The urine metabolome differs between lean and overweight Labrador Retriever dogs during a feed-challenge.

Obesity in dogs is an increasing problem and better knowledge of the metabolism of overweight dogs is needed. Identification of molecular changes related to overweight may lead to new methods to improve obesity prevention and treatment. The aim of the study was firstly to investigate whether Nuclear Magnetic Resonance (NMR) based metabolomics could be used to differentiate postprandial from fasting urine in dogs, and secondly to investigate whether metabolite profiles differ between lean and overweight dogs in fasting and postprandial urine, respectively. Twenty-eight healthy intact male Labrador Retrievers were included, 12 of which were classified as lean (body condition score (BCS) 4-5 on a 9-point scale) and 16 as overweight (BCS 6-8). After overnight fasting, a voided morning urine sample was collected. Dogs were then fed a high-fat mixed meal and postprandial urine was collected after 3 hours. Metabolic profiles were generated using NMR and 45 metabolites identified from the spectral data were evaluated using multivariate data analysis. The results revealed that fasting and postprandial urine differed in relative metabolite concentration (partial least-squares discriminant analysis (PLS-DA) 1 comp: R2Y = 0.4, Q2Y = 0.32; cross-validated ANOVA: P = 0.00006). Univariate analyses of discriminant metabolites showed that taurine and citrate concentrations were elevated in postprandial urine, while allantoin concentration had decreased. Interestingly, lean and overweight dogs differed in terms of relative metabolite concentrations in postprandial urine (PLS-DA 1 comp: R2Y = 0.5, Q2Y = 0.36, cross-validated ANOVA: P = 0.005) but not in fasting urine. Overweight dogs had lower postprandial taurine and a trend of higher allantoin concentrations compared with lean dogs. These findings demonstrate that metabolomics can differentiate 3-hour postprandial urine from fasting urine in dogs, and that postprandial urine metabolites may be more useful than fasting metabolites for identification of metabolic alterations linked to overweight. The lowered urinary taurine concentration in overweight dogs could indicate alterations in lipid metabolism and merits further investigation.

In vitro biodegradation of cyanotoxins in the rumen fluid of cattle.

BACKGROUND: In countries around the Baltic Sea grazing ruminants have access to and drink, surface water from lakes, rivers and in several coastal regions. The water quality of these naturally occurring reservoirs affects performance and health of livestock. In the Baltic Sea both microcystin (MC) and nodularin (NOD) occurs as cyclic peptides and have hepatotoxic effects. Although cattle obviously have died after consuming contaminated water very little information is available as to how susceptible ruminants are to the toxins produced by cyanobacteria. The critical question as to whether the rumen microflora might constitute a protective shield is unresolved. For this reason our aim is to investigate a possible degradation rate of these toxins in rumen. RESULTS: The ability of rumen microorganisms to degrade certain important cyanotoxins (MC-LR, YR, RR and NOD) was studied in vitro by incubating with rumen fluid at three different concentrations (0.05, 0.5 and 5 µg/mL) for 3 h. The degradation efficiencies were determined by LC-MS (ESI) positive mode. Degradation was observed in the following order MC-RR 36%, NOD 35%, MC-RR 25% and MC-LR 8.9% at lower concentrations within 3 h. However, average degradation was observed at concentration of 0.5 µg/mL. No degradation was observed in higher concentrations for entire 3 h. The present results reveal that the degradation was both dose and time dependent. CONCLUSIONS: In conclusion the present results suggest that the rumen microbial flora may protect ruminants from being intoxicated by Cyanotoxins.

Protective effect of Actiniopteris radiata (Sw.) Link. against CCl4 induced oxidative stress in albino rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Actiniopteris radiata is a herb with great medicinal value and is evaluated for hepatoprotective activity. To investigate the protective effect of ethanolic extract of Actiniopteris radiata (EEAR) on CCl4 induced oxidative stress in male Wistar albino rats. MATERIALS AND METHODS: EEAR were administered for 8 consecutive weeks to rats. Group I - control; Group II - toxin control (30% CCl4); Group III and Group IV received EEAR (250 and 500 mg/kg respectively). Antioxidant status in liver were estimated by determining the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx); as well as by determining the levels of lipid peroxidation (LPO) and reduced glutathione (GSH). In addition, isoenzyme pattern and mRNA expression of the antioxidants were studied. Partial characterization of EEAR was performed by Liquid chromatography-mass spectrometry (LC-MS). RESULTS: CCl4 induced oxidative stress as evidenced from increase in LPO along with reduction of SOD, CAT, GPx and GSH. Treatment with EEAR (250 and 500 mg/kg) mitigated the CCl4 induced oxidative stress. An analysis of the isozyme pattern of these antioxidant enzymes revealed variations in SOD2, CAT, GPx2 and GPx3 in CCl4 treated rats, which were normalized after EEAR treatment. Furthermore, expression of genes for the antioxidant enzymes, were down-regulated by CCl4 treatment, which were reversed by EEAR. The results of partial characterization of EEAR by LC-MS revealed the presence of rutin and other 7 unknown phenolic derivatives. CONCLUSIONS: These findings suggest the protective effect of EEAR against CCl4 induced oxidative stress might be attributed to the presence of flavonoids and phenolic compounds.

The anorectic response to growth hormone in obese rats is associated with an increased rate of lipid oxidation and decreased hypothalamic galanin.

The aim of this study was to demonstrate differential effects of growth hormone (GH) on food intake in lean and obese rats and to investigate whether an anticipated anorectic response in obese rats might be associated with increased lipid oxidation and altered hypothalamic neuropeptide levels. GH (4 mg/kg/day) was administered during 5-21 days to non-obese and obese rats. Whereas GH stimulated food intake in the non-obese rats, the obese animals responded with a significantly (p<0.05) suppressed food intake for 4-5 days. On day 4, the obese rats injected with GH and those injected with vehicle consumed 9.2 ± 0.66 g and 12.7 ± 1.05 g, respectively. The suppression of food intake was associated with significantly (p<0.05) increased lipid oxidation. A similar, but statistically not verified, trend was seen in pair-fed rats not exposed to GH. However, while these animals appeared to economize their energy expenditure, the GH-exposed animals did not, thus creating a significant (p<0.05) difference between these two groups. The increased lipid oxidation and energy expenditure observed in the rats exposed to GH were associated with significantly (p<0.05) decreased levels of hypothalamic galanin (111 ± 33.2 pmol/g vs. those of the pair-fed controls: 228.5 ± 49.4 pmol/g). This difference was, however, not sustained. Thus, on day 21 both hypothalamic galanin and the food intake in the GH group were back to normal. Hypothalamic NPY remained unchanged by GH at all times. In conclusion, the present study suggests that increased lipid oxidation and decreased hypothalamic galanin are components in the mechanism by which GH inhibits food intake in an obese phenotype.

Effect of the cannabinoid receptor-1 antagonist rimonabant on lipolysis in rats.

The cannabinoid receptor 1 antagonist, rimonabant, reduces food intake and body weight, but contradictory findings have been reported as to whether the weight-reducing effect is fully accounted for by the reduced food intake or if rimonabant also mediates a lipolytic effect. In the present study, the effect on weight loss was studied in diet-induced obese rats after 3 days and 3 weeks of exposure to rimonabant, respectively. Induction of lipolysis was examined following acute administration and following 3 weeks of repeated dosing. Rimonabant-treated rats lost significantly more weight than their food-restricted controls. This effect was most pronounced in the beginning of the treatment period. No increase in lipolytic activity was found after 3 weeks of repeated dosing as measured by microdialysis in adipose tissue whereas acute administration of 10mg/kg produced a significant increase in microdialysate levels of glycerol illustrating an acute stimulation of lipolysis. No equivalent increase in glycerol was, however, observed in vitro following incubation of isolated rat adipocytes with rimonabant. This finding excludes a direct lipolytic action of rimonabant on tissue level. Instead, administration of 10mg/kg produced a significant increase in noradrenaline excretion in diet-induced obese rats, suggesting an increase in sympathoadrenal activity. In conclusion, the present study suggests an acute lipolytic effect of rimonabant mediated through activation of the sympathoadrenal system.

Growth hormone and growth hormone secretagogue effects on nitrogen balance and urea synthesis in steroid treated rats.

OBJECTIVES: Growth hormone (GH) reduces the catabolic side effects of steroid treatment via effects on the amino-nitrogen metabolism. Ipamorelin is a synthetic peptide with GH releasing properties. We wished to study the metabolic effects of Ipamorelin and GH on selected hepatic measures of alpha-amino-nitrogen conversion during steroid-induced catabolism. DESIGN: Five groups of rats were included: (1) free-fed controls (2) pair-fed controls (3) prednisolone (delcortol, 4 mg x kg(-1) x day(-1)) (4) prednisolone and GH (1 mg x kg(-1) x day(-1)) (5) prednisolone and Ipamorelin (0.5 mg x kg(-1) x day(-1)). After seven days the hepatic capacity of urea-N synthesis (CUNS) was determined in parallel with measurements of liver mRNA levels of urea cycle enzymes, whole-body N-balance, and N-contents of various organs. RESULTS: Compared to pair-fed controls, prednisolone increased CUNS (p<0.01) as well as the expression of urea cycle genes (p<0.01), and decreased N-balance (p<0.01) as well as organ N-contents (p<0.05). Compared to prednisolone treated animals, co-administration of GH reduced CUNS by 33% (p<0.01), normalized urea cycle gene expression, improved N-balance 2.5-fold, and normalized or improved organ N-contents. In prednisolone treated rats Ipamorelin reduced CUNS by 20% (p<0.05), decreased the expression of urea cycle enzymes, neutralised N-balance, and normalized or improved organ N-contents. CONCLUSION: Accelerated nitrogen wasting in the liver and other organs caused by prednisolone treatment was counteracted by treatment with either GH or its secretagogue Ipamorelin, though at the doses given less efficiently by the latter. This functional study of animals confirms that the GH secretagogue exerts GH related metabolic effects and may be useful in the treatment of steroid-induced catabolism.

Antagonistic targeting of the histamine H3 receptor decreases caloric intake in higher mammalian species.

The main purpose of this study was to examine the effects of a selective histamine H(3) receptor antagonist, NNC 38-1202, on caloric intake in pigs and in rhesus monkeys. The compound was given intragastrically (5 or 15 mg/kg), to normal pigs (n=7) and subcutaneously (1 or 0.1mg/kg) to obese rhesus monkeys (n=9). The energy intake recorded following administration of vehicle to the same animals served as control for the effect of the compound. In addition, rhesus monkey and pig histamine H(3) receptors were cloned from hypothalamic tissues and expressed in mammalian cell lines. The in vitro antagonist potencies of NNC 38-1202 at the H(3) receptors were determined using a functional GTPgammaS binding assay. Porcine and human H(3) receptors were found to have 93.3% identity at the amino acid level and the close homology between the monkey and human H(3) receptors (98.4% identity) was confirmed. The antagonist potencies of NNC 38-1202 at the porcine, monkey and human histamine H(3) receptors were high as evidenced by K(i)-values being clearly below 20 nM, whereas the K(i)-value on the rat H(3) receptor was significantly higher (56+/-6.0 nM). NNC 38-1202, given to pigs in a dose of 15 mg/kg, produced a significant (p<0.05) reduction (55%) of calorie intake compared with vehicle alone, (132.6+/-10.0 kcal/kgday versus 59.7+/-10.2 kcal/kgday). In rhesus monkeys administration of 0.1 and 1mg/kg decreased (p<0.05) average calorie intakes by 40 and 75%, respectively. In conclusion, the present study demonstrates that antagonistic targeting of the histamine H(3) receptor decreases caloric intake in higher mammalian species.

In vivo and in vitro microdialysis sampling of free fatty acids.

Microdialysis is a technique that allows continuous sampling of compounds from the interstitial fluid of different tissues with minimal influence on surrounding tissues and/or whole body function. In the present study, the feasibility of using microdialysis as a technique to sample free fatty acids (FFA) was investigated both in vitro and in vivo, by use of a high molecular weight (MW) cut-off membrane (3 MDa) and a push-pull system to avoid loss of perfusion fluid through the dialysis membrane. The relative recovery was examined in vitro for three different concentrations of radiolabelled oleic acid-BSA solutions (oleic acid:BSA molar ratio 1:1) and for various temperatures and flow rates. The recovery of oleic acid was found to be dependent on the concentration of analyte in the medium surrounding the membrane (17.3%, 29.0% and 30.6% for 50, 100 and 200 microM oleic acid-BSA solutions, respectively). Addition of 0.25% BSA to the perfusion fluid resulted, however, in a concentration-independent recovery of 31.4%, 28.1% and 28.1% for the 50, 100 and 200 microM solutions, respectively. The capability of the method to measure FFA together with glycerol was investigated in vivo in visceral adipose tissue of rats, before and after lipolytic treatment with the beta3-adrenergic agent, BRL37344. BRL37344 caused an increase in both dialysate FFA and glycerol, although the increase was markedly higher for glycerol, amounting to 24.5% and 329.2% increase from baseline, respectively. Subsequent in vitro test of probe performance revealed a decrease in the dialysing properties with regard to FFA, but not glycerol. This suggests that clogging of the membrane pores after 110 min prevented the measurement of the full FFA response in vivo.

Increase of neuronal histamine in obese rats is associated with decreases in body weight and plasma triglycerides.

OBJECTIVE: The purpose of the present study was to examine the metabolic effects of a specific histamine H(3) receptor antagonist, the cinnamic amide NNC 0038-0000-1202 (NNC 38-1202). RESEARCH METHODS AND PROCEDURES: Effects of NNC 38-1202 on paraventricular levels of histamine and acute effects on food intake were followed in normal rats, whereas effects on body weight homeostasis and lipid metabolism were studied in a rat model of diet-induced obesity (DIO). RESULTS: NNC 38-1202, administered as single oral doses of 15 and 30 mg/kg, significantly (p < 0.01) increased paraventricular histamine by 339 +/- 54% and 403 +/- 105%, respectively, compared with basal levels. The same doses produced significant (p < 0.01) reductions in food intake. In DIO rats receiving NNC 38-1202 in a daily dose of 5 mg/kg for 22 days, a decrease in food intake was associated with a significant (p < 0.001) net loss of body weight (-11.0 +/- 4.8 grams), compared with rats receiving vehicle, which gained 13.6 +/- 3.0 grams. Also, NNC 38-1202 significantly (p < 0.05) reduced plasma triglycerides by approximately 42%, in parallel with increases in plasma free fatty acids and beta-hydroxybutyrate levels. Despite reductions in food intake and body weight following administration of NNC 38-1202, no sign of a decrease in energy expenditure was observed, and whole-body lipid oxidation was significantly (p < 0.05) increased in the period after dosing. DISCUSSION: The present study suggests that antagonistic targeting of the histamine H(3) receptor decreases food intake, body weight, and plasma TG levels and, thus, represents an interesting approach to treatment of obesity and associated hyperlipidemia.

Locomotion is the major determinant of sibutramine-induced increase in energy expenditure.

The anti-obesity effect of the serotonin and noradrenaline reuptake inhibitor sibutramine has been attributed to a dual mechanism involving a reduction of food intake and an increase in energy expenditure. This dual action increases the possibilities for induction of a negative energy balance, the principal goal of an anti-obesity treatment. To elucidate the mechanism behind sibutramine-induced increase in energy expenditure, we applied indirect calorimetry combined with monitoring locomotor activity and body temperature. We confirm that sibutramine has both anorectic and thermogenic effects. In addition, we show here that sibutramine also causes a dose-dependent increase in locomotor activity (LMA) of rats, occurring in parallel with increase in energy expenditure. The dose of sibutramine necessary to induce an effect on locomotion and energy expenditure was only marginally higher than the dose sufficient to induce a significant reduction of food intake. The relation between LMA and energy expenditure was similar to that found with d-amphetamine, which causes both hyper-locomotion and increased energy expenditure, but was different from 2,4-dinitrophenol which causes increase in energy expenditure but not in locomotion. The effect of sibutramine (20 mg/kg) on energy expenditure was not inhibited by the non-selective 5-HT receptor antagonist, metergoline (1 mg/kg), or a high dose (20 mg/kg) of the non-selective beta-blocker propranolol, but was blocked by D1 dopamine receptor inhibitor SCH 23390 (0.3 mg/kg). Therefore, we conclude that the effect of sibutramine on energy expenditure in rats is predominantly due to a dopamine-dependent increase in locomotor activity.

Treatment of Obesity Using GH.

The obesity epidemic calls for complementary treatment possibilities in addition to lifestyle changes. One of the important regulators of lipid homeostasis is growth hormone (GH). Clinical trials have tested if GH can reduce obesity in humans. The mechanisms underlying the response to GH administration have also been investigated in animal models of human obesity. A literature search yielded 19 randomized placebo-controlled clinical studies and several animal studies investigating chronic GH treatment of obesity. Significant effects were found in some of the larger trials. One clinical trial showed significantly increased weight loss due to GH treatment, and in seven trials, a significant reduction of fat mass was found. The improvements observed were modest, but even minor improvements have been shown to be beneficial, especially if the reduction in fat mass includes visceral adipose tissue, as was reported in three of six trials. In principle, animal data support the clinical observations although the reduction of fat mass was more dramatic than observed in humans. The mechanisms resulting in lipid mobilization most likely include adipose tissue lipo-protein lipase (LPL) inhibition and antagonization of the anti-lipolytic activity of insulin. By feeding a restricted amount of a high fat diet to GH exposed rats hyper-insulinemia was avoided, loss of body fat was accelerated and metabolic markers were improved. Provision of a diet suitable for the metabolic conditions during GH treatment shows promise for improving metabolic control and can perhaps increase the efficacy and/or widen the therapeutic window of GH.

Growth hormone affects both adiposity and voluntary food intake in old and obese female rats.

OBJECTIVE: To investigate whether the promotion of breakdown of body fat and the increased energy expenditure associated with growth hormone (GH) affect the voluntary food intake of an obese organism. DESIGN: Wistar rats (15 months old) were first fed either a high-fat (HF) or a low-fat (LF) diet for 10 weeks. In the subsequent treatment period, two saline groups continued with either the HF or the LF diet, and rats of three other groups had their diet shifted from HF to LF and were treated with saline, human GH (hGH) or rat GH (rGH). hGH and rGH were given in a dose of 4 mg/kg per day. After 21 days of treatment and registration of food intake, rats were killed, blood was collected and tissues were excised. RESULTS: The HF diet produced a significant (P<0.05) increase in weight of fat pads compared with the LF diet: 69+/-5 g compared with 48+/-2 g. The switch from HF to LF diet combined with injections of saline alone decreased the intake of metabolizable energy, but fat pad weight did not decrease significantly (69+/-5 g compared with 63+/-6 g). The latter value was significantly (P<0.05) decreased (to 37+/-3 g) in groups treated with either hGH or rGH. Both GH treatments increased serum IGF-I and muscle weight, whereas the activity of adipose tissue lipoprotein lipase decreased significantly (P<0.01). During the first 9 days of treatment, food intake was significantly (P<0.01) depressed, from 27+/-1 g/kg per day in control rats to 14+/-2 and 16+/-4 g/kg per day in the hGH and rGH groups respectively. CONCLUSION: This study demonstrates that breakdown of adipose tissue and a transient decrease in voluntary food intake are parallel consequences of GH treatment in old and obese rats, and that the actions of hGH and rGH are very similar.

Reliability of the western ligand blot method for the simultaneous relative estimations of serum insulin-like growth factor binding proteins (IGFBPs) / Confiabilidad del método de \"western ligand blot\" para la determinación simultánea de las proteínas de unión de los factores de crecimiento similares a la insulina (IGFBPs) en suero

It is well established that nutrition is an important regulator of both serum insulin-like growth factor-I (IGF) and its binding proteins (IGFBPs). The Western ligand blot method (WLB) for simultaneous determinations of IGFBPs in serum or plasma sambples was evaluated and validated with emplasis on its reproducible capabilities. After electrophoretic separation and transfer, the membranes were incubated with a mixture of recombinant labeled human (GF-I/IGF-II(rhIGF-II) and band intensities measured by autoradiography. The typical electrophoretic profile for pig serum, as determined with molecular weight markers, showed four mainbands of approximately 42-39,32,30-28 and 24 kDa which seemed to correspond to IGFBP-3, IGFBP-2, IGFBP-1 and IGFBP-4 respectively. Likewise, a trinlet of approximately 42-39 kDa (IGFBP-3), a broad area called OGFBP-30 region (most probably IGFBP-1, -2 and -3 variants) and a third band of 24 kDa IGFBP-4) were seen in rat samples. Determination of IGFBP/2 and 1 in rat serum samples, as two separate bands on 12 por ciento gels was difficult due to their close electrophoretic migration and possibly to the reported lower levels of IGFBP-2 in adult rat serum. Dilutions tested on 0.2 µm nitrocellulose membranes with samples volumes between 0.25 to 1.5 µl(1:10-1:60 dilutions), showed IGFBPs curves with good linearity which suggest first, that there exist a quantitative relation betweem the amount of each protein and densitometric response and second, that the transfer of the proteins was linear across the range of 0.25 to 1.5 µl(1:10-1:60 dilutions). Moreover, the results also suggest that losses were aqually spread and that the proteins retained their binding pro[erties after the process. Reproducibility showed intra-assay coefficients of variation (CVs) of 15 per cent or lower using either a transfer device without cooling system or a combination of a transferdevice with cooling system and manually defined band boundaries. In summary, it was shown that the optimized experimental conditions here described for the WLB method, allow realiable simultaneous measurements of the main pig and rat serum IGFBPs and therefore, could be utilised to detect changes in the profile after dietary manipulations