Maternal cafeteria diet influences kisspeptin (Kiss1), kisspeptin receptor(Gpr54), and sirtuin (Sirt1) genes, hormonal and metabolic profiles, and reproductive functions in rat offspring in a sex-specific manner†.

Kisspeptin (KP, encoded by Kiss1, binding to the Gpr54 receptor) is a neuropeptide conveying information on the metabolic status to the hypothalamic-pituitary-gonadal axis. KP acts together with dynorphin A (encoded by Pdyn) and neurokinin B (encoded by Tac2) to regulate reproduction. KP is crucial for the onset of puberty and is under the control of sirtuin (encoded by Sirt1). We hypothesize that the maternal cafeteria (CAF) diet has adverse effects on the offspring's hormonal, metabolic, and reproductive functions due to sex-specific alterations in the expression of Kiss1, Gpr54, Pdyn, Tac2, and Sirt1 in the hypothalamus, and Kiss1, Gpr54, and Sirt1 in the liver. Rats were fed a CAF diet before pregnancy, during pregnancy, and during lactation. The vaginal opening was monitored. Offspring were sacrificed in three age points: PND 30, PND 35, and PND 60 (females) and PND 40, PND 45, and PND 60 (males). Their metabolic and hormonal status was assessed. mRNA for Kiss1, Gpr54, Pdyn, Tac2, and Sirt1 were measured by real-time PCR in the hypothalamus and/or livers. We found that CAF offspring had lower weight and altered body composition; increased cholesterol and triglyceride levels, sex-specific changes in glucose and insulin levels; sex-dependent changes in Sirt1/Kiss1 mRNA ratio in the hypothalamus; sex-specific alterations in Kiss1 and Sirt1 mRNA in the liver with more diversity in males; and a delayed puberty onset in females. We concluded that the mother's CAF diet leads to sex-specific alterations in metabolic and reproductive outcomes via Kiss1/Gpr54 and Sirt1 systems in offspring.

The paraventricular nucleus of the hypothalamus - the concertmaster of autonomic control. Focus on blood pressure regulation.

The autonomic nervous system regulates internal organs and peripheral circulation, which enables the maintenance of homeostasis in vertebrate species. One of the brain regions involved in autonomic and endocrine homeostasis regulation is the paraventricular nucleus of the hypothalamus (PVN). The PVN is a unique site at which multiple input signals can be assessed and integrated. The regulation of the autonomic system by the PVN and, especially, the sympathetic flow, depends upon the integration of inhibitory and excitatory neurotransmitter action. The excitatory neurotransmitters such as glutamate and angiotensin II, and inhibitory neurotransmitters such as γ­aminobutyric acid and nitric oxide, play a key role in the physiological function of the PVN. Moreover, arginine-vasopressin (AVP) and oxytocin (OXT) are important in the regulation of sympathetic system activity. The PVN is also crucial for maintaining cardiovascular regulation, with its integrity being pivotal for blood pressure regulation. Studies have shown that pre­autonomic sympathetic PVN neurons increase blood pressure and the dysfunction of these neurons is directly related to elevated sympathetic nervous system activity under hypertension. Etiology of hypertension in patients is not fully known. Thus, understanding the role of PVN in the generation of hypertension may help to treat this cardiovascular disease. This review focuses on the PVN's inhibitory and excitatory neurotransmitter interactions that regulate sympathetic system activity in physiological conditions and hypertension.

DNA hypermethylation of Kiss1r promoter and reduction of hepatic Kiss1r in female rats with type 2 diabetes.

Kisspeptin, produced from the brain and peripheral tissues, may constitute an important link in metabolic regulation in response to external cues, such as diet. The kisspeptin system is well described in the brain. However, its function and regulation in the peripheral tissues, especially in relation to metabolic disease and sex differences, remain to be elucidated. As Kiss1 and Kiss1r, encoding for kisspeptin and kisspeptin receptors, respectively, are altered by overnutrition/fasting and regulated by DNA methylation during puberty and cancer, epigenetic mechanisms in metabolic disorders are highly probable. In the present study, we experimentally induced type 2 diabetes mellitus (DM2) in female Wistar rats using high-fat diet/streptozocin. We analysed expression and DNA methylation of Kiss1 and Kiss1r in the peripheral tissues, using quantitative-reverse-transcription PCR (qRT-PCR) and pyrosequencing. We discovered differential expression of Kiss1 and Kiss1r in peripheral organs in DM2 females, as compared with healthy controls, and the profile differed from patterns reported earlier in males. DM2 in females was linked to the increased Kiss1 mRNA in the liver and increased Kiss1r mRNA in the liver and adipose tissue. However, Kiss1r promoter was hypermethylated in the liver, suggesting gene silencing. Indeed, the increase in DNA methylation of Kiss1r promoter was accompanied by a reduction in Kiss1r protein, implying epigenetic or translational gene repression. Our results deliver novel evidence for tissue-specific differences in Kiss1 and Kiss1r expression in peripheral organs in DM2 females and suggest DNA methylation as a player in regulation of the hepatic kisspeptin system in DM2.

Animal Foetal Models of Obesity and Diabetes - From Laboratory to Clinical Settings.

The prenatal period, during which a fully formed newborn capable of surviving outside its mother's body is built from a single cell, is critical for human development. It is also the time when the foetus is particularly vulnerable to environmental factors, which may modulate the course of its development. Both epidemiological and animal studies have shown that foetal programming of physiological systems may alter the growth and function of organs and lead to pathology in adulthood. Nutrition is a particularly important environmental factor for the pregnant mother as it affects the condition of offspring. Numerous studies have shown that an unbalanced maternal metabolic status (under- or overnutrition) may cause long-lasting physiological and behavioural alterations, resulting in metabolic disorders, such as obesity and type 2 diabetes (T2DM). Various diets are used in laboratory settings in order to induce maternal obesity and metabolic disorders, and to alter the offspring development. The most popular models are: high-fat, high-sugar, high-fat-high-sugar, and cafeteria diets. Maternal undernutrition models are also used, which results in metabolic problems in offspring. Similarly to animal data, human studies have shown the influence of mothers' diets on the development of children. There is a strong link between the maternal diet and the birth weight, metabolic state, changes in the cardiovascular and central nervous system of the offspring. The mechanisms linking impaired foetal development and adult diseases remain under discussion. Epigenetic mechanisms are believed to play a major role in prenatal programming. Additionally, sexually dimorphic effects on offspring are observed. Therefore, further research on both sexes is necessary.

Impact of rhizobacterial inoculants on plant growth and enzyme activities in soil treated with contaminated bottom sediments.

The impact of contaminated bottom sediments on plant growth and soil enzyme activities was evaluated in a greenhouse pot study. The sediments were moderately contaminated with zinc and heavily contaminated with polycyclic aromatic hydrocarbons and polychlorinated dibenzo-p-dioxins and furans. The sediments were mixed with soil and planted with either Festuca arundinacea or Tagetes patula. The capacity of two rhizobacterial strains (Massilia niastensis P87 and Streptomyces costaricanus RP92), previously isolated from contaminated soils, to improve plant growth under the chemical stress was tested. Application of sediments to soil was severely phytotoxic to T. patula and mildly to F. arundinacea. On the other hand, the addition of sediments enhanced the soil enzymatic activity. Inoculation with both bacterial strains significantly increased shoot (up to 2.4-fold) and root (up to 3.4-fold) biomass of T. patula. The study revealed that the selected plant growth-promoting bacterial strains were able to alleviate phytotoxicity of bottom sediments to T. patula resulting from the complex character of the contamination.

Long term insight into biodiversity of a smelter wasteland reclaimed with biosolids and by-product lime.

Smelter wastelands containing high amounts of zinc, lead, cadmium, and arsenic constitute a major problem worldwide. Serious hazards for human health and ecosystem functioning are related to a lack of vegetative cover, causing fugitive dust fluxes, runoff and leaching of metals, affecting post-industrial ecosystems, often in heavily populated areas. Previous studies demonstrated the short term effectiveness of assisted phytostabilisation of zinc and lead smelter slags, using biosolids and liming. However, a long term persistence of plant communities introduced for remediation and risk reduction has not been adequately evaluated. The work was aimed at characterising trace element solubility, plant and microbial communities of the top layer of the reclaimed zinc and lead smelter waste heaps in Piekary Slaskie, Poland, 20 years after the treatment and revegetation. The surface layer of the waste heaps treated with various rates of biosolids and the by-product lime was sampled for measuring chemical and biochemical parameters, which are indicative for metals bioavailability as well as for microorganisms activity. Microbial processes were characterised by enzyme activities, abundance of specific groups of microorganisms and identification of N fixing bacteria. Plant communities of the area were characterised by a percent coverage of the surface and by a composition of plant species and plant diversity. The study provides a strong evidence that the implemented remediation approach enables a sustainable functioning of the ecosystem established on the toxic waste heaps. Enzyme activities and the count of various groups of microorganisms were the highest in areas treated with both biosolids and lime, regardless their rates. A high plant species diversity and microbial activities are sustainable after almost two decades from the treatment, which is indicative of a strong resistance of the established ecosystem to a metal stress and a poor physical quality of the anthropogenic soil formed by the treatment.

CB1 receptor activation in the rat paraventricular nucleus induces bi-directional cardiovascular effects via modification of glutamatergic and GABAergic neurotransmission.

We have shown previously that the cannabinoid receptor agonist CP55940 microinjected into the paraventricular nucleus of the hypothalamus (PVN) of urethane-anaesthetized rats induces depressor and pressor cardiovascular effects in the absence and presence of the CB1 antagonist AM251, respectively. The aim of our study was to examine whether the hypotension and/or hypertension induced by CP55940 given into the PVN results from its influence on glutamatergic and GABAergic neurotransmission. CP55940 was microinjected into the PVN of urethane-anaesthetized rats twice (S1 and S2, 20 min apart). Antagonists of the following receptors, NMDA (MK801), ß2-adrenergic (ICI118551), thromboxane A2-TP (SQ29548), angiotensin II-AT1 (losartan) or GABAA (bicuculline), or the NO synthase inhibitor L-NAME were administered intravenously 5 min before S2 alone or together with AM251. The CP55940-induced hypotension was reversed into a pressor response by AM251, bicuculline and L-NAME, but not by the other antagonists. The CP55940-induced pressor effect examined in the presence of AM251 was completely reversed by losartan, reduced by about 50-60 % by MK801, ICI118551 and SQ29548, prevented by bilateral adrenalectomy but not modified by bicuculline and L-NAME. Parallel, but smaller, changes in heart rate accompanied the changes in blood pressure. The bi-directional CB1 receptor-mediated cardiovascular effects of cannabinoids microinjected into the PVN of anaesthetized rats depend on stimulatory glutamatergic and inhibitory GABAergic inputs to the sympathetic tone; the glutamatergic input is related to AT1, TP and ß2-adrenergic receptors and catecholamine release from the adrenal medulla whereas the GABAergic input is reinforced by NO.

Age-specific influences of chronic administration of the fatty acid amide hydrolase inhibitor URB597 on cardiovascular parameters and organ hypertrophy in DOCA-salt hypertensive rats.

BACKGROUND: The endocannabinoid system has been suggested to be up-regulated in hypertension. Fatty acid amide hydrolase (FAAH) is the main hydrolytic enzyme for the encocannabinoid anandamide. The aim of our study was to examine the age-specific influence of the chronic administration of the FAAH inhibitor URB597 on blood pressure (BP), heart rate (HR) and cardiac and renal hypertrophy in hypertensive rats during two critical periods for the development of hypertension. METHODS: Experiments were performed on uninephrectomised 4 (younger) and 6-7 (older) weeks old rats rendered hypertensive by a high salt diet and deoxycorticosterone acetate (DOCA) injections and on normotensive animals (unilateral nephrectomy only). URB597 1mg/kg or its vehicle were injected twice daily for 2 weeks. RESULTS: The DOCA-salt procedure caused comparable increases in BP (but not HR) in both age groups and more strongly increased cardiac and renal hypertrophic indices in younger than in older animals. Chronic URB597 administration reduced BP and HR in older but not in younger rats. In contrast, the inhibitor diminished the cardiac and renal hypertrophy in younger but not in older animals. URB597 did not affect body weight gain, and food and water intake in normotensive or hypertensive rats. CONCLUSION: Two weeks of URB597 administration to DOCA-salt hypertensive rats caused an age-specific reduction in BP, HR and cardiac and renal hypertrophy and did not affect the body weight, and water and food intake. Thus, caution should be taken during studies of FAAH inhibitors because of their potential age-specific effects.

Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA-salt hypertensive rats.

AIMS: This study was performed to examine whether hypertension affects the sympathetic transmission to resistance vessels of pithed rats via inhibitory presynaptic cannabinoid CB1 receptors and whether endocannabinoids are involved in this response. MATERIALS AND METHODS: We compared uninephrectomised rats rendered hypertensive by high salt diet and deoxycorticosterone acetate (DOCA) injections with normotensive animals (uninephrectomy only). Experiments were performed on vagotomised and pithed animals. Increases in diastolic blood pressure (DBP) were induced four times (S1-S4) by electrical stimulation or phenylephrine injection. KEY FINDINGS: Electrical stimulation (0.75Hz, 1ms, 50V, 5 impulses) of the preganglionic sympathetic nerve fibres innervating the blood vessels more strongly increased DBP in normotensive than in DOCA-salt rats. Phenylephrine (0.01µmol/kg) induced similar increases in DBP in both groups. The cannabinoid receptor agonist CP55940 (0.01-1µmol/kg) did not modify the rises in DBP induced by phenylephrine. However, it inhibited the electrically stimulated increases in DBP, more strongly in DOCA-salt than in normotensive animals (maximally by 50 and 30%, respectively). The effect of CP55940 was attenuated by the CB1 antagonist AM251 (3µmol/kg). AM251 enhanced the neurogenic vasopressor response during S4 by itself in hypertensive rats only. URB597 (3µmol/kg), which inhibits degradation of the endocannabinoid anandamide, did not modify the electrically stimulated increases in DBP. SIGNIFICANCE: The function of inhibitory presynaptic CB1 receptors on sympathetic nerves is enhanced in DOCA-salt hypertensive rats. Thus, the CB1 receptor-mediated inhibition of noradrenaline release from the sympathetic nerve fibres innervating the resistance vessels might play a protective role in hypertension.

[Role of endocannabinoid 2-arachidonoylglycerol in the physiology and pathophysiology of the cardiovascular system]. / Znaczenie endokannabinoidu 2-arachidonyloglicerolu w fizjologii i patofizjologii ukladu krazenia.

Cannabinoids, the active ingredients of Cannabis sativa var. indica, have been used by humans as recreational and therapeutic agents for thousands of years. This group of substances also includes synthetic ligands and, synthesized in the body of humans and animals, endocannabinoids. The best known compound classified as an endogenous cannabinoid is anandamide. However, recent studies show that another compound of this group, 2-arachidonoylglycerol (2-AG), also performs many important functions in the organism. 2-Arachidonoylglycerol plays an important role in the regulation of the circulatory system via direct and/or indirect, through their metabolites, effects on blood vessels and/or heart. Accumulating evidence reveals that 2-AG is involved in the pathogenesis of various shocks and atherosclerosis. Thus, it may be a novel attractive therapeutic target. However, because of rapid metabolism and opposite effects dependent on the experimental model, the function of 2-AG still remains to be established.

Mechanisms of endothelium-dependent relaxation evoked by anandamide in isolated human pulmonary arteries.

Endocannabinoids contract, relax or do not affect vessels with different calibre and tone in the pulmonary circulation in four species. The aim of the present study was to determine the mechanisms involved in the anandamide-induced relaxation of human pulmonary arteries (hPAs). Studies were performed in the isolated hPAs pre-constricted with the prostanoid TP receptor agonist, U-46619. To detect fatty acid amide hydrolase (FAAH) expression, Western blots were used. Anandamide concentration dependently relaxed the endothelium-intact hPAs pre-constricted with U-46619. The anandamide-induced relaxation was virtually abolished by removal of the endothelium and strongly attenuated by inhibitors of cyclooxygenases (indomethacin, COX-1/COX-2, and nimesulide, COX-2), nitric oxide synthase (N (G) -nitro-L-arginine methyl ester) given separately or in combination, FAAH (URB597), and the prostanoid IP receptor antagonist, RO1138452. The anandamide-evoked relaxation in the endothelium-intact vessels was attenuated in KCl pre-constricted preparations or by the inhibitor of large-conductance Ca(2+)-activated K(+) channels, iberiotoxin. In experiments performed in the presence of URB597 to exclude effects of anandamide metabolites, the antagonist of the endothelial cannabinoid receptor, O-1918, diminished the anandamide-evoked relaxation whereas the antagonists of cannabinoid CB1, CB2 and vanilloid TRPV1 receptors, AM251, SR144528 and capsazepine, respectively, had no effect. Western blot studies revealed the occurrence of FAAH protein in the hPAs. The present study shows that anandamide breakdown products, cyclooxygenase pathways, nitric oxide, potassium channels and the O-1918-sensitive cannabinoid receptor play a role in the anandamide-induced relaxation of the hPAs with intact endothelium.

EP3 receptor-mediated contraction of human pulmonary arteries and inhibition of neurogenic tachycardia in pithed rats.

BACKGROUND: The aim of our study was (1) the pharmacological characterization of EP(3) receptors in human pulmonary arteries and (2) the examination of the potential involvement of these receptors in the regulation of neurogenic tachycardia in pithed rats. L-826266 served as the EP(3) receptor antagonist. METHODS: Experiments were performed on isolated human pulmonary arteries and pithed rats. RESULTS: The prostanoid EP(1)/EP(3) receptor agonist sulprostone (1 nM - 100 µM) concentration-dependently contracted isolated human pulmonary arteries (pEC50, 6.88 ± 0.10). The EP(1) receptor antagonist SC 19920 (100 µM) did not affect the vasoconstriction induced by sulprostone, the TP receptor antagonist sulotroban (10 µM) only slightly attenuated the effects elicited by sulprostone >>3 µM, whereas L-826266 (10 µM) shifted its concentration-response curve to the right (apparent pA(2) value 6.18; incubation time 0.5 h). In rings exposed to L-826266 (0.1, 1 or 10 µM) for 3 h, a concentration-dependent inhibitory effect against the sulprostone-induced vasoconstriction was obtained, yielding a Schild plot-based pA(2) value of 7.39. In pithed rats, sulprostone (10 - 1,000 nmol/kg), but not the IP/EP(1) receptor agonist iloprost (1-100 nmol/kg), inhibited the electrically evoked increase in heart rate (HR) dose-dependently, maximally by at least 80%. L-826266 (3 µmol/kg) did not affect basal HR and diastolic blood pressure, but reduced the inhibitory effect of sulprostone 1,000 nmol/kg by about 20%. CONCLUSION: EP(3) receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibers supplying the heart of pithed rats strongly inhibit the neurogenic tachycardia.

Differentiations of the effect of NMDA on the spatial learning of rats with 4 and 12 week diabetes mellitus.

This study examines possible interactions between behavioral effects and influence of N-methyl-D-aspartate acid (NMDA) receptors in 4 and 12-week streptozotocin (STZ) induced diabetic rats. Effects of NMDA receptor agonist on spatial learning were tested in control groups of rats and in rats with 4 and 12 weeks diabetes mellitus (DM). Experimental diabetes was induced by a single intravenous injection of streptozotocin at a dose of 65 m/kg, dissolved in citrate buffer. We used the water maze task and examined the acquisition and the retrieval of spatial memory in rats. In our present experiments, we observed that DM had no significant influence on acquisition and retrieval in 4 week diabetic rats on Morris water maze, but impaired examined parameters in 12 week diabetic rats in this test. The NMDA receptor agonist did not influence acquisition but increased recall on water maze in 12 week streptozotocin diabetic rats.

Effect of an NMDA receptor agonist on T-maze and passive avoidance test in 12-week streptozotocin-induced diabetic rats.

This study examined behavioral effects mediated by NMDA (N-methyl-D-aspartic acid) receptors in 12-week streptozotocin (STZ)-induced diabetic rats. Effects of an NMDA receptor agonist on behavior in the open field test, passive avoidance test and T-maze were examined in control groups of rats and in rats with diabetes mellitus (DM). We have used 116 rats for experiments. Experimental type I diabetes was induced by a single intravenous injection of streptozotocin at a dose of 65 mg/kg, dissolved in citrate buffer. Stimulation with the NMDA receptor agonist at a dose of 15 mg/kg was performed 30 min before the experiments. In control rats, NMDA increased the number of crossing and rearings in the open field test, improved acquisition and consolidation processes and did not influence recall in the passive avoidance situation and was ineffective in the T-maze. Diabetes significantly inhibited locomotor and exploratory activity and profoundly impaired acquisition, consolidation and recalling in a passive avoidance, and significantly decreased working memory in T-maze. NMDA treatment of diabetic rats significantly improved memory in passive avoidance and T-maze. The NMDA receptor agonist increased locomotor activity in open field test. The obtained results suggested that stimulation of NMDA receptors had beneficial effects on learning and memory in type I diabetic rats.

Influence of NMDA, a potent agonist of glutamate receptors, on behavioral activity in 4-week streptozotocin-induced diabetic rats.

The study was designed to investigate the effects of NMDA receptor agonist on the behavioral activity in rats with experimental diabetes mellitus (DM). Experimental diabetes was induced by a single intravenous injection of streptozotocin at a dose of 65 mg/kg dissolved in saline. Rats treated with saline (0.9%) served as control. Stimulation of the NMDA glutamatergic receptor was evoked by ip injection of an agonist N-methyl-D-aspartate acid (NMDA), at a dose of 15 mg/kg 30 min before the experiments. Memory motivated affectively was evaluated in the passive avoidance responses. Possible influence of the treatment on locomotor and exploratory activity was tested in open field test. Moreover, the working memory was evaluated in the T-maze test. We observed that NMDA given alone did not have significant influence on motor activity in control rats except for the number of bar approaches, while in rats with DM NMDA significantly increased motor activity in the open field test. In rats with experimental diabetes, NMDA increased acquisition, but it did not have any significant influence on consolidation and recall of a passive avoidance responses. NMDA at the tested dose had no influence on a passive avoidance latency in control rats. In the T-maze test, NMDA increased working memory but only in diabetic rats.