsAging and obesity significantly attenuated the phosphorylation of the insulin cascade kinases Akt (protein kinase B, PKB) and GSK-3β (glycogen synthase kinase 3β) in the hippocampi of the fa/fa rats. Furthermore, the hyperphosphorylation of Tau Ser396 alone and both Tau Ser396 and Thr231 was significantly augmented by aging and obesity, respectively, in the hippocampi of these rats.ConclusionsBoth age-induced and obesity-induced peripheral insulin resistance are associated with central insulin resistance that is linked to hyperTau phosphorylation. Peripheral hyperinsulinemia, rather than hyperglycemia, appears to promote central insulin resistance and the Tau pathology in fa/fa rats.

Proteomic analysis of changes in protein expression in liver mitochondria in apoE knockout mice

The involvement of both apolipoprotein E (apoE) and mitochondria in lipid metabolism is widely recognized, however there is surprisingly scarce data about the putative mitochondrial action(s) of this protein. The aim of the study was to screen the alterations in liver mitochondrial proteome caused by apoE deficiency. We applied 2DE-LC-MS/MS methodology to investigate the changes in liver mitochondrial protein expression in 6-months old apoE(-/-) mice as compared to C57BL/6J controls. ApoE(-/-), but not C57BL/6J mice developed visible atherosclerotic changes in aorta and mild, diffuse steatosis of the liver. Collectively, 18 differentially expressed proteins were identified in mitochondria, related to apoptosis, antioxidant and detoxifying mechanisms of mitochondria, as well as lipid metabolism and transport. In conclusion, differential proteomic approach revealed several lines of proteomic evidence that mitochondrial function in the liver of apoE(-/-) mice could be altered as a result of overlapping of pathological and compensatory changes in expression of proteins.

Co‐Administration of Dextromethorphan and Morphine: Reduction of Post‐Operative Pain and Lack of Influence on Morphine Metabolism

We investigated co-analgesic effect of dextromethorphan in adolescent post-operative patients with idiopathic scoliosis. In a double-blind study, 60 patients with ASA physical status I-II were randomised into two groups. Group dextromethorphan (n = 30; age: 15.9 +/- 2.4 years) was given oral dextromethorphan 30 or 45 mg 1 hr before surgery and 8, 20 and 32 hr after operation. Group placebo (n = 30; age: 16.5 +/- 2.7 years) received placebo at identical times. Post-operative analgesic requirements were assessed using nurse-controlled analgesia system. Pain was assessed using numeric rating scale before first administration of morphine and at 2, 3, 4, 6, 24 and 48 hr after operation. Blood samples were taken 20 min. after the first use of morphine (within 1 hr after operation). The total use of analgesics during surgery was lower in the dextromethorphan group. The dose of morphine providing relief immediately after surgery, as well as total analgesic requirements in the first and second day after surgery did not differ between groups. Subjectively evaluated pain intensity score (numeric rating scale) was lower for the dextromethorphan patients in the first 4 hr, but not later after surgery. Plasma levels of morphine, morphine-6-glucuronide and morphine-3-glucuronide did not differ between groups. Dextromethorphan did not influence morphine glucuronidation, in terms of promotion of formation of any morphine glucuronides. In conclusion, in young patients subjected to spine surgery, addition of dextromethorphan to morphine reduced pain only in early post-operative period. In such patients, co-analgesic action of dextromethorphan was not associated with significant changes in plasma levels of morphine metabolites.

Proteomic analysis of liver mitochondria of apolipoprotein E knockout mice treated with metformin

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance. Metformin, a widely known anti-diabetic drug, used for patients with type 2 diabetes mellitus, is also claimed to be useful in treatment of NAFLD. However, both the clinical efficacy and the putative mechanisms underlying the clinical effects of metformin in treating NAFLD are unclear. Adenosine monophosphate-activated protein kinase (AMPK), the primary molecular target for metformin, is a known regulator of mitochondrial function. Thus, we used a proteomic approach to investigate the effect of metformin on liver mitochondria of apolipoprotein E knockout (apoE(-/-)) mice, an animal model of NAFLD. Two-dimensional electrophoresis coupled with mass spectrometry was applied to study the changes in liver mitochondrial protein expression in 6-month old metformin-treated apoE(-/-) mice as compared to non-treated animals. Collectively, 25 differentially expressed proteins were indentified upon metformin treatment including proteins related to metabolism, oxidative stress and cellular respiration. The most up-regulated protein was glycine N-methyltransferase (GNMT) - an enzyme, whose deficiency was shown to be directly related to the development of NAFLD. Our results clearly point to the strong mitochondrial action of metformin in NAFLD. Up-regulation of GNMT may represent an important mechanism of beneficial action of metformin in NAFLD treatment.

The impact of mitochondrial aldehyde dehydrogenase (ALDH2) activation by Alda-1 on the behavioral and biochemical disturbances in animal model of depression.

The etiology of depression remains still unclear. Recently, it has been proposed, that mitochondrial dysfunction may be associated with development of mood disorders, such as depression, bipolar disorder and anxiety disorders. Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, a small-molecule activator of ALDH2, on depressive- and anxiety-like behaviors in an animal model of depression - the prenatally stressed rats - using behavioral, molecular and proteomic methods. Prolonged Alda-1 administration significantly increased the climbing time, tended to reduce the immobility time and increased the swimming time of the prenatally stressed rats in the forced swim test. Moreover, treatment of prenatally stressed rats with Alda-1 significantly increased number of entries into the open arms of the maze and the time spent therein, as assessed by elevated plus-maze test. Such actions were associated with reduction of plasma 4-HNE-protein content, decrease of TNF-α mRNA and increase of PGC-1α (regulator of mitochondrial biogenesis) mRNA level in the frontal cortex and hippocampus of the prenatally stressed rats as well as with normalization of peripheral immune parameters and significant changes in expression of 6 and 4 proteins related to mitochondrial functions in the frontal cortex and hippocampus, respectively. Collectively, ALDH2 activation by Alda-1 led to a significant attenuation of depressive- and anxiety-like behaviors in the prenatally stressed rats. The pattern of changes suggested mitoprotective effect of Alda-1, however the exact functional consequences of the revealed alterations require further investigation.

The influence of angiotensin-(1-7) Mas receptor agonist (AVE 0991) on mitochondrial proteome in kidneys of apoE knockout mice.

Excessive action of angiotensin II on mitochondria has been shown to play an important role in mitochondrial dysfunction, a common feature of atherogenesis and kidney injury. Angiotensin-(1-7)/Mas receptor axis constitutes a countermeasure to the detrimental effects of angiotensin II on AT1 receptors. The aim of the study was to assess the effects of angiotensin-(1-7) peptidomimetic AVE0991 on the kidney mitochondrial proteome in widely used animal model of atherosclerosis (apoE(-/-) mice). Proteins changed in apoE(-/-) mice belonged to the groups of antioxidant enzymes, apoptosis regulators, inflammatory factors and metabolic enzymes. Importantly, AVE0991 partially reversed atherosclerosis-related changes in apoE(-/-) mice.