Introduction

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Pharmacological evidence, obtained predominantly using selective agonists, have suggested that atypical beta adrenergic receptors are located in rodent GI tissue and function to regulate GI motility (Arch and Kaumann, 1993; Croci et al., 1988; Giudice et al., 1989). That these atypical beta adrenergic receptors may be the same as those that mediate lipolysis in white and brown adipose tissues throughout the body, i.e., the ₃-AR, is supported by the results of mRNA localization methods that identified ₃-AR in adipose and GI tissues of various species (Evans et al., 1996; Granneman et al., 1991; Cohen et al., 1995; Berkowitz et al., 1995), and by the similar relative potencies of selective ₃-AR agonists to mediate adipocyte lipolysis and inhibit motility of GI tissues in vitro (Lezama et al., 1996; Landi et al., 1993; Cohen et al., 1995). It has also been demonstrated that selective ₃-AR cause relaxation in the GI tract of rodents in vivo (Thollander et al., 1996; Giudice et al., 1989; Manara et al., 1995). These results suggest that the effects of selective ₃-AR agonists on GI motility are due to activation of ₃-AR present in the GI tissue. The availability of ₃-AR[KO] and ₃-AR[WAT+BAT] mice (Susulie et al., 1995; Grujic et al., 1997), offered us the unique opportunity to obtain direct proof of the involvement of ₃-AR in regulating GI motility. We describe the effects of several selective ₃-AR agonists on the transit of radiolabelled charcoal through the GI tract, as indicative of GI motility, in normal mice and in these two types of genetically engineered mice. Our results confirm the previous reports that selective ₃-AR agonists are capable of causing a decrease in GI motility in rodents, as well as demonstrate that these agents are indeed acting through ₃-AR. However, our results also suggest that the modulation of GI motility by ₃-AR agonists in vivo can occur exclusively as an indirect consequence of activation of ₃-AR in adipose tissue.

	Materials and Methods

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3-AR[KO] mice, generated using homologous recombination in the FVB/N background (Susulic et al., 1995), as well as ₃-AR[KO] mice genetically engineered with insertion of functional ₃-AR exclusively in white and brown adipose tissues only (₃-AR[WAT+BAT]) (Grujic et al., 1997), were used. Inbred FVB/N WT were purchased from Taconic Farms (Germantown, NY). Male, 9- to 11-wk-old mice were used in all cases.

GI motility studies were performed following the method of Miller et al (1961). Mice were fasted 24 hr before bolus oral administration of 0.25 ml 1% methocel containing 0.5 µCi [⁵¹Cr]sodium (ICN Biomedicals, Inc., Irvine, CA), 5% acacia and 10% charcoal. Forty five minutes after administration of radiotracer, at which time no radiotracer had moved past the small intestine, mice were euthanized by CO₂ asphyxiation and the GI tract was removed. The small intestines were divided into 10 segments of equal length. The radioactivity within each segment, as well as within the stomach, was determined by counting in a gamma counter. The mean cpm ± S.E.M. for the radioactivity retained in the stomach, and the GC of intestinal transit were calculated for each treatment group (n = 5-15 animals). Under these conditions, a decrease in GC corresponds to less rapid intestinal transit of radiotracer (increased intestinal transit time). An increase in stomach retention of radiotracer, together with a decrease in the extent of movement of radiotracer through the small intestine were taken to be indicative of an overall decrease in GI motility. The ₃-AR agonists BRL 35135, CL 316,243, ICI 198,157 and SR 56811A were administered in saline, at 3 mg/kg, by s.c. injection 1 hr before oral administration of charcoal. Clonidine was administered at 0.2 mg/kg. An equal volume of saline was injected into vehicle controls. The ₃-AR agonists were provided through the Department of Medicinal Chemistry, Merck & Co., Rahway, NJ. Clonidine was purchased from Sigma Chemical Co., St. Louis, MO.

For measurement of plasma glycerol levels, heparinized blood was obtained by cardiac puncture after carbon dioxide euthanasia and centrifuged at 3000 × g for 15 min at room temperature. The plasma was stored at 70°C until assay. Plasma glycerol levels were determined spectrophotometrically using a commercially available assay kit [Triglyceride(GPO-Trinder), Sigma Diagnostics, St. Louis, MO].

The in vitro potency of the rodent-specific ₃-AR agonists were quantified in terms of the stimulation of adenylyl cyclase activity, in Chinese hamster ovary cells expressing a cloned rat ₃-AR receptor (Candelore et al. 1996).

Statistical analysis of the data was performed using a two-way analysis of variance analysis on rank-transformed data (Normal-Quantile). Statistical difference between groups was considered to be significant at *P <.05, **P <.01, and highly significant at ***P <.001.

	Results

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Normal GI transit and the effects of clonidine treatment. No significant difference was seen in the stomach retention of radioactive charcoal in saline-treated WT and ₃-AR[KO] mice (table 1). Similarly, normal intestinal transit of radiotracer was not significantly different in the saline-treated WT and ₃-AR[KO] mice. Clonidine administration caused a highly significant increase in retention of radiotracer in the stomach and reduced the extent of intestinal transit of radiotracer (decreased GC) in both WT and ₃-AR[KO] mice when compared to vehicle-treated controls.

View this table: [in this window] [in a new window]   TABLE 1 Effect of clonidine and 3-AR agonist treatment on GI motility in WT and 3-AR[KO] mice

Effects of ₃-AR agonists on GI transit in WT and ₃-AR[KO] mice. Subcutaneous administration of 3 mg/kg BRL 35135, CL 316,243 or ICI 198,157 caused a significant increase in stomach retention of radiotracer and reduced the extent of intestinal transit of radiotracer in WT mice, but had no effect on either motility parameter in ₃-AR[KO] mice (table 1). SR 56811A was without significant effect on either motility parameter in WT or ₃-AR[KO] mice when administered under these same conditions. The relative order of these compounds to affect overall GI motility in WT mice after a single subcutaneous dose of 3 mg/kg was BRL 35135 > CL 316,243 > ICI 198,157 >>SR 56811A (inactive).

Effects of BRL 35135 on GI transit in WAT/BAT mice. As BRL 35135 exerted the most profound effects on GI transit in WT mice, it was chosen to evaluate the effects of a ₃-AR agonist on GI transit in 3-AR[WAT+BAT] mice. Administration of BRL 35135 to 3-AR[WAT+BAT] mice produced a significant increase in stomach retention of radiotracer together with a significant decrease in the extent of intestinal transit of radiotracer, similar to that seen when WT mice were treated with this compound (table 1). In contrast, BRL 35135 treatment in 3-AR[KO] mice affected neither motility parameter. Although saline-treated 3-AR[WAT+BAT] controls appeared to exhibit a higher retention of radiotracer in the stomach than saline-treated WT controls, the increase was not statistically significant (P = .089). Similarly, the GC values for normal intestinal transit in the saline-treated controls for WT, 3-AR[KO] and 3-AR[WAT+BAT] mice were not significantly different.

Potencies of ₃-AR agonists in an in vitro receptor assay. The potencies of the ₃-AR agonists used in these experiments to stimulate adenylyl cyclase activity in vitro in Chinese hamster ovary cells expressing the cloned rat ₃-AR are shown in table 2. The relative potencies were BRL 35135 = CL 316,243 > ICI 198,157 >> SR 56811A.

View this table: [in this window] [in a new window]   TABLE 2 In vitro potency of 3-AR agonists

Effects of ₃-AR agonists on plasma glycerol levels. The ability of the ₃-AR agonists to induce lipolysis in adipose tissue was demonstrated by the hyperglycerolemia that was evident 60 min after administration of the agonists to nonfasted WT mice in a manner identical to that used for the GI transit studies. Elevations in plasma glycerol levels were highly significant compared to saline-treated controls after administration of BRL 35135, CL 316,243 or ICI 198,157 (table 3). Although preliminary studies in WT mice indicated that plasma glycerol elevation is maximal within 30 to 60 min after ₃-AR agonist challenge and declines rapidly thereafter, plasma glycerol levels were also determined on samples taken at the time of euthanasia of mice undergoing GI transit studies involving BRL 35135 treatment. At this time after treatment of mice with BRL 35135 (i.e., 105 min postcompound administration), plasma glycerol levels were elevated 25% in WT mice and 50% in 3-AR[WAT+BAT] mice, but were not elevated in the 3-AR[KO] mice.

View this table: [in this window] [in a new window]   TABLE 3 Effect of 3-AR agonist treatment on lipolysis in WT mice

	Discussion

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The biochemical pathways which regulate lipid metabolism in response to adrenergic receptor activity are fairly well understood (reviewed by Lafontan and Berlan, 1993). Selective agonists have been used to demonstrate the involvement of the more recently discovered "atypical-" or ₃-AR in regulating metabolic rate and lipolysis in adipose tissue (Arch et al., 1984; Bond and Clarke, 1988; Howe et al., 1992). Based on the relative expression of ₁-, ₂- and ₃-AR mRNA transcripts in white and brown adipose tissue of the mouse, ₃-AR appear to play a dominant role in modulating metabolism in these tissues (Collins et al., 1994). This conclusion is further strengthened by the report that a mouse strain having a selective disruption of the ₃-AR gene is unresponsive to the typical physiological and biochemical changes related to metabolism that occur in normal mice after administration of ₃-AR agonists (Susulic et al., 1995). Although ₃-AR are located mainly in white and brown adipose tissue (Muzzin et al., 1991; Nahmias et al., 1991), ₃-AR have also been identified in GI tract tract tissue (Emorine et al., 1989; Granneman et al., 1991; Bensaid et al., 1993). A role for adrenergic receptors in mediating muscle contractility in a variety of organs has been well documented (Arch and Kaumann, 1993; De Ponti et al., 1996). Selective ₃-AR agonists have been shown to inhibit motility of isolated organs, such as guinea pig ileum, rat colon, intestine and esophageal smooth muscle (Bond and Clarke, 1988; Croci et al., 1988; van der Vliet et al., 1990; deBoer et al., 1995), as well as inhibit GI motility in vivo (Giudice et al., 1989; Croci et al., 1991; Landi et al., 1993; Manara et al., 1996). In addition, a link between ₃-AR and nervous system regulation of gut motility has been proposed (Thollander et al., 1996; Yoshida et al., 1996). However, the mechanism by which ₃-AR might affect muscle contractility has not been fully elucidated.

Our results in mice have confirmed the earlier reports of a role for ₃-AR in the regulation of GI motility. The effect of ₃-AR agonists to stimulate lipolysis in WT mice, as measured by evoked glycerolemia, was consistent with their ability to stimulate adenylyl cyclase activity in cells expressing a cloned rodent ₃-AR receptor. In the in vitro assay, the relative order of activity was BRL 35135 = CL 316,243 > ICI 198,157 >> SR 56811A. A similar rank order of activity for several of these ₃-AR agonists has been reported by Manara et al. (1995) for relaxation of rat colon in vitro. In our experiments, the extent of radiotracer transit through the GI tract of WT mice was decreased after administration of these selective ₃-AR agonists. In addition, we have demonstrated that these agonists failed to evoke lipolysis or have any affect on GI motility in transgenic mice lacking ₃-AR. Thus, all of these ₃-AR agonists effects on both lipolysis and GI motility appear to be mediated exclusively through the ₃-AR in the WT mice. Our results also confirm those of Susulic et al. (1995), who have reported that the ability of the selective ₃-AR agonist, CL 316,243, to cause an increase in adipose lipolysis and whole body metabolism, as well as reduce food intake, is mediated exclusively by ₃-AR, since these effects are absent in transgenic ₃-AR knockout mice.

In our experiments, treatment of WT mice with SR 58611A (3 mg/kg; s.c.) caused no effect on GI motility. SR 58611A is effective in modulating canine and rat colonic motility both in vitro (Croci et al., 1991) and in vivo when compound was given intravenously (De Ponti et al., 1995; Manara et al., 1996). The lack of activity of SR 58611A in our experiments may be attributed to the lower in vitro potency of this compound compared to the other ₃-AR agonists we tested, and perhaps to suboptimal pharmacokinetics using our dosing regimen. The ₂-AR agonist, clonidine, was used as a positive control in our studies, based on its previously reported ability to increase GI transit time (Maugeri et al., 1994; Puig et al. 1996). Clonidine effectively decreased the extent of movement of radiotracer through the GI tract in both the WT and ₃-AR[KO] mice, demonstrating that its mechanism of action was independent of the ₃-AR receptor, and that other pathways for regulation of GI motility remain operational in the ₃-AR[KO] mouse.

The GI effects of agonists selective for the atypical (non-1, 2) beta adrenergic receptor have been linked with the detection of ₃-AR mRNA in these tissues (Berkowitz et al., 1995; Cohen et al., 1995). These reports, together with our results demonstrating that these agonists affect GI motility exclusively through ₃-AR, could readily be interpreted as evidence for a direct effect of ₃-AR receptor activity in GI tissues. However, it is possible that the molecular identification of ₃-AR in these GI tissues may be due to the wide spread distribution of adipose tissue throughout the digestive tract (Evans et al., 1996). Such low-level signals for ₃-AR mRNA, detected in skeletal muscle from 3-AR[WAT+BAT] mice, have been attributed to adipocytes resident within or surrounding this tissue (Grujic et al., 1997). Also, the absolute pharmacological selectivity of synthetic ₃-AR agonists may be reasonably questioned when attributing their effect on GI function to the activity of specific receptor populations. Therefore, we had originally postulated that the use of transgenic mice lacking ₃-AR, and a range of rodent-specific ₃-AR agonists, would validate the presence of ₃-AR in the GI tract and their potential role in modulating GI motility. The differential effect of three synthetic, rodent-specific ₃-AR agonists on GI motility in the WT and 3-AR[KO] mice are consistent with this supposition, as well as attest to the selectivity of these agonists for the ₃-AR. However, the most effective of these ₃-AR agonists, BRL 35135, caused enhanced lipolysis and decreased GI motility to an equivalent extent in both the 3-AR[WAT+BAT] and WT mice. Characterization of the ₃-AR[WAT+BAT] transgenic mouse has shown that ₃-AR are present only in brown and white adipose tissue, and that these mice respond to administration of the selective ₃-AR agonist, CL 316,243, with the full range of increased lipid metabolism and thermogenesis that is seen in normal, WT mice (Grujic et al., 1997). Our results suggest that ₃-AR agonists, or at least BRL 35135, is capable of regulating GI motility indirectly and exclusively as a consequence of its action on ₃-AR in adipose tissue. It is known that, along with up-regulation of lipolysis and glycogenolysis, ₃-AR agonists acutely elicit increased serum insulin levels (Arch and Kaumann, 1993), a response that is absent in the transgenic ₃-AR knockout mouse (Susulic et al., 1995). Both hyperinsulinemia and hyperglycemia have been shown to cause decreased GI motility (Eliasson et al., 1995; Chang et al., 1995). Therefore, it is plausible that ₃-AR agonists are capable of causing reduced GI motility independent of receptors located in the GI tract via mechanisms secondary to their direct effects on adipose tissue, and presumably consequent upon mediators released during a general increase in whole body metabolic activity. Our results do not negate the possibility that ₃-AR are normally present in GI tissue and may also contribute to regulation of GI motility. A comparison of the in vitro responses of GI tissues from WT, 3-AR[KO] and 3-AR[WAT+BAT] mice upon exposure to the these ₃-AR agonists would be highly enlightening on this point.

In summary, the effect of several selective ₃-AR agonists on GI motility were compared in WT, 3-AR[KO] and 3-AR[WAT+BAT] mice. The ability of these agonists to caused a decrease in the extent to which radiotracer moved through the GI tract reflected their ability to stimulate lipolysis in WT mice. None of these agonists were effective in modulating GI motility or evoking lipolysis in mice totally lacking ₃-AR. However, BRL 35135 effectively increased lipolysis and decreased GI motility in 3-AR[WAT+BAT] mice, suggesting that ₃-AR agonists are able to effect GI motility as a consequence of their effects on adipose tissue alone. We postulate that these effects may include the products of increased lipolysis and thermogenesis in adipose tissue, and a change in circulating hormones, such as insulin, associated with regulation of overall body metabolism, and which are known to modulate GI motility. Therefore, our results are further evidence that adipocytes, not unlike other specialized groups of cells, can modulate the physiological responses of other tissues and organs through humoral mechanisms (Spiegelman and Flier, 1996).