Screening of dipeptides having central functions for excitation and sedation.

The naturally-occurring dipeptide carnosine (beta-alanyl-L-histidine) and the tripeptide glutathione (L-gammaglutamyl-L-cysteinylglycine) are found extensively in animal tissues such as brain and skeletal muscle. Central functions for excitation and sedation of them and their derivatives were screened.

Central administration of corticotropin-releasing factor induces thermogenesis by changes in mitochondrial bioenergetics in neonatal chicks.

Corticotropin-releasing factor (CRF), has multiple biological effects and plays a central regulatory role in the hypothalamic-pituitary-adrenal (HPA) axis regulating energy homeostasis that is required for adaptive responses to maintain and support life. Central administration of CRF increases O(2) consumption, CO(2) and heat production resulting in hyperthermia. To determine the precise mechanism for this condition, here we investigated transcripts of candidate genes for thermogenesis and their up-regulator (avian uncoupling protein (avUCP), avian adenine nucleotide translocator (avANT) and avian peroxisome-proliferator-activated receptor-gamma co-activator-1alpha) and mitochondrial bioenergetics (gene transcripts for mitochondrial fatty acid (FA) transport and oxidation enzymes; carnitine-palmitoyl-transferase (CPT)-I; CPT-II, long-chain acyl CoA dehydrogenase (LCAD), 3-hydroxyacyl CoA dehydrogenase (3HADH) and citrate synthase (CS), and enzyme activities of 3HADH and CS) that might explain the bioenergetic basis of CRF-induced increased thermogenesis. Neonatal chicks (Gallus gallus) with and without i.c.v. injection of CRF (42 pmol) were kept at thermoneutral temperature (30 degrees C) for 3 h. Central administration of CRF increased the core temperature and plasma NEFA level of chicks compared with the control. This CRF-induced increased thermogenesis was not accompanied by enhancement of avUCP and/or avANT gene transcripts and was associated with increased FA oxidation in tissue specific manner: increase in gene transcript levels of CPT-I, CPT-II, LCAD, 3HADH and CS, and increase in enzyme activities of 3HADH and CS were observed in liver and heart while no such changes were observed in skeletal muscle. In conclusion, these results suggest that CRF-induced increased thermogenesis in neonatal chicks was not accompanied by enhancement of gene transcripts of mitochondrial putative thermogenic proteins, and was induced by tissue specific increase in mitochondrial FA transport and beta-oxidation enzymes.

Effect of Garcinia cambogia extract on serum leptin and insulin in mice.

In this study we examined the effects of 3.3% Garcinia cambogia extract on 10% sucrose loading in mice for 4 weeks. Treatment was found to have no effect on body weight, fat pad weight or serum glucose level. On the other hand, serum total cholesterol, triglycerides, NEFA were observed. Levels of serum insulin and leptin, as well as the leptin/WAT ratio, were lower in the treated mice than in the control. These findings suggested that G. cambogia extract efficiently improved glucose metabolism and displayed leptin-like activity.

Intracerebroventricular injection of glucagon-like peptide-1 decreases monoamine concentrations in the hypothalamus of chicks.

1. We measured the concentrations of dopamine (DA), norepinephrine (NE), epinephrine (E) and 5-hydroxytryptamine (5-HT) in the hypothalamus of 21-d-old male brown-egg layer-type chicks after intracerebroventricular injection of glucagon-like peptide-1 (GLP-1). 2. The monoamine concentrations of the whole hypothalamus, paraventricular nucleus and lateral hypothalamic area were not significantly affected by GLP-1. 3. However, concentrations of DA, NE and E, but not 5-HT, in the ventromedial hypothalamic nucleus (VMH) were significantly decreased by GLP-1. 4. These observations suggest that the anorexigenic effect of GLP-1 involves catecholaminergic systems in the VMH in the chick.

Induction of food intake by a noradrenergic system using clonidine and fusaric acid in the neonatal chick.

To clarify noradrenergic systems on food intake of the neonatal chicks, we examined the effects of i.c.v injection of clonidine (CLON), an alpha2-receptor agonist, and fusaric (5-butylpicolinic) acid (FA), a dopamine (DA)-beta-hydroxylase (DBH) inhibitor. Although a high dose (250 ng) of CLON induced a narcoleptic response and reduced food intake, food intake at 30 min post-injection was enhanced by lower doses (25 and 50 ng) of CLON. Central administration of FA (25, 50 and 100 microg) increased food intake in a dose-dependent fashion. It is suggested that feeding behavior is stimulated by low levels of CLON and decreased by further production of norepinephrine (NE), and FA may play the disturbance of sleeping and then enhance food intake.

Effects of dietary cholestyramine on the utilisation of diets containing medium or long chain triacylglycerols by chicks.

1. The effects of graded dietary concentrations of cholestyramine (CSTY, a bile acid binding polymer), which prevents micelle formation and bile acid reabsorption, on the lipid and energy metabolism of chicks given diets containing different dietary concentrations of medium chain triacylglycerol (MCT) and long chain triacylglycerol (LCT) were investigated. 2. MCT- or LCT-supplemented diets containing 100 or 200 g oil/kg diet and 0, 10 or 20 g CSTY kg were fed to 7 d old chicks for 10 d. As dietary CSTY concentration increased, a reduction in the metabolisable energy value was observed for both dietary lipid sources. Consequently, fat and energy retentions were also reduced as the dietary CSTY content increased.

[Functional MRI in search of basal ganglia].

We investigated activation of the globus pallidus using conventional gradient echo sequence with a 1.5 tesla equipment. In accordance with complex finger tapping in the left hand, an increase in signal intensity from bilateral globus pallidus was attained, being grossly predominated on right side. On the other hand, activations in primary motor cortex were elicited mainly on right side, thereby being correctly accorded in attitude of on-off changes in signal intensity. fMRI in basal ganglia faces severer condition in detecting real activation, owing to influences to signal recording by ventricular pulsation, accumulation of iron by age, and differences in activation by the tasks applied. This study, however, suggests that fMRI of basal ganglia is a promising technique for mapping brain function in regard to correlations between cerebral cortex and deep structures.

Intracerebroventricular injection of mammalian and chicken glucagon-like peptide-1 inhibits food intake of the neonatal chick.

Glucagon-like peptide-1 (GLP-1), structurally similar to glucagon, is synthesized from a larger precursor, preproglucagon, and has been postulated to be a novel incretin. Recently, it was reported that central administration of GLP-1 decreased food intake in rats. The amino acid sequences of GLP-1 are identical in all mammals, and chicken GLP-1 exhibits a high homology with mammalian GLP-1. The aim of this study was to elucidate whether central injection of mammalian or chicken GLP-1 inhibits food intake in the chick, and to compare their effects. Intracerebroventricular administration of mammalian and chicken GLP-1 strongly inhibited food intake of chicks. However, the suppressive effect of both GLP-1 on food intake was similar. These results show that GLP-1 with a variety of amino acid sequences may be the most potent inhibitor of food intake in the chicken.

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

Dynamics of cerebral blood flow and metabolism in patients with cranioplasty as evaluated by 133Xe CT and 31P magnetic resonance spectroscopy.

OBJECTIVE: Prolonged improvement in neurological and mental disorders has been seen after only cranioplasty in patients initially treated with external decompression for high intracranial pressure. The objective was to evaluate, using 133Xe CT and 31P magnetic resonance spectroscopy (MRS), how restoring the bone itself can influence cerebral blood flow and cerebral energy metabolism after high intracranial pressure is attenuated. METHODS: Seven patients (45-65 years old) who had undergone external decompression to prevent uncontrollable intracranial hypertension after acute subarachnoid haemorrhage were evaluated. Cerebral blood flow and metabolic changes were evaluated before and after cranioplasty. RESULTS: The ratio of phosphocreatine to inorganic phosphate (PCr/Pi), which is a sensitive index of cerebral energy depletion, was calculated and beta-ATP was measured. The cerebral blood flow value in the thalamus was normalised, from 44 (SD 9) to 56 (SD 8) ml/100 g/min (P < 0.01) and the value in the hemisphere increased from 26 (SD 3) to 29 (SD 4) ml/100 g/min on the side with the bone defect. The PCr/Pi ratio improved greatly from 2.53 (SD 0.45) to 3.01 (SD 0.24) (P < 0.01). On the normal side, the values of cerebral blood flow and PCr/Pi increased significantly (P < 0.01) after cranioplasty, possibly due to transneural suppression. The pH of brain tissue was unchanged bilaterally after cranioplasty. CONCLUSION: Cranioplasty should be carried out as soon as oedema has disappeared, because a bone defect itself may decrease cerebral blood flow and disturb energy metabolism.

Involvement of (n-6) essential fatty acids and prostaglandins in liver lipid accumulation in Japanese quail.

OBJECTIVE: To investigate the involvement of (n-6) essential fatty acids, such as linoleic acid [18:2(n-6)] or gamma-linolenic acid [18:3(n-6)], and of prostaglandins on liver lipid accumulation in Japanese quail. DESIGN: Effects of graded amounts of aspirin, which inhibits prostaglandin synthesis, on liver weight were determined in experiment 1. Experiment 2 was designed to clarify the effect of dietary essential fatty acid sources and inhibition of prostaglandin synthesis on the liver fat and fatty acid profile. ANIMALS: Female Japanese quail. PROCEDURE: In experiment 1, from 1 to 3 weeks of age, birds were fed ad libitum the essential fatty acids-free or linoleic acid-adequate (2%) diets with graded amounts of aspirin (0, 0.1, 0.2, and 0.4%). In experiment 2, from 1 to 4 weeks of age, birds were fed the same amount of essential fatty acids-free, linoleic acid-adequate, or gamma-linolenic acid (0.4%) diets with (0.2%) or without aspirin. RESULTS: In experiment 1, in groups given the essential fatty acids-free diet, liver weight increased with an increase in dietary aspirin concentration. In experiment 2, gamma-linolenic acid completely prevented liver triacylglycerol and cholesterol accumulation induced by the essential fatty acids-free diet. Aspirin treatment significantly lowered plasma prostaglandin F2 alpha concentration, but did not affect liver lipid concentrations. In groups fed the essential fatty acids-free diets, however, aspirin treatment increased liver weight and liver triacylglycerol concentration by 20 and 40%, respectively. CONCLUSIONS: gamma-Linolenic acid or its metabolites, but not linoleic acid itself, are important factors in reducing fatty liver in Japanese quail with the essential fatty acids-deficient condition.

Role of dietary gamma-linolenic acid in liver lipid metabolism in Japanese quail.

1. The importance of linoleic acid (18:2n-6) itself and of dietary gamma-linolenic acid (18:3n-6) as essential fatty acids (EFA) in Japanese quail were investigated with regard to liver lipid metabolism. Experimental diets were made by adding of 0, 2 or 4 g gamma-linolenic acid/kg, or 20 g linoleic acid/kg to an n-6 EFA-free diet. From 3 to 6 weeks of age, birds were fed equal amounts of experimental diets. 2. Liver weight and lipid content in birds fed the 2 and 4 g gamma-linolenic acid/kg diet were significantly lower than those in birds fed the gamma-linolenic acid-free diet. However, no significant difference was observed between the gamma-linolenic acid- and linoleic acid-supplemented diets. 3. In birds fed the 4 g gamma-linolenic acid/kg diet, the proportion of arachidonic acid in the liver lipid was similar to that in quail fed the 20 g linoleic acid/kg diet, implying a conversion rate from linoleic acid to gamma-linolenic acid of approximately 20% of whole body content. 4. It is concluded that linoleic acid itself is not essential for Japanese quail and that at least 2 g/kg of gamma-linolenic acid in the diet completely prevents liver enlargement accompanied by lipid accumulation.

Improvement of dietary protein utilisation in chicks by medium chain triglyceride.

1. To improve the influence of variation of ME intake, an experiment was carried out using equalized feeding to investigate the comparative effects on protein and energy utilisation in chicks of diets containing medium chain triglyceride (MCT) and long chain triglyceride (LCT). Experimental diets were given at 3 different food intakes, namely, 100, 120 or 147 g/bird/10 d. The diets contained MCT or LCT on an isoenergetic basis. Maize oil and caprylic acid triglyceride respectively, were used as LCT and MCT sources. 2. Body weight gain and food efficiency of chicks significantly increased with the supplement of dietary MCT compared with dietary LCT at all food intakes. Protein retention and the efficiency of protein utilisation (protein retained/protein intake) at all food intakes also significantly increased with dietary MCT, while body fat and fat retention were significantly reduced. Chicks fed the LCT-supplemented diet, on the other hand, had a lower protein retention, but significantly higher fat retention. The value for energy retention and the efficiency of energy utilisation (energy retained/ME intake) were not significantly different between MCT- and LCT-supplemented diets. 3. It was concluded that supplementing MCT to the chick diet would improve body weight gain and protein utilisation while regulating fat deposition compared to the LCT supplemented diet, under equalized feeding conditions.

Feeding behaviour in chickens given diets containing medium chain triglyceride.

1. The effect of dietary medium chain triglyceride (MCT) on short-term food intake was compared with the effect of long chain triglyceride (LCT) in chickens. Maize oil was used as the LCT while glyceryl tricaprylate (C 8) and glyceryl tricaprate (C 10) were used as MCT. Cumulative food intake was determined during the 6 h after the start of feeding. 2. Chicks were given diets containing 200 g C 8/kg diet, 200 g C 10/kg diet or 200 g LCT/kg diet in experiment 1. As early as 30 min after feeding, cumulative food intake in both MCT-supplemented diets decreased significantly compared with the diet containing LCT. 3. To determine if endogenous cholecystokinin (CCK) was responsible for the decrease in food intake caused by MCT, birds were injected with the CCK-A receptor antagonist devazepide (DVZ, 1 mg/kg BW) before diet presentation. DVZ had no effect on food intake with either LCT- or MCT-supplemented diets. 4. In experiment 3, chicks were given a choice between either diets containing LCT and C 8, LCT and C 10, or C 8 and C 10 to confirm whether or not the palatability of the diets was influenced by the dietary fat sources. There was no difference in food intake between C 8 and C 10-supplemented diets. However, chicks preferred the LCT-supplemented diet compared with either of the diets containing MCT.

Mineral balance and bone histology in spontaneously hypertensive rats.

SHRs (9 weeks old) were fed diets with or without extra NaCl (2%) and were given water or saline (1%) for 7 weeks. Food and liquid intakes were measured weekly. Blood pressure was determined at 9, 11, 13, 15, and 16 weeks of age. The values for body weight gain and food intake were not influenced by any treatment. Liquid intake greatly increased with the surplus of dietary NaCl. The blood pressure increased with age in all treatments. The increase in blood pressure was enhanced by the addition of NaCl to diet and water. Urinary potassium and calcium excretions were enhanced by added dietary NaCl and saline. Calcium content in the femoral bone was not changed by any treatment, although the number of osteoclast and the area of bone marrow were increased by saline supplementation.

Energy utilisation of medium chain triglyceride in comparison with long chain triglyceride in growing chicks.

1. To evaluate the relative efficiency of energy utilisation of medium chain triglyceride (MCT) compared to long chain triglyceride (LCT), Single Comb White Leghorn 7-d-old male chicks were allocated into 4 experimental groups and one control of 5 birds per group. The chicks in the control group (Group 1) were given 5 g basal diet each day for 10 d. The birds in groups 2 and 3 received 5 g basal diet/d supplemented with MCT to provide 33.5 kJ GE/d and 67.0 kJ GE/d, and those in groups 4 and 5 were supplemented with LCT at 31.9 kJ GE/d and 63.8 kJ GE/d, respectively. Energy retained as protein and fat, and total energy retentions were measured. 2. No significant differences were found in energy retention as protein between the four energy supplemented groups but significant differences in fat and total energy retentions were found between the different dietary energy contents but not between energy source. Net energy of MCT for production was calculated as 16.0 kJ/g which corresponds to about 74% of that of LCT (22.8 kJ/g).

Medium-chain triacylglycerols enhance release of cholecystokinin in chicks.

Whether medium-chain triacylglycerols (MCT) affect the plasma concentration of cholecystokinin (CCK) and crop-emptying rate in chicks was investigated after 0, 30, 60, 90, 120 or 180 min of diet intubation. Triacylglycerol sources used were corn oil [containing long-chain triacylglycerols (LCT)], glyceryl tricaprate and glyceryl tricaprylate at a level of 200 g/kg diet. Plasma CCK concentration was significantly enhanced in chicks given the two MCT treatments, but not in those given the LCT treatment, after 30 min feeding relative to the initial level. At all time points, chicks fed the diet containing LCT had significantly lower plasma CCK concentrations than those fed MCT, and chicks fed glyceryl tricaprate had higher concentrations than those fed glyceryl tricaprylate. Dietary MCT sources significantly delayed diet passage from the crop compared with dietary LCT. These results indicate that MCT are more potent stimulators of CCK secretion in chicks than LCT.

Influence of dietary protein concentrations or of duodenal amino acid infusion on cholecystokinin release in goats.

1. Whether dietary protein levels or duodenal infusion of amino acids alters the release of cholecystokinin (CCK) in blood plasma of goats was investigated in three experiments. The CCK determination was done by radioimmunoassay with specific CCK-8 antibody. The male adult Shiba goat, a miniature Japanese native goat, was used. 2. In Experiment 1, four goats were offered a diet containing 4.94 g crude protein (CP)/kg BW0.75 for the first 7 days. They were then given a diet containing 5.86 g CP/kg BW0.75 for 7 days and thereafter 6.79 g CP/kg BW0.75 for the following 7 days. On the last day of each experimental period, blood samples were taken from the jugular vein at zero (before feeding), 30, 60, 120, 240 or 360 min after the feeding of the diet. Plasma CCK levels were not affected by dietary protein levels and time after feeding. 3. Influence of phenylalanine or tryptophan (2 mmol/20 ml) infusion into the duodenum was investigated by a 3 x 3 latin square in Experiment 2. Plasma CCK level was determined at 15 min intervals for 1 hr. Both phenylalanine and tryptophan gradually enhanced plasma CCK concentrations with time after infusion. 4. Branched-chain amino acids such as leucine and isoleucine were supplemented intraduodenally in Experiment 3 as in Experiment 2. No significant change in plasma CCK levels was observed.

Effect of dietary medium chain triglyceride on protein and energy utilisation in growing chicks.

1. Protein, fat and energy retentions of chicks fed on diets containing medium chain triglyceride (MCT) and long chain triglyceride (LCT), at 100 and 200 g fat/kg diet, were investigated. Maize oil was used as the LCT source. The MCT used was glyceryl tricaprylate. 2. Body weight gain and food intake were decreased with the diets containing MCT and these effects were greater at the higher fat concentration. Protein retention was also reduced by dietary MCT, although the efficiency of protein utilisation (protein retained/protein intake) was not altered. 3. The values for fat and energy retentions were significantly lower in the chicks fed on the MCT-supplemented diets than in those receiving the LCT-containing diets. Dietary ME values and efficiencies for energy utilisation (energy retained/ME intake) were also reduced by dietary MCT.

Influence of dietary sorbose on lipogenesis in gold thioglucose-injected obese mice.

1. The influence of dietary sorbose on food intake and fatty acid synthesis of the liver and epididymal white adipose tissue (EWAT) was investigated in gold thioglucose (GTG)-injected obese mice from 12 to 14 weeks of age. 2. Sorbose was supplemented to a semi-purified diet at a level of 200 g/kg diet at the expense of sucrose. 3. On the last day of the experiment, fatty acids synthesis in the liver and EWAT was measured using an i.p. injection [1-14C]sodium acetate. 4. The decreases in body weight and food intake by dietary sorbose in GTG-injected obese mice were greater than those in control mice. 5. Lipid content and fatty acid synthesis in the liver and EWAT of control mice were not influenced by dietary sorbose. 6. In GTG-injected obese mice, the reduction of food intake by dietary sorbose suppressed fatty acid synthesis and lipid deposition in both liver and EWAT.