Nitric oxide and the release of lipoprotein lipase from white adipose tissue

Background/Aim: Lipoprotein lipase (LPL) is the main enzyme responsible for the distribution of circulating triacylglycerides in tissues. Its regulation via release from active sites in the vascular endothelium is poorly understood. In a previous study we reported that in response to acute immobilization (IMMO), LPL activity rapidly increases in plasma and decreases in white adipose tissue (WAT) in rats. In other stress situations IMMO triggers a generalized increase in nitric oxide (NO) production. Methods/Results: Here we demonstrate that in rats: 1) in vivo acute IMMO rapidly increases NO concentrations in plasma 2) during acute IMMO the WAT probably produces NO via the endothelial isoform of nitric oxide synthase (eNOS) from vessels, and 3) epididymal WAT perfused in situ with an NO donor rapidly releases LPL from the endothelium. Conclusion: We propose the following chain of events: stress stimulus / rapid increase of NO production in WAT (by eNOS) / release of LPL from the endothelium in WAT vessels. This chain of events could be a new mechanism that promotes the rapid decrease of WAT LPL activity in response to a physiological stimulus.

Metabolic alterations and increased liver mTOR expression precede the development of autoimmune disease in a murine model of lupus erythematosus

Although metabolic syndrome (MS) and systemic lupus erythematosus (SLE) are often associated, a common link has not been identified. Using the BWF1 mouse, which develops MS and SLE, we sought a molecular connection to explain the prevalence of these two diseases in the same individuals. We determined SLE- markers (plasma anti-ds-DNA antibodies, splenic regulatory T cells (Tregs) and cytokines, proteinuria and renal histology) and MS-markers (plasma glucose, non-esterified fatty acids, triglycerides, insulin and leptin, liver triglycerides, visceral adipose tissue, liver and adipose tissue expression of 86 insulin signaling-related genes) in 8-, 16-, 24-, and 36-week old BWF1 and control New-Zealand-White female mice. Up to week 16, BWF1 mice showed MS-markers (hyperleptinemia, hyperinsulinemia, fatty liver and visceral adipose tissue) that disappeared at week 36, when plasma anti-dsDNA antibodies, lupus nephritis and a pro-autoimmune cytokine profile were detected. BWF1 mice had hyperleptinemia and high splenic Tregs till week 16, thereby pointing to leptin resistance, as confirmed by the lack of increased liver P-Tyr-STAT-3. Hyperinsulinemia was associated with a down-regulation of insulin related-genes only in adipose tissue, whereas expression of liver mammalian target of rapamicyn (mTOR) was increased. Although leptin resistance presented early in BWF1 mice can slow-down the progression of autoimmunity, our results suggest that sustained insulin stimulation of organs, such as liver and probably kidneys, facilitates the over-expression and activity of mTOR and the development of SLE.

Nitrogen metabolism in Zucker rats is affected by moderate reduction, but not by moderate incerase in dietary protein

Marked changes in the content of protein in the diet affects the rat"s pattern of growth, but there is not any data on the effects to moderate changes. Here we used a genetically obese rat strain (Zucker) to examine the metabolic modifications induced to moderate changes in the content of protein of diets, doubling (high-protein (HP): 30%) or halving (low-protein (LP): 8%) the content of protein of reference diet (RD: 16%). Nitrogen, energy balances, and amino acid levels were determined in lean (L) and obese (O) animals after 30 days on each diet. Lean HP (LHP) animals showed higher energy efficiency and amino acid catabolism but maintained similar amino acid accrual rates to the lean RD (LRD) group. Conversely, the lean LP (LLP) group showed a lower growth rate, which was compensated by a relative increase in fat mass. Furthermore, these animals showed greater efficiency accruing amino acids. Obesity increased amino acid catabolism as a result of massive amino acid intake; however, obese rats maintained protein accretion rates, which, in the OHP group, implied a normalization of energy efficiency. Nonetheless, the obese OLP group showed the same protein accretion pattern as in lean animals (LLP). In the base of our data, concluded that the Zucker rats accommodate their metabolism to support moderates increases in the content of protein in the diet, but do not adjust in the same way to a 50% decrease in content of protein, as shown by an index of growth reduced, both in lean and obese rats.

Modulation in Wistar rats of blood corticosterone compartmentation by sex and prior exposure to a cafeteria diet

In the metabolic syndrome, glucocorticoid activity is increased, but circulating levels show little change. Most of blood glucocorticoids are bound to corticosteroid-binding globulin (CBG), which liver expression and circulating levels are higher in females than in males. Since blood hormones are also bound to blood cells, and the size of this compartment is considerable for androgens and estrogens, we analyzed whether sex or eating a cafeteria diet altered the compartmentation of corticosterone in rat blood. The main corticosterone compartment in rat blood is that specifically bound to plasma proteins, with smaller compartments bound to blood cells or free. Cafeteria diet increased the expression of liver CBG gene, binding plasma capacity and the proportion of blood cell-bound corticosterone. There were marked sex differences in blood corticosterone compartmentation in rats, which were unrelated to testosterone. The use of a monoclonal antibody ELISA and a polyclonal Western blot for plasma CBG compared with both specific plasma binding of corticosterone and CBG gene expression suggested the existence of different forms of CBG, with varying affinities for corticosterone in males and females, since ELISA data showed higher plasma CBG for males, but binding and Western blot analyses (plus liver gene expression) and higher physiological effectiveness for females. Good cross- reactivity to the antigen for polyclonal CBG antibody suggests that in all cases we were measuring CBG.The different immunoreactivity and binding affinity may help explain the marked sex-related differences in plasma hormone binding as sex-linked different proportions of CBG forms.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Depression-like behavior is dependent on age in male SAMP8 mice

Aging is associated with an increased risk of depression in humans. To elucidate the underlying mechanisms of depression and its dependence on aging, here we study signs of depression in male SAMP8 mice. For this purpose, we used the forced swimming test (FST). The total floating time in the FST was greater in SAMP8 than in SAMR1 mice at 9 months of age; however, this difference was not observed in 12-month-old mice, when both strains are considered elderly. Of the two strains, only the SAMP8 animals responded to imipramine treatment. We also applied the dexamethasone suppression test (DST) and studied changes in the dopamine and serotonin (5-HT) uptake systems, the 5-HT2a/2c receptor density in the cortex, and levels of TPH2. The DST showed a significant difference between SAMR1 and SAMP8 mice at old age. SAMP8 exhibits an increase in 5-HT transporter density, with slight changes in 5-HT2a/2c receptor density. In conclusion, SAMP8 mice presented depression-like behavior that is dependent on senescence process, because it differs from SAMR1, senescence resistant strain.

A radiochemical method for carbamoyl-phosphate synthetase I: application to rats fed a hyperproteic diet

A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium was that incorporated into carbamoyl-phosphate, since all 14C-CO2 from bicarbonate was eliminated. The method is rapid and requires only a low pressure supply of CO2 to remove the excess substrate. The reaction is linear up to 10 min using homogenate dilutions of 1:20 to 1:200 (w/v). Rat liver activity was in the range of 89±8 nkat/g. Hyperproteic diet resulted in a significant 1.4-fold increase. The design of the method allows for the processing of multiple samples at the same time, and incubation medium manipulation is unnecessary, since the plastic incubation vial and its contents are finally counted together.

Evaluation of a piezoelectric system as an alternative to electroencephalogram/ electromyogram recordings in mouse sleep studies.

STUDY OBJECTIVES: Traditionally, sleep studies in mammals are performed using electroencephalogram/electromyogram (EEG/EMG) recordings to determine sleep-wake state. In laboratory animals, this requires surgery and recovery time and causes discomfort to the animal. In this study, we evaluated the performance of an alternative, noninvasive approach utilizing piezoelectric films to determine sleep and wakefulness in mice by simultaneous EEG/EMG recordings. The piezoelectric films detect the animal's movements with high sensitivity and the regularity of the piezo output signal, related to the regular breathing movements characteristic of sleep, serves to automatically determine sleep. Although the system is commercially available (Signal Solutions LLC, Lexington, KY), this is the first statistical validation of various aspects of sleep. DESIGN: EEG/EMG and piezo signals were recorded simultaneously during 48 h. SETTING: Mouse sleep laboratory. PARTICIPANTS: Nine male and nine female CFW outbred mice. INTERVENTIONS: EEG/EMG surgery. MEASUREMENTS AND RESULTS: The results showed a high correspondence between EEG/EMG-determined and piezo-determined total sleep time and the distribution of sleep over a 48-h baseline recording with 18 mice. Moreover, the piezo system was capable of assessing sleep quality (i.e., sleep consolidation) and interesting observations at transitions to and from rapid eye movement sleep were made that could be exploited in the future to also distinguish the two sleep states. CONCLUSIONS: The piezo system proved to be a reliable alternative to electroencephalogram/electromyogram recording in the mouse and will be useful for first-pass, large-scale sleep screens for genetic or pharmacological studies. CITATION: Mang GM, Nicod J, Emmenegger Y, Donohue KD, O'Hara BF, Franken P. Evaluation of a piezoelectric system as an alternative to electroencephalogram/electromyogram recordings in mouse sleep studies.

Beaming into the rat world: enabling real-time intereaction between rat and human each at their own scale

Immersive virtual reality (IVR) typically generates the illusion in participants that they are in the displayed virtual scene where they can experience and interact in events as if they were really happening. Teleoperator (TO) systems place people at a remote physical destination embodied as a robotic device, and where typically participants have the sensation of being at the destination, with the ability to interact with entities there. In this paper, we show how to combine IVR and TO to allow a new class of application. The participant in the IVR is represented in the destination by a physical robot (TO) and simultaneously the remote place and entities within it are represented to the participant in the IVR. Hence, the IVR participant has a normal virtual reality experience, but where his or her actions and behaviour control the remote robot and can therefore have physical consequences. Here, we show how such a system can be deployed to allow a human and a rat to operate together, but the human interacting with the rat on a human scale, and the rat interacting with the human on the rat scale. The human is represented in a rat arena by a small robot that is slaved to the human"s movements, whereas the tracked rat is represented to the human in the virtual reality by a humanoid avatar. We describe the system and also a study that was designed to test whether humans can successfully play a game with the rat. The results show that the system functioned well and that the humans were able to interact with the rat to fulfil the tasks of the game. This system opens up the possibility of new applications in the life sciences involving participant observation of and interaction with animals but at human scale.

Beaming into the rat world: enabling real-time intereaction between rat and human each at their own scale

Immersive virtual reality (IVR) typically generates the illusion in participants that they are in the displayed virtual scene where they can experience and interact in events as if they were really happening. Teleoperator (TO) systems place people at a remote physical destination embodied as a robotic device, and where typically participants have the sensation of being at the destination, with the ability to interact with entities there. In this paper, we show how to combine IVR and TO to allow a new class of application. The participant in the IVR is represented in the destination by a physical robot (TO) and simultaneously the remote place and entities within it are represented to the participant in the IVR. Hence, the IVR participant has a normal virtual reality experience, but where his or her actions and behaviour control the remote robot and can therefore have physical consequences. Here, we show how such a system can be deployed to allow a human and a rat to operate together, but the human interacting with the rat on a human scale, and the rat interacting with the human on the rat scale. The human is represented in a rat arena by a small robot that is slaved to the human"s movements, whereas the tracked rat is represented to the human in the virtual reality by a humanoid avatar. We describe the system and also a study that was designed to test whether humans can successfully play a game with the rat. The results show that the system functioned well and that the humans were able to interact with the rat to fulfil the tasks of the game. This system opens up the possibility of new applications in the life sciences involving participant observation of and interaction with animals but at human scale.

Effects of a post-weaning cafeteria diet in young rats: metabolic syndrome, reduced activity and low anxiety-like behaviour.

Among adolescents, overweight, obesity and metabolic syndrome are rapidly increasing in recent years as a consequence of unhealthy palatable diets. Animal models of diet-induced obesity have been developed, but little is known about the behavioural patterns produced by the consumption of such diets. The aim of the present study was to determine the behavioural and biochemical effects of a cafeteria diet fed to juvenile male and female rats, as well as to evaluate the possible recovery from these effects by administering standard feeding during the last week of the study. Two groups of male and female rats were fed with either a standard chow diet (ST) or a cafeteria (CAF) diet from weaning and for 8 weeks. A third group of males (CAF withdrawal) was fed with the CAF diet for 7 weeks and the ST in the 8th week. Both males and females developed metabolic syndrome as a consequence of the CAF feeding, showing overweight, higher adiposity and liver weight, increased plasma levels of glucose, insulin and triglycerides, as well as insulin resistance, in comparison with their respective controls. The CAF diet reduced motor activity in all behavioural tests, enhanced exploration, reduced anxiety-like behaviour and increased social interaction; this last effect was more pronounced in females than in males. When compared to animals only fed with a CAF diet, CAF withdrawal increased anxiety in the open field, slightly decreased body weight, and completely recovered the liver weight, insulin sensitivity and the standard levels of glucose, insulin and triglycerides in plasma. In conclusion, a CAF diet fed to young animals for 8 weeks induced obesity and metabolic syndrome, and produced robust behavioural changes in young adult rats, whereas CAF withdrawal in the last week modestly increased anxiety, reversed the metabolic alterations and partially reduced overweight.