INTRODUCTION

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SURGICAL BODY FAT REMOVAL (lipectomy) leads to compensatory fat deposition in a variety of small rodents that are seasonal breeders [i.e., Siberian hamsters (Phodopus sungorus sungorus), Refs. 17-21; Syrian hamsters (Mesocricetus auratus), Ref. 12; and ground squirrels (Spermophilus lateralis), Ref. 7]. In Siberian hamsters, the restoration of body fat stores after lipectomy is fat pad specific. For example, the inguinal (I) white adipose tissue (WAT) pads of long day-housed hamsters show compensatory mass increases in response to epididymal WAT (EWAT) removal, but EWAT does not increase its mass in long day-housed IWAT-lipectomized (IWATx) hamsters (17). These latter data suggest that EWAT may be resistant to the regulatory signals that restore body fat content in response to surgically induced body fat deficits. However, we have only tested the response of the more internally located EWAT fat pads to removal of the externally located subcutaneous IWAT fat pads. It therefore remains to be determined whether the EWAT of Siberian hamsters lacks responsiveness to total body fat deficits in general or whether EWAT specifically may be unresponsive to IWAT removal. By removal of EWAT from one side only, it can be determined whether EWAT is responsive to the removal of a similarly located and functionally equivalent WAT pad (i.e., its contralateral mate) or whether it is as unresponsive to EWAT deficits as it is to IWAT excision.

The unilateral removal of fat pads can also address questions about the laterality of body fat compensation. That is, by creating side-specific lipid deficits, we can test whether the body fat compensation that occurs is confined to the side from which body fat is removed or whether it is exhibited bilaterally. The potential for a lateralized compensatory fat pad mass response to unilateral lipectomies exists because of the unilateral postganglionic sympathetic nervous system (SNS) innervation of WAT in Siberian hamsters and the involvement of the SNS in the naturally occurring fat pad mass decreases associated with short day exposure in this species (32). If, however, a bilateral compensatory response to a unilateral lipid deficit were observed, this would suggest the existence of a more general neurally mediated or humoral response.

By pairing unilateral EWAT or IWAT excision with bilateral or sham EWAT-lipectomized (EWATx) and IWATx, varying amounts of fat can be removed. This enables us to determine the relationship between the magnitude of compensatory mass increases and the amount of fat removed, ultimately providing for a more quantitative approach in determining how tightly body fat may be regulated. Using a combination of EWATx and IWATx, we also can test whether the compensation that occurs in nonexcised fat pads after lipectomy depends predominantly on the absolute amount of fat removed or whether the location of the removed fat affects the ability to compensate as well as the pattern of compensatory mass increases.

Therefore the purpose of the present study was to answer the following questions. 1) Is there a compensatory increase in EWAT mass after removal of its contralateral counterpart? 2) Do the compensatory increases in WAT pad mass occur in a side-specific manner (i.e., unilaterally) after unilateral lipectomy? 3) Is the compensatory increase in WAT pad mass after partial lipectomy proportional to the amount of body fat removed? This was accomplished in experiment 1 by performing bilateral, left unilateral, right unilateral, or sham EWAT removal. In experiment 2, various combinations of unilateral or bilateral EWATx and/or IWATx were done. Twelve weeks after surgery, nonexcised fat pad mass and cellularity were measured to determine the compensatory responses of Siberian hamsters to the various combinations of fat pad removal.

	METHODS

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Animals and Housing

Surgical Procedures

Experiment 1. At ~3 mo of age, male Siberian hamsters were single housed. After acclimation to single housing, they were divided into five groups matched for body mass and percent body mass change (n = ~10/group). One group (week 0, unoperated) was killed at week 0 to provide baseline measurements of testes, EWAT, IWAT, and retroperitoneal WAT (RWAT) mass and fat pad cellularity. The remaining four groups underwent bilateral EWATx (~5% of total body fat; unpublished observations), left EWATx, right EWATx, or sham surgery. Pentobarbital sodium anesthesia (~50 mg/kg) was used for all surgeries. EWATx was performed by making a single abdominal incision through which both EWAT pads were accessed. Care was taken to minimize disruption of the blood supply to the testes. The peritoneum was closed with silk sutures and the skin was closed with wound clips. Nitrofurazone powder was applied to the skin. Sham surgery only differed in the lack of fat pad removal. For unilateral EWATx hamsters, sham EWATx was conducted on the contralateral nonexcised EWAT pad.

Experiment 2. Ninety-three 2.5- to 3.0-mo-old hamsters were divided into three groups, a bilateral IWATx group, a unilateral IWATx group, and a sham IWATx group (sham). Surgery groups were matched for body mass. Unlike the EWATx hamsters in experiment 1, these animals remained in group housing before surgery and for several days of recuperation after surgery, because IWATx hamsters have a significantly greater survival rate when group housed (unpublished observations). IWAT pads (~10% of total body fat; unpublished observations) were removed via blunt dissection through bilateral dorsal incisions. For sham surgery, IWAT was dissected from the skin but not from underlying muscle. After ~1 wk of recovery, the bilateral IWATx animals were further divided into bilateral or unilateral EWATx surgery groups, and the unilateral IWATx hamsters were further divided into bilateral, unilateral, or sham EWATx groups. These groups were also balanced for body mass. EWATx surgeries were conducted as described for experiment 1, and the hamsters were single housed after these latter surgeries. Note that when IWAT or EWAT was removed unilaterally in this experiment, it was removed from the left side of the body, and the nonexcised fat pad (right IWAT or EWAT) underwent sham surgery, so that for each animal all four fat pads (right and left IWAT and EWAT) were either removed or sham-operated. The final groups included in experiment 2 were as follows: 1) bilateral EWATx and bilateral IWATx (Bi IWATx + Bi EWATx, n = 17); 2) bilateral IWATx and unilateral EWATx (Bi IWATx + Uni EWATx, n = 19); 3) unilateral IWATx and bilateral EWATx (Uni IWATx + Bi EWATx, n = 15); 4) unilateral IWATx and unilateral EWATx (Uni IWATx + Uni EWATx, n = 14); 5) unilateral IWATx (Uni IWATx, n = 10); and 6) sham IWATx and sham EWATx (n = 13).

Food Intake, Body Mass, and Terminal Measures

Statistical Analysis

	RESULTS

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Body Mass and Composition

Food Intake

Fat Pad Mass and Cellularity

EWAT. There was no significant EWAT regrowth after EWATx. There also were no significant compensatory mass increases or cellularity changes by the nonexcised EWAT fat pad of unilateral EWATx hamsters (Fig. 1, Table 1) or by the nonexcised EWAT pads of bilateral or unilateral IWATx hamsters (Fig. 1, Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Epididymal white adipose tissue (EWAT) mass (means ± SE) expressed as percentage of mass of corresponding EWAT pad of hamsters undergoing sham surgery (sham). Groups are identified by shading of hamster representations so that dark fill within upper quadrants indicates inguinal fat pad removal and dark fill within lower quadrants indicates EWAT removal. When a single bar is shown for a group, it represents the intact EWAT pad for that group. There was no statistically significant compensation (P > 0.05) by intact EWAT after removal of its contralateral and/or removal of inguinal white adipose tissue (IWAT).

IWAT. Similar to unilateral EWATx, unilateral IWATx did not produce significant compensatory mass or cellularity changes in the nonexcised contralateral fat pad (P < 0.05; Fig. 2, Table 2). However, when both IWAT pads were intact, total IWAT mass was increased by unilateral and bilateral EWATx. Bilateral EWATx hamsters had larger left and right IWAT pads relative to those of sham and week 0 hamsters (P < 0.05 for each), whereas unilateral EWATx hamsters showed side-specific compensatory mass increases (P < 0.05). That is, left EWATx hamsters had larger left, but not right, IWAT pads than sham hamsters. Although right EWATx hamsters did not show statistically significant fat pad mass increases, they were the only hamsters with both IWAT pads intact to overcome the inherent difference in left and right IWAT mass (present findings; Youngstrom and Bartness, unpublished observations) and thus to have a right IWAT pad that was as large as the left. Furthermore, right EWATx hamsters had larger IWAT fat cells overall compared with those of sham hamsters (P < 0.05; Table 1). This increase was due to a nonsignificant increase of 73% in right (0.05 < P < 0.10) but not left IWAT adipocyte size (8% decrease) relative to comparable fat cells from sham hamsters (Table 1). It should be noted that although the differences between left and right IWAT cellularity were statistically nonsignificant in every group, the number of animals that had left and right fat pads processed separately for cellularity determination was relatively small (n = 3 or 4; Table 1). Finally, comparisons between the compensatory responses (percent sham values) of the left and right IWAT pads showed that left EWATx hamsters tended, albeit to a statistically nonsignificant degree, to have greater compensation in left IWAT than in the right IWAT (P = 0.06), whereas right EWATx and bilateral EWATx hamsters had significantly greater compensation in the right IWAT than in the left IWAT pad (P < 0.05 for both; Fig. 1).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   IWAT mass (means ± SE) expressed as a percentage of mass of corresponding left or right IWAT pad of sham hamsters. Groups are defined by hamster representations as described in Fig. 1. a Statistically significant (P < 0.05) differences in fat pad mass vs. corresponding left or right IWAT pad of sham hamsters. b Statistically significant difference in compensatory response of right IWAT pads vs. left IWAT pads within a group.

RWAT. In contrast to IWAT, RWAT did not show significant compensatory mass or cellularity changes after EWATx alone (P > 0.05 for each; Fig. 3), with the exception of increases in the size of RWAT adipocytes from left EWATx hamsters relative to week 0 baseline and sham hamsters (Table 1). However, all groups from which bilateral or unilateral IWATx and EWATx were combined had greater left, right, and total RWAT masses than did sham hamsters (P < 0.05 for all; Fig. 3). The total fat removed by lipectomy was a significant positive predictor of compensation by this fat pad (P < 0.05, n = 102), so that for every gram of fat removed, RWAT was predicted to show compensatory increases of ~15% above sham values. Aside from the RWAT adipocyte size increase in left EWATx hamsters, there were no statistically significant increases in RWAT adipocyte cellularity after lipectomy. However, the groups from which the largest amount of fat was removed (i.e., Bi IWATx + Uni EWATx and Bi IWATx + Bi EWATx) tended to have a greater, but statistically nonsignificant, number of RWAT adipocytes compared with sham hamsters (0.05 < P < 0.10 for both groups; Table 2). Although there were no statistically significant compensatory increases in the left or right RWAT mass or cellularity of unilateral IWATx hamsters, this group differed from all other groups in that its left RWAT, ipsilateral to unilateral IWAT removal, was larger than its right RWAT (P < 0.05; Fig. 3). However, the laterality effect of unilateral EWATx did not reach statistical significance (P = 0.07).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Retroperitoneal white adipose tissue (RWAT) mass (means ± SE) expressed as a percentage of mass of corresponding left or right RWAT pad of sham hamsters. Groups are defined by hamster representations as described in Fig. 1. a Statistically significant (P < 0.05) differences in fat pad mass vs. corresponding left or right RWAT pad of sham hamsters. b Statistically significant differences in compensatory response of left RWAT pads vs. right RWAT pads within a group.

DWAT. Consistent with previous findings, DWAT showed slight, but statistically nonsignificant, increases in mass after lipectomy (Fig. 4). Although neither left nor right DWAT mass was increased to a significant degree relative to that of sham hamsters, unilateral EWATx resulted in a significant laterality effect such that the left DWAT of right EWATx hamsters was significantly larger than its right counterpart (Fig. 4; P < 0.05). DWAT cellularity was unaffected by lipectomy (P > 0.05 for cell size and number; Table 2). Despite the relatively small compensatory response of DWAT, the slight compensation observed in this fat pad was positively (~12% per gram fat removed) and significantly associated with the total fat removed (P < 0.05, n = 101).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Dorsoscapular white adipose tissue (DWAT) mass (means ± SE) expressed as a percentage of mass of corresponding left or right DWAT pad of sham hamsters. Groups are defined by hamster representations as described in Fig. 1. There were no statistically significant differences in left or right DWAT pad mass of lipectomized hamsters compared with sham hamsters (P > 0.05). b Statistically significant differences in compensatory response of left DWAT pad vs. right DWAT pad within a group.

Testes. Testicular damage was associated with EWATx, but not IWATx. As a result, all EWATx groups had smaller testes than did sham hamsters (P < 0.05 for all; means ± SE, bilateral EWATx = 0.468 ± 0.015, unilateral EWATx = 0.757 ± 0.026, sham EWATx = 0.934 ± 0.029). In unilateral EWATx hamsters only the testis from the excised side was smaller than that of sham hamsters. There was no significant association between paired testis mass and fat pad compensation (P > 0.05 for each fat pad).

	DISCUSSION

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Decades ago, Kennedy (14) hypothesized that total body fat was regulated; however, strong support for this hypothesis and direct tests of it have only recently been achieved through studies using the partial surgical lipectomy paradigm. In the present study, we used various combinations of unilateral and bilateral EWATx and IWATx to test the laterality and fat pad-specific nature of body fat compensation. We found that lipid deposition after unilateral lipectomy can occur in a side-specific manner. In addition, we found that EWAT pads were as unresponsive to removal of their contralateral fat pad counterparts as they were to removal of IWAT, in terms of fat pad mass compensation.

In the present study, unilateral fat pad excision resulted in slightly greater compensatory mass increases ipsilateral to fat removal in RWAT and IWAT after unilateral IWATx and EWATx, respectively. However, the unilateral fat pad compensation only was observed when a relatively small amount of fat was removed (i.e., unilateral EWATx or IWATx). Thus larger deficits in total body fat may override these unilateral effects by triggering additional mechanisms that promote more generalized compensatory lipid deposition. Given our previous findings of unilateral SNS and sensory innervation of WAT in Siberian hamsters (27, 32), the side-specific effects observed after unilateral lipectomies suggest that neural mechanisms may be involved in body fat compensation after partial lipectomy. Thus, although the lateralization effects observed after unilateral EWATx or IWATx were not of great magnitude, coupled with WAT SNS and sensory innervation, their existence should provide the impetus for further investigation into a possible role of neural mechanisms in body fat regulation.

The finding that the nonexcised EWAT pad did not compensate for removal of its contralateral mate 3 mo after unilateral lipectomy contrasts with the reported increase in intact EWAT mass 3 days after unilateral excision of EWAT in rats (6). Our finding is consistent, however, with longer term studies in ground squirrels (9) and laboratory rats (24) in which intact EWAT does not compensate 6 and 8 mo, respectively, after unilateral EWAT removal. This lack of an increase in mass or adipocyte size and/or number in the contralateral intact pads of unilateral EWATx hamsters in the present study suggests that the hypertrophy we have previously observed in the EWAT adipocytes remaining after bilateral EWATx in this species (21) was not an attempt at compensation. Instead, this hypertrophy more likely was due to the normal deposition of lipid into a greatly reduced number of fat cells. Thus, although EWAT is the most responsive of the fat pads in terms of the degree and rapidity of the mass loss that occurs in this species after initial short day exposure and is the earliest fat pad to begin restoring its mass after prolonged short day exposure (4), it appears to be generally unresponsive to lipectomy in that it fails to show compensatory mass increases after removal of internal or subcutaneous fat (present findings; 17, 19). In addition, we have previously shown that when the short day-induced decrease in lipid stores is experimentally accelerated by IWATx, EWAT does not show a reduction in its rate of fat pad mass loss as do other WAT pads (19). EWAT also is relatively unresponsive to obesity-producing lesions of the paraventricular nucleus of the hypothalamus (PVN; 20) and to lipid depletion through food restriction in this species (20). Thus the processes that produce natural body fat losses and gains in response to the photoperiod may be different from those that restore body fat after surgical lipectomy. Alternatively, the lack of response of EWAT to manipulations that are not coupled with testicular changes (i.e., IWATx, PVN lesions, and food deprivation) may be due to the close functional relationship between this fat pad and the testes.

The significant predictive value of the amount of body fat removed on fat pad compensation in the two nonexcised WAT depots, RWAT and DWAT, and the lack of correlation between the amount of body fat removed and the carcass lipid present at week 12 suggest that total body fat content is regulated in a rather precise manner. This relationship is especially impressive when one considers that removal of a greater amount of fat not only increases the difficulty of restoring body fat content (because of the greater amount of lipid deposition necessary to overcome lipectomy-induced body fat deficit) but also decreases the size of the storage depots available for lipid deposition.

Although EWATx produced some testicular damage, it is unlikely that such damage was responsible for the compensatory response of fat pads to lipectomy. First, bilateral EWATx does not produce significant decreases in serum testosterone levels (unpublished observations). Second, unlike laboratory rats (31) or Syrian hamsters (26), Siberian hamsters decrease body and lipid mass after castration and increase these measures after testosterone treatment (2, 18, 28, 29). Thus, even if there was a lipectomy-induced deficit in testicular androgen secretion, this would oppose compensatory increases in body fat stores after lipectomy. Finally, the nonsignificant association between testes mass and fat pad compensation in the present experiments further supports the notion that EWATx-associated testicular damage neither enhances nor diminishes the compensatory response.

In the present study, we have extended our knowledge of the hypothesized regulatory system responsible for the recovery of lipid stores after lipectomy, concluding that the pattern of body fat recovery not only is fat pad specific but may also be dependent on the side from which fat is removed. The laterality effects observed in the present study, though not robust, were very consistent within IWAT, the most responsive of the compensating fat pads; our findings therefore add new evidence to that recently accumulated (27, 32; Youngstrom and Bartness, unpublished observations) implicating neural mechanisms in the assessment and/or restoration of lipid content. Thus total body fat content appears to be rather precisely maintained through a regulatory system that may be capable of sensing the location of body fat removal (left vs. right) and that, with great flexibility, stores excess lipid in nonexcised depots in an effort to restore total body fat content.

Perspectives

As for the output component of this system, our present findings suggest that multiple mechanisms, which may include changes in WAT SNS activity, are responsible for the total compensatory response after lipectomy. Given that the maintenance of seasonally appropriate body fat content may be crucial to both the survival and the reproductive fitness of seasonal breeders, it should not be surprising that a number of redundant mechanisms, including neural, hormonal, metabolic, and behavioral changes, probably underlie the restoration of total body fat after lipectomy specifically, and the apparent regulation of total body fat generally.