An in vitro human muscle preparation suitable for metabolic studies. Decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects.

We have developed an in vitro muscle preparation suitable for metabolic studies with human muscle tissue and have investigated the effects of obesity and non-insulin-dependent diabetes mellitus (NIDDM) on glucose transport. Transport of 3-O-methylglucose and 2-deoxyglucose was stimulated approximately twofold by insulin in muscle from normal nonobese subjects and stimulation occurred in the normal physiological range of insulin concentrations. In contrast to insulin stimulation of 3-O-methylglucose and 2-deoxyglucose transport in muscle from normal, nonobese subjects, tissue from morbidly obese subjects, with or without NIDDM, were not responsive to insulin. Maximal 3-O-methylglucose transport was lower in muscle of obese than nonobese subjects. Morbidly obese patients, with or without NIDDM, have a severe state of insulin resistance in glucose transport. The novel in vitro human skeletal muscle preparation herein described should be useful in investigating the mechanism of this insulin resistance.

Increase in transforming growth factor-beta in the brain during infection is related to fever, not depression of spontaneous motor activity.

When viral infection occurs, this information is transmitted to the brain, and symptoms such as fever and tiredness are induced. One of the causes of these symptoms is the secretion of proinflammatory cytokines in blood and the brain. In this study, the i.p. administration of polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA, to rats was used as an infection model. Poly I:C decreased spontaneous motor activity (SMA) 2 h after i.p. administration, and this decrease was maintained thereafter. The concentration of active transforming growth factor-beta (TGF-beta) in cerebrospinal fluid (CSF) increased 1 h after the administration. This increase occurred earlier than those in the concentrations of other proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), in serum. The intracisternal administration of an anti-TGF-beta antibody partially inhibited fever induced by poly I:C administration; however, this treatment did not affect the decrease in SMA. Furthermore, intracisternal administration of TGF-beta raised the body temperature. These results indicate that TGF-beta in the brain, which was increased by poly I:C administration, is associated with fever but not with a decrease in SMA.

Influence of folate-binding protein from bovine milk on the absorption of folate in gastrointestinal tract of rat.

The absorption of [14C]pteroylglutamic acid (PteGlu) bound to the folate-binding protein of bovine milk was investigated in rat gastrointestinal tract in vivo and in situ. When bound [14C]PteGlu was given to rat intragastrically via oral intubation, a considerable amount of PteGlu was released from folate-binding protein under the acidic conditions in stomach, and it recombined with folate-binding protein in jejunum in vivo. Compared with free PteGlu, bound PteGlu was more gradually absorbed in small intestine, but finally the total amount of bound PteGlu absorbed was almost the same as that of free PteGlu. In all experiments in situ, bound PteGlu was only slightly absorbed in jejunum, where free PteGlu was rapidly absorbed under the same conditions. On the other hand, the absorption rates of the two forms of PteGlu were almost similar to each other in ileum. These results suggest that PteGlu bound to folate-binding protein is absorbed by a manner different from that of free PteGlu in rat gastrointestinal tract.

Localization of pancreatic enzyme secretion-stimulating activity and trypsin inhibitory activity in zymogen granule of the rat pancreas.

The intracellular localization of pancreatic enzyme secretion-stimulating activity in rat pancreas was investigated. We found and purified a pancreatic enzyme secretion-stimulating peptide from rat bile/pancreatic juice. The peptide is trypsin-sensitive (showing temporary trypsin inhibitory activity), and it is hypothesized that it acts as a trypsin-sensitive mediator in the feedback regulation of diet-induced pancreatic enzyme secretion. The zymogen granule fraction was purified 5-fold by ultracentrifugation by the Percoll density gradient method. The purity of the zymogen granule fraction was determined from the specific amylase activity and electron microscopic morphology. The specific enzyme activities of amylase and trypsin and the trypsin inhibitory activity increased in parallel during the purification, and the pancreatic enzyme secretion-stimulating activity was also localized in the zymogen granule fraction. These results suggest that the pancreatic enzyme secretion-stimulating peptide originates from the acinar cells, and that it is secreted through exocytosis of zymogen granules into the small intestine, its ratio to trypsin thus remaining constant. This idea supports our hypothesis that the stimulating peptide acts as a mediator for the feedback regulation of pancreatic enzyme secretion by trypsin.

Molecular cloning and characterization of genes encoding rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and pancreatic secretory trypsin inhibitor (PSTI).

The genes encoding a rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and its structurally related peptide, rat pancreatic secretory trypsin inhibitor (PSTI), have been isolated and sequenced. The two genes share extremely high sequence similarity in the 5' flanking regions, suggesting that these regions may be responsible for the characteristic coordinate expression of the two peptides.

Monitor peptide gene expression is increased by exogenous CCK in the rat pancreas and in a rat pancreatic acinar cell line (AR4-2J).

Monitor peptide (CCK-releasing peptide) mRNA increased on the administration of CCK in rat pancreas and the AR4-2J pancreatic cell line. Subcutaneous injection of CCK into rats at 8 h intervals increased the level of monitor peptide mRNA in the pancreas. Concomitant injection of CCK antagonist CR-1409 strongly decreased it. The monitor peptide mRNA was also increased by CCK in AR4-2J cells and was decreased by the antagonist. These findings suggest that the plasma CCK induced by prolonged intake of a high protein diet may be responsible for the adaptative increase in the monitor peptide as well as exocrine proteases in the pancreas.

Differentiation of intestinal epithelial cell line (IEC-18) by an acid extract of rat small intestine.

A factor which may induce differentiation of intestinal epithelial cell lines in vitro was found in an acid extract of adult rat small intestine. The addition of a partially purified acetic acid extract of rat small intestine to IEC-18 cell culture dishes increased sucrase activity within 48 h. Thymidine incorporation markedly decreased within 24 h. Significant development of microvilli-like structures was observed on the acid extract-treated IEC-18 cells, compared with controls. This activity of rat acid extract was heat-stable and the apparent molecular weight of the factor was 400-800. These findings suggested that the factor may be related to the epithelial differentiation of rat small intestinal crypt cells.

Expression of the putative membrane fatty acid transporter (FAT) in taste buds of the circumvallate papillae in rats.

The putative membrane fatty acid transporter (FAT) protein and its mRNA, originally expressed in adipose tissue, were found in the tongue of rats. Northern blot analysis showed a significant expression of FAT mRNA in the epithelial layer of circumvallate papillae. Immunohistochemical staining revealed that immunoreactivity for FAT is specifically localized in the apical part of taste bud cells, possibly gustatory cells, in the circumvallate papillae.

TRPV1 activation and induction of nociceptive response by a non-pungent capsaicin-like compound, capsiate.

Capsiate is a capsaicin-like ingredient of a non-pungent cultivar of red pepper, CH-19 sweet. To elucidate the mechanisms underlying the non-pungency of capsiate, we investigated whether capsiate activates the cloned capsaicin receptor, TRPV1 (VR1). In patch-clamp experiments, capsiate was found to activate TRPV1 expressed transiently in HEK293 cells with a similar potency as capsaicin. Capsiate induced nociceptive responses in mice when injected subcutaneously into their hindpaws with a similar dose dependency as capsaicin. These data indicate that the non-pungent capsiate is an agonist for TRPV1 and could excite peripheral nociceptors. In contrast to this, capsiate did not induce any significant responses when applied to the skin surface, eye or oral cavity of mice, suggesting that capsiate requires direct access to nerve endings to exhibit its effects. Capsiate was proved to have high lipophilicity and to be easily broken down in normal aqueous conditions, leading to less accessibility to nociceptors. Another highly lipophilic capsaicin analogue, olvanil, was similar to capsiate in that it did not produce irritant responses when applied to the skin surface, although it could activate TRPV1. Taken together, high lipophilicity and instability might be critical determinants for pungency and so help in understanding the effects of capsaicin-related compounds.

Evidence for an essential arginyl residue in bovine milk gamma-glutamyltransferase.

Treatment of bovine milk gamma-glutamyltransferase with 2,3-butanedione in borate buffer markedly inactivates its gamma-glutamyltransferase activity. Inactivation is prevented by a combination of the gamma-glutamyl donor and acceptor substrates, glutathione, and glycylglycine, but less effectively by only one of them. Serine plus borate of maleate provides no protection against the inactivation. Amino acid analysis of the enzyme treated with butanedione in the presence and absence of the protecting substrate combination indicates that complete inactivation correlates with the modification of a single arginyl residue per molecule. The residue modified is associated with the smaller subunit of the two equal subunits which comprise the enzyme. The butanedione-treated enzyme retains a hydrolytic activity, another but less significant catalytic function of the enzyme. The results indicate that the arginyl residue is involved in recognizing the anionic moiety of the acceptor and in binding it to the acceptor site located on the smaller subunit of the enzyme.

An adjustable-current swimming pool for the evaluation of endurance capacity of mice.

A new forced-swimming apparatus for determining maximum swimming time in mice was devised for use in the evaluation of the endurance capacity of Std and ddY and CDF1 mice after various diet and drug treatments. With the apparatus, a water current is generated by circulating water with a pump in a swimming pool. A spout and suction slit were contrived to generate a constant current while the strength of the current is regulated by a valve. The decrease in the leg-kicking intervals of mice accompanying the increase in the current speed confirmed that the workload is adjustable by regulation of the current speed. Compared with the number of forelimb strokes, that of the hindlimb kicks was greater. The swimming time until fatigue was observed to decrease with increasing current speed in the two strains of mice. As biochemical indexes, the blood lactate and muscle glycogen levels corroborated the correlation between current speed and increase in workload. These results indicate that the apparatus employed in the present study is suitable for the evaluation of the endurance capacity of mice and that is useful for detecting the effects of dietary differences and drug pretreatments on this capacity.

Opioidergic contribution to conditioned place preference induced by corn oil in mice.

We previously reported that voluntary intake of corn oil in the light box showed place preference in the conditioned place preference (CPP) test in mice. In the present study, we investigated the contribution of opioidergic systems to the corn oil-induced CPP in mice. Acquisition of the place preference by corn oil intake was blocked by i.p. injections of an opioid mu antagonist, naloxone (0.1 and 0.3 mg/kg), and delta antagonists, 7-benzylidenenaltrexone (0.5 mg/kg) and naltriben (0.5 mg/kg) 15 min before conditioning. The opioid kappa agonist U-50488H (1 and 3 mg/kg i.p.) also blocked corn oil-induced CPP. Naloxone (1 mg/kg, i.p.) and naltriben (0.5 mg/kg, i.p.) did not affect corn oil intake in the home cage. However, 7-benzylidenenaltrexone (0.5 mg/kg, i.p.) and U-50488H (1 mg/kg i.p.) decreased and increased the corn oil intake, respectively. These results suggested that the rewarding effects of corn oil in the CPP test are at least partially mediated via opioidergic systems through mu and delta receptors. Further, we showed that an opioid kappa agonist reduced the rewarding effects of corn oil in the CPP test in mice, although it increased corn oil intake.

Effect of diazepam binding inhibitor (DBI) on the fluid intake, preference and the taste reactivity in mice.

We have reported that a diazepam binding inhibitor (DBI)-like peptide is released by the aversive quinine stimuli 'Chem. Senses 25 (2000) 739'. To determine the effect of DBI on the fluid intake, we injected a DBI peptide fragment into the fourth ventricle in mice. DBI suppressed the intake of 5% sucrose, water and 0.9 mM quinine-HCl and the preference for 0.05% saccharin. Administration (i.p.) of flumazenil, a benzodiazepine receptor antagonist, 20 min before the injection of DBI (i.c.v.) antagonized the suppressive effect of DBI on the intake and the preference for saccharin. We also studied the dose dependency of the effect of DBI on the intake of 5% sucrose. Injection of DBI in excess of 3 microg suppressed the intake of 5% sucrose in mice. Furthermore, injection of DBI (i.c.v.) increased the aversive response to 0.9% NaCl in the taste reactivity in mice. These results suggest that DBI affect the preference to food.

Effects of oil intake in the conditioned place preference test in mice.

We investigated the effects of corn oil intake in the conditioned place preference (CPP) test in mice. Voluntary intake of corn oil in the light box in the CPP test showed place preference, but its peroral administration 60 min before conditioning did not show either place preference or aversion. Acquisition of the place preference by corn oil intake was blocked by i.p. injection of SCH 23390 (0.03 mg/kg) and haloperidol (0.1 mg/kg), but not by that of (+/-)-sulpiride (100 mg/kg), (-)-sulpiride (100 mg/kg), and L-741, 626 (1 mg/kg) 15 min before conditioning. These results suggest that stimulation of corn oil in the oral cavity, but not its postingestive effects, have positive reinforcing effects and the stimulation of corn oil is at least partly mediated via dopaminergic systems through the D(1) receptors. Moreover, the present results suggest that the CPP test is useful for the study of preferable stimulation and rewarding effects of food intake.

Transforming growth factor-beta activated during exercise in brain depresses spontaneous motor activity of animals. Relevance To central fatigue.

Intracerebroventricular administration into sedentary mice of the high molecular mass fraction of cerebrospinal fluid (CSF) from exercise-exhausted rats produced a decrease in spontaneous motor activity [K. Inoue, H. Yamazaki, Y. Manabe, C. Fukuda, T. Fushiki, Release of a substance that suppresses spontaneous motor activity in the brain by physical exercise, Physiol. Behav. 64 (1998) 185-190]. CSF from sedentary rats had no such effect. This suggests the presence of a substance regulating the urge for motion as a response to fatigue. A bioassay system using hydra, a freshwater coelenterate, showed an activity indistinguishable from transforming growth factor-beta (TGF-beta) in the CSF from exercise-fatigued rats, while not in that from sedentary rats. The increase in the concentration of active TGF-beta in the CSF from exercise-fatigued rat was also ascertained by another bioassay system using mink lung epithelial cells (Mv1Lu). Injection of TGF-beta into the brains of sedentary mice elicited a similar decrease in spontaneous motor activity in a dose-dependent manner. Increasing the exercise load on rats raised both the levels of active TGF-beta and the activity of depression on spontaneous motor activity of mice in the CSF of rats. Taken together, these results suggest that exercise increases active TGF-beta in the brain and it creates the feeling of fatigue and thus suppresses spontaneous motor activity.

Pancreatic enzyme secretion mediated by novel peptide: monitor peptide hypothesis.

A new model is proposed for pancreatic enzyme secretion in response to food protein intake in rats. We have found a novel peptide in rat bile-pancreatic juice, which exhibits a trypsin-sensitive, cholecystokinin (CCK)-releasing activity. The amino acid sequence of the peptide purified from rat bile-pancreatic juice is very similar to that of a conservative region in pancreatic secretory trypsin inhibitor (PSTI). The peptide loses its CCK-releasing activity during trypsin digestion, but food protein intake prevents this trypsin digestion. Results of a reconstitution experiment indicate that pancreatic enzyme secretion in response to food protein intake only occurs as a result of interaction between trypsin and our purified peptide. Also, a peptide-specific antibody abolished the response. These findings lead us to hypothesize that this peptide acts as an intraduodenal mediator for CCK release in response to food protein intake.

Hydra biological detection of biologically active peptides in rat cerebrospinal fluid.

The Hydra bioassay system utilizes a tentacle ball formation (TBF), a component of the feeding response of hydra, elicited by S-methyl-glutathione. TBF is modulated by many biologically active peptides in a specific way to individual peptides, and is useful in investigating biologically active peptides in a complex biological sample. We applied the hydra bioassay to explore a possible biologically active substance responsible for the decrease in the motor activity of the mice. The suppression of the CSF obtained from rats after exhaustive exercise was marked lower than that of sedentary rats. Addition of transforming growth factor-beta (TGF-beta), which is the only substance known to nullify TBF, to CSF of the sedentary rat reproduced this change in the suppression of the TBF. This system is useful to screen active peptides in small amounts of biological samples containing very low concentrations of peptides.

Preference for vegetable oils in the two-bottle choice test in mice.

We evaluated in mice their preference for different vegetable oils using the two-bottle choice test. Mice showed preference for all vegetable oils (at 1% concentration in emulsion) tested, including corn oil, canola oil, and mixed vegetable oil, compared to a control fluid without oil. Mice also significantly preferred highly palatable sucrose solutions (5-20%) in this test. One percent corn oil was selected to the same extent as were these sucrose solutions. Mixtures of 1% corn oil and several concentrations of sucrose were preferentially selected as compared to corn oil or sucrose alone. The concentration-response curves of oil-sucrose mixtures vs sucrose (0.5-20% concentrations) were similar to those of sucrose vs control solutions. These results suggest that 1% corn oil and sucrose at the concentrations tested did not affect each other's preference. Chronic intake of 20% sucrose solution, but not 100% corn oil, changed the preference for lower concentrations of each, and both induced remarkable increases in body weight of mice. In conclusion, we showed that mice preferred vegetable oils, and this preference compared favorably with highly palatable sucrose solutions and was not changed by chronic intake of corn oil.

Preference for corn oil in olfactory-blocked mice in the conditioned place preference test and the two-bottle choice test.

We studied the effects of olfactory stimuli on preference for corn oil in mice. In the conditioned place preference test, voluntary intake of 100% corn oil by both olfactory normal and ZnSO4-induced olfactory-blocked (anosmic) mice resulted in their place preference for the corn oil-related box. In the olfactory normal mice, place preference was also observed by voluntary intake of linoleic acid as well as of corn oil. In the two-bottle choice test, normal mice showed significant preference for test fluids that contained corn oil at all concentrations (1-10%) tested relative to vehicle alone. However, the lower concentrations (1 and 3%) of corn oil were not preferred in the anosmic mice. These results suggested that stimuli other than olfaction contributed to the rewarding effects of corn oil, but at lower concentrations olfactory stimuli might act as a signal for the oil.