The submandibular glands (SMGs) were removed in male genetically obese yellow Ay mice at 24 weeks of age. The mice lost weight postoperatively unlike the sham-operated controls. Furthermore, the coat color changed after sialoadenectmy from yellow to black on part of the back. The pattern and tone of the black regions varied from mouse to mouse. The black color was conspicuous 4 to 8 weeks postoperatively, then gradually faded, but was still noticeable for at least 5 months. In Ay mice, the agouti gene is overexpressed, and the pleiotropic effects of excess agouti protein are believed responsible for their obesity and yellow coat color; agouti antagonizes both the hypothalamic melanocortin-4 receptor responsible for obesity and the skin melanocortin-1 receptor responsible for yellow pigmentation. Our observations suggest that a factor or factors in the SMG are essential for the expression of two separable phenotypes. The SMG presumably affects the expression level or action or both of agouti to alter the phenotypes.
INTRODUCTION
The genetically obese yellow mouse (Ay) is a result of the dominant mutation of the agouti gene, which encodes a 131 amino acid protein, agouti (Bultman et al., 1992). The two phenotypes, obesity and yellow coat color, are believed to be derived from pleiotropic effects of agouti due to overexpression of the agouti gene (Miltenberger et al., 1997). However, the factors that control the agouti gene or agouti protein remain to be determined.
We have reported that body weight gain in Ay mice is retarded after the submandibular glands (SMGs) are removed (Yamashita et al., 1996a). In an attempt to clarify the mechanism of weight loss, we found that in mice sialoadenectomized at 24 weeks of age the weight loss was accompanied by a change in dorsal coat color from yellow to black. This phenomenon has intrigued us, because sialoadenectomy apparently affected two separable phenotypes and suggests that the SMG controls the expression of agouti.
MATERIALS AND METHODS
The C57BL/6J-Ay mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and bred with their lean black siblings at our laboratory in stocks propagating induced mutants. Eight male siblings of similar age, derived from five mothers, were divided randomly into sialoadenectomy and sham-operation groups of four mice each. Siblings from the same mother, however, were placed into different groups. All mice were operated on under ether anesthesia at 24 weeks of age as described previously (Yamashita et al., 1996a), housed in individual plastic cages (18 × 12 × 11 cm), and kept in a vivarium maintained at 23 ± 2°C and 65 ± 5% relative humidity with a 13:11 hr light:dark cycle with light on at 07:00. Mice were given free access to standard laboratory chow (MF; Oriental Yeast, Tokyo, Japan) and water.
RESULTS AND DISCUSSION
The coat color of all four sialoadenectomized mice changed from yellow to black on part of the back. Although specifying when the black pigmentation started is difficult, the black color was conspicuous 4 to 8 weeks after sialoadenectomy. The pattern and tone of black color varied among the mice (Fig. 1). Mouse 1 showed the color change not only over a large area of the back but also in the skin around the mouth. Mouse 2 had a small black spot only on the central part of the back. The intensity and extent of black pigmentation in mouse 3 and 4 were intermediate between those in mice 1 and 2. However, no color changes were observed in any of the four sham-operated mice throughout the 7-month observation period.
The black color gradually faded, though it was still recognizable 5 or more months later. Since no regeneration of the SMGs was observed, it is possible that some other organ might have compensated for the removed SMGs.
All four sialoadenectomized mice lost weight after the operation: their body weights 4 and 6 months after operation ranged from 35.6 to 39.8 g and from 41.5 to 47.9 g, respectively compared with from 44.7 to 48.8 g before sialoadenedtomy. In contrast, the weights of all four control mice increased from 41.5 to 47.9 before sham operation to 48.8 to 58.9 g after 6 months. These results are similar to those of our previous study (Yamashita et al., 1996a).
The obesity and yellow coat color Ay mice are believed to result from pleiotropic effects of excessive agouti protein: agouti antagonizes both the hypothalamic melanocortin-4 receptor and the skin melanocortin-1 receptor (Miltenberger et al., 1997). Our observations suggest that a factor or factors in the SMG are essential for the production of the two phenotypes. The SMG presumably plays a role in controlling the level or action or both of agouti to alter the phenotypes. Sialoadenectomy in Ay mice may improve the expression of agouti.
The SMG produces epidermal growth factor (EGF), which is released into both the bloodstream and the saliva (Nexø et al., 1981). EGF injected into the cerebral ventricle depresses food intake in rats (Plata-Salamán, 1988). We have confirmed that EGF administered intraperitoneally also affects feeding behavior in mice (Yamashita et al., 1996b). Therefore, EGF produced by the SMG may regulate energy balance related to the function of agouti.
We have reported that sialoadenectomy does not affect obesity in ob/ob mice (Yamashita et al., 1996a), which become obese due to lack of leptin (Zhang et al., 1994). Thus, the system mediated by the SMG is either independent of the system related directly to leptin or contributes to a downstream factor of the leptin system.
Interestingly, sialoadenectomy at 24 weeks produced black pigmentation in all four mice in the present experiment; however, in our previous studies of weight loss, we did not consistently observe black pigmentation when sialoadenectomy was performed at other times. Further studies with mice sialoadenectomized at various ages are required to clarify the contribution of the SMGs to the agouti expression.