Because histidine is a precursor for the synthesis of histamine, a neurotransmitter that suppresses feeding in rats (Ookuma et al. 1993, Sakata et al. 1990), short-term feeding in rats may be at least partially controlled by the histaminergic pathway driven by histidine (Mercer et al. 1994, Sheiner et al. 1985). Because manipulation of brain histamine concentrations affects food and water intake, and high protein diets or histidine preloads increase plasma and brain concentrations of histidine and histamine (Schwartz et al. 1972, Sheiner et al. 1985), it is assumed that there is a linkage among dietary and plasma histidine, brain histidine and histamine, and ingestive behavior.

There is some evidence that histidine preloads delivered by intraperitoneal (IP)⁴ injection into rats reduces food intake (Orthen-Gambill 1988, Sheiner et al. 1985) and increases water intake (Anderson et al. 1994). However, the physiological relevance of histidine as a regulator of food and water intake has not been addressed fully. The present study was designed to evaluate the precursor role of histidine in the regulation of short-term food intake in rats by blocking the histaminergic pathway using (+)-chlorpheniramine (CPA), an H1-receptor antagonist that induces feeding in rats (Sakata et al. 1988), and -fluoromethylhistidine (FMH), an irreversible inhibitor of histidine decarboxylase that prevents the synthesis of histamine from histidine (Orthen-Gambill and Salomon 1992).

MATERIALS AND METHODS

Rats were given a 25% protein powder diet (Table 1). All of the dietary components were purchased from Teklad Test Diets (Teklad Mills, Madison, WI) except for cornstarch (Nacan Products, Toronto, Canada) and corn oil (Mazola, Best Foods, Toronto, Canada). Food was provided in 250-mL glass jars (7.6 cm high) with trap safety metal (stainless steel) inside. Food cups were also equipped with spill-proof lids (~4.5-cm opening). Brown cardboard paper (Adelco Supply, Toronto, Canada) was placed under the cages to collect food spillage for weighing.

Table 1. Diet composition1 [View Table]

All treatments were based on the individual rat's body weight and were dissolved in 9 g/L saline. For IG delivery, a stainless steel gavage needle (size 16) (Harvard Apparatus, Quebec, Canada) was attached to a 10-mL plastic syringe. For IP delivery, 26G3/8 needles and 5-mL plastic syringes were used.

Tap water was provided in plastic graduated containers. For the measurement periods (1800-2100 h), 10-mL plastic syringes were sealed with parafilm. The piston was removed and a spout, inserted within a rubber stopper (size 0, 1.3 cm), was placed at the end of the syringe container. For the rest of the time (2100-1800 h), 50-mL plastic containers were used.

For each treatment, half of the rats were randomly assigned the treatment and the other half to the control on d 1. On d 2, the procedure was reversed. Day three was a washout day. The procedure continued in this way until rats were given all treatments for each experiment.

Rats were weighed on each treatment day at 1300 h. For all experiments, rats received treatment at 1730 h, except for drug treatments, which were given before this time as specified in the sections below for Experiments 4-8. At 1730 h, all water containers were removed and spillage paper was placed underneath the cages. The first water measurement was taken at 1800 h. Water intake was measured every hour for 3 h (0-1, 1-2 and 2-3 h) after the initial reading. Food intake and spillage were measured every hour for the first 3 h (0-1, 1-2 and 2-3 h) and after 14 h of feeding (3-14 h). Food and water intake was measured for the first 3 h because the conversion of histidine to histamine in the brain has been shown to be within this time frame (Schwartz et al. 1972). The experimental protocol was approved by the University of Toronto's Animal Care Committee.

Because CPA given IP at 1 mg/kg (2.6 µmol/kg) had no effect on food intake at any time period, CPA at 1 mg/kg (0.5 mL) was given IP at 1700 h in conjunction with His-HCl at 375 mg/kg (1.5 mL) at 1730 h. Rats (n = 19) weighed an average of 306 g on the first day and 311 g on the last day of the experimental period.

Injections (0.5 mL) of FMH were given IP at 1300 h (5 h before feeding) and injections (1.5 mL) of His-HCl at 1730 h. Rats in Group A were given saline injections on d 1 (at 1300 and 1730 h) and FMH injections on d 2 (FMH at 1300 h, saline at 1730 h). Rats in Group B were given His-HCl injections on d 1 (saline at 1300 h, His-HCl at 1730 h), saline injections on d 2 (at 1300 and 1730 h) and the combination of FMH (at 1300 h) and His-HCl (at 1730 h) on d 3. Thus, d 2 served as control for both the His-HCl treatment on d 1 and for the FMH plus His-HCl treatment on d 3.

RESULTS

Table 2. Experiment 1: Effect of dose and route of administration of histidine on food and water intake in rats1,2 [View Table]

Compared with the control treatment, food intake was lower (P < 0.01) after IP injection of 250 mg/kg of His-HCl during 0-14 h of feeding. Water intake after the histidine treatment was 33% higher than control (P < 0.01) during 0-1 h but not affected at any other time. Given IG, 250 mg/kg of His-HCl failed to affect food intake during any of the time periods. After histidine treatment, water intake was 45% greater (P < 0.01) during 0-1 h and 33% greater (P < 0.05) during 0-2 h compared with the control treatment.

Food intake after His-HCl at 375 mg/kg given IP was lower than that for the control treatment during 0-1 (P < 0.05), 0-2 (P < 0.01), 0-3 (P < 0.01) and 0-14 h (P < 0.05) of feeding by 17, 25, 17 and 4%, respectively. Water intake was 23% higher after treatment during 0-1 h (P < 0.01) but not affected over measurements for 0-2 or 0-3 h. In contrast, IG delivery of the same dose failed to affect food intake over these times. Water intake, however, was 23% higher (P < 0.05) during 0-1 h but was not affected by treatment during any other time.

His-HCl at 750 mg/kg IP lowered food intake (P < 0.01) during 0-1, 0-2, 0-3 and 0-14 h by 85, 72, 67 and 27%, respectively. Water intake was less (P < 0.01) after treatment by 50, 57 and 59% for the 0-1, 0-2 and 0-3 h intervals, respectively. Administered IG, His-HCl at 750 mg/kg failed to alter feeding. Water intake was higher by 55% (P < 0.01) during 0-1 h, and during 0-2 h by 31% (P < 0.05) compared with the control treatment.

The highest dose of His-HCl (1500 mg/kg), administered IG, suppressed food intake (P < 0.01) during 0-1 h, 0-2, 0-3 and 0-14 h. Water intake was significantly higher (P < 0.01) during 0-1, 0-2 and 0-3 h by 57, 58 and 41%, respectively.

Table 3. Experiment 2: Effect of pH and energy content of histidine preloads on food and water intake in rats1,2 [View Table]

Saline solution at pH 3.79 did not alter food or water intake at any time.

D-Glucose at 202.3 mg/kg had no effect on food intake during these intervals (Table 3). Cumulative water intake was lower (P < 0.05) by 13 and 16% during 0-2 h and 0-3 h, respectively.

Table 4. Experiment 3: Effect of nitrogen content of histidine preloads on food and water intake in rats1,2 [View Table]

L-Alanine at 159.4 mg/kg failed to alter food intake during any time interval (Table 4). However, after the alanine treatment, water intake was significantly higher (P < 0.05) during 0-2 h and 0-3 h, by 16 and 19%, respectively, compared with the control treatment.

Table 5. Experiment 4: Effect of chlorpheniramine (CPA) on the response of food and water intake to histidine in rats1,2 [View Table]

The 5 mg/kg dose of CPA suppressed food intake during 0-1 (P < 0.05), 0-2 (P < 0.01) and 0-3 h (P < 0.01) by 27, 29 and 21%, respectively. Water intake was not affected by this treatment.

The 1 mg/kg dose of CPA failed to alter feeding but reduced water intake during 0-1 (P < 0.01) and 0-2 h (P < 0.05) by 28 and 18%, respectively.

The combination of CPA (1 mg/kg) and His-HCl (375 mg/kg) resulted in lower (P < 0.01) food intake during 0-1, 0-2, 0-3 and 0-14 h (P < 0.05) by 24, 27, 18 and 7%, respectively, compared with the control treatment. There was no effect of treatment on water intake during any of the time periods.

Table 6. Experiment 5: Effect of -fluoromethylhistidine (FMH) on the response of food and water intake to histidine loads in rats1,2 [View Table]

The delivery of FMH alone failed to alter food intake during 0-1 or 0-2 h but increased food intake over 0-3 (P < 0.05) and 0-14 h (P < 0.01), both by 19%. Water intake was lower than the control treatment at 0-2 (P < 0.01) and 0-3 h (P < 0.05) by 18 and 14%, respectively.

The combination of FMH and His-HCl resulted in lower (P < 0.05) food intake over 0-1 h by 24%, but higher (P < 0.05) food intake for the time of 0-3 and 0-14 h by 10 and 13%, respectively. Water intake was not affected.

Table 7. Experiments 6 and 7: Effects of -fluoromethylhistidine (FMH) on response of food intake to histidine in rats1,2 [View Table]

Table 8. Experiments 6 and 7: Effect of -fluoromethylhistidine (FMH) on water intake after histidine in rats1,2 [View Table]

Because food intake was not affected by time of injection, the data for the two times were pooled. Again, the pooled analysis showed FMH did not affect food intake during the times examined. Water intake was reduced by 28 and 18% during 0-1 (P < 0.01) and 0-2 h (P < 0.05), respectively (Table 8).

For the rats in Group B (FMH, 1000 h), His-HCl alone suppressed (P < 0.01) food intake from 0-1, 0-2 and 0-3 h by 32, 26 and 16%, respectively (Table 7). Water intake was suppressed (P < 0.05) during 0-3 h by 15% (Table 8). The combination of FMH at 1000 h and His-HCl resulted in a 4% higher food intake during 0-14 h (P < 0.05). Water intake was not affected by the combination treatment.

Because a comparison of the effect of histidine injections in Groups A and B showed no difference at any time, the results were pooled. On the basis of pooled data, His-HCl suppressed (P < 0.01) food intake from 0-1, 0-2 and 0-3 h by 41, 28 and 21%, respectively (Table 7). Water intake was higher for 0-1 h (P = 0.05) (Table 8).

In addition, a comparison of the effect of the combination of His-HCl and FMH for Groups A and B showed no difference for water intake at any time based on a t test; thus the data were pooled. The results showed that the combination of the treatments had no effect on water intake at any time (Table 8).

DISCUSSION

Clearly, histidine affected food intake in rats. In general, food intake was reduced during the first 3 h after treatment with His-HCl at 375 mg/kg (providing histidine at 277.5 mg/kg). In contrast, the IG administration of His-HCl decreased food intake only at 1500 mg/kg, which is six times higher than the lowest concentration of an effective IP treatment (250 mg/kg) (Table 2).

The greater sensitivity of food intake to amino acids given IP compared with IG has been shown for other amino acids that serve as neurotransmitter precursors. Several times higher doses were required for tryptophan and tyrosine (Ng and Anderson 1992) and phenylalanine (Bialik et al. 1989) to suppress food intake by the IG compared with the IP route of administration. Initially, this was explained on the basis of the observation that plasma concentrations for tryptophan and tyrosine were higher after IP than IG administration of comparable doses. However, even when treatments of tryptophan were given IG to increase plasma and brain concentrations to the same level as lower doses of IP, the effect on feeding was still much weaker (Ng and Anderson 1992). Part of the explanation may be based on the observation of the presence of amino acid receptors in the small intestine (Jeanningros 1982). It is possible that amino acids in the peritoneal cavity stimulate these receptors to send signals to the splanchnic or other neurons that innervate the mesentery (Ng and Anderson 1992).

Water, but not food intake was equally affected by comparable IG and IP treatments. Water intake for the first hour was increased after both IG and IP administrations of His-HCl at 250 mg/kg. This was surprising because IG treatments affected food intake only at a very high dose (1500 mg/kg). Part of the explanation for the different effect of IG treatment on food and water intake is perhaps due to the mechanisms by which histamine is thought to affect feeding and drinking. Water intake is controlled by both peripheral and central mechanisms (Leibowitz 1979), whereas the role of histamine in the regulation of feeding is limited to the brain, more specifically, the hypothalamus (Sakata 1991). Possibly the IG treatment has a major effect only at the level of the gut. Histidine is readily converted to histamine in the gastric mucosa (Dimaline et al. 1990) and can directly signal H1- or H2-receptors that exist in that region (Bertaccini and Coruzzi 1992), which in turn causes an increase in water intake (Leibowitz 1979). The same does not apply to regulation of food intake because only H1-receptors in the hypothalamus have been directly associated with food intake (Fukagawa et al. 1989) and histamine in the periphery cannot cross the blood-brain barrier (Snyder et al. 1964).

Histidine appears to have a direct effect as the intact amino acid, possibly converted to histamine, and not through other components of the His-HCl preload. Histidine suppression of food intake was clearly beyond what could be accounted for by its energy, pH or nitrogen content.

The effect of the energy and nitrogen content of His-HCl on water intake is less clear. Water intake was altered by the glucose and the alanine preloads (Tables 3 and 4), but these preloads do not appear responsible for the effect of histidine on water intake. Neither of the preloads increased water intake during the first hour as observed after His-HCl (375 mg/kg) in Experiments 1 and 2 (Tables 2 and 3). Furthermore, 202.3 mg/kg of D-glucose suppressed total water intake during the 0-2 and 0-3 h of water intake (Table 3), whereas the 159.4 mg/kg dose of L-alanine induced drinking during the same time intervals (Table 4).

As in Experiment 1, the response to food intake in Experiments 2 and 3 was independent of drinking in rats. This observation supports the notion that drinking and feeding alterations by histidine are regulated through independent mechanisms.

The precursor hypothesis is supported indirectly by these experiments in that the time frame for the suppression of food intake after His-HCl matched the time required for the uptake of histidine and synthesis of histamine in the brain. In Experiments 2 and 3, the cumulative food intake for the first 3 h was reduced by 1.4 g (26%) and 3.2 g (70%), respectively (Tables 3 and 4). This reduction accounted for 82 and 100% of reduction in food intake over the 14 h of feeding in the two experiments, respectively. Therefore, the main effect of histidine on food intake occurred during the first 3 h, which is consistent with the time frame of change in hypothalamic histidine and histamine after histidine injections. Specifically, IP injections of 500 mg/kg of His-HCl increased hypothalamic concentrations of histidine and histamine by 83 and 50%, respectively (Schwartz et al. 1972). The hypothalamic concentrations of histidine peaked 1 h after injection, whereas histamine concentrations were increased by 50% 90 min after injections.

Because results from Experiments 1, 2 and 3 (Tables 2, 3 and 4) showed that 375 mg/kg of His-HCl administered IP brought about consistent responses in food and water intake, this dose was used in the experiments aimed at determining its mechanism of action. Evidence that histidine reduces food intake in part by its action on the histaminergic system, presumably by increasing histamine synthesis, was provided in Experiments 5-7 in which FMH was used to prevent histamine synthesis. However, perhaps because of its nonspecific action on other neurotransmitter pathways, the lack of effect of CPA in Experiment 4 failed to support the precursor hypothesis.

The failure of CPA to increase food intake, rather than decrease it as observed here, or to block histidine-induced suppression of food intake was unexpected and is perhaps explained by the route of administration. Several feeding studies have used CPA to block H1-receptors in the brain (Ookuma et al. 1988, Sakata et al. 1988). In all studies, infusion of CPA led to an increase in food intake in rats. However, almost all of the evidence for CPA blockade of histaminergic suppression of food intake is based on direct applications of this drug into the brain (Ookuma et al. 1988, Sakata et al. 1988). Only one report in the literature provided the rationale for giving CPA IP in the present studies, but the evidence was indirect. 1-Deoxy-D-glucosamine, a glucose derivative, decreases food intake of rats and is believed to act by increasing histamine release centrally (Kang et al. 1993). When given IP, CPA at 10 mg/kg reversed the hypophagia. Unfortunately, the effect of CPA alone was not reported. For this reason, a feeding dose-response study for CPA was conducted, but none of the doses selected resulted in an increase in food intake as expected (Table 5). Further studies have also failed to find a dose that increases food intake (unpublished data).

In addition to route of administration and the possibility that a suitable dose was not identified, there are several other possible explanations for failure of CPA to increase food intake or reverse histidine suppression of food intake. CPA stimulates other neurotransmitter systems such as the adrenergic and the serotonergic pathways (Lidbrink et al. 1971), which are known to cause a decrease in feeding (Anderson 1994).

The data from FMH treatments, however, provide some evidence that histamine synthesis is involved in mediating the effect of histidine on food intake. First, in Experiment 5, FMH injected 5 h before feeding increased food intake during 0-3 and 0-14 h (Table 6), which is the expected response to reduced hypothalamic histamine (Ookuma et al. 1993). Although in Experiment 6, FMH injected at 8 or 5 h before feeding did not affect food intake, the pooled data were at least partially indicative that FMH increased food intake (Table 7). Second, FMH blocked or reduced the decrease in food intake suppression observed after His-HCl (Table 9). When FMH was given before histidine, food intake was significantly greater than after histidine alone during 0-1, 0-2, 0-3 and 0-14 h, on the basis of pooled data from Experiments 5, 7A and 7B.

Table 9. Effect of -fluoromethylhistidine (FMH) on histidine-induced suppression of food and water intake in rats [View Table]

These effects of FMH on food intake were consistent with previous feeding studies investigating the effect of central manipulations of histamine (Ookuma et al. 1993, Sakata et al. 1990). For example, infusion of 2.24 µmol of FMH into the third cerebroventricle of the rat brain increased feeding frequency, duration and meal size in the early light period (Ookuma et al. 1993).

During the first hour of feeding, however, FMH did not totally block the effect of histidine on food intake, reversing histidine-induced suppression of food intake by 0.5 (28%), 0.8 (47%) and 1.3 g (48%) in Experiments 5, 7A and 7B (Tables 6 and 7). It is possible that either the histamine synthesis was not fully inhibited or that more than one mechanism is involved in histidine suppression of food intake. Although FMH (20 mg/kg) has been reported to reduce whole-brain concentrations by 63% 3 h after injections in rats (Turon et al. 1991), the histamine rate of turnover in the hypothalamus is lower than in other regions of the brain (Hough et al. 1984).

The evidence accumulated from the current experiments on water intake supports a partial role for the histaminergic influence on the dipsogenic effect of histidine in the first hour of feeding. Delivery of FMH alone suppressed water intake during the first hour (Table 8), which is expected from reduced histaminergic activity. Histidine alone increased water intake in some experiments, but FMH blocked drinking induced by His-HCl during the first and second hours (Table 9). These results are consistent with previous reports that dexbrompheniramine, an H1-antagonist, partially prevented drinking stimulated by subcutaneous injections of histamine (20 mg/kg) (Kraly 1983). It has also been observed that histamine injections stimulate drinking (Leibowitz 1979), but this is the first report that FMH blocked the dipsogenic effect of a histidine preload.

Base-line concentrations of histamine are clearly important in the regulation of drinking during the first hour of feeding. Water intake was reduced by FMH alone during 0-1 h in Experiments 5, 7A and 7B by 15, 30 and 22% (Tables 6 and 8), respectively. Other time periods were not affected by the treatments; this was not surprising because His-HCl did not consistently affect water intake at any other time except the first hour.

Although CPA at 1 mg/kg failed to affect food intake, this dose suppressed drinking during 0-1, 0-2 and 0-3 h by 28, 18 and 10%, respectively (Table 5). Although the results from this study are the first report that FMH suppresses drinking in rats, the effect of CPA on decreasing water intake observed here is consistent with the literature. Injection of H1- and H2-receptor antagonists abolished 60% of the food-related drinking by the rats, delayed the latency to drink after a meal and reduced drinking before a meal (Kraly and Specht 1984).

The effect on water intake of CPA at 5 and 10 mg/kg doses, compared with 1 mg/kg, was less clear. The 5 mg/kg CPA treatment had no effect on water intake at any time, whereas the 10 mg/kg treatment reduced water intake over 0-1, 0-2 and 0-3 h (Table 5). The lack of effect of CPA at 5 mg/kg was surprising although the suppression of water intake by 10 mg/kg was expected. However, the effect of CPA at 10 mg/kg is difficult to interpret because the treatment reduced food intake, which has an effect on drinking (De Castro 1989).

Despite the support for the precursor hypothesis provided by these data, the physiological relevance of dietary histidine in the regulation of food and water intake remains unclear. There is some evidence suggesting that diet may affect the histaminergic pathway. For example, protein-deficient diets have been shown to increase brain concentrations of histidine and histamine (Mercer et al. 1989, Pao and Dickerson 1975). In addition, inhibition of histamine synthesis by FMH increased feeding of a low protein diet (4%) (Mercer et al. 1994). However, in Experiment 1, the lowest dose of His-HCl found to affect food intake in the first hour of feeding was 375 mg/kg; the dose that affected food intake over the full 14 h of feeding was 250 mg/kg of His-HCl (Table 2). These doses of His-HCl contained 185 and 278 mg/kg of pure histidine, ~ two to three times the dietary intake of histidine within an average meal of a 25% protein diet (80 mg/kg).

The possibility, however, that dietary histidine plays a role in regulating food and water intake cannot be excluded by the present data. It is possible that another physiological signal is necessary to activate histaminergic neurons and thus increase their sensitivity to precursor availability. Other systems sensitive to neurotransmitter amino acid precursors also show this dependence on activated neurons. For example, tryptophan at 100 mg/kg IG did not affect feeding at any time (Ng and Anderson 1992), but 60 mg/kg of tryptophan fed in combination with 1 g of carbohydrate significantly altered food selection and brain serotonin concentrations (Li and Anderson 1984).

In conclusion, the results from this work suggest that histidine given IP and IG affects food and water intake in rats. Results also support the linkage between histidine, histamine and food intake. However, the physiological importance of dietary histidine remains unresolved.

ACKNOWLEDGMENT